The management of unresectable or metastatic gastrointestinal stromal tumors (GISTs) has previously been difficult as they are resistant to conventional chemotherapy and radiation. The development of imatinib mesylate has made a major impact on the management of advanced GISTs. It is apparent that there are sanctuary sites such as the central nervous system where imatinib does not achieve adequate concentrations. We describe the case of a man with metastatic GIST who experienced multiple cerebral relapses of disease while systemic disease progression appeared to be controlled by imatinib.A 47-year-old man presented in July 1999 with a jejunal GIST with multiple hepatic metastases. The jejunal primary was resected and after unsuccessful cytoreductive chemotherapy, the liver metastases were also resected in December 1999. The patient subsequently relapsed in August 2001 with symptomatic hepatic, subcutaneous gluteal, left choroidal and right ocular metastases all confirmed on CT and PET scanning. Biopsy confirmed recurrent GIST. MRI and lumbar puncture excluded central nervous system involvement. The patient was commenced on imatinib 400 mg bd in September 2001 through a clinical trial.The symptoms improved with objective PET and CT scan response until December 2002 when the patient developed a right-sided foot drop. MRI scan showed a left parasagittal tumor which was resected and confirmed histologically to be metastatic GIST. Imatinib was ceased pre-operatively due to the trial protocol but recommenced in February 2003 on a compassionate use program. The left parasagittal metastasis recurred and required subsequent re-excision in September 2003 and January 2004. Control of the systemic GIST was temporarily lost on reduction of the dose of imatinib (due to limited drug supply) but on increasing the dose back to 800 mg per day, systemic disease was stabilized for a period of time before generalised progression occurred.This case illustrates that the brain can be a sanctuary site to treatment of GISTs with imatinib. Maintaining dosing of imatinib in the face of isolated sites of disease progression is also important, as other metastatic sites may still be sensitive. This oral drug is also active in all phases of chronic myeloid leukemia (CML) as it also targets the bcr-abl tyrosine kinase. It is apparent that there are sanctuary sites such as the central nervous system where imatinib does not achieve adequate concentrations. We describe the case of a man with metastatic GIST who experienced multiple cerebral relapses of disease while systemic disease progression appeared to be controlled by imatinib.Gastrointestinal stromal tumors (GISTs) are rare mesenchymal gastrointestinal tumors which can have an aggressive course. Management of these tumors apart from surgical resection has been difficult in the past because they are resistant to conventional chemotherapy  and radiA 47-year-old man presented in July 1999 with melena. A small bowel series and CT abdomen showed a jejunal mass and a 5 × 5 cm complex hepatic mass. A biopsy of the liver lesion revealed a spindle cell tumor. Laparotomy was performed, and the 7.5 cm jejunal mass was resected. A total of 4 liver lesions were noted, but not resected. The histopathology confirmed a gastrointestinal stromal tumor with clear resection margins. He subsequently had 4 cycles of attempted cytoreductive chemotherapy using doxorubicin and dacarbazine. A repeat CT scan showed progression of the liver metastases. An extended right hemihepatectomy was performed in December 1999 with successful excision of all 4 liver metastases.The patient remained well until review in August 2001 when he complained of lethargy, right upper quadrant abdominal pain, diplopia and blurred vision in the left eye. Examination revealed weakness of the right lateral rectus ocular muscle, an amelanotic left choroidal lesion, hepatomegaly and a subcutaneous nodule in the gluteal region. An enhancing lesion in the infero-lateral region of the right globe was confirmed on CT which was thought to be the cause of the ocular muscle weakness. The CT scan also demonstrated a recurrence of multiple hepatic metastases. There were no intracerebral lesions. The gluteal lesion was biopsied and confirmed recurrence of GIST (CD117 positive). A positron emission tomogram (PET) scan also confirmed disease recurrence in the same distribution. Lumbar puncture and MRI scan of the brain excluded the presence of leptomeningeal or cerebral metastases. The patient was commenced on imatinib mesylate  400 mg bd in September 2001 as part of a clinical trial. He felt much improved by November 2001 when a repeat CT abdomen showed stable disease but PET scan now showed no FDG uptake. By January 2002 his diplopia had completely resolved but the left-sided choroidal lesion remained unchanged on fundoscopy. The buttock lesion had also resolved.th January 2003 with complete resection of the lesion. Histopathology demonstrated metastatic GIST (CD117 positive) , the patient's dose of imatinib mesylate was reduced to 400 mg per day in March 2003. Six weeks later, his diplopia had returned and a progress CT abdomen demonstrated a minor progression of the liver lesions. His liver function tests remained normal. A subsequent PET scan again showed no abnormal uptake despite disease progression on the CT scan. His dose of imatinib mesylate was increased initially to 600 mg per day with resolution of the diplopia.By May 2003 his foot drop had worsened and his dose of imatinib mesylate was increased back to 800 mg per day. Despite the increase in dose, his foot drop worsened. A repeat CT brain demonstrated a recurrence of the cerebral metastasis with surrounding vasogenic oedema in the previous site of resection. A repeat abdominal CT showed no significant change in the size of the liver metastasis and mild shrinking of the nodule in the buttock.th September 2003. A small residual area of tumor was seen on the postoperative scan. Abdominal imaging two months later showed that two of the liver lesions had increased in size with the other areas stable. He was not a candidate for further hepatic resection as there was insufficient liver reserve due to the past surgery. Radiofrequency ablation was declined as the patient did not have symptoms referable to the area.Due to limited treatment options available for the cerebral metastasis, a re-resection of the cranial metastasis was offered. A repeat MRI of the brain in late July 2003 confirmed the presence of the left parasagittal lesion with surrounding edema but no mass effect. Repeat craniotomy and incomplete debulking of the parasagittal metastasis was performed on the 9The patient remained well until December 2003 when he experienced symptoms of headaches, worsening diplopia, right foot drop and left arm weakness. MRI scan confirmed recurrence of the cerebral metastasis with extension across the falx cerebri. He declined cranial radiotherapy treatment at the time and instead underwent a third resection of the lesion in January 2004 where incomplete debulking was achieved with early improvement of his limb weakness. In April 2004 the patient enrolled into a randomised placebo controlled clinical trial of a novel multi-kinase inhibitor SU-11248 (Pfizer) for the treatment of imatinib refractory GIST. However his condition slowly deteriorated and he died in July 2004.This case illustrates a man who has had evidence of presumed ocular involvement by the GIST that initially responded to imatinib who had a cranial relapse while the systemic disease initially remained controlled. This would imply that the central nervous system could be a sanctuary site where the imatinib mesylate does not achieve adequate levels. Preclinical mice models of CML have shown development of central nervous involvement on imatinib despite systemic disease control with cerebral spinal fluid levels being 155 times lower than plasma . This iskit and clinical outcomes of GISTs on imatinib [502 and Tyr503 This may be marker for malignant course of the disease as 71% have a highly malignant course and 59% arise exclusively in the small intestine [There appears to be a relationship between the presence of different activating kinase mutations of imatinib . The exontestine . The difntestine . The absThe central nervous system can be a sanctuary site to treatment of GIST with imatinib mesylate. Prolonged control of the recurrences in this location was achieved by repeated resections. This patient also highlights the importance of maintaining dosing of imatinib mesylate in the face of isolated sites of disease progression, as other metastatic sites may still be sensitive. Cessation and lowering of the dose of the drug led to temporary loss of systemic control in this case.David Goldstein and Paul Waring have both consulted and spoken on behalf of Novartis Pharma on imatinib and GIST.BH, DY, DG and GC participated in the clinical care of the case. PW and VB performed the mutation analysis and prepared the histopathology images. BH and DY drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Prostate cancer continues to occur in over 230,000 men each year. Although the majority of these will be diagnosed in the early stages, there remains a proportion who will either be diagnosed in late stage disease or develop progressive disease. In patients with advanced disease, research has recently focused on using chemotherapy for symptom management and palliation. Given that the focus is not on cure, the effect of chemotherapy on quality of life is of utmost importance. The present article will 1) summarize the current chemotherapeutic studies that have included a quality of life component, with a particular focus on pain and fatigue, 2) discuss the issue of distress in advanced prostate cancer patients treated with chemotherapy, and 3) suggest future research directions.From the studies that have investigated quality of life, it appears that several chemotherapeutic agents reduce pain and fatigue, although the development of fatigue is often the dose-limiting factor of some agents. The assessment of overall quality of life has occurred in several studies, however, an examination into the impact of chemotherapy on functional status and interpersonal relationships has not been studied. Finally, in contrast to the numerous studies in early stage prostate cancer patients, the presence and effect of distress in chemotherapy-treated prostate patients has not been examined. As such, increased attention is needed to quality of life during phase I-III chemotherapy trials. Prostate cancer will be diagnosed in an estimated 230,110 men during 2004 [For men with late stage disease, chemotherapy is increasingly being examined as a treatment option, although the goal is usually palliative in nature and may not extend length of survival ,3. ChemoThe inclusion of QOL endpoints in chemotherapy trials with cancer patients started in earnest during the last decade, with the majority of studies assessing the impact of chemotherapeutic agents on symptoms such as pain and fatigue. Since then, the inclusion of QOL endpoints has become a more common outcome in chemotherapy studies, although many continue to neglect the psychological component, focusing instead on the occurrence of symptoms and their impact on physical QOL.A comprehensive search of English-language articles in PubMed was conducted to identify studies that had assessed quality of life and/or emotional distress as part of chemotherapeutic trials in advanced cancer patients Table . The keyIn one of the first studies to examine the impact of chemotherapy on pain, Tannock, Osoba, Stockler et al.  randomizPain was also assessed in several studies utilizing docetaxel either alone or in combination with estramustine in androgen-independent prostate cancer -9. In onIn a recent study, docetaxel was administered intravenously on a daily basis for 6 consecutive weeks followed by a 2-week break between each of up to 4 cycles . The autReductions in pain have also been observed in studies using suramin and epirubicin. In a study by Small et al. , compariThe use of the more commonly known platinum compounds  have also demonstrated beneficial efforts on pain reduction. In advanced prostate cancer patients treated with a carboplatin/epirubicin/etoposide regimen, pain relief was observed in 44% of the patients .The overall point of these studies is that for prostate cancer patients with advanced disease, there are a variety of chemotherapeutic agents that have beneficial palliative effects, specifically pain reduction. Despite this, there is no definitive palliative pain regimen in this population and no defined algorithm for pain management using chemotherapy.Several studies have investigated the relationship between chemotherapeutic agents and fatigue in prostate cancer patients. Among the agents studied are the taxanes, specifically docetaxel and paclitaxel. In phase I studies of docetaxel and estramustine, the presence of fatigue has been used to identify the dose-limiting factor ,22. CurrFatigue was also an endpoint in a well-designed study that assessed the effect of 25 mg/m2 epirubicin administered intravenously on a weekly basis compared to 100 mg/m2 administered every 4-weeks . After fIn contrast to the numerous QOL studies of early-stage prostate cancer patients, beyond pain and fatigue, the assessment of QOL in advanced stage prostate cancer patients is relatively lacking. By and large there are no studies assessing the common areas of physical well-being  or psychological well-being , and social or family well-being in chemotherapy treated patients. The notable exception is a study of the QOL of 44 hormone refractory prostate cancer patients and their partners by Kornblith et al. . In a phIn contrast to the various studies that have investigated the presence of emotional distress, depression, and anxiety in early stage prostate cancer patients, there is remarkably little concerning these issues in those with advanced cancer. Indeed, a Pubmed search using the terms "distress AND advanced prostate cancer" resulted in only 12 studies. Of these, only 3 studies specifically focused on advanced prostate cancer -27, withChemotherapy is being examined with greater frequency in the treatment of advanced prostate cancer. Since the goal of treatment for these individuals is more likely to be palliation than cure, understanding the impact on QOL is that much more important. For example, understanding the impact on QOL can assist clinicians and patients in decision-making by providing data on whether symptom benefit obtained outweighs the toxicities from treatment.Thus far, several studies have identified improvements in pain and fatigue as a result of chemotherapeutic protocols, although in some studies, the presence of fatigue is a dose-limiting factor. Disappointing is the lack of focus on the more global aspects of quality of life, the impact on physical, psychological, social, and family well-being. How the treatment of the individual affects their relationships with others  has yet to be studied in this population. Also yet to be studied is the impact on emotional well-being. The presence of distress in cancer patients is well-documented , and it


Video capture virtual reality (VR) uses a video camera and software to track movement in a single plane without the need to place markers on specific bodily locations. The user's image is thereby embedded within a simulated environment such that it is possible to interact with animated graphics in a completely natural manner. Although this technology first became available more than 25 years ago, it is only within the past five years that it has been applied in rehabilitation. The objective of this article is to describe the way this technology works, to review its assets relative to other VR platforms, and to provide an overview of some of the major studies that have evaluated the use of video capture technologies for rehabilitation. Two major goals of rehabilitation are the enhancement of functional ability and the realization of greater participation in community life. These goals are achieved by intensive intervention aimed at improving sensory, motor, cognitive and higher level-cognitive functions on the one hand, and practice in everyday activities and occupations to increase participation on the other hand ,2. InterVirtual reality typically refers to the use of interactive simulations created with computer hardware and software to present users with opportunities to engage in environments that appear to be and feel similar to real world objects and events -8. UsersThe objective of this article is to briefly describe the use of VR in rehabilitation, and then emphasize the unique attributes of the video capture VR to rehabilitation, including an overview of some of the major studies that have evaluated the use of this technology for rehabilitation.Virtual reality has a number of well-known assets, which make it highly suitable as a rehabilitation intervention tool . These aDuring the mid to late 1990s, virtual reality technologies first began to be developed and studied as potential tools for rehabilitation assessment and treatment intervention . The lisGiven the variety of VR platforms and the diverse clinical populations that may benefit from VR-based intervention, it is helpful to view the VR experience as a multidimensional model that appears to be influenced by many parameters. A conceptual model was developed within the context of terminology established by the International Classification of Functioning, Disability and Health (ICF)  and the The "Interaction Space" denotes the interaction that occurs when the client performs within the virtual environment, experiencing functional or game-like tasks of varying levels of difficulty, i.e., the activity component according to the ICF terminology. This interaction is influenced by user characteristics, which include personal factors , body functions  and structures . It is also influenced by the characteristics of VR platform and its underlying technology  that presents the virtual environment and the nature and demands of the task to be performed within the virtual environment.It is during the interaction process that sensations and perceptions related to the virtual experience take place; here the user's sense of presence is established, and the process of assigning meaning to the virtual experience as well as the actual performance of virtual tasks or activities occurs. The sense of presence enables the client to focus on the virtual task, separating himself temporarily from the real world environment. This is an important requirement when motor and, especially, cognitive abilities and skills are trained or restored. The concept of meaning is also thought to be an essential factor that enhances task performance and skills in rehabilitation in general ,3, and tTwo outer circles, the "Transfer Phase" and the "Real World" denote the goal of transferring skills and abilities acquired within the "Interaction Space" and eliminating environmental barriers in order to increase participation in the real world . The "Transfer Phase" may be very rapid and accomplished entirely by the client or may take time and need considerable guidance and mediation from the clinician. The entire process is facilitated by the clinician whose expertise helps to actualize the potential of VR as a rehabilitation tool.Virtual environments are experienced with the aid of special hardware and software for input (transfer of information from the user to the system) and output (transfer of information from the system to the user). The selection of appropriate hardware is important since its characteristics may greatly influence what is taking place in the Interaction Space, i.e., the way users respond  to a virtual environment . The outIn addition to specialized hardware, application software is also necessary. In recent years, off-the-shelf, ready-for-clinical-use VR software has become available for purchase. However, more frequently, special software development tools are required in order to design and code an interactive simulated environment that will achieve a desired rehabilitation goal. In many cases, innovative intervention ideas may entail customized programming to construct a virtual environment from scratch, using traditional programming languages.Video capture VR consists of a family of camera-based, motion capture platforms that differ substantially from the HMD and desktop platforms in wider use. When using a video-capture VR platform, users stand or sit in a demarcated area viewing a large video screen that displays one of a series of simulated environments. Users see themselves on the screen, in the virtual environment, and their own natural movements entirely direct the progression of the task, i.e., the user's movement is the input. The result is a complete engagement of the user in the simulated task. A single video camera converts the video signal of the user's movements wherein the participant's image is processed on the same plane as screen animation, text, graphics, and sound, which respond in real-time. This process is referred to as "video gesture", i.e., the initiation of changes in a virtual reality environment through video contact. The user's live, on-screen video image responds at exactly the same time to movements, lending an intensified degree of realism to the virtual reality experience. Video capture provides both visual and auditory feedback with the visual cues being most predominant.Myron Krueger  was the  in 1996, together with a suite of interactive, game-type environments. This platform makes use of a chroma key-based setup so that the existing background is subtracted and replaced by a simulated background. GX VR has enjoyed considerable success around the world in numerous entertainment and educational facilities including science museums and entertainment parks. During the past five years it has also begun to be adapted for use in rehabilitation and has generated great interest in clinical settings (see below). GX VR currently offers a wide variety of gaming applications including, Birds & Balls, wherein a user is required to touch balls of different colors; if the touch is "gentle", the balls turn into doves whereas an abrupt touch causes them to burst. In another application, a soccer game, the user sees himself as the goalkeeper whose task it is to prevent balls from entering the goal area  platform In the late 1990s two other commercial companies developed video-capture gaming platforms, Reality Fusion's GameCam and Intel's Me2Cam Virtual Game System . Both of. This is an off-the-shelf, low-cost gaming application, which provides the opportunity to interact with virtual objects that can be displayed on a standard TV monitor [Somewhat later, Sony developed its very popular EyeToy application designed to be used with the PlayStation II platform  monitor . As withThe EyeToy application includes many motivating and competitive environments which may be played by one user or more than one user sequentially in a tournament fashion. With GX VR, two users can compete together simultaneously  as well as combine their efforts to create different visual effects without a competitive component . which enables therapists to adapt levels of difficulty and record performance outcomes [The potential of these platforms for rehabilitation was readily apparent despite the fact that they were originally developed for entertainment and gaming purposes. Indeed, VividGroups's GX platform was first applied without adaptations within a clinical setting by Cunningham and Krishack  who usedoutcomes .Video-capture VR differs from other platforms in a number of ways that have great relevance for its use as a tool for rehabilitation evaluation and intervention. Some of these characteristics appear to be advantageous whereas others may limit the utility of video-capture VR.Video-capture VR provides users with a mirror image view of themselves actively participating within the environment. This contrasts with other VR platforms such as the HMD which provides users with a "first person" point of view, or many desktop platforms in which the user is represented by an avatar. The use of the user's own image has been suggested to add to the realism of the environment and to the sense of presence . It alsoThe user in video-capture VR does not have to wear or support extraneous devices such as an HMD, glove or markers in order to achieve a substantial intensity of immersion within the virtual environment. This eliminates a source of encumbrance that would likely hinder the motor response of patients with neurological or orthopedic deficits. Although the newer HMDs and stereoscopic glasses are considerably less cumbersome than previous models, little information is available regarding their use by individuals undergoing cognitive or motor rehabilitation.This characteristic relates to how the user controls objects within the virtual environment. As indicated above, rather than relying on a pointing device or tracker, interaction within video-capture based environments is accomplished in a completely intuitive manner via natural motion of the head, trunk and limbs. Not only is the control of movement more natural, but, in the case of the chroma key GX VR, a "red glove" option (or any object with a distinct color) may be used to restrict system response to one or more body parts as deemed suitable for the attainment of specified therapeutic goals. For example, when it is appropriate to have the intervention directed in a more precise manner, a client may be required to repel projected balls via a specific body part . Or, when intervention is more global, the client will not use the red glove option and thus be able to respond with any part of the body. The ability to direct a client's motor response to be either specific or global makes it possible to train diverse motor abilities such as the range of motion of different limbs and whole body balance training.A limitation of currently available video capture platforms is the reliance on visual and auditory feedback and the absence of a haptic interface that would provide participants with real-time indications of contact with the virtual stimuli. Such feedback could serve as an important addition when used in therapy since the balls, for example, could be rendered to appear to have progressively greater mass, making the task more or less difficult. It would also add an additional element of realism to the gaming experience, and ensure that feedback to participants was more realistic. This could be accomplished to some degree via a quasi-haptic effect that might use vibration to simulate a true haptic interface . For example, small buzzers may be affixed to the tips of the digits. Touching a virtual ball in the Vivid GX Birds & Balls application would generate a low amplitude, high frequency "buzz". In contrast, repelling a larger ball in the Soccer application would generate a high amplitude, low frequency "buzz".Video-capture VR may be implemented while users stand, sit, or even walk on a treadmill. For example, the same environment may thus be suitable for training standing balance of a patient who had a stroke, sitting balance of an individual with an incomplete quadriplegic spinal cord injury, and balance during treadmill locomotion of an individual with a paraplegic spinal cord injury.Moreover, one or more users may participate within the same environment. In some applications, the ability to have two "rival" users interact simultaneously within the same game or task adds an element of competitiveness that may be motivating. Of greater importance is the ability of the therapist to support a client or use handling techniques in order to facilitate active movement while the client interacts with the virtual stimuli. The therapist can be concealed behind the client in order not to be seen in the VE, or can join the client within the virtual environment.Another limitation of the currently available video capture VR platforms is that they may be operated with only one camera. This means that all tasks must be performed within a single plane. In the case of the typical coronal plane setup where the camera is positioned to face the user, any functional movement that takes place in the sagittal or transverse planes is disregarded. Virtual scenarios must therefore be carefully designed such that a meaningful task can be performed despite the restriction to uniplanar movement. Moreover, care must be taken when analyzing the kinematic trajectories since any out-of-plane motion will not be recorded. It is encouraging to note that three dimensional, functional environments will likely soon become available .Although video-capture platforms have only begun to be used for rehabilitation applications within the last five years, there are already results from a number of research groups who have studied its utility with different patient populations. In this section we highlight the major studies that provide evidence that this technology appears to be suitable for use in rehabilitation. The evidence concerning participant sense of presence, enjoyment, usability and performance are summarized as reported by studies of single platforms and by studies that compared different VR platformsNone of the studies carried out to date have reported any significant occurrence of cybersickness-type side effects when using video-capture VR. Rand et al.  explicitSeveral studies examined the influence of video capture platform of the user's sense of presence and level of enjoyment. Rand et al.  in theirThe results of these studies showed that a high sense of presence and level of enjoyment can be achieved in a video capture VR platform. They also demonstrate that user characteristics such as age influence the sense of presence.In another study, Rand et al.  comparedRand at al.  reportedThe GX VR platform has consistently generated high levels of presence and enjoyment across a wide range of clinical populations and ages including adults with paraplegic spinal cord injury , stroke The measures of performance used by video-capture VR studies to date include response times to presented virtual stimuli, percent success with which a given game is performed , a subjective report on how much effort the user has felt while in the environment. The chroma key video capture platforms such as GX and IREX also provide a relatively gross measure of limb kinematics. Whether these data have sufficient precision and resolution to warrant their inclusion in a research study remains to be investigated .Sveistrup, McComas and colleagues have used the IREX platform for balance retraining. Following six weeks of training at an intensity of three sessions per week, improvement was found for all 14 participants in both the VR and control groups . HoweverKizony et al.  performeIn a second study Kizony et al.  examinedRand et al.  used a vWeiss at al. , in a stPerformance within two games (Kung-Foo and Wishy-Washy) was measured while three different groups, young adult participants, healthy senior participants and individuals who were several years post-stroke, used several of the EyeToy games . PerformThe performance results described above highlight the interplay between the user and VR platform characteristics, and emphasize the importance of taking these characteristics into consideration while using VR in rehabilitation. Moreover, they demonstrate the sensitivity of the VR performance measures in their capacity to differentiate between levels of participant ability.Due to the motivating nature of the game-like environments, it is important to determine how much effort healthy subjects and those with disabilities expend while engaged in these tasks. In a study of healthy young adults, the participants using the GX platform perceived the highest level of exertion while playing Soccer, less for Birds & Balls and still less for a third game, Snowboard where only weight transfer was needed . When diUsing the IREX platform, Sveistrup et al.  performeCunningham & Krishack  presenteAnalysis of conventional and video capture VR treatment for SCI by specialists in rehabilitation highlighted several key differences between the two methods of intervention . First, One of the newest developments in video-capture VR is the simulation of more functional environments. Rand et al.  have creSony's EyeToy Wishy Washy application involves the cleaning of successive dirty windows via wiping movements of the hand and arms. Most recently, VividGroup has developed a laundry application . These moves towards more functional applications are encouraging.Evidence from the literature has demonstrated the feasibility, usability and flexibility of video-capture VR, and there is little doubt that this technology provides a useful tool for rehabilitation intervention. The results of presence questionnaires, reports of user satisfaction, and the sensitivity to differences in user ability as functions of age, gender and disability are all strong indicators of the suitability of this tool. A short video-clip, taken from a local news report of applications of video-capture VR for stroke, illustrates the extremely positive response of one user to the use of this technology (see Video 1).To date, as indicated by the studies reviewed above, video capture VR shows great promise for a variety of therapeutic goals including intervention for cognitive and motor rehabilitation, functional activities and leisure opportunities. The general assets of virtual reality summarized above combined with several assets that are unique to video-capture VR, are compelling arguments for the inclusion of this technology in the repertoire of tools available in clinical settings.Market demand, user interest and improvements in technology have led to the availability of a number of different video-capture platforms. There is no doubt that these platforms are valuable as intervention tools during the rehabilitation of patients with neurological and musculoskeletal disorders. Motivated patients would be encouraged to practice movements in a repetitive manner thereby improving their condition, an achievement that is not easy to attain via conventional therapy . CurrentStudies comparing these two platforms have shown that presence, enjoyment, usability and performance were equivalent under many conditions and for diverse users. Thus, despite the EyeToy's limitations, its low cost, user-friendly interface and simple setup requirements makes it highly attractive to therapists. It may be readily acquired for use in any clinical setting, and even be purchased for use at home to provide regular, intensive therapy after discharge from hospital.Nevertheless, it is clear that the EyeToy is not suited for use with the most severely impaired users. The currently available games seem to have a broad appeal for users of different ages but an open architecture that permits adaptations of existing applications and development of new environments appears to be a basic requirement to make this platform truly functional as a clinical tool. A system for generating an outcomes report comparable to the IREX platform would also be of great benefit for clinicians. Additional low-cost video-capture platforms are currently under development . Moreover, video-capture platforms that will provide three dimensional, functional environments will likely soon become available .In contrast to the EyeToy's closed architecture, VividGroup's IREX platform provides a user-friendly interface that a therapist may use to specify a much greater range of levels of difficulty. Their SDK (Software Development Kit) provides programmers with the ability to further adapt existing applications such as the standard set of games  and to dVideo 1: This video clip shows a patient who had a stroke using the VividGroup VR system for cognitive and motor rehabilitation.Click here for file


The history of one of the world's first electronic archives for scientific journals JSTOR is successful for reasons its founders did not intend. Bill Bowen's inspired vision was of a solution to libraries' ever-voracious demands for space to house paper volumes. The idea was that libraries could save space by removing volumes available in electronic format. Few libraries have discarded the volumes digitised in JSTOR, but many libraries without the paper volumes have been able to offer their users access to the important journal runs JSTOR has digitised. Paper holdings have not decreased dramatically, but electronic holdings have increased. So a space-saving service became an access service.As an access service, JSTOR is a creation of its time. Understandable though the decision to use page images may have been eight years ago, future user-friendly access requires searching capabilities across full-text, which page images cannot supply. Likewise, the decision to digitise the back-runs of around 100—now 218—paper journals was a bold decision at the time, but the future for access to journal literature lies in electronic versions of thousands rather than hundreds of titles, both current and retrospective. When we reach that point, JSTOR will still have a valued place in the content on offer, but it is difficult to see JSTOR providing thousands rather than a few hundred titles. Its technical solutions and financial models look dated as both subscription-based and open-access publishers improve their services to authors and to readers. As the number of journal articles accessible over the networks increases, JSTOR will be seen as a small-scale pioneer from which we learned valuable lessons.Roger Schonfeld ends his very detailed description of JSTOR with a chapter on ‘Lessons Learned’, many of which are relevant to current access initiatives. The need for grant funding to launch any such initiative has to be accompanied by a sound business plan to ensure long-term economic viability. JSTOR has achieved that transition, and its success provides a model for others. Much of the credit must go to JSTOR's enterprising president, Kevin Guthrie, who found the quickest way through the maze of conflicting advice—much of which could have resulted in JSTOR's reaching a deadend—and convinced the library and publishing communities to buy into a product that was only a promise. Meeting user needs for easy access to high-quality content was the key to the fulfilment of that promise. JSTOR's public image is of quality in depth—long runs of core journals—and that image has to become the hallmark of the new open-access initiatives as they develop.It is understandable that some mistakes were made on the way. The difficulty that JSTOR financial planning had in coming to terms with consortial purchases delayed its growth as an access service. Although selling to consortia of academic libraries may not have improved JSTOR's financial position in the short-term, consortia are a route to spreading access and therefore securing longer-term financial stability . Some opportunities were also delayed—not lost—through too slow an adaptation of the JSTOR purchasing model for selling outside the United States, the United Kingdom being the exception. The UK deal was with JISC, the Joint Information Systems Committee of the UK Higher Education Funding Councils, acting more as a negotiating agent than a consortium, and this model could have been applied in other countries. More countries would have valued access to JSTOR earlier, but the approach to non-US deals had to be imaginative. For all vendors, there has to be an understanding of the political, social, economic, and educational structure of the country into which the product is being sold, an understanding that takes time to acquire but that pays dividends. Open-access publishers do not have to sell their product to users of their journals, but local knowledge is essential in ‘selling’ their services to authors. The globalisation of publishing has combined with the globalisation of the networks and with the globalisation of research to provide opportunities for high-quality research conducted outside North America and Western Europe to be published in peer-reviewed open-access journals more readily than in the traditional subscription-based journals.Roger Schonfeld's book draws out many of the significant points about JSTOR's place in the history of electronic publication through a minute examination of the process leading to JSTOR as it is today. There is so much detail in the book that the reader may feel that its comprehensiveness cannot be questioned, but one small omission of which I have personal knowledge makes me question the value of so much detail. The omission concerns the interest by my institution, University College London, in joining JSTOR before the JISC deal was considered. Not a detail of world-shattering significance, but it does illustrate the fact that outside the United States, as well as within, the early interest in JSTOR came from individual institutions rather than from consortia. I sympathise with Roger Schonfeld in attempting to write such a comprehensive history, but what is the point of appearing to be comprehensive when comprehensiveness is an impossible goal? Would a briefer history have been just as valuable?Leaving aside quibbles and caveats about the book and about JSTOR, this remains a fascinating and instructive history of an important and ground-breaking initiative. Bill Bowen's vision may not have developed in quite the way he expected, but the ‘bottom-line’ is that the vision did become a successful reality. The problem of ever-expanding libraries has not gone away in the ten years since JSTOR was conceived, but the ultimate solution—the availability of electronic content—has become closer, and JSTOR's success has encouraged others to develop services that are more in accord with 2003 than 1993. One lesson Roger Schonfeld does not draw out is the pace of change in electronic publishing, and if so much has been achieved since 1993, what promise is held out by the next ten years'!


Autotaxin , originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity.We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion.L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches. Lysophosphosphatidic acid (LPA) is both an intracellular and an extracellular signaling molecule that affects biological processes such as cell proliferation, rescue from apoptosis, cell migration, neurite retraction, wound healing, platelet aggregation and vascular remodeling . As a cyATX was originally purified as a potent tumor cell motogen , an effeATX is a member of the nucleotide pyrophosphatase and phosphodiesterase (NPP) family of enzymes. The NPPs are part of the superfamily of alkaline phosphatases, metalloenzymes in which the active site is characterized by histidine residues coordinated around central divalent cations and by a serine, threonine, or cysteine residue, which is utilized to form an intermediate during the reaction ,15. SiteHistidine has been implicated as a requirement for many metalloenzymatic reactions, presumably by virtue of its imidazole moiety. Reversible reagents such as diethylpyrocarbonate, which react with imidazole, inhibit these enzymes . Such fiATX, isolated as a motility-stimulating protein, is a member of the NPP family of metalloenzymes. Both its motogenic and its enzymatic activities appear to require the same active site since both are abolished when T210  or any oFig. ATX was pre-incubated with 200 nM LPC with or without addition of various concentrations of L-histidine, then heated to abolish its enzymatic activity  and adde++ and Zn++ were markedly effective in restoring enzymatic activity to LPLD in EDTA-treated plasma. In fact, addition of Co++ to EDTA-treated heparinized plasma resulted in a recovery from 37% of the untreated value in the presence of EDTA alone to 154% in the presence of Co++ and EDTA. Since histidine plays an important structural role in the catalytic process, we determined first that addition of free histidine has an inhibitory effect upon ATX-catalyzed hydrolysis of both nucleotide and lyso-phospholipid substrates. After establishing the concentration requirements for histidine inhibition, we next examined the capacity of different metal cations to reverse this inhibition. Finally, we determined how histidine and metal cations affect the kinetics of the LPLD reaction.The PDE activity of the NPPs has been shown to be dependent upon the presence of metal ions . TokumurAs seen in Fig. ++ nor Ca++ significantly reversed the inhibitory effect of histidine although Ca++ alone enhanced both reactions by at least 30%. However, Zn++ and Co++ each significantly overcame inhibition caused by free histidine. In the case of Zn++, the presence of the cation alone increased both reactions by up to 50% above control, and virtually abolished the inhibition caused by histidine, with recoveries of 90 – 100% of the activity measured in the presence of Zn++ alone. In the case of Co++, the cation alone increased the PDE activity by 50% and the LPLD activity by 300%; however, histidine still inhibited PDE by ~70% and LPLD by 50% compared to adding Co++ alone. Zn++, therefore, appears to be the most effective cation in reversing the inhibition by histidine. It should also be noted that none of the cations at the levels tested had any inhibitory effects upon the enzymatic reactions of ATX. Histidine therefore inhibits a process that is required for the hydrolysis of both nucleotides and phospholipids. This may be a rate-limiting cation-dependent step in their common reaction mechanism [We tested the effects of a number of divalent cations in both the presence and absence of histidine upon the PDE and LPLD activities of ATX Fig. . Neither++ reversed the inhibitory effect of histidine upon ATX enzymatic reactions, we tested its effect upon the histidine-induced inhibition of ATX- and LPA-stimulated motility. As shown in Fig. ++, or both together had no effect upon LPA-induced motility. In contrast, ATX-induced motility was affected in a more complex manner. Histidine (20 mM) alone inhibited ATX-induced motility by ~65%, while 0.25 mM Zn++ alone increased this same motility by ~30%. When these same concentrations of histidine and zinc were added together to an ATX-stimulated motility assay, the histidine inhibition was largely reversed with greater motility than seen with ATX alone, though not quite up to levels seen with ATX + Zn++. However, statistical analysis of these results revealed no statistically significant difference between ATX + Zn++ + histidine vs. ATX + Zn++. In contrast, there was a statistical significance between ATX + Zn++ + histidine vs. ATX + histidine (p < 0.001).Since Zn++, alone or in addition to histidine. Zn++ alone had no significant effect on the Vmax or Km of the reaction. In the presence of histidine, Zn++ reversed the inhibition, restoring Vmax to untreated levels. It might be noted that the pattern of inhibition seen when histidine is added is typical of a non-competitive inhibitor.Fig. ++), the possibility arose that the imidazole ring of histidine acts as a weak chelation agent, competing with the enzymatic active site for binding to the metal ion. EDTA has been shown to inhibit the nucleotide phosphodiesterase and pyrophosphatase activities of PC-1/NPP1 [++ and with Zn++.Because the histidine-induced inhibition of ATX activities could be reversed by addition of appropriate metal ions  for both LPLD and PDE activities, while an equimolar concentration of Ca++ resulted in ~75% recovery of control activity.In a series of preliminary experiments (data not shown), we found that, under the conditions of our enzymatic assays, addition of 10 – 20 mM Na++ [++ resulted in a 70 – 80% recovery compared to control values, while adding up to 5 mM Ca++ had no significant effect upon phenanthroline-induced inhibition of activity. As can be seen in Fig. Addition of 0.5 mM 1,10-phenanthroline also profoundly inhibited the LPLD activity of ATX  in about 15 – 20 min, and 1,10-phenanthroline inhibited cellular adhesion at concentrations utilized for inhibition of LPLD and PDE activity. Therefore, we were unable to obtain motility data in the presence of these agents.Testing the effect of these chelating agents on motility was problematic because chelation agents have multiple, complex effects upon living cells. For example, NaA number of histidine-derived agents, as well as histamine and imidazole Fig. , were teAs seen in Fig. Among the five histidine analogs found to inhibit ATX LPLD activity, the relative inhibitory activity is: D-histidine = L-histidine > histidine methyl ester >> N-methyl histidine > histidinamide. The effects of all of these agents are reversed by zinc. In contrast, glycine, L-alanine, imidazole, histidinol, and N, N-dimethyl histidine had no significant effect on activity; and histamine appeared to be slightly stimulatory. Most of these histidine analogs, including D-histidine, histidine methyl ester, histidinamide, N, N-dimethyl histidine, and histamine, were toxic to cells. N-methyl histidine resulted in reduced adhesion to our gelatin-coated membranes, precluding an effective motility assay. Only imidazole could easily be tested for its effect on ATX-stimulated motility. At 10–20 mM concentrations, imidazole had no significant effect on ATX-stimulated motility (data not shown).++ to the motility assays. Pre-incubation of ATX and LPC in the presence of L-histidine, followed by heat-killing the ATX, also resulted in much decreased activity, suggesting that L-histidine inhibits ATX-stimulated motility by ablating its LPLD activity. This hypothesis was in fact confirmed both by direct enzymatic studies in which L-histidine inhibited both PDE and LPLD activities and by the failure of ATX, in the presence of L-histidine, to produce an inhibitor of LPA-stimulated motility. Again, addition of Zn++ abrogated this inhibition of enzyme activity. These data provide further evidence that the LPLD activity of ATX, which results in the production of the mediator LPA, is essential for ATX stimulation of cellular motility.We have shown that L-histidine, at concentrations that are innocuous to cells, can inhibit the motility-stimulating action of ATX upon two tumor cell lines derived from human melanoma (A2058) and human ovarian carcinoma (SKOV-3). This inhibition is largely abrogated by addition of 0.25 mM Zn++, Mg++, Zn++ and Co++ to reverse the histidine-induced inhibition of ATX enzymatic activities. Neither Ca++ nor Mg++ reversed the inhibitory effect of histidine, whereas Co++ alone stimulated a 50% increase in PDE and a 300% increase in LPLD activities of ATX. Histidine partially reversed both of these Co++-stimulated increases: PDE activity was reduced to below uninhibited levels of product while LPLD activity, though reduced by 50% compared to treatment with Co++ alone, remained at levels above untreated controls. The mechanism by which cobalt activates ATX remains unexplained; however, Co++ has long been reported to replace one or more zinc cations in a variety of zinc-bound metalloenzymes [The role of metal cations in the activation of ATX was further investigated by first comparing the capacity of Caoenzymes -23, someoenzymes ,25.++ at concentrations as low as 0.25 mM abrogated the inhibitory effects of 10 mM histidine upon both the PDE and LPLD activities of ATX. This 40:1 ratio of ligand to cation appears to be inconsistent with a simple stoichiometric coordination of the cation with the metal-binding imidazole moiety. This would predict that the maximum number of ligand groups to coordinate with a Zn++ would be six. Our result is, therefore, difficult to explain as simple chelation in solution between cations and histidine. Perhaps, Zn++ acts upon ATX in a manner that blocks the entry of histidine into the active site. For example, Zn++ might coordinate with an electronically available locus in proximity with the active site of ATX or it might induce conformational changes in ATX that lowers the affinity of free histidine for the active site. Another possibility is that Zn++ replaces another metal ion, resulting in a binding conformation with a lower affinity for L-histidine. Interestingly, 0.25 mM Zn++ appeared to have a slightly stimulatory effect upon ATX-induced motility, but not LPA-induced motility. This suggests that Zn++ might act in a way that stabilizes ATX in an active configuration.In contrast, Zn++ or Zn++ plus Mg++ incorporated into their catalytic site, although a number of other metal requirements have been documented [4-EDTA and 1,10-phenanthroline both inactivate ATX, confirming that it is a metalloenzyme. Na4-EDTA complexes with a variety of cations, including transition metals as well as alkali and alkaline earth metals, and is thought to inactivate metalloenzymes by removing the metal cation from its binding site. Under the conditions of our assays, the IC50 of EDTA was ~5 mM. This inhibition was completely reversed by addition of equimolar Zn++ and significantly reversed (about 75% recovery) by addition of equimolar Ca++. In contrast, 1, 10-phenanthroline has more complicated and diverse mechanisms of action. When utilized with metalloenzymes, it can form mixed complexes with the metal ions as well as other cationic sites on the protein, resulting in inactivation of the enzyme but not necessarily removal of the metal cation [++ (approximately 1:10) to reverse its inhibitory effect on ATX. Based on all of our data with metal cations, it appears that Zn++, Co++ and perhaps Ca++ can function as the metal moiety in at least one of the two metal binding sites [Many members of the alkaline phosphatase superfamily of metalloenzymes have Zncumented ,26. The l cation . Like hing sites  of the a++-binding groups in matrix metalloproteinase inhibitors indicated that slight changes to the Zn++-binding groups can result in major changes in efficacy of inhibition [In order to determine what portion of the histidine molecule was responsible for its inhibitory activity, we utilized a number of commercially available histidine analogs. These analogs were predominantly substituted on the alpha carboxyl and alpha amino groups of histidine; but they also included histamine, which lacks the alpha carboxyl group altogether, and imidazole, the weakly chelating ring that makes up the major portion of the histidine side chain. Except for D-histidine, none of the other imidazole-containing compounds were as inhibitory as L-histidine. Among the 5 agents found to have significant inhibitory activity, the relative potency is as follows: D-histidine = L-histidine > histidine methyl ester >> N-methyl histidine > histidinamide. Methylation of the alpha amino group of histidine resulted in a stepwise loss of potency, with a single methylation (N-methyl histidine) resulting in about a 40% reduction in activity and a double methylation  not significantly different from untreated control reactions. Since these two compounds should retain the positive charge properties of the native alpha amino group, this could be a steric effect. Similarly, change in the carboxyl group (particularly amidylation or reduction) also decreased the inhibitory potency, though that of esterified histidine methyl ester was only slightly reduced. Interestingly, imidazole itself, a moiety postulated to coordinate with the cation at the active site of ATX, was not effective at the tested concentrations. Likewise, amino acids, which lacked imidazole on their side chains , were also ineffective as inhibitors. Clearly, three functional groups of histidine are required for full inhibitory activity: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Recent work with Znhibition , suggest++ metal cations. Our data suggest that the L-histidine-induced inhibition of the ATX enzymatic activities is predominantly non-competitive, i.e. Vmax is decreased significantly, while Km is not significantly affected. Furthermore, the mechanism of action of L-histidine does not appear to be simple stoichiometric chelation of the metal cations within a metalloenzyme but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. At concentrations required for an inhibitory effect, most available histidine analogs resulted in loss of cell viability. Free L-histidine, therefore, appears to be unique in its ability to inhibit LPLD, and hence regulate a major source of LPA, in living systems. Since LPA has significant pathological effects, the regulation of its formation is of considerable interest. Potential problems with L-histidine as a therapeutic agent include its lack of potency and its conversion in vivo to histamine. While there are known inhibitors of histidine decarboxylase, such as the polyphenols of green tea [ATX, the major source of plasma LPA, has been implicated in a number of physiological and disease-related processes , includireen tea , potent,L-histidine inhibits the LPLD activity of ATX at millimolar concentrations, reducing its capacity to produce its major mediator, LPA.4-EDTA, and 1,10-phenanthroline were from Sigma-Aldrich . N-methyl histidine was from Bachem Bioscience .LPA (18:1) and S1P were purchased from Biomol Research Laboratories, Inc. . LPC (18:1), SPC, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 4-aminoantipyrene (4-AAP), horseradish peroxidase, choline oxidase, p-nitrophenyl-TMP, L-histidine, D-histidine, imidazole, histamine, histidinamide, histidine methyl ester, N,N-dimethyl histidine, histidinol, NaA2058  and SKOVHighly purified recombinant human ATX (vATX), cloned from an MDA-MB-435 cell library, was prepared from a Vaccinia viral lysate, as described previously, through the concanavalin A-agarose step .6 cells/ml in DMEM supplemented with 1 mg/ml bovine serum album (DBA). Ten min before the start of the assay, appropriate concentrations of potential inhibitors were added to the different treatment groups. Chemotaxis was assayed as described previously in detail [Cells were detached from their flasks with a brief exposure to 0.05% trypsin and 0.02% versene and then resuspended at 2 × 10n detail , utilizi3 or HCl, as appropriate. The inhibitor was added to the reaction mixture; then, reactions were initiated by adding ATX.Enzyme activities were determined using a modification of the previously described assays . Briefly2, 0.3 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), 0.5 mM 4-aminoantipyrene (4-AAP), 5.3 U/ml horseradish peroxidase, and 2 U/ml choline oxidase was added to the 50 μl reaction mixture and incubated for 20 min at 37°C. Absorbance was read at 555 nm and converted to nmoles of choline by comparison to a choline standard curve (A555 × 17 = nmoles).For determination of 5'-nucleotide PDE activity, reactions were stopped by the addition of 450 μl 0.1 N NaOH and the nitrophenol product was detected by reading the absorbance at 410 nm (A410 × 64 = nmoles). In order to determine LPLD activity, released choline was detected as follows: a 450 μl cocktail containing 50 mM Tris-HCl (pH 8), 5 mM CaClATX AutotaxinDBA DMEM with 1% bovine serum albuminDMEM Dulbecco's Modified Essential MediumLPA Lysophosphatidic acidLPC LysophosphatidylcholineLPLD Lysophospholipase DNPP Nucleotide pyrophosphatase and phosphodiesterasePDE PhosphodiesterasepNp-TMP p-Nitrophenyl-TMPS1P Sphingosine-1-phosphateSPC SphingosylphosphorylcholineTC carried out the enzymatic studies, EK performed cellular studies with A2058 cells, MP performed cellular studies with SKOV-3 cells, and RWB carried out background studies and fine-tuned methodology. ES originated the idea of studying the effect of L-histidine on LPLD activities. LAL and MLS analyzed and interpreted data. MLS drafted and coordinated the manuscript between authors. All authors participated in reading and refining the manuscript drafts.


Protein evolution and protein classification are usually inferred by comparing protein cores in their conserved aligned parts. Structurally aligned protein regions are separated by less conserved loop regions, where sequence and structure locally deviate from each other and do not superimpose well.Our results indicate that even longer protein loops can not be viewed as "random coils" and for the majority of protein families in our test set there exists a linear correlation between the measures of sequence similarity and loop structural similarity. Results suggest that distance matrices derived from the loop (dis)similarity measure may produce in some cases more reliable cluster trees compared to the distance matrices based on the conventional measures of sequence and structural (dis)similarity.We show that by considering "dissimilar" loop regions rather than only conserved core regions it is possible to improve our understanding of protein evolution. Globular proteins are considered to be structurally similar if their regular secondary structure elements can be superimposed well and are connected in the same order. The loop regions connecting secondary structures demonstrate less regularity in their conformations even though short loops linking specific secondary structures can be classified into distinct classes -6. The sRecently it has been observed that structural variation in the core of homologous proteins is linearly correlated with sequence changes ,9. As waTo check this hypothesis we performed an analysis of structural variation in the loop regions within different homologous protein families using a recently introduced new measure of loop similarity . This neTo select sets of homologous proteins the Conserved Domain Database (CDD) version 1.62 was taken, which can be accessed at . The CDDEach CDD family was decomposed into a set of pairwise structure-structure alignments. Structural alignments were computed by the VAST algorithm  and only- at least one structure in a pair had X-ray resolution of greater than 3.0 Å- the Blast E-value calculated for the VAST alignment exceeded 0.01- at least one structure in a pair contained a chain discontinuous domain inconsistently aligned between VAST and CDD- at least one structure in a pair contained more than 25% of its nonaligned loops with missing residues.To ensure that protein families span a wide range of sequence similarity, all families were examined and those having less than 30% sequence identity span were not considered in further analysis. The redundancy between protein families was checked by using the procedure implemented in the CDART algorithm  and not To measure the sequence similarity between homologous proteins from the same family we used a Blast bitscore normalized by the alignment length. Among structure similarity measures used in this paper, two of them, RMSD and alignment-based Hausdorff measure (AHM) were computed by comparing the proteins in structurally aligned regions, while the loop-based Hausdorff measure (LHM) quantified the difference in the loop regions.A = {a1,..., am} and B = {b1,..., bn} be finite point sets in a Euclidean space. The Hausdorff distance between the sets A and B is then defined by:The root mean squared deviation (RMSD) was calculated using the superposition algorithm due to McLachlan . The AHMdH  = max {min j d,..., min j d, min i d,..., min i d}     (1)d denote the usual Euclidean distance between the points. In other words, the Hausdorff distance between the sets A and B is the smallest distance such that every point ai ∈ A is within this distance of some point bj ∈ B and vice versa. Hausdorff distance can be calculated under the assumption that the Cα atoms for both structures are in a common coordinate frame which is defined by the structural alignment between two domains. The Hausdorff measure for loops (LHM) was calculated as an average of Hausdorff distances over all loops in the protein pair, where ns is the number of aligned secondary structure elements:Here the terms The "loop" was defined as a region between two consecutive aligned secondary structure elements and:hi = 0, if the i-th loop regions do not have any unaligned residues;hi = dH , where Ai contains the set of Cα coordinates of non-aligned residues in the i-th loop of the first structure in a pair, the last aligned residue from the preceding aligned region and the first aligned residue from the following aligned region. Similarly, Bi is defined for the second structure in a pair. The sets  are defined to include two aligned residues so that the measure can be defined even if one of the sets of non-aligned residues is empty. The Hausdorff measure for the structurally aligned regions (AHM) was defined similarly. In this case, instead of the sets that contain the coordinates for the Cα atoms in the loops, we use the coordinates for the Cα atoms in the aligned segments and average over the number of aligned segments.The correlation analysis between the measures of sequence and structural similarity, linear/nonlinear regression analyses and cluster analysis were performed using Splus version 6. Pearson (ρ) and Spearman correlation coefficients were calculated to quantify the accuracy of linear correlation. The P-value under the null hypothesis that the correlation coefficient between two variables is equal to zero has been estimated and those families with the P-values less than 0.01 were considered as having statistically significant correlation. The cluster analysis was done using the complete linkage clustering  where thTables ) and nonlinear  models . In the overall test set only 12 families have r2 – ratio smaller than 0.9  indicating that for these cases adding the non-linear term improves the performance of modeling by about 10%.The dependence of structural similarity on sequence similarity in some cases can be more accurately described by the nonlinear regression model taking into account higher order quadratic terms. To quantify how much the nonlinear terms improve the data fitting, we use the ratio of squared correlation coefficient for linear . The sequence-structure correlation for loop regions of this family is not statistically significant (the Pearson correlation coefficient is close to zero) whereas the sequence-structure correlation for the protein core is very high (ρ = -0.85 with AHM). Indeed, different proteins of this family show distinctly different loop conformations and evolutionary analysis of ADF family argued that the insertions present in the vertebrate ADF/cofilins (and not present in non-vertebrate cofilins) might be important for nuclear function of mammalian cofilins . TherefoAHM = -0.90) and loop regions (ρLHM = -0.94). On the other hand, the protein core structure of the RnaseA family changes very little with sequence whereas the loop structure gradually diverges as sequence becomes more and more dissimilar .Among families with particularly high LHM correlation are the families of Xylose isomerase (Xylose_isom), Class I Histocompatibility antigen , Protein tyrosine phosphatase (PTPc) and others. Figure AHM = -0.48, ρLHM = -0.78), using different measures of sequence and structural similarity. Figure To understand whether significant sequence-structure correlation for loop regions has an underlying biological meaning, we performed a cluster analysis of proteins from two diverse families, Ribonuclease A (RnaseA), and SH2 domain  although angiogenin has a very weak enzymatic activity and is a tumor-growth promoter while Rana ribonuclease P-30 has ribonuclease activity and antitumor effects. In contrast to the RMSD cluster tree, distance matrices based on the loop (dis)similarity measure correctly cluster the representatives of the five major groups of the Ribonuclease family as per Rosenberg et al [The RnaseA family represents a very interesting example to study as it is characterized by considerably different catalytic efficiency and substrate preferences among family members and the different aspects of its activity is not well understood. Although cysteines that form disulfide bonds, catalytic histidines and lysine residues are mostly structurally and sequence conserved, there is a great variability in sequence between other regions of RnaseA proteins ,24. We crg et al . Althougrg et al .SH2 domains represent phosphor-tyrosyl peptide binding modules which are found in many signaling proteins. The specificity of phosphate interaction with a protein has been attributed to the hydrophobic pocket which is mostly formed by two loop regions . Our anaHere we have presented an analysis of how the structure of protein loops changes in evolution as homologous proteins diverge from each other. We showed that for the majority of protein families there exists a statistically significant linear correlation between measures of sequence similarity and average loop structural similarity. This in turn suggests that loops change in evolution via a stepwise insertion or deletion process and clearly one can not portray even longer loop regions as "irregular conformations" or "random coils". Indeed, our results imply that, in general, loops are under constant evolutionary constraints which, apparently, are weaker than those for a protein core but still strong enough to preserve the loop overall structure. Since loops do not contribute much to the protein core stability, these constraints predominantly arise from the importance of loops in interacting with ligands, other proteins and cells, as well as a possible role of loops in protein folding.Modeling of insertion and deletion events in evolution poses a lot of difficulties and protein evolution is usually reconstructed based only on the aligned regions of proteins. We demonstrated that loop regions which usually correspond to the non-aligned protein regions can be very important in inferring the phylogenetic history of a protein family. Moreover, it was shown, that sometimes sequence and structure similarity measures comparing proteins in their core are not sensitive enough to detect subtle (dis)similarities between the subfamilies. Loop-based measures which emphasize the dissimilarities between different protein members can shed light on the evolutionary relationships between homologous proteins.AP and TM contributed equally to this paper.


The evidence on early childhood strongly suggests the need to shift child health policy from the current focus on social welfare to a socio-ecologically based approach. This paper reviews three governing instruments, exhortation, expenditure and regulation, that have been used by governments in Australia and discusses the relative effectiveness of these approaches in shifting the child health policy paradigm. There can be no keener revelation of a society's soul than the way it treats its children .Research evidence has demonstrated that the experiences of early childhood can have a profound lifelong impact on a child's health, wellbeing and competence . The impEducation, literacy and other social determinants of health can influence the coping skills of children, which provide the basis of learning, behaviour and health throughout life . PovertyConsistent with the increasing evidence, many governments have identified support in early childhood as a lifelong determinant of health, wellbeing and competence, as a matter for policy development, initiating actions to ensure comprehensive child development strategies for their societies. This approach requires a whole of government response, integrating health, welfare, education and other relevant parts of government. The evidence suggests that healthy public policy for infants, children and their parents is dependent on understanding of the socio-ecological factors supported by integrated, multidisciplinary and intersectoral policy and programs.'Sure Start', has been positively reviewed by the UK Audit Office and is considered by many to be the standard for the whole of government approach .NSW has also successfully translated much of the evidence into coordinated service planning and delivery at the regional level. Paediatric networks, associated with the Area Health Services, were established in 1997 and today provide designated primary, secondary and tertiary level services for families with children aged 0 to 5 years . In addiFamilies First has been guided by a series of policy and practice guidelines. Recently, an independent review of Families First implementation within three regions,  found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill, with considerable effort to develop structures and processes that sustain interagency collaboration [The implementation of boration . This reRegulation involves the imposition of requirements to meet specific obligations. Often regulation is seen to exist within legislation outlining strict rules of behaviour. However, in health policy guidelines are considered effective means of imposing regulation, recognising the inherent uncertainty in safe practice in health care . The impHowever, it is yet to be seen whether the policy will be supported with the necessary resources for compliance. While regulation involves shifting costs of compliance from government to other participants, enforcement and monitoring can be expensive and difficult . WithoutA variety of instruments have been used by government to change the child health policy paradigm from that focused on social welfare to 'healthy' public policy predicated on a socio-ecological foundation. NSW has chosen regulation to implement a child health policy framework that is built upon the international evidence of the effectiveness of integrated, multidisciplinary and intersectoral policy and programs. A little slower to change paradigms, Victoria has established demonstration programs through targeted expenditure, without an overarching whole-of-government child health policy framework. Nationally, there is a move to change the paradigm to a broader definition of child health almost exclusively through exhortation.Families First implementation found that the system changes required to build and strengthen service networks for families needed more than agreement and goodwill to develop the necessary structures and processes – a suggestion that without other approaches to structural reform, exhortation is unlikely to be successful.Instrument choice is influenced by a variety of factors. The use of exhortation by the Commonwealth Government is a relatively risk-free easy approach, which can counteract the divided accountabilities among federal and state governments in the area of health and social services. Exhortation is easy to implement; it is the least coercive and relies on voluntary goodwill. While it may be successful in building a common understanding, and even this is debatable, an independent review of the The expenditure policy of Victoria illustrates an approach that is compromised by the lack of an underlying agreed evidence-based policy framework. This lack of coordinated government approach is reflected in conflicting expenditure policy in this area, with the potential to confound outcomes.While the regulatory approach of NSW suggests bold steps to change the paradigm, in fact, because regulation is not subjected to the same level of scrutiny of other instruments, such as expenditure, and even exhortation , it is aThe evidence for a new policy paradigm is strong. But the use of these different policy instruments underscores the lack of shared understanding and policy agenda. Oberklaid suggests that while there are similarities in the rhetoric throughout Australia, there has been relatively little investment in child health . The chaThe authors declare that they have no competing interests.


Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment.Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry.Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes.We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype. With the improved long-term outcome of allograft recipients in the cyclosporine or tacrolimus era, malignant tumors have become increasingly important. Malignant tumours develop in 15–20% of graft recipients after 10 years, and thus contribute substantially to the morbidity and mortality of these patients [During the last years, the novel immunosuppressive drug mycophenolate mofetil (MMF) has been introduced into the clinical protocol to overcome severe side effects associated with cyclosporine or tacrolimus. Meanwhile, it has become part of the immunosuppressive regimen after liver, kidney or heart transplantation . Still, MMF effects are based on the inhibition of inosine monophosphate dehydrogenase (IMPDH) and prevention of guanosine monophosphate synthesis from inosine monophosphate, a rate-limiting step in the purine biosynthesis in lymphocytes. Consequently, MMF blocks the proliferation and clonal expansion of T and B lymphocytes, and prevents the generation of cytotoxic T cells, as well as other effector T cells . AdditioThe integrins constitute a family of transmembrane receptor proteins composed of heterodimeric complexes of noncovalently linked alpha and beta chains. Integrins function in cell-to-cell and cell-to-extracellular matrix (ECM) adhesive interactions and transduce signals from the ECM to the cell interior and vice versa. For various types of cancers, different changes in integrin expression are closely associated with tumor growth and metastasis. Based on the knowledge that MMF modulates integrin expression, we postulated that MMF might not only suppress leukocyte recruitment to the donor graft, but also prevent integrin-dependent tumor dissemination. To explore how far MMF might serve as a metastasis-blocking agent, we investigated the beta1 integrin subunit expression pattern of colon, kidney, pancreas and prostate tumor cells before and after MMF treatment, as well as MMF effects on tumor cell adhesion to human endothelium in vitro.The present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium.2 incubator.Kidney carcinoma Caki I cells, pancreatic carcinoma DanG cells and colonic adenocarcinoma HT-29 cells G were obtained from the tumor cell bank of Johannes Gutenberg University, Mainz, Germany. Prostate carcinoma DU-145 cells were purchased from DSMZ . Tumor cells were grown and subcultured in RPMI1640 medium  supplemented with 10% FCS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified, 5% CO4 cells/scan). Cell cultures with a purity > 95% were serially passaged. Subcultures from passages 2–4 were selected for experimental use.Endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. HUVEC were grown in Medium 199 , 10% fetal calf serum , 10% pooled human serum , 20 μg/ml endothelial cell growth factor , 0.1% heparin , 100 ng/ml gentamycin (Gibco) and 2% 1 M HEPES-buffer . To control the purity of HUVEC cultures, cells were stained with fluorescein isothiocyanate (FITC)- labelled monoclonal antibody against Factor VIII-associated antigen  and analyzed microscopically or by FACscan  channel histogram analysis; 1 × 10Tumor cells were pretreated with MMF  . Before adding the MMF-treated tumor cells to HUVEC (monolayer adhesion assay), cell cultures were washed to remove MMF from the medium. Results were compared to untreated controls.Viability of tumor cells in presence of MMF was assessed by propidium iodide dsDNA-intercalation or quantitative fluorescence analysis of enzyme-catalyzed fluorescein-diacetate metabolism.6 tumor cells of each entity/well were carefully added to the HUVEC monolayer for 60 min. Subsequently, non-adherent tumor cells were washed off using warmed (37°C) Medium 199. The adherent cells were fixed with 1% glutaraldehyde and counted in five different fields (5 × 0.25 mm2) using a phase contrast microscope (20 × objective) to calculate the mean cellular adhesion rate.HUVEC were transferred to six-well multiplates  in complete HUVEC-medium. When confluency was reached, 0.5 × 104 cells/scan) and expressed as mean fluorescence units (MFU).Tumor cells were washed in blocking solution  and then incubated for 60 min at 4°C with the FITC-conjugated monoclonal antibody anti-alpha2beta1 , anti-alpha4beta1 , anti-alpha5beta1 , anti-alpha6beta1 , or with the PE-conjugated monoclonal antibody anti-alpha1beta1 , or anti-alpha3beta1 . Integrin expression of tumor cells was then measured using a FACscan  channel histogram analysis; 1 × 10A mouse IgG1-FITC was used as an isotype control for FITC conjugated antibodies. To evaluate background staining of PE conjugated antibodies, goat anti mouse IgG-PE was used .To analyze integrin beta1 distribution on the cell membrane, tumor cells were transferred to round cover slips (pretreated with 2% 3-aminopropyl-triethoxysilan) placed in a 24 well multiplate. Upon reaching confluency, cell cultures were washed and fixed in cold (-20°C) methanol/acetone (60/40 v/v). Subsequently, cells were incubated for 60 min with unconjugated anti-integrin monoclonal antibodies. Indocarbocyanine  conjugated goat-anti-mouse IgG was then added as the secondary antibody. To prevent photobleaching of the fluorescent dye, cover glasses with stained cells were taken out of the wells and the residual liquid was removed. These were then embedded in an antifade reagent / mounting medium mixture  and mounted on slides. The slides were viewed using a confocal laser scanning microscope  with a plan-neofluar ×100 / 1.3 oil immersion objective.2 growth area; Falcon Primaria, Becton Dickinson) and cultured with or without MMF. Total RNA was extracted by using RNeasy kit  and RNA samples were then treated with 80 U/ml of Rnase-free Dnase I  for 60 min at 37°C, to eliminate amplifiable contaminating genomic DNA. Subsequently, samples were incubated for 10 min at 65°C to inactivate Dnase. Complementary DNA was synthesized from 1 μg of total RNA per sample with a 60 min incubation at 42°C, using the Moloney murine leukaemia virus reverse transcriptase  and oligo-(dT) priming (Boehringer Mannheim). Amplification was carried out using gene specific primers and Platinum-Taq polymerase (Invitrogen) in a Mastercycler Gradient thermocycler . Reactions were performed in the presence of 0.5 μl cDNA, with an initial incubation step at 95°C for 2 min. Cycling conditions consisted of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec over a total of 30 cycles. The reaction was completed by another 10 min incubation step at 72°C. The specific sequences for sense and anti-sense primers are shown in table mRNA expression of beta1 integrins was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Tumor cells were seeded in 50 ml culture flasks  > alpha2beta1 (175.7 ± 24.3) > alpha6beta1 (54.8 ± 9.2) > alpha1beta1 (27.3 ± 3.2).Caki I: alpha3beta1 (601.3 ± 82.0) > alpha1beta1 (139.2 ± 24.0) > alpha5beta1 (22.9 ± 4.2).HT-29: alpha3beta1 (202.0 ± 33.8) > alpha6beta1 (119.6 ± 14.5) > alpha2beta1 (69.2 ± 9.0) > alpha1beta1 (25.4 ± 3.4).DU-145: alpha3beta1 (942.5 ± 112.9) > alpha2beta1 (95.4 ± 12.4) > alpha5beta1 (69.1 ± 8.9) > alpha6beta1 (50.9 ± 7.2) > alpha1beta1 (31.4 ± 4.8).Mean IgG1-FITC isotype control was 8.6 ± 1.8 MFU, mean IgG1-PE isotype control was 9.9 ± 1.7 MFU.Analysis of the mRNA expression level confirmed the flow cytometry data (see below).The distribution pattern of those integrins which were predominantly expressed on the respective tumor cell line was further explored by confocal microscopy figure . alpha2bMMF evoked distinct alterations of the beta1 integrin expression pattern figure . MMF onlTo allow a clear interpretation of the strong effects of MMF on adhesion and beta1 integrin surface expression of HT-29 and DU-145 cells, MMF evoked alterations of gene activity was also evaluated in these cell lines figure . ControlAlthough MMF has become part of the standard regimen after organ transplantation its impact on tumor development and dissemination is still not clear.Our adhesion experiments demonstrate that MMF down-regulates binding of tumor cells to endothelium, which might argue for anti-tumoral properties of this compound. Notably, HT-29 and DU-145 cells responded well to MMF (-70% adhesion reduction), while Caki I and DanG tumor cells were influenced only modestly. From a clinical viewpoint, distinct adhesion-blocking properties of MMF might be limited to colon and prostate carcinoma cells. Interestingly, HT-29 cells were more susceptible to MMF than DU-145 cells: 0.1 μM MMF was sufficient to significantly diminish adhesion of HT-29 cells, whereas 1 μM MMF was necessary to evoke maximum effects on DU-145 cells. The different sensitivity of the tumor cell lines to MMF might be caused by an unequal metabolic activity, coupled with variable IMPDH levels. Recent data have shown that the level of expression of IMPDH mRNA and protein differ among several cell lines , and thaIt should also be considered that MMF might switch on/off different intracellular signaling cascades in colon versus prostate tumor cells. Indeed, adhesion blockade of HT-29 cells was accompanied by reduced alpha3beta1 and alpha6beta1 surface expression, while adhesion blockade of DU-145 cells was accompanied by a dose-dependent up-regulation of integrins alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1 on the cell membrane.Studies on integrin receptors presented evidence that beta1 integrin expression by colon carcinoma cells qualifies these cells to successfully adhere to secondary sites. Recent experiments have demonstrated that colon cancer cells adhere to endothelial cells via beta1 integrins and that addition of beta1 integrin blocking antibodies reduces tumor cell adhesion ,16. Basehigh and alpha3beta1low expressing prostate carcinoma cells were injected into immunocompromised SCID mice, only those cells with a drastically reduced integrin level were found to form tumors at the primary sites and to be highly invasive and metastatic [A converse scenario might be created during prostate carcinogenesis, as levels of beta1 integrins have been found reduced in neoplastic versus normal prostate tissue ,19, and tastatic . This isWhen discussing the relevance of integrins in tumor recurrence and malignancy, we should keep in mind that integrin receptors serve as mechanistic binding as well as differentiation triggering elements. Therefore, up-regulation of the same integrin type might either lead to enhanced cell adhesion by coupling the receptor to its ligand, or to a reduced cell adhesion by activating integrin driven differentiation signals. Based upon our in vitro assay, we conclude that MMF blocks adhesion of colon and prostate carcinoma cells by two different mechanisms: a) Loss of alpha3beta1 and alpha6beta1 surface expression directly contributes to the reduced adhesive behaviour of HT-29 cells, b) Enhancement of integrin beta1 subtypes might cause re-differentiation of DU-145 cells towards a low-adhesive phenotype. However, it still remains to be determined if MMF indeed acts as a differentiation inducing drug in prostate tumor cells. Beside the hypothesis that beta1 upregulation might activate differentiation inducing signals, selective inhibition of tumor-promoting pathways should also taken into consideration.Presumably, down-regulation of alpha3beta1 and alpha6beta1 surface expression on HT-29 tumor cells might be caused by inhibition of receptor glycosylation and/or receptor de novo synthesis. The latter hypothesis seems to be more likely because MMF's effects at the cell surface were also observed at the mRNA level. This was not the case with DU-145 cells where MMF evoked up-regulation of membranous beta1 integrins was not paralleled by similar modifications of the beta1 integrin coding mRNA. There is still no clear concept why MMF causes integrin up-regulation in one tumor entity but down-regulation in another entity, both coupled with reduced tumor cell adhesiveness. Presumably, HT-29 and DU-145 tumor cells might be equipped with different enzyme systems, the intracellular signaling cascade might be activated differentially in colon versus prostate tumor cells, or sensitivity of specific pathways to MMF might differ between both tumor types.Speculatively, alterations of post-translational events might change the receptor surface presentation in prostate carcinoma cells. Elegant experiments by Liang and coworkers demonstrated that over-expression of alpha5beta1 or beta1 integrin induced the decrease of protein kinase B (PKB) phosphorylation and subsequent accumulation of cyclin-dependent kinase inhibitor p21 . A yeastThe present study indicates that MMF possesses anti-tumoral properties particularly to colon and prostate carcinoma cells. Alterations of the beta1 integrin profile are responsible for blocking tumor cell adhesion to vascular endothelium. MMF might also act on further adhesion proteins which are relevant for tumor recurrence and dissemination. An in vitro study published recently refers to the sLeX-selectin pathway targeted by MMF . CD44 glThe author(s) declare that they have no competing interests.TE performed parts of the in vitro studies, contributed toward the design of the study and drafted the manuscript. JM carried out confocal microscopy, BR and IN performed FACS-analyses. IM designed PCR primers and carried out the PCR studies. WDB contributed to the manuscript design and finalisation. DJ participated in the conception and design of the study. RAB carried out the adhesion assays, participated in the conception and design of the study and its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: