Virtual environments (VE) are a powerful tool for various forms of rehabilitation. Coupling VE with high-speed networking [Tele-Immersion] that approaches speeds of 100 Gb/sec can greatly expand its influence in rehabilitation. Accordingly, these new networks will permit various peripherals attached to computers on this network to be connected and to act as fast as if connected to a local PC. This innovation may soon allow the development of previously unheard of networked rehabilitation systems. Rapid advances in this technology need to be coupled with an understanding of how human behavior is affected when immersed in the VE.This paper will discuss various forms of VE that are currently available for rehabilitation. The characteristic of these new networks and examine how such networks might be used for extending the rehabilitation clinic to remote areas will be explained. In addition, we will present data from an immersive dynamic virtual environment united with motion of a posture platform to record biomechanical and physiological responses to combined visual, vestibular, and proprioceptive inputs. A 6 degree-of-freedom force plate provides measurements of moments exerted on the base of support. Kinematic data from the head, trunk, and lower limb was collected using 3-D video motion analysis.Our data suggest that when there is a confluence of meaningful inputs, neither vision, vestibular, or proprioceptive inputs are suppressed in healthy adults; the postural response is modulated by all existing sensory signals in a non-additive fashion. Individual perception of the sensory structure appears to be a significant component of the response to these protocols and underlies much of the observed response variability.The ability to provide new technology for rehabilitation services is emerging as an important option for clinicians and patients. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies. Quantification of individual perceptual styles in the VE will support development of individualized treatment programs. The virtual environment can be a valuable tool for therapeutic interventions that require adaptation to complex, multimodal environments. Visual imaging is one of the major technological advances of the last decade. Although its impact in medicine and research is most strongly observed in the explosion of PET and fMRI studies in recent years , there hLet us first define what we consider a VE and consider the signals that need to be transmitted for such a system to operate remotely (TeleImmersion). VE is immersion of a person in a computer generated environment such that the person experiences stereovision, correct perspective for all objects regardless of their motion, and objects in the environment move in a natural fashion with subject motion. To achieve theses characteristics, certain technology must be utilized. To provide stereovision, slightly different images must be presented to the right and left eyes with little if any cross talk between the two images. In some systems this is provided by using field sequential stereo in combination with liquid crystal shutter glasses (StereoGraphics, Inc). In this system the right liquid crystal lens is clear while the left is opaque and the perspective scene generated on the screen is that for the right eye. Then the left eye lens is clear and the right is opaque and the left eye's view is displayed. This method of producing stereo has found its way into projection based systems ,5 and deRegardless of the system used, to keep all the stereo objects in the correct perspective and to keep them from being distorted when the person moves in the environment, it is necessary to track the movements of the person so that the computer can calculate a new perspective image given the reported location of the person's head/eyes. The tracking systems that are used to do this are varied. The most commonly used of these are the 6-degrees of freedom (DOF) magnetic tracking systems (Ascension, Inc and Polhemus, Inc.). With these systems a small sensor cube is placed on the subject and the location of the sensor within the magnetic field is detected. When the sensor is place on the head or glasses of the person the orientation of the head and therefore the location of the eyes can be presumed. Other non-magnetically based systems use a combination of acoustic location to delineate position and acceleration detection to obtain body coordinates in space. The combination results in 6 DOF for the location information (InterSense, Inc). Other systems use cameras to track the person and then transform this information to the 6-DOF needed to maintain a proper image in the VE .So far we have confined our discussion to visual objects and have not considered the use of haptic or other forms of information to be integrated into the VE system . To provIn networked VEs several types of data need to be transmitted between collaborating sites: 1. the main data-set itself (this often consists of 3D geometry); 2. the changes to the data-set (these occur when collaborating users modify the geometry in some way – perhaps by moving the object or deforming it); 3. the virtual representation of the remote collaborator (this often is referred to as an avatar); 4. the video and/or audio channel (that facilitates face-to-face conversation.) Video has limited use in stereoscopic projection-based VEs because the large shutter glasses that the viewer uses to resolve the stereo tends to hide the viewers face from the camera. Furthermore most stereoscopic projection systems operate in dimly lit rooms which are usually too dark for effective use of video.The common model for data sharing in networked VEs is to have most of the main data-set replicated across all the sites and transmit only incremental changes. Furthermore the main data-set is often cached locally at each of the collaborating sites to reduce the need for having to retransmit the entire data-set each time the application is started. Classically TCP (Transmission Control Protocol – the protocol that is widely used on the Internet for reliable data delivery) has been the default protocol used to distribute the data-sets. TCP works well in low-bandwidth below 10 Mb/s) or short distance  networks. However for high-bandwidth long-distance networks, TCP's conservative transmission policy thwarts an application's attempt to move data expediently, regardless of the amount of bandwidth available on the network. This problem is known as the Long Fat Network (LFN) problem  Mb/s or , howeverChanges made to the 3D environment need to be propagated with absolute reliability and with minimal latency and jitter. Latency is the time it takes for a transmitted message to reach its destination. Jitter is the variation in the latency. Fully reliable protocols like TCP have too much latency and jitter because the protocol requires an acknowledgment to verify delivery. Park and Kenyon  have shoth of a second) by a more recent update.The virtual representation of a remote collaborator (avatar) is often captured as the position and orientation of the 3D tracking devices that are attached to the stereoscopic glasses and/or 3D input device (e.g. a wand). With simple inverse kinematics one is able to map this position and orientation information onto a 3D geometric puppet, creating lifelike movements . The 3D Audio and video data are similar in property to the avatar data in that they usually comprise an unending stream that is best transmitted via UDP to minimize latency and jitter. Often video and audio packets are time stamped so that they can be synchronized on the receiving end. When more than two sites are involved in collaboration it is more economical to send audio/video via multicast. In multicast the sender sends the data to a specific device or machine that then copies the data to the various people that are subscribers to the data. For example, a user send their data to a multicast address and the routers that receive the data send copies of the data to remote sites that are subscribed to the multicast address. One drawback of multicast is that it is often disabled on routers on the Internet as one can potentially inundate the entire Internet. An alternative approach is to use dedicated computers as "repeaters" that intercept packets and transmit copies only to receivers that are specifically registered with the repeater. This broadcast method tends to increase the latency and jitter of packets, especially as the number of collaborators increases.QoS refers to a network's ability to provide bandwidth and/or latency guarantees. QoS is crucial for applications such as networked VE, especially those involving haptics or tele-surgery, which are highly intolerant of latency and jitter. Early attempts to provide QoS (such as Integrated Services and Differentiated Services) have been good research prototypes but have completely failed to deploy across the wider Internet because telecommunications companies are not motivated to abide by each others QoS policies. It has been argued that QoS is unnecessary because in the future all the networks will be over-provisioned so that congestion or data loss that result in latency and jitter, will never occur. This has been found to be untrue in practice. Even with the enormous increase in bandwidth accrued during the dot-com explosion, the networks are still as unpredictable as they were a decade ago. Ample evidence is available from the online gaming community which often remarks about problems with bandwidth, latency and jitter during game sessions . These gFrustrated by the lack of QoS on the Internet, there is growing interest in bypassing the traditional routed Internet by using the available dark fiber in the ground. Dark fiber is optical fiber that has not yet been lit. Currently it is estimated that only about 5–10% of the available fiber has been lit, and each fiber has several terabits/s of capacity. The dot-com implosion has made this dark fiber and wavelengths of light in the fiber, very affordable. The newly emerging model is to construct a separate customer-owned network by purchasing or leasing the fiber from a telecommunications company, and installing one's own networking equipment at the endpoints. A number of federally supported national and international initiatives have been underway for the last few years to create customer-controlled networks explicitly for the scientific community. These include the National Lambda rail , StarLigThe ability to use virtual technology for rehabilitation is a function of cost, availability, and the kind of applications that can best utilize the network and provide rehabilitation services. Thus far, tele-rehabilitation research has focused on the use of low speed and inexpensive communication networks. While this work is important, the potential of new high-speed networks has not gathered as much attention. Consequently, we have little but imagined scenarios of how such networks might be utilized. Let us consider the case where a high-speed network connects a rehabilitation center and a remote clinic. The question is what kind of services can be provided remotely.The scenario that we envision is one where patients are required to appear at a rehabilitation center to receive therapy. Our scenario could work in several conditions. For example, a therapist at one location may want an opinion about the patient from a colleague at another location or, perhaps, the therapist can only visit the remote location once per week and with virtual technology the daily therapy could still be monitored by the therapist remotely. In our imagined condition we have a therapist at a rehabilitation center with VE, haptic and video devices and software to help analyze the incoming data (i.e., data mining) feeding to a remote clinic with identical equipment connected together through a dedicated high speed network. As displayed in Fig. A system as described above is possible today although expensive. The network characteristics that would be needed for each information channel would be as follows. A high-bandwidth connection would be needed for video and audio streamed to the plasma displays at each location, in addition to the high bandwidth a low latency and jitter connection would be needed for the Varrier Display system (VE). For a force feedback haptic device communicating between the patient and the therapist, a low network bandwidth could be used but the latency and jitter need to be low.After all possible consideration of how to best construct the virtual system, the next concern is how to associate the complex stimuli with the behavior of interest. The relative influence of particular scene characteristics, namely field of view (FOV), scene resolution, and scene content, are critical to our understanding of the effects of the VE on our response behaviors  and the Expectation of the visual scene characteristics will also influence responses in a VE. When subjects had some knowledge of the characteristics of a forthcoming visual displacement most reduced their postural readjustments, even when they did not exert active control over the visual motion . Thus weThe challenge is to determine whether the subject has become immersed in the environment, i.e., has established a sense of presence in the environment (see paper by Riva in this issue), and then to establish the correlation between the stimulus and response properties. The experience within the VE is multimodal, requiring participation of all sensory pathways as well as anticipatory processing and higher order decision making. Consequently, it is difficult to attribute resultant behaviors to any single event in the environment and responses across participants may be very variable. We have united an immersive dynamic virtual environment with motion of a posture platform  to recorIn our laboratory, a linear accelerator (sled) that could be translated in the anterior-posterior direction was controlled by D/A outputs from an on-line PC. The sled was placed 40 cm in front of a screen on which a virtual image was projected via a stereo-capable projector (Electrohome Marquis 8500) mounted behind the back-projection screen. The wall in our system consisted of back projection material measuring 1.2 m × 1.6 m. An Electrohome Marquis 8500 projector throws a full-color stereo workstation field (1024 × 768 stereo) at 200 Hz [maximum] onto the screen. A dual Pentum IV PC with a nVidia 900 graphics card created the imagery projected onto the wall. The field sequential stereo images generated by the PC were separated into right and left eye images using liquid crystal stereo shutter glasses worn by the subject . The shutter glasses limited the subject's horizontal FOV to 100° of binocular vision and 55° for the vertical direction. The correct perspective and stereo projections for the scene were computed using values for the current orientation of the head supplied by a position sensor (Flock of Birds, Ascension Inc.) attached to the stereo shutter glasses (head). Consequently, virtual objects retained their true perspective and position in space regardless of the subjects' movement. The total display system latency from the time a subject moved to the time the new stereo image was displayed in the environment was 20–35 ms. The stereo update rate of the scene (how quickly a new image is generated by the graphics computer in the frame buffer) was 60 stereo frames/sec. Flock of birds data was sampled at 120 Hz.The scene consisted of a room containing round columns with patterned rugs and painted ceiling Fig. . The colSubjects gave informed consent according to the guidelines of the Institutional Review Board of Northwestern University Medical School to participate in this study. Subjects had no history of central or peripheral neurological disorders or problems related to movements of the spinal column  and a minimum of 20/40 corrected vision. All subjects were naive to the VE.We have tested 7 healthy young adults (aged 25–38 yrs) standing on the force platform (sled) with their hands crossed over their chest and their feet together in front of a screen on which a virtual image was projected. Either the support surface translated ± 15.7 cm/sec (± 10 cm displacement) in the a-p direction at 0.25 Hz, or the scene moved ± 3.8 m/sec (± 6.1 m displacement) fore-aft at 0.1 Hz, or both were translated at the same time for 205 sec. Trials were randomized for order. In all trials, 20 sec of data was collected before scene or sled motion began (pre-perturbation period). When only the sled was translated, the visual scene was visible but stationary, thus providing appropriate visual feedback equivalent to a stationary environment.Three-dimensional kinematic data from the head, trunk, and lower limb were collected at 120 Hz using video motion analysis . Infrared markers placed near the lower border of the left eye socket and the external auditory meatus of the ear  were used to define the Frankfort plane and to calculate head position. Other markers were placed on the back of the neck at the level of C7, the left greater trochanter, the left lateral femoral condyle, the left lateral malleolus, and on the translated surface. Markers placed at C7 and the greater trocanter were used to calculate trunk position, and shank position was the calculated from the markers on the lateral femoral condyle and the lateral malleolus.For trials where the sled moved, sled motion was subtracted from the linear motion of each segment prior to calculating segmental motion. Motion of the three segments was presented as relative segmental angles where motion of the trunk was removed from motion of the head to determine the motion of the head with respect to the trunk. Motion of the shank was removed from motion of the trunk to reveal motion of the trunk with respect to the shank. Motion of the shank was calculated with respect to the sled.The response to visual information was strongly potentiated by the presence of physical motion. Either stimulus alone produced marginal responses in most subjects. When combined, the response to visual stimulation was dramatically enhanced Fig. , perhapsUsing Principal Component Analysis we have determined the overall weighting of the input variables. In healthy young adults, some subjects consistently responded more robustly when receiving a single input, suggesting a proprioceptive (see S3 in Fig. Results from experiments in our laboratory using this sophisticated technology revealed a non-additive effect in the energy of the response with combined inputs. With single inputs, some subjects consistently selected a single segmental strategy. With multiple inputs, most produced fluctuating behaviors. Thus, individual perception of the sensory structure was a significant component of the postural response in the VE. By quantifying the relative sensory weighting of each individual's behavior in the VE, we should be better able to design individualized treatment plans to match their particular motor learning style. Developing treatment interventions in the virtual environment should carry over into the physical world so that functional independence will be increased for many individuals with physical limitations. In fact, there is evidence that the knowledge and skills acquired by disabled individuals in simulated environments can transfer to the real world -31.The ability for us to use this technology outside the area of research labs and bring these systems to clinics is just starting. However, the cost is high and the applications that can best be applied to rehabilitation are limited. The cost of such systems might be mitigated if this technology allowed therapists and patients to interact more frequently and/or resulted in better patient outcomes. Such issues are under study now at several institutions. This brings us to the idea of tele-rehabilitation, which would allow therapy to transcend the physical boundaries of the clinic and go wherever the communication system and the technology would allow . For exaThe ability to provide rehabilitation services to locations outside the clinic will be an important option for clinicians and patients in the near future. Effective therapy may best be supplied by the use of high technology systems such as VE and video, coupled to robots, and linked between locations by high-speed, low-latency, high-bandwidth networks. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies.The ability to provide rehabilitation services to locations outside the clinic is emerging as an important option for clinicians and patients. Effective therapy may best be supplied by the use of high technology systems such as VE and video, coupled to robots, and linked between locations by high-speed, low-latency, high-bandwidth networks. The use of data mining software would help analyze the incoming data to provide both the patient and the therapist with evaluation of the current treatment and modifications needed for future therapies. Although responses in the VE can vary significantly between individuals, these results can actually be used to benefit patients through the development of individualized treatments programs that will raise the level of successful rehabilitative outcomes. Further funding for research in this area will be needed to answer the questions that arise from the use of these technologies.


The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource  Although the HIV-1 genome contains only 9 genes, it is capable of generating more than 19 gene products. These products can be divided into three major categories: structural and enzymatic (Gag, Pol, Env); immediate-early regulatory (Tat, Rev and Nef), and late regulatory (Vif, Vpu, Vpr) proteins. Tat, Rev and Nef are synthesized from small multiply-spliced mRNAs; Env, Vif, Vpu and Vpr are generated from singly-spliced mRNAs, the Gag and Gag-Pol precursor polyproteins are synthesized from full-length mRNA. The matrix (p17), capsid (p24) and nucleocapsid (p7) proteins are produced by protease cleavage of Gag and Gag-Pol, a fusion protein derived by ribosomal frame-shifting. Cleavage of Nef generates two different protein isoforms; one myristylated, the other non-myristylated. The viral enzymes (protease, reverse transcriptase, RNase H and integrase) are formed by protease cleavage of Gag-Pol. Alternative splicing, together with co-translational and post-translational modification, leads to additional protein variability [Phylogenetic analysis, on its own, provides little information about the conformational, immunological and functional properties of HIV-1 proteins, but instead, focuses on the evolution and historical significance of sequence variants. To understand the clinical significance of genetic variation, sequence analysis needs to be combined with methods that assess change in the structural and biological properties of HIV-1 proteins. At present, information and tools for the systematic analysis of HIV-1 proteins are limited, and are scattered across a wide-range of online resources ,3. To faWe have categorized the Proteomics Resource into the following main subject headings Figure  &3:HIV Proteome – Information about structure and sequence, as well as references and tutorials, for each of the HIV-1 proteins  proteins using information available in public databases and tools ; the other from singly-spliced mRNA (p16) [MEPVDPRLEPWKHPGSQPKTA-21; the hydrophobic residues are highlighted in bold, and polar residues are italicized) at the N-terminus of the protein; the cysteine-rich disulphide bond region (22-CTNCYCKKCCFHCQVC-37); the core, basic and glutamine-rich region (49-RKKRRQRRRAHQNSQTHQASLSKQ-72) that is important for nuclear localization and TAR-binding activity, and the RGD cell-attachment site that binds to cellular integrins. In addition to being expressed in HIV-1-infected cells, Tat is also released into the extracellular fluid where it acts as a growth factor for the development of Kaposi's Sarcoma. Additional information about Tat and its protein-protein interactions can be found on the proteome page of the BioAfrica website located at .In the HIV-1 Proteome section, each of the 19 HIV-1 proteins has a webpage that is divided into six parts: "general overview", "genomic location", "domains/folds/motifs", "protein-macromolecule interactions", "primary and secondary database entries", and "references and recommended readings" Figure . The oveHXB2 , sequencB2 [HXB2 , and proB2 [HXB2 ,6  . ImportaPost-translational cleavage of the Gag, Gag-Pol and Nef precursor proteins occurs at the cell membrane during virion packaging, and is essential to the production of infectious viral particles. Drugs that inhibit this process, the protease inhibitors (PIs), are the most potent antiretroviral agents currently available. Thus it is important to collect information, not only on the sequence of protease enzymes from different HIV-1 subtypes, but also on the natural polymorphisms and resistance mutations that may effect their catalytic activities, drug responsiveness, substrate specificities, and cleavage site characteristics. Studies have shown that resistance mutations in the protease of subtype B are associated with impaired proteolytic processing and decreased enzymatic activity, and that compensatory mutations at Gag and Gag-Pol cleavage sites can partially overcome these defects . These fThe cleavage site section of the BioAfrica webpage is the direct extension of a recent publication in the Journal of Virology describing the location and variability of protease cleavage sites  Figure . TogetheHXB2 isolate, which, in addition to a premature stop codon, contained no cAMP/cGMP, PKC or CKII phosphorylation sites.The HIV-1 Protein Data-Mining Tool contains twelve sequence analysis techniques for assessing protein variability among different strains of HIV-1 Figure . These tThe HIV-1 BLAST Structure Tool facilitates the analysis of HIV-1 protein structure by allowing for rapid retrieval of archived structural data stored in the public databases Figure . Users mThe HIV-1 Proteomics Tools Directory is divided into two web pages. The initial webpage is a concise compilation of some of the most commonly used protein-specific Internet resources Figure . This "bThe impending rollout of antiretroviral therapy to millions of HIV-1-infected people in sub-Saharan Africa provides a unique opportunity to monitor the efficacy of non-B treatment programs from their very inception, and to obtain critical new information for the optimization of treatment strategies that are safe, affordable and appropriate for the developing world. An integral part of this massive humanitarian effort will be the collection of large amounts of clinical and laboratory data, including genetic information on viral subtype and resistance mutations, as well as routine CD4+ T-cell counts and viral load measurements. The mere collection of this data, however, does not ensure that it will be used to its maximum potential. To achieve full benefit from this explosive source of new information, the data will need to be appropriately collated, stored, analyzed and interpreted.[The rapidly emerging field of Bioinformatics has the capacity to greatly enhance treatment (and vaccine) efforts by serving as a bridge between Medical Informatics and Experimental Science. By correlating genetic variation and potential changes in protein structure with clinical risk factors, disease presentation, and differential response to treatment and vaccine candidates, it may be possible to obtain valuable new insights that can be used to support and guide rationale decision-making, both at the clinical and public health levels. The HIV-1 Proteomics Resource, described in this report, is an initial first step in the development of improved methods for extracting and analyzing genomics data, converting it into biologically useful information related to the structure, function and physiology of HIV-1 proteins, and for assessing the role these proteins play in disease progression and response to therapy. The Resource, developed at the Molecular Virology and Bioinformatics Unit of the Africa Centre of Health and Population Studies, is a centralized user-friendly database that is easily accessed through the BioAfrica website at .AA – Amino AcidBLAST – Basic Local Alignment Search ToolCKII – casein kinase IICTLs – cytotoxic T-lymphocytesDIP – Database of Interacting ProteinsDNA – deoxyribonucleic acidEnv – envelope glycoproteinGag – group-specific antigen polyproteinGIF – Graphics Interchange FormatHIV – Human Immunodeficiency VirusHIV-1 – Human Immunodeficiency Virus Type-1HTTP – Hypertext Transfer ProtocolLTR – long-terminal repeatmRNA – messenger RNANCBI – National Center for Biotechnology InformationNef – negative factorPDB – Protein Data BankpI – isoelectric pointPIs – protease inhibitorsPKC – protein kinase CPol – polymerase polyproteinRev – ART/TRS anti-repression transactivator proteinRNA – ribonucleic acidRNase H – ribonuclease HTat – transactivating regulatory proteinVif – virion infectivity factorVpr – viral protein RVpu – viral protein UThe author(s) declare that they have no competing interests.RSD created and maintains BioAfrica's HIV proteomics resource, HIV proteome section, proteomics tools directory, HIV-1 protein data-mining tool and HIV structure BLAST tool; performed protein sequence and structural model analyses; and wrote the manuscript.TDO conceived and maintains the BioAfrica website, and continues to oversee its rapid expansion; created the cleavage sites section; and participated in the design and implementation of the HIV proteomics resource.CS participated in the design of the HIV proteomics resource, with an emphasis on the proteomics tools directory.SD participated in the design and creation of the HIV proteome section, with an emphasis on the HIV-1 Tat protein.MG participated in the design of the HIV proteomics resource, with an emphasis on the HIV proteome section.SC supervised the project, and participated in the design and implementation of the HIV proteomics resource.All authors read and approved the final manuscript.A table containing a comparative summary of potential functional motifs (cysteine-rich region, myristoylated Asparagine, amidation, cAMP- and cGMP- dependent kinase phosphorylation, Protein Kinase C phosphorylation, and Casein Kinase II phosphorylation) in the HIV-1 Tat proteins of subtypes B and C, as identified using PROSITE.Click here for file


This paper analyzes the effect of the mean-square error principle on the optimization process using a Special Case of Hopfield Neural Network (SCHNN).The segmentation of multidimensional medical and colour images can be formulated as an energy function composed of two terms: the sum of squared errors, and a noise term used to avoid the network to be stacked in early local minimum points of the energy landscape.Here, we show that the sum of weighted error, higher than simple squared error, leads the SCHNN classifier to reach faster a local minimum closer to the global minimum with the assurance of acceptable segmentation results.The proposed segmentation method is used to segment 20 pathological liver colour images, and is shown to be efficient and very effective to be implemented for use in clinics. Segmentation is an important step in most applications that use medical image data. For example, segmentation is a prerequisite for quantification of morphological disease manifestations and for radiation treatment planning ,2, for cA Number of algorithms based on approaches such as histogram analysis, regional growth, edge detection and pixel classification have been proposed in other articles of medical image segmentation. In recent years, Artificial Neural Networks (ANNs) have been proposed as an attractive alternative solution to a number of pattern recognition problems. In our previous works , we haveHopfield network for the optimization applications consists of many interconnected neuron elements. The network minimizes an energy function of the form:Vk is the output of the kth neuron, Ik is the bias term, and Tkl is the interconnection weight between the kth and lth neurons. The energy function used in the segmentation problem is slightly different from the one defined by Hopfield and the arguments are given in [where N is the number of neurons, given in .The results that have been obtained in  were preThe segmentation problem of an image of N pixels is formulated in  as a parRkl is the Mahalanobis distance measure between the kth pixel and the centroid of class l, Rkl is also equivalent to the error committed when a pixel k is assigned to a class l. The index n in  is the power or weight of the considered error in the energy function of the segmentation problem, and Vkl is the output of the klth neuron. Nkl is a N × M vector of independent high frequency white noise source used to avoid the network being trapped in early local minimums. The term c(t) is a parameter controlling the magnitude of noise which is selected in a way to provide zero as the network reaches convergence. The minimization is achieved by using SCHNN and by solving the motion equations satisfying:where Ukl is the input of the kth neuron, and μ(t) is a scalar positive function of time, used as heuristically motivated stopping criterion of SCHNN, and is defined as in [where ed as in  by:β(t) = t(Ts - t)     (4)t is the iteration step, and Ts is the pre-specified convergence time of the network which has been found to be 120 iterations [kth pixel and the centroid of class l given by:where erations . The netXk is the P-dimensional feature vector of the kth pixel (here P = 3 with respect to the RGB color space components),  is the P-dimensional centroid vector of class l, and Σl is the covariance matrix of class l. The segmentation algorithm is described as follows [where  follows .Step 1 Initialize the input of the neurons to random values.Step 2 Apply the following input-output relation, establishing the assignment of each pixel to only and only one class.Step 3 Compute the centroid  and the covariance matrix Σl of each class l as follows:nl is the number of pixels in class l, and the covariance matrix is then normalized by dividing each of its elements by .where Step 4 Update the inputs of each neuron by solving the set of differential equations in (2) using Eulers approximation:Step 5 if t <Ts, repeats from Step 2, else terminated.For this study, a total of 20 liver tissue sections were provided by the pathological division of National Cancer Center in Tokyo. These sections were taken using needle biopsy, stained with hematoxylin and then magnified with an optical microscope. Figure Ts values between 30 and 120 iterations. Similar curves were obtained for the rest of the images of the dataset. As it is illustrated in Figure Ts = 120 iterations gives the optimal solution, the same as it is with MRI data [Figure MRI data .Rkl in the cost function (2), we have provided a simple modification to the above algorithm as follows:In order to study the effect of the weight of the Mahalanobis distance Step 1 Use the same random initialization N × M matrix, as input of the neurons, when minimizing the energy function (1) with different error's weight n.This condition is added to the algorithm in order to make sure that the random field does not have any effect on the generated results.Step 2 trough Step 5 remain the same.n in equation (2). As aforementioned, the pre-specified convergence time of SCHNN is fixed to Ts = 120 iterations. However, we can clearly see from Figure n in Equation (2), the same convergence point or a close position is reached in half the time of the one reached with n = 2 and Ts = 120 iterations. So, this raises the following question: what is the type of relation between the variable n in (2) and the pre-specified convergence time Ts?Figure n that corresponds to the optimum solution with Ts = 120 iterations. From Figure n = 6 gives the optimum solution with Ts = 120. Similar figures to Figure Before answering this question, it is essential to know at this level what is the best value of n, in Equation (2). When both (Ts and n) used together they give a local optima in the energy landscape of SCHNN. Figure n in Equation (2) that are obtained with Ts values 120, 60, and 30. We realized that the curves corresponding to Ts = 120 and Ts = 60 intersect in their optimum solutions obtained with n = 6, and the two curves are similar when n is in the range 5–10. However, the curve corresponding to Ts = 30, shows higher error at convergence of all values of n.In order to study the effect of the pre-specified time, we repeated the above experiments with different Ts values. We realized that each value of Ts corresponds to a value of n in (2) when it takes the value of six where SCHNN gives an optimum and acceptable results that agree with the pathological experts point of views. However, with other values of n, the random initialization may affect the solution of the problem, or in other words, may affect the error of the SCHNN at convergence as shown in Figure In order to see the effect of the random initialization on the results of the algorithm described in section 3, we have executed the same algorithm with different initialization matrices and the curves of the convergence values of SCHNN corresponding to these initializations are shown in Figure We analyzed the effect of considering the mean-square error in formulating the segmentation problem of multidimensional medical images. We have shown, empirically, that considering an integer power equal to six, of the error in the energy function of the problem, helped SCHNN to converge twice as fast as the same optimal solution obtained with the mean-square error algorithm. This result is promising to make our segmentation method useful for a Computer Aided Diagnosis (CAD) system for liver cancer and the like. In our future work, we will study deeply the effect of the random initialization and its effect on the segmentation result and on the SCHNN classifier.The author(s) declare that they have no competing interests.Rachid carried out the theoretical study, the sequence alignment and drafted the manuscript. Mohammed participated in the design of the study and performed the analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level.The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses.We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins.We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function. The gastrointestinal tract is the initial barrier to dietary constituents and is important in regulating nutrient entry, as well as keeping non-nutrients out. Additionally, the hepatobiliary system acts as an additional filter to rapidly excrete such non-nutrients into bile, thus keeping the net retention of these potential toxins low. Mammals have evolved many mechanisms in the gastrointestinal tract to select out usable dietary constituents from those that maybe potentially toxic to the body. It is apparent that the ATP-binding cassette proteins (ABC proteins/transporters) are the machinery that mediate the ATP-dependent transport of a wide variety of substrates that range from xenobiotics to peptide fragments . A subsede novo synthesis and hepatobiliary secretion. Sitosterolemia, a rare autosomal recessive disorder of sterol metabolism results in the disruption of dietary sterol entry and hepatobiliary sterol secretion [STSL, are known to cause this disease [in vitro and in vivo data, these 'half-transporters' are proposed to function as obligate heterodimers. In vitro experiments have shown that both proteins are needed to be co-expressed for apical expression and that these may function as mutual chaperones in the ER for maturation [In vivo experiments in mice have not been consistent. Using the Abcg5/Abcg8 double knockout mice, Graf et al has shown that by inoculating them with adenoviral constructs for Abcg5 and Abcg8 that both are required for expression of both proteins [et al and our group have constructed mouse models deficient in either Abcg5 [One of these processes involves the regulation of sterol entry and excretion. Whole body cholesterol homeostasis is a tightly regulated process, involving dietary absorption, ecretion -6. Under disease -9. Basedturation ,11. In ver Abcg5  or Abcg8er Abcg5  that shoIn this report, we examined the location of these two proteins using cellular fraction and immunohistochemical analyses of human liver, gallbladder and small intestine. We found a general concordance of co-expression of both proteins, but we also noted that ABCG5 and ABCG8 could be found in plasma membranes, as well as in intracellular membrane locations independent of each other. Additionally, deglycosylation of human liver membranes with peptidyl N-glycosidases did not alter the mobility of the proteins after SDS-PAGE, suggesting that these proteins may not be glycosylated in human liver. This differential localization suggests that perhaps ABCG5 and ABCG8 may have functions independent of each other, as well as functioning as heterodimers.Tissue aquistion. Human liver donated for transplantation, but deemed unsuitable for transplantation on inspection by the transplant service  was obtained in accordance with IRB approval. As soon as the liver was deemed unsuitable  pieces were either snap-frozen in liquid nitrogen and stored at -80°C or liquid nitrogen until use, or placed in ice-cold 2-methylbutane and stored in liquid nitrogen. Samples from more than nine different donors were used in these studies. Additionally, human gallbladders and segments of proximal small intestine were obtained from patients under going either laproscopic cholecystectomy or pancreatoduodenectomy (Whipple procedure). These tissues were directly taken from the operating room in normal saline on ice to be processed directly for frozen sectioning.Anti-membrin, anti-transferrin, and anti-calnexin antibodies were obtained from Stressgen (Victoria, BC Canada), anti-caveolin antibody from BD Bioscience (San Diego, CA, USA), anti-MDR1 that also detects MDR2/3 (C219) from Centocor Inc. , anti-MRP2 (cMOAT) from Chemicon International (Temecula, CA, USA) and secondary antibodies were purchased from Jackson Immuno research (West Grove, PA, USA). Polyclonal rabbit anti-sera to human ABCG5 and ABCG8 peptides were generated in-house, using a 20-peptide immunogen from human ABCG5 (576–587, FQKYCSEILVVNEFYGNFTC, GenBank Accession number NP_071881) and a 22-peptide sequence from human ABCG8 (608–629, SRRTYKMPLGNLTIAVSGDKIL, GenBank Accession number NP_071882). The anti-sera were further purified using peptide affinity columns and stored at a concentration of 0.8 mg/ml in Immuno Pure Binding Buffer (Pierce, Rockford, IL, USA). For peptide blocking experiments the peptides were dissolved in DMSO , incubated with the corresponding peptide for 1 1/2 hours at 37°C then used for immunoblotting as described below.g for 10 minutes, the pellet containing any undisrupted cells and nuclear debris were re-homogenized with one-half the initial volume of homogenization buffer, centrifuged at 1000 g for 10 minutes and this process was repeated once more. Supernatants were pooled and subjected to centrifugation at 100,000 g for 40 minutes. The resulting pellet, deemed the crude membrane fraction, was used as the starting material for Western blotting and fractionation experiments.Crude total membrane isolation was carried out with minor modifications as previously described . All theHuman liver crude total membrane proteins were re-suspended in 30% Nycodenz solution (Nycodenz in 5 mM Tris-HCl pH 7.5 and 1 mM EDTA). This suspension was loaded on top of a 40% Nycodenz solution cushion in an ultracentrifuge tube, overlaid by consecutive 23%, 20%, 15% and 10% Nycodenz solutions and subjected to centrifugation at 39,000 rpm for 16 hours at 4°C in a SW41 rotor . After centrifugation, 800–1000 μl fractions were sequentially removed from the top, combined with two volumes of homogenization buffer (see above) and centrifuged at 39,000 rpm at 4°C for 40 minutes to remove the Nycodenz. The resulting pellets were re-suspended in buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 0.1% SDS, 1 mM PMSF, 20 μg/μl of leupeptin and 1 μg/μl of aprotinin), the protein content determined by the method of Lowry and fractions analysed by SDS-PAGE. Equal amounts of protein (25 μg) per lane were loaded.The procedure for membrane fractionation was essentially as described for the Nycodenz fractionation, except for the homogenization buffer used (25 mM Tris pH6.8, 150 mM NaCl, 1 mM PMSF, 20 μg/μl of leupeptin and 1 μg/μl of aprotinin). The sucrose density gradient fractionation was modified as previously described . Human l® Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Inc. Boston, MA, USA).Proteins resolved by SDS-PAGE were transferred onto nitrocellulose membranes. Membranes were then blocked for 1 hour in 5% dry milk in PBS-T  and incubated with primary antibody against either ABCG5 or ABCG8 in 5% milk in PBS-T overnight at 4°C. Blots were washed three times for 5 minutes in TBS-T  with 150 mM NaCl, incubated with goat-anti-rabbit conjugated HRP antibodies (1:10000 dilution), washed for three times 5 minutes and developed with Western LightningSnap-frozen liver, gallbladder and intestine tissues were used to cut 8 μm thick frozen sections, air-dried for 30 minutes onto glass slides and kept at -80°C until used. The slides were stained with hematoxylin, rinsed with PBS three times, fixed for 10 minutes with cooled methanol at -20°C and rinsed with PBS three times. The slides were treated with blocking solution (10% donkey serum in 0.1 M glycine/PBS) for 30 minutes at room temperature and incubated with primary antibody overnight at 4°C. The slides were washed with PBS and incubated with secondary antibody (goat-anti-rabbit conjugated with Cy3™ or rhodamine or FITC) for 20–30 minutes at room temperature, rinsed with PBS three times and examined under an Olympus BX-5 confocal microscope with Fluoview.Peptide antibodies were raised against human ABCG5 and human ABCG8 and affinity purified prior to use (see Methods). The immunogen peptides used for the antibodies were selected since they were sequences that lay outside of the predicted transmembrane domains and based upon antigenicity.Western blotting experiments Figure  showed tCrude total membrane proteins from human liver were fractionated by Nycodenz gradient centrifugation and examined for localization markers by western blot analyses Figure . After NTo examine whether ABCG5 and ABCG8 were associated with membrane rafts, total membrane proteins from human liver were solubilized with ice-cold 1% Triton X-100 detergent and fractionated by sucrose density gradient centrifugation Figure . FractioThus, ABCG5 and ABCG8 have significant overlap with each other suggesting co-localization. However, these proteins did not seem to co-localize with any specific membrane marker except transferrin, when two different methods of fractionation were utilized.It has been shown previously that ABCG5 and ABCG8 are expressed only in the liver and intestine . To furtSerial sections of human gall bladder were incubated with each antibody and pre-immune serum was used as a negative control. Both ABCG5 and ABCG8 were detected in the epithelium of gall bladder mucosa Figure  and 5B. Both ABCG5 and ABCG8 were detected in the apical surfaces of the enterocytes in biopsy samples of the small intestine , the apical patterns of expression of these proteins in both the liver and intestine would seem to suggest that these proteins seem to traffic to these specialized membranes normally. In the absence of an independent method, and the lack of a direct assay for function, whether these proteins form an active heterodimer can not be resolved at present.Finally, although rodent sterolins are glycosylated and In summary, we report the first immunolocalization of ABCG5 and ABCG8 in human liver, gall bladder and intestine. Our data show that these proteins are located in membranes and can have an apical expression in all of these tissues. Biochemical, as well as immunolocalization studies show that while both proteins co-localize in general, they can also seem to have expression patterns that may be independent of each other.None declared.ELK and MHL performed the experiments, KDC and DBA provided the liver and surgical samples respectively SBP was responsible for supervision, data analyses and obtaining funding for these experiments. ELK and SBP wrote the paper.The pre-publication history for this paper can be accessed here:


The present paper expresses the author's views about the practical utility of Health Behavior Theory for health behavior intervention research. The views are skeptical and perhaps even a bit exaggerated. They are, however, also based on 20-plus years of in-the-trenches research focused on improving health behavior practice through research.The author's research has been theoretically driven and has involved measurement of varying variables considered to be important theoretical mediators and moderators of health behavior. Regretfully, much of this work has found these variables wanting in basic scientific merit. Health Behavior Theory as we have known it over the last 25 years or so has been dominated by conceptualizations of behavior change processes that highlight cognitive decision-making. Although much of health behavior practice targets what people do rather than what they think, the logic of focusing on thoughts is that what people think about is the key to what they will do in the future, and that interventions that can measure and harness those processes will succeed to a greater extent than those that do not. Unfortunately, in the author's experience, the premise of cognitive theories has fallen short empirically in a number of ways. The cognitive schemata favored by most health behavior theories are difficult to measure, they do not predict behavioral outcomes very well, there is little evidence that they cause behavior, and they are hard to change directly.It is suggested that health behavior researchers reconsider their use of these theories in favor of models whose variables are more accessible to observation and experimental manipulation and that most importantly have strong empirical support. The author has been conducting research on behavioral treatment of obesity for about 25 years. During that time, the dominant conceptual models guiding intervention development have been cognitive behavior models that have their origin in psychological theory. Those most often cited include the Health Belief Model , ProtectWeinstein's summary is illustrative of the fact that Health Behavior Theory has tended to be particularly interested in understanding people's motivation to change behavior rather than ability to change. Moreover, motivation is thought to be the result of a relatively complex, but logical, interpretation of large quantities of information about self and environment. The theories that Weinstein reviewed deal almost exclusively with behavioral decision processes in people's minds. They have few if any terms relating to how information gets into peoples minds or how subsets of it receive more or less attention. Broader health behavior theories such as Social Cognitive Theory or the Transtheoretical model have addressed issues and variables outside the person to a greater extent, but the fundamental interest in and belief in psychological variables as the key force in determining health behavior remains.The implications of the focus of health behavior theory on psychological determinants of behavioral decision-making for my own research area of interest, obesity treatment, are several. One is the inclusion of measures of psychological characteristics in most research protocols . A second is the inclusion of treatment elements that specifically target psychological perceptions and processes independent of the diet and physical activity behaviors that actually produce weight change . A third is the belief that psychological reactions to treatment experiences themselves are very important and deserve independent attention. Common behavioral prescriptions for weight-loss goals and frequency of self-weighing are exemplary .The problem with the emphasis on cognitive variables in weight-control research is that they have so far failed to meet fundamental scientific criteria for empirical verification. Thus, they also have not led to a better understanding of the weight-loss process, have not improved our ability to predict weight-loss outcomes, and have not led to improvement in treatment methods. In some cases it is even arguable that they have made treatment worse. I will illustrate these problems with results from my own research.Like most behavioral researchers in the obesity area, I have attempted to measure elements of health behavior theory in every obesity intervention project I have ever conducted. I have assessed weight-loss goals, behavioral and weight-loss self-efficacy, psychological well-being, perceived barriers to diet and physical activity change, stages-of-change, and perceived social support. How well have empirical examinations of these factors fared as predictors of success in weight control?We have examined the predictive value of self-efficacy assessments in several of our studies and describe the results from three of these here in more detail -10. In tThe second study examined mood and situational self-efficacy in 55 men and 58 women before and after a 16-week weight-loss treatment with a 1-year follow-up . Women hThe third study examined predictors of weight change over a 2-year period in 460 men and 1172 women who received a low-intensity weight-loss intervention delivered through their HMO . The selOur overall conclusion from the analyses described above, as well as others not pursued in as great detail, is that self-efficacy is a weak predictor of weight loss and is inconsistent across study populations and gender. It tends to increase with weight loss. However, treatment-induced increases in efficacy are not predictive of longer-term weight-loss success.We have also attempted to measure barriers to adherence to weight-control behaviors in many of our studies -14. The Goal-setting has long been of interest to health behavior theory and in recent years has attracted attention in weight-loss research when it was realized that most people who enter weight-loss treatments want to lose a lot more weight than is realistic given the potency of current weight-loss methodologies . When asPerceived social support is another psychological factor thought to influence health behavior decision-making. We have measured social support in a variety of ways in our studies, ranging from single-item questions to multipaged assessments attempting to differentiate among informational, instrumental, and emotional support. The results, unfortunately, have closely paralleled those we have seen with other assessments of barriers to adherence. Assessments of social support prior to treatment do not predict weight loss. Average reports of social support tend to parallel weight loss itself. When people lose weight they report more social support. When they regain, they report less. In other words, perceptions of social support are not predictive of success in weight-loss treatments.Self-monitoring of health behavior is incorporated into many health behavior theories, usually as part of a person's assessment of achieved outcomes. Although self-monitoring is usually considered a positive element in the adoption of health behavior, in obesity treatment frequent self-monitoring of weight has tended to be down-played or even discouraged on the grounds that disappointing results (i.e., less than desired weight change) may undermine motivation. This is another example in which health behavior theory may have indirectly led to incorrect treatment recommendations. In weight-loss treatments, active discouragement of frequent self-observation of weight has become popular based on the premise that more frequent weighting will cause psychological stress and lower self-efficacy. Recently, we have examined the relationship between frequency of self-weighing and body weight in both clinical and population samples and have found, somewhat to our surprise, that frequency of self-weighing is one of the strongest single predictors of body weight cross-sectionally, and change in the frequency of self-weighing is one of the strongest predictors of weight change [Linde JA, Jeffery RW and French SA, unpublished data]. The direction of predictions, however, is opposite that derived from theory. People who weigh themselves more weigh less and are more successful in losing weight.A final failure of current health behavior theory to prove useful in weight-control research is a recent examination of the relationship between a stage-of-change measure adopted from Prochaska and short- and long-term weight loss . CategorOur most recent effort to utilize health behavior theory in obesity intervention research is a study that attempted to examine the effectiveness of experimentally-induced outcome expectancies on weight loss [Finch EA, Linde JA, Jeffery RW, Rothman AJ and King CM, unpublished data]. Obese men and women participated in an 8-week weight-loss program with 18-month follow-up in which they were assigned to one of two expectancy groups. The optimistic group was told that focusing exclusively on the positive benefits of weight loss would be valuable in ensuring that they remained motivated in their weight-loss efforts and was given assignments during weekly group sessions and homework between sessions to reinforce this optimistic mindset. A "balanced" expectancy group received the instructions that focusing on both the positive and negative aspects of weight loss, a balanced approach, would be most conducive to maintaining weight-loss motivation. This group also received assignments to reinforce their message. Results of this study indicated that the expectation induction was successful initially but difficult to maintain in the face of real weight-loss experience. We were also unable to show that experimentally-induced expectations influenced weight-loss success.To summarize the findings described above, I have had considerable difficulty over the last 25 years in confirming that the psychosocial variables favored by health behavior theory are of much value for obesity intervention research. They do not predict weight loss well, either as mediators or moderators. There is little evidence to support the idea that targeting them for intervention improves weight-loss outcomes. It is, of course, arguable that the weak findings relating to health behavior theory variables are due in large part to methodological weaknesses, either in measurement tools and/or their frequency of measurement. I would argue, however, that 25 years is long enough to wait for improved methods and that it is time to look elsewhere for variables that better predict weight-change outcomes and that, therefore, may form a better basis for improving future treatments.Given the lack of success finding support for cognitive mediators of behavior change in weight loss, one might surmise that progress in improving weight-loss interventions over the last 20 years must have been dreary indeed. Somewhat surprisingly, however, that is not the case. In fact, the short-term (6 to 12 months) success of weight-loss treatments has approximately doubled over that time and several variables have been identified that reliably enhance treatment outcomes. It has been clearly shown experimentally that increasing treatment length , prescriThe argument above about the practical limitations of many popular theories of health behavior is not meant to be a call to abandon theory. Behavior scientists have amassed much useful information about the principles underlying human behavior that should be valuable for health behavior interventions. Much is known about human perception, learning, motivation, and responsiveness to environmental opportunities and contingencies. Health behavior intervention lies at the interface between people and their environment. Interventionists change aspects of the environment  with the intention of producing changes in how people behave. What is needed to advance health behavior intervention is theory that addresses relationships between modifiable aspects of the environment and behavior. There is no doubt that cognitive processes are involved in these relationships. However, the extent to which current theories capture this is questionable. Data now available suggest that easily obtainable information about people's cognitive processes adds little to our ability to predict the results of interventions. Thus, it may be wise to pay more attention to applied theories like classical behavior theory , communiNone declared.


Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands.Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section.These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences. Most proteins can be grouped, on the basis of similarities in their amino acid sequences, into a limited number of protein families. Proteins or protein domains that belong to a particular family usually share functional attributes and are derived from a common ancestor. Highly conserved sequences in protein families are generally important for the function of a protein and/or for the maintenance of its 3-dimensional structure. Within the last decade, the sensitivity of sequence searching techniques has been improved by profile or motif-based analysis, which uses information derived from multiple sequence alignments to construct and search for sequence patterns -4. By stBy doing a keyword search, the protein sequences mined out from different databases is highly varied owing to different levels of redundancy. This could be due to the different strengths and weaknesses underlying the analysis algorithms used in different databases. The usage of the broad range signatures existing in databases, for the retrieval of blue copper proteins like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein brings out different kinds of copper proteins and a lot more of unrelated proteins. A search once again becomes necessary for sorting out the required blue copper proteins. The usage of pattern database would be more selective as it can identify family members based on the conserved functional region patterns. Keeping these broad spectrum signatures in mind, more specific and targeted protein signatures for each of the blue copper proteins was designed. The diagnostic success of these specified signatures over the wide range signatures mentioned lies in the number of true positives picked over the minimal or nil false positives picked from the non redundant databases.Blue copper proteins, which are also known as cupredoxins, are small, soluble proteins 10 – 14 kDa) whose active site contains a type 1-copper . All the – 14 kDaThe use of active site patterns or signatures is very rapidly becoming one of the essential tools of sequence analysis ,15. Althth position, cysteine at the 74th position, histidine at 79th position and methionine at the 74th position are bound to the copper atom involved in electron transport chain.The eukaryotic blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, umecyanin, uclacyanin, and cusacyanin. The prokaryotic blue copper proteins were rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, auracyanin, amicyanin and blue nitrite reductase. Plastocyanins are found both in eukaryotes and prokaryotes. Table The number of sequences retrieved for a protein from different databases by keyword search are tabulated in Table The signatures already available for each of the blue copper proteins retrieved from the Prosite motif database are listed in Table As shown in 'Appendix 1 see ' the newThe members of a protein family can be identified by collecting the matching sequences to profile or motif databases. Protein signatures are sequence motifs diagnostic to a protein family indicating function. Signatures are matched to protein sequences in the non redundant databases and is scored using a dynamic programming algorithm which permits permeability in gap distance and residue type . GeneratBy doing a keyword search, we get varied results from the different databases as indicated in Table The protein signatures in a way can be compared to primers used for amplification. The more specific and concise a primer, the more specific is the amplification, similarly more specific the protein signature more significant are the picks from the non redundant databases. Specific signatures in a way reduce the time taken to pool related sequences from the abundantly available sequences from the non redundant databases. For example as shown in Table Signatures designed around the functionally important regions of a protein are valuable for annotation. In this study, specific signatures were designed around the active site regions of each of the blue copper proteins plantacyanin, plastocyanin, uclacyanin, stellacyanin, rusticyanin, sulfocyanin, amicyanin, halocyanin, pseudoazurin, azurin and nitrite reductase. These will be very useful for annotating uncharacterized proteins as blue copper proteins. Further, because of their high specificity to each subclass, they can be used in classifying the various subtypes of blue copper proteins.The blue copper proteins were distinguished based on the source of origin as prokaryotic and eukaryotic. The active site residues of the eukaryotic and prokaryotic blue copper proteins, which bind the copper metal atom, were identified from the Protein Data Bank and tabulated.The name of each of the blue copper protein was given as a query in the keyword searches at NCBI, SwissProt, TrEMBL, PIR, EMBL and PDB databases to check for the number of sequences retrieved from each database and the results were tabulated.The signatures already existing for each of these blue copper proteins were identified from the Prosite motif database and tabulated. These already existing signatures were used as query patterns in the PIR Motif/Peptide match and a search was made against the PIR-nREF database. The PIR-nREF database consists of sequences from the PIR [282,987 sequences], SwissProt [146,720 sequences], TrEMBL [1,069,649 sequences], GenPept [1,724,186 sequences], Ref Seq [756,736 sequences] and PDB [24,863 sequences]. The number of protein sequences matching the query  was retrieved and the query results were tabulated.Each of the blue copper proteins from different sources were chosen from the different sequence databases and aligned using the multiple sequence alignment tool ClustalW. Conserved regions, which include the amino acids bound to the copper, reflecting the active site imparting a vital biological role of electron transport were chosen to design signatures. In regions showing 100% conserved sequences they were identified as conserved peptides. An example of how the functional protein signatures were designed is shown for plastocyanin in Appendix 1 see . The sigPG, AVG and SA participated in the design and coordination of the study. AVG carried out the bioinformatics search, the designing of the signatures and drafted the manuscript. PG and SA read and approved the final manuscript.Designing protein signatures: Illustrated example Plastocyanin. Plastocyanin sequences of eukaryotic and prokaryotic origin were retrieved from the PDB and SwissProt databases. The eukaryotic sequences were subjected to a ClustalW multiple sequence alignment. Signatures were designed based on the conserved pattern around the active site region [copper binding to four amino acids in plastocyanin]. The same procedure was adopted for plastocyanin sequences of prokaryotic origin. The newly designed signatures were used as queries in the Pattern/peptide match search at the PIR database [Protein Information Resource]. The numbers of plastocyanin sequences retrieved are tabulated in Table 6. The results were compared with the already existing signatures for plastocyanins and the number of sequences that these signatures picked up from the PIR database [data shown in Table 4 & 5].Click here for file


E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated.E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains.By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify  E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants [E. sakazakii in production environments from food  factories and households isolated the organism with varying frequency from nearly all environments examined, strongly indicating, that this is a widespread organism [E. sakazakii has been isolated from a wide range of foods, including cheese, meat, vegetables, grains, herbs and spices and UHT milk, but most of the literature concentrates on the presence the organism in dried infant formula milk [et al. [E. sakazakii isolated from patients and dried infant formula involved in 2 hospital outbreaks using a combination of typing methods. Although the levels of contamination seemed low, the authors could show, that with an initial concentration of 1 CFU/ml, reconstituted formula stored at room temperature would take approx. 10 hrs to reach 107 cells per 100 ml and even sooner in formula held on 35 – 37°C. Since then, many case reports have described this epidemiologic correlation [ infants ,2. Morta infants . The surorganism . Up to nula milk -8. In 19relation .E. sakazakii include the observation of yellow pigmentation of colonies and the testing for glucosidase activity. Based on this latter biochemical activity commercially selective chromogen media have recently been developed such as the Druggan-Forsythe-Iversen agar, DFI  and the Enterobacter Sakazakii Isolation Agar, ESIA (AES Laboratoire, France).Cultural standard identification procedures for E. sakazakii [E. sakazakii strains of different origin. Finally, based on this new sequence data a specific PCR system was developed for E. sakazakii detection.Besides, molecular assays have often proven to be useful as they offer a alternative means to rapidly and specifically identify organisms from a wide variety of sources. Although PCR detection methods are now widely used in identification of microorganisms, only one PCR system, based on a single available full-length 16S rRNA gene sequence of type strain ATCC 29544 [GenBank: AB004746], was published up to now for akazakii . HoweverE. sakazakii strains from various sources (five fruit powder isolates, three human isolates, two production environment isolates, one milk isolate, one baby food isolate, one milk powder isolate) and of E. sakazakii type strain ATCC 51329 were determined. This sequence data has been deposited in the GenBank [AY752936 – AY752943, AY803186 – AY803192]. Thereafter, a sequence comparison was performed between the 16S rRNA genes of the fourteen E. sakazakii strains and the E. sakazakii type strain ATCC 29544 by calculation of a distance matrix using the ARB programme. Thirteen E. sakazakii strains used in the study exhibited 99.4 to 100% sequence similarity to the E. sakazakii type strain ATCC 29544 [GenBank: AB004746]. Meanwhile similarity between strain ATCC 51329 and the strain ATCC 29544 was significantly lower (97.9%).As a starting point, the full-length 16S rRNA sequences of thirteen E. sakazakii species. In figure E. sakazakii type strain ATCC 29544, whereas the E. sakazakii strain ATCC 51329 forms a second branch within this group. The subtree was calculated using the TREEPUZZLE tool within the ARB package exhibiting a "consensus tree" from different calculation methods. The value on each branch is the percent occurrence of the branching order in 500 bootstrapped trees. The 92% bootstrap value for the E. sakazakii ATCC 51329 branch strongly supports the theory about the second lineage.Phylogenetic affiliation of the newly obtained sequences to tree_jul04_1450 comprising >28.000 almost full-length (>1449 nucleotides) 16S rRNA gene sequences revealed the presence of two phylogenetically distinct lineages within the E. sakazakii 16S rRNA gene . Within these regions substitutions ranging from two to seven bases are present in all strains investigated and are most pronounced in E. sakazakkii strain ATCC 51329, thus providing the bases for the formation of the second phylogenetic branch. In table E. sakazakii strains investigated in the study in relation to the E. sakazakii typestrain ATCC 29544, E. cloacae typestrain ATCC13047 and E. coli type strain ATCC 11775. Recently, a number of E. sakazakii partial sequences were deposited into the public database [E. sakazakii species (data not shown). However, for a reliable pylogenetic analyses determination of at least 1000 nucleotides is recommended [E. sakazakii species in view of clinical and food safety aspects needs further evaluation.Further qualitative inspection of the sequences revealed that, besides of the presence of a number of single base substitutions, at least four "hot spots" of polymorphisms can be observed along the E. sakazakii. They were first evaluated using PROBE-MATCH tool of the ARB software. Additionally, BLAST searches were performed against the non-redundant database (nr) of EMBL/GENBANK. Both primers seemed to be specific. Amplification conditions in the assay were optimized by taking an annealing temperature of 60°C for 1 min.In a second step, based on our new sequence information, we designed a specific primer pair for the amplification of the 16S rRNA gene of strains within the two phylogenetic lineages of E. sakazakii strains as well as to differentiate E. sakazakii from other closely related Enterobacteriacae was assessed. This was done by applying the assay to purified genomic DNA template from isolates of 45 known E. sakazakii strains form different origin and 28 non E. sakazakii strains. A gel-based example of the screen is shown in figure E. sakazakii isolates and non-target microorganisms were analyzed. The rest of the results from the screen are summarized in table E. sakazakii isolates tested. There were no amplification products observed when the non E. sakazakii strains controls were similarly analyzed.We then sought to evaluate the utility of the PCR assay next. In particular the assay's ability to correctly identify different E. sakazakii isolates and non-target microorganisms were analyzed. First the system was not able to detect all the E. sakazakii strains. By using the PROBE_MATCH tool included in the ARB software it can be retrieved, that one of the primers (Esak3) used in the Keyser system exhibits four mismatches at the 3 prime end to the 16S rRNA gene sequence of E. sakazakii strain ATCC 51329, thus providing a possible explanation for the negative amplification result with this strain. Moreover, this system also gives positive results with some non-target organisms (e.g. E. cloacae, S. liquefaciens, S. fucaria, S. Enteritidis).For comparison the various strains were also similarly analyzed using the PCR system described by Keyser et al. . The resE. sakazakii lineages (strain ATCC 51329 and strain ATCC 29544). In both strains a detection limit of 10 pg was determined (figure B. cereus ATCC 10876, L. acidophilus ATCC 13651, S. aureus ATCC 25923 and E. faecium DSM 2918 were used. There were no significant influences on assay's performance observed in the presence of up to 200 ng non-target DNA in the reaction mixture (data not shown).To determine the detection limit of the newly developed assay, PCR was performed on decreasing amounts of purified DNA template from representatives of the two E. sakazakii species. Based on this new sequence data we have developed a 16S rRNA targeting PCR assay, which allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other non-target microorganisms was shown. This PCR system provides a valuable tool for rapid identification of this food borne pathogen.By phylogenetic analysis of the new provided full-length 16S rRNA sequence data we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii strains, with isolates from human, food and environmental origin, as well as 28 non E. sakazakii strains from selected species were included in this study (Table E. sakazakii strains (E. sakazakii ATCC 51329, E. sakazakii 1084 fruit powder isolate, E. sakazakii 954 fruit powder isolate, E. sakazakii 858 fruit powder isolate, E. sakazakii 759 fruit powder isolate, E. sakazakii 236 fruit powder isolate, E. sakazakii FSM393 baby food isolate, E. sakazakii FSM33 milk isolate, E. sakazakii 265 milk powder isolate, E. sakazakii FSM468 production environment isolate, E. sakazakii 266 production environment isolate, E. sakazakii ES4 human isolate, E. sakazakii ES11 human isolate, E. sakazakii ES Vo7/24922 human isolate) were sequenced. Afterwards, all strains were used for validation of the specificity of the new developed PCR identification system.Overall, 47 R Tissue Kit (Qiagen AG, Switzerland) in accordance with the suppliers' protocol.The strains were grown on blood agar plates under appropriate conditions. DNA was extracted from the grown colonies using the DNeasy2), 2.5 mM MgCl2, 200 μM dNTPs each and 3 U Expand High Fidelity enzyme mix.For 16S rRNA gene amplification, reaction mixtures  containing primers 616V (5' AGA GTT TGA TYM TGG CTC 3') and 630R (5' CAK AAA GGA GGT GAT CC 3')  at 10 pmThe amplification was performed in a T3 thermocycler (Biometra, Germany). The PCR conditions were: 2 min at 95°C, 10×  followed by 15× . Cycling was completed by a final elongation step at 72°C for 7 min. After PCR the reaction products were separated on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light. In cases where sequencing was desired, the correct size (approx 1500 bp) products were excised from the gel and purified using the MinElute™ gel extraction kit (Qiagen, Switzerland). The products were thereafter sequenced. Sequencing reactions were performed using a modified Sanger method and the Big-Dye chemistry from Applied Biosystems on an ABI 3730 capillary DNA Analyzer (Applied Biosystems, USA) employing the same primer pair as for amplification of the 16S rRNA gene (616V/630R) and additional internal primers for "walking reactions". Sequencing was performed by Microsynth .16S rRNA gene sequences of fourteen newly sequenced strains were added to an alignment of about 28'000 almost full length small subunit rRNA sequences by using the alignment tool of ARB program package [E. sakazakii S16 rRNA gene targeting primers Esakf (5' GCT YTG CTG ACG AGT GGC GG 3') and Esakr (5' ATC TCT GCA GGA TTC TCT GG 3') were designed and synthesized . This primer pair binds to conserved regions (E. coli position 88 – 107 (Esakf) and 1017 – 998 (Esakr)) in the S16 rRNA gene sequences giving an amplicon of 929 bp.The following specific Taq reaction mixture, 2 U Taq polymerase (Promega, Madison, WI),) and 200 μM dNTPs each. Thermal cycling was carried out by using an initial denaturation step of 94°C for 2 min, followed by 29 cycles of denaturation at 94°C for 30 sec. annealing temperature for 1 min and elongation at 72°C for 1 min 30 sec. Cycling was completed by a final elongation step at 72°C for 5 min. Amplification conditions were optimized by gradually increasing the annealing temperature in the assay from 52°c to 64°C. The reaction products were resolved on a 1.5% agarose gel followed by ethidium bromide staining and examination under UV light.For amplification, reaction mixtures  containing primer Esakf and Esakr at a concentration of 10 pM were prepared by using 10 × E. sakazakii ATCC 51329 [GenBank: AY752937], E. sakazakii 1084 fruit powder isolate [GenBank: AY803192], E. sakazakii 954 fruit powder isolate [GenBank: AY752938], E. sakazakii 858 fruit powder isolate [GenBank: AY752936], E. sakazakii 759 fruit powder isolate [GenBank: AY752939], E. sakazakii 236 fruit powder isolate [GenBank: AY752943], E. sakazakii FSM393 baby food isolate [GenBank: AY752941], E. sakazakii FSM33 milk isolate [GenBank: AY752940], E. sakazakii 265 milk powder isolate [GenBank: AY803191], E. sakazakii FSM468 production environment isolate [GenBank: AY752942], E. sakazakii 266 production environment isolate [GenBank: AY803190], E. sakazakii ES4 human isolate [GenBank: AY803186], E. sakazakii ES11 human isolate [GenBank: AY803187], E. sakazakii ES Vo7/24922 human isolate [GenBank: AY803189].AL and TT carried out the experimental part of the study. RS carried out the conception of the study. All authors participated in production and approval of the manuscript.