Epilepsy is a neurological disorder, characterized by recurrent unprovoked seizures which have a high impact on the individual as well as on society as a whole. In addition to the economic burden, epilepsy imposes a substantial burden on the patients and their surroundings. Patients with uncontrolled epilepsy depend heavily on informal care and on health care professionals. About 30% of patients suffer from drug-resistant epilepsy. The ketogenic diet can be a treatment of last resort, especially for children. The beneficial effect of the ketogenic diet has been proven, but information is lacking about its cost-effectiveness. In the current study we will evaluate the (cost-) effectiveness of the ketogenic diet in children and adolescents with intractable epilepsy.In a RCT we will compare the ketogenic diet with usual care. Embedded in this RCT will be a trial-based and model-based economic evaluation, looking from a societal perspective at the cost-effectiveness and cost-utility of the ketogenic diet versus usual care. Fifty children and adolescents (aged 1-18) with intractable epilepsy will be screened for eligibility before randomization into the intervention or the usual care group. The primary outcome measure is the proportion of children with a 50% or more reduction in seizure frequency. Secondary outcomes include seizure severity, side effects/complaints, neurocognitive, socio-emotional functioning, and quality of life. Costs and productivity losses will be assessed continuously by a prospective diary and a retrospective questionnaire. Measurements will take place during consults at baseline, at 6 weeks and at 4 months after the baseline period, and 3, 6, 9 and 12 months follow-up after the 4 months consult.The proposed research project will be the first study to provide data about the cost-effectiveness of the ketogenic diet for children and adolescents with intractable epilepsy, in comparison with usual care. It is anticipated that positive results in (cost-) effectiveness of the proposed intervention will contribute to the improvement of treatment for epilepsy in children and adolescents and will lead to a smaller burden to society.NTR2498).The study has been registered with the Netherlands Trial Registry ( Epilepsy is a brain disorder characterized predominantly by recurrent and unpredictable interruptions of normal brain function. Seizures are transient occurrences of signs and/or symptoms due to such abnormal excessive or synchronous neuronal activities in the brain . DiffereIn the Netherlands, the point prevalence of epilepsy in children and adolescents between 0-18 years is 4.77 boys, and 4.55 girls per 1,000 persons. The yearly incidence of epilepsy in the Netherlands for children and adolescents between 0-18 years is 0.58 per 1,000 boys and 0.53 per 1,000 girls . The ketCurrently the scientific and clinical attention paid to the role of the ketogenic diet is negligible. This means that a ketogenic diet is often overlooked and underutilized as a treatment option for children with intractable epilepsy. An important reason for this is that relatively few children and their parents can comply with the stringent diet. Therefore, physicians are often reluctant to initiate the diet. To offer more children the opportunity to benefit from a ketogenic diet, we suggest that the diet should be initiated and monitored under strictly controlled circumstances in order to maximize compliance.The beneficial effect of a ketogenic diet has been studied in multiple observational studies -20, reviThe aims of our prospective study are to provide more evidence about the effectiveness and to investigate the cost-effectiveness of treatment with a ketogenic diet KD in comparison with usual care in children and adolescents with drug-resistant epilepsy who are not eligible for epilepsy surgery.The design and methods of a randomized controlled trial (RCT) evaluating the (cost-) effectiveness of a ketogenic diet in comparison with usual care in children and adolescents with intractable epilepsy are described in this paper. This study has been approved by the ethics committee of the Academic Medical Centre Utrecht, the Netherlands.We defined the following research questions:(1) What are the effects of the ketogenic diet in comparison with usual care with respect to changes in seizure frequency and seizure severity, side effects/complaints, neurocognitive, socio-emotional functioning, and quality of life?The economic evaluation will consist of a trial-based and a model-based economic evaluation study.(1) From a social perspective, is the ketogenic diet, in comparison with usual care, preferable in terms of costs, effects and utilities?(2) What is the incremental cost-effectiveness ratio (ICER) of the ketogenic diet in comparison with usual care?(1) In comparison with usual care, is the ketogenic diet preferable in terms of costs and utilities during the remaining life expectancy of the study population?The proposed study is a prospective RCT. The subjects will be randomized to either the ketogenic diet or to usual care. The trial flow of the proposed subject enrolment and randomization procedures are shown graphically in Figure The patient population consists of children and adolescents with drug-resistant epilepsy. Patients are eligible to participate if they meet the following criteria: age between 1 and 18 years old; having uncontrolled seizures; not eligible for epilepsy surgery; no fatty acid oxidation disorders and related diseases; no diabetes and hyperinsulinism; no prolonged QT-time syndrome; no hypercholesterolemia or hypertriglyceridemia; no severe liver, kidney or pancreas diseases; no renal tubular acidosis; no severe behavioral disorder; no malnutrition; no treatment with topiramate or acetazolamide and no positive family history or other risk factors for kidney stones or acidosis.http://www.ketogeenmenu.nl. Parents and potential candidates who are considering participating in the study will have at least one week to decide, before signing informed consent.Potential participants are screened for exclusion criteria by their own attending paediatrician, neurologist or paediatric neurologist. Children who are eligible to be treated with a ketogenic diet are subsequently referred to the multidisciplinary ketogenic diet team at the epilepsy centre Kempenhaeghe in the Netherlands. Eligible children and their parents will first receive information by telephone before getting an invitation for an information visit. During the information visit eligibility is checked by the multidisciplinary team. If they are interested, parents, adolescents and/or children can find information about the study on the website A 50% reduction in seizure frequency is considered clinically relevant and is defined as success. Assuming a minimum detectable difference in success rate of 35% between the ketogenic diet group and usual care, and assuming that alpha = 5% and power = 80%, we need 22 children for each group. Taking dropouts into account, we need 50 children in total. A drop-out is defined as a child who drops out of the study before the first consultation with the neurologist; this is scheduled 6 weeks after initiating the KD or 6 weeks after randomization to usual care.Randomization will occur halfway through the 4 week baseline period. We will use a software package (ALEA) to support the online patient registration and randomization which will be based on the minimization method. Patients will be stratified according to age , having a PEG/tube or not, and whether the child is living at a residential centre or attends the epilepsy centre as an outpatient while she/he lives at home. In view of the nature of the treatments, blinding of the patients and researchers is not possible.Patients assigned to the ketogenic diet group will be hospitalized in the tertiary epilepsy center for 1 week. During this week the ketogenic diet is introduced by a dietician. The anti-epileptic drugs the children and adolescents use at the time of inclusion in the study will be continued without changes during the study . The initial calorie prescription for the ketogenic diet is based on an average between the pre-diet intake and the recommendations for energy requirements, taking into account current and previous weight and height, recommended caloric requirements and levels of physical activity. The dietician will decide together with the parents whether the classical diet or the medium-chain triglyceride (MCT) diet is introduced. Sometimes the MCT diet is not possible and the classical ketogenic diet (3:1 or 4:1 ratio) is advised. Children with a PEG/tube feed are also treated with the ketogenic diet. The diet is then adjusted to a fluid version. It is possible to decrease the titration of anti-epileptic drugs in responders from six months after the initiation of the ketogenic diet.Patients randomized to usual care will continue to take their anti-epileptic drugs and no changes will be made to the anti-epileptic drugs treatment. Since a ketogenic diet is a treatment of last resort, the children in the usual care group will also receive a ketogenic diet after a delay of four months. The controls will be treated and monitored according to good clinical practice; however, this is not part of our proposed study.The timeline of the study is shown in Figure Patients in the usual care group will visit the multidisciplinary team  at time points T0, T2, and T3 and will have a neurocognitive assessment at T0 and T3. In case of drop-out, a visit with the multidisciplinary team and a neurocognitive assessment will take place.Several instruments will be used to assess the effects of the ketogenic diet on the primary outcome (seizure frequency) and on secondary outcomes .A seizure calendar is used to record seizures. Types of seizures are described and labelled in accordance with ILAE classification. The number and type of seizures are recorded on the dates they occur.The National Hospital Seizure severity scale (NHS3)  is a strSubjective complaints are assessed using the SIDAED (Side effects of Anti-Epileptic Drug), a list of 46 items with possible anti-epileptic drug-related complaints. The items included in the SIDAED form 10 categories: general Central Nervous System (CNS), behavior (increased irritability), depressive symptoms, changes in cognitive function, motor problems and coordination, visual complaints, headache, cosmetic and dermatological complaints, gastrointestinal complaints, and sexuality and menses. For each item the parents rate the severity of the complaint on a four-point Likert scale . In addition, the duration of the complaints is scored .Peabody Picture Vocabulary testThe Peabody Picture Vocabulary test (PPVT-III-NL: Dutch version) is an individually administered measure of receptive vocabulary . The tesBeery Developmental Visual-Motor Integration testThe Beery Developmental test of Visual-Motor Integration (Beery-VMI) is a widely used assessment of psychomotor development. It measures problems with visual perception, motor coordination, and visual motor integration. The 30-item test can be administered from the age of 2 years. The Beery VMI was standardized on a national US sample of 1,737 individuals age 2 to 18 years (2010) and 1,021 adults ages 19-100 (2006), and has proven reliability and validity .Fepsy computerized testThe Fepsy is a neurocognitive computerized test battery consisting of different subtests. For this study we use the simple auditory and visual reaction times, in which the stimulus exposure endures until a push-button (space-bar) response is given . Simple Kaufman Assessment BatteryThe Kaufman Assessment Battery for Children II (K-ABC II) is used to investigate the level of information processing capacities of children beginning at the age of 2 1/2 years . Two subActigraphy® model AW4: Cambridge Neurotechnology Ltd., United Kingdom) will be used to assess motor activity as a measure of alertness. As some of the children in the design are not testable with the described cognitive tests we decided to use an assessment of motor activity in a standardized situation. A wrist watch will be used that translates activity into movement of the watch hand. Registration takes place in a standardized way during the first 10 minutes of the Peabody Picture Vocabulary test and the first 5 minutes of the K-ABC-II test. When patients are not eligible to perform the neurocognitive assessment, the wrist watch will be used for 15 minutes during the visit at the nurse practitioner's office while watching a DVD with songs and pictures.During the administration of the PPVT-III and the K-ABCII test, an actigraphic device  is a DSM-IV oriented questionnaire which is used to assess four domains of behavioral and social emotional dysfunction. These domains are: Attention Deficit Hyperactivity Disorders, Oppositional Defiant behavior and Conduct Disorders, Anxiety and Depression, and Autism spectrum disorders. The questionnaire consists of 72 items describing problem behavior .SDQ QuestionnaireThe Strengths and Difficulties Questionnaire (SDQ) is a brief behavioral screening questionnaire that provides balanced coverage of children and young people's behaviors, emotions, and relationships . The SDQPOMS QuestionnaireThe Profile of Mood States (POMS) has been developed to identify and assess seven transient, fluctuating affective mood states: Tension-Anxiety, Depression-Dejection, Anger-Hostility, Vigour-Activity, Fatigue-Inertia, Friendliness, and Confusion-Bewilderment . The queThe Personal Adjustment and Role Skills Scale (PARS)The Personal Adjustment and Role Skills Scale, 3rd edition (PARS-III) was specifically developed to measure psychosocial adjustment in children with chronic physical illness . This inThe Hague Restrictions in Childhood Epilepsy Scales (HARCES)The HARCES is a questionnaire consisting of 11 items used to assess impairments in daily functioning that are related to epilepsy, e.g. how much influence does the epilepsy have on your child's freedom of functioning in the house? Items are answered on a 4-point scale ranging from "no impairment" to "very much impairment" .The Credibility and Expectancy Questionnaire (CEQ)  is a mulEuroQol5) possible health states. 'Unconscious' and 'dead' have been added to these states, resulting in a total of 245 states. Furthermore, the EQ-5 D consists of a visual analogue scale (VAS) ranging from zero  to 100 .Living with uncontrolled seizures has a negative impact on the quality of life of the child and his/her parents. Therefore, we will measure generic quality of life of both the children and their parents. For this purpose we will use the EuroQol instrument for children of 12 years and older developed by the EuroQol group . For youTAPQOL or TACQOLQoL will also be assessed using the TAPQOL  for children aged between 1-5 years  and the The economic evaluation will be performed from the societal perspective, which implies that all relevant costs and effects will be taken into account. The economic evaluation compares costs and effects of the ketogenic diet in comparison with usual care. A Cost-Effectiveness Analysis (CEA) and a Cost-Utility Analysis (CUA) will be performed. Outcomes of interest for the CEA and the CUA will be the reduction of seizures and improvement in the Quality Adjusted Life Years (QALYs) respectively. The QALY is a measure of disease burden, including both the quality (utilities) and the quantity of life lived. Total societal costs will be calculated based on the Dutch guidelines for cost calculations in health care. We distinguish four cost categories: intervention costs, health care sector costs, costs for the patient and family, and productivity costs. For this study, three instruments are used to measure the costs and utilities.The diary is used to identify all relevant cost aspects with respect to health care sector costs and patient and family costs . Each diA retrospective questionnaire and the patient modules of the PROductivity and DISease Questionnaire (PRODISQ)  will be Both generic quality of life, as well as utilities, will be derived by means of the EuroQol, as mentioned before. Utility values will be calculated for all health states of the EuroQol, using preferences from the UK general population value set which provides an algorithm as a series of decrements from 1, the value of full health. Values of the UK population for the scoring function of the health states described by the EQ-5D were measured with the Time Trade Off (TTO) technique  on a random sample of approximately 3000 members of the adult population of the UK . The utiOur primary (base-case) analyses will be performed according to the intention-to-treat principle, including data from all participants regardless of whether they received the intervention or not. For the analyses we will use SPSS statistical software. Respondents for whom at least 75% of the data per measurement instrument are available will be included in the analysis. Missing data on an item level will be handled using SPSS missing value regression analysis. Completely missing measurements will be handled using multiple imputation. Between-group differences in proportions (dichotomous variables) will be tested using the Chi-square test and between-group differences in means (continuous variables) will be tested using Student's t-test for independent samples. In addition, a multivariate regression analysis will be performed with the covariates of sex, age, severity of disease, duration of disease etc. The accuracy of the findings will be expressed in terms of 95% confidence intervals.A baseline analysis will be performed to examine the comparability of groups at baseline for both costs and outcomes. If necessary, methods will be applied to control for differences in baseline . A KolmoThe robustness of the ICER will be checked by non-parametric bootstrapping. Bootstrap simulations will also be conducted in order to quantify the uncertainty around the ICER, yielding information about the joint distribution of cost and effect differences. The bootstrapped cost-effectiveness ratios will be plotted subsequently in a cost effectiveness plane. The choice of treatment depends on the maximum amount of money that society is prepared to pay for a gain in effectiveness, which is called the ceiling ratio. Therefore, the bootstrapped ICERs will also be depicted in a cost-effectiveness acceptability curve showing the probability that the ketogenic diet is cost-effective using a range of ceiling ratios. In addition, to demonstrate the robustness of our base-case findings, multi-way sensitivity analyses will be performed .The time horizon of the Cost Utility Analysis (CUA) will be extrapolated towards the remaining life expectancy of the study population. The CUA is of major importance since the impact of a ketogenic diet on seizure reduction, costs, and QoL reaches beyond the 4-month study period of the clinical study. A Markov Monte Carlo decision analytic model will be developed to calculate lifetime incremental costs and incremental QALYs of treatment with a ketogenic diet in comparison with treatment with anti-epileptic drugs only. Markov models assume that a patient is always in one of a finite number of health states. All events are represented as transitions from one state to another. A first-order Monte Carlo evaluation of a Markov model determines the prognoses of a large number of individual patients. The time horizon of the analysis is divided into cycles. Each patient begins in an initial state. During each cycle, the patient may make a transition from one state to another according to the laws of chance, as dictated by the transition probabilities. After the first patient has completed the simulation, another patient begins in the initial state and a new simulation is performed. This process is repeated a very large number of times, and each simulation generates a quality adjusted survival time and costs. Monte Carlo analysis, as opposed to a Markov cohort model without memory, offers the possibility to flag subjects in order to track their characteristics and disease histories; this is a very flexible approach to modelling variability within a population. The model will combine the results of the clinical study and data from the medical literature. In the modelling study we will also perform probabilistic sensitivity analysis to test parameter uncertainty and to construct cost-effectiveness acceptability curves. Future costs and effects will be discounted according to the Dutch guidelines for cost calculations in health care .Our design is aimed at assessing the effectiveness and cost-effectiveness of a ketogenic diet among children and adolescents with intractable epilepsy. It is a prospective RCT comparing the ketogenic diet with usual care.The ketogenic diet can be a treatment of last resort for patients with intractable epilepsy. This alternative therapy is used only in the minority of children who could potentially benefit from it. In order to optimize therapy for children with uncontrolled seizures, the ketogenic diet should be prescribed for more children with intractable epilepsy.Compliance with a ketogenic diet is difficult due to its restrictive nature. Unfortunately, non-compliance limits the intended effect and increases the costs to society, resulting in a less favorable cost-effectiveness ratio. In order to overcome this problem, the children and adolescents with uncontrolled epilepsy who are on the ketogenic diet will be monitored according to a strict standardized protocol. Therefore it is likely that the efficiency of patient care will improve. However, a formal assessment of the diet's cost-effectiveness has not yet been performed and is the focus of the present proposal.Growing up with seizures affects the child's personality and cognitive development and interferes with many aspects of everyday life, including learning at school, leisure and occupational activities. Epilepsy has been shown to have a high impact on quality of life when children have intractable seizures and additional disabilities.This study will provide information about the cost-effectiveness of the ketogenic diet, its effects on clinical outcomes, on neurocognitive functioning and on quality of life. Our study is the first study assessing the cost-effectiveness of a ketogenic diet.The authors declare that they have no competing interests.RK is investigator of the cost-effectiveness part of the study and is the main author of the manuscript. DL is investigator, and grant applicator of the clinical part of the study and wrote these parts of the manuscript. DP is supervisor and grant applicator of the modelling part of the study and wrote these parts of the manuscript. AK is a local statistician and arranged the randomization procedure. JH is coordinator of the neurocognitive research. AA is professor of neuropsychology and supervisor. SE is supervisor and grant applicator of the cost-effectiveness analysis part of the study. MM is supervisor, grant applicator and senior advisor. All authors read, edited and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/10/prepub


This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a  a priori polarity within the Golgi apparatus or directionality of vesicular traffic. The Golgi cisternae inevitably lose active proteins that specify vesicle fusion, the SNARE molecules, as they age, thus breaking the symmetry between compartments and establishing the “seed” for directional vesicular transport. This small decrease in SNARE concentration in older cisternae is then further self-enhanced by the progressively more directional vesicular transport of SNAREs. Competition of enzymes for incorporation into predominantly retrograde-fusing vesicles in turn generates overlapping but distinct stationary enzyme peaks. Applying these general mechanisms of fusion asymmetry and competitive vesicle loading to the actual situation in the stacked mammalian Golgi, we reproduced the experimentally observed distributions of the two SNARE pairs that operate in the Golgi, and enzyme peaks in cis, medial and trans cisternae. We believe that our study attempts the first self-consistent explanation for the polarity in the Golgi stack.We have developed a quantitative model to address a fundamental question in cell biology: How does the Golgi apparatus, an organelle composed of multiple cisternae that exchange vesicles, steadily maintains its inhomogeneous protein composition in the face of ongoing cisternal aging and replacement, and cargo entry and exit. We do not assume any  The individual cisternae are enriched in glycosylation and other enzymes, which form distinct but overlapping gradients with peaks in the cis to trans, it becomes modified by Golgi enzymes in an assembly-line fashion. Efficient and correct cargo processing depends on the distribution of glycosidases, glycosyltransferases and other enzymes within the different Golgi sub-compartments in their expected order of function cis face of the Golgi. The cargo exits the Golgi in transport carriers that emerge from the trans most cisterna when it disintegrates, thus maintaining the Golgi apparatus at a steady state. Individual cisternae mature by shedding their characteristic Golgi enzymes and at the same time acquiring Golgi resident proteins from the more trans cisterna As anterograde cargo traverses the Golgi apparatus from It has been shown that Golgi resident proteins shuttle between the cisternae in vesicles cis Golgi while proteins that are poor competitors can only enter vesicles after the good competitors have been depleted, and thereby end up in more trans cisternae. While this model explains steady enzyme segregation, it is based on an unexplained premise, namely, that the Golgi-enzyme containing vesicles preferentially fuse with the younger rather than the older cisternae.Glick et al. provided one piece of explanation with a simple model according to which competition of Golgi proteins for incorporation into retrograde-destined vesicles accounts for their sorting within the Golgi cisternae Soluble N-ethyl-maleimide-sensitive factor Attachment protein Receptors (SNAREs), integral membrane proteins that reside in the vesicle and target membrane cis-to- trans decrease is indeed observed for Golgi Q-SNAREs cis-to- trans, Fusion of vesicles with acceptor membranes is specified by a priori asymmetry within the Golgi apparatus. The transport is mediated by 2 cognate SNARE pairs, which compete with each other and with other Golgi residents for incorporation into a single vesicle type. The retrograde directionality of vesicular flux is triggered by the temporal decrease of the concentration of cisternal SNAREs, which occurs via loss of SNARE-containing vesicles, including the recycling of COPI vesicles from the Golgi to the ER, decay, and inhibition of SNARE molecules. As a result, cisternal age becomes a distinguishing factor: trans cisternae are older than cis cisternae and thus contain fewer SNAREs. A small distinction in SNARE concentrations provides the seed for a cistrans gradient, which becomes self-enhanced by vesicular transport of the SNAREs. The steady SNARE gradient controls a predominantly retrograde vesicular flux in which Golgi enzymes with stronger affinities for the coated vesicles cycle predominantly between the cis cisternae and the ER, while weaker-binding enzymes only enter vesicles from later cisternae and exhibit less ER retrieval.We present a model of inter-cisternal vesicular transport in which we do not assume any We assume thatcis end of the stack, while the most mature cisterna dissolves and disappears from the trans end of the stack. The new cisterna is formed by coalescence of ER-derived vesicles and contains fixed concentrations of SNAREs and enzymes.The Golgi consists of a stack of cis (less mature) and trans (more mature) cisternae, and with the progenitor. Indeed, in the stacked mammalian Golgi, coil-coiled vesicular tethering factors which span the distance between adjacent cisterna are thought to grab vesicles even prior to their release from the donor cisterna and prevent them from reaching more distant cisternae Saccharomyces cerevisiae.Along with cisternal progression, vesicles containing SNAREs and Golgi enzymes continuously bud from each cisterna. We assume that the vesicles provide local transport and can only fuse with the neighboring SNAREs and Golgi enzymes are uploaded into a vesicle via competitive binding to a fixed number of vesicular sites. We assume that the vesicular transport results primarily in the movement of cargo without any significant change in the volume and budding surface area of the cisternae. This is supported by observations that the size of all cisternae is similar Functioning of the model hinges on two general principles: Establishment and maintenance of a directed retrograde vesicular flux and sorting of the vesicular cargo via competition for binding sites.cistrans gradient in cisternal SNAREs.To reveal the universality of the proposed self-establishing mechanism of vesicular traffic directionality we first consider the simplest possible setup, a single cognate SNARE pair and vesicle type. We assume that the rate of vesicular fusion is proportional to the product of the concentrations of the SNAREs present in vesicles and cisternae, respectively. The precise nature of SNARE molecules does not have to be specified here. We can even consider the SNAREs as mere proxy for fusion-specifying factors. The probability for a vesicle to fuse with a given cisterna depends solely on the cisternal concentration of compatible SNAREs, and cisternae with higher SNARE concentration have a higher probability to absorb vesicles. A retrograde vesicular flux thus requires a cistrans SNARE gradient is the observation that all systems, living and otherwise, function with a loss. As Golgi cisternae mature they inevitably lose active SNARE molecules. Such a decay of active SNAREs breaks the symmetry between the otherwise identical cisternae in a systematic way: The older trans cisternae contain less SNAREs than the younger cis cisternae. The SNARE loss can occur by escape of SNARE-carrying vesicles that fuse with the ER thus recycling their content. However, some of the cisternal SNARE decay is likely due to irreversible loss that requires some new SNARE synthesis to replenish the system.We propose that key to a robust cis rather than trans cisternae, thus initiating the directed vesicular transport. As SNAREs are transported retrograde, their cistrans gradient is further enhanced. When the vesicular flux becomes balanced by the anterograde transport of SNAREs due to cisternal maturation, the system comes to a steady state. Indeed, we show both numerically and analytically that the seed gradient, created by the temporal decay of SNAREs, is self-enhancing nhancing . This isWe note that at steady state the vesicular flux does not depend on the concentration of SNAREs in the vesicles: Lower concentrations of vesicular SNAREs are compensated by a higher steady state number of vesicles. Naturally, a vesicle should contain a minimum number of vesicular SNARE molecules to ensure any fusion at all.The calculation of the steady state SNARE gradient and vesicular flow are presented in the cis, medial and trans cisternae, and with strong, intermediate and weak affinities for vesicular binding sites, respectively , exhibits a somewhat lower enzyme concentration as it does not receive any incoming retrograde vesicular traffic.We find that the distribution of enzymes radically depends on whether vesicles originating from the first cisterna can exit the Golgi and fuse with its produced . Enzymestrans cisterna than the weaker-binding enzymes. As a consequence of their higher concentration, the stronger-binding enzymes do not get sufficiently removed from younger cisternae to achieve their cis-Golgi peak and at the same time they do not give their weaker competitors any chance to enter the retrograde transport vesicles in the later cisternae. Hence, all enzymes peak at the trans face of the Golgi. Thus, recycling of enzymes to the ER is necessary for establishing the cis-medial- trans enzyme segregations. At the same time, we observe that the steady state SNARE distribution and resulting intra-Golgi vesicular flux is only weakly affected by the presence or absence of ER-recycling. This is because both scenarios feature an inherent loss mechanism, which breaks the intra Golgi conservation of SNAREs.On the other hand, if none of the enzyme-carrying vesicles can escape to the ER, the cisternal distribution of all enzymes converges onto a single peak form , In the Golgi, only the In addition to the two Golgi SNAREs, we consider a third v-SNARE, which mediates the fusion of Golgi-derived vesicles with the ER. It is ERS24, which thus has a dual function as part of a t-SNARE complex in intra Golgi transport and as v-SNARE in Golgi-to-ER transport. The corresponding ER t-SNARE does not leave the ER and is therefore not considered here Apart from the SNARE specifications, we implemented a similar set of minimal assumptions as for the single SNARE scenario:The rate of vesicular fusion with Golgi cisternae is determined by both All SNAREs and Golgi enzymes compete for the same binding sites in the vesicles. This is in agreement with the findings for ER-derived COPII vesicles, the only instance where cargo-competition for coat binding has been elucidated to date However, our model reproduces the enzyme segregation as well in the case when the enzymes compete only with each other and not with SNAREs for vesicular binding sites, as shown in cis and trans neighbors of the progenitor cisterna and the progenitor cisterna itself. In addition to fusing with Golgi cisternae, vesicles also fuse with the ER with a rate that is controlled by the product of the concentrations of the vesicular ER v-SNARE and a fixed concentration of ER t-SNAREs. Due to the expansive volume of the ER that brings it in proximity to the entire Golgi apparatus, we assume that all vesicles can fuse with the ER independent of their originating cisterna.Vesicles fuse locally, i.e. with the The age-dependent decrease (loss) of the cisternal concentration of the NARE see .cis cisternae. These vesicles have a high probability to fuse with the ER due to a substantial concentration of the ER SNAREs. These vesicles also recycle a good fraction of strong-binding cis enzymes, and a part of medial enzymes to the ER. The recycling of cistrans t-SNARE gradient to the trans Golgi. In more mature cisternae where the cis-enzymes are depleted, the vesicles incorporate the weaker-binding molecules, such as medial and, to a lesser extent, trans enzymes and trans-most cisternae bud vesicles that contain predominantly trans enzymes and trans-most cisterna in We found a good qualitative agreement between our results and the experimentally observed concentration profiles. With the proper choice of dissociation constants ,  t-SNARcis-enzymes, trans enzymes is indeed born out by numerous experimental observations in yeast and mammalian cells. Cis but not trans Golgi markers accumulated in the ER upon an acute ER-exit block Our prediction that Based on the SNARE dissociation constants that yielded the experimentally observed protein gradients  we furthS. cerevisiae where individual Golgi cisternae are scattered throughout the cytoplasm So far we assumed that vesicles only fuse with the immediate neighbors of their progenitor cisternae. A stacked Golgi, however, is not a requirement for Golgi asymmetry and cisternal maturation, which are also observed in S. cerevisiae must include a factor that considers fusion preferences related to cisternal age although it might be less stringent than the nearest neighbor limitation of a Golgi stack. Golgi scattering occurs when novel cisternae emerge from multiple, short-lived transitional ER (tER) sites rather than from a single, stable tER ad hoc generating a series of maturing Golgi cisternae that remain separate from those generated in parallel by other tER sites. Evidence from a recent study by Nakano et al in S. cerevisiae supports this prediction: When due to altered ER-morphology the motility of Golgi elements away from the ER-exit site(s) is impeded, cis and trans Golgi elements could be seen in close proximity to each other and to ER-exit sites We suggest therefore, that a realistic description of fusion probability in cis side while dissolving the oldest cisterna as secretory and lysosomal cargo exit at the trans end. Age is indeed the distinguishing feature of individual Golgi cisternae that we identify as the key to symmetry breaking. As cisternae mature the concentration of their functional SNAREs decreases, thereby providing the seed for a cistrans cisternal gradient of fusion factors for transport vesicles. This SNARE gradient causes the predominantly retrograde direction of vesicular flux that retrieves Golgi resident proteins, such as the SNAREs themselves and enzymes, from older to younger cisternae and back to the ER. The vesicular transport of SNAREs further enhances their gradient until a steady state between the retrograde vesicular and anterograde cisternal progression is reached. Both the seed gradient and cisternal maturation are indispensable for this outcome.We present a simple model that explains the establishment and maintenance of directed vesicular flow and concentration gradients in the Golgi apparatus, an organelle system that undergoes constant rejuvenation by adding a new cisterna at the cargo-entering trans cisternae is not sufficient to create a seed gradient. ii) Experimental evidence for one such late Golgi SNARE, Gos28, are compatible with the notion that its loss occurs through degradation: The levels of Gos28 can go up as much as 40% when the availability of its chaperone GATE-16 is increased, preventing Gos28's proteolytic degradation trans) but also when present at the same membrane (i.e. in cis), where most of them are disrupted under energy expenditure by the NSF/cis- to trans-Golgi  Retrieval to the ER alone can account for the loss of the SNAREs present mostly in the young cisternae. However, the retrieval to the ER of the Golgi SNAREs from the medial and ient see . Cognatecis, medial and trans cisternae. One finding from our simulations is that the differential distribution of Golgi proteins can only be achieved when the vesicles are allowed to recycle back to the ER. This is in good agreement with experimental observations cis and trans Golgi enzymes can be monitored by dual color time-lapse microscopy Once the retrograde vesicular flux is established, different affinities of Golgi enzymes for the vesicles explain the enzyme peaks in ur model .de novo formation, or considered a complete stack of identical cisternae when turning on the vesicular flux and SNARE loss mechanism, in each case the same steady state was reached.Our simulations are insensitive to a broad spectrum of initial conditions. Regardless of whether we started with a single cisterna and added new cisternae one by one as it would occur during Golgi cis, medial, and trans peaks of Golgi enzymes and the actual 2 SNARE pair scenario were reproduced. The dissociation constants for binding of SNAREs and enzymes to vesicular sites had to be tuned within a 10% precision. The actual values of the dissociation constants are of the same order as protein concentrations, which is quite common for protein-protein interactions and appears to be evolutionally attainable An important question is the relevance and specificity of constants used for the modeling. Naturally, the range of admissible constants narrows as one reproduces more detailed and specific scenarios. Our first observation, that a temporal loss of SNAREs results in directed vesicular flux, is very general and holds for virtually any set of constants see , 12). Thtrans and medial Golgi enzymes from Golgi cisternae and their dispersion into vesicles that are apparently unable to fuse cis face  with a fixed concentration of t-SNAREs which can fuse vesicles (see When the retrograde vesicular transport is counterbalanced by the anterograde cisternal progression, the enzyme distribution reaches its steady state. The nature of the steady state depends on the boundary conditions imposed on the cles see , while ucles see .cis, medial, and trans types of enzymes. For brevity of equations, we use the universal notations cis and trans SNAREs Putting together the two mechanisms considered above, we introduce a realistic model of Golgi transport. It describes the evolution of 8 distinct types of molecules: The escape of a fraction of vesicles from the Golgi to the ER provides one part of a loss mechanism necessary for seeding the gradient of t-SNAREs. Yet we do not exclude the possibility of other mechanisms of t-SNARE decay, so the To model the vesicular transport in yeast, we used an equation similar to


Exocytosis, the process in which material is transported from the cell interior to the extracellular space, proceeds through a complex mechanism. Defects in this process are linked to a number of serious illnesses including diabetes, cancer, and a range of neuropathologies. In neuroendocrine cells, exocytosis involves the fusion of secretory vesicles, carrying signaling molecules, with the plasma membrane through the coordinated interplay of proteins, lipids, and small molecules. This process is highly regulated and occurs in a complex three-dimensional environment within the cell precisely coupled to the stimulus. The study of exocytosis poses significant challenges, involving rapidly changing, nano-scale, protein–protein, and protein–lipid interactions, at specialized sites in the cell. Over the last decade our understanding of neuroendocrine exocytosis has been greatly enhanced by developments in fluorescence microscopy. Modern microscopy encompasses a toolbox of advanced techniques, pushing the limits of sensitivity and resolution, to probe different properties of exocytosis. In more recent years, the development of super-resolution microscopy techniques, side-stepping the limits of optical resolution imposed by the physical properties of light, have started to provide an unparalleled view of exocytosis. In this review we will discuss how advances in fluorescence microscopy are shedding light on the spatial and temporal organization of the exocytotic machinery. In vitro biochemical studies have described many of the individual stages of SNARE protein action; through the formation of a stable binary intermediate, the zippering of the SNARE complex, the formation of cis and trans complexes, and established the SNAREs as the minimal machinery required for exocytosis  has been widely used to examine the molecular motion of SNARE proteins on the plasma membrane . By selermediate , 13. FRAnization –37. FCS nization . This arThe proteins responsible for driving the process of exocytosis are un-resolvable due to their size and high density in neuroendocrine cells using diffraction-limited microscopes. In the last 5 years there has been a revolution in cell imaging with the introduction of super-resolution microscope techniques, which have pushed the ability to resolve objects through supramolecular imaging down to single molecule detection  Figure . These tThese super-resolution techniques have been utilized to investigate the organization of the plasma membrane secretory machinery. Over the last decade it has become clear that the plasma membrane SNAREs, syntaxin, and SNAP-25 are not uniformly distributed over the plasma membrane, but instead are observed to exists in a clustered morphology , 48–53. Although super-resolution techniques provide unparalleled resolution they are limited to describing the position of protein molecules with no information on interaction status. However, it is possible to probe interactions of molecules over distances below 10 nm using Förster resonance energy transfer (FRET) microscopy . This haThe development of new microscopy techniques with ever higher temporal and spatial resolution has mirrored the requirements and advancements of the field of neuroendocrine secretion. This biological system serves as an excellent test bed for many of the new emerging technology as a result of our already considerable understanding and also the specific problems and opportunities offered. The next big step is to combine super-resolution, high speed, and functional imaging into single experiments. The iterative process of technological advancement and new biological findings will continue until the ultimate experiment of being able to watch a complete single fusion event with sub-molecular resolution of multiple protein components is achieved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Electrospray Ionization (ESI) is a soft ionization technique extensively used for production of gas phase ions (without fragmentation) of thermally labile large supramolecules. In the present review we have described the development of Electrospray Ionization mass spectrometry (ESI-MS) during the last 25 years in the study of various properties of different types of biological molecules. There have been extensive studies on the mechanism of formation of charged gaseous species by the ESI. Several groups have investigated the origin and implications of the multiple charge states of proteins observed in the ESI-mass spectra of the proteins. The charged analytes produced by ESI can be fragmented by activating them in the gas-phase, and thus tandem mass spectrometry has been developed, which provides very important insights on the structural properties of the molecule. The review will highlight recent developments and emerging directions in this fascinating area of research. So the only way to analyze the protein mass was to digest the protein and then the analysis of the digest mixture by FAB-mass spectrometry.The basic concepts of chemistry originated from the quantitative estimation  of the constituents in a chemical reaction during the period of Lavoisier more than 200 years ago. Since then the analytical measurement of masses of the samples continuously evolved through the gravimetric analysis to weighing a single atom/molecule using the modern instrument called mass spectrometer. In mass spectrometry, a particular state of matter called gaseous ionic state is studied by transferring the analytes from condensed phase to the gas phase followed by their ionization. The success of the study of gas-phase ion chemistry and its application has been driven by the continuous advancement of the mass spectrometric technique since the studies were performed by Thomson . As a rem/z values of the resulting ions become lower and fall in the mass ranges of all common mass analyzers. Thus ESI became very useful in the production of gas-phase ions from large biologically important macromolecules like proteins and nucleic acids, and their subsequent mass spectrometric analysis for structural characterization as well as their rapid identification on the basis of molecular mass, a very specific property of the analyte. Gradually a systematic analysis of proteins with the mass spectrometry as the central tool led to a discrete subject called “Proteomics,” one of the fastest growing research areas in the chemical sciences  is applied to the m/z value to honor Thomson. Very often the symbol “u” (unified atomic mass) or Da  is also used as the molecular mass unit.Generally the ions derived by ES process are multiply charged, and the analyte remains intact (no fragmentation) when appropriate instrumental conditions are used. In positive ion mode  the charging generally occurs via protonation , but in negative ion mode  charging occurs via deprotonation of the analyte. The mechanism of charging has been discussed later. Since the charging of the analyte occurs by transfer of protons, the ionic species detected are not the true molecular ions (which are formed by the loss or gain of the electron), but they are more preferably protonated or deprotonated molecules . When a m/z values) related to the same molecule? The procedure of finding the charge and mass of the analyte through the ESI-mass spectrum is called “Mathematical Charge Deconvolution” /(n − 1) = m2. Since there are only two unknown parameters M and n, a minimum of two equations are required to find out the values of M and n. So the above two equations can easily be employed to determine the value of n (charge state) and M (deconvoluted mass). One can think the observed multiple peaks as the multiple mass assessment of the same molecule. A more accurate mass of the analyte can be derived by averaging the calculated mass for each peak. The above algorithm works well for the pure analyte and when the analyte produces the successive charge states of the difference unity. But problems arise when the analyte produces metaliated as well as protonated species in gas phase or skips some charge state in between two observed charge states. Therefore, several refined procedures have been developed to cope with these requirements .The charges (say protons in positive ion mode) in the droplets are distributed on its surface with equidistant spacing to minimize the potential energy . There acharge residue model (CRM)  whi whiR) ase is the elementary charge, Z is the charge number, ε0 is the permittivity of the surrounding medium, NA is the Avogadro's number, ρ is the density of the water, M is the molecular weight of the protein, and γ is the surface tension of the solvent (water). A plot (solid line) shown in where R ≤ 10 nm, the ion emission dominates over Rayleigh fission. Thus unlike CRM, this mechanism does not require the production of very small droplets (R  ≈ 1 nm) that contains only one analyte molecule. This mechanism is called ion evaporation mechanism/model as proposed by Iribarne and Thomson . Th. Th+, K+ In our recent study we have showen that the relative volatility of the charged particles  and the surface accessibility of the polar residues of the protein towards the charged particles are important factors in the CSD of the protein in gas phase . The surAmmonium acetate buffer solution is very often used in the mass spectrometric analysis of proteins see  since unn et al.  and Pescn et al.  also sugThe above equations ( and 9)))9)) suggSeveral physical properties of the solvent like polarity (dielectric constant) , gas-pham/z) when the 50% solvent fraction is changed from water to methanol, to acetonitrile, to isopropanol. This is also the order of increasing gas-phase basicities of these solvents. However, the effect is relatively small for these solvents, possibly due to their limited concentration (in gas phase) inside the electrospray interface. But the addition of small amounts of diethylamine (0.4%) in the aspirating solution results in dramatic shifts to lower charge, presumably due to preferential proton transfer from the higher charge state ions to diethylamine. These results clearly show that the maximum charge states and charge state distributions of ions formed by electrospray ionization are influenced by solvents that are more volatile than water and the gas-phase basicity of the solvent too − is deficient one hydrogen relative to M−•). Multiple hydrogen deficiencies are denoted by multiple prime symbols to the left of the letter are used . When a e letter .  Figuree letter . Both thSimilarly the charge remote ring cleavage of the bile acids also occurs, resulting in ring opening, loss of neutral ethylene, and double-bond formation on the product ions. Griffiths et al. have developed a systematic nomenclature see  to interAfter the discovery of the ESI-MS, its application areas rapidly expanded from large macromolecules to small organic and inorganic molecules. Nowadays the ion signals in the ESI-mass spectra are providing a clear and deep perception about the analytes properties far beyond the conventional mass and structural properties of the analytes. Here we will discuss in brief about certain applications of the ESI-MS.Since mass is a very specific property of a molecule, determination of the molecular weight with high precision allows to solve many problems in proteomics. Over the past twenty years, ESI-mass spectrometry has emerged as a powerful tool in the life science to determine the identity –222, qua Sometimes intact charged protein generated by ESI is introduced into the mass analyzer and are subjected to gas-phase fragmentation, and this type of approach is referred to as the “top-down” strategy of protein analysis . Although the ESI-MS technique is not sensitive to probe the local conformation of the polypeptide chain, that is, the secondary structure of the protein, the technique can successfully interpret the three-dimensional conformation of the proteins . As disc ESI-MS can also successfully detect the posttranslational modifications (PTMs) , 237 and Disulphide bonds (a kind of PTM) play important roles in the structure and biological activity of the cystinyl proteins. So the determination of the disulphide bond linkage between two adjacent or closely spaced cysteine residues is an integral part of the structural characterization of the proteins. Several mass spectrometry-based strategies have been developed to map the disulphide bond linkages in the proteins –247. Mos+, Rb+, and Cs+, uracil, thymine, and their homologues form self-assembled quintet structures that are stabilized by hydrogen bonding and ion-dipole interactions in the gas phase [β-lactoglobulin) and ligand (fatty acids) in vacuum has also been reported lately [Since ESI is a sufficiently soft ionization technique, the noncovalent complexes of the analytes formed in solution can representatively, transferred to the gas phase when appropriate instrumental conditions are used , 248–250as phase . ESI-MS as phase , 257, pras phase , proteinas phase , and proas phase  complexeas phase , which das phase . Althougd lately . The corIt is very difficult to ionize the hydrophobic proteins by ESI because of their inherent insolubility in the buffers compatible with electrospray. For this reason the ESI-mass spectrometry has not been applied to intact membrane protein complexes. But recently Barrera et al. have been successful to transfer some hydrophobic membrane protein complexes in vacuum via ESI by encapsulation in a solution phase detergent micelle .The ESI-MS has also been used to study the complexes between polyether  and protonated peptides , 265. ThSince ESI-MS is a sensitive, robust, and reliable tool for studying the femtomole samples in microlitre volumes, it has become an increasingly important technique in the clinical laboratory for structural study or quantitative measurement of metabolites in a complex biological sample . For exa3OD, ND3, etc.) for well-defined reaction intervals in the collision cell [The application of the ESI-MS is not only restricted to the structural characterization, but it has also been extended recently to the study of the molecular dynamics. For this type of application, the analyte ions produced by the ESI-ion source are trapped and subjected to the ion-molecule reaction (H/D exchange) with some gaseous deuterated molecules possessing exchangeable deuterium  can also be monitored by the H/D-exchange experiments , 283. VaThe applications of ESI-MS have also been explored in synthetic organic and organometallic chemistry to study the reactive intermediates and the mechanisms –292. ForDesorption electrospray ionization mass spectrometry (DESI-MS) is relatively a new ambient surface analysis method. The development of imaging mass spectrometry by DESI has been described recently –299, andThe development of electrospray ionization mass spectrometry has enabled us to solve a wide range of biochemical and mechanistic problems as discussed above. Perhaps it is one of the instruments that has continuously evolved over the last three decades both in application and modification of instrument design. For example, the transition of source design from microspray to nanospray has improved the sensitivity. Introduction of high-resolution mass analyzer (FT-ICR) amended the accuracy and redefined the applicability. Yet in several aspects, the understanding of the ESI-MS remains vague to date. One of the major issues is the exact mechanism of ion formation. How is the gas-phase ion formation guided mechanistically? Is the perception of CRM and IEM enough to interpret the ion formation? It is still unclear about the analyte behavior (structure and dynamics) inside the charged droplets produced by ES process. If the proteins change their structure inside the charged droplet, then the charge state distribution (CSD) should reflect the instantaneous conformation of the protein inside the charged droplet not in the original solution. Again, as discussed in the present review, the CSD is not influenced by a specific parameter but governed by multiple parameters. So the precise understanding of the cumulative effect of those different parameters on the protein CSD would no doubt help to earn more quantitative insights about structural and chemical behavior of the proteins.So far a little attention was invoked on the detector characterization and improvement compared to that of the ion source and mass analyzer. So, the characterization and improvement of the detector would likely be the next step in the development of mass spectrometry which would enable us to give the answer whether the ion signal intensity is governed by the molecular conformation of the analyte or not. The application of the ESI-MS got a noteworthy dimension in biochemistry laboratories after its success in protein sequencing. The sequencing is based on the idea and information of gas-phase ion fragmentation chemistry of the peptides in the tandem mass spectrometry. But the tandem mass spectra of the peptides are mostly dominated by anomalous fragment ions than the “sequence informative ions.” So the characterization of those anomalous fragments and accordingly the modification of sequencing algorithms would facilitate the sequencing of proteins/peptides more precisely and rapidly using mass-spectrometric technique.


The prevalence of colon cancer has seen strong demand in screening for colorectal neoplasia, and this has drawn considerable attention to the technological advances in Computed Tomographic Colonography (CTC). With the assistance of an oral contrast agent, an imaging technique known as Electronic Cleansing (EC), can affect virtual cleaning of the computed tomography (CT) images, to remove fecal material that is tagged by the agent. Technical problems can arise with electronic cleansing however, when the air lumen causes distortions to the tagged regions which result in partial volume effects.Combining the simple image arithmetic of an electronic cleansing algorithm, with a vertical motion filter at the fluid level of the bowel, artifacts such as those caused by an air lumen are eliminated. Essentially, the filter becomes a vector for that carries the measurement of vertical motion to neutralise the artifact that is causing partial volume effects. Results demonstrate that despite its simplicity, this technique offers accuracy and is able to successfully maintain the normal intra-colonic structure, while supporting digital leaning of tagged residual material appearing on the colon wall. In the developed world, colorectal cancer represents one of the most prevalent cancer that causes death -3. WhileThe reason for this lack of participation is due to the perception an invasive conventional colonoscopy holds for individuals; these require sedation and a rigorous bowel preparation . Also knThe advantages to be enjoyed with CTC over a conventional colonoscopy are many . CTC is Numerous mathematical models based on statistical data analysis, and adaptive morphological operations have been applied in a bid to cure the problems EC experiences with artifacts in the form of partial volume effects as a result of the air lumen and tagged regions ,12. ThesThis paper suggests an algorithm to eliminate these artifacts from clinical practice. A vertical motion filter will be configured to the fluid level in the bowel and combined with simple image arithmetic. The artifact of partial volume defects will be reduced or eliminated totally, as the filter becomes the vector for vertical motion. The processing speed of this technique is rapid, simple, and successfully maintains a normal anatomical structure for the purposes of EC.This novel method of image processing for EC eliminates the artifacts in partial volume defects caused at the boundary between the air lumen and the regions tagged by the contrast agent.Upon receiving approval from the Institutional Ethical Review Board, the authors performed a CTC colon cancer screening on a patient with a colonic abnormality. In order to enhance the intensity of stool and fluid for electronic cleansing, a bowel preparation was performed prior to the examination.The patient was asked to ingest diluted contrast material at each meal the day prior to that of the procedure. Apart from barium-based contrast agents to affect opacification of the bowel homogenously, additional solutions were also taken by the patient to liquidise and enhance the stool under imaging.At a concentration of 40% w/v , 20ml of barium contrast agent was administered with 800ml water after each meal the day prior. In addition, the patient was given a routine bowel preparation that included 50g of magnesium citrate in 150ml  after dinner, and two tablets of Sennoside  were taken at bedtime as a laxative accelerant. Finally, a suppository  was administered to the patient upon waking.2 gas insufflations of the colon had occurred using CO2 EFFICIENT Endoscopic Insufflator .While the patient was in both supine and prone positions, CT colonography was performed after bowel opacification, and also after COThe CT scan was captured by using a Multi-slice Helical CT scanner  and was performed using the following acquisition protocol:120 kVpVariable tube currentExposure Control Noise level at SD 200.5 second gantry rotationHelical pitch of HP151 mm collimation x 16 rows1 mm image reconstruction intervalElectronic cleansing (EC) was completed on software developed using the MATLAB framework . The program was installed on a standard PC .In order to provide a cleansed model for Virtual Colonoscopy, electronic cleansing (EC) is used to identify the colonic material that is revealed by a contrast agent. Technical difficulties arise however, with partial volume defects being caused by the boundary between the air lumen and the tagged regions.Basically, a template is made of the air material in order to reproduce the partial volume effect of the peripheral edge. Then using the vertical motion filter vector, the vertically spread pixels of the template are de-blurred. The high density tagged materials are substituted with the template of air material using texture synthesis by non-parametric sampling . The paUsing the air material template to reduce the artifact boundary between the air lumen and the tagged regions, the vertical motion filter was applied to the resulting image. The use of the filter reduces the vertical spread of the pixels. The length of the filter kernel is a three row vector array , and the default theta of the filter direction is +90 or -90, which happens to correspond to the image artifact caused by the partial volume effect.The algorithm suggested in this study produces a smooth edge along the colon wall and reduces the artifact created by the by the boundary between the air lumen and the tagged regions . Simple Cleansed images show fine soft tissue area without any trace of tagged material. True polyps however, are clearly illuminated on the CTC due to the barium tagging of stools adhering to the colon wall that increases their resolution . The maiRecently, 3D endoluminal rendering for polyp detection in CT colonography has become possible , and theThe only limitation of this developed algorithm is that it does not support a multi-directional filter in order to reduce the complex shape of partial volume defects. Also, due to the lack of uniform density variance inside the fecal residue, this algorithm failed to cleanse low-density residual fluid and uneven tagging residue. Clearly, the algorithm needs an improved processing speed along with maintaining accurate normal anatomical structure.It is suggested that simple image processing for electronic cleansing (EC) is based on both an air material template that reproduces the partial volume effect, and also the vertical motion filter. In the thresholding level for 3-D surface displays, the EC algorithm removed only tagged materials without removing the artifact. Accordingly, this approach may need further confirmation by subsequent research utilising a larger amount of stool tagging data in the CTC.


We present a case of painful radial nerve palsy following application of a humeral lengthening frame. At re-operation, the radial nerve was found to be compressed against a distal pin. This was re-sited providing immediate pain relief and a gradual resolution of the radial nerve palsy. Pain in association with a nerve palsy should alert the clinician to the possibility of nerve compression that may benefit from urgent decompression. Neuropathic pain, arising from a lesion of the nervous system, is defined as persistent intractable pain disproportional between the extent of the lesion and the severity of the pain. It is associated with sensory and/or motor disturbance . NeurostA 10-year-old girl underwent application of a humeral lengthening frame of her right humerus to correct severe humeral shortening gradually. The shortening was secondary to osteomyelitis of the right proximal humerus aged 10 weeks. This had left her with a 10-cm discrepancy between the lengths of the right and left humeri, resulting in significant functional difficulties. The lengthening frame was applied with the elbow extended without any intra-operative complications. In recovery, her right hand was warm and well perfused with full sensation and movement of the wrist and small hand joints. On the first post-operative day, however, she complained of severe burning pain in her right arm and hand requiring significant opiates for relief and a pain team review. Examination revealed evidence of a partial right radial nerve palsy; weakness of right wrist extension, an inability to extend the fingers and diminished sensation on the dorsum of the first webspace. She displayed evidence of hyperalgesia with an exaggerated response to pin-prick on the dorsum of the first webspace. There was no mechanical allodynia. All other hand and wrist movements and sensation were intact.An ultrasound scan of the right arm was performed. This revealed no haematoma or collection. The radial nerve was not visualised.On the second post-operative day, her symptoms decreased slightly. However, on the third post-operative day, her symptoms did not settle further and the patient was taken back to theatre for exploration of the radial nerve due to ongoing severe pain. The radial nerve was found to be in continuity without any bruising or contusion. Distally, it was found to pass tightly against the most distal external fixator pin such that, with the elbow flexed the nerve became tightly pressed against the pin. The distal pin was removed and re-sited slightly proximal.Post-operatively, there was complete resolution of the right arm pain and hyperalgesia. Sensation to the first webspace recovered on the second post-operative day with motor function returning more gradually. There was full recovery of radial nerve motor function at 4 months post-operatively.In 1943, Seddon  introducNeurapraxia can occur with or without pain. Normal sensory function is the product of an actively maintained equilibrium between neurons and their environment . Any disTransient nerve compression, stretch or distortion, such as the radial neuropathy of ‘Saturday night palsy’, may result in transient nerve palsy but is typically not associated with pain. Closed nerve injuries may be managed by observation for a few months or by early exploration of the nerve. The decision is based on an assessment of the probability that the nerve has been disrupted or is under compression. Chronic nerve compression, distortion and ischaemia results in pain, termed neurostenalgia. Neurostenalgia is neuropathic pain provoked by the continuing action of the agent responsible for the lesion of the nerve, which is anatomically intact. Removal of the agent results in resolution of the pain. Chronic nerve compression can result from a wide variety of aetiologies and can equally produce a wide variety of symptoms and signs, such as muscle weakness and altered sensation in the distribution of the affected nerve. Careful history taking, examination and investigation are essential in guiding the clinician as to the cause. In all cases, the presence of intractable pain should alert the clinician to a compressive cause.A famous case of neurostenalgia was that of the first Lord Nelson who suffered constant stump pain years after amputation of his right upper limb. Loosening and discharge of a suture round the median nerve years later resulted in a sudden resolution of the pain . Birch aIn this case, the patient reported persistent burning pain and an exaggerated response to pin-prick sensation following application of her humeral lengthening frame 1 day post-operatively. The cause of the stimulation was confirmed as the most distal external fixator pin compressing the radial nerve because complete and immediate pain resolution occurred when the pin was re-sited. The variation in the level of symptoms was likely to have been due to a change in elbow position when the elbow was in a semi-flexed position, the nerve was compressed against the pin but with the arm fully extended the pressure was reduced. Nociceptor fibres, widely distributed in skin, muscle, joints and the walls of blood vessels, pass to fine myelinated A-delta fibres and non-myelinated C fibres. Stimulation of A-delta fibres results in sharp, well-localised pain. Stimulation of C fibres causes a dull, burning and poorly localised pain that typically is delayed [We would recommend that clinicians consider neurostenalgia in all cases of nerve palsy associated with severe pain. This is particularly pertinent post-operatively when pain out of proportion to the procedure associated with nerve dysfunction should make the clinician consider nerve compression. A diagnosis of neurostenalgia warrants early exploration. It suggests a cause for the pain, which needs to be removed to resolve the intractable pain and prevent irreversible changes in the affected nerve.


Spatial context in images induces perceptual phenomena associated with salience and modulates the responses of neurons in primary visual cortex (V1). However, the computational and ecological principles underlying contextual effects are incompletely understood. We introduce a model of natural images that includes grouping and segmentation of neighboring features based on their joint statistics, and we interpret the firing rates of V1 neurons as performing optimal recognition in this model. We show that this leads to a substantial generalization of divisive normalization, a computation that is ubiquitous in many neural areas and systems. A main novelty in our model is that the influence of the context on a target stimulus is determined by their degree of statistical dependence. We optimized the parameters of the model on natural image patches, and then simulated neural and perceptual responses on stimuli used in classical experiments. The model reproduces some rich and complex response patterns observed in V1, such as the contrast dependence, orientation tuning and spatial asymmetry of surround suppression, while also allowing for surround facilitation under conditions of weak stimulation. It also mimics the perceptual salience produced by simple displays, and leads to readily testable predictions. Our results provide a principled account of orientation-based contextual modulation in early vision and its sensitivity to the homogeneity and spatial arrangement of inputs, and lends statistical support to the theory that V1 computes visual salience. One of the most important and enduring hypotheses about the way that mammalian brains process sensory information is that they are exquisitely attuned to the statistical structure of the natural world. This allows them to come, over the course of development, to represent inputs in a way that reflects the facets of the environment that were responsible. We focus on the case of information about the local orientation of visual input, a basic level feature for which a wealth of phenomenological observations are available to constrain and validate computational models. We suggest a new account which focuses on the statistics of orientations at nearby locations in visual space, and captures data on how such contextual information modulates both the responses of neurons in the primary visual cortex, and the corresponding psychophysical percepts. Our approach thus helps elucidate the computational and ecological principles underlying contextual processing in early vision; provides a number of predictions that are readily testable with existing experimental approaches; and indicates a possible route for examining whether similar computational principles and operations also support higher-level visual functions. Contextual influences collectively denote a variety of phenomena associated with the way information is integrated and segmented across the visual field. Spatial context strongly modulates the perceptual salience of even simple visual stimuli, as well as influencing cortical responses, as early as in V1 At least two main lines of theoretical inquiry have addressed these influences from different perspectives. First, computational models have related perceptual salience to low-level image features However, although collectively covering a huge range of computational, psychophysical, and neural data, these two theoretical approaches have not been unified. To this end, we introduce a computational model of the statistical dependencies of neighboring regions in images, rooted in recent advances in computer vision We focused in particular on the dependencies between orientations across space, optimizing model parameters based on natural scenes. We used the resulting model to simulate neural and perceptual responses to stimuli used in physiological and perceptual experiments to test orientation-based surround modulation. Note that we did not fit the model to experimental data from individual cells or subjects, but rather compared the qualitative behavior of a model trained on ecologically relevant stimuli with general properties of early visual surround modulation.There is a wealth of experimental results on surround modulation, some classical, and some still subject to debate. We chose to model a set of findings that we intend to be canonical examples of both sorts of results. We included data that have been the subject of previous theoretical treatments, but paid particular attention to phenomena that lie at the boundary between integration and segmentation. Indeed, one claim from our approach is that subtleties at this border might help explain some of the complexities of the experimental findings. We make predictions for regions of the stimulus space that have not yet been fully tested in experiments.In sum, we show that the statistical principles introduced can account for a range of neural phenomena that demand tuned surround suppression as well as facilitation, and encompass V1 as a salience map We first illustrate a class of characteristic statistical dependencies across space in natural scenes, and we outline a model of such dependencies. We provide implementation details and model equations, and describe how model parameters were optimized for an ensemble of natural scenes. Further, we explain how the model relates to contextual modulation in the visual cortex. We specifically focus on relating the statistical model of images to V1 neural firing rates, and also illustrate how this constitutes a generalized form of divisive normalization.output. We concentrated on the statistical dependencies between V1-like filters , whose respective Gaussian variables are multiplied by a common mixer 1k and 2k are more likely to be large together. Conversely, when 1k and 2k will likely be small together. This generates precisely the type of dependency observed in C.We extended a well-known probabilistic model of the variance dependence of We then introduced a variant of the GSM see also  that couk, and the corresponding Gaussian variables by S, and the corresponding Gaussian variables by We implemented the model with a bank of 72 bandpass oriented filters or RFs taken from the first level of a steerable pyramid lity see .http://neuroscience.aecom.yu.edu/labs/schwartzlab/code/standard.zip). Mathematical details are provided at the end of The parameters governing RF interactions – i.e. the covariance matrices and the prior probability of each component of the MGSM – were optimized by maximizing the likelihood of an ensemble of natural scenes (downloadable from http://www.eecs.berkeley.edu/Research/Projects/CS/vision/bsds), and did not renormalize the covariances. The training was repeated 3 times for each image from random starting points. Training on individual images converged to similar values each time, except for one image on which it did not converge. This image was therefore excluded from further analysis.However, to explore the effects of cardinal axis biases and variability across different scenes, we also trained the model separately on each of 40 natural images from the Berkeley Segmentation Dataset  several orders of magnitude smaller than the smallest value observed for the other term under the square root. n; it remains approximately constant over the range of values of For a given input stimulus, we first collected the outputs of center RFs Eventually, we inferred the posterior probability, The inferred model responses  thus constitute a form of divisive normalization −1C) exerts less normalization than a pair of RFs with the same variances but small covariance. In addition, an RF with large variance  weights less than one with small variance. In the visual cortex, the effect of such weights may be represented indirectly in the strength of excitatory connections that are set with development; indeed the higher correlations, in the model, between overlapping RFs, as well as non-overlapping collinear RFs, are qualitatively in agreement with the known specificity and anisotropy of horizontal and feedback connections However, our model generalizes standard divisive normalization in two substantial ways. First, consider the effect of covariance. Normalization allows cells to discount a global stimulus property that is shared across RFs, i.e. contrast in simple divisive normalization, or the mixer value in the GSM. The mixer corresponds to RFs that are statistically coordinated and tend to be high or low together in their absolute value. However, in the generative model, large outputs from RFs that often respond together (i.e. RFs with large covariance or linear correlations) could be generated either by a large value of the mixer or by similar draws from the correlated Gaussians; whereas similar, large outputs from RFs that rarely respond together  are more likely to have been generated by a large value of the mixer. Therefore linearly correlated RFs should contribute less to the estimate of the mixer . This arises in the model since the covariance matrices learned from scenes weight the contribution of the RFs to the normalization signal Second, Equation 2 uses a stimulus-dependent normalization pool, since, for any given input stimulus, only RFs that are inferred as being statistically coordinated and thus to share a common mixer, are jointly normalized . The same RFs can be coordinated for some stimuli and not for others.The computation involved, which in the model is distinct from the evaluation of the corresponding normalization signals (Equation 6), does not necessarily have to be segregated in the biological system, and may be achieved by a number of neural mechanisms. One possibility may be that the normalization signals are computed by inhibitory interneurons that pool the outputs of distinct subpopulations: the different firing thresholds or diversity across types of interneurons For a given input stimulus, we inferred the posterior co-assignment probabilities of each mixture component, Note also that, differently from standard divisive normalization which is inherently suppressive, our model encompasses both surround suppression and facilitation as summarized in k and S under each of the five possible mixture components, weighted by their prior probabilities kn and Sn represent the number of RFs in the center and surround groups respectively, and n = kn+Sn; The full distribution of the RFs variables under the MGSM is given by the mixture model:Similarly, we can derive the distribution conditional on http://neuroscience.aecom.yu.edu/labs/schwartzlab/code/standard.zip). The parameters to be estimated were the covariance matrices associated to the different components of the model, collectively denoted by old). This was obtained via Bayes rule:The parameters governing RF interactions – i.e. the covariance matrices and the prior probability of each component of the MGSM – need to be optimized for an ensemble of natural scenes. The training data were obtained by randomly sampling 25000 patches from an ensemble of 5 natural images from a database of standard images used in image compression benchmarks .First, the model uses a normalization pool  that depends on the input stimulus : when center and surround RFs outputs are statistically coordinated , then both are included in the normalization pool for the center; whereas, when they are independent, the surround RFs are excluded from the normalization pool. For a given input stimulus, we measured the degree of homogeneity (and therefore involvement of the surround RFs) by the probability of co-assignment. Intuitively, this probability is larger for stimuli that elicit similar outputs for center and surround RFs; and this indeed turns out to be true of Bayesian inference in our model. In addition, the probability tends to be larger for high contrast. The grating patch of C when the surround is co-assigned, affect the normalization signal, since in C−1 (see Equation 6). Thus, the normalization signal can be reduced, and the model response enhanced, when the covariance between RFs is large or when a given RF variance is larger  entirely from the natural scenes, and then fixed them. We first evaluated the model by comparing its response (Equation 3) to presentations of different forms and sizes of sinusoidal gratings with those described in previous neurophysiological studies of spatial contextual modulation. Physiology experiments have made extensive use of gratings to show that stimuli presented in regions of visual space that do not drive the neuron (i.e. the surround) can still strongly modulate the responses to a stimulus presented within the RF.First, we tested gratings of variable size and contrast whose orientation and spatial frequency match the chosen RF. Experiments in cat and macaque monkey V1 The contrast-dependence of size tuning has previously been ascribed to divisive normalization We next addressed three sets of data related to the orientation tuning of surround modulation , for whiWe first verified that the model produces contrast-invariant orientation tuning curves . We thenWe also explored the model behavior as a function of the stimulus contrast and the sizes of the central grating patch and surround annulus see . GeneralThe responses of the model  depended0k. Model responses showed the same effect, since stimuli with small orientation differences between center and surround led to large co-assignment probability for the surround group oriented 45 degrees from vertical  gratings that also covered the surround. Chen et al. We then addressed the spatial organization of surround modulation, to illustrate the interplay between the assignment process and the spatial structure of correlations learned by the model from natural images. Different regions of the RF surround can produce different levels of modulation when stimulated with grating patches oriented similarly to the center . Walker et al. To elucidate the origins of this response of the model, we systematically tested a larger range of contrast and size values than has so far been experimentally examined . First, Our model explains the observed spatial asymmetry of modulation by the form of the covariance matrices learned from natural scenes. In the mixture component with dependent center and vertical surround groups, the variances of the vertical center RF and its collinear neighbors were similar, and higher than the variances of the parallel neighbors . Recall ices see  and wereices see  and moduCollinear facilitation with grating stimuli has been observed in V1, although with the contrast not matched between center and surround; for instance, Polat et al. In addition to the effect we discussed, Cavanaugh et al. However, that there is a considerable scatter of positional biases due to cell-to-cell variability in The data presented so far provided an illustration of the biological significance of the two key components of the model, i.e. the assignment and the covariances. As a control, we presented the same stimuli to a reduced model that ignored the assignments and assumed that center RFs were always normalized by the vertical surround group. Such reduced model produced a much weaker contrast-dependence of size tuning of the surround compared to We then asked whether the same statistically motivated computational principles underlie perceptual salience: local image regions are deemed salient when e.g. they can be detected more readily, appear to have higher luminance, or attract human gaze more often compared to the remaining parts of the image  than parallel sets of bars (Par), relative to a homogeneous texture (Txt) of iso-oriented bars , and the neighboring rows of parallel vertical bars above and below (Par). For such bars, only a portion of the surround has the same orientation as the center, leading to overall less surround activation at the boundary than inside the homogenous regions /Txt and (Par-Txt)/Txt, respectively. The larger collinear enhancement was due only in part to the stronger RF linear outputs for collinear stimuli (since surround bars encroached on the center RF) and was largely attributable to the covariance matrices learned from scenes. As explained in Col and Par was reduced for In addition, we observed a larger salience enhancement for the middle row of bars, relative to the homogeneous region, when they were collinear  than parThe two examples above are to some extent combined in the border effect in stimuli such as We have addressed spatial contextual influences in early vision by introducing a statistical model that is rich enough to capture the coordination that exists amongst spatially distributed V1-like RFs in natural scenes. Our model took explicit account of the spatial heterogeneity of scenes. Inference in the model amounted to a generalized form of divisive normalization from each RF's surround. The model reproduced a host of V1 neural surround nonlinearities, including both suppression and facilitation, such as RF expansion at low contrast ; a numbeMany important mechanistic accounts of contextual interactions have included ideas about excitatory and inhibitory connections and their links to divisive normalization, e.g. In addition, surround modulation was more often engaged when stimulating collinear than parallel regions of the surround; but, when surround modulation was engaged, collinear responses were enhanced relative to parallel. These behaviors, due to the form of the covariances learned from natural scenes, may also find a basis in the known specificity and anisotropy of horizontal e.g.  and feedTwo main statistical innovations were critical for capturing the full set of biological data. First, our model provides a formal treatment of the idea of statistical homogeneity vs heterogeneity of visual inputs. We proposed that V1 is sensitive to whether center and surround responses to a given stimulus are inferred to be dependent or independent based on the statistical structure of natural scenes. This sensitivity is actually required to capture the stronger statistical dependence apparent within spatially extended visual objects. In our model, it implies that surround modulation is fully engaged by stimuli comprising extended single objects, but disengaged by stimuli with entirely independent center and surround.This (dis)engagement proved to be crucial to obtain the neural effects in The flexibility of the normalization pool also implied that the model could exhibit surround facilitation. While a strong (and co-assigned) surround was suppressive, a weak (and co-assigned) surround was facilitative, when compared to responses in the absence of surround (not co-assigned). The magnitude of the modulation was larger for weaker center RFs outputs . We suggThe second key statistical feature in our model, namely the RFs covariance matrices, reflects the structure of linear correlations found in natural images. For image patches that the model determined to be homogenous, we observed larger variance for the central RF and its collinear neighbors, and larger covariance between the collinear neighbors, than for any other surround location . This imAt high contrast, collinear surrounds suppress targets more than parallel surrounds. The model reproduced this characteristic  because Furthermore, the model generated salience maps that exhibited collinear enhancement and texture border assignment with simple bar displays . These sOur statistical modeling relates most closely to a number of recent powerful approaches in computer vision and statistical learning which have not been directly applied to contextual effects in neuroscience. These include hierarchical models for unsupervised learning of statistical structure in images e.g. , extensiThe model of Schwartz et al. Certain other statistically-minded approaches to computer vision have addressed the implications for biological vision. Schwartz and Simoncelli A natural extension of our approach would be to optimize the sampling of visual features and test how well the model predicts V1 responses to complex scenes; and their effects on perceptually assessable factors such as salience detection under controlled manipulation of the assignments, and gaze patterns on natural images Figure S1The natural images used to train the model.(TIF)Click here for additional data file.Figure S2Orientation tuning of the center is contrast-invariant. Model tuning curves at different contrast are approximately scaled versions of each other, a characteristic feature of V1 tuning. This holds across different stimulus sizes, both smaller and larger than the receptive field size.(EPS)Click here for additional data file.Figure S3Perceptual pop-out in the model depends on the bars length, separation, and contrast. The red box correspond to the configuration used in (TIF)Click here for additional data file.Text S1Equations and examples of the linear filters used in the simulations.(PDF)Click here for additional data file.Text S2Mathematical details on the training algorithm.(PDF)Click here for additional data file.Text S3Extended simulation results on surround orientation tuning as a function of contrast, and center and surround stimulus size.(PDF)Click here for additional data file.Text S4Additional simulation results on the positional bias. Cardinal axis effects and variability across different natural images can produce a scatter of positional biases consistent with that observed in V1 populations.(PDF)Click here for additional data file.