Neurodegenerative diseases involve the progressive loss of neurons, and a pathological hallmark is the presence of abnormal inclusions containing misfolded proteins. Although the precise molecular mechanisms triggering neurodegeneration remain unclear, endoplasmic reticulum (ER) stress, elevated oxidative and nitrosative stress, and protein misfolding are important features in pathogenesis. Protein disulphide isomerase (PDI) is the prototype of a family of molecular chaperones and foldases upregulated during ER stress that are increasingly implicated in neurodegenerative diseases. PDI catalyzes the rearrangement and formation of disulphide bonds, thus facilitating protein folding, and in neurodegeneration may act to ameliorate the burden of protein misfolding. However, an aberrant posttranslational modification of PDI, S-nitrosylation, inhibits its protective function in these conditions. S-nitrosylation is a redox-mediated modification that regulates protein function by covalent addition of nitric oxide- (NO-) containing groups to cysteine residues. Here, we discuss the evidence for abnormal S-nitrosylation of PDI (SNO-PDI) in neurodegeneration and how this may be linked to another aberrant modification of PDI, S-glutathionylation. Understanding the role of aberrant S-nitrosylation/S-glutathionylation of PDI in the pathogenesis of neurodegenerative diseases may provide insights into novel therapeutic interventions in the future. This may subsequently mediate cell death via activation of the UPR and reduced ERα stability [PDI has been shown to be S-glutathionylated at two of its four active cysteine sites  . S-glutalular NO , triggerlular NO . S-glutalular NO . In addir NO [α) . The PDIormation . Howevertability . Howevertability . There atability . Therefotability  (Figure tability .S-glutathionylation of PDI was proposed to be an upstream signalling event triggering misfolded protein accumulation and UPR induction , 211. AsPDIs are a large family of chaperones and foldases which have complex yet still inadequately described functions with emerging roles in neurodegenerative diseases. Whilst S-nitrosylation plays a normal physiological role in signalling pathways, aberrant modification is triggered during conditions of elevated nitrosative and oxidative stress. Accumulating evidence suggests that SNO-PDI plays a role in the pathogenesis of neurodegenerative diseases such as AD, PD, and ALS, and this may exacerbate neurodegeneration via a number of mechanisms. However, most of the available reports are correlative in nature and therefore more direct approaches examining the contribution of S-nitrosylation of PDI family members to neurodegeneration are warranted. S-nitrosylation is also linked to another previously described modification of PDI, S-glutathionylation, although the S-glutathionylation of PDI and its role in neurodegenerative diseases have not been elucidated. Whilst PDI family members are conventionally regarded as being ER localized, they are also present and catalytically active in several other cellular locations, which is likely to be particularly important in disease as few proteins associated with neurodegeneration are found in the ER. Finally, cell surface PDI, which reduces NO allowing it to pass through the plasma membrane, may lead to the production of SNO proteins and therefore also contribute to the pathogenesis of neurodegenerative diseases. The broad involvement of PDIs in human neurodegenerative diseases highlights the need for a better understanding of how they become inactivated by posttranslational modification, which is crucial to evaluate their use as possible targets for disease intervention.


Stroke remains the number one cause of disability in the adult population. Despite scientific progress in our understanding of stroke pathology, only one treatment (tissue plasminogen activator or tPA) is able to afford benefits but to less than 3% of ischemic stroke patients. The development of experimental dietary supplement therapeutics designed to stimulate endogenous mechanisms that confer neuroprotection is likely to open new avenues for exploring stroke therapies. The present review article evaluates the recent literature supporting the benefits of dietary supplementation for the therapy of ischemic stroke. This article focuses on discussing the medical benefits of NT-020 as an adjunct agent for stroke therapy. Based on our preliminary data, a pre-stroke treatment with dietary supplementation promotes neuroprotection by decreasing inflammation and enhancing neurogenesis. However, we recognize that a pre-stroke treatment holds weak clinical relevance. Thus, the main goal of this article is to provide information about recent data that support the assumption of natural compounds as neuroprotective and to evaluate the therapeutic effects of a dietary supplement called NT-020 as in a stroke model. We focus on a systematic assessment of practical treatment parameters so that NT-020 and other dietary supplementations can be developed as an adjunct agent for the prevention or treatment of chronic diseases. We offer rationale for determining the optimal dosage, therapeutic window, and mechanism of action of NT-020 as a dietary supplement to produce neuroprotection when administered immediately after stroke onset. We highlight our long-standing principle in championing both translational and basic science approaches in an effort to fully reveal the therapeutic potential of NT-020 as dietary supplementation in the treatment of stroke. We envision dietary supplementation as an adjunct therapy for stroke at acute, subacute, and even chronic periods. Stroke is the number one cause of disability in the adult population . DespiteCurrently, research on dietary supplementation to alleviate vascular dieases, ischemic damage and to increase neuronal plasticity is gaining popularity among laboratories that seek a reliable therapy to prevent neurodegeneration or to even slow the onset of brain pathologies. There is a well supported body of literature about the positive medicinal effects of dietary supplementation. In a review article by Arab and Liebeskird, epidemiologic studies were evaluated to clarify the effects of dietary supplementation of tea catechins in animal models of stroke and in humans. The results showed that across studies, there were highly consistent neuroprotective effects when ingesting tea catechins prior or shortly after ischemic insult. Ingestion of tea catechins was effective in reducing stroke volume after the ischemic insults in all subjects including humans . MoreoveThe treatment of stroke represents current unmet medical conditions of significance around the world. The therapy for stroke is limited. Other than one recombinant protein therapy directed at the dissolution of thrombi in affected blood vessels in adults following stroke, tumor plasminogen activator or tPA, no specific treatment is available for either focal cerebral ischemia or global ischemic event. Small molecule therapies such as anti-platelet drugs, anti-coagulants, and statins act as prophylactics and have no immediate benefit following an acute attack . The numUntil recently, the non-regenerative capability of the adult damaged brain was an accepted scientific dogma. However, accumulating evidence over the last decades indicates that neurogenesis occurs during adulthood, and that neurons and astrocytes can be generated from isolated cells of the adult mammalian central nervous system (CNS) . There a®, which is a proprietary blend plus individual chemicals such as vitamin D3 . NT-020 is an anti-inflammatory, antioxidant proprietary formula that contains polyphenols from blueberry extract, catechins from green tea, aminoacids such as carnosine, and vitamin D3. As a nutritious diet formula, it was shown previously to act synergistically to promote neurogenesis in adult rats and to exert functional benefits in a stroke model [in vitro. Primary cell neurons were cultured with LMN cream, hydrogen peroxide, and Aβ1–42 to asses the antioxidant benefits against toxic insults. Results by western blot analysis revealed that the LMN diet salvages cell death associated with either hydrogen peroxide or toxic amyloid beta insult [Regenerative medicine has emerged as a new scientific field advancing stem cell therapy for treating brain disorders, with emphasis on either transplanting exogenous stem cells or amplifying endogenous stem cells via dietary suppplementation ,17. Our ke model . By “nutke model . Studieske model . In 2009a insult . In a dia insult .Interestingly, the reverse model of diet restriction and even fasting has been recently reported to influence neurogenesis ,27. In p2, and electrolyte levels across groups, suggesting that changes in physiological functions did not contribute to the observed neuroprotection. In addition, stroke animals treated with blueberry, spinach, or spirulina exhibited significantly lower caspase-3 activity in the ischemic hemisphere. These data demonstrate that diet supplementation with blueberry, spinach, or spirulina attenuated stroke-induced cerebral infarction and apoptosis, and general locomotor impairments. However, the exact mechanism underlying such neuroprotection remains to be determined. Although not in a stroke model, short-term supplementation with blueberry (one of the components of NT-020) has been shown to increase hippocampal neurogenesis [Wang and colleagues  recentlyogenesis , which cogenesis .Recent studies on dietary supplementation have focused their attention on essential fatty acids for the prevention and treatment of cardiovascular diseases. In a human study of carotid plaques, the intra plaque levels of omega-6 fatty acids and omega-3 fatty acids were evaluated in order to determine whether or not there is a decrease of these fatty acids in systematic patients with carotid plaques. Concentrations of these two fatty acids were evaluated using ELISA and it was found that there was a significant intra-plaque omega-3 fatty acids decrease in symptomatic patients. Omega-6 fatty acids levels did not show any differences relative to asymptomatic patients . In the i.e., increased BDNF levels) signaling pathways was observed [The reverse diet manipulation paradigm involving caloric restriction and intermittent fasting has also been found to protect against stroke ,30,38. Hobserved . These sobserved  indicatei.e., high caloric intake), stands as beneficial stroke adjunct therapy. However, upon careful examination of these studies, it is clear that the mechanisms mediating the diet-induced neuroprotection warrant further examination. In our diet supplementation paradigm, we will pursue the neurogenesis mechanism, which is suggested by [These two lines of laboratory evidence support our general hypothesis that diet manipulation, via supplementation of nutritious diet extracts/compounds or by restricting unhealthy diet , which is likely characterized by an imbalance of necessary nutrients in the brain microenvironment. Supporting evidence shows that exposure to unhealthy diets such as high fat diets are detrimental to the endogenous regenerative capacity after an ischemic insult. Also, after a long-term exposure to high fat diets, there is an increase of the pro-inflammatory cytokine TNF-α in the brain, activated migroglia, APP levels, and macrophage activation [Our data and others, strongly support the diet supplementation paradigm model, to enhance neurogenesis in the adult rat stroke model due to its ability to provide nutrients that balance the homeostasis of the brain’s milieu. Moreover, our rationale for focusing on diet manipulation in elucidating the effects of nutritious diet on neurogenesis is largely based on the existing reverse paradigm of “unhealthy diet restriction”  as a neutivation ,43.The diet supplementation paradigm will generate new information about neurogenesis in the stroke brain that should complement the diet restriction model, and advance the entire field of diet manipulation for stroke. We hypothesize here that our proposed diet supplementation strategy will achieve the desirable neuroprotection that increases cell proliferation beneficial to stroke outcome. This review paper makes a case in extending our scientific platform for determining the benefit of NT-020 diet supplementation in stroke. We will determine the optimal dosage and therapeutic window post-injury, and examine the mechanism underlying NT-020 neuroprotection in defining a clinical development strategy for adjunct treatment in ischemic stroke. This paper will establish important Phase I background information that will guide us to develop a Phase II study for further product development, safety studies and clinical trials.in vivo studies showing the therapeutic efficacy of NT-020 to induce neurogenesis and to reduce stroke-induced behavioral and histological deficits in a pre-injury treatment paradigm. These data, combined with the published reports of neuroprotective effects of individual dietary components of NT-020 in animal models of stroke [This section establishes the feasibility of achieving the goals of the proposed paradigm advancing the therapeutic potential of NT-020 supplementation as a post-stroke treatment based on the accomplishment of major discovery-driven milestones, namely, f stroke , Parkinsf stroke , and agif stroke , form thn = 8) or vehicle (n = 7). The highest dosages of the components on NT-020 (135 mg/kg) were used and adjusted accordingly, for in vivo administration [in vitro cell studies [Two groups of adult male Sprague-Dawley rats initially received 135 mg/kg of NT-020  and EBST  . However, NT-020-treated stroke animals showed significantly less abnormalities in both motor and neurologic tests compared to vehicle-treated stroke animals (at post-stroke testing: p < 0.05). Reductions of 11.8% and 24.4% in EBST motor asymmetry and Bederson neurologic dysfunction, respectively, were detected in NT-020-treated stroke animals compared to vehicle-treated stroke animals. These results indicate that pre-stroke treatment with NT-020 significantly ameliorated motor and neurological abnormalities.ANOVA revealed significant treatment effects in both Bederson ( 0.0001) . Pair-wiFollowing the behavioral testing at day 14 post-stroke, all animals were euthanized in order to evaluate the cerebral infarction using the glial fibrillary acidic protein (GFAP) immunostaining . This rot-test revealed that NT-020 reduced the glial scar/ischemic area damage in the striatum (p < 0.0005) (Student striatum  comparedstriatum . A signi 0.0005) . These hin vitro also occurs in vivo, which could serve as the mechanistic explanation for the observed behavioral and histological protection. Alternate brain sections obtained from the same NT-020- or vehicle-treated stroke animals above were processed for BrdU immunostaining  to reveal cell proliferation. Analyses of BrdU labeling were focused at the neurogenic (SVZ) and the nonneurogenic striatum. The rationale for initially focusing on SVZ is to reveal that NT-020 could indeed increase cell proliferation in a neurogenic site. Although an increase in cell proliferation in SVZ could be used as a reasonable index to support a mechanism for NT-020-mediated neuroprotection, we further tested this hypothesis by subsequently analyzing BrdU labeling in the ischemic striatum to reveal whether newly formed cells from the SVZ are able to migrate towards the site of injury. This migration potential would reveal to us stronger evidence of NT-020’s therapeutic efficacy as such data would demonstrate the compound’s potential to not only increase cell proliferation, but also to facilitate proper homing of newly formed cells to the appropriate injured brain site.This next set of data was generated to determine whether the NT-020-induced cell proliferation seen F3,16 = 18.03, p < 0.0001), with at least a one-fold increment in the number of BrdU-positive cells in the NT-020-treated stroke brains compared to vehicle-treated stroke brains (p < 0.0005) , with at least a three-fold increase in the number of BrdU-positive cells in the NT-020-treated stroke brains compared to vehicle-treated stroke brains (p < 0.0001)  . Similar 0.0001) . In cont 0.0001) , there wi.e., ischemic striatal penumbra) used for cell migration studies above were double-labeled with the neuronal marker doublecortin or glial marker GFAP. Immunofluorescence microscopy revealed widespread double-labeling of cells with BrdU and doublecortin in NT-020-treated stroke brains, whereas only a few cells double-labeled with both BrdU and doublecortin in vehicle-treated stroke brains  (p < 0.0001)  . In cont 0.0001) . These din vivo. The dose of blueberry alone to produce neuroprotection in a previous study [In summary, these data  indicateus study  is 100 tThe overall design of our envisioned translational experiments is geared towards the examination of the neuroprotective effects of diet supplementation in a post-stroke paradigm. Three functional outcomes that are deemed critical to the demonstration of the optimal regimen of NT-020 for stroke include: (1) assessing behavioral neuroprotection of NT-020 by examining stroke-induced motor and neurologic functions of stroke animals; (2) evaluating histological neuroprotection of NT-020 by measuring stroke-induced cerebral infarction; and (3) identifying the mechanism underlying NT-020 neuroprotection by immunohistochemically characterizing newly formed cells or cells that are undergoing neurogenesis, as well as those cells differentiating into neurons, astrocytes or oligodendrocytes.Guidance on the experimental design of this translational study draws largely from the demonstration that pre-stroke treatment of NT-020 attenuated stroke-induced behavioral and histological deficits . The ovei.e., 5-min delay). Although it is possible that a short-delay (1–3 h) post-stroke may prove more beneficial than the 5-min delay, the functional effects of NT-020 may be time-dependent, in that administration of the compound at long delay post-stroke will display waning of beneficial effects. However, based on our data [i.e., newly formed cells have migrated to the site of injury and have differentiated into neuronal cells. Thus, NT-020 treatments proximal to stroke onset will likely produce robust behavioral effects compared to long delay NT-020 treatments, but if neurogenesis indeed plays a significant role in NT-020 neuroprotection, the delayed NT-020 treatments will also produce functional effects at later post-stroke period of behavioral evaluation.The timing of the diet supplement spirulina may be effective up to 24 h post-stroke . Capturiour data , NT-020 For assessment of mechanism of action, there is a need to recognize that stroke compromises BBB ,41,52,53In addition to anti-inflammatory effects and BBB repair, NT-020 at optimal dosage and optimal treatment onset is thought to afford neurogenesis as a major pathway of its neuroprotection. In particular, NT-020 treatment is envisioned to significantly enhance neurogenesis, characterized by increased neurogenesis at least in the SVZ and in the ischemic striatum, compared to vehicle-treated stroke animals. Based on published data , pre-strWhile the long-term goal is to develop NT-020 as an adjunct agent, the short-term goal is to focus solely on evaluating the efficacy of NT-020 alone. Once NT-020 neuroprotection in the post-stroke paradigm is validated, subsequent experiments can proceed by assessing combination of NT-020 with the established stroke treatment of tPA. A clinical scenario wherein NT-020 extends the therapeutic window of tPA beyond three hours is desirable. In addition, combining NT-020 with experimental therapies, such as stem cell transplantation and neurotrophic factor therapy, may enhance the therapeutic benefits of these strategies for stroke.The behavioral evaluation of NT-020 may initially focus on motor and neurologic tasks, as these simple tasks will be the most sensitive assays if NT-020 does indeed show neuroprotection. Following demonstration of amelioration of stroke-induced motor and neurologic impairments, subsequent evaluations may focus on NT-020’s potential to attenuate stroke-induced cognitive deficits, using tasks such as Morris water maze and radial arm maze. Indeed, aged rats fed with diet supplements display improvement in cognitive function . In a sti.e., post-stroke treatment should offer direct clinical applications. If NT-020 is proven effective when only delivered at post-stroke periods, this will extend NT-020 as a prophylactic agent to being an adjunct treatment in stroke patients who have not used diet supplementation prior to stroke. Since most stroke patients are likely not taking diet supplementation prior to stroke, testing the efficacy of NT-020 in such post-stroke paradigm will provide a solid evidence of its clinical utility as an adjunct treatment. In addition, in order to ensure the highest quality of NT-020 and batch-to-batch consistency, a GMP-licensed manufacturing facility has been contracted to produce quality-controlled and quality-assured clinical grade dietary supplement.The clinical relevance of a post-stroke paradigm in testing the efficacy of NT-020 cannot be understated. Although the optimal diet supplementation regimen in the clinic is to treat prophylactically patients who are at risk of stroke and also to continue such treatment after stroke, evaluating NT-020’s efficacy in a more stringent stroke model, Equally as important as demonstrating efficacy, monitoring any potential adverse effects in the higher dosages of NT-020 will be clinically relevant. However, a wider range of dosages will be necessary to fully determine the safety and toxicity of NT-020. Such safety and toxicity studies can be addressed after determination of NT-020 efficacy in the post-stroke paradigm. To this end, we refer to our recent call, resonating the position of several others in the field, to adhere to STAIR (Stroke Therapy Academic Industry Roundtable) and STEPS (Stem cell Therapeutics as an Emerging Paradigm in Stoke) ,61,62 crFinding a novel therapy for stroke is an urgent clinical need. This review article provides scientific evidence that dietary supplementation may afford neuroprotection for stroke. Whereas the laboratory evidence supports a pre-stroke treatment with dietary supplementation for producing neuroprotection in stroke, extending this treatment at post-stroke period will increase its clinical relevance. A systematic evaluation in a post-stroke model will help determine the optimal dosage and therapeutic window of NT-020 as a dietary supplement for stroke therapy. A further understanding on the mechanisms of action underlying dietary supplementaion, notably via anti-inflammatory and pro-neurogenic pathways will also facilitate in optimizing the treatment regimen. The use of dietary supplementation as an adjunct therapy is likely to improve stroke outcome.


Plasma d-dimer levels have been associated with the status of tumor progression or oncological outcomes in cancer. Although there are many evidences suggesting the involvement of procoagulant trend in musculoskeletal sarcoma, no clinical data on d-dimer levels and oncological outcome of musculoskeletal sarcoma has been reported.In this study, we included a total of 85 patients who were diagnosed with musculoskeletal sarcoma and treated at our institute. Plasma d-dimer levels were determined before performing any clinical intervention, including open biopsy, chemotherapy, radiotherapy or tumor resection. We evaluated the effect of d-dimer levels and other clinicopathological factors on oncological outcomes of patients.Upregulation of plasma d-dimer levels proved to be an independent risk factor for metastasis and lethal outcome of patients with musculoskeletal sarcoma.Upregulation of plasma d-dimer levels were indicated poor oncological outcome in metastasis and total survival rate of musculoskeletal sarcoma patients. Hence d-dimer levels may be a helpful marker for evaluating the tumor progression status and prognosis of musculoskeletal sarcoma. Deterioration in the hemostatic status is one of the significant physiological changes induced by malignant condition. The close relationship between cancer and thrombosis has been clinically well established. Indeed, the risk of venous thromboembolism is higher in cancer patients than in non-cancer patients .Various kinds of procoagulant factors such as malignant condition itself, chemotherapy, long rest period, pathological fracture, orthopedic surgery, and reconstruction by prosthesis or plastic surgery, have been associated with musculoskeletal sarcoma. Indeed, the incidence of venous thromboembolism caused by systemic activation of clotting-fibrinolytic system in musculoskeletal sarcoma patients is considerably high -5.Direct or collateral evidences suggested the involvement of procoagulant molecular mechanisms in musculoskeletal sarcoma. Some examples are as follows. 1) Tissue factor (TF) is a key factor in thrombin generation/fibrin formation and regulates procoagulant activity in many cancer tissues  Tissue f,7. This D-dimer, a degradation product of cross-linked fibrin blood clots, is an indicator of fibrin concentration. Upregulation of plasma d-dimer levels has been reported in several procoagulant pathophysiological conditions, including cancer. Recent studies revealed that plasma d-dimer levels could be used to determine tumor stage/grade ,14-17, dWe designed a retrospective uncontrolled study based on data obtained from medical records. The inclusion criteria for this study were as follows: 1) musculoskeletal sarcoma diagnosed and treated at our institute between 2006 and 2010; (2) patients who had undergone standard oncological resection ; (3) ade musculosAdjuvant and neoadjuvant systemic chemotherapy were performed in less than 65 years old patients with high grade sarcoma. The treatment regimens were selected based on the histological findings of the patients -25. Radi® D-Di , which was performed on the STA-R® coagulation analyzer) was peformed [Plasma d-dimer levels were assessed before performing any kind of intervention for tumor, including chemotherapy, radiotherapy, open biopsy, or tumor resection. For the measurement of d-dimer levels, a latex agglutination assay , histological grade, extracompartment extension, tumor size, surgical margin, and d-dimer levels on referral.Statistical analysis was performed using the receiver operating characteristic (ROC) curve analysis, Kaplan-Meier methods, log-rank tests, and Cox proportional hazards model with JMP . The differences were considered significant when p < 0.05. For multivariate analysis, covariates with a p value of less than 0.05 were retained in the final model. The study was approved by the institutional review board of the authors institution.The d-dimer levels ranged from 0.2 to 8.3 μg/ml . In order to determine the cut off value of d-dimer levels in this analysis, we performed ROC curve analysis Figure . The areNext, the effect of independent variables, including d-dimer levels, on the 2 endpoints, namely, metastasis and total survival, was analyzed by using survival analysis model. The result of univariate analysis suggested that elevated d-dimer levels (p = 0.002) Figure  and histMusculoskeletal sarcoma is a group of rare heterogeneous tumors of the mesenchymal lineage. Innovation in treatment modality, including theory in determining safety margin, limb salvage procedure and systemic chemotherapy have improved oncological outcomes of musculoskeletal sarcoma over the past 3 decades . AccumulD-dimer is a degradation product of cross-linked fibrin blood clots and indicates fibrin concentration. We have previously shown that plasma d-dimer levels were closely related to the histological grade of musculoskeletal tumor . HoweverMusculoskeletal malignancy is characterized by heterogeneity in tumor site and patients age, which may be independent from its biological properties that are regulated by genetic change. Our previous data  and analU test), suggesting that the application of chemotherapy might serve as a confounding bias. Thus, we did not include application of adjuvant chemotherapy as independent risk factor in survival analysis. In addition, limited number of patients received radiotherapy, and thus, indication of radiotherapy was not considered as risk factor.In this study, chemotherapy was indicated strictly on the basis of grade/histological subtype of the tumor and patient age. The results of preliminary statistical analysis suggested a strong association between tumor grade and indication of adjuvant chemotherapy  and between age and indication of adjuvant chemotherapy  from the Ministry of Health, Labor and Welfare of Japan.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/250/prepub


HiTRACE has been used extensively for quantitating data from RNA and DNA structure mapping experiments, including mutate-and-map contact inference, chromatin footprinting, the Eterna RNA design project and other high-throughput applications. However, HiTRACE is based on a suite of command-line MATLAB scripts that requires nontrivial efforts to learn, use and extend. Here, we present HiTRACE-Web, an online version of HiTRACE that includes standard features previously available in the command-line version and additional features such as automated band annotation and flexible adjustment of annotations, all via a user-friendly environment. By making use of parallelization, the on-line workflow is also faster than software implementations available to most users on their local computers. Free access:  Capillary electrophoresis (CE) is one of the most powerful and widely used nucleic acid separation techniques available . Its conIn chemical-probing-based RNA structure inference studies, a chemical reagent modifies the RNA of interest, either cleaving it or forming a covalent adduct with it. Commonly used reagents include hydroxyl radicals ,6, dimetAnalyzing a large number of electrophoretic traces from a high-throughput structure-mapping experiment is time-consuming and poses a significant informatics challenge. It requires a set of robust signal-processing algorithms for accurate quantification of the structural information embedded in the noisy traces. Current software methods for CE analysis include capillary automated footprinting analysis (CAFA) , ShapeFihttp://www.mathworks.com).Although these programs are all useful for semi-automated CE data analysis, they suffer from certain limitations. CAFA has effective peak-fitting capabilities but lacks alignment and annotation features, thus necessitating laborious efforts for analyzing multiple capillaries. ShapeFinder and QuShape provide sophisticated signal alignment, annotation and peak fitting capabilities but have limited cross-capillary analysis capabilities, making these tools less efficient for analyzing data from large-scale experiments involving hundreds of capillaries. FAST is highly optimized for rapid band annotation but relies on the proprietary Applied Biosystems (ABI) utility programs, which has made it difficult to customize and extend to various experimental scenarios. HiTRACE is feature rich and has been extensively used for high-throughput structure mapping studies such as the mutate-and-map strategy ,20,21, cHiTRACE-Web is based on the HiTRACE workflow and thus inherits all of its core functionalities for high-throughput CE analysis. Going one step further, HiTRACE-Web provides an integrated and interactive on-line interface. In addition to the standard features previously available, HiTRACE-Web presents additional features such as automated band annotation and adjustment. By making use of multi-core server processors, HiTRACE-Web also runs faster than the previous off-line implementations available to most users on their local computers. To the best of the authors’ knowledge, HiTRACE-Web is the first on-line tool for high-throughput CE data analysis.HiTRACE-Web takes as input a set of nucleic acid structure mapping profiles obtained from a number of capillaries. A profile represents the intensity of a nucleic acid sample in a capillary as a function of electrophoretic time. The peaks in a profile appear as bands in a gray-scale image. Band/peak locations represent individual residues of a nucleic acid sequence. Commonly used ABI sequencers  can separate the products of hundreds of nucleotides, and we assume that each CE profile contains hundreds of bands. The final output of HiTRACE-Web is a set of aligned and annotated profiles with quantified band areas in text and image formats. To deliver the output, HiTRACE-Web works in multiple stages interleaved with user checkpoints: preprocessing, profile alignment, band annotation, peak fitting and quantification. In preprocessing, the user needs to provide information on capillary channel compositions. To facilitate the analysis process, it is common in typical CE experiments to co-load samples with a reference ladder, which fluoresces in a different color. Multiple spectral channels thus exist in each capillary, and the user specifies which channels contain the structure mapping signal and the reference  (Step A.1).The next step in preprocessing is to select the range of analysis, as typical profiles carry information only within a subset of the entire electrophoresis run. HiTRACE-Web uses edge-detection techniques  to selecThe second stage in HiTRACE-Web is alignment. Profiles need to be aligned to each other because the products in different capillaries are subject to slightly different electrophoretic conditions, and their detection times are then shifted and scaled compared with each other. Owing to the finite number of capillaries, an ABI sequencer can handle at a time (e.g. 96 for ABI3730), CE experiments using hundreds of capillaries consist of multiple batches. We designate the first capillary in each batch as the reference and use it for within-batch alignment. There, we align each profile to the reference by finding the optimal shift and scaling amounts that maximize its correlation to the reference (part of Step B.1). After the within-batch alignment, we carry out additional alignment procedures: between-batch adjustment for global alignment (part of Step B.1), adjustment of smooth baseline (Step B.2) and dynamic-programming-based piece-wise-linear alignment for fine-tuning local disagreements   and specifies the sequence probed and the type of chemical modifications applied to each capillary (Step C.2). The user can currently select among nine options: three types of chemical reagents , four types of dideoxynucleotides , no modification or ‘other’. HiTRACE-Web provides a convenient graphical interface for selecting signal and reference channels and specifying capillary modification types. This specification provides HiTRACE-Web with information on where bands should appear in the data, e.g. primarily at A and C positions for DMS modification of RNA data , and witFollowing the profile alignment is the band annotation procedure (Step D.1), which refers to the process of mapping each band in an electrophoretic profile to a position in the nucleic acid sequence. For verification, visual inspection of band annotation results is normally inevitable to certain extent, but the manual band annotation step takes significant human efforts when there are a large number of profiles. To address this issue, HiTRACE-Web provides an automated band annotation functionality, which allows users to complete initial assignments of hundreds of bands in hundreds of profiles in the order of seconds. More details of the automated band assignment algorithm are beyond the scope of this server-focused paper and will be described elsewhere. HiTRACE-Web also provides an interactive interface to adjust the band annotation manually so that users can correct any suboptimal assignments.http://rmdb.stanford.edu/submit) or structure server for secondary structure model estimation (http://rrmdb.stanford.edu/structureserver)  to approximate a profile as a sum of Gaussian curves, each of which is centered at the intensity peak location. The peak amplitudes are selected in the least-squares sense by using a standard optimization technique, thus minimizing the deviations of the Gaussian model from the CE profiles . As the eserver)  and linkad hoc assumptions , background subtraction and final normalization to the user. These post-processing tasks are carried out differently by independent groups who have made distinct umptions ,26,27, aumptions , owing thttp://httpd.apache.org/) for basic web services and the MySQL Server  for internal data management. For client-side programming, we used CodeIgniter , an open-source web application framework and coded the web pages in PHP  and JavaScript  with jQuery  and jQuery user-interface plugins. Interactive user-interface components also use the canvas elements defined in the HTML5 standard . We used Ajax [http://en.wikipedia.org/wiki/Ajax_(programming)] for asynchronous data transfers between the client and server sides. On the server side, we used Gearman (http://gearman.org), an open-source application framework, to schedule multiple requests. Each user request is handled by a worker written in PHP. The worker creates template code in MATLAB that contains input parameters and links to the user data. The worker also forks the MATLAB interpreter so that it can execute the template code. Most of the time-consuming operations are multi-threaded, and the current HiTRACE-Web server runs on a 48-core machine . In this environment, processing 52 capillaries (with 60 bands per capillary) takes a few minutes including manual adjustments. HiTRACE-Web supports most of the widely used web browsers including Google Chrome , Mozilla Firefox , Apple Safari  and Microsoft Internet Explorer .The HiTRACE-Web server uses the Apache HTTP Server  between band intensities quantified with HiTRACE-Web, and those quantified with HiTRACE indicates lack of any major systematic deviations induced by HiTRACE-Web. To test the consistency in band quantification between different users, we let two independent users carry out quantification of the same data using HiTRACE-Web and HiTRACE, as shown in 2R of 0.9993 and 0.9986; P < 0.001). The data used are from round 44 of the massively parallel RNA design project Eterna , an online server for rapid analysis of large collections of profiles obtained from high-throughput CE experiments. Recent generations of high-throughput DNA and RNA structure mapping studies give hundreds of CE profiles each containing hundreds of bands. To isolate signals from such a large collection of profiles, we previously developed a signal-processing pipeline that consists of multiple stages including preprocessing, alignment, annotation and band quantification. HiTRACE-Web now enables users to follow the whole pipeline through a user-friendly, integrative and interactive web environment. It is our hope that HiTRACE-Web can contribute to large-scale structure inference studies based on chemical probing and CE separation by providing an effective and easily accessible data analysis framework.We developed HiTRACE-Web ]; a CONACyT pre-doctoral scholarship to P.C. (in part); Burroughs-Wellcome Foundation Career Award at the Scientific Interface to R.D. for computational work (in part); and the National Institutes of Health [R01 GM102519 to R.D. (in part)]. Funding for open access charge: Seoul National University.Conflict of interest statement. None declared.


Comparison of successful Stop-trials (Sstop)>Go, and unsuccessful Stop-trials (Ustop)>Sstop between the three beverage groups enabled the identification of alcohol effects on functional neural circuits supporting inhibitory behaviour and error processing. Alcohol impaired inhibitory control as measured by the Stop-signal reaction time, but did not affect other aspects of VSST performance, nor performance on the VSST_C. The low alcohol dose evoked changes in neural activity within prefrontal, temporal, occipital and motor cortices. The high alcohol dose evoked changes in activity in areas affected by the low dose but importantly induced changes in activity within subcortical centres including the globus pallidus and thalamus. Alcohol did not affect neural correlates of perceptual processing of infrequent cues, as revealed by conjunction analyses of VSST and VSST_C tasks. Alcohol ingestion compromises the inhibitory control of action by modulating cortical regions supporting attentional, sensorimotor and action-planning processes. At higher doses the impact of alcohol also extends to affect subcortical nodes of fronto-basal ganglia- thalamo-cortical motor circuits. In contrast, alcohol appears to have little impact on the early visual processing of infrequent perceptual cues. These observations clarify clinically-important effects of alcohol on behaviour.Alcohol impairs inhibitory control, including the ability to terminate an initiated action. While there is increasing knowledge about neural mechanisms involved in response inhibition, the level at which alcohol impairs such mechanisms remains poorly understood. Thirty-nine healthy social drinkers received either 0.4g/kg or 0.8g/kg of alcohol, or placebo, and performed two variants of a Visual Stop-signal task during acquisition of functional magnetic resonance imaging (fMRI) data. The two task variants differed only in their instructions: in the classic variant (VSST), participants inhibited their response to a “Go-stimulus” when it was followed by a “Stop-stimulus”. In the control variant (VSST_C), participants responded to the “Go-stimulus”  A weakening of inhibitory control  is implicated in the development of alcohol addiction . In adolIn the laboratory, both, the Stop-signal task (SST) , which tBehavioural studies typically demonstrate impairments following the acute administration of alcohol in these measures of inhibitory control but see ,9. SpeciNeuroimaging  studies in healthy volunteers performing SST and Go-No/Go tasks demonstrate the importance of a fronto-basal-ganglia network, encompassing the right inferior frontal gyrus , the dorsolateral prefrontal cortex , the medial prefrontal cortex  and the basal ganglia/thalamus, in response inhibition ,24,25,26A prevalent conceptualization of behavioural inhibition, proposes that the functional processes underlying “Go” and “Stop” responses are largely independent. Excitatory motor processes activated by the Go-signal compete in time with inhibitory processes triggered by the Stop-signal. Upon detection of the Stop-signal, prefrontal cortical regions (IFG or pre-SMA) initiate a counter-command that is relayed to the basal ganglia, where functional nodes associated with “Going” are suppressed in favour of those associated with “Stopping”. Subsequent output from the basal ganglia via global pallidus to the thalamus, blocks the thalamic “Go” signal to motor cortices, and the initiated motoric response is terminated ,29,30,31Recent fMRI studies ,37,38,39Ιntegral components of the ability to inhibit a behavioural response include processes involved in performance monitoring, and more specifically, the ability to process failed inhibitions (i.e. errors) in SST and Go-No/Go tasks. The region most commonly associated with these functions is the dorsal anterior cingulate cortex (ACC) ,41.per se.To date, only two published fMRI studies have assessed the effect of acute alcohol ingestion on regional brain responses during Go-No/Go and SST task performance ,43. SpecThe study of Schuckit and colleagues  examinedThus based on the evidence described so far, acute alcohol leads to measurable behavioural changes on established measures of response inhibition. However, the neural mechanisms by which alcohol compromises response inhibition remain incompletely understood. Possibilities include the direct influence of alcohol on fronto-striatal pathways that normally inhibit inappropriate actions, either by affecting sites within the basal ganglia, or prefrontal components of these networks. Another possibility is that acute alcohol ingestion impairs the early attention-dependent perceptual processing of infrequent Stop-signals that would normally elicit a re-evaluation of behavior, and consequently the inhibition of an inappropriate action: Alcohol in moderate doses can influence the processing of salient and infrequent stimuli  within visual cortical regions ,45. AddiConsequently, the aim of the current study was to examine the neural mechanisms by which alcohol influences motor inhibition (stopping processes) using the SST. We administered two different doses of alcohol (0.4g/kg and 0.8g/kg) to test for dose-response relationships in alcohol’s effects. Furthermore, we employed a visual SST (VSST), in which the Go and Stop stimuli were identical in every respect except in colour, and which utilized a performance-dependent staircase procedure to modulate SOA: The SOA is titrated through incremental increases or decreases, respectively, after correct or incorrect inhibition trials. At shorter SOAs, stopping is easier than at longer SOAs. The staircase procedure ensured that participants were able to successfully inhibit their response on 50% of Stop-trials, and allowed for the calculation of the SSRT, by subtracting the mean SOA from the mean Go-latency. The staircase procedure also ensured that participants would not differ in the number of successful and unsuccessful Stop-trials. However, if alcohol had an effect, participants would differ in the speed of the stopping process (i.e. SSRT), thereby revealing how effective the stopping mechanism is as a function of acute alcohol intoxication.We also employed a control variant of the SST, in which participants were told to continue to respond irrespective of whether the Go-stimulus was followed by a Stop-stimulus. In this control variant (VSST_C), the Stop-signals occurred as frequently as in the typical variant and were perceptually identical to the Stop-signals of the typical variant. The inclusion of this control task allowed us to test whether the effects of alcohol on the SST are mediated by an effect on the mechanisms responsible for processing an infrequent, visually salient stimulus in general.In line with a previous study that examined the effect of alcohol on response inhibition , we addiWe predicted that behaviourally, alcohol would impair inhibitory control as evidenced by an increase in SSRT. In addition, we predicted that this effect of alcohol would be mediated by an effect on components of fronto-striatal networks implicated in the inhibition of an initiated motoric response. If however this effect of alcohol was also mediated by an impairment in the processing of infrequent and perceptually salient stimuli, this would be revealed through fMRI conjunction analyses with the control SST variant. Consistent with evidence from previous studies, alcohol effects on error processing were predicted to be revealed through changes in the engagement of anterior cingulate cortex.The study recruited 42 moderate-to-heavy social drinkers  from the University of Sussex subject pool. Advertisements requested right-handed, English-speaking social drinkers, between 18 and 40 years of age. Inclusion to the study required a weekly alcohol consumption of 10-60 units, as assessed using the Alcohol Use Questionnaire  . ExclusiParticipants were randomly allocated to receive one of three flavoured drinks that contained either a low dose of ethanol , a high dose of ethanol , or no ethanol (Placebo group), under double-blind conditions.Participants took part in an initial baseline/task-familiarization session and a subsequent drink-administration/scanning session (see below for details). During scanning, participants completed two variants of a Visual Stop-signal Task (VSST) alongside other tasks. The two VSST variants were always presented 20-25 minutes after drink administration, when the blood alcohol concentration reaches a plateau .Prior to each session, participants were asked to refrain from drinking alcohol- or caffeine-containing drinks for at least 12 and 4 hours, respectively, and from taking illicit drugs for one week. They were also asked to refrain from consuming a high-fat-content breakfast or lunch prior to testing. Participants were allowed to smoke as they would normally before each session, but smoking was not permitted during testing.A standard breathalyser , with a detection-limit equivalent to 0.01 g/l of alcohol in the bloodstream, was used to measure breath alcohol concentrations (BRaCs) at the start of each session to ensure zero blood alcohol levels. BRaCs were converted to blood alcohol concentrations (BACs), and are reported as BACs throughout the paper, as is standard for alcohol-related studies.Three days before scanning, participants gave fully-informed consent to take part in the study, and provided demographic details  and information regarding their medical history , including estimates of the number of cigarettes they smoked per day. They also completed a number of measures that were used to ensure that the three groups were matched on baseline cognitive ability  , on baseDuring the main scanning session, participants were given their allotted drink and then were placed in the 1.5Tesla Siemens Avanto MRI scanner where they completed the variants of the VSST during acquisition of functional (fMRI) datasets. Subjective feelings associated with alcohol ingestion   were ratAlcohol was administered at a dose of either 0.4g/kg or 0.8g/kg of body weight. Each dose was diluted with tonic water  to create a 500ml drink. The placebo beverage consisted only of the respective volume of tonic water (i.e. 500ml). Six drops of Angostura bitters® were added to the placebo and the alcohol beverages to match the taste of each drink-type, and to disguise the taste of the alcohol. Each drink-type was administered in 10×50ml portions and was consumed at a rate of one portion every 3 minutes. Drinks were prepared by another researcher to ensure the double-blind conditions, and the experimenter was present throughout the drinking procedure.The following measures were administered in the initial familiarisation session, and were used to ensure that the groups were matched on baseline trait impulsivity and cognitive ability, as well as in their drug-use history, their average weekly alcohol-use, and their baseline expectations concerning the effects of alcohol.The Rey Auditory Verbal Learning Test (RAVLT)  is a worDrug Use history Questionnaire (DUQ)  were tested using 3 mixed 2 x 3 ANOVAs with time (baseline vs. scanning session) as the within-subjects factor and group (placebo vs. low-dose vs. high-dose) as the between subjects factor. The factor time was included to ensure that groups did not differ on any VSST index at baseline. Significant time x group interactions were explored further using one-way ANOVAs comparing the performance of the three groups separately at baseline and during the main scanning session. Significant effects were explored further using post-hoc Bonferroni-corrected independent samples t-Tests.In order to ensure that VSST_C Stop- and Go-trials did not differ on latency or accuracy scores, mixed 2x3 ANOVAs were undertaken with trial-type (go vs. stop) as the within-subjects factor and group (placebo vs. low-dose vs. high-dose) as the between subjects factor. Significant group x trial-type interactions were explored further using one-way ANOVAs comparing the performance of the three groups separately during Go-trials and during VSST_C Stop-trials. Significant effects were explored further using post-hoc Bonferroni-corrected independent samples t-Tests.Between VSST-variant analyses” in order to ensure that trials requiring a response  did not differ with respect to latency or accuracy scores. Mixed 3x3 ANOVAs were undertaken with trial-type (Go_ VSST_C vs. Stop_VSST_C vs. Go_VSST) as the within-subjects factor and group (placebo vs. low-dose vs. high-dose) as the between subjects factor. Significant group x trial-type interactions were explored with one-way ANOVAs and post-hoc Bonferroni-corrected t-Tests.We also ran “Task and condition-related changes in neural activity were derived from regional changes in BOLD contrast consequent upon evoked haemodynamic responses. For the analyses of the fMRI data, two independent statistical models, one per VSST variant, were initially computed for each participant in each group. For each model, condition-specific event onsets were convolved with the canonical haemodynamic response function and a high pass filter (128 s) was applied to remove low-frequency artefacts . Events st level models included event onsets of correct Go-trials (Go), successful Stop-trials (Sstop), and unsuccessful Stop-trials (Ustop). Movement regressors, incorrect Go-trials, as well as fixation onsets were included as regressors of no interest.Specifically, the VSST 1st level models included event onsets of correct Go- (Go_VSST_C), and of correct Stop-trials (Stop_VSST_C). Movement regressors, and incorrect Go/Stop trials, as well as fixation onsets were included as regressors of no interest.The control VSST 1We computed the contrasts Sstop>Go (VSST contrast), Ustop>Sstop (VSST-Error contrast), and Stop_VSST_C>Go VSST_C (VSST_C contrast) for each participant. These contrast images were submitted to separate second level random-effects analyses ,61.The VSST the VSST_C contrasts were submitted to two separate random effects analyses using one-sample t-Tests in the placebo group only, in order to examine regions of activation uncontaminated by the effects of alcohol.The VSST_C thresholded SPM map was used as an exclusive mask to the VSST analysis in order to identify clusters that were NOT involved in the processing of infrequent Stop-stimuli.both analyses . Overall in the low-dose group, BACs ranged between 0.35g/l and 0.80g/l before scanning, while in the high-dose group they ranged between 0.80g/l and 2.19g/l before scanning  reported that they thought that they had received placebo. Interestingly, the majority of participants in the placebo group reported being somewhat confident that they had received the low alcohol dose. Participants in the two alcohol groups, however reported being “somewhat” to “very” confident that they had received alcohol, with only 2 participants in the low-dose group guessing placebo, and no participant in the high-dose group guessing placebo. Thus, while blinding was not successful in the two alcohol groups, probably due to the effects of alcohol itself, participants in the placebo group (i.e. the control group) were fully blind to the drink administration.Subjective reports of “lightheadedness” significantly increased following drink-administration in the placebo  and high-dose groups , but not in the low-dose group , explaining a significant time x group interaction F = 4.96, p < 0.05). There was a significant difference between groups pre-drinking  with the group allocated to the low alcohol dose showing the highest ratings. A significant time x group interaction  = 3.73, p < 0.05] for the factor “contented” was due to that participants reported feeling more “content” following administration of the high alcohol dose [t(12)=2.32 p = 0.04], but not following administration of placebo or the low alcohol dose . No other time x group interaction was statistically significant, and we did not find any main effects of group . Mean VAS scores for each group pre- and post-drinking are presented in Stop-accuracy did not differ between alcohol groups in either the baseline or experimental session , and was approximately at 50%, suggesting that the staircase manipulation was effective.There were no significant main effects of group, or time x group interactions with respect to Go-latency or Go-accuracy . However, we did observe a significant main effect of group in SSRT , with participants in the high-dose group displaying higher SSRTs overall relative to the placebo group . No other comparison survived Bonferroni adjustment . This main effect of group was qualified by a significant time x group interaction . Groups did not differ in their baseline SSRT . However, following drink- administration, both the low-dose and the high-dose group showed higher SSRTs when compared to the placebo group . The two alcohol groups did not differ in SSRT . Mean indices derived from the VSST for each group are presented in We found no main effects for trial-type or group, nor trial-type x group interactions with respect to either accuracy or latency . Mean indices derived from the VSST_C for each group are presented in We found no main effects of group, or significant trial-type x group interactions with respect to either accuracy or latency . In addition, accuracy scores were equal across the three trial-types . However, Go-trials in the VSST were significantly slower than both Go- and Stop- trials in the VSST_C variant .Successful stopping relative to successful going (Sstop>Go) was associated with increases in activity within a set of regions including parts of the dorsolateral PFC, the inferior frontal gyrus pars opercularis, medial PFC and pre_SMA, the insula, and sectors within the basal ganglia. When the Sstop>Go contrast was masked exclusively with the Stop_VSST_C>Go VSST_C contrast, clusters within medial PFC, including pre-SMA and dorsolateral PFC , basal ganglia and insular cortex were unaffected, suggesting that these regions were not merely involved in the perceptual processing of infrequent visual cues see . SeveralThe two doses of alcohol resulted predominantly in non-linear changes in activity . Of the The low dose of alcohol resulted in enhanced responses relative to placebo within parahippocampal and occipital clusters , as well as in inferior frontal orbitalis, superior frontal gyri and gyrus rectus/ventral anterior cingulate see  and middThe high dose of alcohol increased activation in superior frontal and inferior frontal pars orbitalis clusters, as well as in lingual gyrus and in the thalamus . In contThe high dose, compared to the low dose of alcohol resulted predominantly in activity reductions , somatosensory and motor cortices  and in the inferior temporal gyrus. By contrast, decreased activations were found in the parahippocampal gyrus, in frontal clusters , and in the thalamus  were observed within prefrontal cortex, parietal, temporal and occipital cortex as well as thalamus and cerebellum . These oThe present study assessed the effects of two acute doses of alcohol on the inhibition of an initiated motoric response, and explored the brain mechanisms associated with these effects. Two versions of a visual Stop-signal task were used. One involved stopping an initiated response at the onset of a Stop-signal, while the other required participants to respond regardless of whether the Stop-signal appeared or not. This latter condition challenged perceptual and attentional processes associated with the presentation of infrequent Stop-signals, yet was uncontaminated by processes of response inhibition.Behaviourally, acute alcohol at both the high, and the low dose significantly impaired inhibitory control, relative to placebo, as evidenced by a significant increase in SSRT in the typical version of the task. Importantly, we titrated the task to maintain the same level of performance accuracy, thus the effects of alcohol were restricted to inhibitory processes, consistent with previous data ,19. In tThe VSST was successful at producing measurable changes in BOLD responses in regions previously implicated in response inhibition. These included regions within the inferior frontal and dorsolateral prefrontal cortex , the right insular cortex, the pre-SMA, and the basal ganglia ,24,25,41Interestingly, the effects of the two alcohol doses on activations associated with successful stopping, appear to be expressed regionally differentiated. Linear reductions in activation, as the dose of alcohol increased, were observed within the inferior temporal cortex. Non-linear changes were observed in clusters within PFC, including middle frontal cortex and paracentral lobule/SMA proper, regions that have been implicated in successful stopping ,24,25. IDuring successful stopping the low dose of alcoholevoked both increases and decreases in activation, relative to placebo, within a set of cortical regions associated with integrative sensorimotor and cognitive processes including parietal, temporal, occipital and prefrontal regions. Particularly noteworthy is the engagement of anterior insular cortex, where activity, reduced by alcohol, was related to successful stopping (not the processing of infrequent cues). This observation is interesting from the perspective of what is known about anterior insula engagement in the representation of emotional and motivationally- salient stimuli . In the The high alcohol dose evoked changes in activity in several of those areas affected by the low dose, including paracentral lobule/SMA proper, but importantly induced changes in activity within subcortical centres. It enhanced activation, relative to the placebo group, in the thalamus, but it decreased it in the global pallidus and in the paracentral lobule/SMA proper. The global pallidus is known to suppress the thalamic “go” output to motor cortices in order for an initiated motoric response to be terminated ,29,30,31Our findings also highlight a functional distinction in the role of the anterior cingulate cortex. Activation within the anterior cingulate was affected in a non-linear way by the two doses of alcohol, but only in the VSST_C contrast (Stop_VSST_C> Go_VSST_C). Previous studies looking at the effects of alcohol on interference tasks also report modulation of activity within this region by alcohol ,73. ThesThe dorsal anterior cingulate cortex has also been discussed in the literature as an area affected by a high dose of alcohol in conjunction with error processing . HoweverTaken together, our data suggest that whereas both the high and low alcohol dose may modulate brain activation in regions involved in ‘representational’ sensorimotor and motor planning processes, only the high dose of alcohol modulates subcortical areas of the fronto-striatal-thalamo-cortical loops, specifically globus pallidus and thalamus, that are arguably more proximate to the final motor response.It is noteworthy that in this study, neither dose of alcohol was found to affect the activity of regions identified in the conjunction analysis of the typical VSST and control tasks, i.e. brain areas engaged by the perceptual processing of infrequent, visually salient signals. This suggests that alcohol does not affect VSST performance through effects expressed at the level of visual sensory representations. Nevertheless, alcohol may still produce deficits in the capacity to shift attention, or potentially reduce overall cognitive/attentional set. This may account in part for the group effects of alcohol observed in the control task on activity within temporal and parietal regions associated with visual attention.Acute alcohol, relative to placebo, can increase cerebral blood-flow in lateral prefrontal, as well as in medial frontal regions ,76. GiveIn summary, the present study provides evidence that alcohol at low and high doses affects motor inhibitory control by modulating activation within prefrontal, parietal, temporal and motor cortical areas. Moreover, the high dose of alcohol additionally affects subcortical nodes within fronto-basal ganglia- thalamo-cortical motor loops known to be involved in initiating and terminating a motoric response. Alcohol had little effect on regions associated with the perceptual processing of an infrequent cue. These findings detail the processes and the underlying neural substrates through which alcohol impairs behavioural control as expressed in motor inhibition, and are relevant to understanding the progression of deficits in self-regulation and inhibition in problem drinking with implications for addictive disorders.


We studied the diagnostic accuracy of bedside lung ultrasound (the presence of a comet-tail sign), N-terminal pro-brain natriuretic peptide (NT-proBNP) and clinical assessment (according to the modified Boston criteria) in differentiating heart failure (HF)-related acute dyspnea from pulmonary (chronic obstructive pulmonary disease (COPD)/asthma)-related acute dyspnea in the prehospital setting.n = 129) and a pulmonary (asthma/COPD)-related acute dyspnea group (n = 89). All patients underwent lung ultrasound examinations, along with basic laboratory testing, rapid NT-proBNP testing and chest X-rays.Our prospective study was performed at the Center for Emergency Medicine, Maribor, Slovenia, between July 2007 and April 2010. Two groups of patients were compared: a HF-related acute dyspnea group (P < 0.05) and (2) Boston modified criteria (P < 0.05). The combination of ultrasound sign and NT-proBNP has 100% sensitivity, 100% specificity, 100% NPV and 100% PPV. With the use of ultrasound, we can exclude HF in patients with pulmonary-related dyspnea who have positive NT-proBNP  and a history of HF.The ultrasound comet-tail sign has 100% sensitivity, 95% specificity, 100% negative predictive value (NPV) and 96% positive predictive value (PPV) for the diagnosis of HF. NT-proBNP  has 92% sensitivity, 89% specificity, 86% NPV and 90% PPV. The Boston modified criteria have 85% sensitivity, 86% specificity, 80% NPV and 90% PPV. In comparing the three methods, we found significant differences between ultrasound sign and (1) NT-proBNP (An ultrasound comet-tail sign alone or in combination with NT-proBNP has high diagnostic accuracy in differentiating acute HF-related from COPD/asthma-related causes of acute dyspnea in the prehospital emergency setting.NCT01235182.ClinicalTrials.gov  The eet al.  first deet al.  describe [et al.  proposed [et al.  combinedThe aim of our study was to determine the diagnostic accuracy of bedside lung ultrasound , NT-proBNP and clinical assessment in differentiating heart failure (HF)-related acute dyspnea from pulmonary (COPD/asthma)-related acute dyspnea in the prehospital setting .This prospective cohort study was performed in the prehospital emergency setting  between July 2007 and April 2010. The study was approved by the Ethical Review Board of the Ministry of Health of Slovenia. During the period of the study, 248 consecutive patients with acute dyspnea were treated by emergency teams . After prehospital care, all patients were admitted  to the University Clinical Center Maribor and followed until discharge.The inclusion criterion for the study was shortness of breath as the primary complaint (defined as either the sudden onset of dyspnea without history of chronic dyspnea or an increase in the severity of chronic dyspnea). Exclusion criteria were age < 18 years, history of renal insufficiency, trauma, severe coronary ischemia (unless patient's predominant presentation was dyspnea) and other causes of dyspnea, comprising pneumonia, pulmonary embolism, carcinoma, pneumothorax, pleural effusion, intoxication (drugs), anaphylactic reactions, upper airway obstruction, bronchial stenosis and gastroesophageal reflux disorder, according to the history, clinical status and additional laboratory tests available in the prehospital setting . Among 248 patients, 218 met the criteria for inclusion in the study. The distribution of all patients is shown in Figure After enrollment, patients' demographic characteristics, symptoms and signs, medical histories, medication use, chest X-rays and standard blood test results  were recorded. Our protocol for clinical assessment of HF-related acute dyspnea  was designed according to the Boston criteria  and the The final hospital diagnosis of HF-related acute dyspnea and pulmonary-related acute dyspnea  was confirmed by cardiologists and/or intensive care physicians in the University Clinical Center Maribor using the reference standard definition for HF and pulmonary diseases in accordance with the previously cited instruments, including chest X-ray, echocardiographic examination, cardiac functional assessment (exercise test), pulmonary function test, complete blood count, biochemistry and invasive investigation or angiography ,13-16.According to these criteria, identification of independent predictors for final diagnosis of acute dyspnea was performed by examination of 27 variables , a 5-mL sample of blood was collected into a tube containing edetate calcium disodium for blinded measurement of NT-proBNP. The level of NT-proBNP was measured using a portable Cardiac Reader device  and recorded according to the special protocol. The test was finished within 15 minutes ,18.et al. [et al. [et al. [et al. [et al. [The bedside thoracic ultrasound was performed according to the protocol described by Cardinale et al. , Volpice [et al.  and Lite [et al. , in whic [et al.  and Volp [et al.  requiresNT-proBNP measurements and ultrasound examinations were performed immediately after the arrival of the patient at the emergency department but before application of medication, thus our results were not altered by treatment. The raters who made the diagnosis  were blinded to the results of NT-proBNP. In addition, the investigators of NT-proBNP did not collaborate in making the final diagnosis. On the other side, prehospital emergency physicians were not blinded to the ultrasound findings, because bedside lung ultrasound represents the routine method for assessment of acute dyspnea in our prehospital emergency unit. To avoid bias, the ultrasound findings were recorded by the emergency physicians but did not affect the diagnosis. The raters who made the diagnosis in the hospital were blinded to the findings of prehospital ultrasound. To our knowledge, no previous study has compared the diagnostic utility of ultrasound examination and NT-proBNP in a prehospital setting.2 test for categorical variables and an unpaired t-test for continuous variables with normal distribution . Odds ratios (ORs) and 95% confidence interval (CIs) were calculated to examine the risk of acute HF (adjusted using multiple logistic regression). Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), positive likelihood ratio (LR+) and negative likelihood ratio (LR-) were estimated for clinical assessment (based on the modified Boston criteria), NT-proBNP, ultrasound examination and a combination of ultrasound with NT-proBNP. The comparison of these four methods was done by using the χ2 test with the Bonferroni correction for multiple comparisons. The area under the receiver-operating curve (AUROC) was also used to determine the diagnostic accuracy of the four methods in differentiating HF-related acute dyspnea from pulmonary-related acute dyspnea. Single areas were calculated and compared with univariate Z-score testing. We compared the areas under different curves using the technique proposed by Hanley and McNeil [et al. [Univariate comparisons were made by using the χd McNeil  and Jann [et al. . StatistThe authors confirm that all patients gave their consent for study participation and potential publication of the study results.P = 0.001). The feasibility of ultrasound examination in the prehospital setting was 100%, and the duration of the examination was always less than 1 minute. For the identification of independent predictors for the final diagnosis of acute dyspnea, we examined 24 variables  in multivariate logistic regression analysis. Ten variables remained significant after analysis . Thirty patients were excluded from the study. The clinical and demographic characteristics of patients are presented in Table is Table . Evident+, LR- and AUROC values are presented for ultrasound examinations , modified Boston criteria , NT-proBNP  and a combination of ultrasound examination with NT-proBNP. In comparing the methods, we found significant differences between ultrasound signs versus NT-proBNP (P < 0.05) and ultrasound signs versus modified Boston criteria (P < 0.05). All 11 patients for whom false-positive results were found using the NT-proBNP method had values higher than 1,000 pg/mL  and a history of HF. In all of these 11 patients, we confirmed the absence of comet-tail signs. With ultrasound, we can exclude HF in pulmonary-related dyspneic patients with positive NT-proBNP results and a history of HF. All five patients for whom false-positive results were found using the ultrasound method had NT-proBNP values less than 1,000 pg/mL  and a history of COPD/asthma. With the value of NT-proBNP, we can exclude HF in ultrasound-positive pulmonary-related dyspneic patients.In Table + infinite, LR- zero, and AUROC 0.99.The combination of ultrasound examination and NT-proBNP was statistically significantly different from the use of single methods. It had values of 100% sensitivity, 100% specificity, 100% NPV, 100% PPV, LROur study demonstrates that ultrasound examination was the best single method for confirming the diagnosis of acute HF in the prehospital setting. Compared with clinical assessment using modified Boston criteria and NT-proBNP testing, lung ultrasound had a significantly better AUROC with regard to diagnostic accuracy. Furthermore, the combination of ultrasound examination and rapid bedside NT-proBNP testing proved to be an even more reliable method for the identification of acute HF and its differentiation from COPD/asthma-related causes of acute dyspnea.et al. [et al. [et al. [et al. [et al. [Acute dyspnea is one of the most common conditions encountered in emergency care settings. Correct diagnosis and treatment are of primary importance, as misdiagnosis can result in deleterious consequences for patients. Timely differentiation of HF from other causes of acute dyspnea  may be difficult. Physical examination, chest radiography, electrocardiography, and standard biological tests often fail to accurately differentiate HF from pulmonary causes of dyspnea ,4-6. Rapet al. ,10, Card [et al.  and Volp [et al. . The com [et al. . Agricol [et al.  studied  [et al.  reportedet al. [+ infinite; and LR- zero. To our knowledge, no previous study has specifically compared the utility of lung ultrasound and NT-proBNP in the out-of-hospital setting, as researchers have focused on the patients in emergency departments and intensive care units. Zechner et al. [The data from our study  suggestr et al.  presentePrehospital emergency physicians offer the earliest treatment of acute dyspnea, which is performed as soon as clinically possible after the event. On the basis of clinical judgment alone, it is sometimes very difficult to distinguish cardiac from pulmonary causes of dyspnea. If prehospital physicians have the tools of rapid NT-proBNP testing and ultrasound at their disposal, the diagnostic dilemmas in differentiating causes of dyspnea are reduced and the treatment possibilities in clinically obscure cases are mainly improved.Ultrasound is currently the only imaging method that can be used in the field. It offers an opportunity to extend and improve out-of-hospital diagnostic possibilities and is useful for prehospital emergency physicians with additional knowledge of point-of-care ultrasound diagnostics. Under special circumstances, it may be used by well-educated paramedics ,26. The This study has methodological limitations. In our analysis, we included only patients with primary HF or COPD/asthma diagnosed in the field, and this limitation decreases the generalizability of this study to other causes of acute dyspnea in the prehospital setting. The primary aim of our study was to determine the diagnostic accuracy of bedside lung ultrasound and NT-proBNP in differentiating HF-related acute dyspnea from COPD/asthma-related acute dyspnea in prehospital settings.Ultrasound examination of the lungs alone or in combination with NT-proBNP testing has high diagnostic accuracy in differentiating acute HF-related from COPD/asthma-related causes of acute dyspnea in prehospital emergency settings. The combination of these two methods helps to improve the diagnostic and treatment possibilities in clinically obscure cases of acute dyspnea in the earliest phases of their appearance. Both methods are simple, non-time-consuming and can be used at bedside or in the field.• Diagnosis of severe, acute dyspnea in the prehospital arena and/or the emergency department can be challenging, but lung ultrasound is proving to be an accurate new diagnostic tool by itself or in combination with other diagnostic modalities.• Pulmonary edema gives specific, diffusely vertical artefact line (B lines and comet-tail signs) patterns on ultrasound, unlike the results found in patients with obstructive diseases or pulmonary emboli .• The question remains how well specific patterns of diffuse B lines on ultrasound scans correlate with levels of NT-pro-BNP and how they help in making the correct diagnosis.• In our study, the combination of ultrasound examination and NT-proBNP had 100% sensitivity, 100% specificity, 100% NPV and 100% PPV for differentiating heart failure as the cause of acute dyspnea compared to pulmonary causes in the prehospital setting.• Both ultrasound examinations and NT-pro-BNP point-of-care assays are quick, accurate and feasible, with high diagnostic accuracy, in the prehospital arena.+: positive likelihood ratio; LR-: negative likelihood ratio; NPV: negative predictive value; NT-proBNP: N-terminal pro-brain natriuretic peptide; PetCO2: partial pressure of end-tidal carbon dioxide; PPV: positive predictive value.AUROC: area under the receiver-operating curve; BNP: brain natriuretic peptide; CHF: congestive heart failure; CI: confidence interval; COPD: chronic obstructive pulmonary disease; CVP: central venous pressure; HF: heart failure; LRThe authors declare that they have no competing interests.PG participated in the design of the study and collected and interpreted the data. KP participated in the design of the study, collected the data and wrote a final version of the manuscript. SM collected the data and participated in the coordination of the study. GŠ designed the study, participated in the data collection, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.


The use of Gene Ontology (GO) data in protein analyses have largely contributed tothe improved outcomes of these analyses. Several GO semantic similarity measureshave been proposed in recent years and provide tools that allow the integration ofbiological knowledge embedded in the GO structure into different biologicalanalyses. There is a need for a unified tool that provides the scientificcommunity with the opportunity to explore these different GO similarity measureapproaches and their biological applications.http://web.cbio.uct.ac.za/ITGOM, which incorporates many differentGO similarity measures for exploring, analyzing and comparing GO terms andproteins within the context of GO. It uses GO data and UniProt proteins with theirGO annotations as provided by the Gene Ontology Annotation (GOA) project toprecompute GO term information content (IC), enabling rapid response to userqueries.We have developed DaGO-Fun, an online tool available atThe DaGO-Fun online tool presents the advantage of integrating all the relevantIC-based GO similarity measures, including topology- and annotation-basedapproaches to facilitate effective exploration of these measures, thus enablingusers to choose the most relevant approach for their application. Furthermore,this tool includes several biological applications related to GO semanticsimilarity scores, including the retrieval of genes based on their GO annotations,the clustering of functionally related genes within a set, and term enrichmentanalysis. During the last decade several Gene Ontology (GO) semantic similarity approaches -10 have Initially, path- or edge-based approaches, which use a distance or the number of edgesbetween terms in the ontology structure, were introduced ,16. For Annotation-based approaches have been widely analyzed, deployed in many biologicalapplications and were shown to outperform path-based models . Most ofThe main use of GO semantic similarity measures is the computation of protein semanticsimilarity or functional similarity between proteins based on their GO annotations. Thecompletion of several genome sequencing projects has generated immense quantities ofsequence data. Subsequently, with the continuous development of new high-throughputmethods the amount of functional data has increased dramatically, justifying thedevelopment of dedicated methods and tools that help extract information from thesedata. GO  has succhttp://neurolex.org/wiki/Category:Resource:Gene_Ontology_Tools) thatfacilitate effective exploration of these measures.As well as different GO semantic similarities, several functional similarity approacheshave been proposed. Some of them depend directly on the GO term IC, referred to asDirect Term- or graph-based approaches, and others are constructed via computation of GOterm semantic similarity measures, referred to as Term Semantic-based approaches. Theformer includes approaches derived from the Jaccard, Dice and universal indices based onthe Tversky ratio model of similarity , referreThese tools include software packages and web-based online tools. Most of the softwarepackages are implemented in the R programming language ,29, amonThe appropriate use of functional similarity measures depends on the applications ,24 sinceThe DaGO-Fun tool integrates GO IC-based semantic similarity measures, allowingresearchers to explore and choose an appropriate measure for their analysis. Theresulting GO similarity scores are retrieved from the DaGO-Fun database implementedusing MySQL and accessible via a web interface. The whole system is implemented using aLAMP (Linux-Apache-MySQL and PHP/Python) platform. This means that the DaGO-Fun tool isimplemented under free software  using a Linux Apache serverwith a database structured in a relational model using MySQL, with the web interfaceimplemented in PHP-HTML.7 proteins with GOannotations and 38 877 GO terms  from the GO database. The current version ofDaGO-Fun uses UniProt and GOA-UniProtKB release 2013-01 of Jan 9, 2013 and GO version1.3499 downloaded on 19-January-2013. The database will be updated using an automatedscheme every three months.The back-end is composed of a set of query processing programs implemented in Python.The user input data are GO terms or UniProt proteins -37 and tWe have implemented two main families of IC-based GO semantic similarity measures:annotation and topology-based families. The annotation-based methods are constrainedby the annotation statistics related to terms, while topology-based measures use theintrinsic topology of the GO DAG. In terms of GO term IC, the DaGO-Fun tool includesboth families and for the topology-based family, the tool implements threeapproaches; Zhang et al. , Wang etThe GO term semantic similarity approaches include traditional Resnik and Linmeasures and two approaches that have been suggested to improve the performance ofthe Lin measure, namely Relevance (SimRel)  and InfoThese annotation-based GO term similarity approaches are combined using statisticalmeasures of closeness, such as average (Avg), maximum (Max), best-match average (BMA)and averaging all the best matches (ABM) for calculating protein functionalsimilarity scores. The difference between ABM and BMA approaches is subtle in theirconception and scores produced by these two approaches differ. The ABM ,24 for tp and q is the mean of thefollowing two values: average of best matches of GO terms annotated to proteinp against those annotated to protein q, and average of bestmatches of GO terms annotated to protein q against those annotated toprotein p, given by the following formula: The Best Match Average (BMA) ,22 for ts and t, X representing themolecular function (MF), biological process (BP) or cellular component (CC) ontologyannotating a given protein r and n=m, which is not often the case in a set of annotated genes orproteins.In equations (1) and (2), A well known issue with all these statistical measures of closeness is that they aresensitive to scores that lie at abnormal distances from the majority of scores, oroutliers. This means that these measures may produce biases which affect proteinfunctional similarity scores . The funX representing the ontology  annotating a given proteinr. Note that these two measures are still to be evaluated and compared tothe existing functional similarity measures.where The DaGO-Fun tool implements 27 functional similarity measures , genename and description. GO term topological features (term parents and level) wereextracted from the GO database. These data are integrated into a MySQL database ofbiological concepts present in DaGO-Fun, and used to produce GO term IC, GO termsemantic similarity and protein functional similarity scores. The GO term IC scoresare integrated into the precompiled dictionaries in the DaGO-Fun tool. The tool isbased on a client-server model and is accessible athttp://web.cbio.uct.ac.za/ITGOM by any user with a standard webbrowser. The user interface in DaGO-Fun allows easy and comprehensive navigation,query and exploration of GO term, protein semantic similarity scores, and includesbiological applications, as shown in Figure Protein annotations were retrieved from GOA-UniProtKB atThe DaGO-Fun tool provides a comprehensive searching scheme. The user selects thetask to be processed, which includes the ontology  under consideration, and chooses the GO semanticsimilarity measure family (annotation or topology-based). After this, he/she canselect one from a list of available models, which is restricted according to theselected family. Finally, some additional options are available only when dealingwith proteins, depending on the user’s choices. If the user selects theannotation-based family then more information is requested about the class (directIC or non direct IC) of the approach selected and how the IC or GO term similarityscores should be combined. The engine changes further steps to guide theuser’s choices by only making available the options relevant to the currentchoice.After selecting appropriate parameters, the user enters their queries in a textarea or from a file, and the size of the input allowed depends on theapplications. Note that the DaGO-Fun tool currently includes four applications,namely: Term and protein semantic similarity measures (IT-GOM), ProteinFuzzy-Identification (GOSP-FIT), Term Fuzzy-Enrichment Analysis (GOSS-FEAT) andProtein Fuzzy-Classification (GOSP-FCT). Here, the fuzzy concept is related to thefact that the results or outputs of a given query are a function of a certainagreement score or level. http://web.cbio.uct.ac.za/ITGOM/tools/itgom.php: up to 3000 pairs ofGO Ids, UniProt protein accessions or gene names can be submitted for GO termsimilarity and functional similarity querying. For GO term IC, the user can enterup to 5000 GO Ids.• For IT-GOM athttp://web.cbio.uct.ac.za/ITGOM/tools/gotspfit.php.• A list of at most 20 GO Ids belonging to the same GO ontologyis recommended when using GOSP-FIT athttp://web.cbio.uct.ac.za/ITGOM/tools/gossfeat.php: a target list ofat most 2000 protein UniProt accessions or gene names is recommended.• For GOSS-FEAT athttp://web.cbio.uct.ac.za/ITGOM/tools/gospfuct.php.• Finally, a list of no more than 200 protein UniProt accessionsor gene names is recommended for GOSP-FCT atThese cut-offs are mainly due to the limitations of the computational resourcesavailable but also to the visualization constraints and algorithm complexity, forexample when running hierarchical clustering in GOSP-FCT.http://www.ebi.ac.uk/QuickGO), and for GO term semantic similarityand information content queries, GO Ids are linked to their characteristics andtheir sub-GO graphs displayed using AmiGO athttp://amigo.geneontology.org. A given concept (protein accession orGO Id) can also be linked to more detailed results related to the concept. Moredetails on the use of the tool are provided in the help page on the website.Comprehensive summary reports generated from the DaGO-Fun tool are made availablein table format. An example of a result report is shown in Figure ℓ genes from a target gene set of size nby chance, knowing that the reference dataset, considered as a backgrounddistribution, contains m such annotated genes out of N genes is given by thefollowing formula: The DaGO-Fun tool uses a binomial test for the retrieval of genes based of their GOannotations (GOSP-FIT) and hyper-geometric test for term enrichment analysis(GOSS-FEAT), adjusted using the Bonferroni multiple testing correction. Note thatusing the hyper-geometric distribution, the p-value, which is the probability ofobserving at least p of observing the GOterm under consideration. In this case, a gene taken at random from the referencedataset is an event with two possible outcomes, namely success (1), if the gene isannotated with the GO term, and failure (0) otherwise. Thus, the probability ofobtaining at least ℓ successes in n trials or observing atleast ℓ genes annotated with the GO term under consideration amongn genes in the target set is given by the following formula: The random variable X represents the number of genes within a given target genesubset, annotated with a given GO term. Note that we are dealing with very largepopulation size , in which case the size of the target gene or protein subset is verysmall compared to the population size. Thus, the p-value can also be approximated byor modeled using the binomial distribution  by takinIn these cases, the lower the p-value, the less likely it is that the observedfrequency of the term is due to chance, the more meaningful the term is in the targetgene set. Thus, GO terms in the dataset under consideration can be ranked based ontheir p-values using the fact that the lower the p-value, the more significant theobserved GO term is.g is in factfuzzy-frequency of this term modeled using GO similarity scoreNote that as the biological applications implemented depend on the agreement level,the frequency of occurrence of a term through a gene or protein Xannotating the gene g and t and s. We say the gene g is not annotated witht or t does not occur through the gene g ifg is fully annotated with t ort fully occurs if g is fuzzy annotated with t ort fuzzy occurs if t in a set of genes ff(t), is calculated using the following formula: t,s:s∈TgX, with S genome using different GO semantic similarity approaches andanalyzed the results obtained. MTB is an intracellular pathogen that causes tuberculosis(TB), one of the most threatening infectious diseases considering the severity of itsimpact on human populations [response tonitrosative stress (GO:0051409), cellular response to antibiotic(GO:0071236), acquisition by symbiont of nutrients from host via siderophores(GO:0052099), cellular lipid metabolic process (GO:0044255), etc. We used theseGO biological process terms as initial data or input for running different biologicalapplications in the DaGO-Fun tool at moderate agreement, unless otherwise stated.In this section we provide and discuss briefly some illustrations of biologicalapplications included in the DaGO-Fun tool, namely GO Term Similarity basedProtein-Fuzzy Identification Tool (GOSP-FIT), GO based Similarity Protein-FuzzyClassification Tool (GOSP-FCT) and GO Semantic Similarity based-Fuzzy EnrichmentAnalysis Tool (GOSS-FEAT). We ran these applications on the ulations . To be sUsing the biological process terms listed above, we ran GOSP-FIT to identify proteinsinvolved in a process similar to the input processes, using the GO-universal metric,Wang et al., Zhang et al, Resnik, Lin and Lin with Li et al. enhancement similaritymeasures. Results are shown in Table Before running other applications, we first identified in the MTB genome all genes orproteins involved in the GO annotations under consideration. A total of 23 proteinshave been identified with 18 proteins  for GO:00051409, one protein (P65720) for GO:0071236, 2 for GO:0044255, and 2  for GO:0052099. We used theseproteins as input data for running GOSP-FCT using hierarchical clustering under thecustomized agreement level. Results are depicted in Figure et al and Resnik approaches and results are shown in Table response tonitrosative stress GO:0051409. This application suggests that theGO-universal approach may constitute an effective solution to the GO metric problemfor the next generation of functional similarity metrics [Finally, we ran GOSS-FEAT, taking as the target set a list of 18 proteins annotatedto GO:0051409 in order to identify the most statistically relevant biologicalprocesses in which these proteins are involved. We used the GO-universal metric, Wang metrics .As mentioned previously, there have been numerous tools developed for producing GOterm and protein semantic similarity scores. These include web interfaces andsoftware tools very often implemented in the R programming language. These tools,together with functional similarity measures they support, are shown in Table In terms of input size, the G-SESAME and FuSSiMeg web tools accept only one pair ofGO terms or proteins. The ProteInOn tool may take up to 1000 GO terms or proteinsaccording to its authors, for which the tool outputs all pairs of similarity scores,and the FunSimMat tool has unlimited input size. We aim to let the DaGO-Fun toolcalculate results for as many user inputs as possible, however, because oflimitations in computational resources, we have to balance the maximum number of GOterms, and GO term and protein pairs for each user query. Thus, the DaGO-Fun toolaccepts up to 5000 GO terms when retrieving GO term IC scores, in which case the toolwill display only 10 of them per page, but all GO term features can be retrieved bydownloading them in a text file. For GO term semantic similarity scores as well asfor protein functional similarity scores, the user can enter at most 3000 pairs.Entries beyond the maximum limitations will be ignored. Unfortunately if you havecases where your data exceeds these limitations, it is necessary to divide the inputdata, run the DaGO-Fun tool separately, and merge the results at the end of theprocess. Alternatively you can contact the authors who are willing to collaborate andrun large data sets for analysis.We have developed the DaGO-Fun tool, a customized web-based GO semantic similarityresource. This userfriendly online interface produces GO term information content (IC),GO term semantic similarity and protein functional similarity scores, which may assistexperimental and computational biologists in several applications involving proteinanalyses at the functional level. These include gene list enrichment, protein functionprediction and comparison, clustering genes or proteins based on their GO annotationinformation, and ranking disease candidate proteins or identification of novel diseasecandidate proteins. This tool will be updated quarterly (every three months) using anautomated scheme in order to remain up to date to meet requirements of ever increasingapplications in the biomedical field. The DaGO-Fun tool is freely available, meaningthat one is free to copy, distribute, display and make unrestricted non-commercial useof it under the GNU General Public Licence provided that it is done with appropriatecitation of the tool and its components.Despite the wide range of IC-based GO semantic similarity applications and the existenceof several approaches to meet requirements of these applications, there was no toolavailable that integrates all these IC-based approaches. Thus, researchers had toimplement these approaches themselves, use different tools for different approaches, ordownload the individual software packages, making extraction and comparison of thesescores difficult and time-consuming. The DaGO-Fun tool overcomes these issues, providingeasy retrieval of IC-based GO term semantic similarity and protein functional similarityscores within a large protein annotation dataset from GOA-UniProtKB. It ensures that GOsemantic similarity data are conveniently accessible to researchers and can effectivelybe used to investigate functional similarity between proteins based on their GOannotations. In addition, we implemented some biological applications of these semanticsimilarity measures, including protein classification and identification based on theirGO annotations, and term enrichment analysis.Future work includes facilitating the search for functional similarity between sets ofGO terms. In this case, the user will have to provide pairs of sets of GO terms using aspecified key linking the sets. This will undoubtedly improve the flexibility of theDaGO-Fun tool, by allowing users to produce functional similarity scores for their ownpredicted set of genes given their GO annotations. We will assess the relevance of twoIC-term based functional similarity approaches introduced here, namely SimDIC and SimUICand evaluate the use of annotation-based functional similarity approaches in the contextof the GO term IC topology-based family. Finally, we will be expanding the DaGO-Fun toolto include some other applications of GO semantic similarity in protein analyses, suchas protein function prediction, annotation system comparisons, and disease proteinprioritization.http://web.cbio.uct.ac.za/ITGOM, accessible by anyuser with a standard web browser but has only been tested on Mozilla Firefox 20.0. Thewhole system is implemented using a LAMP (Linux-Apache-MySQL and PHP/Python) platform.This means that the DaGO-Fun tool is implemented under free software  using a Linux Apache server with a database structured in a relational modelusing MySQL version 14.14 Distrib 5.5.31, and the web interface is implemented in PHPversion 5.3.10 and standard HTML. The back-end is composed of a set of query processingprograms implemented in Python version 2.7.3.DaGO-Fun is available at The authors declare that they have no competing interests.NJM generated and supervised the project, and finalized the manuscript. GKM designed andimplemented the tool, and wrote the manuscript. Both authors read and approved the finalmanuscript. NJM approved the production of this paper.