Two-Dimensional EspEn: A New Approach to Analyze Image Texture by Irregularity

Image processing has played a relevant role in various industries, where the main challenge is to extract specific features from images. Specifically, texture characterizes the phenomenon of the occurrence of a pattern along the spatial distribution, taking into account the intensities of the pixels for which it has been applied in classification and segmentation tasks. Therefore, several feature extraction methods have been proposed in recent decades, but few of them rely on entropy, which is a measure of uncertainty. Moreover, entropy algorithms have been little explored in bidimensional data. Nevertheless, there is a growing interest in developing algorithms to solve current limits, since Shannon Entropy does not consider spatial information, and SampEn2D generates unreliable values in small sizes. We introduce a proposed algorithm, EspEn (Espinosa Entropy), to measure the irregularity present in two-dimensional data, where the calculation requires setting the parameters as follows: m (length of square window), r (tolerance threshold), and ρ (percentage of similarity). Three experiments were performed; the first two were on simulated images contaminated with different noise levels. The last experiment was with grayscale images from the Normalized Brodatz Texture database (NBT). First, we compared the performance of EspEn against the entropy of Shannon and SampEn2D. Second, we evaluated the dependence of EspEn on variations of the values of the parameters m, r, and ρ. Third, we evaluated the EspEn algorithm on NBT images. The results revealed that EspEn could discriminate images with different size and degrees of noise. Finally, EspEn provides an alternative algorithm to quantify the irregularity in 2D data; the recommended parameters for better performance are m = 3, r = 20, and ρ = 0.7

Development of therapeutic glycopolymers for the treatment of peripheral neuropathies and viral infections

Immune mediated neuropathies are a group of heterogenous disorders affecting the peripheral nervous system. Characteristic for these diseases is the involvement of an immune response against autoantigens on axonal membranes or surrounding myelin. In a variety of peripheral neuropathies autoantibodies target glycan or peptide epitopes in the nodal and paranodal region. These polyneuropathies can have chronic or acute manifestations but generally respond to immunotherapies. Most treatment options however target the immune system unspecifically and can cause serious adverse effects. In some cases, patients do not respond to treatment or even show deterioration. In anti-MAG neuropathy and multifocal motor neuropathy the antigen-specific pathogenic autoantibodies have been well described and therapeutic intervention is aimed at reducing antibody levels or interfering with its effector functions. A recently developed glycopolymer-based therapeutic approach for the treatment of anti-MAG neuropathy (PPSGG) specifically targets these disease-causing autoantibodies by presenting multiple carbohydrate mimetic copies of its antigen on a biodegradable poly-L-lysine (PLL) scaffold. In this thesis we review the importance of a reduction of autoantibody levels for clinical improvement in anti-MAG neuropathy, discuss the mechanism of action of PPSGG, evaluate its safety profile, and develop new glycopolymers to treat related peripheral neuropathies and viral infections. Despite clinical evidence for the pathogenicity of anti-MAG IgM autoantibodies, the significance of antibody titers as a predictive factor for response to therapy remains controversial. Current literature does not provide conclusive evidence on the association between reduced anti-MAG IgM titers and clinical improvement of neuropathic symptoms. We performed a retrospective study to test our hypothesis that changes in antibody titers are correlated with clinical response in anti-MAG neuropathy patients. We included 50 studies involving 410 anti-MAG neuropathy patients undergoing immunotherapy in our analysis and characterized relative change in anti-MAG IgM titers, paraprotein levels, and total IgM prior, during, or post-treatment. Patients were categorized according to their response to treatment into “responders”, “non-responders”, or “acute deteriorating” and the studies were qualified as “supportive” or “not supportive”. 40 studies supported the hypothesis that “responders” showed relative reduction of anti-MAG IgM titers and “non-responders” did not show significant change in antibody titers, confirming our hypothesis. In an experimental study we further investigated the pharmacodynamic properties of PPSGG and tested its inhibitory potential on peripheral nerves. The polymer selectively prevented binding of anti-MAG IgM from patient sera to non-human primate sciatic nerves. In an immunological mouse model, PPSGG showed superiority in removing anti-MAG IgM antibodies compared to B-cell depletion with an anti-CD20 antibody (analogous to Rituximab). Safety evaluation with human and murine peripheral blood mononuclear cells (PBMC) showed no interaction. Furthermore, no increase in systemic inflammatory markers was observed in mice or in human PBMC ex vivo after treatment. We investigated the binding characteristics of PPSGG and anti-MAG IgM as well as pharmacokinetic properties to understand the mode of action of the glycopolymer. First, physicochemical and morphological characteristics of the polymer were investigated. The linear rod-shaped PPSGG showed an approximate length of 100 nm, a hydrodynamic radius of around 60 nm, and a highly negative charge ( 46 mV ). Because of its high negative charge density, it was readily taken up by liver-and spleen-resident macrophages through scavenger receptors, explaining the previously observed short half-life in mice (approx. 17 min). No large aggregate formation in vitro and no immune complex deposition in murine liver, spleen, kidney, or brain was observed. Despite the extensive uptake in Kupffer cells in the liver, it did not exhibit hepatotoxic effects in a human hepatic tissue ex vivo. In the presence of anti-MAG IgM, PPSGG preferentially formed complexes in a 1:1 or 1:2 stoichiometry in vitro, supporting previously reported dose titration experiments with an immunological mouse model. Analogous to the approach for the treatment of anti-MAG neuropathy, we developed a glycopolymer displaying GM1 carbohydrate mimetics on a PLL backbone to treat anti-GM1 mediated peripheral neuropathies. A series of ten mimetics was synthesized and conjugated to the PLL backbone for testing with MMN patient sera. The specificity and inhibitory potential of the glycopolymers was assessed by competitive ELSIA. After screening of 22 MMN patient samples we identified three interesting candidates for further evaluation in functional assays. One of most active glycopolymers was carrying the natural GM1 carbohydrate epitope and was excluded for further selection, because the aim of the project was to develop simplified glycomimetcs that retain or increase binding affinity while simultaneously reducing synthetic complexity. One of the two remaining candidates showed high temperature sensitivity and could not inhibit anti-GM1 antibody binding to GM1 at physiological temperatures. The remaining glycopolymer, composed of the natural GM1 core epitope with only a replacement of the terminal glucose by a tyramine moiety, prevented anti-GM1 antibody binding to terminal axonal networks in vitro and ex vivo using an animal model for acute motor axonal neuropathy. Being confronted with the surge of the COVID-19 pandemic and the rapid progress in scientific discoveries and understanding of SARS-CoV-2, we applied our expertise in glycoplymer development to tackle SARS-CoV-2 infections. Increasing evidence was pointing at the involvement of DC-SIGN in the pathogenesis of COVID-19. Interaction of viral spike protein with DC-SIGN was reported to lead to internalization of the virus by immune cells, thus presenting an alternative entry receptor for SARS-CoV-2, independent of ACE2. Infection of immune cells is involved in exaggerated immune response in severe COVID-19. It was later reported that DC-SIGN levels were increased in severe COVID-19 patients showing elevated levels of proinflammatory macrophages, inflammatory cytokines and chemokines. Inhibition of the interaction between DC-SIGN and spike protein might serve as a strategy to prevent these severe disease courses. We demonstrated that mannose-functionalized PLL glycopolymers efficiently inhibit the attachment of spike protein to DC-SIGN presenting cells with picomolar affinity in a competitive setting. Pre-treatment of the cells lead to prolonged receptor internalization and protected the cells for up to 6 h from virus binding. Moreover, DC-SIGN acts as a receptor for multiple viruses and we could additionally demonstrate effective inhibition of HIV and Ebola viral glycoprotein biding to DC-SIGN presenting cells. This host-directed approach might not only be applicable for multiple unrelated viral infections but could be unaffected by the rapidly mutating variants of SARS-CoV-2. In a follow-up study we reported the discovery of a new class of potent glycomometic DC SIGN ligands from a library of triazole-based mannose analogs. Structure-based optimization yielded a glycomimetic ligand with over 100-fold improved binding affinity compared to methyl α D mannopyranoside. Multivalent display of the ligand on PLL was able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN expressing cells, as well as DC-SIGN mediated trans-infection of ACE2 expressing cells by SARS-CoV-2 spike protein expressing viruses in nanomolar concentrations

2015 IMSAloquium, Student Investigation Showcase

We want to express our gratitude for the generosity and steadfast support of all the experts and leaders who have nurtured These collaborative partnerships are the strength of our SIR program.https://digitalcommons.imsa.edu/archives_sir/1014/thumbnail.jp

06. 2015 IMSAloquium Student Investigation Showcase

https://digitalcommons.imsa.edu/class_of_2015/1004/thumbnail.jp

Applications of EMG in Clinical and Sports Medicine

This second of two volumes on EMG (Electromyography) covers a wide range of clinical applications, as a complement to the methods discussed in volume 1. Topics range from gait and vibration analysis, through posture and falls prevention, to biofeedback in the treatment of neurologic swallowing impairment. The volume includes sections on back care, sports and performance medicine, gynecology/urology and orofacial function. Authors describe the procedures for their experimental studies with detailed and clear illustrations and references to the literature. The limitations of SEMG measures and methods for careful analysis are discussed. This broad compilation of articles discussing the use of EMG in both clinical and research applications demonstrates the utility of the method as a tool in a wide variety of disciplines and clinical fields

Trauma, Tumors, Spine, Functional Neurosurgery

This book is written for graduate students, researchers, and practitioners who are interested in learning how the knowledge from research can be implemented in clinical competences. The first section is dedicated to deep brain stimulation, a surgical procedure which is the paramount example of how clinical practice can take advantage from fundamental research. The second section gathers four chapters on four different topics and illustrates how significant is the challenge to translate scientific advances into clinical practice because the route from evidence to action is not always obvious. It is hoped that this book will stimulate the interest in the process of translating research into practice for a broader range of neurosurgical topics than the one covered by this book, which could result in a forthcoming more comprehensive publication

Transthyretin familial amyloid polyneuropathy: novel therapeutics derived from drug repurposing and new insights in diagnosis through proteomic analysis of clinical samples

La transtirretina (TTR) és una proteïna tetramèrica amiloidogènica (55 kDa) present al plasma humà i responsable del transport de la hormona T4 i del retinol a través de la proteïna d’unió a retinol (RBP). La proteïna TTR està associada amb diverses amiloïdosis, concretament la polineuropatia amiloide familiar (FAP), la cardiomiopatia amiloide familiar (FAC) i l’amiloïdosi senil sistèmica (SSA). La variabilitat associada a la TTR es deu tant a mutacions puntuals al gen codificant per aquesta com a modificacions post-traduccionals (PTMs) al residu Cys-10. Les PTMs més comuns associades a la Cys-10 de la TTR són la S-Sulfonació (S-Sulfo), la S-Glicinilcisteinilació (S-CysGly), la S-Cisteinilació (S-Cys) i la S-Glutationilació (S-GSH). Es creu que dites PTMs associades a la Cys-10 podrien jugar un paper biològic important en l’inici i procés patològic de les diferents amiloïdosis lligades a TTR. Hem tractat les amiloïdosis lligades a TTR des de dues perspectives diferents i) Intervencions terapèutiques i ii) Diagnòstic i monitorització de FAP. Referent a la primera part del projecte, hem portat a terme el cribratge de 41 possibles inhibidors de fibril·logènesis seleccionats mitjançant estratègies bioinformàtiques de repurposing de fàrmacs. Com a resultat de l’estudi, s’han trobat 4 nous estabilitzadors del tetràmer de TTR i, per tant, nous candidats pel tractament d’amiloïdosis lligades a TTR. Pel que fa a l’aproximació diagnòstica d’aquest treball, hem desenvolupat una metodologia per a la quantificació de PTMs en mostres de sèrum, així com per a la determinació dels nivells de TTR en aquest, tant en individus sans (wt) com en individus portadors de TTR amiloïdogènica (mutació V30M). Dita metodologia consisteix en una primera etapa d’enriquiment en TTR mitjançant immunoprecipitació, seguit de l’anàlisi de la TTR per espectrometria de masses de i) la proteïna intacta i ii) els pèptids de TTR portadors de les PTMs d’interès mitjançant l’anàlisi dirigit per LC-MS. L’anàlisi de les mostres de sèrum per la combinació d’ambdues estratègies aporta informació sobre la quantificació relativa i absoluta de les diferents PTMs presents a la TTR. Ha sigut possible mostrar que els mètodes basats en proteïna intacta es troben esbiaixats per algunes de les PTMs, donat que assumeixen un factor de resposta constant per les diferents isoformes. Contràriament, el nou mètode de LC-MS dirigit permet la quantificació absoluta de les diferents PTMs i els nivells totals de TTR (wt i mutant). La metodologia reportada ha sigut aplicada en l’anàlisi de dos grups de mostres clíniques. Com a resultat de l’estudi de mostres humanes de pacients de FAP en els diferents estadis de la malaltia, suggerim de forma preliminar les isoformes S-GSH i S-CysGly com a biomarcadors de progressió de la malaltia, permetent la diferenciació entre pacients en estadi 0 i 1 i, per tant, indicant l’aparició de la malaltia. Mitjançant l’anàlisi de mostres de pacients de FAP a diferents temps després de sotmetre’s a un transplantament de fetge (LT) i de pacients receptors de transplantament de fetge dominó provinent d’individus portadors de la mutació V30M, hem caracteritzat la progressió de la relació wt:V30M, així com l’evolució dels nivells de PTMs a la Cys-10, des de la intervenció fins a 9 anys després. Addicionalment, hem observat diferències significatives en els nivells de S-GSH i S-CysGly en comparar pacients de LT i DLT, resultats anàlegs als obtinguts en la comparació d’individus wt (sans) i pacients de FAP en estadi 0.La transtirretina (TTR) es una proteína tetramérica amiloidogénica (55 kDa) presente en el plasma humano y la responsable del transporte de la hormona T4 y el retinol, a través de la proteína de unión al retinol (RBP). La proteína TTR está asociada con varias amiloidosis, concretamente la polineuropatía amiloide familiar (FAP), la cardiomiopatía amiloide familiar (FAC) y la amiloidosis senil sistémica (SSA). La variabilidad encontrada en la TTR se debe tanto a mutaciones puntuales encontradas en el gen que codifica para ésta como a modificaciones post-traduccionales (PTMs) en el residuo Cys-10. Las PTMs más comunes asociadas a la Cys-10 de la TTR son la S-Sulfonación (S-Sulfo), la S-Glicinilcisteinilación (S-CysGly), la S-Cisteinilación (S-Cys) y la S-Glutationilación (S-GSH). Se cree que dichas PTMs asociadas a la Cys-10 podrían jugar un papel biológico importante en el inicio y proceso patológico de las distintas amiloidosis ligadas a TTR. Hemos abordado las amiloidosis ligadas a TTR desde dos perspectivas distintas i) Intervenciones terapéuticas y ii) Diagnóstico y monitorización de FAP. Respecto a la primera parte del proyecto, hemos llevado a cabo el cribado de 41 posibles inhibidores de fibrilogenesis seleccionados mediante estrategias bioinformáticas de repurposing de fármacos. De este modo, se han encontrado 4 nuevos estabilizadores del tetrámero de TTR y por tanto, nuevos candidatos para el tratamiento de amiloidosis ligadas a TTR. En relación a la aproximación diagnóstica de este trabajo, hemos desarrollado una metodología para la cuantificación de PTMs en muestras de suero, así como para la determinación de los niveles de TTR en éste, tanto en individuos sanos (wt) como en individuos portadores de TTR amiloidogénica (mutación V30M). Dicha metodología consiste en una primera etapa de enriquecimiento en TTR mediante immunoprecipitación, seguido por el análisis de ésta mediante espectrometría de masas de i) la proteína TTR intacta y ii) de los péptidos de TTR portadores de las PTMs de interés mediante análisis dirigido por LC-MS. El análisis de muestras de suero mediante la combinación de ambas estrategias aporta información sobre la cuantificación relativa y absoluta de las distintas PTMs en TTR. Ha sido posible mostrar que los métodos basados en proteína intacta se encuentran sesgados para algunas de las PTMs, dado que asumen un factor de respuesta constante para las distintas isoformas. Por el contrario, el nuevo método de LC-MS dirigido permite la cuantificación absoluta de las distintas PTMs y los niveles totales de TTR (wt y mutante). La metodología reportada ha sido aplicada en el análisis de dos grupos de muestras clínicas. Como resultado del estudio de muestras humanas de pacientes de FAP en los distintos estadios de la enfermedad, sugerimos de forma preliminar las isoformas S-GSH y S-CysGly como biomarcadores de progresión de la enfermedad, permitiendo la diferenciación entre pacientes en estadio 0 y 1 y, por lo tanto, indicando la aparición de la enfermedad. Mediante el análisis de muestras de pacientes de FAP a distintos tiempos después de someterse a un trasplante de hígado (LT) y de pacientes receptores de trasplante de hígado dominó proveniente de individuos portadores de la mutación V30M, hemos caracterizado la progresión del ratio wt:V30M así como la evolución de los niveles de PTMs en la Cys-10, des de la intervención hasta 9 años después. Adicionalmente, hemos observado diferencias significativas en los niveles de S-GSH y S-CysGly en comparar pacientes de LT y DLT, resultados análogos a los obtenidos en la comparación de individuos wt (sanos) y pacientes de FAP en estadio 0.Transthyretin (TTR) is an amyloidogenic tetrameric protein (55kDa) present in human plasma, transporting T4 hormone and retinol, through the retinol binding protein (RBP). TTR is associated with several amyloidosis, namely familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Variability of TTR is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys-10, the most common PTMs being the S-Sulfonation (S-Sulfo), S-Glycinylcysteinylation (S-CysGly), S-Cysteinylation (S-Cys) and S-Glutathionylation (S-GSH). It is thought that PTMs at Cys-10 may play an important biological role in the onset and pathological process of amyloidosis related to TTR. We have aimed TTR amyloidosis from two different perspectives i) Therapeutic interventions and ii) FAP diagnosis and monitoring. Regarding the first branch of the project, we have performed the screening of a library of 41 possible fibrillogenesis inhibitors selected by a bioinformatic repurposing workflow, finding 4 new TTR tetramer stabilizers and thus, new potential candidates for TTR amyloidosis treatment. Concerning the clinical approach of this work, we have developed a methodology for quantification of PTMs in serum samples, as well as for the determination of serum TTR levels, from healthy (wt) and TTR-amyloidotic (V30M mutation) individuals. It involves an enrichment step by immunoprecipitation followed by mass spectrometry analysis of (i) the intact TTR protein and (ii) targeted LC-MS analysis of peptides carrying the PTMs of interest. Analysis of serum samples by the combination of the two methods affords complementary information on the relative and absolute amounts of the selected TTR PTM forms. It is shown that methods based on intact protein are biased for specific PTMs since they assume constant response factors, whereas the novel targeted LC-MS method provides absolute quantification of PTMs and total TTR variants. The reported methodology has been applied to two different sets of clinical samples. As a result of the study of human samples of FAP patients at different disease stages, we preliminary pointed out S-GSH and S-CysGly isoforms as biomarkers of disease progression, allowing the differentiation between FAP stage 0 and 1 and therefore indicating disease onset. Through the analysis of a time series from FAP patients having undergone liver transplantation (LT) and from domino liver transplantation (DLT) recipients from V30M carriers, we have characterized the progression of the wt:V30M ratios, as well as the evolution of the Cys-10 PTMs, from transplantation and up to 9 years after. Additionally, we have observed significant differences in the levels of S-GSH and S-CysGly when comparing liver and domino liver transplanted patients, analogous to the results obtained in the comparison of wt individuals and FAP stage 0 patients