Alterations in the Structure, Composition, and Organization of Galactosaminoglycan-Containing Proteoglycans and Collagen Correspond to the Progressive Stages of Dupuytren's Disease.

Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.

The glycosaminoglycan-binding chemokine fragment CXCL9(74-103) reduces inflammation and tissue damage in mouse models of coronavirus infection.

Introduction: Pulmonary diseases represent a significant burden to patients and the healthcare system and are one of the leading causes of mortality worldwide. Particularly, the COVID-19 pandemic has had a profound global impact, affecting public health, economies, and daily life. While the peak of the crisis has subsided, the global number of reported COVID-19 cases remains significantly high, according to medical agencies around the world. Furthermore, despite the success of vaccines in reducing the number of deaths caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a gap in the treatment of the disease, especially in addressing uncontrolled inflammation. The massive recruitment of leukocytes to lung tissue and alveoli is a hallmark factor in COVID-19, being essential for effectively responding to the pulmonary insult but also linked to inflammation and lung damage. In this context, mice models are a crucial tool, offering valuable insights into both the pathogenesis of the disease and potential therapeutic approaches. Methods: Here, we investigated the anti-inflammatory effect of the glycosaminoglycan (GAG)-binding chemokine fragment CXCL9(74-103), a molecule that potentially decreases neutrophil transmigration by competing with chemokines for GAG-binding sites, in two models of pneumonia caused by coronavirus infection. Results: In a murine model of betacoronavirus MHV-3 infection, the treatment with CXCL9(74-103) decreased the accumulation of total leukocytes, mainly neutrophils, to the alveolar space and improved several parameters of lung dysfunction 3 days after infection. Additionally, this treatment also reduced the lung damage. In the SARS-CoV-2 model in K18-hACE2-mice, CXCL9(74-103) significantly improved the clinical manifestations of the disease, reducing pulmonary damage and decreasing viral titers in the lungs. Discussion: These findings indicate that CXCL9(74-103) resulted in highly favorable outcomes in controlling pneumonia caused by coronavirus, as it effectively diminishes the clinical consequences of the infections and reduces both local and systemic inflammation.

Biomarkers of endothelial glycocalyx damage are associated with microvascular dysfunction in resuscitated septic shock patients.

BACKGROUND: Microvascular dysfunction plays a central role in organ dysfunction during septic shock. Endothelial glycocalyx (eGC) damage could contribute to impaired microcirculation. The aim was to assess whether several eGC-damaged biomarkers are associated with microvascular dysfunction in resuscitated septic shock patients. METHODS: This cross-sectional study included resuscitated septic shock patients (N = 31), and a group of healthy individuals (N = 20). The eGC damage biomarkers measured were syndecan-1 (SDC-1), soluble CD44 (CD44s), hyaluronic acid (HYAL) in blood sample; sulfated glycosaminoglycans (GAGs) in urine sample; and thrombomodulin (TBML) in blood sample as biomarker of endothelial cell damage. Microcirculation was assessed through sublingual videocapillaroscopy using the GlycoCheck™, which estimated the perfused vascular density (PVD); the perfused boundary region (PBR), an inverse parameter of the eGC thickness; and the microvascular health score (MVHS). We defined a low MVHS (<50th percentile in septic patients) as a surrogate for more impaired microvascular function. RESULTS: The SDC-1, CD44s, TBML and GAGs levels were correlated with impaired microvascular parameters (PVD of vessels with diameter < 10 µm, MVHS and flow-adjusted PBR); p < 0.05 for all comparisons, except for GAGs and flow-adjusted PBR. The SDC-1 [78 ng/mL (interquartile range (IQR) 45-336) vs. 48 ng/mL (IQR 9-85); p = 0.052], CD44s [796ρg/mL (IQR 512-1995) vs. 526ρg/mL (IQR 287-750); p = 0.036], TBML [734ρg/mL (IQR 237-2396) vs. 95ρg/mL (IQR 63-475); p = 0.012] and GAGs levels [0.42 ρg/mg (IQR 0.04-1.40) vs. 0.07 ρg/mg (IQR 0.02-0.20); p = 0.024]; were higher in septic patients with more impaired sublingual microvascular function (low MVHS vs. high MVHS). CONCLUSION: SDC-1, CD44s, TBML and GAGs levels were associated with impaired microvascular function in resuscitated septic shock patients.

Cycle of the sexual segment of the kidney: histological insights into the role of the urinary tract in the reproduction of male Notomabuya frenata (Squamata: Scincidae).

The kidneys of male Squamata have an important reproductive function as some portions of the nephron may undergo hypertrophy, characterizing the sexual segment of the kidney (SSK). Although its function is still not completely understood, it is believed that the secretions produced by the SSK may act in the maintenance of spermatozoa. In this study, we investigated the reproductive biology of males of Notomabuya frenata based on the seasonal variation of the SSK. We performed macroscopic and microscopic evaluations of the male reproductive tract of museum specimens to characterize the SSK cycle. The nephron portion in which hypertrophy was observed was the collecting duct with secretory granules accumulation in the apical portion. SSK hypertrophy was observed in all seasons, with the tubule diameter in autumn differing from spring and the epithelium height showing no variation. Alcian Blue reacted positively to acid mucopolysaccharides in all seasons. Periodic acid-Schiff's reacted positively to neutral mucopolysaccharides in all seasons, except autumn. Both stains reacted only in the collecting duct. In addition, spermatozoa were found in the lumen of the SSK of one specimen examined. Cycle of the SSK varied seasonally as does the chemical composition of the secretions produced by the collecting duct. The reflux of spermatozoa into SSK may indicate that (1) these secretions act in sperm maintenance, and (2) possibly there is communication between the seminal and urinary ducts.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Alterations of Plasmatic Biomarkers of Neurodegeneration in Mucopolysaccharidosis Type II Patients Under Enzyme Replacement Therapy.

Mucopolysaccharidosis type II (MPS II) is a disorder caused by a deficient activity of iduronate-2-sulfatase, a lysosomal enzyme responsible for degrading glycosaminoglycans (GAGs). The abnormal storage of GAGs within lysosomes disrupts cellular homeostasis and leads to a severe symptomatology. Patients present neuropsychiatric impairment characterized by mental retardation and impaired cognition. The aim of this study was to quantify four neurodegeneration biomarkers in plasma: brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF-AA), neural cell adhesion molecule (NCAM) and cathepsin-D, as well as to identify possible correlations with urinary GAGs in seven patients undergoing treatment with ERT (Elaprase® 0.5 mg/kg of body weight). Patients with both severe and attenuated forms of MPS II showed signs of neurodegeneration in neuroimaging exams. Patients have a decrease in BDNF and PDGF-AA concentrations, and an increase in NCAM level compared to controls. No alterations in cathepsin-D concentration were seen. GAGs levels were higher in patients than in controls, but no significant correlations between GAGs and biomarkers were observed. These results evidence that patients have neurodegeneration and that monitoring these biomarkers might be useful for assessing this process. To this date, this is the first work to analyze these plasmatic markers of neurodegeneration in patients.

Effect of glycosaminoglycans on the structure and composition of articular cartilage and bone of broilers.

This study aimed to assess the influence of glycosaminoglycan (chondroitin and glucosamine sulfates) supplementation in the diet of broilers on the expression of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 2 (TIMP-2) genes, the synthesis of proteoglycans, collagen type II and chondrocytes, bone and cartilage macroscopy, bone mineral densitometry, bone breaking strength and mineral profile. A completely randomized design was carried out in a 3 × 3 factorial scheme (3 levels of chondroitin sulfate: 0.00, 0.05, and 0.10%; and 3 levels of glucosamine sulfate: 0.00, 0.15, and 0.30%), totaling 9 treatments. At 21 and 42 d of age, broilers were slaughtered, and tibias and femurs were collected for evaluation. There was an interaction (P < 0.05) of sulfates for the expression of MMP-9 and its inhibitor TIMP-2 in femur articular cartilage, as well as for the number of chondrocytes, collagen type II and proteoglycans in tibia articular cartilage, bone and cartilage macroscopy and mineral profile (P < 0.05), with better results obtained with the inclusion of chondroitin and/or glucosamine sulfates in the feed. In conclusion, chondroitin and glucosamine sulfates can be used in broiler diets in order to favor the development of the structure of the locomotor system (bones and joints), thus preventing locomotion problems.

Chondrotoxic effects of tranexamic acid and povidone-iodine on the articular cartilage of rabbits.

PURPOSE: To evaluate the chondrotoxic effects of intra-articular use of TXA 20 mg/kg and/or 0.35% PVPI on knee joint cartilage in an experimental model of rabbits. METHODS: Forty-four male New Zealand adult rabbits were randomly assigned to four groups (control, tranexamic acid (TXA), povidone-iodine (PVPI), and PVPI + TXA). The knee joint cartilage was accessed through an arthrotomy and exposed to physiological saline SF 0.9% (control group), TXA, PVPI, and PVPI followed by TXA. Sixty days after surgical procedure, the animals were sacrificed and osteochondral specimens of the distal femur were obtained. Histological sections of cartilage from this area were stained with hematoxylin/eosin and toluidine blue. The following cartilage parameters were evaluated by the Mankin histological/histochemical grading system: structure, cellularity, glycosaminoglycan content in the extracellular matrix, and integrity of the tidemark. RESULTS: The isolated use of PVPI causes statistically significant changes in cartilage cellularity (p-value = 0.005) and decrease glycosaminoglycan content (p = 0.001), whereas the isolated use of TXA decreased significantly the glycosaminoglycan content (p = 0.031). The sequential use of PVPI + TXA causes more pronounced alterations in the structure (p = 0.039) and cellularity (p = 0.002) and decreased content of glycosaminoglycans (p < 0.001) all with statistical significance. CONCLUSION: Data suggest that intra-articular use of tranexamic acid 20 mg/kg and intraoperative lavage with 0.35% povidone-iodine solution for three min are toxic to the articular cartilage of the knee in an experimental in vivo study in rabbits.

Untargeted LC-HRMS metabolomics reveals candidate biomarkers for mucopolysaccharidoses.

BACKGROUND: Mucopolysaccharidoses (MPSs) are inherited genetic diseases caused by an absence or deficiency of lysosomal enzymes responsible for catabolizing glycosaminoglycans (GAGs). Undiagnosed patients, or those without adequate treatment in early life, can be severely and irreversibly affected by the disease. In this study, we applied liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics to identify potential biomarkers for MPS disorders to better understand how MPS may affect the metabolome of patients. METHODS: Urine samples from 37 MPS patients (types I, II, III, IV, and VI; untreated and treated with enzyme replacement therapy (ERT)) and 38 controls were analyzed by LC-HRMS. Data were processed by an untargeted metabolomics workflow and submitted to multivariate statistical analyses to reveal significant differences between the MPS and control groups. RESULTS: A total of 12 increased metabolites common to all MPS types were identified. Dipeptides, amino acids and derivatives were increased in the MPS group compared to controls. N-acetylgalactosamines 4- or 6-sulfate, important constituents of GAGs, were also elevated in MPS patients, most prominently in those with MPS VI. Notably, treated patients exhibited lower levels of the aforementioned acylaminosugars than untreated patients in all MPS types. CONCLUSIONS: Untargeted metabolomics has enabled the detection of metabolites that could improve our understanding of MPS physiopathology. These potential biomarkers can be utilized in screening methods to support diagnosis and ERT monitoring.

Hematology and serum biochemistry of broilers fed diets supplemented with chondroitin and glucosamine sulfates

The objective of this study was to evaluate the hematology and serum biochemistry of broilers fed diets supplemented with chondroitin and glucosamine sulfates. An experiment was laid out in a completely randomized design with a 3 × 3 factorial arrangement (three levels of chondroitin sulfate: 0, 0.05 and 0.10%; and three levels of glucosamine sulfate: 0, 0.15, and 0.30%), with each treatment involving six replicates of 30 birds. Hematology (red blood cells, hemoglobin, hematocrit, total plasma protein [TPP], thrombocytes, white blood cells, eosinophils, monocytes, heterophils, and lymphocytes) and serum biochemistry (totalinteraction between sulfates influenced (p < 0.05) total calcium by 21 days, ionic calcium by 21 and 42 days, and phosphorus, chlorides, and sodium by 42 days. Supplementation with chondroitin and glucosamine sulfates in the broilers' diet favored their immune system and mineral metabolism, increasing serum concentrations of calcium, phosphorus, and sodium. serum protein [TSP], albumin, globulins, aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT], alkaline phosphatase [AP], total calcium, ionic calcium, phosphorus, sodium, potassium, and chlorides) variables were evaluated at 21 and 42 days. Data were subjected to analysis of variance. When the means differed significantly by the F-test, orthogonal analysis was performed to test the linear and quadratic effects of chondroitin and glucosamine sulfate levels. Glucosamine sulfate reduced the number of monocytes linearly, by 42 days (p = 0.0399). There was an interaction effect between the sulfates on total white blood cells (p = 0.0099) and lymphocytes (p = 0.0004) by 21 days. Chickens supplemented with 0.10% chondroitin sulfate showed a linear increase in white blood cells (p = 0.0287) and lymphocytes (p = 0.0144) with the addition of glucosamine sulfate. Chondroitin sulfate supplementation increased serum albumin linearly (p = 0.0099) and TSP quadratically (p = 0.0140), by 21 days. Glucosamine sulfate induced a quadratic response (p < 0.05) in albumin by 42 days, with the lowest value found with the inclusion of 0.06%. Glucosamine sulfate reduced chlorides linearly (p = 0.0237) by 21 days and increased calcium linearly (p = 0.0012) by 42 days. The interaction between sulfates influenced (p < 0.05) total calcium by 21 days, ionic calcium by 21 and 42 days, and phosphorus, chlorides, and sodium by 42 days. Supplementation with chondroitin and glucosamine sulfates in the broilers' diet favored their immune system and mineral metabolism, increasing serum concentrations of calcium, phosphorus, and sodium.(AU)

Objetivou-se avaliar a hematologia e a bioquímica sérica de frangos de corte suplementados com sulfatos de condroitina e de glucosamina na ração. Foi conduzido um experimento em delineamento inteiramente casualizado, em esquema fatorial 3 x 3 (três níveis de sulfato de condroitina: 0; 0,05 e 0,10%; e três níveis de sulfato de glucosamina: 0; 0,15 e 0,30%), cada tratamento com seis repetições de 30 aves. Foram avaliadas as variáveis de hematologia (hemácias, hemoglobina, hematócrito, proteínas plasmáticas totais [PPT], trombócitos, leucócitos, eosinófilos, monócitos, heterofilos e linfócitos) e bioquímica sérica (proteínas séricas totais [PST], albumina, globulinas, aspartato aminotransferase [AST], gama glutamiltransferase [GGT], fosfatase alcalina [FA], cálcio total, cálcio iônico, fósforo, sódio, potássio e cloretos) aos 21 e 42 dias. Os dados foram submetidos à análise de variância. Quando as médias diferiram significativamente pelo teste F, a análise ortogonal foi realizada para testar os efeitos lineares e quadráticos dos níveis dos sulfatos de condroitina e de glucosamina. Observou-se efeito linear decrescente (p = 0,0399) do sulfato de glucosamina na quantidade de monócitos aos 42 dias. Houve interação dos sulfatos para leucócitos totais (p = 0,0099) e linfócitos (p = 0,0004) aos 21 dias. Frangos suplementados com 0,10% de sulfato de condroitina mostraram um aumento linear dos leucócitos (p = 0,0287) e dos linfócitos (p = 0,0144) com a inclusão de sulfato de glucosamina. A suplementação com sulfato de condroitina aumentou linearmente (p = 0,0099) a albumina sérica e afetou de forma quadrática (p = 0,0140) as PST aos 21 dias. O sulfato de glucosamina demonstrou um efeito quadrático (p < 0,05) sobre a albumina aos 42 dias, o menor valor foi encontrado para a inclusão de 0,06%, respectivamente. O sulfato de glucosamina reduziu linearmente (p = 0,0237) os cloretos aos 21 dias e aumentou linearmente (p = 0,0012) o cálcio total aos 42 dias. Verificouse interação (p < 0,05) dos sulfatos para cálcio total aos 21 dias, cálcio iônico aos 21 e 42 dias e para fósforo, cloretos e sódio aos 42 dias. A suplementação com os sulfatos de condroitina e de glucosamina na ração de frangos de corte favoreceram o sistema imune e o metabolismo de minerais, com aumento nas concentrações séricas de cálcio, fósforo e sódio.(AU)

Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.

Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is a lysosomal disease caused by deficient degradation of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate due to the deficiency of the enzyme iduronate-2-sulfatase. The main treatment for MPS II is the administration of the recombinant form of the enzyme, in a process known as enzyme replacement therapy (ERT). Oxidative damage can contribute to the pathophysiology of MPS II and treatment with ERT can reduce the effects of oxidative stress. For a better understanding of pathophysiology of MPS II, we evaluated biomarkers of mitochondrial dysfunction, DNA (Deoxyribonucleic acid) damage, antioxidant defenses, reactive species production and lysosomal size in IDS-deficient HEK 293 cells and investigate the in vitro effect of genistein and coenzyme Q10 (CoQ) on these biomarkers. An increase in the production of reactive species was demonstrated, as well as an increase in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Also, an increase in lysosomal volume and oxidative damage to DNA were verified. There was no evidence of a change in mitochondrial function in this cell model. In the HEK 293 (human embryonic kidney 293) knockout (KO) HP10 cell model we found that genistein at concentrations of 25 and 50 µm decreased in vitro the production of reactive species and the activity of the SOD enzyme, showing an antioxidant protective effect. Still, in these cells we verified that the coenzyme Q10 in the concentrations of 5 and 10 µm decreased in vitro the activity of the SOD enzyme and in the concentration of 10 µm decreased in vitro the DNA damage, also demonstrating antioxidant protection. In conclusion, MPS II knockout cells demonstrated oxidative stress and DNA damage and genistein, as well as coenzyme Q10, have been shown to have an important protective effect in vitro against these oxidative damages.

Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.

Efficient CRISPR/Cas9 nickase-mediated genome editing in an in vitro model of mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.

Plasma proteome responses in zebrafish following λ-carrageenan-Induced inflammation are mediated by PMN leukocytes and correlate highly with their human counterparts.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

Delivery and assessment of a CRISPR/nCas9-based genome editing system on in vitro models of mucopolysaccharidoses IVA assisted by magnetite-based nanoparticles.

Mucopolysaccharidosis IV A (MPS IVA) is a lysosomal disorder caused by mutations in the GALNS gene. Consequently, the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate accumulate in the lysosomal lumen. Although enzyme replacement therapy has shown essential advantages for the patients, several challenges remain to overcome, such as the limited impact on the bone lesion and recovery of oxidative profile. Recently, we validated a CRISPR/nCas9-based gene therapy with promising results in an in vitro MPS IVA model. In this study, we have expanded the use of this CRISPR/nCas9 system to several MPS IVA fibroblasts carrying different GALNS mutations. Considering the latent need to develop more safety vectors for gene therapy, we co-delivered the CRISPR/nCas9 system with a novel non-viral vector based on magnetoliposomes (MLPs). We found that the CRISPR/nCas9 treatment led to an increase in enzyme activity between 5 and 88% of wild-type levels, as well as a reduction in GAGs accumulation, lysosomal mass, and mitochondrial-dependent oxidative stress, in a mutation-dependent manner. Noteworthy, MLPs allowed to obtain similar results to those observed with the conventional transfection agent lipofectamine. Overall, these results confirmed the potential of CRISPR/nCas9 as a genome editing tool for treating MPS IVA. We also demonstrated the potential use of MLPs as a novel delivery system for CRISPR/nCas9-based therapies.

Initial Recognition and Attachment of the Zika Virus to Host Cells: A Molecular Dynamics and Quantum Interaction Approach.

The zika virus (ZIKV), transmitted to humans from the bites of Aedes Aegypti and Aedes Albopictus mosquitoes produces Zika fever and neurodegenerative disorders that despite affecting millions of people, most recently in Africa and the Americas, has been declared a neglected tropical disease by the World Health Organization. In this work, atomistic molecular dynamics simulations followed by rigorous analysis of the intermolecular interactions reveal crucial aspects of the initial virus⋯cell molecular recognition and attachment, events that trigger the infectious cycle. Previous experimental studies have shown that Dermatan Sulfate (DS) and Chondroitin Sulfate A (CSA), two glycosaminoglycans which are actually epimers to each other and that are structural constituents of receptors expressed in cell membranes, are the preferred anchorage sites, with a marked preference for DS. Our calculations rationalize this preference from a molecular perspective as follows: when free of the virus, DS has one sulfate group that does not participate in intramolecular strong hydrogen bonds, thus, it is readily available to interact with the envelope protein of the virus (Zika-E), then, after formation of the complexes, Zika-E⋯DS exhibits ten strong salt brides connecting the two fragments against only six salt bridges and two hydrogen bonds in Zika-E⋯CSA. Our results complement the current view of the interaction between the virus and the receptor glycosoaminoglycans revealing that the negatively charged carboxylate groups in CSA and DS are just as important as the sulfates because of the formation of equally strong salt bridges with the positively charged Arginine and Lysine aminoacids in the envelope protein of the virus.

Decellularization of porcine esophageal tissue at three diameters and the bioscaffold modification with EETs-ECM gel.

Damaged complex modular organs repair is a current clinical challenge in which one of the primary goals is to keep their biological response. An interesting case of study it is the porcine esophagus since it is a tubular muscular tissue selected as raw material for tissue engineering. The design of esophageal constructs can draw on properties of the processed homologous extracellular matrix (ECM). In this work, we report the decellularization of multilayered esophagus tissue from 1-, 21- and 45-days old piglets through the combination of reversible alkaline swelling and detergent perfusion. The bioscaffolds were characterized in terms of their residual composition and tensile mechanical properties. The biological response to esophageal submucosal derived bioscaffolds modified with ECM gel containing epoxyeicosatrienoic acids (EETs) was then evaluated. Results suggest that the composition (laminin, fibronectin, and sulphated glycosaminoglycans/sGAG) depends on the donor age: a better efficiency of the decellularization process combined with a higher retention of sGAG and fibronectin is observed in piglet esophageal scaffolds. The heterogeneity of this esophageal ECM is maintained, which implied the preservation of anisotropic tensile properties. Coating of bioscaffolds with ECM gel is suitable for carrying esophageal epithelial cells and EETs. Bioactivity of EETs-ECM gel modified esophageal submucosal bioscaffolds is observed to promote neovascularization and antiinflammatory after rabbit full-thickness esophageal defect replacement.

Cell-surface glycosaminoglycans regulate the cellular uptake of charged polystyrene nanoparticles.

Engineered nanoparticles approaching the cell body will first encounter and interact with cell-surface glycosaminoglycans (GAGs) before reaching the plasma membrane and becoming internalized. However, how surface GAGs may regulate the cellular entry of nanoparticles remains poorly understood. Herein, it is shown that the surface GAGs of Chinese hamster ovary cells perform as a charge-based barrier against the cellular internalization of anionic polystyrene nanoparticles (PS NPs). In contrast, cationic PS NPs interact favorably with the surface GAGs and thereby are efficiently internalized. Anionic PS NPs eventually reaching the plasma membrane bind to scavenger receptors and are endocytosed by clathrin-mediated and lipid raft/cholesterol-dependent mechanisms, whereas cationic PS NPs are primarily internalized via clathrin-mediated endocytosis and macropinocytosis. Upon the enzymatic shedding of surface GAGs, the uptake of anionic PS NPs increases while that of cationic PS NPs is dramatically reduced. Interestingly, the diminished uptake of cationic PS NPs is observed only when heparan sulfate, but not chondroitin sulfate, is cleaved from the cell surface. Heparan sulfate therefore serves as anchors/first receptors to facilitate the cellular entry of cationic PS NPs. These findings contribute to advance the basic science of nanoparticle endocytosis while also having important implications for the use of engineered nanocarriers as intracellular drug-delivery systems.

Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy.

OBJECTIVE: To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II. STUDY DESIGN: Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue. RESULTS: Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10 years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity in vitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers. CONCLUSIONS: Patients with the neuronopathic form and lack of IDS protein expression were most at risk to develop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity in vitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels.