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Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of "heparanome" and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin-spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.

Supplementary 1: Table 1 - Sequences of shRNA used in the study. Supplementary 2: Table 2 - Sequences of primers used in the study. Supplementary 3: mRNA level of SULFs after shRNA knockdown. Supplementary 4: mRNA expression of HS biosynthetic enzymes

AIMS To evaluate the concentration of hyaluronan acid and proliferation/cellular death in mammary gland of ovariectomized female rat after estroprogestative therapy. MATERIALS AND METHODS Forty ovariectomized female rats were divided into four groups with 10 animals/each: OG (vehicle); EG: (Estradiol, 7 days of treatment), PG (Progesterone acetate, 23 days of treatment), and EPG: (Estradiol, 7 days of treatment, and next Progesterone acetate, 23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary gland removed, then, a fragment was immersed in acetone to quantifying of the hyaluronan acid biochemical method (ELISA-Like fluorometric assay), and a fragment fixed for 24 h in 10% formaldehyde in phosphate-buffered saline (PBS) processed for immunohistochemistry method for detection of the cell marker proliferation (Ki67) and cellular marker death by DNA fragmentation the TUNEL method. RESULTS The estradiol-treatment alone (EG) or associated with progesterone (EPG) affected the concentration of hyaluronan acid, increased cell proliferation, and decreased cell death compared to OG and PG (p

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload.

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

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for β-hairpin peptides.

Last month in Egypt at the United Nations Climate Change Conference, Brazil’s president-elect Luiz Inácio Lula da Silva reaffirmed his pledge to make Brazil a global leader in addressing climate change and deforestation. However, when Lula takes the reins on 1 January, he will step into a situation that is quite different from when last he was president (2003–2010). At that time, he prioritized science and education in all government actions and guided Brazil to a prosperous social state and sustainable economy. This time, he will face a much different local and global scenario. The world is still going through an unprecedented health crisis, and like other countries, Brazil needs to establish new ways of tackling the consequential social, educational, environmental, and economic problems. This will be especially challenging given that since 2016, the country has taken the opposite direction of most governments, cutting investments in education at all levels and in science, technology, and innovation (ST&I). The question is how Lula can immediately address the serious poverty and hunger crisis in Brazil while also restoring the environment and a competitive and equitable economy to the country.

Objective: To report the stabilization of urinary glycosaminoglicans (GAG) excretion and clinical improvements in patients with mucopolysaccharidosis type I (MPS I) under an alternative dose regimen of laronidase of 1.2 mg/kg every other week. Methods: We participated in a dose-optimization trial for laronidase in MPS-I patients using four alternative regimens: 0.58 mg/kg every week, 1.2 mg/kg every two weeks, 1.2 mg/kg every week and 1.8 mg/kg every other week (EOW). After the trial ended, the patients resumed the recommended dose and regimen of 0.58 mg/kg every week. Under this regimen, some patients presented difficulties in venous access and were unable to commute weekly to the treatment center. Therefore, we used an alternative regimen that consisted of 1.2 mg/kg EOW in eight patients. A retrospective study of medical records of MPS-I patients who underwent both enzyme replacement therapy (ERT) regimens, of 0.58 mg/kg every week and 1.2 mg/kg EOW, was done. Results: Patients remained clinically stable under the alternative regimen, did not present elevation of urinary GAG nor any adverse event. Conclusions: The switch of dose regimen to 1.2 mg/kg EOW of laronidase was safe, and did not cause any clinical worsening in patients who had been previously under standard dose ERT.

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.

Background Heparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes. Methods Lymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR. Results The medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer. Conclusions The results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes.

Background: The disease caused by hepatitis C virus (HCV) is asymptomatic, silent, and progressive liver disease. In HCV-infected patients the increase in serum HA is associated with the development of hepatic fibrosis and disease progression. Methods: HCV-RNA detection was performed in all serological samples of blood donors that tested positive using HCV Ultra ELISA. Determination of hyaluronan (HA) was performed in positive HCV samples using ELISA-like fluorometric method. The HA content was compared to HCV viral load, genotype of the virus, liver fibrosis as well as ALT and GGT liver biomarkers. Results: Persistently normal ALT (&lt, 40 U/L) and GGT (&lt, 50 U/L) serum levels were detected in 75% and 69% of the HCV-Infected blood donors, respectively. Based on ROC analysis, the HA value &lt, 34.2 ng/mL is an optimal cut-off point to exclude HCV viremia (specificity = 91%, NPV = 99%). Applying HA value ≥34.2 ng/mL significant liver fibrosis (≥F2) can be estimated in 46% of the HCV-infected blood donors. HA serum level (≥34.2 ng/mL) associated with a high ALT level (&gt, 40 U/mL) can correctly identify HCV infection and probable liver fibrosis (sensitivity = 96% and specificity = 90%) in asymptomatic blood donors. Conclusions: A high level of HA (≥34.2 ng/mL) in association with ALT (≥40 U/L) in serum can provide a good clinical opportunity to detect HCV-infected asymptomatic persons that potentially require a liver biopsy confirmation and antiviral treatment to prevent the development of advanced liver fibrosis or cirrhosis.

Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65 kDa isoform and that post-translation processing forms an active 50 kDa enzyme. In the present study, we are interested in investigating whether heparinase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

Dorival Mendes Rodrigues-Junior,1,2 Maria Fernanda de Andrade Pelarin,1 Helena Bonciani Nader,1 André Luiz Vettore,3 Maria Aparecida Silva Pinhal1,4 1Department of Biochemistry, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; 2Institute of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden; 3Department of Biological Science, Universidade Federal de São Paulo (UNIFESP), Diadema, Brazil; 4Department of Biochemistry, Faculdade de Medicina do ABC, Santo André, BrazilCorrespondence: Dorival Mendes Rodrigues-JuniorLaboratório de Biologia Molecular do Câncer, UNIFESP, Rua Pedro de Toledo, 669 – 11° Andar, São Paulo, SP 04039-032, BrazilEmail dorivalmrjr@gmail.comMaria Aparecida Silva PinhalDepartment of Biochemistry, Universidade Federal de São Paulo (UNIFESP), Rua Três de Maio, 100, Vila Clementino, São Paulo, SP 04044-020, BrazilEmail maspinhal@yahoo.com.brIntroduction: Multiple myeloma (MM) remains an incurable disease, and patient survival requires a better understanding of this malignancy’s molecular aspects. Heparanase (HPSE) is highly expressed in aggressive MM cells and related to tumor growth, metastasis, and bortezomib (BTZ) resistance. Thus, targeting HPSE seems to be a promising approach for MM treatment, and because microRNAs (miRNAs) have emerged as potential regulators of HPSE expression, the use of extracellular vesicles (EVs) can allow the efficient delivery of therapeutic miRNAs.Methods: We used prediction algorithms to identify potential miRNAs that regulate negatively HPSE expression. RT-qPCR was performed to assess miRNAs and HPSE expression in MM lines (U266 and RPMI-8226). Synthetic miRNA mimics were electroporated in MM cells to understand the miRNA contribution in HPSE expression, glycosaminoglycans (GAGs) profile, cell proliferation, and cell death induced by BTZ. EVs derived from HEK293T cells were engineered with miRNAs to evaluate their therapeutic potential combined with BTZ.Results: It revealed a direct association between BTZ sensitivity, HPSE, and miR-1252-5p expressions. Moreover, overexpression of miR-1252-5p significantly reduced HPSE expression and HPSE enzymatic activity in MM cells. The higher level of miR-1252-5p was correlated with a reduction of cell viability and higher sensitivity to BTZ. Further, EVs carrying miR-1252-5p increased MM cells’ sensitivity to BTZ treatment.Conclusion: These results showed that miR-1252-5p could negatively regulate HPSE in MM, indicating the use of EVs carrying miR-1252-5p as a potential novel BTZ sensitization approach in MM cells.Keywords: bortezomib, cancer, extracellular vesicles, exosomes, heparanase, multiple myeloma, microRNA

Heparan sulfate proteoglycans (HSPGs) play important roles in cancer initiation and progression, by interacting with the signaling pathways that affect proliferation, adhesion, invasion and angiogenesis. These roles suggest the possibility of various strategies of regulation of these molecules. In this review, we demonstrated that the anticancer drugs can regulate the heparan sulfate proteoglycans activity in different ways: some act directly in core protein, and can bind to a specific type of HSPG. Others drugs interact with glycosaminoglycans chains, and others can act directly in enzymes that regulate HSPGs levels. We also demonstrated that the HSPGs drug targets can be divided into four groups: monoclonal antibodies, antitumor antibiotic, natural products, and mimetics peptide. Interestingly, many drugs demonstrated in this review are approved by FDA and is used in cancer therapy (Food and Drug Administration) like trastuzumab, panitumumab, bleomycin and bisphosphonate zoledronic acid (ASCO) or are in clinical trials like codrituzumab and genistein. This review should help researchers to understand the mechanism of action of anticancer drugs existing and also may inspire the discovery of new drugs that regulate the heparan sulfate proteoglycans activity.

Endothelial cells (ECs) are subjected to physical forces such as shear stress (SS) induced by blood flow that leads to significant changes in morphology, physiology and gene expression. The abnormal mechanical forces applied in the cardiovascular system can influence the development of conditions and diseases such as thrombosis, hypertension and atherosclerosis. This study investigated the expression of glycosaminoglycans (GAGs), proteoglycans and extracellular matrix molecules in ECs exposed to normal and altered SS. ECs were exposed to SS of 12 dyn/cm2 (artery physiological condition) and 4 dyn/cm2 (artery pathological condition). Subsequently, ECs were subjected to immunofluorescence, qPCR, GAG biosynthesis analyses and cell-based assays. SS induced changes in ECs morphology. There were other pathological consequences of altered SS, including inhibited adhesion, stimulation of migration and capillary-like tube formation, as well as increases of GAG synthesis. We observed higher expression of syndecan-4, perlecan, decorin, fibronectin and collagen III α1 and growth factors, including VEGF-A and TGFβ-1. ECs exposed to SS displayed extracellular matrix remodeling as well as expression of cell-matrix and cell-cell interaction molecules. This study contributes to the understanding of how vascular biology is affected by mechanical forces and how these molecules can be affected in cardiovascular diseases.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca

Background: Ion channels are pharmacological targets for antiarrhythmic action, and drugs currently used for this purpose are generally not specific to a site of action and may act on several channels and even trigger proarrhythmic phenomena. Trisulfate disaccharide (TD) is an heparin fragment known to act on the sodium calcium exchanger (NCX), reducing intracellular calcium in overload situations and reversing arrhytmias, but its action on other ionic currents is unknown. Objective: To evaluate by patch clamp the action of TD at different concentrations in NCX and ionic currents in situations of intracellular calcium overload. Materials and Methods: Adult rat myocytes were obtained from a sample from ventricles. Currents were measured using the whole-cell variant of the patch clamp method. Creation of voltage clamp pulses and data acquisition was controlled by a computer with pClamp software. Peak inward current amplitude was measured for ion currents. For Na/Ca exchange current a ramp voltage protocol was employed. Three different concentrations of Cai (300nM, 400nM and 600nM) were used in separate experiments. One drug concentration was applied per cell (10, 30 and 100 micromolar each). The current sensitive to 5mM nickel was taken as the Na/Ca exchange current. The effects of TD on the INa, L-type Ca, and the potassium currents, transiente outward current (Ito), inwardly rectifying potassium current (IK1), and sustained current (Isus) recorded from adult rat ventricular myocytes were also examined in the same conditions. Results: TD concentration-dependently increased the inward Na/Ca exchange current in all intracellular calcium concentration. The effects of TD on the INa, L-type Ca, and the potassium currents, Ito, IK1 and Isus was associated with less than 30% mean reduction on any current at the highest concentration of TD tested (100 micromolar) and still below the positive block controls for different channels that is above 40% block. Conclusion: TD acts on NCX under different concentrations used, without affecting other ionic currents, suggesting specificity in the mechanism of action and possibly not exerting a pro-arrhythmic activity, this effect being desirable for its possible use in reversal of cardiac arrhythmias.

The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 μg.ml−1, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control or influence crucial biological processes. Attempts to describe its structure-function relationships with HS binding proteins in a classical ‘lock and key’ type manner, however, have been unsuccessful. HS chains are synthesized in a non-template driven process in the ER and Golgi apparatus, involving a large number of enzymes capable of fine-tuning structures. Changes in the localization of HS-modifying enzymes throughout the Golgi, rather than protein expression levels, were found to correlate with changes in the structure of HS. Following brefeldin A treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Further, shortly after treatment with heparin, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS trisulfated disaccharide content. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings highlight that the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking processes are critical prerequisite for the complete delineation of HS biosynthesis.

Mucopolysaccharidosis type I (MPS I) is caused by genetic deficiency of &alpha, l-iduronidase and impairment of lysosomal catabolism of heparan sulfate and dermatan sulfate. In the brain, these substrates accumulate in the lysosomes of neurons and glial cells, leading to neuroinflammation and neurodegeneration. Their storage also affects lysosomal homeostasis-inducing activity of several lysosomal proteases including cathepsin B (CATB). In the central nervous system, increased CATB activity has been associated with the deposition of amyloid plaques due to an alternative pro-amyloidogenic processing of the amyloid precursor protein (APP), suggesting a potential role of this enzyme in the neuropathology of MPS I. In this study, we report elevated levels of protein expression and activity of CATB in cortex tissues of 6-month-old MPS I (Idua -/- mice. Besides, increased CATB leakage from lysosomes to the cytoplasm of Idua -/- cortical pyramidal neurons was indicative of damaged lysosomal membranes. The increased CATB activity coincided with an elevated level of the 16-kDa C-terminal APP fragment, which together with unchanged levels of &beta, secretase 1 was suggestive for the role of this enzyme in the amyloidogenic APP processing. Neuronal accumulation of Thioflavin-S-positive misfolded protein aggregates and drastically increased levels of neuroinflammatory glial fibrillary acidic protein (GFAP)-positive astrocytes and CD11b-positive activated microglia were observed in Idua -/- cortex by confocal fluorescent microscopy. Together, our results point to the existence of a novel CATB-associated alternative amyloidogenic pathway in MPS I brain induced by lysosomal storage and potentially leading to neurodegeneration.

In this chapter, we will emphasize the importance of heparan sulfate proteoglycans (HSPG) in controlling various physiological and pathological molecular mechanisms and discuss how the heparanase enzyme can modulate the effects triggered by HSPG. Additionally, we will also navigate about the existing knowledge of the possible role of heparanase-2 in biological events. Heparan sulfate is widely distributed and evolutionarily conserved, evidencing its vital importance in cell development and functions such as cell proliferation, migration, adhesion, differentiation, and angiogenesis. During remodeling of the extracellular matrix, the breakdown of heparan sulfate by heparanase results in the release of molecules containing anchored glycosaminoglycan chains of great interest in heparanase-mediated cell signaling pathways in various physiological states, tumor development, inflammation, and other diseases. Taken together, it appears that heparanase plays a key role in the maintenance of the pathology of cancer and inflammatory diseases and is a potential target for anti-cancer therapies. Therefore, heparanase inhibitors are currently being examined in clinical trials as novel cancer therapeutics. Heparanase-2 has no enzymatic activity, displays higher affinity for heparan sulfate and the coding region alignment shows 40% identity with the heparanase gene. Heparanase-2 plays an important role in embryogenic development however its mode of action and biological function remain to be elucidated. Heparanase-2 functions as an inhibitor of the heparanase-1 enzyme and also inhibits neovascularization mediated by VEGF. The HPSE2 gene is repressed by the Polycomb complex, together suggesting a role as a tumor suppressor.

Proteoglycans are glycosylated proteins which have covalently attached highly anionic glycosaminoglycans. They can be located on the extracellular matrix, cell membrane or intracellular granules. To date, few studies have reported the presence of proteoglycans in human dental pulp.The aim of this study was, therefore, to analyze the expression of lumican, versican and glypican proteoglycans in deciduous and permanent human dental pulp by real-time polymerase chain reaction (q-PCR) and immunofluorescence.Healthy human dental pulps were used: 13 from permanent teeth (group 1) and eight from deciduous teeth (group 2). Versican, lumican and glypican (glypican-1 to 6) gene expressions were quantitatively evaluated by real-time PCR technique, using the expression of the endogenous gene GAPDH as control. Pulp sections were submitted to immunostaining procedure with fluorescence labelling, the tissues being fixed and incubated with well-characterized monoclonal and polyclonal antibodies against proteoglycan epitopes, including anti-versican and anti-lumican. Comparisons among the groups of the quantitative scores for each proteoglycan were analyzed using the t-test and ANOVA (P 0.05).The real-time PCR analysis showed expression of versican and lumican proteoglycans in the two groups, with significant predominance of lumican gene (P = 0.03). Considering the glypican genes, glypican-3 was the proteoglycan most significantly expressed in permanent pulps (P 0.001), while glypican-2 was not expressed in this tissue. The immunofluorescence quantification exhibited no significant differences between lumican and versican among the pulps and groups.The lumican gene was more expressed than versican and glypican-3 was the isoform more expressed in permanent pulp compared to deciduous.

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schroederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012–0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01–0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B.

The occurrence of a natural and unmodified highly sulfated chondroitin sulfate from Litopenaeus vannamei heads (sCS) is herein reported. Its partial digestion by Chondroitinases AC and ABC together with its electrophoretic migration profile revealed it as a highly sulfated chondroitin sulfate despite its average molecular weight being similar to CSA. Using orthogonal 1D/2D NMR experiments, the anomeric signals (δ 4.62/106.0) corresponding to unusual 2,3-di-O-Sulfo-GlcA (∼36%), U33S (δ 4.42/84.1, ∼63%) and U22S (4.12/80.1, ∼50%) substitutions were confirmed. In addition, non-sulfated GlcA (δ 4.5/106.3) linked to 4-O- (A14S, 36%) or 6-O-Sulfo (A16S, 28%) GalNAc (δ 4.64/103.5) was observed. Although the biological role of sCS in shrimp is unknown, its influence on hemostasis was also demonstrated. The sCS identification brings to light new questions about the hierarchical model of GAGs biosynthesis and contributes to the better understanding of the subtle relationship between GAGs structure and function.

Background Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. Methods Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. Results Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 μM and Ki = 0,59 μM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1β secretion in Caco-2 cells. Conclusions BF4 is a potent and selective inhibitor of PC1/3. General significance BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

Summary Objective To investigate the effects of different strontium ranelate (SrR) doses alone or in combination with low-intensity and high-frequency mechanical vibration (MV) on articular cartilage in ovariectomized rats. Design Fifty 6-month-old female Wistar rats underwent ovariectomy (OVX) and after 3 months were divided into: control group ( Control ); SrR 300 mg/kg/day ( SrR300 ); SrR 625 mg/kg/day ( SrR625 ); MV ; SrR 625 mg/kg/day plus MV ( SrR625 + MV ). The vehicle and the SrR were administered by gavage 7 days/week and vibration (0.6 g/60 Hz) was performed for 20 min/day, 5 days/week. Bone mineral density (BMD) and body composition were evaluated by densitometry. Changes in cartilage were assessed 90 days after treatment by histomorphometry; immunohistochemistry analysis evaluating cell death (caspase-3), tumor necrosis factor-α (TNF-α), metalloproteinase 9 (MMP-9) and type II collagen; Osteoarthritis Research Society International (OARSI) grading system and glycosaminoglycans (GAGs) analyses. Results SrR-treated groups exhibited a lower OARSI grade, a smaller number of chondrocyte clusters, increased levels of chondroitin sulfate (CS) and decreased expression of caspase-3. Additionally, compared to all the groups, SrR300 exhibited increased levels of hyaluronic acid (HA). Vibration applied alone or in combination accelerated cartilage degradation, as demonstrated by increased OARSI grade, reduced number of chondrocytes, increased number of clusters, elevated expression of type II collagen and cell death, and was accompanied by decreased amounts of CS and HA; however, MV alone was able to reduce MMP-9. Conclusions SrR and vibration modulate distinct responses in cartilage. Combined treatment accelerates degeneration. In contrast, SrR treatment at 300 mg/kg/day attenuates osteoarthritis (OA) progression, improving cartilage matrix quality and preserving cell viability in ovariectomized rats.

Background Leptospirosis is a widespread zoonotic disease that causes reproductive losses and/or hepatorenal failure in a number of animal species. Wild reservoirs of the disease, such as rodents, harbor the causative bacterium, Leptospira spp., in their kidneys and contaminate the environment by excreting infected urine. In this study, we tested small wild mammals, environmental water, and livestock in the Cumberland Gap region of southeastern Appalachia for the presence of pathogenic Leptospira or leptospiral antibodies. Methods/Results Small wild mammals (n = 101) and environmental water samples (n = 89) were screened by a real time quantitative PCR that targets the pathogenic Leptospira-specific lipl32 gene. Kidneys from 63 small wild mammals (62.37%) and two water sources (2.25%) tested positive for leptospiral DNA. To identify the infecting leptospiral species in qPCR-positive water and kidney samples, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. L. kirschneri and L. interrogans were the leptospiral species carried by small wild mammals. Furthermore, sera from livestock (n = 52; cattle and horses) were screened for leptospiral antibodies using microscopic agglutination test (MAT). Twenty sera (38.46%) from livestock had antibodies to one or more serovars of pathogenic Leptospira spp. Conclusions In conclusion, results from our study show exposure to leptospiral infection in farm animals and the presence of this zoonotic pathogen in the environmental water and kidneys of a significant number of small wild mammals. The public health implications of these findings remain to be assessed., Author summary Leptospirosis, a zoonotic disease caused by Leptospira, affects people and animals, including dogs, cows and horses. Many species of small mammals, and particularly rodents, are believed to be natural reservoirs of the pathogen across numerous geographical ranges with similar climatic conditions. The Cumberland Gap region (CGR) in southern Appalachia has several climatic risk factors for leptospirosis. With this in mind, we tested surface water, kidneys of small wild mammals, and blood from cows and horses in the CGR for the presence of leptospiral DNA or antibodies. Notably, more than 60% of tested small mammals carried leptospires in their kidneys. Furthermore, we found that 2 of the 89 tested environmental water samples contained leptospiral DNA, and a significant percentage of cows and horses had leptospiral antibodies. These findings will advance our understanding of the ecology of leptospirosis in the region, which will lead to stakeholder awareness, and development of preventive interventions with broad applicability within and outside this region of southern Appalachia.

The detailed structure of a further Chondroitin Sulfate from Litopenaeus vannamei shrimp (sCS) is described. The backbone structure was established by 1H/13C NMR, which identified 3-O-sulfated GlcA, 4-O-sulfated GalNAc, 6-O-sulfated GalNAc, and 4,6-di-O-sulfated GalNAc residues. GlcA is linked to GalNAc 4,6 di S and GlcA 3S is linked to GalNAc 4S, GalNAc 4,6 di-S and GalNAc6S residues. The anticoagulant properties of this sCS were evaluated by activated partial thromboplastin time, anti-IIa, anti-Xa and anti-heparin cofactor II-mediated activities, and sCS failed to stabilise antithrombin in a fluoresence shift assay. The anti-inflammatory effect of sCS was explored using a model of acute peritonitis, followed by leukocyte count and measurement of the cytokines, IL-1β, IL-6 and TNF-α. The compound showed low clotting effects, but high anti-IIa activity and HCII-mediated thrombin inhibition. Its anti-inflammatory effect was shown by leukocyte recruitment inhibition and a decrease in pro-inflammatory cytokine levels. Although the biological role of sCS remains unknown, its properties indicate that it is suitable for studies of multi-potent molecules obtained from natural sources.

Introduction: Enoxaparin (Enox) is used in cardiovascular emergency and coronary angiography due to its effects on coagulation mechanisms. However, its effect on changing the structure of the exchange-inhibiting peptide present in the sodium-calcium exchanger, leading to the acceleration of its function and withdrawing calcium from the cell in overload situations could lead to a new therapeutic function, where the intracellular calcium overload would be deleterious, as in cardiac arrhythmias and myocardial infarction. Objective: To analyze the action of Enox on cardiac arrhythmias induced by ischemia and reperfusion (IR) in isolated rat hearts. Materials and method. Adult Wistar rats were sacrificed, hearts isolated and placed under perfusion in Langhendorf Model with Krebs solution (KS) and monitoring of the electrocardiogram. After 30 minutes in sinus rhythm, ischemia was performed with discontinuation of the KS for 5 min and then reconnected. They were divided into 2 groups: Control (n = 10), with IR without medication and Enox Group, with infusion of 2mg / kg, in the cannula that perfuses the heart, soon after initiation and support for 30 seconds of Ventricular Tachycardia (VT) or Torsade de Pointes (TP). In case of reversal of the arrhythmia, a new arrhythmia induction will be attempted with the same protocol. Monitoring is continued until asystole or completion scheduled for 60 minutes of experimentation. Results: The control group presented VT in 70% of hearts and total atrioventricular block (TAVB) in 30% of hearts, followed by asystole after 7 minutes in 100% of hearts, and no further attempt of ischemia was possible. In the Enox group, 60% of VT, 30% of TP and 10% of TAVB were observed. All arrhythmias were reversed between 10 and 15 seconds after infusion of Enox, with return and maintenance of sinus rhythm after medication. In this group, a new attempt was made to induce arrhythmia due to IR without success after 10 minutes of interruption of the arrhythmia by Enox and sinus rhythm was maintained until the interruption of the experimental protocol. Conclusion: Enoxaparin reverses arrhythmias triggered by IR, with its effect prolonging and protecting new events after infusion, as well as decreasing organ mortality in isolated hearts of rats.

Metastasis is responsible for the majority of deaths among patients with malignant melanoma. Despite recent advances, the majority of current and modern therapies are ineffective and/or financially unfeasible. Thus, in this study, we investigated two low‑cost highly‑diluted natural complexes (HDNCs) that have been shown to be effective against malignant melanoma in a murine model in vivo. The aim of this study was to determine the mechanisms through which these HDNCs directly affect melanoma cells, either alone or in an artificial tumor microenvironment, suppressing the metastatic phenotype, thus explaining previous in vivo effects. For this purpose, HDNC in vitro treatments of B16‑F10 melanoma cells, alone or in co‑culture with Balb/3T3 fibroblasts, were carried out. Molecular biology techniques and standard functional assays were used to assess the changes in molecule expression and in cell behaviors related to the metastatic phenotype. Melanoma progression features were found to be regulated by HDNCs. Molecules related to cell adhesion (N‑cadherin, β1‑integrin and CD44), and migration, extracellular matrix remodeling and angiogenesis were modulated. The cell migratory, invasive and clonogenic capacities were reduced by the HDNCs. No loss of cell proliferation or viability were observed. On the whole, the findings of this study indicate that HDNCs directly reprogram, molecularly and functionally, melanoma cells in vitro, modulating their metastatic phenotype. Such findings are likely to be responsible for the attenuation of tumor growth and lung colonization previously observed in vivo.

In 1977, plastination was unveiled, which replaced tissue fluid with a curable polymer. Today preservation via plastination of various animal and plant tissues, organs, and whole bodies is an extremely useful technique to display such and help educate vast arrays of both allied science students and the lay public across the planet. The diversity of applications of plastination techniques seems to be without limits. In fact, the only real limitation to plastination is one's imagination! The size of plastinates during the early years of plastination was comparatively small and dictated primarily by the size of the available plastination kettle/chamber, 35 L Heidelberg plastination kettle (49 cm H × 34.5 cm diam.). In the 1990s larger chambers were designed and slowly became available:150-210 cm (long) × 65-80 cm (wide) and 83-92 cm (high). Today a few large vacuum chambers are in service which will accommodate whole bodies of man and domestic or exotic animals. Today, at least two gigantic chambers are available to impregnate massive specimens. These are 3.5 m × 2 m × 1.5 m (Dalian) and 4 m × 3 m × 2.2 m (Guben). Also, the need for larger quantities of acetone and impregnation mix, not to mention the great increase in specimen preparation time, makes this a major investment. The "cold temperature process" is used to impregnate these massive creations. The room temperature technique could be used. The same four plastination steps are necessary for larger and massive specimens. Besides their tremendous size, the slippery silicone polymer is a reckoning force.

Introduction Heparin obtained from bovine intestinal mucosa is being considered for reintroduction in cardiovascular, surgical, and interventional procedures. Although the anticoagulant potency of ...

Endometrium extracellular matrix provides a wide range of signals at different cellular levels, like cell death and proliferation, which can be important for regulating menses and reducing the proliferative processes. The objective of this study is to evaluate hyaluronic acid concentration, the enzymes of hyaluronic acid synthases in the endometrium of patients with polycystic ovary syndrome (PCOS) and eumenorrheic women. A total of 60 endometrial samples from 30 patients with PCOS and 30 women with regular menstrual cycles in the proliferative phase, attended at Gynecology Division of Clinical Hospital of the FMUSP (HC-USP). Profile determination and the concentration of hyaluronic acid was performed by the biochemical method of the fluorimetric assay (ELISA-like). Its location in the endometrial tissue as well as the dosage of enzymes synthases (HAS1, HAS2 and HAS3) was done by immunohistochemistry and western blotting. Statistical analyses were performed with one-way ANOVA, followed by the Bonferroni test. Regarding hyaluronic acid synthases, there was a higher HAS1 and HAS2 reactivity and lower HAS3 reactivity in the PCOS endometrium compared to women with regular menstrual cycles in the proliferative phase. We suggest that PCOS patients have different composition of hyaluronic acid in relation to a regular cycle in the proliferative phase.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

Proteolytic enzymes mediate the activation or inactivation of many physiologic and pathologic processes. The PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome) encodes a metallopeptidase, which is mutated in patients with a prevalent form (1:20,000) of inherited rickets-X-linked hypophosphatemia (XLH). XLH shows growth retardation, hypophosphatemia, osteomalacia, and defective renal phosphate reabsorption and metabolism of vitamin D. Most PHEX studies have focused on bone, and recently we identified osteopontin (OPN) as the first protein substrate for PHEX, demonstrating in the murine model of XLH (Hyp mice) an increase in OPN that contributes to the osteomalacia. Besides its role in bone mineralization, OPN is expressed in many tissues, and therein has different functions. In tumor biology, OPN is known to be associated with metastasis. Here, we extend our PHEX-OPN studies to investigate PHEX expression in a squamous cell carcinoma (SCC) cell line and its possible involvement in modulating OPN function. Real-time PCR showed PHEX-OPN co-expression in SCC cells, with sequencing of the 22 exons showing no mutation of the PHEX gene. Although recombinant PHEX hydrolyze SCC-OPN fragments, unlike in bone cells, SCC-PHEX protein was not predominantly at the plasma membrane. Enzymatic activity assays, FACs and immunoblotting analyses demonstrated that membrane PHEX is degraded by cysteine proteases and the decreased PHEX activity could contribute to inappropriate OPN regulation. These results highlight for the first time PHEX in tumor biology.

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis.

Objective: To evaluate and compare the behavior of glycosaminoglycans (GAGs) in Dupuytren disease (DD). Methods: This is an experimental study with 23 patients diagnosed with DD. Tissue collected through fasciectomy with incision type Brunner or McCash were evaluated by electrophoresis for identification of GAGs. The quantification was carried out by immunofluorescence and dosage of proteins for different types of glycosaminoglycans. The results were expressed in percentage and statistically evaluated. Results: A significant increase was observed through eletrophoresis in GAGs, as compared to the control (p

The cell's resistance to cell death by adhesion loss to extracellular matrix (anoikis), contributes to tumor progression and metastasis. Various adhesion molecules are involved in the anoikis resistance, including the syndecan-4 (SDC4), a heparan sulfate proteoglycan (HSPG) present on the cell surface. Changes in the expression of SDC4 have been observed in tumor and transformed cells, indicating its involvement in cancer. In previous works, we demonstrated that acquisition of anoikis resistance resistance by blocking adhesion to the substrate up-regulates syndecan-4 expression in endothelial cells. This study investigates the role of SDC4 in the transformed phenotype of anoikis resistant endothelial cells. Anoikis-resistant endothelial cells (Adh1-EC) were transfected with micro RNA interference (miR RNAi) targeted against syndecan-4. The effect of SDC4 silencing was analyzed by real-time PCR, western blotting and immunofluorescence. Transfection with miRNA-SDC4 resulted in a sequence-specific decrease in syndecan-4 mRNA and protein levels. Furthermore, we observed a reduction in the number of heparan and chondroitin sulfate chains in the cell extract and culture medium. The SDC4 silencing led to downregulation of proliferative and invasive capacity and angiogenic abilities of anoikis-resistant endothelial cells. Compared with the parental cells (Adh1-EC), SDC4 silenced cells (SDC4 miR-Syn-4-1-Adh1-EC e miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to collagen and laminin and also in the apoptosis rate. Moreover, transfection with miRNA-SDC4 caused a decrease in the number of cells in the S phase of the cell cycle. This is accompanied by an increase in the heparan sulfate synthesis after 12 h of simulation with fetal calf serum (FCS). SDC4 silencing cells are more dependent of growth factors present in the FCS to synthesize heparan sulfate than parental cells. Similar data were obtained for the wild-type cell line (EC). Our results indicated that downregulation of SDC4 expression reverses the transformed phenotype of anoikis resistant endothelial cells. These and other findings suggest that syndecan-4 is suitable for pharmacological intervention, making it an attractive target for cancer therapy.

Trastuzumab (Tmab), an antibody for breast cancer, was incorporated in Langmuir monolayers with different lipidic compositions to investigate the drug action in lipidic interfaces of pharmaceutical interest. Tmab caused all lipid films to expand as confirmed with by surface pressure-area isotherm, proving its incorporation. It also affected the compressional and structural properties as observed by in-plane elasticity curves and polarization modulation reflection-absorption infrared spectroscopy (PM-IRRAS), respectively. Although Tmab did not change significantly the compressional modulus for dipalmitoylphosphatidylcholine (DPPC) monolayers, it decreased it for the mixtures of DPPC with cholesterol. In contrast, for dipalmitoylphosphoethanolamine (DPPE), Tmab increased the compressional modulus for both monolayers, pure DPPE or mixed with cholesterol. While Brewster Angle Microscopy showed discrete distinctive morphological patterns for the monolayers investigated, PM-IRRAS showed that Tmab caused an increased number of gauche conformers related to the CH2 stretching mode for the lipid acyl chains, suggesting molecular disorder. Furthermore, the antibody kept the β-sheet structure of the polypeptide backbone adsorbed at the lipid monolayers although the secondary conformation altered according to the film composition at the air-water interface. As a result, the results suggest that the membrane lipid profile affects the adsorption of Tmab at lipid monolayers, which can be important for the incorporation of this drug in lipidic supramolecular systems like in liposomes for drug delivery and in biomembranes.