A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting.

Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacry-late) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modeling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a nonlinear function, allowing us to tune the interaction by varying the radius and degree of polymerization. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36, and CD81.

Efficacy and safety of cold versus hot snare polypectomy for small (5-9 mm) colorectal polyps: a multicenter randomized controlled trial.

BACKGROUND: Resection techniques for small polyps include cold snare polypectomy (CSP) and hot snare polypectomy (HSP). This study compared CSP and HSP in 5-9 mm polyps in terms of complete resection and adverse events. METHODS: This was a multicenter, randomized trial conducted in seven Spanish centers between February and November 2019. Patients with ≥ 1 5-9 mm polyp were randomized to CSP or HSP, regardless of morphology or pit pattern. After polypectomy, two marginal biopsies were submitted to a pathologist who was blinded to polyp histology. Complete resection was defined as normal mucosa or burn artifacts in the biopsies. Abdominal pain was only assessed in patients without < 5 mm or > 9 mm polyps. RESULTS: 496 patients were randomized: 237 (394 polyps) to CSP and 259 (397 polyps) to HSP. Complete polypectomy rates were 92.5 % with CSP and 94.0 % with HSP (difference 1.5 %, 95 % confidence interval -1.9 % to 4.9 %). Intraprocedural bleeding occurred during three CSPs (0.8 %) and seven HSPs (1.8 %) (P = 0.34). One lesion per group (0.4 %) presented delayed hemorrhage. Post-colonoscopy abdominal pain presented similarly in both groups 1 hour after the procedure (CSP 18.8 % vs. HSP 18.4 %) but was higher in the HSP group after 5 hours (5.9 % vs. 16.5 %; P = 0.02). A higher proportion of patients were asymptomatic 24 hours after CSP than after HSP (97 % vs. 86.4 %; P = 0.01). CONCLUSIONS: We observed no differences in complete resection and bleeding rates between CSP and HSP. CSP reduced the intensity and duration of post-colonoscopy abdominal pain.

The multiple functions of the co-chaperone stress inducible protein 1.

Stress inducible protein 1 (STI1) is a co-chaperone acting with Hsp70 and Hsp90 for the correct client proteins' folding and therefore for the maintenance of cellular homeostasis. Besides being expressed in the cytosol, STI1 can also be found both in the cell membrane and the extracellular medium playing several relevant roles in the central nervous system (CNS) and tumor microenvironment. During CNS development, in association with cellular prion protein (PrPc), STI1 regulates crucial events such as neuroprotection, neuritogenesis, astrocyte differentiation and survival. In cancer, STI1 is involved with tumor growth and invasion, is undoubtedly a pro-tumor factor, being considered as a biomarker and possibly therapeutic target for several malignancies. In this review, we discuss current knowledge and new findings on STI1 function as well as its role in tissue homeostasis, CNS and tumor progression.

The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis.

Within the brain, endothelial cells lining the blood vessels meticulously coordinate the transport of nutrients, energy metabolites and other macromolecules essential in maintaining an appropriate activity of the brain. While small molecules are pumped across specialised molecular transporters, large macromolecular cargos are shuttled from one side to the other through membrane-bound carriers formed by endocytosis on one side, trafficked to the other side and released by exocytosis. Such a process is collectively known as transcytosis. The brain endothelium is recognised to possess an intricate vesicular endosomal network that mediates the transcellular transport of cargos from blood-to-brain and brain-to-blood. However, mounting evidence suggests that brain endothelial cells (BECs) employ a more direct route via tubular carriers for a fast and efficient transport from the blood to the brain. Here, we compile the mechanism of transcytosis in BECs, in which we highlight intracellular trafficking mediated by tubulation, and emphasise the possible role in transcytosis of the Bin/Amphiphysin/Rvs (BAR) proteins and glycocalyx (GC)-a layer of sugars covering BECs, in transcytosis. Both BAR proteins and the GC are intrinsically associated with cell membranes and involved in the modulation and shaping of these membranes. Hence, we aim to summarise the machinery involved in transcytosis in BECs and highlight an uncovered role of BAR proteins and the GC at the brain endothelium.

On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias.

The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor-related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.

Designing peptide nanoparticles for efficient brain delivery.

The targeted delivery of therapeutic compounds to the brain is arguably the most significant open problem in drug delivery today. Nanoparticles (NPs) based on peptides and designed using the emerging principles of molecular engineering show enormous promise in overcoming many of the barriers to brain delivery faced by NPs made of more traditional materials. However, shortcomings in our understanding of peptide self-assembly and blood-brain barrier (BBB) transport mechanisms pose significant obstacles to progress in this area. In this review, we discuss recent work in engineering peptide nanocarriers for the delivery of therapeutic compounds to the brain: from synthesis, to self-assembly, to in vivo studies, as well as discussing in detail the biological hurdles that a nanoparticle must overcome to reach the brain.

Cyclopamine sensitizes glioblastoma cells to temozolomide treatment through Sonic hedgehog pathway.

AIM: Glioblastoma is an extremely aggressive glioma, resistant to radio and chemotherapy usually performed with temozolomide. One of the main reasons for glioblastoma resistance to conventional therapies is due to the presence of cancer stem-like cells. These cells could recapitulate some signaling pathways important for embryonic development, such as Sonic hedgehog. Here, we investigated if the inhibitor of the Sonic hedgehog pathway, cyclopamine, could potentiate the temozolomide effect in cancer stem-like cells and glioblastoma cell lines in vitro. MAIN METHODS: The viability of glioblastoma cells exposed to cyclopamine and temozolomide treatment was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while the induction of apoptosis was assessed by western blot. The stemness properties of glioma cells were verified by clonogenic and differentiation assay and the expression of stem cell markers were measured by fluorescence microscopy and western blot. KEY FINDINGS: The glioblastoma viability was reduced by cyclopamine treatment. Cyclopamine potentiated temozolomide treatment in glioblastoma cell lines by inducing apoptosis through activation of caspase-3 cleaved. Conversely, the combined treatment of cyclopamine and temozolomide potentiated the stemness properties of glioblastoma cells by inducing the expression of SOX-2 and OCT-4. SIGNIFICANCE: Cyclopamine plays an effect on glioblastoma cell lines but also sensibilize them to temozolomide treatment. Thus, first-line treatment with Sonic hedgehog inhibitor followed by temozolomide could be used as a new therapeutic strategy for glioblastoma patients.

Membrane Elastic Properties During Neural Precursor Cell Differentiation.

Neural precursor cells differentiate into several cell types that display distinct functions. However, little is known about how cell surface mechanics vary during the differentiation process. Here, by precisely measuring membrane tension and bending modulus, we map their variations and correlate them with changes in neural precursor cell morphology along their distinct differentiation fates. Both cells maintained in culture as neural precursors as well as those plated in neurobasal medium reveal a decrease in membrane tension over the first hours of culture followed by stabilization, with no change in bending modulus. During astrocyte differentiation, membrane tension initially decreases and then increases after 72 h, accompanied by consolidation of glial fibrillary acidic protein expression and striking actin reorganization, while bending modulus increases following observed alterations. For oligodendrocytes, the changes in membrane tension are less abrupt over the first hours, but their values subsequently decrease, correlating with a shift from oligodendrocyte marker O4 to myelin basic protein expressions and a remarkable actin reorganization, while bending modulus remains constant. Oligodendrocytes at later differentiation stages show membrane vesicles with similar membrane tension but higher bending modulus as compared to the cell surface. Altogether, our results display an entire spectrum of how membrane elastic properties are varying, thus contributing to a better understanding of neural differentiation from a mechanobiological perspective.

Glioblastoma Factors Increase the Migration of Human Brain Endothelial Cells In Vitro by Increasing MMP-9/CXCR4 Levels.

BACKGROUND/AIM: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. MATERIALS AND METHODS: We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. RESULTS: Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. CONCLUSION: GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4.

Glioblastoma Therapy in the Age of Molecular Medicine.

Glioblastoma (GBM) is the most common and fatal primary malignant brain tumor. Despite advances in the understanding of the biology of gliomas, little has changed in the treatment of these tumors in the past decade. Phase III clinical trials showed no benefit for the use of bevacizumab in newly diagnosed patients, leading to a renewed search for new antiangiogenic drugs, as well as immunotherapeutic approaches, including checkpoint inhibitors, chimeric antigen receptor T cells, and intracerebral CpG-oligodeoxynucleotides. The emerging role of infiltrating microglia and macrophages, and of metabolic alterations, is also being taken into account in preclinical research and drug development. In this review, we discuss progress in the search for new therapeutic strategies, particularly approaches focusing on the tumor microenvironment.

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/ß-Catenin Signaling.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/ß-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial ß-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1ß gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/ß-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

Cellular and molecular mechanisms of glioblastoma malignancy: Implications in resistance and therapeutic strategies.

Glioblastoma (GB) is the more frequent and malignant brain tumour. In spite of all efforts, the median overall survival of GB patients remains approximately 15 months under therapy. The molecular biology underlying GB is complex, which highlight the need of specific treatment strategies. In fact, the deregulation of several molecular signalling pathways, the existence of the blood-brain barrier (BBB), that makes almost all the chemotherapeutic agents inaccessible to the tumour site, and the existence of a population of stem-like cells known to be responsible for tumour recurrence after therapy, can contribute to GB chemoresistance. In the present review, we summarize the reliable factors responsible for the failure of the most important chemotherapeutic agents in GB. Specifically, we describe the utmost important characteristics of the BBB, as well as the genetic, molecular and transcription factors alterations that lead to tumour malignancy, and ultimately their impact on stem-like cell plasticity modulation. Recently, nanocarriers have attracted increasing attention in brain- and tumour-targeted drug-delivery systems, owing to their potential ability to target cell surface specific molecules and to cross the BBB delivering the drug specifically to the tumour cells, improving efficacy and thus reducing non-specific toxicity. In this sense, we will lastly highlight the therapeutic challenges and improvements regarding GB treatment.

Microglia/Astrocytes-Glioblastoma Crosstalk: Crucial Molecular Mechanisms and Microenvironmental Factors.

In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prominence in several diseases of the central nervous system, especially in glioblastoma (GB), the most malignant primary brain tumor that leads to poor clinical outcomes. Studies showed that microglial cells or astrocytes play a critical role in promoting GB growth. Based on the recent findings, the complex network of the interaction between microglial/astrocytes cells and GB may constitute a potential therapeutic target to overcome tumor malignancy. In the present review, we summarize the most important mechanisms and functions of the molecular factors involved in the microglia or astrocytes-GB interactions, which is particularly the alterations that occur in the cell's extracellular matrix and the cytoskeleton. We overview the cytokines, chemokines, neurotrophic, morphogenic, metabolic factors, and non-coding RNAs actions crucial to these interactions. We have also discussed the most recent studies regarding the mechanisms of transportation and communication between microglial/astrocytes - GB cells, namely through the ABC transporters or by extracellular vesicles. Lastly, we highlight the therapeutic challenges and improvements regarding the crosstalk between these glial cells and GB.

Glioblastoma entities express subtle differences in molecular composition and response to treatment.

Glioblastoma (GBM) is a grade IV astrocytoma. GBM patients show resistance to chemotherapy such as temozolomide (TMZ), the gold standard treatment. In order to simulate the molecular mechanisms behind the different chemotherapeutic responses in GBM patients we compared the cellular heterogeneity and chemotherapeutic resistance mechanisms in different GBM cell lines. We isolated and characterized a human GBM cell line obtained from a GBM patient, named GBM11. We studied the GBM11 behaviour when treated with Tamoxifen (TMX) that, among other functions, is a protein kinase C (PKC) inhibitor, alone and in combination with TMZ in comparison with the responses of U87 and U118 human GBM cell lines. We evaluated the cell death, cell cycle arrest and cell proliferation, mainly through PKC expression, by flow cytometry and western blot analysis and, ultimately, cell migration capability and f-actin filament disorganization by fluorescence microscopy. We demonstrated that the constitutive activation of p-PKC seems to be one of the main metabolic implicated on GBM malignancy. Despite of its higher resistance, possibly due to the overexpression of P-glycoprotein and stem-like cell markers, GBM11 cells presented a subtle different chemotherapeutic response compared to U87 and U118 cells. The GBM11, U87, U118 cell lines show subtle molecular differences, which clearly indicate the characterization of GBM heterogeneity, one of the main reasons for tumor resistance. The adding of cellular heterogeneity in molecular behaviour constitutes a step closer in the understanding of resistant molecular mechanisms in GBM, and can circumvents the eventual impaired therapy.

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells.

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition.

PURPOSE: Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. METHODS: The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of ß3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. RESULTS: We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of ß3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. CONCLUSION: From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Microglia in Cancer: For Good or for Bad?

Glioblastoma is a malignant tumor of astrocytic origin that is highly invasive, proliferative and angiogenic. Despite current advances in multimodal therapies, such as surgery, radio- and chemotherapy, the outcome for patients with glioblastoma is nearly always fatal. The glioblastoma microenvironment has a tremendous influence over the tumor growth and spread. Microglia and macrophages are abundant cells in the tumor mass. Increasing evidence indicates that glioblastoma recruits these cell populations and signals in a way that microglia and macrophages are subverted to promote tumor progression. In this chapter, we discuss some aspects of the interaction between microglia and glioblastoma, consequences of this interaction for tumor progression and the possibility of microglial cells being used as therapeutic vectors, which opens up new alternatives for the development of GBM therapies targeting microglia.

[A comparison of medical versus surgical treatment in Barrett's esophagus acid control]. / Estudio comparativo del tratamiento médico frente al quirúrgico en el control ácido del esófago de Barrett.

INTRODUCTION: Barrett's oesophagus (BE) is an oesophageal injury caused by gastroesophageal acid reflux. One of the main aims of treatment in BE is to achieve adequate acid reflux control. OBJECTIVE: To assess acid reflux control in patients with BE based on the therapy employed: medical or surgical. METHODS: A retrospective study was performed in patients with an endoscopic and histological diagnosis of BE. Medical therapy with proton pump inhibitors (PPI) was compared with surgical treatment (Nissen fundoplication). Epidemiological data and the results of pH monitoring (pH time <4, prolonged reflux >5min, DeMeester score) were evaluated in each group. Treatment failure was defined as a pH lower than 4 for more than 5% of the recording time. RESULTS: A total of 128 patients with BE were included (75 PPI-treated and 53 surgically-treated patients). Patients included in the two comparison groups were homogeneous in terms of demographic characteristics. DeMeester scores, fraction of time pH<4 and the number of prolonged refluxes were significantly lower in patients with fundoplication versus those receiving PPIs (P<.001). Treatment failure occurred in 29% of patients and was significantly higher in those receiving medical therapy (40% vs 13%; P<.001). CONCLUSIONS: Treatment results were significantly worse with medical treatment than with anti-reflux surgery and should be optimized to improve acid reflux control in BE. Additional evidence is needed to fully elucidate the utility of PPI in this disease.

[Vitamin D deficiency in chronic liver disease, clinical-epidemiological analysis and report after vitamin d supplementation]. / Déficit de vitamina D en la enfermedad hepática crónica, análisis clínico epidemiológico y tras aporte vitamínico.

INTRODUCTION: Vitamin D (VD) is known to have multiple extra-skeletal health functions. There is emerging interest in exploring the relationship between vitamin D and chronic liver disease (CLD). OBJECTIVES: To determine the prevalence of VD deficiency in patients with CLD in our setting and to assess whether VD supplementation influences plasma levels and is associated with improved liver function. MATERIAL AND METHODS: We conducted a study in 2 phases. First, we analysed clinical and epidemiological characteristics in 94 patients with CLD; second, different doses of calcifediol (25-OH-VD) were administered to patients with VD deficiency (<20ng/mL) and insufficiency (20-30ng/mL). Plasma concentrations and liver function (Child-Pugh and MELD) at the end of treatment were compared with baseline data. RESULTS: Deficient or insufficient VD levels were found in 87% of the patients, with an average concentration of 18.8ng/mL. Levels were lower in patients with cirrhosis (15.9ng/mL) (P=.002) and in alcoholic liver disease. VD levels were inversely proportional to the degree of liver function: Child A (16.52ng/mL) vs C (7.75ng/mL). After VD supplementation, optimal serum levels were achieved in 94% of patients and significant improvements were observed in platelet count, albumin levels (P<.05) and functional status assessed by the Child-Pugh scale (P<.05). CONCLUSION: Given the high prevalence of VD deficiency or insufficiency, the need for screening should be considered in the population with CLD. VD supplementation could be safe and effective.