Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Gene silencing using small interfering RNA (siRNA) is a promising new approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers.In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-beta-cyclodextrin, genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-beta-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrin-dependent and caveolin-dependent endocytosis vs only caveolin-dependent endocytosis in T98G cells.Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Cosaka, María Luz. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

Ana Paula Perez1, Cecilia Mundiña-Weilenmann2, Eder Lilia Romero1, Maria Jose Morilla11Programa de Nanomedicinas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Buenos Aires, 2Centro de Investigaciones Cardiovasculares, CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, ArgentinaBackground: Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined.Materials: 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured.Results: The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA.Conclusion: The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.Keywords: nucleic acids, gel, dendrimers, mucosa, olfactory bul

Design of a case management model for people with chronic disease (Heart Failure and COPD). Phase I: modeling and identification of the main components of the intervention through their actors: patients and professionals (DELTA-ICE-PRO Study

Background Chronic diseases account for nearly 60% of deaths around the world. The extent of this silent epidemic has not met determined responses in governments, policies or professionals in order to transform old Health Care Systems, configured for acute diseases. There is a large list of research about alternative models for people with chronic conditions, many of them with an advanced practice nurse as a key provider, as case management. But some methodological concerns raise, above all, the design of the intervention (intensity, frequency, components, etc). Methods/Design Objectives: General: To develop the first and second phases (theorization and modeling) for designing a multifaceted case-management intervention in people with chronic conditions (COPD and heart failure) and their caregivers. Specific aims: 1) To identify key events in people living with chronic disease and their relation with the Health Care System, from their point of view. 2) To know the coping mechanisms developed by patients and their caregivers along the story with the disease. 3) To know the information processing and its utilization in their interactions with health care providers. 4) To detect potential unmet needs and the ways deployed by patients and their caregivers to resolve them. 5) To obtain a description from patients and caregivers, about their itineraries along the Health Care System, in terms of continuity, accessibility and comprehensiveness of care. 6) To build up a list of promising case-management interventions in patients with Heart Failure and COPD with this information in order to frame it into theoretical models for its reproducibility and conceptualization. 7) To undergo this list to expert judgment to assess its feasibility and pertinence in the Andalusian Health Care. Design: Qualitative research with two phases: For the first five objectives, a qualitative technique with biographic stories will be developed and, for the remaining objectives, an expert consensus through Delphi technique, on the possible interventions yielded from the first phase. The study will be developed in the provinces of Almería, Málaga and Granada in the Southern Spain, from patients included in the Andalusian Health Care Service database with the diagnosis of COPD or Heart Failure, with the collaboration of case manager nurses and general practitioners for the assessment of their suitability to inclusion criteria. Patients and caregivers will be interviewed in their homes or their Health Centers, with their family or their case manager nurse as mediator. Discussion First of a series of studies intended to design a case-management service for people with heart failure and COPD, in the Andalusian Health Care System, where case management has been implemented since 2002. Accordingly with the steps of a theoretical model for complex interventions, in this study, theorization and intervention modeling phases will be developed.This research was carried out with the support of one research grant, awarded by the Regional Health Ministry of Andalusia (Exp. 0222/2008

Competencias de Comunicación y Liderazgo en el grado de Comunicación Audiovisual

[ES] La pretensión de esta comunicación es mostrar la experiencia educativa transversal en la adquisición de competencias con alumnos de primer curso del grado de Comunicación Audiovisual. Se trata de una experiencia enmarcada en una praxis amplia de proyectos transversales en la Universidad Francisco de Vitoria, que responde a la voluntad de diálogo entre las disciplinas y a una misión compartida de formación integral del alumno.Gambarini Duarte, MF.; Del Valle Morilla, A.; Alejos Bermejo, JM.; León Fernandez, R. (2019). Competencias de Comunicación y Liderazgo en el grado de Comunicación Audiovisual. En INNODOCT/18. International Conference on Innovation, Documentation and Education. Editorial Universitat Politècnica de València. 389-397. https://doi.org/10.4995/INN2018.2018.8783OCS38939

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy

<p>Abstract</p> <p>Background</p> <p>Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new <it>Halorubrum tebenquichense </it>strains isolated from grey crystals (<it>GC</it>) and black mood (<it>BM</it>) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form <it>BM </it>(ARC-BM) and from <it>GC </it>(ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined.</p> <p>Results</p> <p>DNA G+C content (59.5 and 61.7% mol <it>BM </it>and <it>GC</it>, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that <it>BM </it>and <it>GC </it>isolates were two non-previously described strains of <it>H. tebenquichense</it>. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response.</p> <p>Conclusion</p> <p>We herein report the finding of new <it>H. tebenquichense </it>non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.</p

The Development of the Bengamides as New Antibiotics against Drug-Resistant Bacteria

The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.Supported by grants RTI2018-098296-BI00 (Ministerio de Ciencia e Innovación), PI19/01478 from Instituto de Salud Carlos III (ISCIII) (FEDER), P20_00540 (Andalusian Government and FEDER), K99GM138758 and R35GM136286 (National Institute of General Medical Sciences of the National Institutes of Health), A-CTS-666-UGR20 (University of Granada) (FEDER), CTS-107 (Andalusian Government) and 2021-GRIN-30998 (University of Castilla-La Mancha). Partial funding for open access charge: Universidad de Málag

Determination of essential biomarkers in lung cancer: a real-world data study in Spain.

Background: The survival of patients with lung cancer has substantially increased in the last decade by about 15%. This increase is, basically, due to targeted therapies available for advanced stages and the emergence of immunotherapy itself. This work aims to study the situation of biomarker testing in Spain. Patients and Methods: The Thoracic Tumours Registry (TTR) is an observational, prospective, registry-based study that included patients diagnosed with lung cancer and other thoracic tumours, from September 2016 to 2020. This TTR study was sponsored by the Spanish Lung Cancer Group (GECP) Foundation, an independent, scientific, multidisciplinary oncology society that coordinates more than 550 experts and 182 hospitals across the Spanish territory. Results: 9,239 patients diagnosed with stage IV non-small cell lung cancer (NSCLC) between 2106 and 2020 were analysed. 7,467 (80.8%) were non-squamous and 1,772 (19.2%) were squamous. Tumour marker testing was performed in 85.0% of patients with non-squamous tumours vs 56.3% in those with squamous tumours (p-value <0.001). The global testing of EGFR, ALK, and ROS1 was 78.9%, 64.7%, 35.6% respectively, in non-squamous histology. PDL1 was determined globally in the same period (46.9%), although if we focus on the last 3 years it exceeds 85%. There has been a significant increase in the last few years of all determinations and there are even close to 10% of molecular determinations that do not yet have targeted drug approval but will have it in the near future. 4,115 cases had a positive result (44.5%) for either EGFR, ALK, KRAS, BRAF, ROS1, or high PDL1. Conclusions: Despite the lack of a national project and standard protocol in Spain that regulates the determination of biomarkers, the situation is similar to other European countries. Given the growing number of different determinations and their high positivity, national strategies are urgently needed to implement next-generation sequencing (NGS) in an integrated and cost-effective way in lung cancer

Determination of essential biomarkers in lung cancer : a real-world data study in Spain with demographic, clinical, epidemiological and pathological characteristics

Background The survival of patients with lung cancer has substantially increased in the last decade by about 15%. This increase is, basically, due to targeted therapies available for advanced stages and the emergence of immunotherapy itself. This work aims to study the situation of biomarker testing in Spain. Patients and methods The Thoracic Tumours Registry (TTR) is an observational, prospective, registry-based study that included patients diagnosed with lung cancer and other thoracic tumours, from September 2016 to 2020. This TTR study was sponsored by the Spanish Lung Cancer Group (GECP) Foundation, an independent, scientific, multidisciplinary oncology society that coordinates more than 550 experts and 182 hospitals across the Spanish territory. Results Nine thousand two hundred thirty-nine patients diagnosed with stage IV non-small cell lung cancer (NSCLC) between 2106 and 2020 were analysed. 7,467 (80.8%) were non-squamous and 1,772 (19.2%) were squamous. Tumour marker testing was performed in 85.0% of patients with non-squamous tumours vs 56.3% in those with squamous tumours (p-value < 0.001). The global testing of EGFR, ALK, and ROS1 was 78.9, 64.7, 35.6% respectively, in non-squamous histology. PDL1 was determined globally in the same period (46.9%), although if we focus on the last 3 years it exceeds 85%. There has been a significant increase in the last few years of all determinations and there are even close to 10% of molecular determinations that do not yet have targeted drug approval but will have it in the near future. 4,115 cases had a positive result (44.5%) for either EGFR, ALK, KRAS, BRAF, ROS1, or high PDL1. Conclusions Despite the lack of a national project and standard protocol in Spain that regulates the determination of biomarkers, the situation is similar to other European countries. Given the growing number of different determinations and their high positivity, national strategies are urgently needed to implement next-generation sequencing (NGS) in an integrated and cost-effective way in lung cancer

Lymphangioleiomyomatosis biomarkers linked to lung metastatic potential and cell stemness

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM