Nuevas estrategias de imagenología molecular en cáncer

La imagenología molecular es una disciplina que abarca innumerables herramientas. Entre sus aplicaciones se encuentra el diagnóstico in vivo del cáncer con agentes que reconocen marcadores tumorales y que a su vez poseen óptimas características para obtención de imágenes. Asimismo, proporciona información clínicamente fundamental, permitiendo una correcta selección del tratamiento a seguir y el monitoreo de sus efectos. Detalles como la localización, tamaño, morfología y cambios estructurales de un área afectada pueden ser detectados a través de los métodos convencionales de imagen. Sin embargo, técnicas de imagenología molecular como la SPECT, PET y FMT, entre otras, son cada vez más utilizadas. Debido a su característica no invasiva la imagen molecular permite evaluar la patología en su contexto, siendo clave para entender el proceso tumoral, sin perturbar el ambiente y proporcionando información adicional a los métodos convencionales. La estadificación tumoral, detección de metástasis, cirugía guiada, así como la cuantificación de una lesión son diferentes aplicaciones en este área. En este contexto, es muy importante el conocimiento de marcadores tumorales y el desarrollo de nuevos agentes de imagenología molecular específicos de estos. Los aptámeros son oligonucleótidos (DNA o RNA) que tienen la característica de reconocer su blanco con alta afinidad y especificidad. Similar a los anticuerpos en estas características, los aptámeros presentan ventajas que los hacen interesantes para su aplicación como agentes de imagen o terapia. El Sgc8-c es una secuencia truncada del aptámero sgc8, el cual muestra una unión específica y con alta afinidad al receptor PTK7. Sgc8-c tiene solamente 41 bases y una Kd de 0.78 nM para este receptor. Si bien este receptor está presente en células normales, su sobreexpresión ha sido observada en cáncer de colon, tumores gástricos, cáncer de pulmón, próstata, mama e incluso en metástasis. Si bien, su participación en la oncogénesis parece ser clara, aun no es un receptor extensamente caracterizado. El presente trabajo consistió en la investigación y desarrollo del aptámero Sgc8-c como agente de imagenología molecular en cáncer. Para ello Sgc8-c fue modificado en su extremo 5 con HYNIC, DOTA y se marcó con Tecnecio-99m y Galio-67 respectivamente. Asimismo el aptámero Sgc8-c fue marcado con Alexa647, para su evaluación como agente de imagen óptica en el infrarrojo cercano. Todos los agentes desarrollados fueron evaluados fisicoquímicamente y frente a diferentes líneas celulares tumorales. Finalmente, las características de biodistribución y farmacocinética fueron estudiadas y tres modelos tumorales en ratón fueron seleccionados para realizar imágenes. Resumen 3 Molecular imaging is a discipline that involves many tools. One of its major applications is the in vivo diagnosis of cancer, using molecular probes that recognize tumor markers and have optimal characteristics for imaging. It also provides clinically important information, allowing the correct choice of treatment options and monitoring their effects. Details such as the location, size, morphology and structural changes of an affected area can be detected by conventional imaging methods. Moreover, molecular imaging techniques such as SPECT, PET and FMT, among others, are increasingly used. Due to its minimal invasive characteristics, molecular imaging allows the evaluation of the pathology in context, being key to understanding tumor process without disturbing the environment and providing additional information to conventional methods. Tumor staging, detection of metastasis, guided surgery, as well as quantification of disease are different applications in this area. In this context, knowledge of tumor markers and the development of new targeted molecular imaging agents is very important. Aptamers are oligonucleotides (DNA or RNA), which have the characteristics to recognize their target with high affinity and specificity. Similar to antibodies in their molecular recognition properties, aptamers offer advantages that make them interesting for use as imaging agents or therapy. Sgc8-c is a truncated aptamer sequence, which exhibits specific binding and high affinity for the receptor PTK7. The Sgc8-c aptamer has only 41 bases, but its Kd for its receptor is 0.78 nM. Although this receptor is present in normal cells, its overexpression has been observed in colon, gastric tumors, lung, prostate, breast cancer and even their metastases. While participation in oncogenesis seems clear, the receptor needs to be characterized more extensively in vivo as a valid biomarker. The work presented describes the research and development of aptamer Sgc8-c as a molecular imaging agent for cancer. Sgc8-c was modified at its 5 'end with radiometal chelators HYNIC and DOTA and labeled with technetium-99m and gallium-67 respectively. Also, the Sgc8-c aptamer was labeled with the near infrared dye Alexa647 for evaluation as an optical imaging agent. All agents were developed and evaluated physicochemically against different tumor cell lines. Finally, the pharmacokinetics and biodistribution characteristics of the labeled Sgc8-c aptamer were studied and three mouse tumor models were selected for imaging

Metastatic and non-metastatic melanoma imaging using Sgc8-c aptamer PTK7-recognizer

Existe información complementaria en: https://doi.org/10.1038/s41598-021-98828-6Melanoma is one of the most aggressive and deadly skin cancers, and although histopathological criteria are used for its prognosis, biomarkers are necessary to identify the different evolution stages. The applications of molecular imaging include the in vivo diagnosis of cancer with probes that recognize the tumor-biomarkers specific expression allowing external image acquisitions and evaluation of the biological process in quali-quantitative ways. Aptamers are oligonucleotides that recognize targets with high affinity and specificity presenting advantages that make them interesting molecular imaging probes. Sgc8-c (DNA-aptamer) selectively recognizes PTK7-receptor overexpressed in various types of tumors. Herein, Sgc8-c was evaluated, for the first time, in a metastatic melanoma model as molecular imaging probe for in vivo diagnostic, as well as in a non-metastatic melanoma model. Firstly, two probes, radio- and fluorescent-probe, were in vitro evaluated verifying the high specific PTK7 recognition and its internalization in tumor cells by the endosomal route. Secondly, in vivo proof of concept was performed in animal tumor models. In addition, they have rapid clearance from blood exhibiting excellent target (tumor)/non-target organ ratios. Furthermore, optimal biodistribution was observed 24 h after probes injections accumulating almost exclusively in the tumor tissue. Sgc8-c is a potential tool for their specific use in the early detection of melanoma.ANII: POS_NAC_2017_1_14036

Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damagedSpanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015 to J.A.T.]; Fundación Ramón Areces (Institutional Grant to the Centro de Biologıa Molecular Severo Ochoa); Spanish Ministry of Economy and Competitiveness (predoctoral fellowships to M.V.V and M.A.O-B.); Universidad Autónoma de Madrid (predoctoral fellowship to M.G-F.); Consejo Superior de Investigaciones Cientıficas (JAE-Doc contract to M.S.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015]Peer Reviewe

Terapia con luz en el tratamiento de la hidradenitis supurativa.

Resumen Antecedentes: la terapia con luz es una alternativa en el tratamiento de las lesiones causadas por la hidradenitis supurativa, ya sea utilizando fotosensibilizador o no, aplicándola intralesional o tópica. Objetivos: evaluar la eficacia, efectividad y seguridad de las distintas modalidades de terapia con luz en el tratamiento de la hidradenitis supurativa, mediante una revisión sistemática. Material y métodos: se han considerado los trabajos realizados en pacientes con hidradenitis supurativa, mayoritariamente series de casos y revisiones sistemáticas. Se han identificado los estudios mediante una búsqueda electrónica en las siguientes bases de datos: MEDLINE, EMBASE, Universidad de York, Cochrane Database of Systematic Reviews, Cochrane Skin Group, Centre of Evidence based dermatology de la Universidad de Nottingham, TESEO. Adicionalmente, se han buscado los trabajos registrados en webs electrónicas: ClinicalTrials.gov, Registro Español de Ensayos Clínicos, Clinicaltrialsregister.eu. Resultados: se incluyeron 6 series de casos y 1 revisión sistemática; 1 estudio utilizaba láser nd:YAG, 1 estudio luz intensa pulsada, y 6 TFD, con un total de 133 pacientes tratados. Los resultados estadísticamente significativos de la terapia con luz, se observan en la medición del índice de calidad de vida dermatológica (DQLI), con una reducción de su puntuación comparando antes y después de cada tratamiento; observándose así: disminución de 21 puntos en 4 sesiones de TFD tópica con ALA (Andino R(1)); 89,3% de mejora en dicha puntuación con TFD intralesional con AM (Agut-Busquet E(2)) y disminución de 19.3 puntos después de la cirugía más TFD con ALA (Bu W(3)). En cuanto a la efectividad medida mediante la escala de Sartorius se objetiva: mejoría de las lesiones de hidradenitis del 77,3% con TFD tópica con AM (Fadel M.A(4)); respuesta completa entre el 37% y 76,3% de los pacientes tratados con TFD intralesional con ALA al 5% (Suárez MJ(5)) y de los pacientes tratados con TFD intralesional con ALA al 1% (Valladares LM(6)); reducción del 12% inmediatamente después de la terapia, 10% a los 3 y 6 meses y 3% a los 12, en el tratamiento con luz intensa pulsada (Highton L(7)); después de 3 meses de tratamiento con láser nd:YAG (Tierney E(8)) reducción del 65.3% sobre todas las regiones anatómicas, 73.4% inguinal, 62.0% axilar y 53.1% inframamaria. En general, el riesgo de sesgos fue elevado y la calidad de las publicaciones baja. Conclusión: se necesitan ensayos clínicos controlados y aleatorizados que confirmen la eficacia de cada uno de estos tratamientos, ya se TFD, láser o luz intensa pulsada y estandaricen el protocolo más adecuado para poder garantizar un adecuado nivel de evidencia en términos de eficacia y seguridad

T908 polymeric micelles improved the uptake of Sgc8-c aptamer probe in tumor-bearing mice: A co-association study between the probe and preformed nanostructures

Aptamers are oligonucleotides that have the characteristic of recognizing a target with high affinity and specificity. Based on our previous studies, the aptamer probe Sgc8-c-Alexa647 is a promising tool for molecular imaging of PTK7, which is an interesting biomarker in cancer. In order to improve the delivery of this probe as well as create a novel drug delivery nanosystem targeted to the PTK7 receptor, we evaluate the co-association between the probe and preformed nanostructures. In this work, preformed pegylated liposomes (PPL) and linear and branched pristine polymeric micelles (PMs), based on PEO–PPO–PEO triblock copolymers were used: poloxamer F127® and poloxamines T1307® and T908®. For it, Sgc8-c-Alexa647 and its co-association with the different nanostructures was exhaustively analyzed. DLS analysis showed nanometric sizes, and TEM and AFM showed notable differences between free-and co-associated probe. Likewise, all nanosystems were evaluated on A20 lymphoma cell line overexpressing PTK7, and the confocal microscopy images showed distinctness in cellular uptake. Finally, the biodistribution in BALB/c mice bearing lymphoma-tumor and pharmacokinetic study revealed an encouraging profile for T908-probe. All data obtained from this work suggested that PMs and, more specifically T908 ones, are good candidates to improve the pharmacokinetics and the tumor uptake of aptamer-based probes.Fil: Castelli, Romina. Universidad de la República; UruguayFil: Ibarra, Manuel. Universidad de la República; UruguayFil: Faccio, Ricardo. Universidad de la República; UruguayFil: Miraballes, Iris. Universidad de la República; UruguayFil: Fernández, Marcelo. Universidad de la República; UruguayFil: Moglioni, Albertina Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Cabral, Pablo. Universidad de la República; UruguayFil: Cerecetto, Hugo. Universidad de la República; UruguayFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Calzada, Victoria. Universidad de la República; Urugua

Conocimientos, actitudes y prácticas sobre la malaria en la población indígena guna de la comarca de Madungandí, Panamá, 2012

Introduction: Knowledge, attitudes and practices surveys allow to determine the degree of knowledge on the management of malaria in a given population, as well as the attitudes and practices that contribute or not to its transmission.Objective: To identify the knowledge, attitudes and practices that favor or not the transmission of malaria in the indigenous Guna population of Madungandí.Materials and methods: A cross-sectional study was conducted by applying a survey to the heads of the families in a sample of 40% of households in three communities with high malaria incidence. Local Guna residents and translators were part of the research team that applied the questionnaires. The statistical analysis was performed in Epi-Info 6.04.Results: The age range of those surveyed was between 20 and 70 years. All responders indicated that they belonged to and spoke the language of the Guna ethnic group, 64% were male and 30% were illiterate. Half (51%) of the responders declared they had suffered malaria at least once in the last eight years, and 89% accepted that malaria was a health problem. Sixty-three per cent responded that their traditional doctors, “inadule”, cured malaria and 7.0 % practiced the “pipe smoking” and “cocoa burn” rituals to prevent the disease.Conclusion: Considering the limited knowledge about malaria and its vector, as well as the willingness to collaborate shown by the Guna population, it is essential to initiate educational and participative programs to improve control and prevention activities in the communities aimed at achieving a reduction in malaria incidence in the Madungandí indigenous region.Introducción. Las encuestas sobre comportamientos, actitudes y prácticas permiten determinar el grado de conocimiento de la población sobre la malaria o paludismo, así como las actitudes y prácticas que contribuyen a su transmisión.Objetivo. Identificar los conocimientos, actitudes y prácticas que favorecen la transmisión de la malaria en la población indígena guna de Madungandí.Materiales y métodos. Se hizo un estudio transversal mediante una encuesta a los jefes de familia de una muestra de 40 % de las viviendas en tres comunidades con alta incidencia de malaria. La encuesta se hizo con encuestadores gunas e integrantes del equipo de investigación, y con la ayuda de un traductor. El análisis se hizo en Epi-Info 6.04.Resultados. El rango de edad de los encuestados fluctuaba entre los 20 y los 70 años. Todos los encuestados indicaron pertenecer a la etnia guna y hablar su lengua; 64 % eran del sexo masculino y 30 % eran analfabetas. La mitad de los encuestados (51 %) manifestó haber enfermado de malaria, por lo menos, una vez en los últimos ocho años y 89 % reconoció la enfermedad como un problema de salud. El 63 % manifestó que el médico tradicional “inadule” curaba la malaria, y 78 % practicaba la “fuma de la pipa” y la “quema del cacao” para prevenirla.Conclusión. Dado el limitado conocimiento sobre la malaria y su vector, así como la actitud de colaboración de la población guna, es necesario iniciar programas educativos y de participación comunitaria para mejorar las prácticas de prevención y control en las comunidades, y disminuir así la incidencia de la enfermedad en la comarca de Madungandí

Preliminary in vivo characterization of a theranostic aptamer: Sgc8-c- DOTA-67Ga

Nucleic acid aptamers can recognise their target with high affinity and specificity, and their potential as molecular imaging agents and use in theranostics are being explored. Compared with antibodies, aptamers can be easily synthesized and chemically modified, rendering them a valuable tool for in vivo approaches. Herein, we investigated a 41nt DNA aptamer as a theranostic agent for lymphoma and melanoma. This aptamer exhibits specific binding and high affinity for the PTK7 receptor that is overexpressed in many cancer cells. A 5’-amino-derivative of the Sgc8-c aptamer was bound to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid) and labelled with the radionuclide 67Ga, forming the aptamer probe Sgc8- c-DOTA-67Ga. Different conditions during synthesis, purification and identification of the intermediate and final radiolabelled probe, were examined. Aptamer modification and radiolabelling were performed with high yields, resulting in a probe that was stable in neutral buffered solution. Binding to PTK7 was studied in CCRFCEM, A20 and B16F1 cell lines, and in purified PTK7-1 receptor, to confirm specificity. The in vitro cell lines showed different levels of uptake, and the signal increased over time. In vivo binding properties were studied in A20 and B16F10 tumour-bearing mice and images were acquired using X-rays and gamma imaging modalities for both models. Preliminary results in both tumour models showed good aptamer uptake by tumour. Hepatobiliar metabolism was observed with Sgc8-c-DOTA-67Ga and no signal was detected in normal tissue. In summary, these results support the utility of labelled aptamers as theranostic agents in different imaging modalities and theranostic

In vivo evaluation of Sgc8-c aptamer as a molecular imaging probe for colon cancer in a mouse xenograft model

Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications

Zn(II) detection in biological samples with a smart sensory polymer

We have developed a new sensory material for the rapid and inexpensive determination of Zn(II), and we have carried out a proof of concept for the determination of Zn(II) in biological samples. The interaction with Zn(II) generates an OFF-ON fluorescence process on the material, which can be recorded both with a fluorimeter and with a smartphone by analyzing the RGB components of the taken photographs. This sensory material is prepared with 99.75% of commercially available monomers and contains 0.25% of a sensory monomer based on a quinoline structure. The sensory motifs are chemically anchored to the polymeric structure, and, accordingly, no migration of organic substances from the material occurs during the sensing process. Our method has been tested with freshly prepared Zn(II) aqueous solutions, but also with biological samples from exudates of chronic wounds. The proposed methodology provides limits of detection (LOD) of 13 and 27 ppb when employing a water-soluble polymer (WsP) and a hydrophilic polymeric film (HP), respectively, using emission spectroscopy. The measurements have been contrasted with ICP-MS as the reference method, obtaining reliable data. This study is the starting point toward a larger investigation with patients, which will address the challenge of establishing a direct relationship between the concentration of zinc(II), other cations and also of amino acids, with the protease activity and, finally, with the state/evolution of chronic wounds. In this context, the proposed sensory material and others we are now working with will act as a simple and cheap method for this purpose.FEDER (Fondo Europeo de Desarrollo Regional), and both the Spanish Ministerio de Economía, Industria y Competitividad (MAT2017-84501-R) and the Consejería de Educación—Junta de Castilla y León (BU061U16