Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. FCT-MCTES is also acknowledged for 2020.07660.BD for BBO. Open access funding provided by FCT|FCCN (b-on).info:eu-repo/semantics/publishedVersio

Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells

The authors would like to acknowledge the financial support of the NCCR Bioinspired Materials through the Swiss National Science Foundation, the Adolphe Merkle Foundation. Aura Maria Moreno-Echeverri and Barbara Rothen-Rutishauser acknowledge the Swiss National Science Foundation (SNSF) (project 310030_192056/1). Publisher Copyright: Copyright © 2023 Susnik, Bazzoni, Taladriz-Blanco, Balog, Moreno-Echeverri, Glaubitz, Brito Oliveira, Ferreira, Viana Baptista, Petri-Fink and Rothen-Rutishauser.Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs. Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC. Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC. Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery.publishersversionpublishe

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine

Epidermal growth factor alters silica nanoparticle uptake and improves gold-nanoparticle-mediated gene silencing in A549 cells

Introduction: Delivery of therapeutic nanoparticles (NPs) to cancer cells represents a promising approach for biomedical applications. A key challenge for nanotechnology translation from the bench to the bedside is the low amount of administered NPs dose that effectively enters target cells. To improve NPs delivery, several studies proposed NPs conjugation with ligands, which specifically deliver NPs to target cells via receptor binding. One such example is epidermal growth factor (EGF), a peptide involved in cell signaling pathways that control cell division by binding to epidermal growth factor receptor (EGFR). However, very few studies assessed the influence of EGF present in the cell environment, on the cellular uptake of NPs.Methods: We tested if the stimulation of EGFR-expressing lung carcinomacells A549 with EGF affects the uptake of 59 nm and 422 nm silica (SiO2) NPs. Additionally, we investigated whether the uptake enhancement can be achieved with gold NPs, suitable to downregulate the expression of cancer oncogene c-MYC.Results: Our findings show that EGF binding to its receptor results in receptor autophosphorylation and initiate signaling pathways, leading to enhanced endocytosis of 59 nm SiO2 NPs, but not 422 nm SiO2 NPs. Additionally, we demonstrated an enhanced gold (Au) NPs endocytosis and subsequently a higher downregulation of c-MYC.Discussion: These findings contribute to a better understanding of NPs uptake in the presence of EGF and that is a promising approach for improved NPs delivery

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Narcissism and the strategic pursuit of short-term mating : universal links across 11 world regions of the International Sexuality Description Project-2.

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating

Narcisismo y búsqueda estratégica del emparejamiento a corto plazo a través de las culturas: Enlaces omnipresentes a través de 11 regiones mundiales del Proyecto de la descripción de la sexualidad internacional 2

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating.Estudios previos, en primer lugar a través de las muestras de culturas occidentales, han documentado asociaciones sistemáticas del narcisismo subclínico con múltiples indicadores de estrategias del emparejamiento a corto plazo (p. ej. sociosexualidad ilimitada, infidelidad, caza de pareja). En este estudio se han usado respuestas de la encuesta transcultural de 30.470 personas de 53 naciones de 11 regiones mundiales (América del Norte, América del Sur/América Central, Europa del Norte, Europa del Oeste, Europa del Este, Europa del Sur, Oriente Próximo, África, Asia del Sur/Sudoeste de Asia, Asia del Este y Oceanía) para evaluar si el narcisismo (medido por el Inventario de Personalidad Narcisista; NPI) se asocia panuniversalmente con los indicadores del emparejamiento a corto plazo, tanto en la dirección, como en la intensidad. Los resultados sugieren que el narcisismo (incluidos muchos aspectos suyos medidos por el NPI) tiene las mismas asociaciones básicas con los rasgos de personalidad relacionados con el sexo (p. ej. extraversión alta) y con los resultados sexuales claves (p. ej. búsqueda más activa de las estrategias del emparejamiento a corto plazo) a través de las 11 mayores regiones mundiales del PDSI 2. La discusión se enfoca en las implicaciones y limitaciones del estudio actual

Avaliação do potencial antigênico da candidata a vacina antimalárica GMZ2.6c e seus componentes, Proteína 3 da Superfície do Merozoíto (MSP-3), Proteína Rica em Glutamato (GLURP) e Antígeno de Pré-fertilização 48/45 (Pfs48/45), em populações expostas à malária na Amazônia brasileira

Made available in DSpace on 2018-03-07T16:41:41Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) barbara_baptista_ioc_mest_2017.pdf: 6124125 bytes, checksum: 807b7fac3978141c5aa510a2d6e43b3a (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A malária é causada por protozoários do gênero Plasmodium. Estima-se que ocorram 212 milhões de casos e 300 mil mortes decorrentes de malária anualmente, principalmente devido à infecção por P. falciparum. Assim, uma vacina eficaz é possivelmente a arma potencial para reduzir de forma drástica essas estatísticas. Entretanto, embora mais de 30 antígenos tenham sido identificados como candidatos a vacina, até o momento nenhum deles gerou uma perspectiva sólida de que uma vacina possa estar disponível nos próximos anos. Torna-se assim fundamental a pesquisa de novos candidatos vacinais. A GMZ2.6c é uma proteína recombinante que contém fragmentos de 3 antígenos candidatos a vacina contra P. falciparum, MSP-3, GLURP e Pfs48/45. Ensaios clínicos realizados na África mostraram que a GMZ2, contendo MSP-3 e GLURP, é bem tolerada, segura e imunogênica e que os anticorpos induzidos foram específicos e funcionais, capazes de controlar o crescimento in vitro do P. falciparum. Importa, entretanto, verificar se o mesmo potencial é apresentado pelas populações-alvo no Brasil, uma vez que os parasitos circulantes aqui são geneticamente distintos daqueles na África e também porque a constituição genética da população pode influenciar na resposta imune aos antígenos vacinais. No presente trabalho, nos propomos a avaliar a resposta imune humoral contra a GMZ2.6c e seus componentes (MSP-3, GLURP e Pfs48/45) em indivíduos residentes em áreas endêmicas de malária da Amazônia Brasileira. O estudo foi concentrado nas cidades de Cruzeiro do Sul (Acre), Mâncio Lima (Acre) e Guajará (Amazonas). Foram coletadas 124 amostras em Cruzeiro do Sul (grupo CZS), 88 em Mâncio Lima (grupo ML) e 87 em Guajará (grupo GJ) Também, foram coletadas amostras de 53 indivíduos sem história de malária, que foram usadas como controles de área. Foi avaliada a resposta imune contra a proteína recombinante GMZ2.6c e os fragmentos individuais. Para mapeamento epitópico, foram utilizados 4 peptídeos correspondentes a diferentes regiões da MSP-3, 13 da GLURP e 2 de Pfs48/45. A pesquisa de anticorpos IgG, IgM, IgE e IgA contra as proteínas recombinantes e o mapeamento de epítopos B da GMZ2.6c foram realizadas pela técnica de ELISA. A pesquisa de anticorpos IgG1, IgG2, IgG3 e IgG4 foi realizada em todas as amostras que apresentaram anticorpos IgG. Nossos dados mostraram que a maior parte dos indivíduos dos grupos CZS (84%), ML (74%) e GJ (72%) apresentavam anticorpos que reconheciam a GMZ2.6c e que esses anticorpos foram principalmente da classe IgG. Dentre os fragmentos da GMZ2.6c, a GLURP-R0 foi a mais amplamente reconhecida nos três grupos estudados e os anticorpos dirigidos a esta região foram, preferencialmente, da classe IgG. As subclasses de IgG que predominaram foram as citofílicas (IgG1 e IgG3), fato importante considerando que a aquisição de uma imunidade protetora está associada aos níveis de IgG1 e IgG3. O mapeamento epitópico da GMZ2.6c mostrou que os epítopos imunodominantes foram P11 e P3, derivados da GLURP-R0. Em conjunto, nossos dados mostraram que a proteína GMZ2.6c é amplamente reconhecida por anticorpos nas populações estudadas e que entre os componentes individuais da GMZ2.6c, a GLURP se revelou mais imunogênica durante a infecção naturalMalaria is caused by blood-borne apicomplexan parasites of the genus Plasmodium. According to the latest estimates approximately 212 million cases and 300 000 deaths due to malaria occur worldwide, mainly due P. falciparum infection. Thus, an effective vaccine is possibly the potential weapon to drastically reduce these statistics. However, although more than 30 antigens have been identified as vaccine candidates, so far none have generated a solid prospect that a vaccine may be available in the next years. The search for new vaccine candidates is therefore fundamental. GMZ2.6c is a recombinant protein containing fragments of 3 P. falciparum vaccine candidate, MSP-3, GLURP and Pfs48/45. Clinical trials performed in Africa have shown that GMZ2, containing MSP-3 and GLURP, is well tolerated, safe and immunogenic and that antibodies induced were able to control the in vitro P. falciparum growth. However, it is important to verify if this potential is observed in individuals living in Brazil, since circulating parasites are genetically different from those in Africa and the genetic background of the population can influence the immune response to the vaccine antigens. In this work, we evaluated the humoral immune response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in individuals living in malaria endemic areas in the Brazilian Amazon. This study was carried out in Cruzeiro do Sul (Acre), Mâncio Lima (Acre) and Guajará (Amazonas) A total of 124 samples were collected in Cruzeiro do Sul (CZS group), 88 in Mâncio Lima (ML group) and 87 in Guajará (GJ group). Samples from 53 individuals with no previous malaria episode were collected and used as area controls. IgG, IgM, IgE and IgA antibodies against GMZ2.6c and the individual fragments were evaluated by ELISA. For epitope mapping, 4 peptides corresponding to different regions of MSP-3, 13 of GLURP and 2 of Pfs48/45 were used. IgG1, IgG2, IgG3 and IgG4 antibodies against recombinant proteins were analyzed in all samples that presented IgG antibodies. Our data showed that most individuals in the CZS (84%), ML (74%) and GJ (72%) groups had antibodies that recognized GMZ2.6c and that these antibodies were mainly of the IgG class. Among the fragments of GMZ2.6c, GLURP-R0 was the more widely recognized in the three groups studied and the antibodies against this region were mainly of the IgG class. The predominant IgG subclasses were cytophilic (IgG1 and IgG3), an important fact considering that the acquisition of a protective immunity against malaria is associated with IgG1 and IgG3 levels. The epitope mapping of GMZ2.6c showed that the immunodominant epitopes were P11 and P3 from GLURP-R0. Our data showed that the GMZ2.6c protein is widely recognized by antibodies in the studied populations and that among the individual components of GMZ2.6c, GLURP was more immunogenic during natural infectio