Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

DNA aptamers selection for Staphylococcus aureus cells by SELEX and Cell-SELEX.

BACKGROUND: Staphylococcus aureus is the most common bacteria found in skin, soft tissues, bone, and bone prostheses infections. The aim of this study was to select DNA aptamers for S. aureus to be applied in the diagnosis of bacteria. METHODS AND RESULTS: We used SELEX (Systematic Evolution of Ligands by EXponencial Enrichment) for peptidoglycan followed by cell-SELEX with S. aureus cells as target. Four sequences showed significantly higher binding to S. aureus distinguishing it from the control cells of other significant microbial species: Escherichia coli, Candida albicans, Streptococcus pyogenes and Streptococcus pneumoniae. In particular, ApSA1 (Kd = 62.7 ± 5.6 nM) and ApSA3 (Kd = 43.3 ± 3.0 nM) sequences combined high affinity and specificity for S. aureus, considering all microorganisms tested. CONCLUSIONS: Our results demonstrated that these aptamers were able to identify peptidoglycan in the S. aureus surface and have great potential for use in the development of radiopharmaceuticals capable to identify S. aureus infectious foci, as well as in other aptamer-based methodologies for bacteria diagnosis.

Adipose-derived stem/stromal cell secretome modulates breast cancer cell proliferation and differentiation state towards aggressiveness.

It is becoming increasingly evident that mesenchymal stem/stromal cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor proliferation, angiogenesis, invasion, and metastasis, as well as mediate therapeutic resistance. Consequently, understanding the regulatory mechanisms of ASCs that influence the tumor microenvironment may provide an avenue for further treatment. To understand the role of the ASC secretome in breast cancer cell proliferation, death, and phenotype alteration, adipose-derived stem cell-conditioned medium (mASC) was used to cultivate MCF-7 and MDA-MB-231 cells. These breast cancer cells in mASC showed a shorter doubling time, higher frequency of EdU positivity, and higher levels of phosphorylated histone 3. In addition, increased expression of cyclin B1 was observed, suggesting that proliferation was induced. The mASC was also able to increase apoptosis in MCF-7 cells, which was confirmed by caspase-7 activation. The number of tumor-initiating cells (CD44+ CD24-/low) and migration capacity were increased in cells cultivated in mASC. These data collectively suggest that ASC-conditioned medium can induce selective pressure by increasing cell proliferation, giving rise to a more aggressive phenotype in MCF-7 and MDA-MB-231 cells. Our study provides a foundation for further elucidation of the precise mechanism underlying ASCs in breast cancer cells and the modulation of ASCs in potential therapeutic uses.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Human adipose-derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing.

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

DNA aptamers selection for carcinoembryonic antigen (CEA).

Carcinoembryonic antigen (CEA) is a glycoprotein antigen generally used for diagnosis, prognosis and treatment monitoring of several types of tumors, including colorectal cancer. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain aptamers specific to CEA for use as radiopharmaceuticals in colorectal cancer diagnosis. Five aptamers were selected through the Systematic Evolution of Ligands by EXponencial Enrichment (SELEX) and tested using T84 (CEA+) and Hela (CEA-) cells. Apta 3 and Apta 5 showed the best results presenting high specificity and affinity for T84 cells, with dissociation constants (Kd) of 60.4 ± 5.7 nM and 37.8 ± 5.8 nM, respectively. These results indicate that Apta 3 and Apta 5 are promising candidates for identifying tumor cells that overexpress CEA.

Pneumonia associada à ventilação mecânica: conhecimento dos profissionais de saúde acerca da prevenção e medidas educativas / Ventilator-associated pneumonia: the knowledge of health professionals towards prevention and educational measures / La neumonía asociada a la ventilación mecánica: el conocimiento de los profesionales de la salud acerca de las medidas de prevención y educación

Objetivo: Avaliar o conhecimento dos profissionais de saúde sobre a prevenção da Pneumonia Associada à Ventilação Mecânica (PAVM) em pacientes críticos internados nas Unidades de Terapia Intensiva (UTIs) e, promover educação permanente (EP) para profissionais das UTIs sobre prevenção de PAVM. Métodos: Estudo transversal, quanti-qualitativo. Os dados foram coletados, entre agosto e outubro de 2015, através de um questionário e analisados através dos Softwares Microsoft Excel 2013 e Epi Info 7. Participaram da pesquisa 28 profissionais de saúde. Resultados: 43% afirmou ter conhecimento sobre bundle de prevenção; 36% citaram já terem participado de algum treinamento sobre a temática; 96% manifestou interesse em receber algum treinamento específico; apenas 25% responderam corretamente a pressão ideal do cuff; 96% afirmou avaliar, diariamente, a retirada da sedação. Após a análise, foi realizada uma EP com os profissionais

Objetivo: Evaluar los conocimientos de los profesionales sanitarios en la prevención de la neumonía asociada a la ventilación mecánica (NAV) en pacientes críticamente enfermos en unidades de cuidados intensivos (UCI) y promueven la formación permanente (EP) para los profesionales de la UCI sobre la prevención de la NAV. Métodos: Estudio transversal, cuantitativo y cualitativo. Los datos fueron recogidos entre agosto y octubre de 2015, mediante un cuestionario y analizados mediante el software de Microsoft Excel 2013 y Epi Info 7. Búsqueda de 28 profesionales de la salud participaron. Resultados: el 43% afirmó tener conocimiento sobre la prevención del haz; 36% informó haber participado en algún tipo de formación sobre el tema; 96% expresó su interés en recibir formación específica; sólo el 25% respondió correctamente la presión del manguito ideales; 96% dijo que evaluaban la eliminación diaria de la sedación. Después del análisis, EP se realizó con los profesionales. Conclusión: Se ha demostrado que existe una debilidad en el conocimiento de los profesionales en la prevención de la NAV

Objective: The study's purpose has been to assess the knowledge of health professionals with regards to the prevention of ventilator-associated pneumonia (VAP) in critically ill patients admitted to Intensive Care Units (ICUs), and also to promote Continuing Education (CE) for ICUs' professionals on VAP prevention. Methods: It is a cross-sectional study with a both quantitative and qualitative approach. Data were collected from August to October 2015 through a questionnaire, and subsequently analyzed by the Microsoft Excel 2013 and the Epi Info 7 softwares. The study was carried out by 28 health professionals. Results: 43% reported being knowledgeable about prevention bundle; 36% mentioned that they had participated in some training on the topic; 96% showed some interest in receiving specific training; Only 25% gave the correct answer with regards to the ideal cuff pressure; 96% said they assessed daily withdrawal from sedation. After analysis, a CE was performed with the professionals. Conclusion: It was evidenced that there is some insubstantiality in the professionals' knowledge concerning the VAP prevention

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.

Ca2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP3), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipases C (PLCs), that leads to Ca2+ release from endoplasmic reticulum by InsP3 receptors (InsP3R). Ca2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca2+ levels have specific biological effects that differ from those of Ca2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16INK4A+ and p21Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.

Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem/Stromal Cells Accelerate Migration and Activate AKT Pathway in Human Keratinocytes and Fibroblasts Independently of miR-205 Activity.

Mesenchymal stem/stromal cells (MSCs) are promising tools in cell therapy. They secrete extracellular vesicles (EVs) that carry different classes of molecules that can promote skin repair, but the mechanisms are poorly understood. Skin wound healing is a complex process that requires the activity of several signaling pathways and cell types, including keratinocytes and fibroblasts. In this study, we explored whether adipose tissue MSC-derived EVs could accelerate migration and proliferation of keratinocytes and fibroblasts, activate the AKT pathway, and promote wound healing in vivo. Furthermore, we evaluated if EV effects are miR-205 dependent. We found that MSC EVs had an average diameter of 135 nm. Keratinocytes and fibroblasts exposed to EVs exhibited higher levels of proliferation, migration, and AKT activation. Topical administration of EVs accelerated skin wound closure. Knockdown of miR-205 decreased AKT phosphorylation in fibroblasts and keratinocytes, whereas migration was decreased only in keratinocytes. Moreover, knockdown of miR-205 failed to inhibit AKT phosphorylation in fibroblasts and keratinocytes exposed to EVs. About the mechanism of EV effects, we found that incubation with EVs prevented inhibition of AKT activation by miR-205 knockdown, suggesting that EVs activate AKT independently of miR-205. In conclusion, we demonstrated that EVs are a promising tool for wound healing.

Meanings about mothering by women in treatment for drug use / Sentidos produzidos sobre a maternagem por mulheres em tratamento por uso de drogas

Abstract There is a growing number of women who use drugs. Furthermore, the responsibility for family care is still attributed solely to women. This study aimed to describe the meanings constructed regarding mothering by women in treatment for drug use. This qualitative and descriptive study, with a social constructionist intelligibility, was developed with women in treatment at the Centro de Atenção Psicosocial - Álcool e Drogas of Ribeirão Preto, Brazil. Eight life story thematic interviews were carried out. The analysis resulted in four themes: 1) Mothering as the woman's choice; 2) Learning with drug consumption experiences and using this to warn and educate children; 3) Drug consumption interfering in mothering and 4) Drug consumption understood in different ways throughout the treatment. The meanings attributed to mothering consist of moral and gender discourses regarding the responsibilities attributed to women and the difficulties solely attributed to their drug use.

Resumo É crescente o número de mulheres que consomem drogas. Além disso, ainda se atribui unicamente à mulher a responsabilidade pelo cuidado familiar. Neste estudo, objetivou-se descrever os sentidos construídos a respeito da maternagem por mulheres em tratamento devido ao uso de drogas. Caracteriza-se como um estudo qualitativo, descritivo e exploratório, com inteligibilidade construcionista social. Foi desenvolvido com mulheres em tratamento no Centro de Atenção Psicossocial - Álcool e Drogas de Ribeirão Preto, SP. As oito entrevistas de história de vida temática resultaram em quatro eixos temáticos: 1) Maternagem como escolha da mulher; 2) Experiências do consumo como aprendizado para si e como cuidado/alerta e ensino aos filhos; 3) O consumo de substâncias interferindo na maternagem; e 4) Consumo compreendido de diferentes formas ao longo do tratamento. Os sentidos atribuídos à maternagem são constituídos por discursos morais e de gênero sobre as responsabilidades ditas da mulher e sobre dificuldades consideradas unicamente femininas pelo uso de drogas.

Toxoplasma gondii superinfection and virulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination.

UNLABELLED: The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection. IMPORTANCE: Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains.

The endless race between Trypanosoma cruzi and host immunity: lessons for and beyond Chagas disease.

Infection with the protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, is characterised by a variable clinical course - from symptomless cases to severe chronic disease with cardiac and/or gastrointestinal involvement. The variability in disease outcome has been attributed to host responses as well as parasite heterogeneity. In this article, we review studies indicating the importance of immune responses as key determinants of host resistance to T. cruzi infection and the pathogenesis of Chagas disease. Particular attention is given to recent studies defining the role of cognate innate immune receptors and immunodominant CD8+ T cells that recognise parasite components - both crucial for host-parasite interaction and disease outcome. In light of these studies we speculate about parasite strategies that induce a strong and long-lasting T-cell-mediated immunity but at the same time allow persistence of the parasite in the vertebrate host. We also discuss what we have learned from these studies for increasing our understanding of Chagas pathogenesis and for the design of new strategies to prevent the development of Chagas disease. Finally, we highlight recent studies employing a genetically engineered attenuated T. cruzi strain as a vaccine shuttle that elicits potent T cell responses specific to a tumour antigen and protective immunity against a syngeneic melanoma cell line.

Recruitment and endo-lysosomal activation of TLR9 in dendritic cells infected with Trypanosoma cruzi.

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-kappaB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.