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  • 1
    Language: English
    In: Methods, 01 January 2019, Vol.152, pp.55-64
    Description: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally modulate gene expression and orchestrate a wide range of biological and pathological processes. The use of high-throughput screening technologies, in particular microscopy-based screenings (also known as high-content screenings), coupled with genome-wide libraries for modulation of miRNA levels, allow for comprehensive functional analysis of each member of the miRNome in different phenotypic cell-based assays. The wealth of information obtained from such screenings spans across various fields of research, including cancer, cardiovascular, cell reprogramming, and infection biology. Here, we provide an overview of the rationale for performing screenings using synthetic libraries of miRNA mimics and inhibitors, and of the microscopy-based miRNA screenings performed to date. Moreover, a list of resources available for such endeavor is provided. Finally, we describe a detailed procedure for a case study where microscopy-based screening using a library of miRNA mimics was performed to identify miRNAs that control infection of epithelial cells by the bacterial pathogen . The methodologies described here can be easily adapted for screenings addressing other biological questions.
    Keywords: Microrna ; Microscopy-Based Screening ; High-Content Microscopy ; Microrna Libraries ; Bacterial Infection ; Salmonella ; Chemistry ; Anatomy & Physiology
    ISSN: 1046-2023
    E-ISSN: 1095-9130
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  • 2
    In: EMBO Journal, 03 December 2018, Vol.37(23), pp.n/a-n/a
    Description: While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress‐dependent inhibition of binding to host cells. Interestingly, stress caused by intracellular replication also results in remodeling of the host cell membrane, and , which precludes re‐infection by this and other non‐motile pathogens. In contrast, Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress‐responsive cell‐autonomous defense mechanism that protects epithelial cells from infection by non‐motile bacterial pathogens. Stress‐induced host membrane remodeling constitutes a novel cell‐autonomous defensive mechanism that protects epithelial cells from infection by and other non‐motile bacterial pathogens. Host oxidative stress strongly reduces S. flexneri binding to epithelial cells. Stress leads to host membrane remodeling, via activation of the acid sphingomyelinase by the MAPK p38 pathway, resulting in the formation of ceramide domains. Intracellular Shigella replication induces remodeling of the host cell membrane, in vitro and in vivo. Stress‐induced host membrane remodeling precludes re‐infection by non‐motile pathogens; motile pathogens are able to overcome this barrier through flagellar motility. Host membrane remodeling is a cell‐autonomous defense mechanism that protects epithelial cells from infection by .
    Keywords: Acid Sphingomyelinase ; Host Stress Response ; Membrane Remodeling ; Salmonella ; Shigella
    ISSN: 0261-4189
    E-ISSN: 1460-2075
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  • 3
    Language: English
    In: Clinical and Developmental Immunology, Sept-Oct, 2012
    Description: Abnormalities in monocytes and in peripheral blood dendritic cells (DC) subsets have been reported in systemic lupus erythematosus (SLE). We aim to clarify the tolerogenic or inflammatory role of these cells based on ICOSL or IFN-a and chemokine mRNA expression, respectively, after cell purification. The study included 18 SLE patients with active disease (ASLE), 25 with inactive disease (ISLE), and 30 healthy controls (HG). In purified plasmacytoid DC (pDC) was observed a lower ICOSL mRNA expression in ASLE and an increase in ISLE; similarly, a lower ICOSL mRNA expression in monocytes of ALSE patients was found. However, a higher ICOSL mRNA expression was observed in ASLE compared to HG in myeloid DCs. Interestingly, clinical parameters seem to be related with ICOSL mRNA expression. Regarding the inflammatory activity it was observed in purified monocytes and CD[14.sup.-/low] CD[14.sup.+] DCs an increase of CCL2, CXCL9, and CXCL10 mRNA expression in ASLE compared to HG. In myeloid DC no differences were observed regarding chemokines, and IFN-[alpha] mRNA expression. In pDC, a higher IFN-[alpha] mRNA expression was observed in ASLE. Deviations in ICOSL, chemokine, and IFN-[alpha] mRNA expression in peripheral blood monocytes and dendritic cells subpopulations in SLE appear to be related to disease activity.
    Keywords: Lupus -- Development And Progression ; Systemic Lupus Erythematosus -- Development And Progression ; Messenger Rna ; Interferon ; Medical Research ; Dendritic Cells
    ISSN: 1740-2522
    Source: Cengage Learning, Inc.
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  • 4
    Language: English
    In: Clinical and Developmental Immunology, Sept-Oct, 2012
    Description: Abnormalities in monocytes and in peripheral blood dendritic cells (DC) subsets have been reported in systemic lupus erythematosus (SLE). We aim to clarify the tolerogenic or inflammatory role of these cells based on ICOSL or IFN-a and chemokine mRNA expression, respectively, after cell purification. The study included 18 SLE patients with active disease (ASLE), 25 with inactive disease (ISLE), and 30 healthy controls (HG). In purified plasmacytoid DC (pDC) was observed a lower ICOSL mRNA expression in ASLE and an increase in ISLE; similarly, a lower ICOSL mRNA expression in monocytes of ALSE patients was found. However, a higher ICOSL mRNA expression was observed in ASLE compared to HG in myeloid DCs. Interestingly, clinical parameters seem to be related with ICOSL mRNA expression. Regarding the inflammatory activity it was observed in purified monocytes and CD[14.sup.-/low] CD[14.sup.+] DCs an increase of CCL2, CXCL9, and CXCL10 mRNA expression in ASLE compared to HG. In myeloid DC no differences were observed regarding chemokines, and IFN-[alpha] mRNA expression. In pDC, a higher IFN-[alpha] mRNA expression was observed in ASLE. Deviations in ICOSL, chemokine, and IFN-[alpha] mRNA expression in peripheral blood monocytes and dendritic cells subpopulations in SLE appear to be related to disease activity.
    Keywords: Lupus -- Development And Progression ; Systemic Lupus Erythematosus -- Development And Progression ; Messenger Rna ; Interferon ; Medical Research ; Dendritic Cells
    ISSN: 1740-2522
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2011, Vol.6(2), p.e16947
    Description: BACKGROUND: Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13); between the 4(th) and 7(th) day (n = 33); and after one week from the fracture (n = 10). Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th)-7(th) days after fracture. Sclerostin expression diminished during the first days after fracture. CONCLUSIONS: The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: JOM, 2009, Vol.61(1), pp.38-41
    Description: Polymeric nanocomposites, which are hybrids of polymers and modified inorganic clay with organic surfactants, are extremely attractive in both science and industry. These materials present improvements in such polymer properties as modulus, heat capacity, thermal stability, flame resistance, and so on. Research has been conducted in recent decades to obtain high-quality materials that can be used in applications like food packing, car components, and combustible cells. Polymeric nanocomposites present many advantages in relation to composites due to the quantity of filler added to the polymer and also to the improved properties. In a composite, the quantity of filler must be as high as possible (i.e., over 30%). In the polymeric nanocomposite the quantity of filler varies from 1% to 5% because of the nanosize of the particles. These nanoparticles often have a large surface area that results in improved polymer-matrix properties.
    Keywords: Nanotechnology -- Research ; High Density Polyethylene -- Properties ; Composite Materials -- Properties;
    ISSN: 1047-4838
    E-ISSN: 1543-1851
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  • 7
    Language: English
    In: Plant physiology, August 2016, Vol.171(4), pp.2371-8
    Description: Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.
    Keywords: Catharanthus -- Metabolism ; Cell Separation -- Methods ; Flow Cytometry -- Methods ; Secologanin Tryptamine Alkaloids -- Metabolism ; Vinblastine -- Metabolism
    ISSN: 00320889
    E-ISSN: 1532-2548
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  • 8
    Language: English
    Keywords: Ciências Exactas E Naturais ; Natural Sciences ; Ciências Exactas E Naturais ; Natural Sciences
    Source: Networked Digital Library of Theses and Dissertations
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  • 9
    Language: English
    In: Free Radical Biology and Medicine, 20 May 2018, Vol.120, pp.S84-S84
    Description: To access, purchase, authenticate, or subscribe to the full-text of this article, please visit this link: http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.278 Byline: InA*s Castro (1,2,3), Vanessa Lopes-Rodrigues (1,2), C.P.R. Xavier (1,2), M.H. Vasconcelos (1,2,4) Drug resistance is a major clinical problem in cancer treatment. Thus, it is important to establish cell line models to better understand and counteract this problem. The aim of this study was to establish and characterize a drug resistant acute myeloid leukemia cell line, as a sub-clone from a drug sensitive parental cell line. The resistant cell line (HL60--734 VR) was established by treating the parental sensitive cell line (HL60) with increasing concentrations of doxorubicin during several months. Dose-response curves were performed to confirm that the HL60--734 VR cell line was resistant to doxorubicin and to three other drugs (etoposide, cisplatin and daunorubicin). The resistant phenotype was not lost upon drug removal for 1 month. Resistant cells did not express the drug efflux pumps P-gp or BCRP, neither had alterations in apoptotic proteins such as Bax, XIAP, p53, Caspase-3 and Bcl-2. Resistant cells showed alterations in cell cycle profile and presented increased expression of a DNA repair protein ([gamma]-H2AX) and also of proteins involved in drug metabolism (CYP3A and CYP2J2), suggesting that these alterations might be associated with the drug resistant phenotype in these cells. Author Affiliation: (1) i3S - Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, Portugal (2) Cancer Drug Resistance Group, IPATIMUP -- Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal (3) FMUP -- Faculty of Medicine of the University of Porto, Porto, Portugal (4) FFUP -- Faculty of Pharmacy of the University of Porto, Porto, Portugal
    Keywords: Biology ; Anatomy & Physiology
    ISSN: 0891-5849
    E-ISSN: 1873-4596
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  • 10
    Language: English
    In: International Journal of Cardiology, 01 January 2014, Vol.170(3), pp.324-330
    Description: We evaluated the impact of hypertension on the left ventricular mass regression in aortic stenosis after aortic valve replacement. We prospectively studied 135 patients with severe aortic stenosis at baseline and 1 year after surgery. In 32 patients we analyzed myocardial gene expression of collagen types I and III, connective tissue growth factor, transforming growth factor-β1, metalloproteinase-2 and its tissue inhibitor and compared its levels vs controls. Seventy-six patients (56.3%) had a history of hypertension. Hypertensive patients were older, had higher Euroscore-II and NYHA class, with no differences in stenosis severity. At 1 year follow-up there was a median decrease of mass index of 14.2% (P25–75: − 4.3%–30.4%; p 〈 0.001). Mass regression was significantly higher in patients without hypertension, with a median decrease of 25.9% (P25–75: 12.0%–38.7%) vs 5.4% (P25–75: − 12.5%–20.1%; p = 0.001), despite similar increase in effective orifice area and no differences in valvuloarterial impedance. After 1 year, higher baseline left ventricular mass index (p = 0.005) and the absence of hypertension (p = 0.002) or diabetes (p = 0.041) were the only independent predictors of mass regression higher than the median. Comparing with controls, aortic stenosis patients had an increased expression of collagen types I and III, but only hypertensive patients had higher relative expression of collagen type I vs III. In hypertensive patients TIMP2 expression was up-regulated and correlated with higher baseline left ventricular mass index (r = 0.61; p = 0.020). In aortic stenosis, hypertension impairs mass regression one year after valve replacement, independently of total afterload. Differences in the expression of extracellular matrix remodeling genes might contribute to this finding.
    Keywords: Aortic Stenosis ; Hypertension ; Aortic Valve Replacement ; Left Ventricular Mass ; Reverse Remodeling ; Extracellular Matrix ; Medicine
    ISSN: 0167-5273
    E-ISSN: 1874-1754
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