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1.
Srabani Bhaumik Jean Boyer Chaitali Banerjee Samantha Clark Noemi Sebastiao Elizabeth Vela Penny Towne 《Journal of cellular biochemistry》2020,121(12):4974-4990
In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells. 相似文献
2.
Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP. 总被引:1,自引:0,他引:1 下载免费PDF全文
A Teplyakov P Sebastiao G Obmolova A Perrakis G S Brush M J Bessman K S Wilson 《The EMBO journal》1996,15(14):3487-3497
NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination. 相似文献
3.
Cristina Bocanegra Sara Gallego Jacobo Mendioroz Milagros Moreno Elena Sulleiro Fernando Salvador Nicolau Sikaleta Arlette Nindia Daniel Tchipita Morais Joromba Sebastiao Kavaya Adrián Sánchez Montalvá Teresa López Israel Molina 《PLoS neglected tropical diseases》2015,9(10)
Introduction
Schistosomiasis remains a public health major problem and little is known in many areas, mainly in Sub-Saharan AfricaObjectives
To assess the burden and risk factors of schistosomiasis and intestinal parasitic helminthes in the children of Cubal, Angola, and to compare different diagnostic approaches for urinary schistosomiasis under field conditions.Methods
A cross-sectional study was conducted. Urine and faeces samples of school children were microscopically studied. A random sample of children was obtained from an alphabetically arranged list of children, taking one of two children. Urine dipstick, colorimetric test and macrohaematuria were considered as indirect diagnostic methods and compared to direct urine examination. Possible risk factors for the infection were sex, age, distance to the river and previous treatment with praziquantel; the assessment was performed using Chi-square test.Results
A total of 785 (61.18%) children showed S. haematobium eggs in urine; children living within 500 meters from the river had a higher odds for infection: Odds ratio 1.97 (1.45–2.7 CI 95%); urine dipstick showed sensitivity of 96% and specificity of 61.3%, with a positive predictive value; colorimetric test showed sensitivity of 52.5%, specificity of 74.6% and a positive predictive value of 77%. Proteinuria was present in 653 (51.1%) children, being more frequent in children with S. haematobium in urine (75.2%); 32 of 191 stool samples (16%) showed the presence of other intestinal parasites and 8 (4%) for S. haematobium.Conclusions
Prevalence of urinary schistosomiasis in our study area is much higher than the national average, considering it as a high-risk community. Proximity to a source of water was a risk factor for the infection. Indirect tests, as urine dipstick and colorimetric test, were useful tools for diagnosis, due to ease of use and low cost. Proteinuria was a common finding, probably showing an early structural damage due to schistosomiasis in this group of children. 相似文献4.
Bischoff KM de Rezende ST Larson TM Liu S Hughes SR Rich JO 《Biotechnology letters》2011,33(10):2013-2018
Acremonium zeae, one of the most prevalent fungal colonists of preharvest corn, possesses a suite of hemicellulolytic activities including xylanase, xylosidase, and arabinofuranosidase. Two enzymes with arabinofuranosidase activity were purified from cell-free culture supernatants of A. zeae grown on oat spelt xylan. A 47 kDa enzyme (AF47) was optimally active at 37 °C and pH 6.0, and had a specific activity for 4-nitrophenyl-α-L-arabinofuranoside (4NPA) of 6.2 U/mg. A 30 kDa enzyme (AF30) was optimally active at 50 °C and pH 4.5, and had a specific activity for 4NPA of 12.4 U/mg. AF47 hydrolyzed 4-nitrophenyl-β-D-xylopyranoside, 4-nitrophenyl-β-D-glucopyranoside, and 4-nitrophenyl-β-D-cellobioside, as well as producing reducing sugars from corn fiber, wheat, and oat spelt arabinoxylan. AF30 had little detectable activity on the 4-nitrophenyl substrates, except for 4NPA, but activity on arabinoxylans from corn fiber, wheat, and oat spelt was at least 7-fold higher than AF47, with specific activities of 109, 358, and 153 U/mg, respectively. A combination of the two enzymes released 61 and 88% of the total arabinose from corn fiber and wheat arabinoxylans. The arabinofuranosidases produced by A. zeae may have industrial application for the enzymatic hydrolysis of recalcitrant lignocellulosic feedstocks such as corn fiber and wheat straw. 相似文献
5.
The synthesis of four different types of oxadiazoles containing a terminal acetylenic group is described. Reaction of these oxadiazoles with various azidoglycosides via a copper-catalyzed [3+2] cycloaddition ('click chemistry') afforded the corresponding glycosyl-triazole linked 1,2,4-oxadiazoles in good yields. 相似文献
6.
Andrea M.C. Joaquim Chris C.L. Wyatt Jolanta Aleksejūnienė Sebastiao L.A. Greghi Luiz F. Pegoraro H. Asuman Kiyak 《Gerodontology》2010,27(4):258-265
doi: 10.1111/j.1741‐2358.2009.00340.x A comparison of the dental health of Brazilian and Canadian independently living elderly Objective: To compare the dental status of Brazilian and Canadian elderly populations with respect to socioeconomic and quality of life factors. Materials and methods: A total of 496 adults aged 60–75 years, having four or more teeth, and physically and cognitively suitable for a clinical oral examination were included. Subjects answered questions concerning their lifestyle and completed the Geriatric Oral Health Assessment Index (GOHAI) questionnaire. Results: In all populations, the majority were females, aged between 60 and 65 years and married. Although the Canadian New Immigrant population had lower mean income, they had more remaining teeth (23.04 ± 6.1), more functional teeth (sound and restored teeth) (14.92 ± 5.7), more sound teeth (15.40 ± 7.6), but more carious teeth (2.97 ± 3.0). The Brazilian population had higher numbers of restored teeth (12.26 ± 6.8) and fewer remaining teeth (17.80 ± 7.6). In all populations, females, married and younger (60–65 years old) adults were more likely to retain 20 or more teeth. The mean GOHAI scores were similar for Canadians (40.55 ± 5.7) and Canadian New Immigrants (39.28 ± 6.5), but were higher than that among Brazilians (31.97 ± 8.9). Conclusions: The numbers of remaining teeth were related to greater education and higher income status for Brazilian and Canadian populations. However, Canadian New Immigrants with lower income and education retained more teeth than the other populations. 相似文献
7.
Joshua C. Rosen Jessica Weiss Nhu-An Pham Quan Li Sebastiao N. Martins-Filho Yuhui Wang Ming-Sound Tsao Nadeem Moghal 《Translational oncology》2021,14(10)
Gain-of-function Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations occur in 25% of lung adenocarcinomas, and these tumors are challenging to treat. Some preclinical work, largely based on cell lines, suggested KRASmut lung cancers are especially dependent on the nuclear export protein exportin-1 (XPO1), while other work supports XPO1 being a broader cancer dependency. To investigate the sensitivity of KRASmut lung cancers to XPO1 inhibition in models that more closely match clinical tumors, we treated 10 independently established lung cancer patient-derived tumor xenografts (PDXs) with the clinical XPO1 inhibitor, Selinexor. Monotherapy with Selinexor reduced tumor growth in all KRASmut PDXs, which included 4 different codon mutations, and was more effective than the clinical MEK1/2 inhibitor, Trametinib. Selinexor was equally effective in KRASG12C and KRASG12D tumors, with TP53 mutations being a biomarker for a weaker drug response. By mining genome-wide dropout datasets, we identified XPO1 as a universal cancer cell dependency and confirmed this functionally in two KRASWT PDX models harboring kinase drivers. However, targeted kinase inhibitors were more effective than Selinexor in these models. Our findings support continued investigation of XPO1 inhibitors in KRASmut lung adenocarcinoma, regardless of the codon alteration. 相似文献
8.
Kenneth M. Bischoff Donald T. Wicklow Douglas B. Jordan Sebastiao T. de Rezende Siqing Liu Stephen R. Hughes Joseph O. Rich 《Current microbiology》2009,58(5):499-503
Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte
Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme
activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following
SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase
(0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and β-glucosidase (0.85 U/mg)
activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free
culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and
wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.
Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information
and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 相似文献
9.
10.
Amanda J. Craig Teresa Garcia-Lezana Marina Ruiz de Galarreta Carlos Villacorta-Martin Edgar G. Kozlova Sebastiao N. Martins-Filho Johann von Felden Mehmet Eren Ahsen Erin Bresnahan Gabriela Hernandez-Meza Ismail Labgaa Delia DAvola Myron Schwartz Josep M. Llovet Daniela Sia Swan Thung Bojan Losic Amaia Lujambio Augusto Villanueva 《PLoS genetics》2021,17(6)