Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms

A QUANTITATIVE MODEL FOR NONRANDOM GENERALIZED TRANSDUCTION, APPLIED TO THE PHAGE P22–<i>SALMONELLA TYPHIMURIUM</i> SYSTEM

ABSTRACT A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from cotransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of cotransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic equations for cotransduction frequencies of markers located within one phage length. The method allows a relatively precise determination of distances between markers, positions of transducing particle ends and deletion or insertion lengths. The approach is applied to the trp-cysB-pyrF and aroC-hisT-purF-dhuA regions of the Salmonella typhimurium chromosome.</jats:p

Por Don Fernando del Valle ... en el pleyto con Alfonso Bocache ... sobre delito de alzamiento, y con Ivan de Casanova, y Pedro de Menviela... [Bernardo de Castro y Azeuedo]

2 ej. de la misma obraPort. con grab. calc. de Muñoz representando a la Virgen con NiñoInic. grab.Enc. Perg.Sign.: A-G2, H

Abstract 452: MIC-1 and Endoglin are protein serum biomarkers capable of increasing the clinical diagnostic specificity of the PSA test

Abstract PSA test and digital rectal examination have been routinely used to screen for prostate cancer (PCa). PSA screening revolutionized the management of PCa, especially with regards to early detection. Before the PSA test was available, ∼20% of men were diagnosed with PCa that had already spread to the bone. Today, that number is around 4%. However, it is estimated that 56% of the cases are overdiagnosed resulting in overtreatment and exposing the patients to serious unnecessary treatment morbidities. Also the rate of false-positives is incredibly high (60-80%), since PSA is not cancer specific. It is critical to discriminate clinically significant PCa patients requiring definitive treatment from those who would safely undergo active surveillance monitoring. The aim of this project is to identify and incorporate multiple serum protein biomarkers into an assay that will enhance the clinical diagnostic specificity of the PSA screening test. Using ELISA, we analyzed protein serum levels of Endoglin, IL-8 and MIC-1 in samples obtained from 50 PCa patients and 50 non-cancer individuals. ROC curve analysis was used to determine if these biomarkers were capable of separating the 2 groups. Multivariate logistic regression (MLR) was used to identify a panel of biomarkers that combined to PSA would increase the test specificity. MIC-1 was the only biomarker capable of discriminating PCa from normal cases (AUC = 0.76; p&amp;lt;0.0001). Endoglin protein levels were significantly different (p = 0.0010) between the groups, although the AUC was borderline (AUC = 0.69) to our cut-off (AUC&amp;gt;0.7) it suggests a good potential for this protein as a biomarker in PCa diagnosis. IL-8 did not show significant differences (p = 0.1828) in the levels detected between cancer and normal samples. The expression of these proteins were also evaluated for their prognostic association with clinical-pathological features: Gleason Score (GS), T and N stage. MIC-1 protein levels was capable to separate GS 3+3 and 3+4 from those 4+3 or higher (AUC = 0.73), suggesting that this biomarker might be associated with more aggressive cases. Using MLR, we identified Endoglin, MIC-1 and PSA as a multiple biomarker panel that enhanced specificity of the PSA screening test. The panel resulted in an assay with 88% specificity and 86% sensitivity that is slightly higher than PSA alone (82% specificity; 84% sensitivity). Our results suggest that MIC-1 is a strong candidate as a biomarker for cancer screening and disease aggressiveness. We also showed that when PSA test is combined with the presence of MIC-1 and Endoglin in serum, it resulted in an assay with higher specificity and sensitivity than when these biomarkers are evaluated alone. To validate these results, protein serum levels of Endoglin, MIC-1 and IL-8 will be determined in a bigger cohort of normal and PCa patients. Other biomarkers are also being evaluated. Citation Format: Luciane T. Kagohara, Ji Li, Christian P. Pavlovich, Christine Davis, Leslie Mangold, Guangjing Zhu, Colm Morrissey, Alan W. Partin, Wlodeck Mandecki, Robert W. Veltri. MIC-1 and Endoglin are protein serum biomarkers capable of increasing the clinical diagnostic specificity of the PSA test. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 452.</jats:p