Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Binding thermodynamics of paromomycin, neomycin, neomycin-dinucleotide and -diPNA conjugates to bacterial and human rRNA.

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity.

RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains.

Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1-4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1-RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins' ability to interact with RNA as demonstrated by ion mobility-mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1-RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the ß-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1-RRM2-CPE complex that illustrates the experimental data.

A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials.

An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(ß) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.

Clinical interest of Streptococcus bovis isolates in urine / Significado clínico de los aislados de Streptococcus bovis en orina

Introduction. Streptococcus bovis includes variants related to colorectal cancer and non-urinary infections. Its role as urinary pathogen is unknown. Our objective was to assess the presence of urinary infection by S. bovis, analysing the patients and subsequent clinical course. Material and Methods. Observational study, with longitudinal data collection, performed at our centre between all the cultures requested between February and April 2015. Clinical course of the patients and response to treatment were analysed. Results. Two thousand five hundred and twenty urine cultures were analysed, of which 831 (33%) had a significant microbial count. S. bovis was isolated in 8 patients (0.96%). In 75% of these cases the urine culture was requested because of urinary tract infection symptoms; the remaining 25% because of fever of uncertain source; during the follow-up period no evidence of cancer or endocarditis was detected. S. gallolyticus subspecie pasteurianus was the only variant observed (100%). The clinical response to initial treatment was favourable in all cases. Conclusions. S. bovis bacteriuria may have clinical significance, especially when S. gallolyticus subspecies pasteurianus is isolated in cases with underlying urinary tract disease (AU)

Introducción. Streptococcus bovis comprende multitud de variantes de especie relacionados con infecciones no urinarias y cáncer colorrectal. Su papel como patógeno urinario es desconocido. Nuestro objetivo fue valorar la presencia de infección urinaria por S. bovis, analizando los pacientes y su evolución clínica posterior. Material y métodos. Estudio observacional, con obtención de datos longitudinal, realizado en nuestro centro entre todos los urocultivos solicitados durante entre los meses de febrero y abril de 2015. Se analizó la evolución clínica y la respuesta al tratamiento. Resultados. Se analizaron 2.520 urocultivos, de los que en 831 (33%) hubo un recuento microbiano significativo. Se aisló S. bovis en 8 (0,96%) pacientes. En el 75% de estos casos el urocultivo fue solicitado por clínica de infección del tracto urinario. El 25% restante por fiebre sin foco evidente clínicamente, no objetivando historia de cáncer y/o endocarditis durante el periodo seguimiento. La única variante presente fue S. gallolyticus subespecie pasteurianus (100%). La respuesta clínica al tratamiento inicial fue favorable en todos los casos. Conclusiones. La bacteriuria por S. bovis puede tener significación clínica, sobre todo cuando se aísla S. gallolyticus subespecie pasteurianus, en pacientes con patología previa del aparato urinario (AU)

Anuria tras biopsia del injerto renal: intervención multidisciplinar / Anuria after renal graft biopsy: Multidisciplinary intervention

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El paciente trasplantado renal en urgencias / Renal transplant patient in emergency department

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