Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes

Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo

<p>Abstract</p> <p>Background</p> <p>Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or <it>in vivo</it>. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins <it>in vivo</it>. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions.</p> <p>Results</p> <p>Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, <it>in vivo</it>, without any cross talk between the two intein systems.</p> <p>Conclusions</p> <p>Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, <it>in vivo</it>, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.</p

Análisis del comportamiento de un firme reciclado en central en caliente con alta tasa tras un año en servicio

En 2008-2009 el Ministerio de Fomento llevó a cabo la rehabilitación del firme de la autovía A-5 en la provincia de Cáceres, en el tramo comprendido entre el límite con la provincia de Toledo y Almaraz. La rehabilitación se ejecutó mediante un reciclado en caliente en central con una tasa de fresado del 40 %, de manera que para la reposición de la capa de base se extendió una mezcla bituminosa en caliente del tipo AC 22 base G-R40, cuyo ligante fue un betún con rejuvenecedores diseñado al efecto. Se utilizó para la fabricación de la mezcla una central de tipo discontinuo con una capacidad de 200 t/h. Los ensayos realizados durante las obras arrojaron unos resultados que cumplían las exigencias de las especificaciones del Proyecto, y el resultado final fue absolutamente satisfactorio. Un año después de la puesta en obra de la mezcla reciclada se ha querido analizar en detalle su comportamiento a través de ensayos sobre testigos extraídos en la campaña que se ha realizado ahora expresamente para ello. Por un lado se ha pretendido comparar los resultados obtenidos en los testigos de esta campaña con los que se obtuvieron durante la ejecución de las obras. Por otro lado, se ha procedido a comparar el comportamiento de la mezcla reciclada con el de la mezcla convencional colocada en la misma obr

Exploiting protected maleimides to modify oligonucleotides, peptides and peptide nucleic acids

This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols

High-Resolution Whole-Mount In Situ Hybridization Using Quantum Dot Nanocrystals

The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mount in situ hybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D

We Have Something to Say: Youth Participatory Action Research as a Promising Practice to Address Problems of Practice in Rural Schools

The purpose of this article is to highlight a critical approach for practice, youth participatory action research, that can be used to invite rural youth to collaborate with school administrators, educators, and community leaders to identify and examine challenges, while building upon the strengths of a school and community to address challenges. Our youth participatory action research project was a collaboration between adult researchers and five students from a rural high school to examine and address postsecondary education access challenges. The adult and student researchers developed and implemented two evidence-based products: (a) a conference and (b) a resource corner in the school library. Student co-researchers demonstrated an increased commitment to the project, development of postsecondary education knowledge, and development as leaders during the project. Our project demonstrates evidence of youth participatory action research being an effective approach to address problems of practice in rural education

Why volubility can predict the success of cochlear implantation

BackgroundWe sought to identify potential communication markers predicting the success of cochlear implantation, that might be observed within the first year of life. According the last ten years literature review volubility can be considered as a potentially important vocal measure predicting later language development.AimsThe present review aims to review existing evidence related with: (i) why volubility posits a plausible marker of cochlear implantation success in infancy, and (ii) presents the clinical usefulness of volubility data in predicting later language trajectory. Methods Rate of vocalization or volubility measured in terms of frequency of syllable production and it is clearly affected by parental interactivity. A low percentage of volubility can be predictive of significant communication impairment. Vocalization growth during the first year of life, as demonstrated in publications examining sound production characteristics of normally hearing (NH) and hearing impaired (HI) infants fitted with CI, were reviewed. Results Literature results revealed differences in linguistic performance among NH and CI infants which are typically attributed to auditory deprivation. Infants received late CI, produce fewer syllables (low volubility) and exhibit late-onset babbling, especially those who underwent the procedure as late as the age of 12 months or thereafter. Early recipients (implanted before the age of 12-months) related with more vocalizations, which is thought to stem from CI-initiated auditory feedback. In sum, total syllables produced (volubility) demonstrate the developmental trajectory of language acquisition which in turn is a crucial factor related with the success of cochlear implantation.ConclusionContemporary findings collectively endorse volubility as a plausible criterion of differentiation between successful and non-successful early CI. It is argued that volubility measures predict language development and, in doing so, carry vast implications on designing efficient clinical assessment and intervention practices

Endocrine and molecular investigations in a cohort of 25 adolescent males with prominent/persistent pubertal gynecomastia

Pubertal gynecomastia is a common condition observed in up to 65% of adolescent males. It is usually idiopathic and tends to regress within 1–2 years. In this descriptive cross-sectional study, we investigated 25 adolescent males with prominent (>B3) and/or persistent (>2 years) pubertal gynecomastia (P/PPG) to determine whether a hormonal/genetic defect might underline this condition. Endocrine investigation revealed the absence of hormonal disturbance for 18 boys (72%). Three patients presented Klinefelter syndrome and three a partial androgen insensitivity syndrome (PAIS) as a result of p.Ala646Asp and p.Ala45Gly mutations of the androgen receptor gene. The last patient showed a 17α-hydroxylase/17,20-lyase deficiency as a result of a compound heterozygous mutation of the CYP17A1 gene leading to p.Pro35Thr(P35T) and p.Arg239Stop(R239X) in the P450c17 protein. Enzymatic activity was analyzed: the mutant protein bearing the premature stop codon R239X showed a complete loss of 17α-hydroxylase and 17,20-lyase activity. The mutant P35T seemed to retain 15–20% of 17α-hydroxylase and about 8–10% of 17,20-lyase activity. This work demonstrates that P/PPG had an endocrine/genetic cause in 28% of our cases. PAIS may be expressed only by isolated gynecomastia as well as by 17α-hydroxylase/17,20-lyase deficiency. Isolated P/PPG is not always a ‘physiological’ condition and should thus be investigated through adequate endocrine and genetic investigations, even though larger studies are needed to better determine the real prevalence of genetic defects in such patients