New molecular detection tools adapted to emerging rhinoviruses and enteroviruses

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2′-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

NANO(BIO)SENSORES ELECTROQUIMICOS Y PLASMÓNICOS: DISEÑO, CARACTERIZACIÓN Y APLICACIONES PARA LA CUANTIFICACION DE BIOMARCADORES DE ALTO IMPACTO

En Argentina, en consonancia con el resto del mundo, la Nanotecnología es considerada un área estratégica. Sin embargo, las investigaciones en Nanobiotecnología todavía constituyen un área de vacancia. El uso de nanomateriales para desarrollar plataformas bioanalíticas que permitan la construcción de biosensores ofrece múltiples ventajas y una promisoria perspectiva de aplicación en diversas áreas. En la actualidad, los laboratorios de análisis clínicos, la industria farmacéutica y alimentaria, y los laboratorios de control bromatológico y ambiental requieren de metodologías analíticas que proporcionen resultados exactos, reproducibles, rápidos, sensibles y selectivos empleando pequeños volúmenes de muestra, con un mínimo consumo de reactivos y una producción de deshechos limpia y escasa. Las investigaciones en nanobiosensores se encuentran dirigidas hacia el logro de estas metas. Uno de los grandes desafíos es lograr biosensores miniaturizados con potencialidad para el desarrollo de dispositivos de medición descentralizada (“point of care”) y la detección simultánea de multianalitos. Aún cuando se han hecho innumerables desarrollos en los casi 50 años de vida de los biosensores, todavía hay numerosos interrogantes por dilucidar. La modificación con nanomateriales juega un rol preponderante en los transductores tanto en los electroquímicos como en los plasmónicos. El uso de películas delgadas de Au para SPR modificadas con grafeno u óxido de grafeno, es un campo de una enorme potencialidad y sin embargo es muy poco explotado, por lo que reviste gran importancia. En lo referido a la capa de biorreconocimiento, se trabajará con moléculas capaces de establecer interacciones de bioafinidad, como los anticuerpos y también moléculas que son muy poco usadas en nuestro país y en Latinoamérica como ADN, aptámeros, PNA y lectinas. RESUMEN: El Objetivo general de este proyecto es desarrollar nuevas plataformas bioanalíticas para la detección de diferentes eventos de bioafinidad a partir de la integración de transductores electroquímicos (EQ) y plasmónicos con materiales nanoestructurados (nanotubos de carbono, nanoláminas de grafeno, nanoalambres metálicos); biomoléculas (ADN, “peptide nucleic acid” (PNA), aptámeros, anticuerpos, lectinas) y polímeros funcionalizados con moléculas bioactivas. Las arquitecturas supramoleculares resultantes estarán dirigidas al desarrollo de biosensores EQ y plasmónicos para la cuantificación de biomarcadores de relevancia clínica y medioambiental. Se funcionalizarán CNT, grafeno, óxido de grafeno, nanoalambres metálicos empleando homopéptidos y proteínas con alta afinidad por cationes metálicos, los que se integrarán a transductores de carbono y oro y biomoléculas de reconocimiento capaces de formar complejos de afinidad (antígeno-anticuerpo, aptámero-molécula blanco, ADN-ADN, PNA-ADN, lectinas-hidratos de carbono, ligandos-cationes metálicos y avidina-biotina). Se sintetizarán y caracterizarán nuevos monómeros y polímeros funcionalizados con moléculas bioactivas y/o grupos rédox empleando diferentes rutas sintéticas. Se desarrollarán genosensores para la detección del evento de hibridación de secuencias de interés médico (cáncer de colon y de mama, tuberculosis); aptasensores para la detección de marcadores proteicos de T. cruzi, enfermedades cardiovasculares y contaminantes catiónicos; inmunosensores para la detección de biomarcadores proteicos relacionados con enfermedades cardiovasculares y cáncer; y biosensores de afinidad con lectinas para la detección de hidratos de carbono. La caracterización de las plataformas y las señales analíticas se obtendrán empleando las siguientes técnicas: voltamperometrías cíclica, de pulso diferencial y de onda cuadrada; stripping; resonancia de plasmón superficial; espectroscopía de impedancia electroquímica; microscopías de barrido electroquímico, SEM, TEM, AFM,SNOM, espectroscopías: UV-vis, FTIR,Raman;RMN, TGA y DSC.

Solid-phase combinatorial chemistry and scintillation proximity assays

The Scintillation Proximity Assay (SPA) is a method that is frequently used to detect and quantify the strength of intermolecular interactions between a biological receptor and ligand molecule in aqueous media. This thesis describes the synthesis of scintillant-tagged-compounds for application in a novel cell-based SPA. A series of 4-functianlised-2,5-diphenyloxazole molecules were synthesised. These 4-functionalised-2,5-diphenyloxazoles were evaluated by Sense Proteomic Ltd. Accordingly, the molecules were evaluated for the ability to scintillate in the presence of ionising radiation. In addition, the molecules were incorporated into liposomal preparations which were subsequently evaluated for the ability to scintillate in the presence of ionising radiation. The optimal liposomal preparation was introduced into the membrane of HeLa cells that were used successfully in a cell-based SPA to detect and quantify the uptake of [14C]methionine. This thesis also describes the synthesis and subsequent polymerisation of novel poly(oxyethylene glycol)-based monomers to form a series of new polymer supports. These Poly(oxyethylene glycol)-polymer (POP) supports were evaluated for the ability to swell and mass-uptake in a variety of solvents, demonstrating that POP-supports exhibit enhanced solvent compatibilities over several commercial resins. The utility of POP-supports in solid-phase synthesis was also demonstrated successfully. The incorporation of (4’-vinyl)-4-benzyl-2,5-diphenyloxazole in varying mole percentage into the monomer composition resulted in the production of chemically functionalised scintillant-containing poly(oxyethylene glycol) polymer (POP-Sc) supports. These materials are compatible with both aqueous and organic solvents and scintillate efficiently in the presence of ionising radiation. The utility of POP-Sc supports in solid-phase synthesis and subsequent in-situ SPA to detect and quantify, in real-time, the kinetic progress of a solid-phase reaction was exemplified successfully.In addition, POP-Sc supports were used successfully both in solid-phase combinatorial synthesis of a peptide nucleic acid (PNA)-library and subsequent screening of this library for the ability to hybridise with DNA, which was labelled with a suitable radio-isotape. This data was used to identify the dependence of the number and position of complimentary codon pairs upon the extent of hybridisation. Finally, a further SPA was used to demonstrate the excellent compatibility of POP-Sc supports for use in the detection and quantification of enzyme assays conducted within the matrix of the POP-Sc support.

Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of human immunodeficiency virus type I Rev, were selected from a random sequence RNA pool. Several of the selected RNAs could bind the free peptide more tightly than a natural RNA ligand, the Rev-binding element. In accord with the hypothesis that protein and nucleic acid binding cusps are functionally similar, interactions between aptamers and the peptide target could be disrupted by sequence substitutions. Moreover, the aptamers appeared to be able to bind peptides with different solution conformations, implying an induced fit mechanism for binding. Just as anti-peptide antibodies can sometimes recognize the corresponding epitope when presented in a protein, the anti-peptide aptamers were found to specifically bind to Rev.

Lividins: Novel antimicrobial peptide homologs from the skin secretion of the Chinese Large Odorous frog, Rana (Odorrana) livida: Identification by “shotgun” cDNA cloning and sequence analysis.

Odorous frogs of the sub-genus Odorrana are of oriental distribution, and are so called due to the foul smell of their defensive skin secretions released from specialized skin glands following stress or predator attack. Here we report the application of a “shotgun” skin secretion cDNA library cloning technique which can rapidly expedite identification of secretion bioactive peptides. From a library constructed from the skin secretion of the Large Chinese Odorous frog, Rana (Odorrana) livida, we have identified four novel peptides whose primary structures were deduced initially from cloned precursors. Subsequently, mature peptides were located in and structurally characterized from reverse phase HPLC fractions of skin secretion. Named lividins 1–4, these were found to be structural homologs of known antimicrobial peptide families from Rana frogs. Rapid identification of novel peptides can thus be rapidly achieved using this non-invasive, non-destructive technology and the extensive similarities revealed between antimicrobial peptide precursor organization and nucleic acid sequences would lend support to the hypothesis that they have a common ancestral origin.

Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were < 100 nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

Immune recognition of self nucleic acids driven by endogenous antimicrobial peptides: role in autoimmunity

Innate immune recognition of extracellular host-derived self-DNA and self-RNA is prevented by endosomal seclusion of the Toll-like receptors (TLRs) in the dendritic cells (DCs). However, in psoriasis plasmacytoid dendritic cells have been found to be able to sense self-DNA molecules in complex with the endogenous cationic antimicrobial peptide LL37, which are internalized into the endosomal compartments and thus can access TLR9. We investigated whether this endogenous peptide can also interact with extracellular self-RNA and lead to DC activation. We found that LL37 binds self-RNA as well as self-DNA going into an electrostatic interaction; forms micro-aggregates of nano-scale particles protected from enzymatic degradation and transport it into the endosomal compartments of both plasmacytoid and myeloid dendritic cells. In the plasmacytoid DCs, the self-RNA-LL37 complexes activate TLR7 and like the self-DNA-LL37 complexes, trigger the production of IFN-α in the absence of induction of maturation or production of IL-6 and TNF-α. In contrast to the self-DNA-LL37 complexes, the self-RNA-LL37 complexes are also internalized into the endosomal compartments of myeloid dendritic cells and trigger activation through TLR8, leading to the production of TNF-α and IL-6, and the maturation of the myeloid DCs. Furthermore, we found that these self nucleic acid-LL37 complexes can be found in vivo in the skin lesions of the cutaneous autoimmune disease psoriasis, where they are associated with mature mDCs in situ. On the other hand, in the systemic autoimmune disease systemic lupus erythematosus, self-DNA-LL37 complexes were found to be a constituent of the circulating immune complexes isolated from patient sera. This interaction between the endogenous peptide with the self nucleic acid molecules present in the immune complexes was found to be electrostatic and it confers resistance to enzymatic degradation of the nucleic acid molecules in the immune complexes. Moreover, autoantibodies to these endogenous peptides were found to trigger neutrophil activation and release of neutrophil extracellular traps composed of DNA, which are potential sources of the self nucleic acid-LL37 complexes present in SLE immune complexes. Our results demonstrate that the cationic antimicrobial peptide LL37 drives the innate immune recognition of self nucleic acid molecules through toll-like receptors in human dendritic cells, thus elucidating a pathway for innate sensing of host cell death. This pathway of autoreactivity was found to be pathologically relevant in human autoimmune diseases psoriasis and SLE, and thus this study provides new insights into the mechanisms autoimmune diseases.

Porcine cathelicidins efficiently complex and deliver nucleic acids to plasmacytoid dendritic cells and can thereby mediate bacteria-induced IFN-α responses.

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine-rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide-DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid-complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

The yeast STM1 protein binds G*G multiplex nucleic acids in vitro and associates with ribosomes in vivo

The formation of triple helical, or triplex DNA has been suggested to occur in several cellular processes such as transcription, replication, and recombination. Our laboratory previously found proteins in HeLa nuclear extracts and in S. cerevisiae whole cell extracts that avidly bound a Purine-motif (Pu) triplex probe in gel shift assays, or EMSA. In order to identify a triplex DNA-binding protein, we used conventional and affinity chromatography to purify the major Pu triplex-binding protein in yeast. Peptide microsequencing and data base searches identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a Pu triplex-binding protein was obtained by EMSA using both recombinant Stm1p and whole cell extracts from stm1Δ yeast. Stm1p had previously been identified as G4p2, a G-quartet DNA- and RNA-binding protein. To study the cellular role and identify the nucleic acid ligand of Stm1p in vivo, we introduced an HA epitope at either the N- or C-terminus of Stm1p and performed immunoprecipitations with the HA.11 mAb. Using peptide microsequencing and Northern analysis, we positively identified a subset of both large and small subunit ribosomal proteins and all four rRNAs as associating with Stm1p. DNase I treatment did not affect the association of Stm1p with ribosomal components, but RNase A treatment abolished the association with all ribosomal proteins and RNA, suggesting this association is RNA-dependent. Sucrose gradient fractionation followed by Western and EMSA analysis confirmed that Stm1p associates with intact 80S monosomes, but not polysomes. The presence of additional, unidentified RNA in the Stm1p-immunoprecipitate, and the absence of tRNAs and elongation factors suggests that Stm1p binds RNA and could be involved in the regulation of translation. Immunofluorescence microscopy data showed Stm1p to be located throughout the cytoplasm, with a specific movement to the bud during the G2 phase of the cell cycle. A dramatically flocculent, large cell phenotype is observed when Stm1p has a C-terminal HA tag in a protease-deficient strain background. When STM1 is deleted in this background, the same phenotype is not observed and the deletion yeast grow very slowly compared to the wild-type. These data suggest that STM1 is not essential, but plays a role in cell growth by interacting with an RNP complex that may contain G*G multiplex RNA. ^

RNA-peptide fusions for the in vitro selection of peptides and proteins

Covalent fusions between an mRNA and the peptide or protein that it encodes can be generated by in vitro translation of synthetic mRNAs that carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end. The stable linkage between the informational (nucleic acid) and functional (peptide) domains of the resulting joint molecules allows a specific mRNA to be enriched from a complex mixture of mRNAs based on the properties of its encoded peptide. Fusions between a synthetic mRNA and its encoded myc epitope peptide have been enriched from a pool of random sequence mRNA-peptide fusions by immunoprecipitation. Covalent RNA-peptide fusions should provide an additional route to the in vitro selection and directed evolution of proteins.

Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA.

Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 (-10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands.

Conserved and variable features of gag structure and arrangement in immature retrovirus particles

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.

Versatility of polymethacrylate monoliths for chromatographic purification of biomolecules

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.