Coordination of Eu3+ ions in siliceous nanohybrids containing short polyether chains and bridging urea cross-links

Infrared and photoluminescence spectroscopies have been used to investigate the local environment of the Eu3+ ions in luminescent sol-gel derived materials-di-ureasils-based on a hybrid framework represented by U(600). This host is composed of a siliceous backbone grafted, through urea cross-links, to both ends of polymer segments incorporating 8.5 oxyethylene repeat units. The active centers have been introduced as europium perchlorate, Eu(ClO4)3. Samples with compositions n = 232, 62, 23, 12, and 6 (where n denotes the ratio of (OCH2CH2) moieties per lanthanide ion) have been examined. The combination of the information retrieved from the analysis of characteristic bands of the FTIR spectra-the perchlorate and the Amide I/Amide II features-with that obtained from the photoluminescence data demonstrates that at compositions n = 232 and 62 the anions are free, whereas the Eu3+ ions are complexed by the heteroatoms of the polyether chains. At higher salt concentration, the cations are bonded, not only to the ClO4 - ions, but also to the ether oxygen atoms of the organic segments and to the carbonyl oxygen atoms of the urea linkages. The dual behavior of U(600) with respect to cation coordination has been attributed to the presence in this nanohybrid of strong hydrogen-bonded urea-urea structures, which, at low salt content, cannot be disrupted, thus inhibiting the formation of Eu3+-O=C(urea) contacts and promoting the interaction between the lanthanide ions and the (OCH2CH2) moieties. The present work substantiates the claim that the activation of the coordinating sites of the di-ureasil framework can be tuned by varying either the guest salt concentration at constant chain length or the length of the.organic segments at constant salt concentration. This relevant property opens challenging new prospects in the fields of application of this class of hybrids. © 2001 American Chemical Society.

Shorter CAG repeat in the AR gene is associated with atypical hyperplasia and breast carcinoma

Background: Previous reports into the role of [CAG]n repeat lengths in the androgen receptor (AR) gene indicate that these may play an important part in the development and progression of breast cancer, however, knowledge regarding benign breast lesions is limited. Patients and Methods: PCR-based GeneScan analysis was used to investigate the [CAG]n repeat length at exon 1 of the AR gene in 59 benign breast lesions (27 fibroadenomas, 18 atypical hyperplasias, and 14 hyperplasias without atypia) and 54 ductal breast carcinomas. Seventy-two cancer-free women were used as a control group. In addition, [CAG]n repeats were evaluated for the presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in a subset of these samples (27 fibroadenomas, 14 hyperplasias without atypia and 22 breast carcinomas). Results: Shorter [CAG]n repeat lengths were strongly correlated with atypical hyperplasias (p=0.0209) and carcinomas (p<0.0001). LOH was found in 1/12 and 4/20 informative cases of hyperplasias without atypia and breast carcinomas, respectively. Three patients with breast carcinoma who had previously presented atypical hyperplasia showed a reduction in the [CAG]n repeat length in their carcinomas. Conclusion: Short [CAG]n repeat length (≤20) polymorphisms are strongly associated with breast carcinomas and atypical hyperplasias. Although non-significant, a subgroup of patients with breast carcinoma and genotype SS showed an association with parameters of worse outcome.

Terminal-repeat retrotransposons with GAG domain in plant genomes: a new testimony on the complex world of transposable elements

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Neuronal differentiation and epithelial integrity: the role of Drosophila Short stop

The nervous system is the most complex organ in animals and the ordered interconnection of neurons is an essential prerequisite for normal behaviour. Neuronal connectivity requires controlled neuronal growth and differentiation. Neuronal growth essentially depends on the actin and microtubule cytoskeleton, and it has become increasingly clear, that crosslinking of these cytoskeletal fractions is a crucial regulatory process. The Drosophila Spectraplakin family member Short stop (Shot) is such a crosslinker and is crucial for several aspects of neuronal growth. Shot comprises various domains: An actin binding domain, a plakin-like domain, a rod domain, calcium responsive EF-hand motifs, a microtubule binding Gas2 domain, a GSR motif and a C-terminal EB1aff domain. Amongst other phenotypes, shot mutant animals exhibit severely reduced dendrites and neuromuscular junctions, the subcellular compartmentalisation of the transmembrane protein Fasciclin2 is affected, but it is also crucially required in other tissues, for example for the integrity of tendon cells, specialised epidermal cells which anchor muscles to the body wall. Despite these striking phenotypes, Shot function is little understood, and especially we do not understand how it can carry out functions as diverse as those described above. To bridge this gap, I capitalised on the genetic possibilities of the model system Drosophila melanogaster and carried out a structure-function analysis in different neurodevelopmental contexts and in tendon cells. To this end, I used targeted gene expression of existing and newly generated Shot deletion constructs in Drosophila embryos and larvae, analyses of different shot mutant alleles, and transfection of Shot constructs into S2 cells or cultured fibroblasts. My analyses reveal that a part of the Shot C-terminus is not essential in the nervous system but in tendon cells where it stabilises microtubules. The precise molecular mechanism underlying this activity is not yet elucidated but, based on the findings presented here, I have developed three alternative testable hypothesis. Thus, either binding of the microtubule plus-end tracking molecule EB1 through an EB1aff domain, microtubulebundling through a GSR rich motif or a combination of both may explain a context-specific requirement of the Shot C-terminus for tendon cell integrity. Furthermore, I find that the calcium binding EF-hand motif in Shot is exclusively required for a subset of neuronal functions of Shot but not in the epidermal tendon cells. These findings pave the way for complementary studies studying the impact of [Ca2+] on Shot function. Besides these differential requirements of Shot domains I find, that most Shot domains are required in the nervous system and tendon cells alike. Thus the microtubule Gas2 domain shows no context specific requirements and is equally essential in all analysed cellular contexts. Furthermore, I could demonstrate a partial requirement of the large spectrin-repeat rod domain of Shot in neuronal and epidermal contexts. I demonstrate that this domain is partially required in processes involving growth and/or tissue stability but dispensable for cellular processes where no mechanical stress resistance is required. In addition, I demonstrate that the CH1 domain a part of the N-terminal actin binding domain of Shot is only partially required for all analysed contexts. Thus, I conclude that Shot domains are functioning different in various cellular environments. In addition my study lays the base for future projects, such as the elucidation of Shot function in growth cones. Given the high degree of conservation between Shot and its mammalian orthologues MACF1/ACF7 and BPAG1, I believe that the findings presented in this study will contribute to the general understanding of spectraplakins across species borders.

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine.

TanDEM-X High Resolution DEMs and Their Applications to Flow Modeling

Lava flow modeling can be a powerful tool in hazard assessments; however, the ability to produce accurate models is usually limited by a lack of high resolution, up-to-date Digital Elevation Models (DEMs). This is especially obvious in places such as Kilauea Volcano (Hawaii), where active lava flows frequently alter the terrain. In this study, we use a new technique to create high resolution DEMs on Kilauea using synthetic aperture radar (SAR) data from the TanDEM-X (TDX) satellite. We convert raw TDX SAR data into a geocoded DEM using GAMMA software [Werner et al., 2000]. This process can be completed in several hours and permits creation of updated DEMs as soon as new TDX data are available. To test the DEMs, we use the Harris and Rowland [2001] FLOWGO lava flow model combined with the Favalli et al. [2005] DOWNFLOW model to simulate the 3-15 August 2011 eruption on Kilauea's East Rift Zone. Results were compared with simulations using the older, lower resolution 2000 SRTM DEM of Hawaii. Effusion rates used in the model are derived from MODIS thermal infrared satellite imagery. FLOWGO simulations using the TDX DEM produced a single flow line that matched the August 2011 flow almost perfectly, but could not recreate the entire flow field due to the relatively high DEM noise level. The issues with short model flow lengths can be resolved by filtering noise from the DEM. Model simulations using the outdated SRTM DEM produced a flow field that followed a different trajectory to that observed. Numerous lava flows have been emplaced at Kilauea since the creation of the SRTM DEM, leading the model to project flow lines in areas that have since been covered by fresh lava flows. These results show that DEMs can quickly become outdated on active volcanoes, but our new technique offers the potential to produce accurate, updated DEMs for modeling lava flow hazards.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Elucidation of Nucleic Acid–Drug Interactions by Tandem Mass Spectrometry

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid–drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.

Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.

Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea.

A short interspersed nuclear element, Mg-SINE, was isolated and characterized from the genome of the rice blast fungus, Magnaporthe grisea. Mg-SINE was isolated as an insertion element within Pot2, an inverted-repeat transposon from M. grisea and shows typical features of a mammalian SINE. Mg-SINE is present as a 0.47-kb interspersed sequence at approximately 100 copies per haploid genome in both rice and non-rice isolates of M. grisea, indicating a common evolutionary origin. Secondary structure analysis of Mg-SINE revealed a tRNA-related region at the 5' end which folds into a cloverleaf structure. Genomic fusions resulting in chimeric Mg-SINEs (Ch-SINEs) composed of a sequence homologous to Mg-SINE at the 3' end and an unrelated sequence at its 5' end were also isolated, indicating that this and other DNA rearrangements mediated by these elements may have a major effect on the genomic architecture of this fungus.

Rolling replication of short DNA circles.

Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiological specificity and activity. Given the selective value of such repeats, it is likely that several different mechanisms have been responsible for their generation. One mechanism that has been shown to generate relatively long tandem repeats (in the kilobase range) is rolling circle replication. In this communication, we demonstrate that rolling circle synthesis in a simple enzymatic system can produce tandem repeats of monomers as short as 34 bp. In addition to suggesting possible origins for natural tandem repeats, these observations provide a facile means for constructing libraries of repeated motifs for use in "in vitro evolution" experiments designed to select molecules with defined biological or chemical properties.

Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The disease gene has been localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The function of the encoded protein remains undetermined. In this study we have characterized mutations in 12 unrelated patients to confirm the identity of the disease gene. We have also revised the coding sequence and genomic structure for the WAS gene. To analyze further the transmittance of the disease gene, we have characterized a polymorphic microsatellite at the DXS6940 locus within 30 kb of the gene and demonstrate the inheritance of the affected alleles in families with a history of WAS.

Evolution of the regular zone of histone H1 in Fabaceae plants

An analysis of the historic H1 subtype, H1-1, in eight legumes belonging to four genera of the tribe Vicieae (Pisum, Lathyrus, Lens, and Vicia), revealed an extended region consisting of the tandemly repeated AKPAAK motifs. We named this region the Regular zone (RZ). The AKPAAK motifs are organized into two blocks separated by a short (two or six amino acids) intervening sequence (IS). The distal block contains six AKPAAK motifs, while the number of repeats in the proximal block varies from six in V. faba to seven in the other species. In V. hirsuta, the first two repeated units of the proximal block are octapeptides AKAKPAAK. The apparent rate of synonymous substitutions in the blocks of RZ is much higher than in the rest of the gene. This can be explained by repeat shuffling within each block. In the C-domain of the orthologous H1 subtype froth Medicago truncatula (tribe Trifolieae), a region corresponding to the RZ of Vicieae species was found. It also consists of two blocks of AKPAAK motifs (four and three repeats in the proximal and distal blocks, respectively). These blocks are separated by a 20-amino acid IS. The first 20 amino acids of the Medicago RZ are not part of AKPAAK repeats. We hypothesise that the RZ has most probably evolved as a result of an expansion of AKPAAK repeats from two separate sites in the C-domain. This process started tens of millions of years ago and was most likely directed by positive selection.

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA. telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of rnRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem beta alpha beta beta alpha beta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.