Radiation effects on bone architecture in mice and rats resulting from in vivo micro-computed tomography scanning.

Recently established techniques for performing in vivo micro-computed tomography (micro-CT) provide the capability of monitoring bone changes in a living animal at various points in time. However, radiation exposure from repeated micro-CT scans may have an effect on skeletal growth in normal or disease-model animals. The purpose of this study is to test a high resolution (approximately 10 microm) in vivo micro-CT protocol on mice and rats used for bone research to understand the impact of micro-CT radiation exposure on bone architecture. Ovariectomy (OVX) or sham-OVX surgery was performed on groups (n=6-8/group) of 12-week-old C3H/HeJ, C57BL/6J, and BALB/cByJ mice, and one strain of rat (Wistar, retired breeders). The right proximal tibiae were scanned at weekly intervals while the contralateral left limbs were not scanned until the endpoint of the protocol. Trabecular and cortical bone morphology was compared between radiated and non-radiated limbs at the endpoint to quantify the radiation effect. No effects of radiation were observed in OVX or sham rats. Lower trabecular bone volume was observed in the radiated limbs (-8 to -20% relative to non-radiated limb) of all mice groups except sham BALB/cByJ mice and normal control C57BL/6J mice, however, the observed effects were much less than the observed effects of ovariectomy ( approximately 40-50% total bone volume reduction, depending on mouse strain), and no interactions between radiation and OVX treatment were observed (p>0.2). Using an internal non-radiated control within each animal is a potential method to elucidate the effect of radiation exposure for any in vivo protocol. Thus, although in vivo micro-CT is a valuable tool for bone-related research, the impact of radiation in skeletally immature mice should be considered, particularly for strains with low bone volume at the measured site.

Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis.

The use of high resolution peripheral quantitative computed tomography (HR-pQCT) and in vivo micro-CT for studies of bone disease and treatment has become increasingly common, and with these methods comes large quantities of data requiring analysis. A simple, robust, and fully-automated segmentation algorithm is presented that efficiently segments bone regions. The dual threshold technique refers to two required threshold inputs that are used to extract the periosteal and endosteal surfaces of the cortex. The proposed method was tested against the gold standard, semi-automated hand contouring, using 45 datasets: mouse, rat, human, and cadaver data from the tibia or radius with nominal isotropic resolutions of 10-82 microm. The performance of the proposed method to segment cortical and trabecular compartments was evaluated qualitatively from visualizations and quantitatively based on morphological measurements. Visual inspection confirmed successful segmentation of all datasets using the new method, with qualitatively better results when applied to the human and cadaver data compared to the gold standard. The dual threshold algorithm was able to extract thin and porous cortices, whereas some clipping and perforations occurred for the gold standard. Morphological parameters measured for segmentation by the proposed method versus the gold standard agreed (95% confidence) for Tb.Th, Tb.Sp, and Tb.N, but not Ct.Th and BV/TV for the human and cadaver datasets. Nonetheless, correlations ranged from 0.95 to 1.00 for all morphological parameters except the cadaver Ct.Th because systematic errors were present. Poor agreement for Ct.Th and BV/TV was due to qualitatively incorrect segmentation by the gold standard when the cortex was thin compared to trabeculae, or operator bias during hand contouring. Since Tb.Th, Tb.Sp, and Tb.N were insensitive to segmentation method, despite operator bias, they are robust parameters for inter-site comparisons. The dual threshold method offers a robust and fully-automated alternative to the gold standard that can efficiently segment bone regions with accurate and repeatable results. The algorithm can be easily implemented since it uses simple image analysis tools. Two input thresholds allow adjustment of the masked output, and are easily determined by trial and error. Using the same input thresholds for similar datasets assures maximal consistency while alleviating time consuming semi-automated contouring.

A potential RNA drug target in the hepatitis C virus internal ribosomal entry site.

Subdomain IlId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IlId, to explore the structural features of this subdomain. The proposed secondary structure of IlId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IlId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA alpha-sarcin/ricin loop was located in the internal loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U.G base pair. The sixth nucleotide was bulged out to allow stacking of this U.G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

The recognition of a noncanonical RNA base pair by a zinc finger protein.

BACKGROUND: The zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence. RESULTS: We constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex. CONCLUSIONS: It is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.

Structural conservation in RNA loops III and VI of the internal ribosome entry sites of enteroviruses and rhinoviruses.

Alignment of the internal ribosome entry sites (IRES) of members of the Enteroviridae-Rhinoviridae (E/R) family reveals a consensus loop sequence of AANCCA closed by a C.G base pair. The consensus sequence was present in two distinct loops in domains III and VI. Four hairpins corresponding to the most common loop sequences, AAUCCA, AAACCA, GAACCA and AUCCA, were synthesized and studied by UV spectroscopy. Although all four oligomers had similar UV melting points their thermodynamic parameters revealed differing stabilities consistent with their loop size. Comparison of the aromatic proton and H1' chemical shifts for the four loop sequences obtained from this and our previous NMR study revealed strikingly similar trends. The pattern of chemical shifts suggest similar solution structures in spite of differences in sequence and loop size. This common structure provides a structural basis for their sequence conservation in E/R IRESes.

Structural characterization of three RNA hexanucleotide loops from the internal ribosome entry site of polioviruses.

Structural characteristics of three RNA hairpins from the internal ribosome entry site of poliovirus mRNAs have been determined in solution by NMR. Complete proton, phosphorus and carbon resonance assignments were made for the three 16 nt hairpins. The loop sequences, 5'-AAUCCA , AAACCA and GAACCA, have been shown to be essential for viral mRNA translation. NOESY spectra for the three oligomers were very similar indicating a common three dimensional structure. Stems were A-type duplexes with C3'-endo sugar pucker. In the loops, sequential base stacking interactions were detected for all bases except between U8/A8 and C9, indicating a turn in the phosphodiester backbone at this point. Only one nucleotide, U8/A8, had a sugar pucker which deviated appreciably from C3'-endo. The final base in the loop, A11, exhibited an unusual gauche (-) gamma angle. An ensemble of 10 structures calculated for one hairpin using restrained molecular dynamics shows that the first three bases of the loop are turned so as to be exposed to the exterior of the molecule, while the remaining three bases are in an orientation approximating a continuation of the stem helix. Structure calculations and NMR relaxation measurements indicate that the loop apex is subject to considerable local dynamics.

Association of branched oligonucleotides into the i-motif.

The unique architecture of branched oligonucleotides mimicking lariat RNA introns [Wallace and Edmons, Proc. Natl. Acad. Sci. USA 80, 950-954 (1983)] was exploited to study compounds that associate as two parallel duplexes with intercalating C/C+ base pairs (i-motif DNA) [Gehring et al. Nature 363, 561-565 (1993)]. The formation of a branched cytosine tetrad was induced by joining the 5'-ends of pair of pentadeoxycytidine strands with a branching riboadenosine (rA) linker. This arrangement causes the orientation of the dC strands to be parallel, and forces the formation of a C/C+ duplex that self-associates into i-DNA. Presence of the i-motif in this structure is supported by thermal denaturation, native gel electrophoresis, CD, and NMR spectroscopy.

Structural characterization of an intramolecular RNA triple helix by NMR spectroscopy.

A chemically synthesized 29-base RNA oligomer, designed to fold to form an intramolecular triple helix at acid pH, has been studied by NMR spectroscopy. The molecule consisted of seven U.A.U or C+.G.C base triples joined by two pyrimidine tetra-loops. The fold was such that the third strand was Hoogsteen base-paired in the major groove of a Watson-Crick paired double helix. The nature and size of the molecule required the use of an assignment strategy using two- and three-dimensional homonuclear methods, complemented by a natural abundance 13C correlation experiment. The assignment of the majority of the exchangeable and non-exchangeable resonances is presented. The data suggest a C3'-endo sugar puckering for all the nucleotides involved in base triples. A preliminary structural model consistent with the NMR data is presented.

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

Spectroscopic evidence for an intramolecular RNA triple helix.

A 29-base RNA oligomer has been chemically synthesized and shown to form an intramolecular triple helix in solution at acidic pH. Assignment of the majority of the exchangeable proton NMR resonances demonstrated the Watson-Crick and Hoogsteen base pairings consistent with folding to form pyrimidine-purine-pyrimidine base triplets. FTIR spectroscopy provided independent evidence of base triplet formation, and indicated a predominately C3'-endo sugar pucker. UV absorption as a function of temperature suggested monophasic melting behaviour, which was confirmed by NMR of the imino protons.

1H-NMR for the safety control of food packaging materials: analysis of extracts from polyolefin samples.

A new approach for the identification of the additives in extracts of packaging plastics, using proton nuclear magnetic resonance (1H-NMR), is presented. The technique can be used in a preliminary step for the determination of contaminants potentially released by the food packaging materials; it may greatly reduce the time required to identify the constituents of the materials, either individually or as functional classes. A classification of the EEC additives is proposed on the basis of chemical shift. 1H-NMR can also be used as a fingerprint technique in the quality control of the food packaging materials.