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Purpose of Review Deep brain stimulation (DBS) is an established treatment in several movement disorders, including Parkinson's disease, dystonia, tremor, and Tourette syndrome. In this review, we will review and discuss the most recent findings including but not limited to clinical evidence. Recent Findings New DBS technologies include novel hardware design (electrodes, cables, implanted pulse generators) enabling new stimulation patterns and adaptive DBS which delivers potential stimulation tailored to moment-to-moment changes in the patient's condition. Better understanding of movement disorders pathophysiology and functional anatomy has been pivotal for studying the effects of DBS on the mesencephalic locomotor region, the nucleus basalis of Meynert, the substantia nigra, and the spinal cord. Eventually, neurosurgical practice has improved with more accurate target visualization or combined targeting. A rising research domain emphasizes bridging neuromodulation and neuroprotection. Recent advances in DBS therapy bring more possibilities to effectively treat people with movement disorders. Future research would focus on improving adaptive DBS, leading more clinical trials on novel targets, and exploring neuromodulation effects on neuroprotection.

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5′-UNR-3′ (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3′ phosphate group of the R residue as well as a hydrogen bond between the 2′-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3′ from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.

Riboswitches are novel gene regulatory elements found in the 5′- and 3′-untranslated regions of mRNAs. They regulate gene expression upon direct and specific interaction with small molecule ligands. In most riboswitches ligand binding triggers conformational changes on the secondary and/or tertiary structure level which result in changes in gene expression levels. While a lot is known about the structural basis for ligand recognition by riboswitch domains the conformational dynamics of riboswitches which are central to their function is less explored. NMR-spectroscopy in solution is well suited for investigating dynamical systems and for characterising conformational changes. We applied NMR-spectroscopy to two smaller synthetic riboswitches to characterize in detail their conformational dynamics in relation to their regulatory functions.

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Riboswitches are highly structured RNA motifs with gene regulatory activity located in the untranslated regions of mRNAs. They either modulate transcription termination or translation initiation through conformational changes triggered by direct interactions with small metabolite ligands. Many naturally occurring riboswitches are large and structurally very complex. In contrast, synthetic riboswitches—tailored gene regulatory elements for synthetic biology applications—are based on small in vitro selected RNA aptamers. Yet, despite a ligand affinity and specificity comparable to their natural counterparts only a few in vitro selected aptamers are regulatory active in vivo. Recently, Suess et al. engineered a riboswitch for the aminoglycoside antibiotic neomycin B by subjecting an in vitro SELEX-pool to an in vivo screening for gene regulatory activity in a yeastbased reporter gene assay. The resulting neomycin B and ribostamycin (Figure 1a) responsive RNA-element (N1) contains only 27 nucleotides in a bulged hairpin secondary structure (Figure 1b)—the smallest riboswitch functional in vivo identified to date. In sequence and secondary structure, N1 differs completely from an in vitro selected but regulatory inactive RNA-aptamer for the same ligand (R23). Instead it partially resembles the ribosomal A-site, the natural target for aminoglycoside antibiotics (Figure 1b). The NMR spectroscopic analysis of the N1 riboswitch complexed with ribostamycin identifies structural determinants for its regulatory activity and suggests a ligand binding mechanism based on conformational capture. Our results provide insights into the modularity of ligand binding sites in RNA and highlight structural and dynamic features N1 shares with the larger naturally occurring riboswitches as well as with other regulatory active aptamers. This knowledge may guide the future design of novel synthetic riboswitches for targeted in vivo applications. Structure of the N1–ligand complex—the “OFF”-state of the riboswitch: N1 represses gene expression upon binding to either neomycin B or the closely related but smaller ribostamycin. NMR spectra of N1 bound to either ligand (Supporting Information Figure S1) indicate that both complexes are formed with similarly high affinity and display a high degree of structural similarity suggesting that the contribution of ring IV of neomycin to the interaction is negligible. Thus, we determined the structure of the N1–ribostamycin complex, because of its superior spectral resolution for the ligand resonances, by NMR spectroscopy (see Table 1). Chemical shift assignments and coordinates have been deposited (BMRB code: 16609, pdb-code: 2kxm). The structure of ribostamycin-bound N1 consists of a continuous helical stem with canonical stacking interactions between the G5:C23 and the G9:C22 base pair despite the presence of a flexible three-nucleotide bulge (C6–U8) and a compactly folded apical hexaloop organized around a U-turn motif (U14–A16) closed by the U13:U18 base pair (Figure 1c–e). Ribostamycin rings I and II are sandwiched between the N1 major groove, in the region from G5:C23 to U13:U18 and A17 protruding from the apical loop (Figure 2). Ring III is located close to the backbone of the 3’-strand (U18 to G20). Simultaneous contacts of the ligand with the G5:C23 base pair below and G9:C22 above the bulge (Figure 2b) clamp together the lower and upper helical stem and thus enforce the uninterrupted coaxial helical stacking across the flexible C6–U8 internal bulge. The bulge itself is not interacting with the ligand. A detailed structural description of the N1– ribostamycin complex is given in the Supporting Information. A comparison of the N1–ribostamycin complex with other aminoglycoside binding RNAs reveals partial similarities to known aminoglycoside binding sub-motifs: The helical stem centered at the U10:U21 base pair is similar to the ribosomal [*] Dr. E. Duchardt-Ferner, S. R. Schmidtke, Prof. Dr. J. W hnert Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany) Fax: (+49)69-798-29527 E-mail: woehnert@bio.uni-frankfurt.de

The neomycin-sensing riboswitch is an engineered riboswitch developed to regulate gene expression in vivo in the lower eukaryote Saccharomyces cerevisiae upon binding to neomycin B. With a size of only 27nt it is the smallest functional riboswitch element identified so far. It binds not only neomycin B but also related aminoglycosides of the 2'-deoxystreptamine class with high affinity. The regulatory activity, however, strongly depends on the identity of the aminoglycoside. As a prerequisite for the structure determination of riboswitch-ligand complexes we report here the (1)H, (15)N, (13)C and partial (31)P chemical shift assignments for the minimal functional 27nt neomycin sensing riboswitch RNA in complex with the 4,5-linked neomycin analog ribostamycin and the 4,6-linked aminoglycoside tobramycin.

We present new measurements of the energy spectra of cosmic-ray (CR) nuclei from the second flight of the balloon-borne experiment CREAM (Cosmic Ray Energetics And Mass). The instrument (CREAM-II) was comprised of detectors based on different techniques (Cherenkov light, specific ionization in scintillators and silicon sensors) to provide a redundant charge identification and a thin ionization calorimeter capable of measuring the energy of cosmic rays up to several hundreds of TeV. The data analysis is described and the individual energy spectra of C, O, Ne, Mg, Si and Fe are reported up to ~ 10^14 eV. The spectral shape looks nearly the same for all the primary elements and can be expressed as a power law in energy E^{-2.66+/-0.04}. The nitrogen absolute intensity in the energy range 100-800 GeV/n is also measured., Comment: 4 pages, 3 figures, presented at ICRC 2009, Lodz, Poland