A Novel NAD-RNA Decapping Pathway Discovered by Synthetic Light-Up NAD-RNAs.

The complexity of the transcriptome is governed by the intricate interplay of transcription, RNA processing, translocation, and decay. In eukaryotes, the removal of the 5'-RNA cap is essential for the initiation of RNA degradation. In addition to the canonical 5'-N7-methyl guanosine cap in eukaryotes, the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD) was identified as a new 5'-RNA cap structure in prokaryotic and eukaryotic organisms. So far, two classes of NAD-RNA decapping enzymes have been identified, namely Nudix enzymes that liberate nicotinamide mononucleotide (NMN) and DXO-enzymes that remove the entire NAD cap. Herein, we introduce 8-(furan-2-yl)-substituted NAD-capped-RNA (FurNAD-RNA) as a new research tool for the identification and characterization of novel NAD-RNA decapping enzymes. These compounds are found to be suitable for various enzymatic reactions that result in the release of a fluorescence quencher, either nicotinamide (NAM) or nicotinamide mononucleotide (NMN), from the RNA which causes a fluorescence turn-on. FurNAD-RNAs allow for real-time quantification of decapping activity, parallelization, high-throughput screening and identification of novel decapping enzymes in vitro. Using FurNAD-RNAs, we discovered that the eukaryotic glycohydrolase CD38 processes NAD-capped RNA in vitro into ADP-ribose-modified-RNA and nicotinamide and therefore might act as a decapping enzyme in vivo. The existence of multiple pathways suggests that the decapping of NAD-RNA is an important and regulated process in eukaryotes.

A technical concept of a computer game for patients with Parkinson's disease - a new form of PC-based physiotherapy.

Background: At present, there are no meaningful and sophisticated computer games that simultaneously allow the treatment of movement disorders such as Parkinson's syndrome. In particular, there are no systems to consider the severity of the disease and the physical skills of the patient. Methods: A computer game using the Microsoft Kinect as markerless sensor for the 3 D recognition of the patient's movement was developed to support the rehabilitation. The scenario of a basketball game was created after determining that the movement like throwing a ball and the correct posture of the body are important. A study based on system usability was performed with 15 patients to evaluate the system. Results: The technical feasibility of a computer-assisted training system for supporting patients with Parkinson's disease has been demonstrated. No markers on the patient are required for movement detection and allow a user-friendly handling. Regarding the usability study, the patients were accepting of such a system and its at-home use and symptoms like 'freezing' and the Pisa syndrome can be treated. Conclusions: The physiotherapist can be assisted by the developed rehabilitation system. An objective measurement of the patient's training progress delivers valuable information to adjust the training sessions for every patient individually. Due to its modular character, the system can also be applied to other diseases or sports injuries and offers the basis for further development.

Is Low-field NMR a Complementary Tool to GC-MS in Quality Control of Essential Oils? A Case Study: Patchouli Essential Oil.

High-field NMR is an expensive and important quality control technique. In recent years, cheaper and simpler low-field NMR has become available as a new quality control technique. In this study, 60 MHz 1H-NMR was compared with GC-MS and refractometry for the detection of adulteration of essential oils, taking patchouli essential oil as a test case. Patchouli essential oil is frequently adulterated, even today. In total, 75 genuine patchouli essential oils, 10 commercial patchouli essential oils, 10 other essential oils, 17 adulterants, and 1 patchouli essential oil, spiked at 20% with those adulterants, were measured. Visual inspection of the NMR spectra allowed for easy detection of 14 adulterants, while gurjun and copaiba balsams proved difficult and one adulterant could not be detected. NMR spectra of 10 random essential oils differed not only strongly from patchouli essential oil but also from one another, suggesting that fingerprinting by low-field NMR is not limited to patchouli essential oil. Automated chemometric evaluation of NMR spectra was possible by similarity analysis (Mahalanobis distance) based on the integration from 0.1 - 8.1 ppm in 0.01 ppm increments. Good quality patchouli essential oils were recognised as well as 15 of 17 deliberate adulterations. Visual qualitative inspection by GC-MS allowed for the detection of all volatile adulterants. Nonvolatile adulterants, and all but one volatile adulterant, could be detected by semiquantitation. Different chemometric approaches showed satisfactory results. Similarity analyses were difficult with nonvolatile adulterants. Refractive index measurements could detect only 8 of 17 adulterants. Due to advantages such as simplicity, rapidity, reproducibility, and ability to detect nonvolatile adulterants, 60 MHz 1H-NMR is complimentary to GC-MS for quality control of essential oils.

Stable phase-shift despite quasi-rhythmic movements: a CPG-driven dynamic model of active tactile exploration in an insect.

An essential component of autonomous and flexible behavior in animals is active exploration of the environment, allowing for perception-guided planning and control of actions. An important sensory system involved is active touch. Here, we introduce a general modeling framework of Central Pattern Generators (CPGs) for movement generation in active tactile exploration behavior. The CPG consists of two network levels: (i) phase-coupled Hopf oscillators for rhythm generation, and (ii) pattern formation networks for capturing the frequency and phase characteristics of individual joint oscillations. The model captured the natural, quasi-rhythmic joint kinematics as observed in coordinated antennal movements of walking stick insects. Moreover, it successfully produced tactile exploration behavior on a three-dimensional skeletal model of the insect antennal system with physically realistic parameters. The effect of proprioceptor ablations could be simulated by changing the amplitude and offset parameters of the joint oscillators, only. As in the animal, the movement of both antennal joints was coupled with a stable phase difference, despite the quasi-rhythmicity of the joint angle time courses. We found that the phase-lead of the distal scape-pedicel (SP) joint relative to the proximal head-scape (HS) joint was essential for producing the natural tactile exploration behavior and, thus, for tactile efficiency. For realistic movement patterns, the phase-lead could vary within a limited range of 10-30° only. Tests with artificial movement patterns strongly suggest that this phase sensitivity is not a matter of the frequency composition of the natural movement pattern. Based on our modeling results, we propose that a constant phase difference is coded into the CPG of the antennal motor system and that proprioceptors are acting locally to regulate the joint movement amplitude.

Phosphine-free Stille-Migita chemistry for the mild and orthogonal modification of DNA and RNA.

An optimized catalyst system of [Pd2 (dba)3 ] and AsPh3 efficiently catalyzes the Stille reaction between a diverse set of functionalized stannanes and halogenated mono-, di- and oligonucleotides. The methodology allows for the facile conjugation of short and long nucleic acid molecules with moieties that are not compatible with conventional chemical or enzymatic synthesis, among them acid-, base-, or fluoride-labile protecting groups, fluorogenic and synthetically challenging moieties with good to near-quantitative yields. Notably, even azides can be directly introduced into oligonucleotides and (deoxy)nucleoside triphosphates, thereby giving direct access to "clickable" nucleic acids.

Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling.

Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.

A modified dinucleotide for site-specific RNA-labelling by transcription priming and click chemistry.

An improved strategy for RNA labelling using an alkyne-carrying dinucleotide is reported. This involves near-quantitative priming by phage RNA-polymerases followed by conjugation of different labels using click chemistry. Moreover, these transcripts bear a ligation compatible 5'-end, and thus through ligation the terminal label can be transformed to an internal one.

Central drive and proprioceptive control of antennal movements in the walking stick insect.

In terrestrial locomotion, active touch sensing is an important source of near-range information. Walking stick insects show active tactile exploration behaviour by continuously sampling the ambient space with their antennae. Here, we identify central and proprioceptive contributions to the control of this behaviour. First, we investigate the potential role of synaptic drive to central neural networks using pilocarpine, an agonist of muscarinic acetylcholine receptors. In an in situ preparation, pilocarpine induced rhythmic antennal movements with a persisting pattern of inter-joint coordination, matching that seen in intact walking animals, albeit with lower cycle frequency. After de-cerebration, stick insects were still able to walk but no longer moved their antennae during walking. Here, pilocarpine still induced antennal movement, suggesting that synaptic drive to central neural networks involved in antennal movement generation occurred in the brain and not in the suboesophageal ganglion. During intact walking, these networks are likely to receive activation by ascending input. Second, we show persistent coupling of both antennal joints during intact walking, with the distal scape-pedicel joint (SP) always leading the proximal head-scape joint (HS). Ablation of joint proprioceptors had no effect on this overall pattern of inter-joint coordination but could affect the magnitude of the phase-lag. Third, we revise the description of antennal hair fields and show that complete ablation of all seven hair fields strongly affects antennal movements. Ablating dorsal hair fields mainly affected the working-ranges of antennal joints: Ablation of the dorso-medial pedicellar hair plate caused a ventral shift of the SP working-range. Ablation of the dorsal scapal hair plate considerably expanded the dorsal HS working-range, and, in combination with ablation of pedicellar hair fields, increased the SP working-range, too. We conclude that the working-ranges of both joints are under proprioceptive control of dorsal antennal hair fields. Thus, both synaptic drive to central neural networks and proprioceptive feedback are involved in the control of active tactile exploration behaviour in stick insects.

Microscale thermophoresis provides insights into mechanism and thermodynamics of ribozyme catalysis.

The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (KD) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse.

An insect-inspired bionic sensor for tactile localization and material classification with state-dependent modulation.

INSECTS CARRY A PAIR OF ANTENNAE ON THEIR HEAD: multimodal sensory organs that serve a wide range of sensory-guided behaviors. During locomotion, antennae are involved in near-range orientation, for example in detecting, localizing, probing, and negotiating obstacles. Here we present a bionic, active tactile sensing system inspired by insect antennae. It comprises an actuated elastic rod equipped with a terminal acceleration sensor. The measurement principle is based on the analysis of damped harmonic oscillations registered upon contact with an object. The dominant frequency of the oscillation is extracted to determine the distance of the contact point along the probe and basal angular encoders allow tactile localization in a polar coordinate system. Finally, the damping behavior of the registered signal is exploited to determine the most likely material. The tactile sensor is tested in four approaches with increasing neural plausibility: first, we show that peak extraction from the Fourier spectrum is sufficient for tactile localization with position errors below 1%. Also, the damping property of the extracted frequency is used for material classification. Second, we show that the Fourier spectrum can be analysed by an Artificial Neural Network (ANN) which can be trained to decode contact distance and to classify contact materials. Thirdly, we show how efficiency can be improved by band-pass filtering the Fourier spectrum by application of non-negative matrix factorization. This reduces the input dimension by 95% while reducing classification performance by 8% only. Finally, we replace the FFT by an array of spiking neurons with gradually differing resonance properties, such that their spike rate is a function of the input frequency. We show that this network can be applied to detect tactile contact events of a wheeled robot, and how detrimental effects of robot velocity on antennal dynamics can be suppressed by state-dependent modulation of the input signals.

Active tactile sampling by an insect in a step-climbing paradigm.

Many insects actively explore their near-range environment with their antennae. Stick insects (Carausius morosus) rhythmically move their antennae during walking and respond to antennal touch by repetitive tactile sampling of the object. Despite its relevance for spatial orientation, neither the spatial sampling patterns nor the kinematics of antennation behavior in insects are understood. Here we investigate unrestrained bilateral sampling movements during climbing of steps. The main objectives are: (1) How does the antennal contact pattern relate to particular object features? (2) How are the antennal joints coordinated during bilateral tactile sampling? We conducted motion capture experiments on freely climbing insects, using steps of different height. Tactile sampling was analyzed at the level of antennal joint angles. Moreover, we analyzed contact patterns on the surfaces of both the obstacle and the antenna itself. Before the first contact, both antennae move in a broad, mostly elliptical exploratory pattern. After touching the obstacle, the pattern switches to a narrower and faster movement, caused by higher cycle frequencies and lower cycle amplitudes in all joints. Contact events were divided into wall- and edge-contacts. Wall contacts occurred mostly with the distal third of the flagellum, which is flexible, whereas edge contacts often occurred proximally, where the flagellum is stiff. The movement of both antennae was found to be coordinated, exhibiting bilateral coupling of functionally analogous joints [e.g., left head-scape (HS) joint with right scape-pedicel (SP) joint] throughout tactile sampling. In comparison, bilateral coupling between homologous joints (e.g., both HS joints) was significantly weaker. Moreover, inter-joint coupling was significantly weaker during the contact episode than before. In summary, stick insects show contact-induced changes in frequency, amplitude and inter-joint coordination during tactile sampling of climbed obstacles.

The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing.

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.

Analysis of pre-mRNA and pre-rRNA processing factor Snu13p structure and mutants.

Snu13p is a Saccharomyces cerevisiae protein essential for pre-messenger RNA splicing and pre-ribosomal RNA processing. Snu13p binds U4 snRNA of the spliceosome and box C/D snoRNAs of the pre-ribosomal RNA processing machinery to induce assembly of each ribonucleoprotein complex. Here, we present structural and biochemical analysis of Snu13p. The crystal structure of Snu13p reveals a region of the protein which could be important for protein interaction during ribonucleoprotein assembly. Using the structure of Snu13p we have designed the first temperature-sensitive mutants in Snu13p, L67W and I102A. Wild-type and mutant Snu13p proteins were assayed for binding to U4 snRNA and U3 snoRNA. Both temperature-sensitive mutants displayed significantly reduced RNA binding compared to wild-type protein. As the temperature-sensitive mutations are not in the known RNA binding region of Snu13p this indicates that these mutants indirectly influence the RNA binding properties of Snu13p. This work provides insight into Snu13p function during ribonucleoprotein assembly.

Slanted joint axes of the stick insect antenna: an adaptation to tactile acuity.

Like many flightless, obligatory walking insects, the stick insect Carausius morosus makes intensive use of active antennal movements for tactile near range exploration and orientation. The antennal joints of C. morosus have a peculiar oblique and non-orthogonal joint axis arrangement. Moreover, this arrangement is known to differ from that in crickets (Ensifera), locusts (Caelifera) and cockroaches (Blattodea), all of which have an orthogonal joint axis arrangement. Our hypothesis was that the situation found in C. morosus represents an important evolutionary trait of the order of stick and leaf insects (Phasmatodea). If this was true, it should be common to other species of the Phasmatodea. The objective of this comparative study was to resolve this question. We have measured the joint axis orientation of the head-scape and scape-pedicel joints along with other parameters that affect the tactile efficiency of the antenna. The obtained result was a complete kinematic description of the antenna. This was used to determine the size and location of kinematic out-of-reach zones, which are indicators of tactile acuity. We show that the oblique and non-orthogonal arrangement is common to eight species from six sub-families indicating that it is a synapomorphic character of the Euphasmatodea. This character can improve tactile acuity compared to the situation in crickets, locusts and cockroaches. Finally, because molecular data of a recent study indicate that the Phasmatodea may have evolved as flightless, obligatory walkers, we argue that the antennal joint axis arrangement of the Euphasmatodea reflects an evolutionary adaptation to tactile near range exploration during terrestrial locomotion.

Tactile efficiency of insect antennae with two hinge joints.

Antennae are the main organs of the arthropod tactile sense. In contrast to other senses that are capable of retrieving spatial information, e.g. vision, spatial sampling of tactile information requires active movement of the sense organ. For a quantitative analysis of basic principles of active tactile sensing, we use a generic model of arbitrary antennae with two hinge joints (revolute joints). This kind of antenna is typical for Orthoptera and Phasmatodea, i.e. insect orders that contain model species for the study of antennal movements, including cricket, locust and stick insect. First, we analyse the significance of morphological properties on workspace and sampling acuity. It is shown how joint axis orientation determines areas out of reach while affecting acuity in the areas within reach. Second, we assume a parametric set of movement strategies, based on empirical data on the stick insect Carausius morosus, and investigate the role of each strategy parameter on tactile sampling performance. A stochastic environment is used to measure sampling density, and a viscous friction model is assumed to introduce energy consumption and, thus, a measure of tactile efficiency. Up to a saturation level, sampling density is proportional to the range or frequency of joint angle modulation. The effect of phase shift is strong if joint angle modulation frequencies are equal, but diminishes for other frequency ratios. Speed of forward progression influences the optimal choice of movement strategy. Finally, for an analysis of environmental effects on tactile performance, we show how efficiency depends on predominant edge direction. For example, with slanted and non-orthogonal joint axis orientations, as present in the stick insect, the optimal sampling strategy is less sensitive to a change from horizontal to vertical edge predominance than with orthogonal and non-slanted joint axes, as present in a cricket.