Sequence-specific Effects Research Articles

Abstract A136: AST-008, a novel TLR9 agonist SNA, induces abscopal antitumor effects in mouse tumor models

Abstract Spherical nucleic acids (SNA) are a novel class of therapeutic agents in which oligonucleotides are densely packed and radially arranged on the surface of liposomal nanoparticles providing a 3D-architecture. This architecture provides increased cellular uptake and nuclease stability compared to linear oligonucleotides that are not in SNA format. AST-008 is an SNA configuration of a Toll-like receptor 9 (TLR9) agonist that induces potent Th1-type immune responses in vitro, and in mice and nonhuman primates. Subcutaneous (SC), intratumoral (IT) and intravenous (IV) administration of AST-008 has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor (CPI) activity in a number of mouse tumor models. Our previous studies have shown that AST-008 induces potent innate and adaptive immune responses and increases tumor infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T-cells with reduced T-regulatory cells in the tumor microenvironment facilitating increased effectiveness of CPI. In the present studies, we have evaluated i) sequence-specific antitumor effects of AST-008 following SC delivery in an EMT-6 breast tumor model, ii) abscopal antitumor effects of a murine version of the TLR9 agonist SNA following SC delivery in a two-flank EMT-6 tumor model, and iii) abscopal antitumor effects of AST-008 following SC and IT administrations in an MC38 colon cancer model. Treatment of EMT-6 breast tumors in mice with SC administration of AST-008 showed dose-dependent tumor growth inhibition that was statistically significant compared with vehicle-treated tumor-bearing mice. A control oligonucleotide-SNA had no effect on tumor growth compared with the vehicle group. In a two-flank EMT-6 breast tumor model, SC administration of TLR9 agonist SNA showed about 59% and 37% tumor growth inhibition at the injected and distant tumors, respectively, compared to the vehicle-treated mice. The tumor growth inhibition was statistically significant at both the injected and uninjected tumors compared to vehicle-treated tumor-bearing mice. In a two-flank MC38 colon cancer model, either SC or IT administration of AST-008 resulted in 69% and 68% tumor growth inhibition, respectively, at the injected tumor. In this study, distant tumor that was not treated with AST-008 showed 59% and 46% tumor growth inhibition for SC and IT routes of administration, respectively. The tumor growth inhibition at both the treated and untreated tumors was statistically significant compared to the vehicle-treated tumors. These data demonstrate sequence-specific antitumor activity of AST-008 and that SC or IT administration at one tumor lesion leads to systemic antitumor activity at distant uninjected tumors. The current results, together with our previous studies in combination with CPI in several tumor models, support the potential utility of the TLR9-mediated innate immune stimulator AST-008 in combination with CPI in cancer patients. AST-008 is currently in clinical development. Citation Format: Ekambar R. Kandimalla, SubbaRao Nallagatla, Bart R. Anderson, Richard Kang. AST-008, a novel TLR9 agonist SNA, induces abscopal antitumor effects in mouse tumor models [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A136.

NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.

Do musicians learn a fine sequential hand motor skill differently than non-musicians?

Do professional musicians learn a fine sequential hand motor skill more efficiently than non-musicians? Is this also the case when they perform motor imagery, which implies that they only mentally simulate these movements? Musicians and non-musicians performed a Go/NoGo discrete sequence production (DSP) task, which allows to separate sequence-specific from a-specific learning effects. In this task five stimuli, to be memorized during a preparation interval, signaled a response sequence. In a practice phase, different response sequences had to be either executed, imagined, or inhibited, which was indicated by different response cues. In a test phase, responses were required to familiar (previously executed, imagined, or inhibited) and unfamiliar sequences. In both phases, response times and response accuracy were measured while the electroencephalogram (EEG) was only registered during the practice phase to compare activity between motor imagery, motor execution, and motor inhibition for both groups. Results in the practice phase revealed that musicians learned the response sequences faster and more accurately than non-musicians although no difference in initiation time was found. EEG analyses revealed similar lateralized activity during learning a motor skill for both groups. Our results from the test phase showed better sequence-a-specific learning effects (i.e., faster response times and increased accuracy) for musicians than for non-musicians. Moreover, we revealed that non-musicians benefit more from physical execution while learning a required motor sequence, whereas sequence-specific learning effects due to learning with motor imagery were very similar for musicians and non-musicians.

Does Transcranial Direct Current Stimulation Affect the Learning of a Fine Sequential Hand Motor Skill with Motor Imagery?

Learning a fine sequential hand motor skill, like playing the piano or learning to type, improves not only due to physical practice, but also due to motor imagery. Previous studies revealed that transcranial direct current stimulation (tDCS) and motor imagery independently affect motor learning. In the present study, we investigated whether tDCS combined with motor imagery above the primary motor cortex influences sequence-specific learning. Four groups of participants were involved: an anodal, cathodal, sham stimulation, and a control group (without stimulation). A modified discrete sequence production (DSP) task was employed: the Go/NoGo DSP task. After a sequence of spatial cues, a response sequence had to be either executed, imagined, or withheld. This task allows to estimate both non-specific learning and sequence-specific learning effects by comparing the execution of unfamiliar sequences, familiar imagined, familiar withheld, and familiar executed sequences in a test phase. Results showed that the effects of anodal tDCS were already developing during the practice phase, while no effects of tDCS on sequence-specific learning were visible during the test phase. Results clearly showed that motor imagery itself influences sequence learning, but we also revealed that tDCS does not increase the influence of motor imagery on sequence learning.

Growth-restricting effects of siRNA transfections: a largely deterministic combination of off-target binding and hybridization-independent competition.

Perturbation of gene expression by means of synthetic small interfering RNAs (siRNAs) is a powerful way to uncover gene function. However, siRNA technology suffers from sequence-specific off-target effects and from limitations in knock-down efficiency. In this study, we assess a further problem: unintended effects of siRNA transfections on cellular fitness/proliferation. We show that the nucleotide compositions of siRNAs at specific positions have reproducible growth-restricting effects on mammalian cells in culture. This is likely distinct from hybridization-dependent off-target effects, since each nucleotide residue is seen to be acting independently and additively. The effect is robust and reproducible across different siRNA libraries and also across various cell lines, including human and mouse cells. Analyzing the growth inhibition patterns in correlation to the nucleotide sequence of the siRNAs allowed us to build a predictor that can estimate growth-restricting effects for any arbitrary siRNA sequence. Competition experiments with co-transfected siRNAs further suggest that the growth-restricting effects might be linked to an oversaturation of the cellular miRNA machinery, thus disrupting endogenous miRNA functions at large. We caution that competition between siRNA molecules could complicate the interpretation of double-knockdown or epistasis experiments, and potential interactions with endogenous miRNAs can be a factor when assaying cell growth or viability phenotypes.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in "enhancerless" self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required.IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.

Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli

BackgroundUP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear.ResultsHere, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants (Kd) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < Kd < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity.ConclusionsThese results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes.

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3-5bp), positioned within 25bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lowerthroughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein.

How effector-specific is the effect of sequence learning by motor execution and motor imagery?

The aim of the present study was twofold. First, we wanted to examine how effector specific the effect of sequence learning by motor execution is, and second, we wanted to compare this effect with learning by motor imagery. We employed a Go/NoGo discrete sequence production task in which in each trial a spatial sequence of five stimuli was presented. After a Go signal the corresponding spatial response sequence had to be executed, while after a NoGo signal, the response sequence had to be mentally imagined. For the training phase, participants were divided into two groups. In the index finger group, participants had to respond (physically or mentally) with the left or right index finger, while in the hand group they had to respond with four fingers of the left or right hand. In a final test phase both execution modes were compared and all trials had to be executed. Response times and the percentage of correct responses were determined to establish learning effects. Results showed that sequence learning effects as assessed in the test phase were independent of the effector used during the training phase. Results revealed the presence of aspecific learning effects in the case of learning a required motor task with an index finger, but sequence-specific learning effects, both due to motor execution and to motor imagery, were not effector specific.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

P.340 - Translating DUX4-targeted RNAi therapy for Facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies. It is a dominant disorder caused by overexpression of the myotoxic gene DUX4. Previously our lab demonstrated proof-of-principle for the use of a DUX4-targeted RNA interference (RNAi)-based gene therapy as a putative treatment for FSHD. To accomplish this we pursued two lead engineered microRNAs as target products for DUX4 inhibition. Both sequences showed strong therapeutic efficacy determined by their ability to suppress pathogenic levels of DUX4, and prevent myopathy in DUX4-expressing mice. The goal of this study was to pursue the next steps toward translation by assessing safety and off-target effects of our lead miRNA target products following dose-escalation. To do this, we delivered adeno-associated viral (AAV) vectors expressing U6 promoter-driven, DUX4-targeted miRNAs to mice, either by local intramuscular or intravascular injections. Over-expression related toxicity (or lack thereof) in multiple organ systems was assessed at 3 weeks and 5 months by an independent veterinary pathologist using a blinded experimental design. One sequence showed time- and dose-dependent toxicity in the skeletal muscle, while the other produced no overt deleterious effects in any organ system. To assess sequence-specific off-target effects, we performed RNA-seq using RNA harvested from human myoblasts transfected with our lead therapeutic miRNAs. Surprisingly, only 4 transcripts were reduced upon miRNA expression, and of these, only 1 contained seed match binding sites for our lead sequence suggesting the promiscuity of the DUX4-targeted miRNAs is not a major concern. Our goal is to translate this work toward a clinical trial, and we are now performing additional vector optimization, safety and efficacy measures in anticipation of pre-IND discussions with FDA in the near future.

A Novel Coarse-Grained Model to Study Liquid-Liquid Phase Separation of Disordered Proteins

Recent studies have led to the discovery of droplets composed of disordered protein chains in a separate liquid-like phase. Such assemblies have been found to be relevant to physiological function as membraneless compartments in living cells including the nucleolus and ribonucleoprotein (RNP) granules as well as many organelles in prokaryotic cells (1). The thermodynamic driving force for this phase separation, however, is still not well characterized. Fused in Sarcoma (FUS) is an RNA binding protein that naturally forms these droplets as RNP granules which aid with DNA repair. The low complexity domain, FUS LC is a fully disordered domain and has been observed to form these droplets on its own in vitro (2). Mutations of FUS can result in transformation of these assemblies into poorly soluble aggregates involved in the pathogenesis of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (3). As the spatiotemporal scales involved in the phase separation of proteins cannot be resolved using fully detailed all-atom models, we develop a simple Cα;-based coarse-grained model that treats each amino acid in the chain as a single interaction site. The model is parameterized to treat each of the twenty naturally occurring amino acids differently in order to capture sequence-specific effects. Development of the proposed model involves top-down comparisons with experimental data available from the recent literature as well as comparisons with atomistic simulations of a single chain. We then use this model to characterize the droplet formation for FUS LC including the proposed transition from liquid-like to solid-like aggregates. We are also able to capture known trends in FUS LC phase separation as a function of salt concentration and disease-related mutations. This model should aid in the molecular understanding of phase behavior of disordered proteins thus aiding in development of therapeutic strategies.1.Brangwynne, C. P.; Eckmann, C. R.; Courson, D. S.; Rybarska, A.; Hoege, C.; Gharakhani, J.; Julicher, F.; Hyman, A. A. Science 2009, 324, 1729-17322.Burke, K. A.; Janke, A. M.; Rhine, C. L.; Fawzi, N. L. Molecular Cell 2015, 60, 231-2413.Patel, A.; Lee H. O.; Jawerth, L.; Dechsel, D.; Hyman, A. A.; Alberti, S. and othersCell 2015, 162, 1066-1077

Intrinsic α helix propensities compact hydrodynamic radii in intrinsically disordered proteins.

Proteins that lack tertiary stability under normal conditions, known as intrinsically disordered, exhibit a wide range of biological activities. Molecular descriptions for the biology of intrinsically disordered proteins (IDPs) consequently rely on disordered structural models, which in turn require experiments that assess the origins to structural features observed. For example, while hydrodynamic size is mostly insensitive to sequence composition in chemically denatured proteins, IDPs show strong sequence-specific effects in the hydrodynamic radius (Rh ) when measured under normal conditions. To investigate sequence-modulation of IDP Rh , disordered ensembles generated by a hard sphere collision model modified with a structure-based parameterization of the solution energetics were used to parse the contributions of net charge, main chain dihedral angle bias, and excluded volume on hydrodynamic size. Ensembles for polypeptides 10-35 residues in length were then used to establish power-law scaling relationships for comparison to experimental Rh from 26 IDPs. Results showed the expected outcomes of increased hydrodynamic size from increases in excluded volume and net charge, and compaction from chain-solvent interactions. Chain bias representing intrinsic preferences for α helix and polyproline II (PPII ), however, modulated Rh with intricate dependence on the simulated propensities. PPII propensities at levels expected in IDPs correlated with heightened Rh sensitivity to even weak α helix propensities, indicating bias for common (φ, ψ) are important determinants of hydrodynamic size. Moreover, data show that IDP Rh can be predicted from sequence with good accuracy from a small set of physicochemical properties, namely intrinsic conformational propensities and net charge. Proteins 2017; 85:296-311. © 2016 Wiley Periodicals, Inc.

Bacterial RNA as a signal to eukaryotic cells as part of the infection process.

The discovery of regulatory RNA has identified an underappreciated area for microbial subversion of the host. There is increasing evidence that RNA can be delivered from bacteria to host cells associated with membrane vesicles or by direct release from intracellular bacteria. Once inside the host cell, RNA can act by activating sequence-independent receptors of the innate immune system, where recent findings suggest this can be more than simple pathogen detection, and may contribute to the subversion of immune responses. Sequence specific effects are also being proposed, with examples from nematode, plant and human models providing support for the proposition that bacteria-to-human RNA signaling and the subversion of host gene expression may occur.

A mechanism for sequence specificity in plant-mediated interactions between herbivores.

Summary Herbivore communities are shaped by indirect plant‐mediated interactions whose outcomes are strongly dependent on the sequence of herbivore arrival. However, the mechanisms underlying sequence specificity are poorly understood.We examined the mechanisms that govern sequence‐specific effects of the interaction between two specialist maize herbivores, the leaf feeder Spodoptera frugiperda and the root feeder Diabrotica virgifera virgifera. In the field, S. frugiperda reduces D. v. virgifera abundance, but only when it arrives on the plant first.In behavioral experiments, D. v. virgifera larvae continued feeding on plants that they had infested before leaf infestation, but refused to initiate feeding on plants that were infested by S. frugiperda before their arrival. Changes in root‐emitted volatiles were sufficient to elicit this sequence‐specific behavior. Root volatile and headspace mixing experiments showed that early‐arriving D. v. virgifera larvae suppressed S. frugiperda‐induced volatile repellents, which led to the maintenance of host attractiveness to D. v. virgifera.Our study provides a physiological and behavioral mechanism for sequence specificity in plant‐mediated interactions and suggests that physiological canalization of behaviorally active metabolites can drive sequence specificity and result in strongly diverging herbivore distribution patterns.

The influence of motor imagery on the learning of a fine hand motor skill.

Motor imagery has been argued to affect the acquisition of motor skills. The present study examined the specificity of motor imagery on the learning of a fine hand motor skill by employing a modified discrete sequence production task: the Go/NoGo DSP task. After an informative cue, a response sequence had either to be executed, imagined, or withheld. To establish learning effects, the experiment was divided into a practice phase and a test phase. In the latter phase, we compared mean response times and accuracy during the execution of unfamiliar sequences, familiar imagined sequences, and familiar executed sequences. The electroencephalogram was measured in the practice phase to compare activity between motor imagery, motor execution, and a control condition in which responses should be withheld. Event-related potentials (ERPs) and event-related lateralizations (ERLs) showed strong similarities above cortical motor areas on trials requiring motor imagery and motor execution, while a major difference was found with trials on which the response sequence should be withheld. Behavioral results from the test phase showed that response times and accuracy improved after physical and mental practice relative to unfamiliar sequences (so-called sequence-specific learning effects), although the effect of motor learning by motor imagery was smaller than the effect of physical practice. These findings confirm that motor imagery also resembles motor execution in the case of a fine hand motor skill.

Non-parametric Algorithm to Isolate Chunks in Response Sequences.

Chunking consists in grouping items of a sequence into small clusters, named chunks, with the assumed goal of lessening working memory load. Despite extensive research, the current methods used to detect chunks, and to identify different chunking strategies, remain discordant and difficult to implement. Here, we propose a simple and reliable method to identify chunks in a sequence and to determine their stability across blocks. This algorithm is based on a ranking method and its major novelty is that it provides concomitantly both the features of individual chunk in a given sequence, and an overall index that quantifies the chunking pattern consistency across sequences. The analysis of simulated data confirmed the validity of our method in different conditions of noise, chunk lengths and chunk numbers; moreover, we found that this algorithm was particularly efficient in the noise range observed in real data, provided that at least 4 sequence repetitions were included in each experimental block. Furthermore, we applied this algorithm to actual reaction time series gathered from 3 published experiments and were able to confirm the findings obtained in the original reports. In conclusion, this novel algorithm is easy to implement, is robust to outliers and provides concurrent and reliable estimation of chunk position and chunking dynamics, making it useful to study both sequence-specific and general chunking effects. The algorithm is available at: https://github.com/artipago/Non-parametric-algorithm-to-isolate-chunks-in-response-sequences.

EP 129. The effect of tDCS over M1 prior to and during implicit motor sequence learning

Motor learning is an essential skill allowing the acquisition of new movement patterns. Transcranial direct current stimulation (tDCS) has been shown to modulate neuronal excitability along with behavioural performance in a polarity-dependent manner. Besides polarity, the effects of tDCS on motor learning also vary with the timing of stimulation. In order to gain a better understanding regarding the optimal timing of tDCS application to modulate implicit motor sequence learning, motor-cortical tDCS was applied prior to and during training on a serial reaction time task (SRTT). The SRTT employs a fixed sequential pattern of button presses allowing the assessment of implicit motor sequence learning. A random pattern served as control condition. 36 healthy subjects were assigned to one of two experiments: tDCS was applied prior to (experiment 1) or during (experiment 2) SRTT training. Anodal vs. cathodal vs. sham tDCS was applied to the left primary motor cortex (M1) for ten minutes in a counterbalanced order. Reaction times of the right hand were measured at Baseline and after SRTT training (End of Acquisition (EoA)). Anodal tDCS prior to SRTT training yielded a beneficial effect on the acquisition of the motor sequence, i.e. reaction times decreased significantly from Baseline to EoA in the sequential pattern. Moreover, at EoA, reaction times were significantly faster following anodal as compared to cathodal tDCS. Reaction times of the random pattern were not differentially modulated by stimulation polarity indicating a sequence-specific effect. In contrast, tDCS applied during SRTT training did not differentially modulate the acquisition of the motor sequence. These results indicate that the timing of tDCS is a crucial parameter yielding distinct effects on implicit motor sequence learning. Anodal tDCS prior to SRTT training resulted in a facilitation emphasising a beneficial effect of increased left M1 excitability for the subsequent acquisition of a new motor sequence. In contrast, modulation of motor-cortical excitability by tDCS during SRTT training yielded no differential effects. This finding implies that tDCS effects may vary with the activation level during stimulation (rest vs. movement).

Targeting insect mitochondrial complex I for plant protection.

SummaryPlant engineered to express double‐stranded RNA (dsRNA) can target the herbivorous insect gene for silencing. Although mounting evidence has emerged to support feasibility of this new pest control technology, field application is slow largely due to lack of potent targets. Here, we show that suppression of the gene encoding NDUFV2, a subunit of mitochondrial complex I that catalyses NADH dehydrogenation in respiratory chain, was highly lethal to insects. Feeding cotton bollworm (Helicoverpa armigera) larvae with transgenic cotton tissues expressing NDUFV2 dsRNA led to mortality up to 80% within 5 days, and almost no larvae survived after 7 days of feeding, due to the altered mitochondrial structure and activity. Transcriptome comparisons showed a drastic repression of dopa decarboxylase genes. Reciprocal assays with Asian corn borer (Ostrinia furnacalis), another lepidopteran species, revealed the sequence‐specific effect of NDUFV2 suppression. Furthermore, the hemipteran lugus Apolygus lucorum was also liable to NDUFV2 repression. These data demonstrate that the mitochondrial complex I is a promising target with both sequence specificity and wide applicability for the development of new‐generation insect‐proof crops.

Sequence-non-specific effects generated by various types of RNA interference triggers

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.