A Protocol for Genome-Wide Analysis of DNA Replication Timing in Intact Root Tips.

DNA replication during S phase in eukaryotes is a highly regulated process that ensures the accurate transmission of genetic material to daughter cells during cell division. Replication follows a well-defined temporal program, which has been studied extensively in humans, Drosophila, and yeast, where it is clear that the replication process is both temporally and spatially ordered. The replication timing (RT) program is increasingly considered to be a functional readout of genomic features and chromatin organization. Although there is increasing evidence that plants display important differences in their DNA replication process compared to animals, RT programs in plants have not been extensively studied. To address this deficiency, we developed an improved protocol for the genome-wide RT analysis by sequencing newly replicated DNA ("Repli-seq") and applied it to the characterization of RT in maize root tips. Our protocol uses 5-ethynyl-2'-deoxyuridine (EdU) to label replicating DNA in vivo in intact roots. Our protocol also eliminates the need for synchronization and frequently associated chemical perturbations as well as the need for cell cultures, which can accumulate genetic and epigenetic differences over time. EdU can be fluorescently labeled under mild conditions and does not degrade subnuclear structure, allowing for the differentiation of labeled and unlabeled nuclei by flow sorting, effectively eliminating contamination issues that can result from sorting on DNA content alone. We also developed an analysis pipeline for analyzing and classifying regions of replication and present it in a point-and-click application called Repliscan that eliminates the need for command line programming.

An efficient and adaptive test of auditory mental imagery.

The ability to silently hear music in the mind has been argued to be fundamental to musicality. Objective measurements of this subjective imagery experience are needed if this link between imagery ability and musicality is to be investigated. However, previous tests of musical imagery either rely on self-report, rely on melodic memory, or do not cater in range of abilities. The Pitch Imagery Arrow Task (PIAT) was designed to address these shortcomings; however, it is impractically long. In this paper, we shorten the PIAT using adaptive testing and automatic item generation. We interrogate the cognitive processes underlying the PIAT through item response modelling. The result is an efficient online test of auditory mental imagery ability (adaptive Pitch Imagery Arrow Task: aPIAT) that takes 8 min to complete, is adaptive to participant's individual ability, and so can be used to test participants with a range of musical backgrounds. Performance on the aPIAT showed positive moderate-to-strong correlations with measures of non-musical and musical working memory, self-reported musical training, and general musical sophistication. Ability on the task was best predicted by the ability to maintain and manipulate tones in mental imagery, as well as to resist perceptual biases that can lead to incorrect responses. As such, the aPIAT is the ideal tool in which to investigate the relationship between pitch imagery ability and musicality.

Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation.

5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

Arabidopsis DNA Replication Initiates in Intergenic, AT-Rich Open Chromatin.

The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2'-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.

Musical imagery depends upon coordination of auditory and sensorimotor brain activity.

Recent magnetoencephalography (MEG) studies have established that sensorimotor brain rhythms are strongly modulated during mental imagery of musical beat and rhythm, suggesting that motor regions of the brain are important for temporal aspects of musical imagery. The present study examined whether these rhythms also play a role in non-temporal aspects of musical imagery including musical pitch. Brain function was measured with MEG from 19 healthy adults while they performed a validated musical pitch imagery task and two non-imagery control tasks with identical temporal characteristics. A 4-dipole source model probed activity in bilateral auditory and sensorimotor cortices. Significantly greater ß-band modulation was found during imagery compared to control tasks of auditory perception and mental arithmetic. Imagery-induced ß-modulation showed no significant differences between auditory and sensorimotor regions, which may reflect a tightly coordinated mode of communication between these areas. Directed connectivity analysis in the θ-band revealed that the left sensorimotor region drove left auditory region during imagery onset. These results add to the growing evidence that motor regions of the brain are involved in the top-down generation of musical imagery, and that imagery-like processes may be involved in musical perception.

Chromatin structure profile data from DNS-seq: Differential nuclease sensitivity mapping of four reference tissues of B73 maize (Zea mays L).

Presented here are data from Next-Generation Sequencing of differential micrococcal nuclease digestions of formaldehyde-crosslinked chromatin in selected tissues of maize (Zea mays) inbred line B73. Supplemental materials include a wet-bench protocol for making DNS-seq libraries, the DNS-seq data processing pipeline for producing genome browser tracks. This report also includes the peak-calling pipeline using the iSeg algorithm to segment positive and negative peaks from the DNS-seq difference profiles. The data repository for the sequence data is the NCBI SRA, BioProject Accession PRJNA445708.

Genome-Wide Analysis of the Arabidopsis Replication Timing Program.

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.

Reduced α-stable dynamics for multiple time scale systems forced with correlated additive and multiplicative Gaussian white noise.

Stochastic averaging problems with Gaussian forcing have been the subject of numerous studies, but far less attention has been paid to problems with infinite-variance stochastic forcing, such as an α-stable noise process. It has been shown that simple linear systems driven by correlated additive and multiplicative (CAM) Gaussian noise, which emerge in the context of reduced atmosphere and ocean dynamics, have infinite variance in certain parameter regimes. In this study, we consider the stochastic averaging of systems where a linear CAM noise process in the infinite variance parameter regime drives a comparatively slow process. We use (semi)-analytical approximations combined with numerical illustrations to compare the averaged process to one that is forced by a white α-stable process, demonstrating consistent properties in the case of large time-scale separation. We identify the conditions required for the fast linear CAM process to have such an influence in driving a slower process and then derive an (effectively) equivalent fast, infinite-variance process for which an existing stochastic averaging approximation is readily applied. The results are illustrated using numerical simulations of a set of example systems.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips.

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

Repliscan: a tool for classifying replication timing regions.

BACKGROUND: Replication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replication timing. RESULTS: To accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods make Repliscan generally applicable and more robust than previous methods that classify regions based on thresholds. CONCLUSIONS: Repliscan is simple and effective to use on organisms with different genome sizes. Even with analysis window sizes as small as 1 kilobase, reliable profiles can be generated with as little as 2.4x coverage.

The influence of visual information on auditory processing in individuals with congenital amusia: An ERP study.

While most normal hearing individuals can readily use prosodic information in spoken language to interpret the moods and feelings of conversational partners, people with congenital amusia report that they often rely more on facial expressions and gestures, a strategy that may compensate for deficits in auditory processing. In this investigation, we used EEG to examine the extent to which individuals with congenital amusia draw upon visual information when making auditory or audio-visual judgments. Event-related potentials (ERP) were elicited by a change in pitch (up or down) between two sequential tones paired with a change in spatial position (up or down) between two visually presented dots. The change in dot position was either congruent or incongruent with the change in pitch. Participants were asked to judge (1) the direction of pitch change while ignoring the visual information (AV implicit task), and (2) whether the auditory and visual changes were congruent (AV explicit task). In the AV implicit task, amusic participants performed significantly worse in the incongruent condition than control participants. ERPs showed an enhanced N2-P3 response to incongruent AV pairings for control participants, but not for amusic participants. However when participants were explicitly directed to detect AV congruency, both groups exhibited enhanced N2-P3 responses to incongruent AV pairings. These findings indicate that amusics are capable of extracting information from both modalities in an AV task, but are biased to rely on visual information when it is available, presumably because they have learned that auditory information is unreliable. We conclude that amusic individuals implicitly draw upon visual information when judging auditory information, even though they have the capacity to explicitly recognize conflicts between these two sensory channels.

Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.

5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.

Melodic Contour Training and Its Effect on Speech in Noise, Consonant Discrimination, and Prosody Perception for Cochlear Implant Recipients.

Cochlear implant (CI) recipients generally have good perception of speech in quiet environments but difficulty perceiving speech in noisy conditions, reduced sensitivity to speech prosody, and difficulty appreciating music. Auditory training has been proposed as a method of improving speech perception for CI recipients, and recent efforts have focussed on the potential benefits of music-based training. This study evaluated two melodic contour training programs and their relative efficacy as measured on a number of speech perception tasks. These melodic contours were simple 5-note sequences formed into 9 contour patterns, such as "rising" or "rising-falling." One training program controlled difficulty by manipulating interval sizes, the other by note durations. Sixteen adult CI recipients (aged 26-86 years) and twelve normal hearing (NH) adult listeners (aged 21-42 years) were tested on a speech perception battery at baseline and then after 6 weeks of melodic contour training. Results indicated that there were some benefits for speech perception tasks for CI recipients after melodic contour training. Specifically, consonant perception in quiet and question/statement prosody was improved. In comparison, NH listeners performed at ceiling for these tasks. There was no significant difference between the posttraining results for either training program, suggesting that both conferred benefits for training CI recipients to better perceive speech.

Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

Intonation processing deficits of emotional words among Mandarin Chinese speakers with congenital amusia: an ERP study.

BACKGROUND: Congenital amusia is a disorder that is known to affect the processing of musical pitch. Although individuals with amusia rarely show language deficits in daily life, a number of findings point to possible impairments in speech prosody that amusic individuals may compensate for by drawing on linguistic information. Using EEG, we investigated (1) whether the processing of speech prosody is impaired in amusia and (2) whether emotional linguistic information can compensate for this impairment. METHOD: Twenty Chinese amusics and 22 matched controls were presented pairs of emotional words spoken with either statement or question intonation while their EEG was recorded. Their task was to judge whether the intonations were the same. RESULTS: Amusics exhibited impaired performance on the intonation-matching task for emotional linguistic information, as their performance was significantly worse than that of controls. EEG results showed a reduced N2 response to incongruent intonation pairs in amusics compared with controls, which likely reflects impaired conflict processing in amusia. However, our EEG results also indicated that amusics were intact in early sensory auditory processing, as revealed by a comparable N1 modulation in both groups. CONCLUSION: We propose that the impairment in discriminating speech intonation observed among amusic individuals may arise from an inability to access information extracted at early processing stages. This, in turn, could reflect a disconnection between low-level and high-level processing.

Common cues to emotion in the dynamic facial expressions of speech and song.

Speech and song are universal forms of vocalization that may share aspects of emotional expression. Research has focused on parallels in acoustic features, overlooking facial cues to emotion. In three experiments, we compared moving facial expressions in speech and song. In Experiment 1, vocalists spoke and sang statements each with five emotions. Vocalists exhibited emotion-dependent movements of the eyebrows and lip corners that transcended speech-song differences. Vocalists' jaw movements were coupled to their acoustic intensity, exhibiting differences across emotion and speech-song. Vocalists' emotional movements extended beyond vocal sound to include large sustained expressions, suggesting a communicative function. In Experiment 2, viewers judged silent videos of vocalists' facial expressions prior to, during, and following vocalization. Emotional intentions were identified accurately for movements during and after vocalization, suggesting that these movements support the acoustic message. Experiment 3 compared emotional identification in voice-only, face-only, and face-and-voice recordings. Emotion judgements for voice-only singing were poorly identified, yet were accurate for all other conditions, confirming that facial expressions conveyed emotion more accurately than the voice in song, yet were equivalent in speech. Collectively, these findings highlight broad commonalities in the facial cues to emotion in speech and song, yet highlight differences in perception and acoustic-motor production.

A developmental study of the effect of music training on timed movements.

When people clap to music, sing, play a musical instrument, or dance, they engage in temporal entrainment. We examined the effect of music training on the precision of temporal entrainment in 57 children aged 10-14 years (31 musicians, 26 non-musicians). Performance was examined for two tasks: self-paced finger tapping (discrete movements) and circle drawing (continuous movements). For each task, participants synchronized their movements with a steady pacing signal and then continued the movement at the same rate in the absence of the pacing signal. Analysis of movements during the continuation phase revealed that musicians were more accurate than non-musicians at finger tapping and, to a lesser extent, circle drawing. Performance on the finger-tapping task was positively associated with the number of years of formal music training, whereas performance on the circle-drawing task was positively associated with the age of participants. These results indicate that music training and maturation of the motor system reinforce distinct skills of timed movement.

Singing emotionally: a study of pre-production, production, and post-production facial expressions.

Singing involves vocal production accompanied by a dynamic and meaningful use of facial expressions, which may serve as ancillary gestures that complement, disambiguate, or reinforce the acoustic signal. In this investigation, we examined the use of facial movements to communicate emotion, focusing on movements arising in three epochs: before vocalization (pre-production), during vocalization (production), and immediately after vocalization (post-production). The stimuli were recordings of seven vocalists' facial movements as they sang short (14 syllable) melodic phrases with the intention of communicating happiness, sadness, irritation, or no emotion. Facial movements were presented as point-light displays to 16 observers who judged the emotion conveyed. Experiment 1 revealed that the accuracy of emotional judgment varied with singer, emotion, and epoch. Accuracy was highest in the production epoch, however, happiness was well communicated in the pre-production epoch. In Experiment 2, observers judged point-light displays of exaggerated movements. The ratings suggested that the extent of facial and head movements was largely perceived as a gauge of emotional arousal. In Experiment 3, observers rated point-light displays of scrambled movements. Configural information was removed in these stimuli but velocity and acceleration were retained. Exaggerated scrambled movements were likely to be associated with happiness or irritation whereas unexaggerated scrambled movements were more likely to be identified as "neutral." An analysis of singers' facial movements revealed systematic changes as a function of the emotional intentions of singers. The findings confirm the central role of facial expressions in vocal emotional communication, and highlight individual differences between singers in the amount and intelligibility of facial movements made before, during, and after vocalization.

Augmenting melodic intonation therapy with non-invasive brain stimulation to treat impaired left-hemisphere function: two case studies.

The purpose of this study was to investigate whether or not the right hemisphere can be engaged using Melodic Intonation Therapy (MIT) and excitatory repetitive transcranial magnetic stimulation (rTMS) to improve language function in people with aphasia. The two participants in this study (GOE and AMC) have chronic non-fluent aphasia. A functional Magnetic Resonance Imaging (fMRI) task was used to localize the right Broca's homolog area in the inferior frontal gyrus for rTMS coil placement. The treatment protocol included an rTMS phase, which consisted of 3 treatment sessions that used an excitatory stimulation method known as intermittent theta burst stimulation, and a sham-rTMS phase, which consisted of 3 treatment sessions that used a sham coil. Each treatment session was followed by 40 min of MIT. A linguistic battery was administered after each session. Our findings show that one participant, GOE, improved in verbal fluency and the repetition of phrases when treated with MIT in combination with TMS. However, AMC showed no evidence of behavioral benefit from this brief treatment trial. Post-treatment neural activity changes were observed for both participants in the left Broca's area and right Broca's homolog. These case studies indicate that a combination of MIT and rTMS applied to the right Broca's homolog has the potential to improve speech and language outcomes for at least some people with post-stroke aphasia.