We identified plenty of RNA primary sequences and secondary structural features that affect editing specificity and efficiency. Although hairpin loop structures contribute importantly to editing, unlike in animals, the primary sequences have more profound influences on editing than secondary structures. Nucleotide preferences at adjacent positions of editing sites are the most important features, especially preferences at the −1 position. Unexpectedly, besides the number of positions with preferred nucleotides, the combination of preferred nucleotides with depleted ones at different positions are also important for editing. Some cis-sequence features have distinct importance for editing specificity and efficiency. Machine learning models built from diverse sequence and secondary structural features can accurately predict genome-wide editing sites but not editing levels, indicating that the cis-regulatory principle of editing efficiency is more complex than that of editing specificity. Nevertheless, our model interpretation provides insights into the quantitative contribution of each feature to the prediction of both editing sites and levels. We found that efficient editing of FG3G34330 transcripts depended on the full-length RNA molecule, suggesting that additional RNA structural elements may also contribute to editing efficiency. Our work uncovers multidimensional cis-regulatory elements important for A-to-I RNA editing in F. graminearum, helping to elucidate the fungal editing mechanism.Adenosine-to-inosine (A-to-I) RNA editing independent of adenosine deaminase acting on RNA (ADAR) enzymes was discovered in fungi recently, and shown to be crucial for sexual reproduction. However, the underlying mechanism for editing is unknown. Here, we combine genome-wide comparisons, proof-of-concept experiments, and machine learning to decipher  RNA editing is an epigenetic phenomenon that alters the RNA sequence so that it differs from the genomic DNA sequence. Types of RNA editing include insertion, deletion, and base modification of nucleotides . The mosFusarium graminearum  family . The ADAcis-acting regulatory elements) have been proposed to regulate ADAR editing, including nucleotides opposing the target adenosine, 5′ and 3′ nearest-neighboring nucleotides next to the editing site, local secondary structure, length and stability of dsRNA structure, and tertiary structure of the RNA substrate , which can vary from very low to nearly 100%. The underlying mechanisms determining which sites within the dsRNA to be edited (editing specificity) and to what levels (editing efficiency) by ADAR enzymes have been extensively investigated in animals. Multiple RNA sequence and structure features   , one of ht (FHB) . Ascospoht (FHB) , 19. F. scovered . Strand-minearum , 20, 21.. crassa . Therefo. crassa , 23.cis-regulatory sequence and structural elements that affect A-to-I RNA editing in F. graminearum by genome-wide comparisons. As a proof-of-concept, we chose 4 representative editing sites to evaluate the importance of different RNA sequence and structure features for their editing levels using a site-specific mutagenesis approach at the native locus. Based on the identified cis-regulatory features, we also used supervised machine learning to build predictive models of RNA editing sites and levels and quantified the contributions of all features to the RNA editing predictions. Our results provide insights into the complex cis-regulation of A-to-I RNA editing and help to elucidate the editing mechanism in fungi.In this study, we dissected the F. graminearum, we generated over 17 Gb strand-specific RNA-seq data from 5 biological replicates of perithecia collected at 6 days post-fertilization (dpf)  to 100%, with a median value of 12%. More than 80% of sites had editing levels <30%, whereas less than 4% had editing levels >60%. The fraction of sites with low editing levels was increased relative to the previous report in F. graminearum (To detect more A-to-I RNA editing sites in on (dpf) . In combon (dpf) , we idenon (dpf) . The num. crassa  but overminearum . The edi10.1128/mbio.01872-22.6TABLE S1Table S1, DOCX file, 0.01 MB.RNA-seq and DNA-seq data used. Download Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01872-22.7TABLE S2Fusarium graminearum. Download Table S2, DOCX file, 0.01 MB.The number of different RNA variant sites detected in Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the F. graminearum showed a conserved sequence motif surrounding the editing sites from −2 to +4 positions , the −1 position of editing sites was strongly enriched for uridine (U) but depleted for the other 3 nucleotides. Whereas the −2 position was slightly enriched for cytidine (C)/U but depleted for A/G, the +1, +3, and +4 positions were slightly enriched for A/G but depleted for C/U  and depleted nucleotides at −2 to +4 positions . The groF. graminearum. It should be noted that the group with preferred nucleotides at more positions were not necessary to have more editing sites. For example, the group with preferred nucleotides at −1, +1, and +3 positions and depleted nucleotides at the other 3 positions contained a larger number of editing sites than that of many groups with preferred nucleotides beyond the −1, +1, and +3 positions  . Consistent with previous observations , over 62< 2e-16) . A simil< 2e-16) . In addi< 2e-16) , and the< 2e-16) . These r10.1128/mbio.01872-22.1FIG S1http://fgbase.wheatscab.com/). Download FIG S1, PDF file, 0.1 MB.Stacked columns showing the percentage of edited sites with marked editing levels and control sites in the 5 types of RNA secondary structure elements predicated from full-length RNA sequences. The full-length RNA sequence of each gene was obtained from FgBase  of predicated RNA secondary structures revealed that the hairpin loops with editing sites had a lower MFE value (more stable) compared to those with control A sites , and the. crassa , suggest10.1128/mbio.01872-22.2FIG S2Cis-regulatory elements of A-to-I RNA editing in Neurospora crassa. (A) Boxplots comparing the editing levels of editing sites with enriched and depleted nucleotides at -2 to +3 positions. (B) Percentage of editing sites with marked editing levels located in hairpin loops. (C) Boxplots comparing the editing level of the editing sites located in hairpin loops and other types of RNA secondary structure elements. (D) Boxplots comparing the minimum free energy (MFE) of predicted hairpin loops for edited and control A sites. (E) Correlation between the mean editing level and the mean MFE. Each dot represents 10% of editing sites located in hairpin loops. The Pearson correlation coefficient and P-value are indicated. P-values in (A), (C), and (D) are from two-tailed Wilcoxon rank-sum tests. Download FIG S2, PDF file, 1.5 MB.Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the t test, P < 0.05), implying that the influence of secondary structure on RNA editing is not as critical as primary sequences.Intriguingly, in comparison with all the editing sites in general, the editing sites with preferred nucleotides at all 6 positions had lower fractions of sites located in hairpin loops . MoreoveF. graminearum, we adopted a recyclable marker module to perform gene deletion and allelic exchange at the native genomic locus (To verify the effect of neighboring nucleotides and secondary structures on RNA editing in ic locus . We seleic locus . The traic locus . All 4 e10.1128/mbio.01872-22.3FIG S3http://fgbase.wheatscab.com/). Coni, conidia collected from 5-day-old CMC cultures; Ger12h and Hyp24h, germlings and vegetative hyphae collected from conidia incubated for 12 and 48 h in liquid YEPD cultures; Inf3d, inoculated spikelets of flowering wheat heads collected 3 days after inoculation; TBI3d, DON-producing hyphae collected from 3-d-old liquid trichothecene biosynthesis induction (TBI) cultures; Sex3d and Sex8d, perithecia harvested from carrot agar plates at 3- or 8-days postfertilization. Download FIG S3, PDF file, 0.04 MB.RNA-seq expression of FG3G34330 gene in different samples. The Transcripts Per Kilobase of exon model per Million mapped reads (TPM) expression value was obtained from FgBase . Bar = 20 μm. Download FIG S4, PDF file, 0.4 MB.Morphology of colonies, perithecia, asci, and ascospores generated by wild type (WT) and FG3G34330 deletion mutant  at the −1 position of editing sites 1, 2, and 4 into the depleted G at the native genomic locus of FG3G34330. Reverse-transcription (RT)-PCR amplification was performed with 6-dpf perithecia of obtained mutant and wild type . Sanger 10.1128/mbio.01872-22.8TABLE S3Table S3, DOCX file, 0.02 MB.Summary of strains used. Download Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P > 0.05), but markedly reduced the editing level of site 4   . Moreove < 0.05) . Altogett test, P < 0.05)  . Particu < 0.05)  and C. TTo determine whether efficient RNA editing depends on intact mRNA molecules, we generated transformants ectopically expressing the full-length and 5′-partial (including the 5′ untranslated region [5′-UTR] and the first 137 bp coding sequences) transcripts of FG3G34330 . In compt test, P < 0.05)   and B. T 1 and 3  and D. STo determine whether the influence of the different lengths of RNA molecules on editing is caused by alterations of secondary structure elements surrounding the editing sites, we predicted the secondary structures for the full-length transcript , transcript without 3′-UTR (−333 to 1047 bp), and 5′ partial transcript (−330 to 137 bp) of FG3G34330. In the predicted secondary structures of the full-length transcript, editing sites 2 and 3 were situated in the hairpin loop while the editing sites 1 and 4 were in the multi-loop . In bothin situ mutant FG3G34330Δ3’UTR had no significant change compared to the wild type   . Therefo10.1128/mbio.01872-22.5FIG S5in situ 3’ UTR deletion mutant (Δ3’UTR). The gene expression level in WT was arbitrarily set to 1. Means and standard deviations of relative expression levels were calculated from 3 biological replicates. Significant differences for pairwise comparison are based on t-test . Download FIG S5, TIF file, 0.3 MB.Relative expression of FG3G34330 alleles in PH-1 (WT), transformants ectopically expressing the full-length transcript (pWT) or transcript without 3’-untranslated region (UTR) (pΔ3’UTR) or with only the 5’-UTR and the first 137 bp coding sequences (p5’part), and Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the s) of 0.75, the model only accounted for 33.8% of the variance (R2) in editing levels  . A classg levels . Therefo10.1128/mbio.01872-22.10TABLE S5Table S5, DOCX file, 0.03 MB.Summary of feature information used for Machine learning. Download Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01872-22.9TABLE S4Table S4, DOCX file, 0.02 MB.Summary of primers used. Download Copyright © 2022 Feng et al.2022Feng et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To quantify the contribution of each feature to the model prediction in the held-out test sets, we used the TreeExplainer SHAP (Shapley Additive exPlanations) algorithm  to interThe third to fourth ranking features were the hairpin loop structure (srd_str_h) and the MFE  and F. TThe combination of enriched nucleotides at downstream +1 to +4 positions (combi_down_EEEE) had a high contribution for both editing site prediction  and editing level prediction . However, a combination of depleted nucleotides at upstream −2 and −1 positions (combi_up_DD) had a high contribution for editing site prediction , but contributed less to editing level prediction (out of the top 20 features)  and F. Icis-regulatory models of RNA editing and highlights the need for comprehensive knowledge of cis-regulatory features to learn more accurate models of RNA editing.By integrating the contributions of individual features in each feature group, we found that the relative importance of different feature groups was consistent for the prediction of the editing sites and levels . The nucF. graminearum (cis-regulatory elements that affect A-to-I RNA editing in F. graminearum. We identified a variety of cis-sequence features important for the specificity and efficiency of RNA editing. Nucleotide preferences (preferred and depleted nucleotides) at −2 to +4 positions of editing sites are the most important features for editing specificity and efficiency, especially the nucleotide preferences at the −1 position. In animals, although adjacent nucleotides beyond nearest neighbors also affect editing specificity, the 5′ and 3′ nearest neighbors are most influential  has a more important role in editing specificity than in efficiency.We have previously identified 26,056 A-to-I editing sites in minearum . In thisluential . In F. gF. graminearum. Unlike in animals where A-to-I editing occurs in dsRNA (stem) structure, the A-to-I editing in F. graminearum preferentially targets the A’s located in hairpin loops. The editing levels of the sites located in hairpin loops are significantly higher than those in other types of RNA secondary structures. The more stable  the hairpin loop structures, the higher the editing levels. Despite their importance, the roles of RNA secondary structures in editing are less important compared with primary sequences. This is the opposite of what happens in animals, where the editing site structure and its stability have the largest contributions to editing efficiency , and poly (A)+ mRNA was enriched with oligo (dT) magnetic beads. Strand-specific RNA-seq libraries were prepared with the NEBNext Ultra Directional RNA Library Prep Kit following the manufacturer’s instruction and sequenced by Illumina HiSeq-2500 with a 2 × 150-bp paired-end read mode at the Novogene Bioinformatics Institute. For each library, more than 20 Mb of high-quality reads were obtained. The RNA-seq reads were submitted to the NCBI SRA database under accession numbers included in http://fgbase.wheatscab.com/). The DNA-seq and RNA-seq reads were mapped to the PH-1 genome using Bowtie 2 (MarkDuplicates from Picard package v1.99 (http://broadinstitute.github.io/picard/). The unduplicated RNA-seq BAM files of all samples were combined into one BAM file and then divided into 2 separated files containing sense-strand and antisense-strand read alignments, respectively, using BamTools v2.4.0 (http://fgbase.wheatscab.com/).Published RNA-seq and DNA-seq data of PH-1 generated in our previous study  were dowBowtie 2  and HISABowtie 2 , respects v2.4.0 . A-to-I s v2.4.0  using mas v2.4.0 . The numhttps://github.com/wangqinhu/NC.edits).The nucleotide preference surrounding the A-to-I editing sites was visualized using WebLogo 3  and Two The gene deletion construct expresses a translational fusion of the hygromycin phosphotransferase (HygR) with the thymidine kinase (TK) from herpes simplex virus 1, which confers resistance to the antibiotic hygromycin and sensitivity to nucleoside analog 5-fluoro-2’-deoxyuridine (Floxuridine) , 44. TheΔFG3G34330 deletion mutant. Transformants resistant to Geneticin were identified and confirmed by PCR assays. All the mutant strains generated and primers used in this study were listed in Allelic fragments of the FG3G34330 gene with desired nucleotide mutations or sequence deletions were generated by overlapping PCR. For allelic exchange at the native locus, the allelic fragments containing about 1-kb upstream and 1-kb downstream homologous arms were transformed into the protoplasts of the FG3G34330 deletion mutant. Floxuridine-sensitive transformants were identified and desired mutations at the native locus were verified by sequencing analysis. Quantitative PCR was used to confirm the obtained mutants without ectopic integration. For ectopic expression of the mutant alleles of FG3G34330, the allelic genes under the control of the native promoter were cloned into the pFL2 plasmid by the yeast gap repair method . The con-ΔΔCT method with the actin gene as an internal control. All the primers used were listed in Total RNA was isolated with the TRIzol reagent (Invitrogen) from 6-dpf perithecia as described. For each strain, at least 3 biological replicates were prepared. cDNA synthesis was performed with FastQuant RT Kit (TIANGEN) following the manufacturer’s instructions. RT-PCR products were gel purified and subjected to direct sequencing. Sequencing of RT-PCR products was done using an automatic DNA sequencer. Relative expression levels of FG3G34330 alleles in different strains were assayed by quantitative real-time RT-PCR using the 2https://www.snapgene.com/snapgene-viewer/). Quantitative analysis of editing level was performed using QSVanalyser (The Sanger sequencing traces were visualized using SnapGene Viewer 4.3 (analyser , which chttps://github.com/XINYUCAO111/machineleaning_of_RNA_editing.Both the classification model and the regression model were constructed by XGBoost v1.6.0 . The non


Results showed that threatening stimuli increase the choice of care, decrease RT, and increase LPP magnitude in centro-parietal areas . Food choice was lower, with increased RT. Furthermore, larger LPP magnitude in centro-parietal cluster was associated with increased RT in the choice of care. Considering the dimensions of attachment, in threatening conditions, while anxiety was not associated with RT and care/food choice, avoidance was associated with an increase in care choice and RT. In conclusion, the specific association of increased RT in care choice with high LPP magnitude centro-parietal cluster may be explained in terms of a functional interference of these areas in the choice of care, but not of food. Further, we postulate that the increased RT of avoidant individuals may reflect a more articulated choice process.In this study, we aimed to test the association of the Late Positive Potential (LPP) response and attachment dimensions in the choice of care/food pictures and its reaction time (RT) in threatening versus neutral conditions. Fifty-two participants (38 females, M More specifically, when a threat activates the attachment system, it automatically motivates the subject to seek physical or symbolic proximity to an attachment figure, which can provide a sense of safety, helping to cope successfully with threats2. According to Bowlby1, interactions with attachment figures are remembered in the form of a schematic internal working model3, and are automatically carried out when processing new information4. These representations consist of both conscious and unconscious memories of interaction with attachment figures, as well as strategies for regulating negative emotions5. In adulthood, the primary attachment strategy is not necessarily the behavior of seeking physical proximity, but it may also consist in the activation of mental representations of their symbolic presence6.Attachment theory explains the children's construction of a preferred relationship with a protective figure to increase survival when in danger and in times of need. The core of the attachment theory is the alleviation of distress and fear from a perceived environmental threat, through a process of co-regulation provided by a caregiver7. An event-related potentials (ERPs) study8 found that avoidant attached adults exhibited a bias toward increased neural activation, showing greater motivational relevance and attention to negative emotional stimuli. This result, which appears in contrast to the deactivation assumption of avoidant individuals, may be explained through a dual process in which avoidance behavior follows awareness of the nature of the stimulus9, necessary in order to activate the avoidant defense.Individual differences in the level of attachment system activation have generally been conceptualized in terms of two dimensions: anxiety and avoidance, which are associated with distinct patterns of emotional distress regulation. When confronted with salient stimuli, individuals with anxious attachment show patterns of hyperactivation, while avoidant attached individuals show patterns of deactivation12. Bradley13 argues that the LPP may represent a neural response indicating that relevance has been detected in the environment and, therefore, emotional content elicits an increase in LPP because it activates aversive or appetitive motivational systems. These findings are consistent with the cognitive models that emphasize the role of schema-driven processing in allocating attention to threatening stimuli14.ERP research has amply demonstrated that the late positive potential (LPP) response is enhanced for salient stimuli and reflects relatively deep encoding and cognitive processing16. A convergence emerges from the studies regarding the role of attentional and memory processes in terms of accessibility to mental concepts. According to priming research, primes often do not cause direct effects, but rather affect the accessibility of mental content related to the primes. The impact of affective priming should depend on the nature of the accessible content and its relation to the current task. Therefore, affective priming could modulate decisions through several pathways, including non-decision time and bias17. Mikulincer et al.18 found that threatening primes led to greater accessibility of mental representations of the attachment figure, as indicated by faster reaction times (RT). Later, Dewitte et al.19 demonstrated that priming with attachment-related threats, automatically activated an attention bias toward the attachment name figure. Conversely, studies of attention to positive stimuli indicate that the magnitude of attentional bias varies in proportion to motivational variables such as relevance and arousal21. The magnitude of attentional bias is also consistent with the incentive salience hypothesis, in which positive stimuli capture attentional resources22. In this perspective, various authors used positive stimuli with a higher level of biological relevance such as nutrition, offspring care, and reproduction23. Relevant negative and positive stimuli are then attention trigger signals and fulfill different and complementary motivational goals. Aversive stimuli generate activation and drive to action, while approach stimuli can play a regulatory function and restore homeostasis24.The processing of emotional information and its regulation has been investigated by several researchersOverall, extensive research described the effect of threatening stimuli on the physiological response as indexed by LPP amplitude as well as on the propensity to seek care. However, there is less experimental evidence on the association between attachment dimensions, LPP amplitude and emotion regulation through comfort seeking, in threatening situations.25. Parental child care pictures (a strong symbolic representation of care), in agreement with attachment theory, were used as the goal-congruent stimulus to the threatening condition. Food, on the other hand, was selected as the alternative choice because of its strong biological reward function, irrespective of the eating style26, for its enhanced attentional effect27 and for its role in the regulation of negative emotional states and restoration of homeostasis30 related to the attachment system31.Therefore, in this work we studied whether care-seeking is the primary choice after exposure to threatening stimuli, and whether the participants' different attachment orientations lead their choice toward other forms of emotion regulation, such as comfort food (henceforth: food)Based on the attachment theory and studies on attentional bias, we expect that in a threatening situation participants will generally choose a stimulus congruent with the negative emotional trigger, such as a care picture. On this line, we also hypothesize that the choice of care, but not the choice of food, will be associated with a shorter RT in comparison to the neutral condition. Further, despite food being an attentional priority and its emotion regulation function, in our experimental setting it should be a secondary choice and therefore associated with a longer RT in comparison to the neutral condition.32. However, due to their threatening information processing9, it is hypothesized that avoidant individuals will show an increase in RT, specifically when choosing care.Based on previous research we also focused on two aspects potentially relevant to the process of choice and its RT. The first concerns attachment representations in the two dimensions of anxiety and avoidance, the second concerns LPP magnitude. We expect that attachment anxious individuals, hypervigilant to emotional stimuli, would be more motivated to seek care, with a shorter RT in comparison to individuals with low anxiety. Regarding avoidant individuals, recent evidences have shown that, despite they rely on attachment deactivation as a defense when coping with threats, when they are certain of the availability of another person, they actually activate the proximity-seeking behaviorFinally, being the LPP an indicator of emotional response, we expect to record an increased magnitude in the threatening condition, and hypothesize that this phenomenon will be associated with an increase in care choice. We also hypothesize that differences in LPP magnitude between the neutral and threatening conditions will be associated with an increase in RT, because of the interference of the high emotional activation on the decision making process.Fifty-two healthy subjects  participated in the study. A written informed consent was obtained from all subjects prior to study entry; the experimental procedure was approved by the Institutional Review Board of the Department of Biomedical Sciences of the University of Sassari and conducted in accordance with the Helsinki Declaration. None of the participants had history and/or current signs/symptoms of neurological and/or psychiatric diseases and current or recent use of any drugs affecting the central nervous system. All participants had normal or corrected-to-normal vision.The study was composed of two experimental sessions: questionnaires administration and EEG recording.33 of the Experiences in Close Relationships Scale revised (ECR-R34). This questionnaire contains 36 relationship-related statements that refer to attachment anxiety and avoidance. The anxiety scale includes eighteen items that reflects an individual’s concern about rejection and abandonment. The avoidance scale includes eighteen items that assesses discomfort with closeness and dependence. Participants responded on a 7-point Likert scale ranging from 1 (strongly disagree) to 7 (strongly agree).Attachment style was measured using the Italian versionα = 0.92, as well as for the avoidance scale, α = 0.91. The mean attachment scores were 3.16 (SD = 1.17) for attachment anxiety and 2.33 (SD = 0.93) for attachment avoidance.In the current sample, Cronbach alphas were high for the anxiety scale, Participants filled in the ECR-R questionnaire and underwent EEG recording in a counterbalanced way: half started with the questionnaire and half started with the recording.EEG recordings were carried out in a quiet, dimly lighted room, while subjects were seated in a comfortable chair and were asked to stay relaxed but alert during the experiment. EEG data was collected using a Neuroscan Synamps amplifier , digitized , band-pass filtered (0.05–100 Hz), and recorded using Scan software . EEG recordings were obtained with standard 32-channels Ag/AgCl electrodes arranged according to the 10–20 international EEG system, and included Fp1, Fp2, F7, F3, Fz, F4, F8, Ft7, Fc3, Fcz, Fc4, Ft8, T3, C3, Cz, C4, T4, Tp7, Cp3, CPz, CP4, Tp8, T5, P3, Pz, P8, T6, O1, Oz, O2. Averaged mastoids (2 electrodes placed over the mastoid bones) served as the reference lead and the ground electrode was placed on FPz. For electrooculogram recordings, electrodes were placed above and below the left eye, as well as on the outer canthi of each eye. Skin impedances were kept below 5 kΩ, and during the experiment the EEG signal was continuously monitored online.35 and ERPLAB36 toolboxes running in the MATLAB environment . Raw EEG signal was epoched  and demeaned (baseline: − 300 to 0 ms). Epochs were visually inspected and excessively noisy ones excluded from analysis (less than 5% for each participant). Residual artifacts were identified using an Independent Component Analysis algorithm (INFOMAX ICA) and eliminated after visual inspection, based on time, frequency, scalp distribution, and amplitude criteria.Off-line EEG processing was performed with EEGLAB R2019b update 737. LPP peak was identified as a positive deflection with latency between 500 and 1000 ms and its magnitude measured by calculating the relative area under curve.For the purposes of the study CPz, Pz, P3 and P4 channels  were selected for analysisVisual stimuli were presented using STIM2 software , on an analog 17’ color monitor at a viewing distance of approximately 60 cm. Pictures occupied about 40° of visual angle horizontally and vertically, and participants were instructed to fixate on the center of the screen. In order to record the picture chosen as well as the RT, subjects were instructed to use a dedicated response pad  to choose between the target pictures. RT was defined as the time elapsed between the stimulus presentation and the key press.38. A total of 60 IAPS pictures were selected for the threatening  and neutral (e.g. domestic objects) conditions. The contents were associated consistently with higher or lower valence or arousal ratings. Mean ratings of valence and arousal were as per Lang et al., 2008 . The 60 pictures had a mean valence of 2.05 (SD = 0.54) and M = 5.07, (SD = 0.32); mean arousal of 6.52 (SD = 0.50) and M = 3.0 (SD = 0.64) for threatening and neutral condition respectively. Valence and arousal were significantly different between the two conditions, F = 264 and F = 438 respectively.For emotional stimulation, the International Affective Picture System (IAPS) ((IAPS): Threatening: 1120, 1304, 3010, 3015, 3030, 3061, 3063, 3064, 3071, 3100, 3102, 3110, 3120, 3130, 3140, 3225, 3230, 3500, 3530, 6313, 6315, 6350, 6370, 6510, 6550, 6560, 6570, 9253, 9414, 9635. Neutral: 7000, 7002, 7004, 7009, 7011, 7012, 7018, 7019, 7020, 7025, 7026, 7032, 7040, 7042, 7050, 7053, 7059, 7061, 7062, 7080, 7090, 7095, 7150, 7175, 7185, 7190, 7205, 7211, 7233, 7235.) was used39) ((BAPS). Care (threatening condition): 1, 5, 6, 8, 9, 12, 14, 15, 16, 17, 25, 27, 29, 30, 31, 32, 34, 36, 39, 40, 41, 43, 47, 48, 49, 52, 53, 54, 55, 61. Care : 2, 3, 4, 7, 11, 13, 19, 20, 21, 22, 23, 24, 26, 28, 35, 37, 38, 42, 44, 46, 50, 51, 56, 57, 58, 59, 60, 62, 63, 64.), were used. The 60 pictures depicted comfort-related scenarios where care was represented . Two random lists of 30 pictures were created: one for the threatening and one for the neutral condition. Mean ratings of perceived care, valence and arousal, were as per Szymanska et al.39: the two lists did not significantly differ on any dimension, all F values < 1.For one of the two choice options, 60 pictures from the The Besançon Affective Picture Set-Adult (BAPS-Adult40 ((Food-Pics). Food (threatening condition): 4, 5, 9, 14, 22, 26, 45, 56, 66, 67, 90, 94, 104, 109, 116, 133, 137, 170, 177, 186, 189, 225, 287, 539, 613, 673, 684, 713, 877, 878. Food : 15, 25, 31, 35, 38, 43, 44, 60, 65, 80, 102, 103, 113, 115, 121, 127, 130, 138, 140, 150, 172, 173, 183, 205, 286, 510, 614, 676, 682, 869.). The pictures were of typical foods  and they were randomly divided into two random lists of 30 items: one for the neutral and one for the threatening condition. Mean ratings of calories, palatability, and craving were as per Blechert et al.40: the two lists did not significantly differ on any dimension, all Fs < 1.For the other of the two choice options, 60 pictures were taken from the Food-Pics_ExtendedFor the 30 filler-trials 90 neutral pictures were selected from the IAPS and Food-Pics_Extended databases.Two hundred and seventy pictures were selected from the following databases to serve as threatening stimuli or target pictures in the experiment:41.The experiment consisted of 90 randomly intermixed trial: 30 trials in the threatening condition; 30 trials in the neutral condition; and 30 filler trials. Each trial began with a gray screen followed by a fixation dot in the center of the screen; the duration of the two pictures varied randomly up to a total duration of 5 s, followed by a prime picture  lasting for 2.5 s, another gray screen lasting for 0.8 s, after which two probe pictures were presented side-by-side Fig. . Both inPrior to the start of the task, participants were adequately informed on the experimental procedure; to ensure consistency, all information on the experimental procedure were administered as part of the visual stimulation protocol . Participants were allowed to familiarize with the experimental procedure with 10 practice trials; all pictures in the practice trials had neutral content, and were not included in the experiment.Repeated measures analyses of variance (ANOVA) with the factors of condition (2) × electrocortical position (4) was performed. Huynh–Feldt corrected degrees of freedom were applied when the assumption of sphericity was not met.To compare the care/food choice and the RT differences, separate two-way repeated-measures ANOVA were performed between the neutral and threatening condition.We also conducted four hierarchical multiple regression analyses (four-step) to assess the association between the choice of care and food and their RT as dependent variables and anxiety attachment, avoidance attachment and LPP magnitude differences between conditions as predictors. LPP magnitude in the centro-parietal cluster, care/food choice and RT scores used in the regression analyses, were determined by subtracting the averages of the neutral condition from the threatening condition.In the first regression analysis, RT differences, attachment anxiety, attachment avoidance, and LPP differences were entered as predictors, and the care choice differences as a dependent variable. In the second regression analysis the care choice differences, attachment anxiety, attachment avoidance, and LPP differences were entered as predictors; while the RT differences as a dependent variable. In the third regression analysis, RT differences, attachment anxiety, attachment avoidance, and LPP differences were entered as predictors, the food choice differences as a dependent variable. In the fourth regression analysis the food choice differences, attachment anxiety, attachment avoidance, and LPP differences were entered as predictors; while the RT differences as a dependent variable.42. The same analysis structure was also applied in the third and fourth regression, using food choice and RT as dependent variables. The hierarchy of inclusion of the remaining variables are common to all regression analysis. Attachment anxiety was entered at Step two because it is a central component of attachment theory under threatening situations. The avoidance dimension of attachment was included in Step three. In step four centro-parietal cluster were entered, along with the other predictors.The order of entry of predictors was based on evidence and theory described earlier. Since RT and care/food choice have been associated with each other, in the first regression analysis (dependent variable: care choice), RT was included first; in the second regression analysis (dependent variable: RT) care choice was included firstExperimental values across the two conditions and their differences are reported in Table F = 394.897, p < 0.001, η2p = 0.886. The main effect of electrocortical position with Huynh–Feldt correction was not significant F = 1.389, p = 0.251. The interaction effect was significant F = 2.873, p = 0.038, η2p = 0.053. (Table The two-way repeated measures ANOVA [2 (condition) × 4 ] showed a significant increase in the threatening condition compared to the neutral condition F,51 = 394F = 28.189, p < 0.001, η2p = 0.356. The interaction effect was significant F = 41.474, p < 0.001, η2p = 0.448  = 1.582, p = 0.214, ns. Whereas in the threatening condition a significant difference emerged F = 63.055, p < 0.001, η2p = 0.553, with the percentage of care choice being about three times as high as food. With regard to the differences in the choice of care between the two conditions , the analysis showed that in the neutral condition participants chose the picture of care in about half of the cases (M = 16.29), while in the threatening condition the percentage increased by over 40% (M = 22.96). Averages of choices between the two conditions express significant differences F = 41.474, p < 0.001, η2p = 0.448. The choice of food mirrored the one of care.The results of the repeated measures 2 × 2 ANOVA revealed the main effects of choice and condition  F = 6.388, p = 0.016, η2p = 0.141. The main effect of the condition was not significant F = 2.530, p = 0.120, ns. The interaction effect was significant F = 12.357, p = 0.001, η2p = 0.241  = 12.264, p < 0.001, η2p = 0.239, while in the neutral condition the differences between care and food were not significant F = 2.843, p = 0.100. It emerged that when participants chose food, the RT significantly increases in threatening with respect to neutral condition F = 11.172, p = 0.002, η2p = 0.223, while when they choose care the RT decrease in the threatening condition, but our result only reached near significance F = 3.399, p = 0.073. It is to be noted that the degrees of freedom in the analysis of RT are lower, because 12 participants never chose food in the threatening condition.The second 2 × 2 repeated analysis of variance yielded a main effect for the choice RT The results of multivariate hierarchical regression that analyzed effects on choice of care are reported in Table 2 = 0.239) which was statistically significant, F = 3.695, p = 0.011.Step one indicates that when participants increased their choice of care in the threatening condition, as compared to the neutral condition, they decreased their RT. The inclusion of the attachment anxiety dimension (step two) provided no contribution to the choice model. Conversely, attachment avoidance contributed positively to the choice of care (step three). The results showed that high avoidance scores were associated with greater increases in care choice in the threatening condition. In step four, the inclusion of the centro-parietal cluster values did not produce a significant increase in the explained variance of the model. In fact, when all four independent variables were included in the regression model, neither attachment anxiety nor centro-parietal cluster were significant predictors of care choice differences. The model including the centro-parietal cluster explained 23.9% of the variance (R2 = 0.350) in care RT, F = 6.321, p < 0.001.Results of the second multivariate hierarchical regression that analyzed the effects on care RT are reported in Table The results of multivariate hierarchical regression that analyzed the effects of food on choice were not statistically significant. None of the independent variables provided a significant contribution to food choice. In addition, the results of multivariate hierarchical regression that analyzed effects on RT choice of food were not statistically significant.The present study was designed to determine, firstly: the differences in LPP magnitude, care/food choices, and RT, between the threatening and the neutral condition; secondly, whether differences in the care choices and their RT were associated with specific attachment dimensions and differences of LPP magnitude in centro-parietal cluster.43. This result allowed testing the association between LPP magnitudes with the behavioral responses (choice and RT), which, in line with our hypothesis, confirms one of the core concepts of the attachment theory posits, i.e. that in a situation perceived as threatening, the motivational drive to seek care is activated44. While in the neutral condition care and food pictures were chosen almost equally, in the threatening condition the increased choice of care would perform an emotion regulation function. Lower RT values in the threatening condition, compared with the food choice, appear to express greater decisiveness when choosing care45.In agreement with previous studies we found that the exposure to the threatening condition produced a larger LPP magnitude in centro-parietal cluster, in comparison with the neutral condition31 required a longer processing time before taking the decision of choosing it. Relevant research has shown that in general RT increases with the cognitive load of the task46, which could be the case of our threatening condition. Aside from the increase in RT, food choice showed no associations with attachment dimensions and LPP magnitude, and appeared to play a secondary role in comparison with care choice. The threatening condition in this study can be considered a stressful condition, and while some studies have associated food intake with acute stress47, other studies have associated it with chronic stress48. It is therefore likely that food, in addition to having a secondary regulatory role, could exert a stronger function in chronic stress than in acute stress, as in our experiment.In the present study, increased RT values were found in the threatening condition, which suggests that food, despite its regulatory function50 and could be interpreted in terms of aversion derived from threatening stimuli, following the neuro-anatomical model of attachment proposed by Long et al.24. Our hypothesis of the association between increased LLP and higher choice of care and increased RT in the threatening condition was partially confirmed. Indeed, increases in LLP in the centro-parietal cluster are associated with an increased, but not significant, choice of care.We also found that in the threatening condition the increased RT in care choice was associated to high LPP magnitudes. While in general the threatening condition promotes decreased RT in choosing care, this appears to be a prerogative of participants with low LPP magnitudes in the centro-parietal cluster. Greater amplitude in the same area in the threatening than neutral condition corresponded to increased RT in the choice of care (but not of food). These findings can be explained by an interference of the ongoing emotional process51. However, previous research did not find a clear association between attachment anxiety and proximity behavior52, showing that anxiously attached individuals (who worry that others will not be available in times of need51), tend to avoid exposure to threatening information53. According to these observations, our results may suggest that anxiously attached individuals will be initially vigilant and then will try to elude the threatening stimulus, in agreement with the vigilance-avoidance theory54.While our data clearly showed that threatening stimuli increase LPP response and increment the choice of care, with respect with the neutral stimuli, we also studied the relationship between care choice, RT and attachment dimensions. The attachment theory has among its main cores the general tendency to seek care in threatening and dangerous situations. This aspect, regardless of the attachment style, is indeed clearly confirmed by our results. Unexpectedly, attachment anxiety did not show any association with increased choice of care and decreased RT in the threatening condition. In fact, anxiously attached individuals preferentially tend to heighten the effect of attachment-related stimuli due to their hypersensitivity, with anxious searching for care and support6, in particular because of the defenses built against the rejections received by their caregivers during the early stages of development55. On the other hand, previous researches have shown that when avoidant individuals are confident in the availability of their significant other, they are able to seek help and lower their defenses56. We found that high attachment avoidance is also associated with increased choice of care in the threatening condition. Indeed, the availability of care pictures may lead avoidant subjects to choose the care picture as a form of regulation, at the cost, however, of a significant increase in RT. The increase in care choice RT in avoidant subjects is in contrast to a general decrease in participants' RT in the threatening condition. This phenomenon has often been explained as greater attentional57 and cognitive engagement in the elaboration (e.g. avoidance defenses) of the choice process15, but also with an increase in distraction time due to an increased focus on the less emotional content of the threatening picture58. These avoidant defenses, , would operate as a cognitive load that increases RT. In other words, low avoidance and lower increased LPP amplitude produce a higher speed of decision.Furthermore, various studies have also shown that avoidant attachment is characterized by a peculiar difficulty in seeking care in situations of needThe first limit of the study we can identify is related to the nature and age of the population under analysis; in fact our sample was composed only of university students, although from different courses. Another limit may be related to the choice of comfort food as the only alternative to care. Although food is well recognized as a strong emotion regulator, other images of emotion regulation stimuli need to be used in future studies. Predictors have been selected based on theoretical assumptions. However other than the care RT criterion, the other criteria were found to be poorly correlated with the predictors. Still, the results in terms of more global models beyond individual correlations, are found to be significant.We did not measure EEG data during the choice process, and this could also be a relevant aspect to be investigated in future studies. Further, functional connections studies should be useful in order to disentangle the role of the different brain areas during threatening emotional stimuli evaluation and emotion regulation choice process.Previous studies have demonstrated that threatening stimuli activate the centro-parietal area of the brain in comparison to neutral stimuli. Our results show that while the choice of care in the threatening condition is generally associated with a decrease in RT, an increase in the centro-parietal amplitude is associated with an increase in care RT. The involvement of the centro-parietal area is shown to be associated with specific regulatory responses, in particular relational ones. It is therefore confirmed that the propensity is to use care (and not food) as a form of emotional regulation, despite the strong role of food in emotional regulation. When considering the dimensions of attachment, anxiety does not show associations neither with the choice of care/food or with decision-making times. On the contrary, avoidance is associated with an increase in the choice of care in the threatening condition and to an increase in RT, in contrast with the decrease in RT shown by the total sample. This data shows how in certain situations the defenses of avoidant subjects can be by-passed, and the increased RT may reflect as a more articulated elaboration of the choice process.


Rectal metastasis of prostate cancer origin is exceedingly rare. Its clinical presentation, endoscopic morphology, and histopathology are similar to primary rectal cancer. Misdiagnosis may lead to inappropriate treatment.We report a case of a gentleman in his 80's with a history of treated prostate cancer T3aN0M0 with radical prostatectomy sixteen years ago. He presented with one-year complaints of altered bowel habits and weight loss. Physical and rectal examination was unremarkable. Colonoscopy manifested some inflammatory changes in the rectum. The pelvis magnetic resonance imaging (MRI) showed an abnormal posterior rectal wall thickening 2 cm above the anal canal. Biopsy confirmed poorly differentiated adenocarcinoma of prostate origin. The staging workup was negative for other distant metastasis. After a multidisciplinary decision, the patient was started on androgen deprivation therapy and given palliative radiotherapy to the rectum. Six weeks later, the patient was stable with mild radiation proctitis.Management of rectal metastasis varies depending on the patient's choice, the extent of metastatic burden, symptoms, age, life expectancy, quality of life and comorbidities. While surgery remains the standard of care, other option including radiotherapy, hormonal therapy and chemotherapy has been documented in the literature with survival of few weeks to 2 years.Delayed rectal metastasis of prostate cancer after radical prostatectomy is a rare entity. Its clinical presentation and endoscopic and histopathological findings of rectal metastasis are similar to primary colorectal cancer, making diagnosis more demanding. •Rectum is a rare isolated site of metastasis from prostate cancer.•Symptoms and endoscopic morphology of rectal metastasis are similar to primary rectal cancer.•Prostate-specific antigen is not always raised in rectal metastasis.•Emergent lower gastrointestinal symptoms in patients with a history of prostate cancer warrants a multidisciplinary approach to reach an accurate diagnosis and prompt treatment.•Management of rectal metastasis depends on patient desire, the severity of symptoms, age, quality of life, life expectancy and comorbidities. This case has been reported in line with the SCARE 2020 criteria and PROCESS guidelines 2A gentleman in his 80's underwent radical prostatectomy and lymphadenectomy in 2005 due to prostate adenocarcinoma pT3aN0M0. In 2007, his prostate-specific antigen (PSA) relapsed and was treated with salvage prostate radiotherapy and androgen deprivation therapy (ADT). Since then, he became incontinent and underwent implantation of an artificial sphincter in 2009. Sixteen years after radical prostatectomy, in September 2021, he presented with one-year history of altered bowel habits and rectal bleeding. He had also lost a notable amount of weight, approximately two stones over a year. On physical examination, Eastern Cooperative Oncology Group (ECOG) performance status was 1–2; his abdomen was soft and non-tender with no palpable masses or visceromegaly. A digital rectal examination (DRE) revealed a smooth feeling rectal mucosa but no perceptible craggy pelvic mass.3PSA was 4.7 microgram per litre (μg/L), and carcinoembryonic antigen (CEA) was normal (2 μg/L). Patient underwent a Faecal Immunochemical Test (FIT) test which was >400 microgram of Haemoglobin per gram of faeces , and went on to have a screening colonoscopy, which showed some inflammatory changes within his rectum, which were thought to be secondary to radiation proctitis but showed no other colorectal pathology . Biopsie4After a multidisciplinary meeting, ADT was commenced and had prescribed palliative radiotherapy 20 Gy in five fractions over a week due to his age for the symptom control.5At the time of submission, patient was reviewed in the clinic six weeks after the completion of radiotherapy, complained of grade 2 radiation proctitis for which symptomatic medications were given and planned for follow-up at 3 months.6Winter CC et al. first described three cases of non-contiguous involvement of the rectosigmoid region The lower gastrointestinal tract is a rare site of metastasis. The incidence of metastasis to the lower gastrointestinal tract is 0.05 % of all metastases n = 7;33.3 %) chronic constipation , abdominal pain , rectal bleeding  and asymptomatic . As these symptoms are customarily associated with primary rectal cancer, patients may initially be mismanaged and have resultant hampers in care n = 5; 23.8 %) 3–15 cm anal verge, ulceration  as in our case and stricture/significant narrowing of rectal lumen . Histopathologically, thirteen (61.9 %) had poorly differentiated adenocarcinoma with PSA immunohistochemical positivity in 8 patients (38.1 %).The median age of prostate cancer patients with rectal metastasis was 73 years (range: 60–90). The median time between primary and rectal metastasis was 6.7 years. Serum PSA was normal in eight (38.1 %) patients at presentation. Total thirteen (61.9 %) were metachronous, and eight were synchronous (38.1 %). Six patients (28.6 %) had other metastases in addition to rectal metastasis. The presentation of rectal metastasis was variable with frequency; obstructive symptoms , anterior resection , Abdominoperineal resection , pelvic exenteration  and colostomy . One (4.8 %) patient experienced anastomotic recurrence salvaged with radiofrequency ablation Management of rectal metastasis was variable depending on the patient's choice, the extent of metastatic burden, symptoms, age, life expectancy, quality of life and comorbidities. Local treatment was mainly in the form of surgery; local excision , Mutahar A, Tunio , Almootazbellah M. Agamy , Daniel Hanratty , Namor Wyn Williams .Mutahar A. Tunio.Not applicable.None to declare.


The replication complex (RC) of SARS-CoV-2 was recently shown to be one of the fastest RNA-dependent RNA polymerases of any known coronavirus. With this rapid elongation, the RC is more prone to incorporate mismatches during elongation, resulting in a highly variable genomic sequence. Such mutations render the design of viral protein targets difficult, as drugs optimized for a given viral protein sequence can quickly become inefficient as the genomic sequence evolves. Here, we use biochemical experiments to characterize features of RNA template recognition and elongation fidelity of the SARS-CoV-2 RdRp, and the role of the exonuclease, nsp14. Our study highlights the 2′OH group of the RNA ribose as a critical component for RdRp template recognition and elongation. We show that RdRp fidelity is reduced in the presence of the 3′ deoxy-terminator nucleotide 3′dATP, which promotes the incorporation of mismatched nucleotides . We find that the nsp10–nsp14 heterodimer is unable to degrade RNA products lacking free 2′OH or 3′OH ribose groups. Our results suggest the potential use of 3′ deoxy-terminator nucleotides in RNA-derived oligonucleotide inhibitors as antivirals against SARS-CoV-2. They replicate solely in the cytoplasm, where the viral RNA is multiplicated, proteins are produced, matured, and assembled to form new viral particles3. Replication of the viral genome starts with the generation of the negative (-) RNA strand, starting from the 3′ end4. During this process, the replication complex (RC) produces full-length genomes but due to employment of the discontinuous transcription mechanism, a set of shorter sub-genomic (sg) mRNAs is also formed. In a proportion of cases, the RC stops at the transcription-regulating sequence (TRS) elements and switches the template, adding the leader fragment from the 5′ end of the genome to a truncated mRNA molecule. TRS sequences are located before each functional open reading frame. Consequently, a subset of mono-cistronic sg mRNAs is produced, and the sequence of the TRS tightly regulates the quantity of each transcript. Resulting RNAs are transcribed again, to yield the plus (+) stranded RNA that serves as a template for protein production. This discontinuous transcription process is very peculiar and not fully understood, but it also constitutes an elegant and compact system allowing for an efficient regulation of protein production.Coronaviruses are amongst the largest known RNA viruses, with a genome size reaching 30,000 nucleotides (nt)8. The structure reveals that the nsp12 polymerase forms a tight complex with nsp7 and nsp8. While the function of nsp7 is not clear, the two copies of nsp8 form extended helices that serve as RNA-binding interfaces; the extended helices may also serve as sliding poles that stabilize the RNA template/product duplex emerging from the nsp12 catalytic core7. This unique feature has been proposed to ensure and enhance processivity for replication of the large SARS-CoV-2 genome. Nps8 was also postulated to possess polymerase/primase activity based on hexadodecameric self-assembly10. This implies that nsp7–nsp8 might have two functions that are structurally completely independent.Recent structural studies have revealed important features of RNA binding by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), comprised of the nsp12 polymerase and the nsp7 and two nsp8 cofactors12. The RdRp has also been reported to exhibit low nucleotide insertion fidelity. Low replication fidelity has been proposed to allow RNA viruses to quickly adapt to different (host) environments and selection pressures. This requires a fine balance between genome diversity and fitness for virulence and evolution14. As a consequence of the poor replication fidelity, the SARS-CoV-2 RC tolerates the incorporation of RdRp inhibitor nucleoside analogs including Favipiravir and Remdesivir, which confer C-to-U and G-to-A transitions15. In response to the low replication fidelity, there exists an exonuclease, nsp14, which is conserved in coronaviruses, and functions to proofread and exclude mismatched nucleotides during the replication process16. Nsp14 is peculiar in that it combines two enzymatic properties: 3′–5′ exonuclease activity and methyltransferase activity, which is responsible for adding a 5′ cap to the nascently transcribed viral RNA to enable its translation by the host machinery. The nsp14 exonuclease activity is strongly enhanced by a protein–protein interaction with the nsp10 cofactor, and is proposed to interact with the RdRp21.The SARS-CoV-2 RC was recently shown to be the fastest RdRp of any known coronavirus, elongating RNA products at 600 nts/secHere, we investigate the molecular features required for RNA recognition and replication fidelity of the SARS-CoV-2 RdRp with naturally-occurring and ribose-modified substrates and oligo-nucleotides. Furthermore, we characterize the exonuclease activity of nsp10–nsp14 proofreading function in the presence of the polymerase. Our results show that the RdRp binds with micromolar affinity to its RNA substrates and that the 2′OH group of the ribose backbone is critical for elongation activity. Chemically stabilized RNA analogues are preferentially bound to nsp12. We find that the RdRp indiscriminately incorporates the adenosine derivative Cordycepin (3′-deoxyadenosine 5′-triphosphate), which results in the premature termination of an elongated RNA product and promotes U:U, U:G, and U:C mismatches. Interestingly, the nsp10–nsp14 exonuclease heterodimer is unable to degrade RNAs possessing terminal 3′ deoxynucleotides. This suggests that these nucleosides could serve as a SARS-CoV-2 antiviral agent through interference with RdRp machinery. The exonuclease activity depends on the RNA sequence and the recognition of free 2′OH and 3′OH ribose groups. Altogether, our results provide insight into the molecular mechanisms and chemical specificity of the SARS-CoV-2 RdRp and the nsp10–nsp14 exonuclease complexes. This opens a possibility to design specific RdRp inhibitors based on oligonucleotide analogues as novel antiviral drugs.7  and RNA substrates possessing modified riboses  , the RdRp did not incorporate nucleotides modified at the 2′ hydroxyl group into an elongated product . Mutation of critical residues for nsp14 activity 27 resulted in no significant degradation of RNA targets relative to a wildtype nsp14 control  and the role of the exonuclease nsp14 of SARS-CoV-2. The RdRp, comprised of the polymerase nsp12, nsp7 and nsp8, works concomitantly to bind RNA substrates and facilitate RNA elongation. Individually, these proteins show no detectable binding affinity or elongation of RNA, unlike other viral RNA-dependent RNA polymerases22. Thus, disruption of RdRp complex formation could serve as a therapeutic target against SARS-CoV-2. While extended duplexed RNA substrates are able to bind the RdRp, and only RNA substrates possessing a single-stranded 5′ template overhang allows the extension of RNA substrates.SARS-CoV-2 shares only 79.5% sequence identity with SARS-CoV-138. Furthermore, our work shows that Cordycepin (3′dATP) is incorporated into elongated products indiscriminately by nsp7,8,12, and as a result, promotes the inclusion of mismatch nucleotides during elongation in vitro. Importantly, we find that recognition of a free 3′OH group is necessary for exonuclease activity by nsp14. Notably, a 2′ OH is also required as 2′-OMe oligos were not degraded by nsp14. Our results offer an alternative mechanism of action for the repurposing of Cordycepin as an antiviral against SARS-CoV-2, where it has been proposed to inhibit viral replication through an interaction with the S and Mpro proteins39. We find that the RdRp directly incorporates Cordycepin into elongated RNA products. The recognition and binding of Cordycepin by the RdRp, as recently investigated through MD simulations, further supports our experimentally-supported mechanism of action40. At low dosage levels, Cordycepin has been shown to be non-toxic43, and with further cellular evaluation, it could be repurposed into an antiviral drug against SARS-CoV-2. This work lays the foundation for the development of novel inhibitors that binds and block the SARS-CoV-2 replication.We further show that the SARS-CoV-2 nsp12 polymerase is characterized by low fidelity, but that not all possible base pair mismatches are tolerated during elongation. RdRp is capable of incorporating U:C, U:G, U:U, C:U, and A:C mismatches when 3′ deoxy-terminator nucleotides are present. Our results show that mismatches involving G:C base pairs rarely occur, possibly associated with to the low GC content of SARS-CoV-2 genesAltogether, our data provide insight into the molecular mechanisms and features required by RdRp  to recognizes and elongate RNA substrates. It sheds new light to the unique roles of the nsp14 exonuclease and nsp10. Finally, our findings suggests that development of novel therapeutic approaches for treatment of SARS-CoV-2 can be based on RNA-derived oligonucleotides with 3′ deoxy-terminator nucleotides to inhibit replication, combined with modifying 2′OH and 3′ OH recognition.2, and 0.03 mg of T7 polymerase. Transcription reactions were run at 37 °C for 1 h, followed by denaturing purification on a 20% urea polyacrylamide gel, run at 10 W for 1 h. The RNA was excised from the gel and extracted using the crush and soak method44. The extracted RNA was equilibrated against 6 volumes of water and then concentrated to 1 mg/mL. All other RNAs used in this study were purchased from IDT as PAGE purified and desalted oligos.DNA template containing the T7 promoter region for the RNAs shown in Supplementary Fig. 2 and 5 mM β-mercaptoethanol), and 5 μL of nsp7,8,12 pre-formed complex . The RNA protein mix was incubated at room temperature for 5 min before the addition of 1 mM  of each rNTP. Cordycepin was purchased from Sigma Aldrich. Reactions were carried out at 37 °C for up to 30 min and quenched with the addition of 50 mM EDTA and 1,000 units of proteinase K, followed by 5 additional min of incubation at 37 °C. 6X denaturing buffer  was then added to the reactions, followed by incubation at 95 °C for 5 min and subsequent loading onto a denaturing 20% urea polyacrylamide gel. Gels were run at 10 W in 1X TBE for 1 h, followed by SYBR-Gold staining and imaging using GelDoku Biorad software. Gels were cropped to show relevant regions in the main figures; full gels are shown in Supplementary Figs. For both elongation and binding shift assays, RNAs were diluted to 20 ng/μL in buffer containing 10 mM Na-HEPES pH 7.4 and 50 mM NaCl, and then snap cooled  prior to incubation with nsp proteins. For nsp7,8,12 complex assembly, 20 μM nsp12 was incubated for 10 min with 60 μM nsp7 on ice, followed by the addition of 60 μM nsp8. The complex was incubated for 30 min on ice and further purified using size exclusion chromatography  with nsp10–nsp14 at equimolar concentrations to nsp7,8,12. For RNA only substrate degradation assays, the optimal nsp10–nsp14 concentration and degradation time for non-elongated RNA substrates was determined in control degradation assay experiments, as depicted in Supplementary Fig. 5. The gene encoding the nsp7 sequence  was ordered from Integrated DNA Technologies  optimized for E.coli expression and cloned using the Slice method45 into the petM5a vector (optimized petM1146) . The constructs of nsp10, comprising the amino acids 4254–4392 and nsp14, comprising the amino acids 5926–6452, of SARS-CoV-2 polyprotein 1ab were optimized for expression in E. coli and ordered from GeneArt; constructs were then subcloned into pETDUet-1 expression plasmid using BamHI and NotI restriction enzymes. Nsp7 plasmid was transformed into E. coli BL21 (DE3) and nsp10 and nsp14 plasmids were transformed into Rosetta (DE3) pLysS competent cells, separately for single expressions and co-transformed to generate the complex of both proteins. Transformed cells were cultured at 37 °C in TB media containing 100 mg/L kanamycin or 100 mg/mL ampicillin for nsp7 and nsp10 or nsp14, respectively. After the OD600 reached 2, the culture was cooled to 18 °C and supplemented with 0.5 mM IPTG. After overnight induction, the cells were harvested through centrifugation, and the pellets were resuspended in lysis buffer  and lysed by sonication. The insoluble material was removed through centrifugation. The fusion protein was first purified by Ni–NTA affinity chromatography. Supernatant was applied to nickel column, 5 mL, and washed extensively with lysis buffer, followed by then 20 mL of buffer supplemented with 20 mM imidazole and eluted with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl2, 10% glycerol, 350 mM imidazole. The N-terminal 6-histidine tag was removed by TEV protease and the solution was dialyzed against a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM MgCl2, 10% glycerol, 10 mM imidazole and 1 mM β-mercaptoethanol. After 16 h, the protein was purified over a nickel column, the flow though was collected (containing the cleaved nsp7 or nsp10 or nsp14) concentrated and applied to a size exclusion chromatography column (S200) pre-equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM TECEP 1 mM MgCl2 for nsp7, nsp10 and nsp14, or with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 2 mM β-mercaptoethanol, for the nsp10–nsp14 complex. To further remove possible impurities from nsp10–14 complex, the sample was diluted to 50 mM NaCl with 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 2 mM β-mercaptoethanol, concentrated and applied to an ion-exchange chromatography column  equilibrated with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2, 2 mM β-mercaptoethanol. The purified protein elutes first, where the impurities are bound to the resin and eluted by sequentially increasing the NaCl concentration. For all the proteins, its purity was assessed by SDS gel (Supplementary Fig. Nsp12 and nsp8 were expressed as previously publishedSupplementary Figures.


The first ribozymes are thought to have emerged at atime whenRNA replication proceeded via nonenzymatic template copying processes.However, functional RNAs have stable folded structures, and such structuresare much more difficult to copy than short unstructured RNAs. Howcan these conflicting requirements be reconciled? Also, how can theinhibition of ribozyme function by complementary template strandsbe avoided or minimized? Here, we show that short RNA duplexes withsingle-stranded overhangs can be converted into RNA stem loops bynonenzymatic cross-strand ligation. We then show that loop-closingligation reactions enable the assembly of full-length functional ribozymeswithout any external template. Thus, one can envisage a potentialpathway whereby structurally complex functional RNAs could have formedat an early stage of evolution when protocell genomes might have consistedonly of collections of short replicating oligonucleotides. However,the compact, folded structure required for catalysis is incompatiblewith the demand for an unstructured RNA as a template for copying.Thus, a fragmentation strategy has been widely explored to assemblefull-length RNAs nonenzymatically, based on the rationale that copyingthe unstructured, constituent fragments individually would be lessproblematic than copying the structured, full-length strands. Theemergence of functional RNAs can be divided into two stages: first,the copying and replication of the short fragments8 and then anassembly process leading to functional RNAs.10 Twostrategies for the assembly process have been explored. The template-directedligation of adjacent oligonucleotides was first reported in 196611 and has been studied in many contexts in subsequentdecades.16 The advantage of this strategy is that it allows many ways to positionligation junctions and template strands , we reasoned those nicked loops might be nonenzymaticallysealed because of the physical proximity of the termini of a nickedbut pre-organized loop. If this could be done without disrupting amultifragment ribozyme assembly  is the sum of the of first-order rates of loop-closingligation and competing hydrolysis. Based on the partition of activatedoligonucleotide between the stem-loop product (16%) and hydrolysis(84%), the actual rate of loop-closing ligation was 0.006 h–1, which is comparable with previously reported rates for nicked duplexligation using 5′-phosphorimidazolide RNAs.15To test the feasibilityof loop-closing ligation, we prepared a 5′-phosphorimidazolide-activatedRNA oligonucleotide (Im-p-AGCGA-3′) and annealed it with apartially complementary 10-mer RNA with a 3′-overhang (5′-N-methylimidazole(N-MeIm), a nucleophilic catalyst26 , to the above reaction and observed a concentration-dependent increasein the reaction rate. The reaction half-life decreased from 40 to0.6 h as the concentration of N-MeIm increased from0 to 200 mM, while ligation yields remained almost constant as expectedfrom similar catalysis of ligation and hydrolysis (Table S1). After systematically exploring other parametersincluding temperature, pH, concentration of MgCl2, andconcentration of NaCl that affect the model reaction (Tables S1–S4), a standard condition forloop-closing ligation  waschosen, in which the reactions proceed to completion in less than10 h (S2).To increase the reaction rate, we added han10 h 2B and S230 we wished to explore the scope of the loop-closing ligation. Usingthe overhang UGCCA as a reference sequence , we investigated the effect of varyingthe length and sequence of the 3′-overhang. Shortening theoverhang length to 2 or 3 nucleotides resulted in very poor ligationyields , while lengthening the overhang to 6-nucleotidesalso decreased the corrected yield . Presumably, a 2- or 3-nucleotide long overhangcannot easily adopt a folded conformation that brings the 5′and 3′ termini into proximity, while the entropic cost of correctlystructuring a longer overhang is increased.28 Changes to the sequence of 4 or 5 nucleotide overhangs significantlyaffect the yield of loop-closing ligation. For example, a UCCA overhanggave a three-fold higher yield than an ACCA overhang ; interestingly, this sameeffect was previously observed for nicked loop acyl transfer.25 In contrast, changing the G at the second positionof the original 5-nucleotide overhang to each of the other three nucleobaseshad no major effect on the ligation yield or rate . However, changing the 3′-terminalA into C or U greatly decreased the yield ,while a change from A to G was well tolerated . These results suggest that the firstand last nucleotides of the overhang are particularly important, withthe former likely being important in allowing a U-turn conformationand the latter in stacking to the last base pair of the stem.Giventhe variety of hairpin loops that are present in functional RNAs,Figure S17). Taken together, our observationsshow that the efficiency of loop-closing ligation is highly dependenton the length and sequence of the stem and the overhang. Changingthe 3′-terminal ribonucleoside into either a 2′- ora 3′-deoxyribonucleoside suppressed the loop-closing ligationseverely . However, in two other cases,we observed predominantly 3′-5′-linked products, whichsuggests that the regioselectivity of loop-closing ligation is sequence-dependent.The introduction of 2′,5′-linkages into functional RNAshas previously been demonstrated to be well tolerated31 and will be addressed further below.Regarding the 2′-5′versus 3′-5′ regioselectivityof the loop-closing ligation, we observed that the new phosphodiesterbonds formed are predominantly 2′,5′-linked in 5 outof 7 tested examples, rather than the canonical 3′,5′-linkage,as determined by comparison to synthetic standards on RNA PAGE analysis and was further verified by incubatingp-RNA-2 and Im-p-RNA-3 in the absence of FAM-labeled RNA-1.Conceptually, the tRNA minihelix can be disconnectedinto three fragments 3A. The RThese results demonstrate that loop-closing ligationcan constructfunctional RNA structures from short oligonucleotides. Three shortRNA oligonucleotides generated the desired tRNA minihelix (P3) andtwo on-pathway intermediate products (P2 and P4). The formation ofthe off-target product (P1) in the full reaction  and a 3′-half fragment (p-HH-3′-RNA)4A, B. Th24 When the loop-closing reaction mixture was diluted by 500-fold or1000-fold (HH-Full was 12 nM or 6 nM), the yields of the cleavageproduct (HH-Pdt) decreased to 79% and 34% . These resultsdemonstrate the effectiveness of loop-closing ligation in constructingfunctional, full-length ribozymes directly in a template-free manner.We also assembled an RNA ligase ribozyme36 Our results suggest that loop-closingligation could have circumvented this problem by enabling the assemblyof primordial ribozymes without external templates or splints. Thisminimizes but does not eliminate the problem of template oligonucleotidesinhibiting ribozyme function because the fragments being assembledmust still be replicated, and thus, their complementary strands mustalso reside within the same protocell. We suggest that biased strandsynthesis could lead to an excess of one strand over its complement;under conditions favorable to strand annealing, the strand in excesswould then exist in solution free of its complementary template. NonenzymaticRNA template copying is known to be biased in favor of purine richstrands.38 Furthermore, the loops and internal bulgesof aptamers and ribozymes tend to be purine-rich,40 supporting the hypothesis that asymmetry in strand synthesis coupledwith loop-closing ligation would favor the assembly of functionalRNAs in primordial protocells. Another possibility arises in our virtualcircular genome scenario,41 in which thereplicating oligonucleotides are fairly short, and under favorableconditions, they are partially base-paired with each other in a largevariety of configurations. In this case, inhibition by complementaryoligonucleotides might be minimized by their pairing with oligonucleotidesfrom the other strand.The tension between the need for the stablesecondary structurein ribozymes and the difficulty posed by the structure for the nonenzymatic copying of RNA templates has long beenrecognized.Another potential problem is that 3′-primerextension mightout-compete loop-closing ligation. However, if loop-closing involvesa 3′-overhang, the 5′-end of the partially complementaryoligonucleotide cannot be extended. Even for loop-closing via a 5′-overhang,a possible solution stems from the fact that the rate of primer extensioncan vary greatly depending on the 3′ nucleotide of the primerand the next two nucleotides of the template. Some of these very slowlyextended sequences may correspond to sequences with fast loop-closingkinetics. Additional experiments will be required to explore thesepossibilities.The assembly of complex RNA structures by loop-closingligationalso decreases the need for efficient postsynthetic folding of thefull-length, single-stranded RNA. Prior to the emergence of RNA chaperones,loop-closing ligation may have provided an internally guided pathfor the self-assembly of complex structures because the self-assemblyprocess relies upon the prior formation of the correct secondary structure.We suggest that such a self-assembly path may have played key rolesin the de novo emergence of structured RNAs at the origin of life.25 The efficiency ofthese reactions would be enhanced if the single-stranded overhangsequence was preorganized in a conformation similar to that of theclosed loop. The observation that certain overhang sequences resultin higher yields of loop-closing ligation . The observation of efficient ribozyme activity (S21) suggests that 2′,5′-linkages in loop regions do nothave a dramatic impact on these two ribozymes. The relatively lowyields of loop-closing ligation in our studies (∼10% observedyield on average) are due in part to the inefficiency of phosphorimidazolideactivation, and in part to the competing hydrolysis reaction. A compatiblein situ activation chemistry that could maintain sets of oligonucleotidesin an activated state44 might drive these ligation reactions closer to completionand thus favor ribozyme assembly by iterated loop-closing ligations.The efficiency of loop-closing ligation could also be increased byusing 3′-amino terminated oligonucleotides, as suggested byour previous work with the splinted ligation of 3′-amino or2′/3′-aminoacylated nucleotides.17The loop-closing ligation chemistry that we have described hereand our previously described (amino)acyl transfer chemistry both requirea close approach of the 5′-end of one strand with the 3′-endof the other strand.ligation 1 stronglactivity 3 and S2145 Notably, it has been demonstrated experimentally that compared tofully random pools of RNA, compact structured pools are superior sourcesof functional RNAs in in vitro selection experiments.47 These results strengthen the potential advantage of having directaccess to structured, single-stranded RNAs for the emergence of RNAfunctions. This proposal is in contrast with the conventional viewof full-length primary sequences forming first followed by folding.In a pool of short oligonucleotides,8 loosely structured assembliesof short oligonucleotides with low-level function would have beenaccessible via dynamic assembly/disassembly of short RNA oligonucleotidesat equilibrium. These loosely structured assemblies could have beenpulled out-of-equilibrium and trapped as more stable structures byloop-closing ligation, thus enabling the emergence of RNA functionsthat were more robust than the original noncovalent assembly of fragments.We suggest the straightforward and economical strategy of assemblingfunctional RNA structures by loop-closing ligation is a plausiblemechanism for the nonenzymatic emergence of ribozymes before a robustprocess for the replication of long RNAs was available.The noncovalent assembly of fragments into partly functionalensemblescan be seen as an evolutionary precursor to the assembly of full-lengthribozymes by loop-closing ligation.


C. elegans, is a molecule that mediates axonal outgrowth and stability. UNC-33/CRMP2 has been hypothesized as a potential drug target for treating Alzheimer’s and other neurodegenerative diseases, which can often be attributed in part to aging. In aging, CRMP2 becomes hyperphosphorylated, which decreases the protein’s functionality, destabilizes the cellular skeleton, and contributes to neurodegeneration. In C. elegans, aging can be slowed by entering dauer diapause; a non-aging developmental stage turned on when the DAF-7/TGFβ signaling pathway is silenced in response to environmental stressors. In our laboratory, we discovered that unc-33 mutants are unable to form dauers in response to environmental stressors, but the mechanism behind this is still unknown. Here, we present a study that investigates whether a mutation in the daf-7 gene which leads to a temperature sensitive constitutive dauer phenotype can rescue phenotypes characteristic of unc-33 mutants. To this end, we created unc-33; daf-7 double mutants and quantified proper dauer formation after exposure to unfavorable environmental conditions. In addition, we tested how the introduction of the daf-7 mutation would affect the locomotion of the double mutants on an agar plate and a liquid medium. Furthermore, we examined axonal elongation of the double mutants using a transgene, juIs76, which expresses GFP in GABAergic motor neurons. Our analysis of unc-33; daf-7 double mutants showed that introducing the daf-7 mutation into an unc-33 mutant rescued dauer formation. However, further studies revealed that the unc-33; daf-7 double mutants had defects in axonal outgrowth of their D-type motor neuron which had been previously seen in unc-33 single mutants and impaired locomotion. Based on these results, we concluded that unc-33 mutants might have a problem suppressing DAF-7 signaling under unfavorable environmental conditions, leading to the activation of reproductive programs and the development of adults instead of dauers.Collapsin response mediator protein-2 (CRMP2) in humans, UNC-33 in  This gene encodes for three different isoforms through alternative splicing, which are UNC-33S, UNC-33M, and UNC-33L  (unc-33(e204) mutants mislocalize UNC-33 proteins to the cell body of neurons instead of the ventral nerve cord and nerve ring where the proteins normally accumulate  and unc-33(mn407) mutants are short and dumpy, exhibit uncoordinated locomotion, and have defects in axonal guidance of sensory and motor neurons (The  UNC-33L . In vert UNC-33L . Hyperph UNC-33L . As for endrites . UNC-33  fertile . The hypD 389 N) . Studiescumulate . As a reof axons . Further neurons .unc-33 mutants are incapable of forming dauers . The TGFβ signaling pathway enables processes such as development, regulation of the immune system, and differentiation of adult stem cells in humans  point mutation results in a temperature sensitive phenotype producing 100% dauers at 25°C ; daf-7 (e1372) and unc-33(e204); daf-7(e1372), to examine their phenotypes. First, we assessed whether the introduction of the daf-7 mutation restores dauer formation by isolating dauers via a treatment with 1% SDS. Next, we characterized the locomotion and axonal outgrowth of these double mutants. Collectively, our results show that the introduction of the daf-7(e1372) mutation successfully restores the production of dauers in unc-33 mutants exposed to unfavorable environmental conditions. Furthermore, we found that adding daf-7(e1372) mutation to an unc-33 mutant background does not rescue the uncoordinated movements in unc-33 mutants, nor does it rescue the axonal elongation defects.To better understand the inability of unc-33(e204) IV, CB1372 daf-7(e1372) III, SP1382 unc-33(mn407) IV, CZ13799 juIs76 [unc-25p::GFP + lin-15(+)] II, and DR466 him-5 (e1490) V were provided by Caenorhabditis Genetics Center (CGC). Strains AMH113 daf-7(e1372) III; unc-33(mn407) IV, AMH115 daf-7(e1372) III; unc-33(e204) IV, AMH32 juIs76 II; unc-33(mn407) IV, AMH34 juIs76 II; unc-33(e204) IV, AMH145 juIs76 II; daf-7(e1372) III; unc-33(mn407) IV, AMH151 juIs76 II; daf-7(e1372) III, AMH155 juIs76 II; daf-7(e1372) III; unc-33(e204) IV were generated in our laboratory.Strains Wild-type N2, CB204 Escherichia coli OP50 lawn (daf-7(e1372) mutation were subject to incubation of 25°C to induce dauer formation. According to Ren and colleagues, daf-7(e1372) produce 100% dauers at 25°C and 5% at 20°C  with 100 ug/ml of proteinase K. Tubes were then placed at −80°C for 20 min, and worm lysis was finalized by incubating tubes for 90 min at 62°C and 15 min at 95°C. Worm lysates were then combined with the appropriate primers  mutation. The hermaphrodites were placed on NGM agar plates containing an OP50 lawn and incubated at 25°C for 6 hours. After 6 hours, gravid hermaphrodites were removed, and the synchronous eggs were incubated at 25°C for 4 days. After 4 days, nematodes were resuspended with 1 mL DI water and transferred into 1.5 mL tubes. Worms were centrifuged for 1 min at 9,221 x g and pellet worms were treated with 1 mL of 1% SDS for 20 min at room temperature on an orbital shaker. After the 20-min incubation, worms were centrifuged for 1 min at 9,221 x g and the supernatant was removed. Pelleted worms were washed 3 times using 1 mL of M9 buffer and centrifuged for 1 min at 9,221 x g at 20°C after each wash. After the last wash, 100 uL of pellet worms were transferred onto an NGM plate with no bacteria and the percent survival was quantified (daf-7(e1372) mutation were grown on NGM agar plates containing a spot of OP50 at 25°C until all OP50 was consumed. Nematodes were then starved for 7 days at 25°C. After starvation, nematodes were exposed to 1% SDS treatment as described, and survival was calculated.Twenty gravid hermaphrodites were used to synchronize nematodes from strains containing the antified . StrainsIn order to measure locomotion, an animal in the L3 or dauer stage was placed onto an NGM agar plate containing an OP50 lawn and was allowed to move for 10 min at room temperature . The disThe motility in liquid of L3 nematodes, dauers, as well as adults that developed after exiting the L3 or the dauer stage was quantified after following these steps. First, 1 mL of deionized water was placed in each well on a 24-well plate at room temperature. Next, individual nematodes were picked from the plate and suspended in one of the wells. After a 10–15 s acclimation period, the body movements within 30 s were recorded. One isolated body movement of head, torso, or tail was considered one thrash. A total of 60 hermaphrodites per strain was quantified.In order to assess axonal outgrowth, nematodes of the L3 stage or dauer stage were placed on an agar pad containing 5 μL of 200 mM Levamisole to immobilize them. These nematodes were then imaged using an Olympus fluoview confocal microscope at a total magnification of 600X, and z-stacks of the dorsal nerve cord were captured. Each animal’s whole dorsal nerve cord was recorded using two z-stack images, one containing the head to the midsection of the worm and the other focusing on the midsection to the tail. Once the z-stacks were captured, the images were then turned into a projection using the Olympus CellSens software, and the projections were then stitched together using a Multiple Image Alignment. Axonal outgrowth in the dorsal nerve cord was analyzed by measuring the number and length of the gaps within the dorsal nerve cord using an ROI polyline in CellSens.post hoc test after testing for homogeneity of variance using the Brown-Forsythe Test. Data that did not pass the Shapiro-Wilk test for normal distribution were analyzed using Kruskal–Wallis’s test with Dunn’s multiple comparison test. All statistical analyses and graphs were generated with Prism, Grand GraphPad.Statistical analysis of data collected that followed a normal distribution was performed using a one-way ANOVA with Tukey’s multiple comparison unc-33 mutants fail to form dauers under starvation conditions  and the temperature-sensitive mutation daf-7(e1372) were generated. Once these double mutant strains were confirmed via genotyping, strains grown in dauer-inducing conditions were exposed to 1% SDS treatment, and their survival was quantified. Strains with the daf-7 mutation were synchronized and grown at 25°C, then exposed to 1% SDS treatment. The results confirmed that unc-33 mutants die after the SDS treatment suggesting that they do not form dauers (daf-7(e1372) incubated at 25°C for 4 days formed 93% ± 8 dauers. Additionally, these results revealed that the double mutant strains unc-33(mn407); daf-7(e1372) and unc-33(e204); daf-7(e1372) form dauers as they are resistant to 1% SDS (unc-33(mn407); daf-7(e1372) and unc-33(e204); daf-7(e1372) to 1% SDS is not significantly different from wildtype nematodes . However, the percent survival of double mutants unc-33(mn407); daf-7(e1372) and unc-33(e204); daf-7(e1372) is different from daf-7(e1372) (p < 0.0001). Together, this data suggests that unc-33 mutants could have a defect that prevents them from inhibiting the synthesis or secretion of DAF-7 in response to environmental stressors, thus failing to form dauers under these conditions.Previous studies have shown that nditions ; howeverm dauers . Wildtypo 1% SDS . One-wayunc-33 mutants have severely uncoordinated movements (daf-7(e1372) mutation into unc-33 mutants rescues uncoordinated locomotion, the movement of all strains was analyzed on two media: NGM plates seeded with E. coli OP50 and wells with liquid media. Strains with the daf-7 mutation were synchronized as dauers, while the other strains without the daf-7 mutation were synchronized to be at the L3 life stage. Analysis of nematodes’ locomotion on NGM plates showed that the severely uncoordinated locomotion characteristics of unc-33 mutants was not rescued by introducing the daf-7(e1372) mutation (p > 0.9999) between the single mutants unc-33(mn407), unc-33(e204), and double mutants unc-33(mn407); daf-7(e1372), and unc-33(e204); daf-7(e1372). In contrast, the distance traveled by wildtype nematodes was significantly more than double mutants unc-33(mn407); daf-7(e1372) (p < 0.0001), and unc-33(e204); daf-7(e1372) (p < 0.0172).Besides defects in dauer formation, ovements . To studmutation . Statistunc-33 mutants was not rescued either. Double mutants unc-33(mn407); daf-7(e1372) and unc-33(e204); daf-7(e1372) dauers showed jerky locomotion and diminished frequencies of body bends which were indistinguishable from those quantified for unc-33 single mutants (daf-7(e1372) dauers, we noticed that after leaving the animals in liquid media for several minutes, they displayed a different behavior. First, dauers were lethargic in liquid media and then they became active by producing significantly more body bends per minute after the initial 30-s recording of thrashes. This phenomenon of two different behaviors in liquid was not observed in wildtype, unc-33 mutants, or any of the unc-33; daf-7 double mutants.In addition, locomotion in liquid was assessed in dauers or the L3 life stage of nematodes. Strains were given a 10-s or 15-s acclimation period followed by a 30-s recording of thrashes. The thrashing rate was then multiplied by two to provide a total recorded time of 1 min. In liquid medium, the uncoordinated locomotion of  mutants . In the unc-33 mutants have defects in axonal outgrowth which result in the uncoordinated movements in unc-33 mutants   . Multipl mutants . The lac mutants .unc-33; daf-7 double mutants dauers still have defects in locomotion, another study was conducted to determine if adult double mutants recovering from the dauer stage would have the same abnormalities. We evaluated the locomotion of adults that developed after exiting the dauer stage for plate locomotion and locomotion in liquid. Strains with the daf-7 mutation were selected by synchronizing them at 25°C to promote dauer formation and then shifted to 20°C to induce them to leave dauer diapause to develop into adults. Strains without the daf-7 mutation were chosen as they entered adulthood. The strains were then placed either on an NGM agar plate or in a liquid medium.After analyses revealed that unc-33; daf-7 double mutants traveled the same distance as their unc-33 single mutant counterparts  allele and p < 0.0036 for unc-33(e204), unc-33; daf-7 double mutants showed a significantly lower thrashing rate in comparison to wildtype animals . Previous research has found that unc-33 mutants are incapable of making dauers (unc-33 mutants can be rescued by the introduction of the daf-7(e1372) mutation indicating that the inability of unc-33 mutants to enter dauer diapause could be related to a defect within the DAF-7 pathway in unc-33 mutants. Studies have shown that UNC-33 is essential for neuronal polarity, microtubule assembly, and formation of axons  mutants prematurely terminate the axonal process of ASI neurons which would normally extend to the nerve ring (unc-33(mn407) mutants found a mislocalization of the sensory chemoreceptor protein ODR-10 from the cilium to axons . The neuro-endocrine signal DAF-7 is predominantly expressed in ASI neurons, chemosensory neurons that detect environmental cues and regulate the progression through the life cycle of the nematode  mutation into an unc-33 mutant background restores dauer formation by conditionally inhibiting DAF-7 and impeding the activation of its downstream pathway  mutation into unc-33 mutants does not restore the uncoordinated locomotion in dauer and adult animals. Further analysis of the dorsal nerve cord of unc-33; daf-7 double mutants demonstrated that axonal outgrowth defects persisted. These findings suggest that knocking out the DAF-7 pathway affects dauer formation but does not impact the restoration of axonal outgrowth or other neuron-specific developmental features impacted by the mutation in the unc-33 locus. These results also suggest that DAF-7 does not play a significant role in the regulation of axonal outgrowth in C. elegans motor neurons. In contrast, expression and axonal localization of UNC-33 was sufficient for restoring coordinated movements and rescuing axonal outgrowth defects in an unc-33(mn407) mutant  mutation to an unc-33 animal is sufficient to conditionally restore dauer formation.Besides its inability to regulate dauer diapause, ovements . In this) mutant . Therefo