Molnupiravir maintains antiviral activity against SARS-CoV-2 variants and exhibits a high barrier to the development of resistance.

Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.

The Long and Winding Road: 25 Years of the National Advanced Driving Simulator.

The National Advanced Driving Simulator is a high-fidelity motion-base simulator owned by the National Highway Transportation Safety Administration and managed and operated by the University of Iowa. Its 25-year history has intersected with some of the most significant developments in automotive history, such as advanced driver assistance systems like stability control and collision warning systems, and highly automated vehicles. The simulator is an application of immersive virtual reality that uses multiprojection instead of head-mounted displays. A large-excursion motion system provides realistic acceleration and rotation cues to the driver. Due to its level of immersion and realism, drivers respond to events in the simulator the same way they would in their own vehicle. We document the history and technology behind this national facility.

Silent failure detection in partial automation as a function of visual attentiveness.

OBJECTIVE: Drivers using level 2 automation are able to disengage with the dynamic driving task, but must still monitor the roadway and environment and be ready to takeover on short notice. However, people are still willing to engage with non-driving related tasks, and the ways in which people manage this tradeoff are expected to vary depending on the operational design domain of the system and the nature of the task. Our aim is to model driver gaze behavior in level 2 partial driving automation when the driver is engaged in an email task on a cell phone. Both congested highway driving, traffic jams, and a hazard with a silent automation failure are considered in a driving simulator study conducted in the NADS-1 high-fidelity motion-based driving simulator. METHODS: Sequence analysis is a methodology that has grown up around social science research questions. It has developed into a powerful tool that supports intuitive visualizations, clustering analysis, covariate analyses, and Hidden Markov Models. These methods were used to create models for four different gaze behaviors and use the models to predict attention during the silent failure event. RESULTS: Predictive simulations were run with initial conditions that matched driver state just prior to the silent failure event. Actual gaze response times were observed to fall within distributions of predicted glances to the front. The three drivers with the largest glance response times were not able to take back manual control before colliding with the hazard. CONCLUSIONS: The simulated glance response time distributions can be used in more sophisticated ways when combined with other data. The glance response time probability may be conditioned on other variables like time on task, time of day, prevalence of the current behavior for this driver, or other variables. Given the flexibility of sequence analysis and the methods it supports (clustering, HMMs), future studies may benefit from its application to gaze behavior and driving performance data.

Multi-sensor driver monitoring for drowsiness prediction.

OBJECTIVE: Driver monitoring systems are growing in importance as well as capability. This paper reports drowsy driving detection models that use vehicular, behavioral, and physiological data. The objectives were to augment camera-based system with vehicle-based and heart rate variability measures from a wearable device and compare the performance of drowsiness detection models that use these data sources. Timeliness of the models in predicting drowsiness is analyzed. Timeliness refers to how quickly a model can identify drowsiness and, by extension, how far in advance of an adverse event a classification can be given. METHODS: Behavioral data were provided by a production-type Driver Monitoring System manufactured by Aisin Technical Center of America. Vehicular data were recorded from the National Advanced Driving Simulator's large-excursion motion-base driving simulator. Physiological data were collected from an Empatica E4 wristband. Forty participants drove the simulator for up to three hours after being awake for at least 16 hours. Periodic measurements of drowsiness were recorded every ten minutes using both observational rating of drowsiness by an external rater and the self-reported Karolinska Sleepiness Scale. Nine binary random forest models were created, using different combinations of data sources and ground truths. RESULTS: The classification accuracy of the nine models ranged from 0.77 to 0.92 on a scale from 0 to 1, with 1 indicating a perfect model. The best-performing model included physiological data and used a reduced dataset that eliminated missing data segments after heartrate variability measures were computed. The most timely model was able to detect the presence of drowsiness 6.7 minutes before a drowsy lane departure. CONCLUSIONS: The addition of physiological measures added a small amount of accuracy to the model performance. Models trained on observational ratings of drowsiness detected drowsiness earlier than those based only on Karolinska Sleepiness Scale, making them more timely in detecting the onset of drowsiness.

Difficulty suppressing visual distraction while dual tasking.

Human beings must often perform multiple tasks concurrently or in rapid succession. Laboratory research has revealed striking limitations in the ability to dual task by asking participants to identify two target objects that are inserted into a rapid stream of irrelevant items. Under a variety of conditions, identification of the second target (T2) is impaired for a short period of time following presentation of the first target (T1). Several theories have been developed to account for this "attentional blink" (AB), but none makes a specific prediction about how processing of T1 might impact an observer's ability to ignore a salient distractor that accompanies T2. Using event-related potentials (ERPs) to track target and distractor processing, we show that healthy young adults are capable of suppressing a salient visual-search distractor (D2) while dual tasking (as measured by the PD component, which has been associated with suppression) but struggle to do so shortly after the appearance of T1. In fact, the impairment was more severe for distractor processing than it was for target processing (as measured by the N2pc component). Whereas, the T2-elicited N2pc was merely delayed during the AB, the distractor PD was reduced in magnitude and was found to be statistically absent. We conclude that the inhibitory control processes that are typically engaged to prevent distraction are unavailable while an observer is busy processing a target that appeared earlier.

Autonomous Shuttle Operating on Highways and Gravel Roads in Rural America: A Demonstration Study.

The safe integration of Automated Driving Systems (ADS) into the nation's on-road transportation system, particularly in rural areas, could vastly improve overall quality of life for a rapidly growing segment of the US population. This paper describes findings from the first half (i.e., three of six phases) of a demonstration project called "ADS for Rural America". The goal of this project is to conduct a series of demonstrations that utilizes an autonomous shuttle to show how older adults (≥65 years old) could be transported from their rural homes to other locations in rural areas, as well as an urban center. This paper examines older adults' perceptions of automation before and after riding in an autonomous shuttle and their ratings of anxiety throughout the ride as they experience particular road types and maneuvers. After riding in the shuttle, older adults expressed decreased suspicion, increased trust, and increased reliability of ADS compared to baseline. Older adults reported low levels of anxiety during the 90 min ride in the shuttle. To promote the adoption and acceptance of ADS, older adults should be exposed to this technology.

High-efficient serum-free differentiation of endothelial cells from human iPS cells.

INTRODUCTION: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. METHOD: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. RESULT: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). CONCLUSION: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

Age-related effects on a novel dual-task Stroop paradigm.

The Stroop task is a traditional measure of cognitive control processes, yet results remain mixed when it comes to assessing age-related differences perhaps in part due to strategies participants use to reduce inhibitory control demands required for success on the task. Thirty-three older adults and 34 younger adults completed a Baseline (traditional, single-task) version of Stroop, followed by two, novel dual-task Stroop variants: Color-Dual (maintain secondary count of prespecified font color regardless the lexical content) and Lexical-Dual (maintain secondary count of prespecified word regardless the font color). With regard to Baseline performance, we predicted an Age x Trial Type interaction in which older adults would be selectively impaired on Incongruent trials compared to younger adults, and this prediction was supported. When we added secondary task demands, we predicted a Trial Type x Dual-Task Type interaction in which performance in the Lexical-Dual condition would be worse than performance in the Color-Dual condition. This prediction was also supported, suggesting that having a secondary task that activated the irrelevant stream of information required more inhibitory control. Finally, we also predicted that Age would interact with Trial Type and Dual-Task Type, which was partially supported in response latencies and more definitively supported in error rates. Overall, our results indicate that Stroop performance is differentially influenced by additional dual-task demands that potentially minimize strategy usage, which has implications for both young and older adult Stroop performance.

Antisaccade Deficits in Schizophrenia Can Be Driven by Attentional Relevance of the Stimuli.

The antisaccade task is considered a test of cognitive control because it creates a conflict between the strong bottom-up signal produced by the cue and the top-down goal of shifting gaze to the opposite side of the display. Antisaccade deficits in schizophrenia are thought to reflect impaired top-down inhibition of the prepotent bottom-up response to the cue. However, the cue is also a highly task-relevant stimulus that must be covertly attended to determine where to shift gaze. We tested the hypothesis that difficulty in overcoming the attentional relevance of the cue, rather than its bottom-up salience, is key in producing impaired performance in people with schizophrenia (PSZ). We implemented 3 versions of the antisaccade task in which we varied the bottom-up salience of the cue while holding its attentional relevance constant. We found that difficulty in performing a given antisaccade task-relative to a prosaccade version using the same stimuli-was largely independent of the cue's bottom-up salience. The magnitude of impairment in PSZ relative to control subjects was also independent of bottom-up salience. The greatest impairment was observed in a version where the cue lacked bottom-up salience advantage over other locations. These results indicate that the antisaccade deficit in PSZ does not reflect an impairment in overcoming bottom-up salience of the cue, but PSZ are instead impaired at overcoming its attentional relevance. This deficit may still indicate an underlying inhibitory control impairment but could also reflect a hyperfocusing of attentional resources on the cue.

Short paired-end reads trump long single-end reads for expression analysis.

BACKGROUND: Typical experimental design advice for expression analyses using RNA-seq generally assumes that single-end reads provide robust gene-level expression estimates in a cost-effective manner, and that the additional benefits obtained from paired-end sequencing are not worth the additional cost. However, in many cases (e.g., with Illumina NextSeq and NovaSeq instruments), shorter paired-end reads and longer single-end reads can be generated for the same cost, and it is not obvious which strategy should be preferred. Using publicly available data, we test whether short-paired end reads can achieve more robust expression estimates and differential expression results than single-end reads of approximately the same total number of sequenced bases. RESULTS: At both the transcript and gene levels, 2 × 40 paired-end reads unequivocally provide expression estimates that are more highly correlated with 2 × 125 than 1 × 75 reads; in nearly all cases, those correlations are also greater than for 1 × 125, despite the greater total number of sequenced bases for the latter. Across an array of metrics, differential expression tests based upon 2 × 40 consistently outperform those using 1 × 75. CONCLUSION: Researchers seeking a cost-effective approach for gene-level expression analysis should prefer short paired-end reads over a longer single-end strategy. Short paired-end reads will also give reasonably robust expression estimates and differential expression results at the isoform level.

Electrophysiological evidence that psychopathic personality traits are associated with atypical response to salient distractors.

Event-related potentials (ERPs) were used to assess the neural mechanisms underlying visual-spatial attention abnormalities associated with psychopathic personality traits. Sixty-nine undergraduates (56 women, 13 men) completed the Psychopathic Personality Inventory-Revised (PPI-R; Lilienfeld & Widows, 2005) and performed two cognitive tasks in which search displays containing a lateralized singleton encircled a fixation point that changed luminance from trial-to-trial. When searching for the singleton as a target, PPI-R scores were uncorrelated with ERP measures of its salience (Ppc), goal-directed selection (N2pc), and working memory evaluation (negative amplitude CDA). In contrast, when responding to the changes in luminance at fixation and ignoring the lateral singleton as a salient distractor, PPI-R Self-Centered Impulsivity factor scores were positively correlated with a potential indicator of distractor suppression (a sustained positive amplitude CDA). These findings provide support for a neurophysiological interpretation of the changes in visual-spatial attention associated with psychopathic personality traits: normal selection of target information accompanied by greater elimination of distractor information at a later visual working memory stage.

People with schizophrenia show enhanced cognitive costs of maintaining a single item in working memory.

BACKGROUND: Working memory (WM) deficits are seen as a core deficit in schizophrenia, implicated in the broad cognitive impairment seen in the illness. Here we examine the impact of WM storage of a single item on the operation of other cognitive systems. METHODS: We studied 37 healthy controls (HCS) and 43 people with schizophrenia (PSZ). Each trial consisted of a sequence of two potential target stimuli, T1 and T2. T1 was a letter presented for 100 ms. After delays of 100-800 ms, T2 was presented. T2 was a 1 or a 2 and required a speeded response. In one condition, subjects were instructed to ignore T1 but respond to T2. In another condition, they were required to report T1 after making their speeded response to T2 (i.e. to make a speeded T2 response while holding T1 in WM). RESULTS: PSZ were dramatically slowed at responding to T2 when T1 was held in WM. A repeated measures ANOVA yielded main effects of group, delay, and condition with a group by condition interaction (p's < 0.001). Across delays, the slowing of the T2 response when required to hold T1 in memory, relative to ignoring T1, was nearly 3 times higher in PSZ than HCS (633 v. 219 ms). CONCLUSIONS: Whereas previous studies have focused on reduced storage capacity, the present study found that PSZ are impaired at performing tasks while they are successfully maintaining a single item in WM. This may play a role in the broad cognitive impairment seen in PSZ.

Oculomotor Inhibition of Salient Distractors: Voluntary Inhibition Cannot Override Selection History.

Several studies have demonstrated that salient distractors can be proactively inhibited to prevent attentional capture. Traditional theories frame attentional guidance effects such as this in terms of explicit goals. However, several researchers have recently argued that that unconscious factors-such as the features of attended and ignored items on previous trials (called selection history)-play a stronger role in guiding attention and can overpower explicit goals. The current study assessed whether voluntary inhibition can overpower selection history. We directly compared both forms of top-down control by measuring the control of eye movements, which offer an unambiguous measure of which location has won the competition for attention. We repeatedly found that selection history overpowered any effects of voluntary goals, such that observers were unable to avoid fixating a salient distractor of a known color if the target had been presented in that color on the previous trial. Moreover, a salient distractor of a particular color captured gaze even when the observer had voluntarily chosen this color to be the distractor color just moments before. Taken together, these experiments suggest that the ability to inhibit a salient color singleton is primarily a result of recent experience and not a result of explicit goals.

The detection of drowsiness using a driver monitoring system.

Objective: Drowsiness is a major cause of driver impairment leading to crashes and fatalities. Research has established the ability to detect drowsiness with various kinds of sensors. We studied drowsy driving in a high-fidelity driving simulator and evaluated the ability of an automotive production-ready driver monitoring system (DMS) to detect drowsy driving. Additionally, this feature was compared to and combined with signals from vehicle-based sensors. Methods: The National Advanced Driving Simulator was used to expose drivers to long, monotonous drives. Twenty participants drove for about 4 h in the simulator between 10 p.m. and 2 a.m. They were allowed to use cruise control and traffic was sparse and semirandom, with both slower- and faster-moving vehicles. Observational ratings of drowsiness (ORDs) were used as the ground truth for drowsiness, and several dependent measures were calculated from vehicle and DMS signals. Drowsiness classification models were created that used only vehicle signals, only driver monitoring signals, and a combination of the 2 sources. Results: The model that used DMS signals performed better than the one that used only vehicle signals; however, the combination of the two performed the best. The models were effective at discriminating low levels of drowsiness from moderate to severe drowsiness; however, they were not effective at telling the difference between moderate and severe levels. A binary model that lumped drowsiness into 2 classes had an area under the receiver operating characteristic (ROC) curve of 0.897. Conclusions: Blinks and saccades have been shown to be predictive of microsleeps; however, it may be that detection of microsleeps and lane departures occurs too late. Therefore, it is encouraging that the model was able to distinguish mild from moderate drowsy driving. The use of automation may make vehicle-based signals useless for characterizing driver states, providing further motivation for a DMS. Future improvements in impairment detection systems may be expected through a combination of improved hardware, physiological measures from unobtrusive sensors and wearables, and the intelligent integration of environmental variables like time of day and time on task.

The Effect of Partial Automation on Driver Attention: A Naturalistic Driving Study.

OBJECTIVE: This naturalistic driving study investigated how drivers deploy visual attention in a partially automated vehicle. BACKGROUND: Vehicle automation is rapidly increasing across vehicle fleets. This increase in automation will likely have both positive and negative consequences as drivers learn to use the new technology. Research is needed to understand how drivers interact with partially automated vehicle systems and what impact new technology has on driver attention. METHOD: Ten participants drove a Tesla Model S for 1 week during their daily commute on a stretch of busy interstate. Drivers were instructed to use Autopilot, a system that provides both lateral and longitudinal control, as much as they felt comfortable while driving on the interstate. Driver-facing video data were recorded and manually reduced to examine glance behavior. RESULTS: Drivers primarily allocated their visual attention between the forward roadway (74% of glance time) and the instrument panel (13%). With partial automation engaged, drivers made longer single glances and had longer maximum total-eyes-off-road time (TEORT) associated with a glance cluster. CONCLUSION: These results provide a window into the nature of visual attention while driving with partial vehicle automation. The results suggest that drivers may be more willing to execute long, "outlier" glances and clusters of glances to off-road locations with partial automation. The findings highlight several important human factors considerations for partially automated vehicles.

Gaze position modulates the effectiveness of forward collision warnings for drowsy drivers.

Advanced driver assistance systems (ADAS) have the potential to prevent crashes and reduce their severity. Forward collision warnings (FCW) are quickly becoming standard across vehicle lineups and may prevent frontal crashes by alerting drivers. Previous research has demonstrated the effectiveness of FCW for distracted drivers, but their effectiveness for other types of impairment remains unknown. Like distraction, drowsiness can impair driver response time and lead to crashes. The goal of the present study was to evaluate the effectiveness of FCW for moderately and severely drowsy drivers using a high-fidelity driving simulator. Drowsy drivers were divided into three warning conditions during a revealed stop vehicle forward collision event: An auditory alert, a haptic seat vibration, and a no warning baseline. Results indicate that FCW were effective at speeding drowsy driver response, but only when the drowsy drivers were looking away from the forward roadway at the onset of the event. These results have important implications for ADAS technology and driver state monitoring systems.

NGmerge: merging paired-end reads via novel empirically-derived models of sequencing errors.

BACKGROUND: Advances in Illumina DNA sequencing technology have produced longer paired-end reads that increasingly have sequence overlaps. These reads can be merged into a single read that spans the full length of the original DNA fragment, allowing for error correction and accurate determination of read coverage. Extant merging programs utilize simplistic or unverified models for the selection of bases and quality scores for the overlapping region of merged reads. RESULTS: We first examined the baseline quality score - error rate relationship using sequence reads derived from PhiX. In contrast to numerous published reports, we found that the quality scores produced by Illumina were not substantially inflated above the theoretical values, once the reference genome was corrected for unreported sequence variants. The PhiX reads were then used to create empirical models of sequencing errors in overlapping regions of paired-end reads, and these models were incorporated into a novel merging program, NGmerge. We demonstrate that NGmerge corrects errors and ambiguous bases better than other merging programs, and that it assigns quality scores for merged bases that accurately reflect the error rates. Our results also show that, contrary to published analyses, the sequencing errors of paired-end reads are not independent. CONCLUSIONS: We provide a free and open-source program, NGmerge, that performs better than existing read merging programs. NGmerge is available on GitHub ( https://github.com/harvardinformatics/NGmerge ) under the MIT License; it is written in C and supported on Linux.

Suppression of angiotensin II-induced pathological changes in heart and kidney by the caveolin-1 scaffolding domain peptide.

Dysregulation of the renin-angiotensin system leads to systemic hypertension and maladaptive fibrosis in various organs. We showed recently that myocardial fibrosis and the loss of cardiac function in mice with transverse aortic constriction (TAC) could be averted by treatment with the caveolin-1 scaffolding domain (CSD) peptide. Here, we used angiotensin II (AngII) infusion (2.1 mg/kg/day for 2 wk) in mice as a second model to confirm and extend our observations on the beneficial effects of CSD on heart and kidney disease. AngII caused cardiac hypertrophy (increased heart weight to body weight ratio (HW/BW) and cardiomyocyte cross-sectional area); fibrosis in heart and kidney (increased levels of collagen I and heat shock protein-47 (HSP47)); and vascular leakage (increased levels of IgG in heart and kidney). Echocardiograms of AngII-infused mice showed increased left ventricular posterior wall thickness (pWTh) and isovolumic relaxation time (IVRT), and decreased ejection fraction (EF), stroke volume (SV), and cardiac output (CO). CSD treatment (i.p. injections, 50 µg/mouse/day) of AngII-infused mice significantly suppressed all of these pathological changes in fibrosis, hypertrophy, vascular leakage, and ventricular function. AngII infusion increased ß1 and ß3 integrin levels and activated Pyk2 in both heart and kidney. These changes were also suppressed by CSD. Finally, bone marrow cell (BMC) isolated from AngII-infused mice showed hyper-migration toward SDF1. When AngII-infused mice were treated with CSD, BMC migration was reduced to the basal level observed in cells from control mice. Importantly, CSD did not affect the AngII-induced increase in blood pressure (BP), indicating that the beneficial effects of CSD were not mediated via normalization of BP. These results strongly indicate that CSD suppresses AngII-induced pathological changes in mice, suggesting that CSD can be developed as a treatment for patients with hypertension and pressure overload-induced heart failure.

High Level of Trait Anxiety Leads to Salience-Driven Distraction and Compensation.

Individuals with high levels of anxiety are hypothesized to have impaired executive control functions that would otherwise enable efficient filtering of irrelevant information. Pinpointing specific deficits is difficult, however, because anxious individuals may compensate for deficient control functions by allocating greater effort. Here, we used event-related-potential indices of attentional selection (the N2pc) and suppression (the PD) to determine whether high trait anxiety is associated with a deficit in preventing the misallocation of attention to salient, but irrelevant, visual search distractors. Like their low-anxiety counterparts ( n = 19), highly anxious individuals ( n = 19) were able to suppress the distractor, as evidenced by the presence of a PD. Critically, however, the distractor was found to trigger an earlier N2pc in the high-anxiety group but not in the low-anxiety group. These findings indicate that, whereas individuals with low anxiety can prevent distraction in a proactive fashion, anxious individuals deal with distractors only after they have diverted attention.

Regulated nuclear accumulation of a histone methyltransferase times the onset of heterochromatin formation in C. elegans embryos.

Heterochromatin formation during early embryogenesis is timed precisely, but how this process is regulated remains elusive. We report the discovery of a histone methyltransferase complex whose nuclear accumulation and activation establish the onset of heterochromatin formation in Caenorhabditis elegans embryos. We find that the inception of heterochromatin generation coincides with the accumulation of the histone H3 lysine 9 (H3K9) methyltransferase MET-2 (SETDB) into nuclear hubs. The absence of MET-2 results in delayed and disturbed heterochromatin formation, whereas accelerated nuclear localization of the methyltransferase leads to precocious H3K9 methylation. We identify two factors that bind to and function with MET-2: LIN-65, which resembles activating transcription factor 7-interacting protein (ATF7IP) and localizes MET-2 into nuclear hubs, and ARLE-14, which is orthologous to adenosine 5'-diphosphate-ribosylation factor-like 14 effector protein (ARL14EP) and promotes stable association of MET-2 with chromatin. These data reveal that nuclear accumulation of MET-2 in conjunction with LIN-65 and ARLE-14 regulates timing of heterochromatin domains during embryogenesis.