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SEquence Evaluation through k -mer Representation (SEEKR) is a method of sequence comparison that uses sequence substrings called k -mers to quantify the nonlinear similarity between nucleic acid species. We describe the development of new functions within SEEKR that enable end-users to estimate P- values that ascribe statistical significance to SEEKR-derived similarities, as well as visualize different aspects of k -mer similarity. We apply the new functions to identify chromatin-enriched lncRNAs that contain XIST -like sequence features, and we demonstrate the utility of applying SEEKR on lncRNA fragments to identify potential RNA-protein interaction domains. We also highlight ways in which SEEKR can be applied to augment studies of lncRNA conservation, and we outline the best practice of visualizing RNA-seq read density to evaluate support for lncRNA annotations before their in-depth study in cell types of interest., (© 2024 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Comparing COVID-19 response strategies across nations is a key step in preparing for future pandemics. Conventional comparisons, which rank individual non-pharmaceutical intervention (NPI) effects, are limited by: (i) a focus on epidemiological outcomes; (ii) NPIs typically being applied as packages of interventions; and (iii) different political, economic and social conditions among nations. Here, we develop a coupled epidemiological-behavioural-macroeconomic model that can transfer NPI effects from a reference nation to a focal nation. This approach quantifies epidemiological, behavioural and economic outcomes while accounting for both packaged NPIs and differing conditions among nations. As a first proof of concept, we take Germany as our focal nation during Spring 2020, and New Zealand and Switzerland as reference nations with contrasting NPI strategies. Our results suggest that, while New Zealand's more aggressive strategy would have yielded modest epidemiological gains in Germany, it would have resulted in substantially higher economic costs while dramatically reducing social contacts. In contrast, Switzerland's more lenient strategy would have prolonged the first wave in Germany, but would also have increased relative costs. More generally, these findings indicate that our approach can provide novel, multifaceted insights on the efficacy of pandemic response strategies, and therefore merits further exploration and development.

Direct encounters, in which two or more individuals are physically close to one another, are a topic of increasing interest as more and better movement data become available. Recent progress, including the development of statistical tools for estimating robust measures of changes in animals' space use over time, facilitates opportunities to link direct encounters between individuals with the long-term consequences of those encounters. Working with movement data for coyotes (Canis latrans) and grizzly bears (Ursus arctos horribilis), we investigate whether close intraspecific encounters were associated with spatial shifts in the animals' range distributions, as might be expected if one or both of the individuals involved in an encounter were seeking to reduce or avoid conflict over space. We analyze the movement data of a pair of coyotes in detail, identifying how a change in home range overlap resulting from altered movement behavior was apparently a consequence of a close intraspecific encounter. With grizzly bear movement data, we approach the problem as population-level hypothesis tests of the spatial consequences of encounters. We find support for the hypotheses that (1) close intraspecific encounters between bears are, under certain circumstances, associated with subsequent changes in overlap between range distributions and (2) encounters defined at finer spatial scales are followed by greater changes in space use. Our results suggest that animals can undertake long-term, large-scale spatial changes in response to close intraspecific encounters that have the potential for conflict. Overall, we find that analyses of movement data in a pairwise context can (1) identify distances at which individuals' proximity to one another may alter behavior and (2) facilitate testing of population-level hypotheses concerning the potential for direct encounters to alter individuals' space use., (© 2024. The Author(s).)

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1 , which also contains a purine-rich region in its 5' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA., (© 2023 Cherney et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Effective personnel scheduling is crucial for organizations to match workload demands. However, staff scheduling is sometimes affected by unexpected events, such as the COVID-19 pandemic, that disrupt regular operations. Limiting the number of on-site staff in the workplace together with regular testing is an effective strategy to minimize the spread of infectious diseases like COVID-19 because they spread mostly through close contact with people. Therefore, choosing the best scheduling and testing plan that satisfies the goals of the organization and prevents the virus's spread is essential during disease outbreaks. In this paper, we formulate these challenges in the framework of two Mixed Integer Non-linear Programming (MINLP) models. The first model aims to derive optimal staff occupancy and testing strategies to minimize the risk of infection among employees, while the second is aimed only at optimal staff occupancy under a random testing strategy. To solve the problems expressed in the models, we propose a canonical genetic algorithm as well as two commercial solvers. Using both real and synthetic contact networks of employees, our results show that following the recommended occupancy and testing strategy reduces the risk of infection 25-60% under different scenarios. The minimum risk of infection can be achieved when the employees follow a planned testing strategy. Further, vaccination status and interaction rate of employees are important factors in developing scheduling strategies that minimize the risk of infection.

The complex network framework has been successfully used to model interactions between entities in Complex Systems in the Biological Sciences such as Proteomics, Genomics, Neuroscience, and Ecology. Networks of organisms at different spatial scales and in different ecosystems have provided insights into community assembly patterns and emergent properties of ecological systems. In the present work, we investigate two questions pertaining to fish species assembly rules in US river basins, a) if morphologically similar fish species also tend to be phylogenetically closer, and b) to what extent are co-occurring species that are phylogenetically close also morphologically similar? For the first question, we construct a network of Hydrologic Unit Code 8 (HUC8) regions as nodes with interaction strengths (edges) governed by the number of common species. For each of the modules of this network, which are found to be geographically separated, there is differential yet significant evidence that phylogenetic distance predicts morphological distance. For the second question, we construct and analyze nearest neighbor directed networks of species based on their morphological distances and phylogenetic distances. Through module detection on these networks and comparing the module-level mean phylogenetic distance and mean morphological distance with the number of basins of common occurrence of species in modules, we find that both phylogeny and morphology of species have significant roles in governing species co-occurrence, i.e. phylogenetically and morphologically distant species tend to co-exist more. In addition, between the two quantities (morphological distance and phylogentic distance), we find that morphological distance is a stronger determinant of species co-occurrences., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tripathi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

COVID-19 lockdowns in early 2020 reduced human mobility, providing an opportunity to disentangle its effects on animals from those of landscape modifications. Using GPS data, we compared movements and road avoidance of 2300 terrestrial mammals (43 species) during the lockdowns to the same period in 2019. Individual responses were variable with no change in average movements or road avoidance behavior, likely due to variable lockdown conditions. However, under strict lockdowns 10-day 95th percentile displacements increased by 73%, suggesting increased landscape permeability. Animals' 1-hour 95th percentile displacements declined by 12% and animals were 36% closer to roads in areas of high human footprint, indicating reduced avoidance during lockdowns. Overall, lockdowns rapidly altered some spatial behaviors, highlighting variable but substantial impacts of human mobility on wildlife worldwide.

The severe shortfall in testing supplies during the initial COVID-19 outbreak and ensuing struggle to manage the pandemic have affirmed the critical importance of optimal supply-constrained resource allocation strategies for controlling novel disease epidemics. To address the challenge of constrained resource optimization for managing diseases with complications like pre- and asymptomatic transmission, we develop an integro partial differential equation compartmental disease model which incorporates realistic latent, incubation, and infectious period distributions along with limited testing supplies for identifying and quarantining infected individuals. Our model overcomes the limitations of typical ordinary differential equation compartmental models by decoupling symptom status from model compartments to allow a more realistic representation of symptom onset and presymptomatic transmission. To analyze the influence of these realistic features on disease controllability, we find optimal strategies for reducing total infection sizes that allocate limited testing resources between 'clinical' testing, which targets symptomatic individuals, and 'non-clinical' testing, which targets non-symptomatic individuals. We apply our model not only to the original, delta, and omicron COVID-19 variants, but also to generically parameterized disease systems with varying mismatches between latent and incubation period distributions, which permit varying degrees of presymptomatic transmission or symptom onset before infectiousness. We find that factors that decrease controllability generally call for reduced levels of non-clinical testing in optimal strategies, while the relationship between incubation-latent mismatch, controllability, and optimal strategies is complicated. In particular, though greater degrees of presymptomatic transmission reduce disease controllability, they may increase or decrease the role of non-clinical testing in optimal strategies depending on other disease factors like transmissibility and latent period length. Importantly, our model allows a spectrum of diseases to be compared within a consistent framework such that lessons learned from COVID-19 can be transferred to resource constrained scenarios in future emerging epidemics and analyzed for optimality., (© 2023 The Authors.)

The population of older adults is projected to increase dramatically as Baby Boomers continue to reach age 65 into 2029. This article discusses key shifts in this demographic, including changes in overall health status and living arrangements, that can aid in defining older adults and their medical needs. It also highlights the changes in dental use patterns and the increase in demand for comprehensive dental services for older adults in recent years. The article focuses on the fact that oral health contributes to overall health and the dental workforce must be prepared to treat older adults in their practices., Competing Interests: Disclosure The authors have nothing to disclose. This article has no commercial or financial conflicts of interest and has no external funding sources for all authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

While biological invasions are recognized as a major threat to global biodiversity, determining non-native species' abilities to establish in new areas (species invasiveness) and the vulnerability of those areas to invasions (community invasibility) is challenging. Here, we use trait-based analysis to profile invasive species and quantify the community invasibility for >1,800 North American freshwater fish communities. We show that, in addition to effects attributed to propagule pressure caused by human intervention, species with higher fecundity, longer lifespan and larger size tend to be more invasive. Community invasibility peaks when the functional distance among native species was high, leaving unoccupied functional space for the establishment of potential invaders. Our findings illustrate how the functional traits of non-native species determining their invasiveness, and the functional characteristics of the invaded community determining its invasibility, may be identified. Considering those two determinants together will enable better predictions of invasions., (© 2023. The Author(s).)

The global extent and temporally asynchronous pattern of COVID-19 spread have repeatedly highlighted the role of international borders in the fight against the pandemic. Additionally, the deluge of high resolution, spatially referenced epidemiological data generated by the pandemic provides new opportunities to study disease transmission at heretofore inaccessible scales. Existing studies of cross-border infection fluxes, for both COVID-19 and other diseases, have largely focused on characterizing overall border effects. Here, we couple fine-scale incidence data with localized regression models to quantify spatial variation in the inhibitory effect of an international border. We take as a case study the border region between the German state of Saxony and the neighboring regions in northwestern Czechia, where municipality-level COVID-19 incidence data are available on both sides of the border. Consistent with past studies, we find an overall inhibitory effect of the border, but with a clear asymmetry, where the inhibitory effect is stronger from Saxony to Czechia than vice versa. Furthermore, we identify marked spatial variation along the border in the degree to which disease spread was inhibited. In particular, the area around Löbau in Saxony appears to have been a hotspot for cross-border disease transmission. The ability to identify infection flux hotspots along international borders may help to tailor monitoring programs and response measures to more effectively limit disease spread., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Vibert et al. (2022) demonstrate that oncogenic transcription factor fusion proteins bind otherwise silent genomic regions, producing RNAs that can be spliced, exported, and translated. These "neogenes" represent possible targets for immunotherapy and may even be universal byproducts of altered transcription in cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Insufficient testing capacity has been a critical bottleneck in the worldwide fight against COVID-19. Optimizing the deployment of limited testing resources has therefore emerged as a keystone problem in pandemic response planning. Here, we use a modified SEIR model to optimize testing strategies under a constraint of limited testing capacity. We define pre-symptomatic, asymptomatic, and symptomatic infected classes, and assume that positively tested individuals are immediately moved into quarantine. We further define two types of testing. Clinical testing focuses only on the symptomatic class. Non-clinical testing detects pre- and asymptomatic individuals from the general population, and a concentration parameter governs the degree to which such testing can be focused on high infection risk individuals. We then solve for the optimal mix of clinical and non-clinical testing as a function of both testing capacity and the concentration parameter. We find that purely clinical testing is optimal at very low testing capacities, supporting early guidance to ration tests for the sickest patients. Additionally, we find that a mix of clinical and non-clinical testing becomes optimal as testing capacity increases. At high but empirically observed testing capacities, a mix of clinical testing and non-clinical testing, even if extremely unfocused, becomes optimal. We further highlight the advantages of early implementation of testing programs, and of combining optimized testing with contact reduction interventions such as lockdowns, social distancing, and masking., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Animal movement is a key ecological process that is tightly coupled to local environmental conditions. While agriculture, urbanisation, and transportation infrastructure are critical to human socio-economic improvement, these have spurred substantial changes in animal movement across the globe with potential impacts on fitness and survival. Notably, however, human disturbance can have differential effects across species, and responses to human activities are thus largely taxa and context specific. As human disturbance is only expected to worsen over the next decade it is critical to better understand how species respond to human disturbance in order to develop effective, case-specific conservation strategies., Methods: Here, we use an extensive telemetry dataset collected over 22 years to fill a critical knowledge gap in the movement ecology of lowland tapirs (Tapirus terrestris) across areas of varying human disturbance within three biomes in southern Brazil: the Pantanal, Cerrado, and Atlantic Forest., Results: From these data we found that the mean home range size across all monitored tapirs was 8.31 km 2 (95% CI 6.53-10.42), with no evidence that home range sizes differed between sexes nor age groups. Interestingly, although the Atlantic Forest, Cerrado, and Pantanal vary substantially in habitat composition, levels of human disturbance, and tapir population densities, we found that lowland tapir movement behaviour and space use were consistent across all three biomes. Human disturbance also had no detectable effect on lowland tapir movement. Lowland tapirs living in the most altered habitats we monitored exhibited movement behaviour that was comparable to that of tapirs living in a near pristine environment., Conclusions: Contrary to our expectations, although we observed individual variability in lowland tapir space use and movement, human impacts on the landscape also had no measurable effect on their movement. Lowland tapir movement behaviour thus appears to exhibit very little phenotypic plasticity in response to human disturbance. Crucially, the lack of any detectable response to anthropogenic disturbance suggests that human modified habitats risk being ecological traps for tapirs and this information should be factored into conservation actions and species management aimed towards protecting lowland tapir populations., (© 2022. The Author(s).)

The polycomb repressive complexes 1 and 2 (PRCs; PRC1 and PRC2) are conserved histone-modifying enzymes that often function cooperatively to repress gene expression. The PRCs are regulated by long noncoding RNAs (lncRNAs) in complex ways. On the one hand, specific lncRNAs cause the PRCs to engage with chromatin and repress gene expression over genomic regions that can span megabases. On the other hand, the PRCs bind RNA with seemingly little sequence specificity, and at least in the case of PRC2, direct RNA-binding has the effect of inhibiting the enzyme. Thus, some RNAs appear to promote PRC activity, while others may inhibit it. The reasons behind this apparent dichotomy are unclear. The most potent PRC-activating lncRNAs associate with chromatin and are predominantly unspliced or harbor unusually long exons. Emerging data imply that these lncRNAs promote PRC activity through internal RNA sequence elements that arise and disappear rapidly in evolutionary time. These sequence elements may function by interacting with common subsets of RNA-binding proteins that recruit or stabilize PRCs on chromatin. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications., (© 2021 Wiley Periodicals LLC.)

The SARS-CoV-2 virus has spread around the world with over 100 million infections to date, and currently many countries are fighting the second wave of infections. With neither sufficient vaccination capacity nor effective medication, non-pharmaceutical interventions (NPIs) remain the measure of choice. However, NPIs place a great burden on society, the mental health of individuals, and economics. Therefore the cost/benefit ratio must be carefully balanced and a target-oriented small-scale implementation of these NPIs could help achieve this balance. To this end, we introduce a modified SEIRD-class compartment model and parametrize it locally for all 412 districts of Germany. The NPIs are modeled at district level by time varying contact rates. This high spatial resolution makes it possible to apply geostatistical methods to analyse the spatial patterns of the pandemic in Germany and to compare the results of different spatial resolutions. We find that the modified SEIRD model can successfully be fitted to the COVID-19 cases in German districts, states, and also nationwide. We propose the correlation length as a further measure, besides the weekly incidence rates, to describe the current situation of the epidemic., Competing Interests: The authors have declared that no competing interests exist.

The population of older adults is projected to increase dramatically as Baby Boomers continue to reach age 65 into 2029. This article discusses key shifts in this demographic, including changes in overall health status and living arrangements, that can aid in defining older adults and their medical needs. It also highlights the changes in dental use patterns and the increase in demand for comprehensive dental services for older adults in recent years. The article focuses on the fact that oral health contributes to overall health and the dental workforce must be prepared to treat older adults in their practices., Competing Interests: Disclosure The authors have nothing to disclose. This article has no commercial or financial conflicts of interest and has no external funding sources for all authors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

RNA-protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.

Nomadic movements are often a consequence of unpredictable resource dynamics. However, how nomadic ungulates select dynamic resources is still understudied. Here we examined resource selection of nomadic Mongolian gazelles (Procapra gutturosa) in the Eastern Steppe of Mongolia. We used daily GPS locations of 33 gazelles tracked up to 3.5 years. We examined selection for forage during the growing season using the Normalized Difference Vegetation Index (NDVI). In winter we examined selection for snow cover which mediates access to forage and drinking water. We studied selection at the population level using resource selection functions (RSFs) as well as on the individual level using step-selection functions (SSFs) at varying spatio-temporal scales from 1 to 10 days. Results from the population and the individual level analyses differed. At the population level we found selection for higher than average NDVI during the growing season. This may indicate selection for areas with more forage cover within the arid steppe landscape. In winter, gazelles selected for intermediate snow cover, which may indicate preference for areas which offer some snow for hydration but not so much as to hinder movement. At the individual level, in both seasons and across scales, we were not able to detect selection in the majority of individuals, but selection was similar to that seen in the RSFs for those individuals showing selection. Difficulty in finding selection with SSFs may indicate that Mongolian gazelles are using a random search strategy to find forage in a landscape with large, homogeneous areas of vegetation. The combination of random searches and landscape characteristics could therefore obscure results at the fine scale of SSFs. The significant results on the broader scale used for the population level RSF highlight that, although individuals show uncoordinated movement trajectories, they ultimately select for similar vegetation and snow cover., Competing Interests: The authors have declared that no competing interests exist.

K-mer based comparisons have emerged as powerful complements to BLAST-like alignment algorithms, particularly when the sequences being compared lack direct evolutionary relationships. In this chapter, we describe methods to compare k-mer content between groups of long noncoding RNAs (lncRNAs), to identify communities of lncRNAs with related k-mer contents, to identify the enrichment of protein-binding motifs in lncRNAs, and to scan for domains of related k-mer contents in lncRNAs. Our step-by-step instructions are complemented by Python code deposited in Github. Though our chapter focuses on lncRNAs, the methods we describe could be applied to any set of nucleic acid sequences.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of X O and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research., (Copyright © 2020 by the Genetics Society of America.)

The Xist lncRNA requires Repeat A, a conserved RNA element located in its 5' end, to induce gene silencing during X-chromosome inactivation. Intriguingly, Repeat A is also required for production of Xist. While silencing by Repeat A requires the protein SPEN, how Repeat A promotes Xist production remains unclear. We report that in mouse embryonic stem cells, expression of a transgene comprising the first two kilobases of Xist (Xist-2kb) causes transcriptional readthrough of downstream polyadenylation sequences. Readthrough required Repeat A and the ∼750 nucleotides downstream, did not require SPEN, and was attenuated by splicing. Despite associating with SPEN and chromatin, Xist-2kb did not robustly silence transcription, whereas a 5.5-kb Xist transgene robustly silenced transcription and read through its polyadenylation sequence. Longer, spliced Xist transgenes also induced robust silencing yet terminated efficiently. Thus, in contexts examined here, Xist requires sequence elements beyond its first two kilobases to robustly silence transcription, and the 5' end of Xist harbors SPEN-independent transcriptional antiterminator activity that can repress proximal cleavage and polyadenylation. In endogenous contexts, this antiterminator activity may help produce full-length Xist RNA while rendering the Xist locus resistant to silencing by the same repressive complexes that the lncRNA recruits to other genes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA's intracellular abundance, i.e. , approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular abundance, especially for lncRNAs that accumulate in focused subcellular regions. Calibrated RT-qPCR may return inaccurate estimates of abundance because individual PCR amplicons spaced across the length of a transcript can vary in their efficiency of reverse transcription. Here, we describe a sequencing-based approach that is straightforward, orthogonal to smFISH and RT-qPCR, and can be used to approximate the intracellular abundance for most expressed long RNAs (lncRNAs and mRNAs) in a cell type of interest. Firstly, the average weight of total RNA per cell for the cell type of interest is estimated by replicate rounds of RNA purification from a known number of cells. Secondly, an rRNA-depletion RNA-Seq protocol is performed after adding spike-in control RNAs to a known quantity of total cellular RNA. Lastly, by comparing read counts per transcript to a standard curve derived from the spiked-in RNAs, the intracellular abundance for each transcript is estimated. The sequencing-based approach provides a powerful complement to existing methods, particularly in situations where it is desirable to quantify the abundance of multiple lncRNAs and/or mRNAs simultaneously.

Encounter rates link movement strategies to intra- and inter-specific interactions, and therefore translate individual movement behavior into higher-level ecological processes. Indeed, a large body of interacting population theory rests on the law of mass action, which can be derived from assumptions of Brownian motion in an enclosed container with exclusively local perception. These assumptions imply completely uniform space use, individual home ranges equivalent to the population range, and encounter dependent on movement paths actually crossing. Mounting empirical evidence, however, suggests that animals use space non-uniformly, occupy home ranges substantially smaller than the population range, and are often capable of nonlocal perception. Here, we explore how these empirically supported behaviors change pairwise encounter rates. Specifically, we derive novel analytical expressions for encounter rates under Ornstein-Uhlenbeck motion, which features non-uniform space use and allows individual home ranges to differ from the population range. We compare OU-based encounter predictions to those of Reflected Brownian Motion, from which the law of mass action can be derived. For both models, we further explore how the interplay between the scale of perception and home-range size affects encounter rates. We find that neglecting realistic movement and perceptual behaviors can lead to systematic, non-negligible biases in encounter-rate predictions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Accurately quantifying species' area requirements is a prerequisite for effective area-based conservation. This typically involves collecting tracking data on species of interest and then conducting home-range analyses. Problematically, autocorrelation in tracking data can result in space needs being severely underestimated. Based on the previous work, we hypothesized the magnitude of underestimation varies with body mass, a relationship that could have serious conservation implications. To evaluate this hypothesis for terrestrial mammals, we estimated home-range areas with global positioning system (GPS) locations from 757 individuals across 61 globally distributed mammalian species with body masses ranging from 0.4 to 4000 kg. We then applied block cross-validation to quantify bias in empirical home-range estimates. Area requirements of mammals <10 kg were underestimated by a mean approximately15%, and species weighing approximately100 kg were underestimated by approximately50% on average. Thus, we found area estimation was subject to autocorrelation-induced bias that was worse for large species. Combined with the fact that extinction risk increases as body mass increases, the allometric scaling of bias we observed suggests the most threatened species are also likely to be those with the least accurate home-range estimates. As a correction, we tested whether data thinning or autocorrelation-informed home-range estimation minimized the scaling effect of autocorrelation on area estimates. Data thinning required an approximately93% data loss to achieve statistical independence with 95% confidence and was, therefore, not a viable solution. In contrast, autocorrelation-informed home-range estimation resulted in consistently accurate estimates irrespective of mass. When relating body mass to home range size, we detected that correcting for autocorrelation resulted in a scaling exponent significantly >1, meaning the scaling of the relationship changed substantially at the upper end of the mass spectrum., (© 2020 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Lineage specification in early development is the basis for the exquisitely precise body plan of multicellular organisms. It is therefore critical to understand cell fate decisions in early development. Moreover, for regenerative medicine, the accurate specification of cell types to replace damaged/diseased tissue is strongly dependent on identifying determinants of cell identity. Long noncoding RNAs (lncRNAs) have been shown to regulate cellular plasticity, including pluripotency establishment and maintenance, differentiation and development, yet broad phenotypic analysis and the mechanistic basis of their function remains lacking. As components of molecular condensates, lncRNAs interact with almost all classes of cellular biomolecules, including proteins, DNA, mRNAs, and microRNAs. With functions ranging from controlling alternative splicing of mRNAs, to providing scaffolding upon which chromatin modifiers are assembled, it is clear that at least a subset of lncRNAs are far from the transcriptional noise they were once deemed. This review highlights the diversity of lncRNA interactions in the context of cell fate specification, and provides examples of each type of interaction in relevant developmental contexts. Also highlighted are experimental and computational approaches to study lncRNAs., (Copyright © 2019 by the Genetics Society of America.)

Background: Speed and distance traveled provide quantifiable links between behavior and energetics, and are among the metrics most routinely estimated from animal tracking data. Researchers typically sum over the straight-line displacements (SLDs) between sampled locations to quantify distance traveled, while speed is estimated by dividing these displacements by time. Problematically, this approach is highly sensitive to the measurement scale, with biases subject to the sampling frequency, the tortuosity of the animal's movement, and the amount of measurement error. Compounding the issue of scale-sensitivity, SLD estimates do not come equipped with confidence intervals to quantify their uncertainty., Methods: To overcome the limitations of SLD estimation, we outline a continuous-time speed and distance (CTSD) estimation method. An inherent property of working in continuous-time is the ability to separate the underlying continuous-time movement process from the discrete-time sampling process, making these models less sensitive to the sampling schedule when estimating parameters. The first step of CTSD is to estimate the device's error parameters to calibrate the measurement error. Once the errors have been calibrated, model selection techniques are employed to identify the best fit continuous-time movement model for the data. A simulation-based approach is then employed to sample from the distribution of trajectories conditional on the data, from which the mean speed estimate and its confidence intervals can be extracted., Results: Using simulated data, we demonstrate how CTSD provides accurate, scale-insensitive estimates with reliable confidence intervals. When applied to empirical GPS data, we found that SLD estimates varied substantially with sampling frequency, whereas CTSD provided relatively consistent estimates, with often dramatic improvements over SLD., Conclusions: The methods described in this study allow for the computationally efficient, scale-insensitive estimation of speed and distance traveled, without biases due to the sampling frequency, the tortuosity of the animal's movement, or the amount of measurement error. In addition to being robust to the sampling schedule, the point estimates come equipped with confidence intervals, permitting formal statistical inference. All the methods developed in this study are now freely available in the ctmmR package or the ctmmweb point-and-click web based graphical user interface., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is cotransfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. We show that CRISPR-Bac can be used to knock down proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery., (© 2019 Schertzer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The marsupial inactive X chromosome expresses a long noncoding RNA (lncRNA) called Rsx that has been proposed to be the functional analog of eutherian Xist Despite the possibility that Xist and Rsx encode related functions, the two lncRNAs harbor no linear sequence similarity. However, both lncRNAs harbor domains of tandemly repeated sequence. In Xist , these repeat domains are known to be critical for function. Using k -mer based comparison, we show that the repeat domains of Xist and Rsx unexpectedly partition into two major clusters that each harbor substantial levels of nonlinear sequence similarity. Xist Repeats B, C, and D were most similar to each other and to Rsx Repeat 1, whereas Xist Repeats A and E were most similar to each other and to Rsx Repeats 2, 3, and 4. Similarities at the level of k -mers corresponded to domain-specific enrichment of protein-binding motifs. Within individual domains, protein-binding motifs were often enriched to extreme levels. Our data support the hypothesis that Xist and Rsx encode similar functions through different spatial arrangements of functionally analogous protein-binding domains. We propose that the two clusters of repeat domains in Xist and Rsx function in part to cooperatively recruit PRC1 and PRC2 to chromatin. The physical manner in which these domains engage with protein cofactors may be just as critical to the function of the domains as the protein cofactors themselves. The general approaches we outline in this report should prove useful in the study of any set of RNAs., (© 2019 Sprague et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Xist requires Repeat-A, a protein-binding module in its first two kilobases (2kb), to repress transcription. We report that when expressed as a standalone transcript in mouse embryonic stem cells (ESCs), the first 2kb of Xist (Xist-2kb) does not induce transcriptional silencing. Instead, Xist-2kb sequesters RNA produced from adjacent genes on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription and can be induced by lncRNAs with similar sequence composition to Xist-2kb. We did not detect sequestration by full-length Xist, but we did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. Xist-2kb associated with SPEN, a Repeat-A binding protein required for Xist-induced transcriptional silencing, but SPEN was not necessary for sequestration. Thus, when expressed in mouse ESCs, a 5' fragment of Xist that contains Repeat-A sequesters RNA from adjacent genes on chromatin and associates with the silencing factor SPEN, but it does not induce transcriptional silencing. Instead, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. We propose that sequestration is mechanistically related to the Repeat-A dependent stabilization and tethering of Xist near actively transcribed regions of chromatin., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SCHLAP1 is a long noncoding RNA that is reported to function by depleting the SWI/SNF complex from the genome. We investigated the hypothesis that SCHLAP1 affects only specific compositions of SWI/SNF. Using several assays, we found that SWI/SNF is not depleted from the genome by SCHLAP1 and that SWI/SNF is associated with many coding and noncoding RNAs, suggesting that SCHLAP1 may function in a SWI/SNF-independent manner.

The functions of most long non-coding RNAs (lncRNAs) are unknown. In contrast to proteins, lncRNAs with similar functions often lack linear sequence homology; thus, the identification of function in one lncRNA rarely informs the identification of function in others. We developed a sequence comparison method to deconstruct linear sequence relationships in lncRNAs and evaluate similarity based on the abundance of short motifs called k-mers. We found that lncRNAs of related function often had similar k-mer profiles despite lacking linear homology, and that k-mer profiles correlated with protein binding to lncRNAs and with their subcellular localization. Using a novel assay to quantify Xist-like regulatory potential, we directly demonstrated that evolutionarily unrelated lncRNAs can encode similar function through different spatial arrangements of related sequence motifs. K-mer-based classification is a powerful approach to detect recurrent relationships between sequence and function in lncRNAs.

Tightly synchronized reproduction in vast wildebeest herds underpins the keystone role this iconic species plays in the Serengeti. However, despite decades of study, the proximate synchronizing mechanism remains unknown. Combining a season-long field experiment with simple stochastic process models, we show that females exposed to playback of male rutting vocalizations are over three times more synchronous in their expected time to mating than a control group isolated from all male stimuli. Additionally, predictions of both mating and calving synchrony based on the playback group were highly consistent with independent data on wildebeest mating and calving synchrony, while control-based predictions were inconsistent with the data. Taken together, our results provide the first experimental evidence that male rutting vocalizations alone could account for the highly synchronized reproduction observed in Serengeti wildebeest. Given anthropogenically driven losses in many areas, a mechanistic understanding of synchrony can highlight additional risks declining wildebeest populations may face.

Home-range estimation is an important application of animal tracking data that is frequently complicated by autocorrelation, sampling irregularity, and small effective sample sizes. We introduce a novel, optimal weighting method that accounts for temporal sampling bias in autocorrelated tracking data. This method corrects for irregular and missing data, such that oversampled times are downweighted and undersampled times are upweighted to minimize error in the home-range estimate. We also introduce computationally efficient algorithms that make this method feasible with large data sets. Generally speaking, there are three situations where weight optimization improves the accuracy of home-range estimates: with marine data, where the sampling schedule is highly irregular, with duty cycled data, where the sampling schedule changes during the observation period, and when a small number of home-range crossings are observed, making the beginning and end times more independent and informative than the intermediate times. Using both simulated data and empirical examples including reef manta ray, Mongolian gazelle, and African buffalo, optimal weighting is shown to reduce the error and increase the spatial resolution of home-range estimates. With a conveniently packaged and computationally efficient software implementation, this method broadens the array of data sets with which accurate space-use assessments can be made., (© 2018 by the Ecological Society of America.)

While many animal species exhibit strong conspecific interactions, movement analyses of wildlife tracking datasets still largely focus on single individuals. Multi-individual wildlife tracking studies provide new opportunities to explore how individuals move relative to one another, but such datasets are frequently too sparse for the detailed, acceleration-based analytical methods typically employed in collective motion studies. Here, we address the methodological gap between wildlife tracking data and collective motion by developing a general method for quantifying movement correlation from sparsely sampled data. Unlike most existing techniques for studying the non-independence of individual movements with wildlife tracking data, our approach is derived from an analytically tractable stochastic model of correlated movement. Our approach partitions correlation into a deterministic tendency to move in the same direction termed 'drift correlation' and a stochastic component called 'diffusive correlation'. These components suggest the mechanisms that coordinate movements, with drift correlation indicating external influences, and diffusive correlation pointing to social interactions. We use two case studies to highlight the ability of our approach both to quantify correlated movements in tracking data and to suggest the mechanisms that generate the correlation. First, we use an abrupt change in movement correlation to pinpoint the onset of spring migration in barren-ground caribou. Second, we show how spatial proximity mediates intermittently correlated movements among khulans in the Gobi desert. We conclude by discussing the linkages of our approach to the theory of collective motion.This article is part of the theme issue 'Collective movement ecology'., (© 2018 The Author(s).)

Animal movement is fundamental for ecosystem functioning and species survival, yet the effects of the anthropogenic footprint on animal movements have not been estimated across species. Using a unique GPS-tracking database of 803 individuals across 57 species, we found that movements of mammals in areas with a comparatively high human footprint were on average one-half to one-third the extent of their movements in areas with a low human footprint. We attribute this reduction to behavioral changes of individual animals and to the exclusion of species with long-range movements from areas with higher human impact. Global loss of vagility alters a key ecological trait of animals that affects not only population persistence but also ecosystem processes such as predator-prey interactions, nutrient cycling, and disease transmission., (Copyright © 2018, The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Many animals undertake movements that are longer scaled and more directed than their typical home ranging behaviour. These movements include seasonal migrations (e.g. between breeding and feeding grounds), natal dispersal, nomadic range shifts and responses to local environmental disruptions. While various heuristic tools exist for identifying range shifts and migrations, none explicitly model the movement of the animals within a statistical framework that facilitates quantitative comparisons. We present the mechanistic range shift analysis (MRSA), a method to estimate a suite of range shift parameters: times of initiation, duration of transitions, centroids and areas of respective ranges. The method can take the autocorrelation and irregular sampling that is characteristic of much movement data into account. The mechanistic parameters suggest an intuitive measure, the range shift index, for the extent of a range shift. The likelihood based estimation further allows for statistical tests of several relevant hypotheses, including a range shift test, a stopover test and a site fidelity test. The analysis tools are provided in an R package (marcher). We applied the MRSA to a population of GPS tracked roe deer (Capreolus capreolus) in the Italian Alps between 2005 and 2008. With respect to seasonal migration, this population is extremely variable and difficult to classify. Using the MRSA, we were able to quantify the behaviours across the population and among individuals across years, identifying extents, durations and locations of seasonal range shifts, including cases that would have been ambiguous to detect using existing tools. The strongest patterns were differences across years: many animals simply did not perform a seasonal migration to wintering grounds during the mild winter of 2006-2007, even though some of these same animals did move extensively in other, harsher winters. For seasonal migrants, however, site fidelity across years was extremely high, even after skipping an entire seasonal migration. These results suggest that for roe deer behavioural plasticity and tactical responses to immediate environmental cues are reflected in the decision of whether rather than where to migrate. The MRSA also revealed a trade-off between the probability of migrating and the size of a home range., (© 2017 The Authors. Journal of Animal Ecology © 2017 British Ecological Society.)