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While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m⁵U54). Here, we uncover contributions of m⁵U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m⁵U in the T-loop (TrmA inEscherichia coli, Trm2 inSaccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m⁵U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells,trm2Δcell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m⁵U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis. 

Ciguatera poisoning (CP) is the most common form of phycotoxin-borne seafood poisoning globally, affecting thousands of people on an annual basis. It most commonly occurs in residential fish of coral reefs, which consume toxin-laden algae, detritus, and reef animals. The class of toxins that cause CP, ciguatoxins (CTXs), originate in benthic, epiphytic dinoflagellates of the genera, Gambierdiscus and Fukuyoa, which are consumed by herbivores and detritivores that facilitate food web transfer. A number of factors have hindered adequate environmental monitoring and seafood surveillance for ciguatera including the low concentrations in which the toxins are found in seafood causing illness (sub-ppb), a lack of knowledge on the toxicity equivalence of other CTXs and contribution of other benthic algal toxins to the disease, and the limited availability of quantified toxin standards and reference materials. While progress has been made on the identification of the dinoflagellate taxa and toxins responsible for CP, more effort is needed to better understand the dynamics of toxin transfer into reef food webs in order to implement a practical monitoring program for CP. Here, we present a conceptual model that utilizes empirical field data (temperature, Gambierdiscus cell densities, macrophyte cover) in concert with other published studies (grazing rates and preference) to produce modeling outputs that suggest approaches that may be beneficial to developing monitoring programs: 1) targeting specific macrophytes for Gambierdiscus and toxin measurements to monitor toxin levels at the base of the food web (i.e., toxin loading); and 2) adjusting these targets across sites and over seasons. Coupling this approach with other methodologies being incorporated into monitoring programs (artificial substrates; FISH probes; toxin screening) may provide an “early warning” system to develop strategic responses to potential CP flare ups in the future. 

Ciguatera Poisoning (CP) is a widespread and complex poisoning syndrome caused by the consumption of fish or invertebrates contaminated with a suite of potent neurotoxins collectively known as ciguatoxins (CTXs), which are produced by certain benthic dinoflagellates species in the genera Gambierdiscus and Fukuyoa. Due to the complex nature of this HAB problem, along with a poor understanding of toxin production and entry in the coral reef food web, the development of monitoring, management, and forecasting approaches for CP has lagged behind those available for other HAB syndromes. Over the past two decades, renewed research on the taxonomy, physiology, and toxicology of CP-causing dinoflagellates has advanced our understanding of the species diversity that exists within these genera, including identification of several highly toxic species (so called “superbugs”) that likely contribute disproportionately to ciguatoxins entering coral reef food webs. The recent development of approaches for molecular analysis of field samples now provide the means to investigate in situ community composition, enabling characterization of spatio-temporal species dynamics, linkages between toxic species abundance and toxin flux, and the risk of ciguatoxin prevalence in fish. In this study we used species-specific fluorescent in situ hybridization (FISH) probes to investigate Gambierdiscus species composition and dynamics in St. Thomas (USVI) and the Florida Keys (USA) over multiple years of sampling (2018-2020). Within each location, samples were collected seasonally from several sites comprising varying depths, habitats, and algal substrates to characterize community structure over small spatial scales and across different host macrophytes. This approach enabled the quantitative determination of communities over spatiotemporal gradients, as well as the selective enumeration of species known to exhibit high toxicity, such as Gambierdiscus silvae. The investigation found differing community structure between St. Thomas and Florida Keys sites, driven in part by differences in the distribution of toxin producing species G. silvae and G. belizeanus, which were present throughout sampling sites in St. Thomas but scarce or absent in the Florida Keys. This finding is significant given the high toxicity of G. silvae, and may help explain differences in fish toxicity and CP incidence between St. Thomas and Florida. 

Abstract Limited dispersal of individuals between generations results in isolation by distance, in which individuals further apart in space tend to be less related. Classic models of isolation by distance assume that dispersal distances are drawn from a thin-tailed distribution and predict that the proportion of the genome that is identical by descent between a pair of individuals should decrease exponentially with the spatial separation between them. However, in many natural populations, individuals occasionally disperse over very long distances. In this work, we use mathematical analysis and coalescent simulations to study the effect of long-range (power-law) dispersal on patterns of isolation by distance. We find that it leads to power-law decay of identity-by-descent at large distances with the same exponent as dispersal. We also find that broad power-law dispersal produces another, shallow power-law decay of identity-by-descent at short distances. These results suggest that the distribution of long-range dispersal events could be estimated from sequencing large population samples taken from a wide range of spatial scales. 

The operational safety of Automated Driving System (ADS)-Operated Vehicles (AVs) are a rising concern with the deployment of AVs as prototypes being tested and also in commercial deployment. The robustness of safety evaluation systems is essential in determining the operational safety of AVs as they interact with human-driven vehicles. Extending upon earlier works of the Institute of Automated Mobility (IAM) that have explored the Operational Safety Assessment (OSA) metrics and infrastructure-based safety monitoring systems, in this work, we compare the performance of an infrastructure-based Light Detection And Ranging (LIDAR) system to an onboard vehicle-based LIDAR system in testing at the Maricopa County Department of Transportation SMARTDrive testbed in Anthem, Arizona. The sensor modalities are located in infrastructure and onboard the test vehicles, including LIDAR, cameras, a real-time differential GPS, and a drone with a camera. Bespoke localization and tracking algorithms are created for the LIDAR and cameras. In total, there are 26 different scenarios of the test vehicles navigating the testbed intersection; for this work, we are only considering car following scenarios. The LIDAR data collected from the infrastructure-based and onboard vehicle-based sensors system are used to perform object detection and multi-target tracking to estimate the velocity and position information of the test vehicles and use these values to compute OSA metrics. The comparison of the performance of the two systems involves the localization and tracking errors in calculating the position and the velocity of the subject vehicle, with the real-time differential GPS data serving as ground truth for velocity comparison and tracking results from the drone for OSA metrics comparison. 

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m⁵U54). Here, we uncover contributions of m⁵U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m⁵U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m⁵U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m⁵U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis. 

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N 2-methylguanosine, N 2, N 2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N 2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.