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In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, owing to its ability to detect multiple modifications within the same full-length native RNA molecules. Although RNA modifications can be identified in the form of systematic basecalling "errors" in DRS data sets, N6 -methyladenosine (m 6 A) modifications produce relatively low "errors" compared with other RNA modifications, limiting the applicability of this approach to m 6 A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the "error" signal of m 6 A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads-especially in shorter RNA fractions-and increased basecalling error signatures at pseudouridine (Ψ)- and N1 -methylpseudouridine (m 1 Ψ)-modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability, and basecalling accuracy in nanopore DRS data sets., (© 2024 Diensthuber et al.; Published by Cold Spring Harbor Laboratory Press.)

Understanding the impacts of, and options for, controlling invasive species is crucial to their management. Wetlands are a widely invaded ecosystem, since dispersal of aquatic species is facilitated by seasonal flooding. This study evaluated the effects of the translocated pondweed Monochoria hastata on fish and rice production in two wetlands of Bangladesh over six years (2017-2022). Fish and rice production were compared between control (negligible M. hastata) and three treatments under different M. hastata management methods comprising manual-, herbicide- and mechanical-treatment. Density of M. hastata increased significantly in all treatment groups over time in both wet and dry seasons. However, M. hastata density was lower by 270% in the dry season than the wet season. For fishes, a negative relationship between M. hastata density and fish production was recorded for snakeheads and catfishes, the most saleable fishes, whereas a mixed pattern was recorded for barbs and minnows across treatments. A positive relationship occurred between the density of M. hastata and production of the most common fish, mud eel, and therefore, the overall fish production increased in all treatment groups. Compared to control plots, rice production was lower in M. hastata infested plot groups. Among the M. hastata infested plot groups, rice production in herbicide-and mechanical-treatment groups was similar but lower than the manual-treatment group. Although manual-treatment plots yielded greater rice production, the weed management cost was also higher. This study provides evidence that translocated M. hastata can be of an invasive nature and impact rice production, not only by reducing yield but also by increasing the production costs through additional management for M. hastata control. Its presence in wetlands in Bangladesh can increase overall fish production due to the overriding influence of increased mud eel yield which has little demand locally but can decrease the species of high demand (e.g. snakehead and catfish). None of the existing control measures are effective in controlling M. hastata. Further research is needed on better management approaches for both agricultural and fish production in areas invaded by M. hastata., 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 © 2024 Elsevier Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC ( Pdx1-Cre , LSL-Kras G12D/+ , LSL-Trp53 R172H/+ ) and poorly metastatic KP fl C ( Pdx1-Cre , LSL-Kras G12D/+ , Trp53 fl/+ ) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KP fl C, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress., (© 2023. The Author(s).)