Direct Sequencing Research Articles

Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduces their respective read counts by more than 85%, resulting in an increase in sequencing depth of 47% on average for long non-coding RNAs. We also apply RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as an increase in non-globin reads by 16% on average. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER’s modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser.

The Novel Long QT Syndrome Type 2-associated F129I Mutation in the KCNH2 Gene Significantly Affects I Kr Through the hERG1 Homomeric and Heteromeric Potassium Channels

Objective: The long QT syndrome type 2 is caused by the loss-of-function mutations in the KCNH2 gene, which encodes hERG1, the voltage-gated potassium channel. The hERG1 channels conduct rapid delayed rectifier K+ currents (I Kr) in the human cardiac tissue. KCNH2 encodes 2 main isoforms—hERG1a and hERG1b, which assemble to form the homomeric or heteromeric hERG1 channels. However, the functional characteristics of the heteromeric hERG1 channels in long QT syndrome type 2 are not clear. In this study, a novel mutation in the N-terminus of hERG1a (F129I) was identified in a proband of long QT syndrome type 2. The purpose of this study was to identify the electrophysiological change of homomeric and heteromeric hERG1 channels with the F129I-hERG1a. Methods: Candidate genes were screened by direct sequencing. F129I-hERG1a was cloned in the pcDNA3.1 vector by site-directed mutagenesis. Then, the wild-type (WT) hERG1a and/or F129I-hERG1a were transiently expressed in the HEK293 cells with or without hERG1b co-expression. The expression levels of the transgenes, cellular distribution of hERG1a and hERG1b, and the electrophysiological features of the homomeric and the heteromeric hERG1 channels with the WT-hERG1a or F129I-hERG1a were analyzed using whole-cell patch-clamp electrophysiology, western blotting, and immunofluorescence techniques. Results: The proband was clinically diagnosed with long QT syndrome type 2 and carried a heterozygous mutation c.385T>A (F129I) in the KCNH2 gene. Electrophysiology study proved that the F129I substitution in hERG1a significantly decreased I Kr in both the homomeric and heteromeric hERG1channels by 86% and 70%, respectively (WT-hERG1a (54.88 ± 18.74) pA/pF vs. F129I-hERG1a (7.34 ± 1.90) pA/pF, P < 0.001; WT-hERG1a/hERG1b (89.92 ± 24.51) pA/pF vs. F129I-hERG1a/hERG1b (26.54 ± 9.83) pA/pF, P < 0.001). The voltage dependence of IKr activation (V½ and k) was not affected by the mutation in both the homomeric and heteromeric hERG1 channels. The peak current densities and the kinetic characteristics of IKr were comparable for both WT/F129I-hERG1a and WT-hERG1a. The channel inactivation and deactivation analysis showed that F129I substitution did not affect deactivation of the homomeric hERG1a channel, but significantly accelerated the deactivation and recovery from inactivation of the heteromeric hERG1a/hERG1b channel based on the time constants of fast and slow recovery from deactivation F129I-hERG1a/hERG1b vs. WT-hERG1a/hERG1b (P < 0.05). Western blotting and immunofluorescence labeling experiments showed that maturation and intracellular trafficking of the F129I-hERG1a protein was impaired and potentially increased the ratio of hERG1b to hERG1a in the F129I-hERG1a/hERG1b tetramer channel, thereby resulting in electrophysiological changes characteristic of the long QT syndrome type 2 pathology. Conclusions: I Kr was significantly reduced in the homomeric and heteromeric hERG1 channels with F129I-hERG1a. The F129I mutation significantly accelerated the deactivation and recovery from inactivation of the heteromeric F129I-hERG1a/hERG1b channel. F129I-hERG1a exhibited impaired maturation and intracellular trafficking, thereby potentially increasing the ratio of the hERG1b to hERG1a stoichiometry in the hERG1 tetrameric channel. These changes demonstrated the importance of the heteromeric hERG1 channel in long QT syndrome type 2 pathophysiology.

Surface material analysis for human papillomavirus detection in nail Bowen's disease caused by HPV type 58.

Over the past few years, cases of human papillomavirus (HPV) infection in nail Bowen's disease have been reported. This disease presents diagnostic challenges due to its similarity to nail malignant melanoma, particularly with respect to the clinical manifestation of black nail streaks. While skin biopsy is usually employed for diagnosis, it is an invasive procedure. We report the case of a 52-year-old healthy Japanese male with a pigmented streak on the nail of the fourth finger of his right hand, which had extended from the central to the lateral nail fold within 4 months. Dermoscopic examination revealed a dark-brown pigmented band with splinter microhemorrhage. Clinically, nail Bowen's disease was suspected. The lesion was excised in strips under local anesthesia. Histopathological examination revealed hyperkeratosis, parakeratosis, papillomatosis, and dyskeratotic cells with atypical nuclei irregularly arranged. Immunohistochemistry using anti-HPV L1 antibody detected HPV-positive cells in the upper epidermis and stratum corneum of the nail matrix. Mucosal high-risk HPV type 58 DNA was detected from brush cytology of the keratotic surface prior to surgery, which was confirmed in formalin-fixed, paraffin-embedded excised samples using polymerase chain reaction (PCR) and subsequent direct DNA sequencing. Our case highlights HPV type 58 as a potential causative agent of nail Bowen's disease and shows that brush cytology of the surface material prior to excision may be a useful and less invasive way for mucosal high-risk HPV detection. PCR analysis of the nail surface could serve as a supplementary diagnostic tool for nail Bowen's disease.

Linking Survivor Stories to Forensic Engineering: How an Interscience Approach Reveals Opportunities for Reducing Tornado Vulnerability in Residential Structures

Abstract When a tornado strikes a permanent or mobile/manufactured home, occupants are at risk of injury and death from blunt force trauma caused by debris-loaded winds and failure of the structure. Mechanisms for these failures have been studied for the past few decades and identified common weaknesses in the structural load path. Also under study in recent decades, much has been learned about how people receive and understand warnings and determine how, when, and if they will shelter in advance. Recent research, for example, shows most people do not shelter until close to impact, after seeing, hearing, or feeling the approaching tornado. To advance beyond these innovations, a new, multi-disciplinary approach was fielded in nine Southeast U.S. tornadoes between 2019 and 2022. For each tornado, 1) wind engineering assessments documented near-surface wind fields, 2) structural engineering assessments documented the primary wind load path for each structure, and 3) social science interviews captured the survivor’s narrative and asked several follow-up questions to assure key items of interest were addressed in each interview. When possible, the team was multi-disciplinary during the interview, enabling survivors to ask questions and better understand their experiences. Most survivors became aware of the approaching tornado with at least a few minutes lead time and most were able to reach a place of refuge. Most survivors recalled sensory experiences during the tornado and about half could describe direction or temporal sequences of damage. A case study of the Cookeville, Tennessee, Tornado of 3 March 2020 illustrates the power of the integrated data assessment.

The Effects of a Direct Instruction Curriculum Sequence on the Reading Comprehension Responses of Early Readers

Reading comprehension is critical for early learners’ academic success. Derived relations are an important feature of reading comprehension within the science of behavior because reading comprehension involves experientially acquired derived stimulus relations (Helou-Care, 2008; Neves in Behavioral Interventions 38:376–400, 2023, Park, 2005; da Silva in Experimental Analysis of Human Behavior Bulletin 24:1–8, 2006). That is, derived relations pertain to a learner demonstrating comprehension by making untrained connections to previously trained stimuli (e.g., words). For example, a learner who is trained that “kitten” is a type of “cat” and that all “cats meow” should then be able to demonstrate the untrained connection that “kittens meow.” The purpose of this study was to investigate the effects of the Corrective Reading Comprehension (CR) curriculum, which employs an instructional sequence involving derived relations, on reading comprehension responses entailing derived relations. Four typically developing first-grade students were the participants in this study and the students were divided into two dyads. The principal researcher instructed each dyad on each of the five lessons of CR until mastery was obtained and then conducted postprobes on the dependent measures that were metaphors, derived relations from sentences, derived relations from letters/numbers, and implicit/explicit comprehension questions. The researchers used a delayed multiple probe design across 1st-grade dyads to assess the effect of five CR lessons on derived relations and reading comprehension responses of the participants. Posttest assessments showed gains for all four participants. The article concludes with a discussion of the main findings, implications for future research, and the limitations of this study.

#314 Systemic lupus erythematous (SLE) and isolated multiple sclerosis: analysis of BAFF—VAR variant on gene TNSF13B

Abstract Background and Aims We report a case of young a woman (33 years old) affected by SLE since 2007. In this year, a renal biopsy showed class IV lupus nephritis and the patient received corticosteroids and immunosuppressant for 3 years. The patient remained thereafter in disease remission with normal renal function, no proteinuria and normal complement levels. In 2013 the patient performed brain MRI for sudden hearing loss. The MRI showed a pattern compatible with isolated multiple sclerosis (RIS), confirmed on subsequent radiographic checks. In 2023, due to the initial onset of renal failure, a new re-staging biopsy showed microscopic aspects compatible with class I/II lupus nephritis (INS/RPS Classification 2004, 2018); the activity index calculated with SLEDAI—2K was +4. A new brain MRI carried out without onset of new symptoms showed a new hyperintense region at the level of the head of the right caudate nucleus and enlargement of the intrasellar CSF content as per the condition of a “partially empty” sella. A genetic variant of the TNFSF13B gene was therefore suspected, since it is involved in encoding the cytochine and drug target B-cell activating factor (BAFF), associated with multiple sclerosis as well as SLE. We searched for the genetic variant in order to undertake target therapy early in our patient. Method We performed PCR amplification and direct sequencing of all coding exons plus all the intron-exon junctions of the analyzed gene, by automatic sequencer with SeqStudio Genetic Analyzer (analytical sensitivity and specificity >99%). Results We found the heterozygous presence of the BAFF-var variant in the 3' UTR region of the TNFSF13B gene (Heterozygous genotype for BAFF-var). This variant involves an insertion/deletion (GCTGT>A) at the level of the 3' UTR region with consequent production of an alternative polyadenylation site (APA). This APA leads to the formation of a shorter transcript (BAFF-var mRNA) which causes greater production of the encoded protein compared to the wild-type transcript (BAFF-WT mRNA). Conclusion The molecular analysis conducted highlighted the presence of the BAFF-var variant in heterozygosis which leads to increased levels of soluble BAFF in the serum, with consequent up-regulation of humoral immunity. It is reported in the literature1 that this variant is associated with both Multiple Sclerosis and SLE and may represent a valid therapeutic target being treatable with the monoclonal antibody Belimumab.

The Relationship between Intelligence and Mitochondrial DNA Polymorphism at Site of 15925 Base Pairs

Intelligence has a huge influences and is very closely related with human’s life. Because intelligence is indefinable, scholars have specified it through testing tools and named it intelligence quotient (IQ). In measuring IQ, researchers have found that IQ has a wide distribution and tried to find out the reasons by assessing the brain's functional level and environmental and genetic factors. One study reported an association between IQ and mitochondrial DNA (mtDNA) sequence 15,925, and this study investigated the association in Koreans. The number of participants was 57 in the mental retardation group without chromosomal abnormalities and 148 in the normal group, respectively. PCR technique and direct sequencing analysis were used to know participant’s mtDNA sequences. At 15,925 base, all participants had only wild type C and no variant type of m.15,925 C > T. We further studied variants in mtDNA sequences 15,926-15,927. At 15,926 base, the variant type of 15,926 C < T was found in only one case (0.7%) in the control group. At the 15,927 base, variant type of 15,927 G < A was found in 9 cases (6.1%) in the control group and 1 case (1.8%) in the retardation group, respectively. As a result, the polymorphisms of mtDNA sequences at bases 15,925-15,927 had no statistical significance in the Korean control and mental retardation group.

PGPg_finder: A comprehensive and user-friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data

Identifying and comparing plant growth-promoting traits (PGPT) within whole-genome and metagenomic sequencing data can significantly advance agricultural research and promote sustainable crop production. This study introduces PGPg_finder, a comprehensive pipeline designed to annotate and compare PGPT from both whole-genome and metagenome sequencing datasets. This pipeline utilizes direct sequence annotation alongside de novo assembly methods to accurately detect PGPT. By cross-referencing sequences from the PLaBAse database, it identifies and quantifies the presence of these genes within the original datasets, facilitating an intuitive comparison of the abundance and distribution of PGPT across various samples. We evaluated the performance of PGPg_finder by analyzing genomes from five rhizobacterial strains: Paenibacillus vini, Paenibacillus polymyxa, Fictibacillus sp., Brevibacillus agri, and Bacillus cereus, and also metagenomic samples from bulk soils subjected to forest-to-pasture conversion in the Amazon rainforest. The genomic workflow revealed several genes associated with substrate utilization, abiotic stress neutralization, phosphate solubilization, and iron acquisition. It also identified genes unique to specific lineages, including those associated with colonization and plant-derived substrate usage in P. polymyxa, quorum sensing response and biofilm formation in P. vini, heavy metal detoxification and nitrogen acquisition in B. agri, and spore production and neutralizing biotic stress in B. cereus. The strain Fictibacillus sp. presented several unique genes related to surface attachment, stress response, xenobiotic degradation, phosphate solubilization, and phytohormone production. The use of PGPg_finder highlights its potential to uncover novel inoculants and strains. The metagenomic workflow distinguished plant-growth promotion gene profiles between soils from the Amazon rainforest and pasture, with the latter showing a profile more aligned with simple carbohydrate consumption, abiotic stress tolerance, motility and chemotaxis, and phosphorus mineralization. Native forests exhibited a profile associated with the degradation of complex organic matter, oxidative stress tolerance, xenobiotic degradation, bactericidal activity, iron acquisition, and volatile pathways. These findings underscore the effectiveness and sensitivity of PGPg_finder in accurately identifying and comparing PGPT genes, highlighting both commonalities and variations across samples. The application of this pipeline has the potential to significantly facilitate the identification of plant growth-promoting microbes.

Molecular Identification of the Gag and Tax Gene of Bovine Leukemia Virus (BLV) in Women with Breast Cancer

Background: Breast cancer is one of the most common cancers in the world particularly among Iranian women. Although the mortality rate decreased in developed countries, there were increased changes in breast cancer incidences. The study aimed at evaluating the correlation between BLV infection and breast cancer in Iran. Methods: In the cross-sectional study, a total of 85 breast cancer tissue samples and adjacent tumor samples (normal tissue) were collected during the year 2022 from women referred to general hospitals in Qom Province, Iran. The nested PCR technique was performed to determine the presence of tax and gag genes of BLV in the collected samples. To confirm the presence of the tax gene of BLV, PCR products of some BLV-positive samples were subjected to direct sequencing. Results: Based on nested PCR technique, tax and gag genes of BLV were detected in 12.9% (11.85) and 3.4% (3.85) of breast cancer tissue samples, respectively. Also, the tax gene was present in 4.7% of the control samples, but the presence of the gag gene was not observed. Most malignant samples of BLV were III grade. The constructed phylogenetic trees for the tax gene of BLV showed most BLV isolates had similar sequences to Iranian isolates. Conclusion: The results of the study by using nested PCR technique, demonstrate a possible relationship between human breast cancer and bovine leukemia virus infection in women of Iran. BLV is one of the main risk factors for breast cancer.

High-throughput nanopore DNA sequencing of large insert fosmid clones directly from bacterial colonies.

Fosmids and cosmids are vectors frequently used in functional metagenomic studies. With a large insert capacity (around 30 kb) they can encode dozens of cloned genes or in some cases, entire biochemical pathways. Fosmids with cloned inserts can be transferred to heterologous hosts and propagated to enable screening for new enzymes and metabolites. After screening, fosmids from clones with an activity of interest must be de novo sequenced, a critical step toward the identification of the gene(s) of interest. In this work, we present a new approach for rapid and high-throughput fosmid sequencing directly from Escherichia coli colonies without liquid culturing or fosmid purification. Our sample preparation involves fosmid amplification with phi29 polymerase and then direct nanopore sequencing using the Oxford Nanopore Technologies system. We also present a bioinformatics pipeline termed "phiXXer" that facilitates both de novo read assembly and vector trimming to generate a linear sequence of the fosmid insert. Finally, we demonstrate the accurate sequencing of 96 fosmids in a single run and validate the method using two fosmid libraries that contain cloned large insert (~30-40 kb) genomic or metagenomic DNA.IMPORTANCELarge-insert clone (fosmids or cosmids) sequencing is challenging and arguably the most limiting step of functional metagenomic screening workflows. Our study establishes a new method for high-throughput nanopore sequencing of fosmid clones directly from lysed Escherichia coli cells. It also describes a companion bioinformatic pipeline that enables de novo assembly of fosmid DNA insert sequences. The devised method widens the potential of functional metagenomic screening by providing a simple, high-throughput approach to fosmid clone sequencing that dramatically speeds the pace of discovery.

Lipoprotein glomerulopathy with markedly increased arterial stiffness successfully treated with a combination of fenofibrate and losartan: a case report

BackgroundLipoprotein glomerulopathy (LPG) is a apolipoprotein E (ApoE)-related glomerular disease and has been associated with type III hyperlipidemia. Without appropriate treatment, chronic kidney disease (CKD) caused by LPG progresses, and approximately half of the patients develop end-stage kidney disease within 1–27 years of disease onset. However, few studies have highlighted the clinical course of cardiovascular diseases (CVDs) in patients with LPG. Herein, we report the first case of LPG in which the CVD risk was assessed using arterial stiffness.Case presentationA 32-year-old Japanese man was referred to our hospital due to persistent proteinuria. Kidney biopsy showed markedly dilated capillary lumens containing pale-stained thrombi, which stained positively with Oil Red O. Electron microscopy revealed the presence of thrombi in the capillary lumen with low electron density and vacuoles of various sizes in part of the thrombi. Toluidine blue and Sudan IV stains were used to stain the thin sections of Epon-embedded tissue samples for electron microscopy. Sudan IV-positive droplets were observed in the capillary lumens, vascular walls, and cytoplasm of tubular cells. Increased serum ApoE concentration was observed. Liquid chromatography-tandem mass spectrometry of laser-microdissected glomeruli from paraffin sections revealed an increase in ApoE. Direct deoxyribonucleic acid sequencing of ApoE revealed a heterozygous ApoE Sendai mutation (Arg145Pro). The patient was finally diagnosed with LPG with heterozygosity for ApoE-Sendai mutation (Arg145Pro). Notably, at the time of diagnosis, he had markedly increased arterial stiffness for his age. Arterial stiffness was measured using brachial-ankle pulse wave velocity (baPWV), which was equivalent to that of a 56-year-old man. After three months of treatment with fenofibrate and losartan, a significant reduction in proteinuria was achieved along with an improvement in baPWV. Furthermore, these effects were maintained despite the lack of decrease in serum ApoE levels.ConclusionHerein, we report the case of a patient with LPG with markedly increased arterial stiffness at the time of diagnosis, in whom combination therapy with fenofibrate and losartan successfully improved proteinuria and arterial stiffness. To the best of our knowledge, this is the first case report of LPG in which CVD risk was assessed using arterial stiffness.

Quantitative profiling N1-methyladenosine (m1A) RNA methylation from Oxford nanopore direct RNA sequencing data

With the recent advanced direct RNA sequencing technique that proposed by the Oxford Nanopore Technologies, RNA modifications can be detected and profiled in a simple and straightforward manner. Majority nanopore-based modification studies were devoted to those popular types such as m6A and pseudouridine. To address current limitations on studying the crucial regulator, m1A modification, we conceived this study. We have developed an integrated computational workflow designed for the detection of m1A modifications from direct RNA sequencing data. This workflow comprises a feature extractor responsible for capturing signal characteristics (such as mean, standard deviations, and length of electric signals), a single molecule-level m1A predictor trained with features extracted from the IVT dataset using classical machine learning algorithms, a confident m1A site selector employing the binomial test to identify statistically significant m1A sites, and an m1A modification rate estimator. Our model achieved accurate molecule-level prediction (Average AUC = 0.9689) and reliable m1A site detection and quantification. To show the feasibility of our workflow, we conducted a study on in vivo transcribed human HEK293 cell line, and the results were carefully annotated and compared with other techniques (i.e., Illumina sequencing-based techniques). We believed that this tool will enabling a comprehensive understanding of the m1A modification and its functional mechanisms within cells and organisms.

Nm-Nano: a machine learning framework for transcriptome-wide single-molecule mapping of 2´-O-methylation (Nm) sites in nanopore direct RNA sequencing datasets

ABSTRACT 2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.

NanoMUD: Profiling of pseudouridine and N1-methylpseudouridine using Oxford Nanopore direct RNA sequencing

Nanopore direct RNA sequencing provided a promising solution for unraveling the landscapes of modifications on single RNA molecules. Here, we proposed NanoMUD, a computational framework for predicting the RNA pseudouridine modification (Ψ) and its methylated analog N1-methylpseudouridine (m1Ψ), which have critical application in mRNA vaccination, at single-base and single-molecule resolution from direct RNA sequencing data. Electric signal features were fed into a bidirectional LSTM neural network to achieve improved accuracy and predictive capabilities. Motif-specific models (NNUNN, N = A, C, U or G) were trained based on features extracted from designed dataset and achieved superior performance on molecule-level modification prediction (Ψ models: min AUC = 0.86, max AUC = 0.99; m1Ψ models: min AUC = 0.87, max AUC = 0.99). We then aggregated read-level predictions for site stoichiometry estimation. Given the observed sequence-dependent bias in model performance, we trained regression models based on the distribution of modification probabilities for sites with known stoichiometry. The distribution-based site stoichiometry estimation method allows unbiased comparison between different contexts. To demonstrate the feasibility of our work, three case studies on both in vitro and in vivo transcribed RNAs were presented. NanoMUD will make a powerful tool to facilitate the research on modified therapeutic IVT RNAs and provides useful insight to the landscape and stoichiometry of pseudouridine and N1-pseudouridine on in vivo transcribed RNA species.

Epitranscriptome insights into Riccia fluitans L. (Marchantiophyta) aquatic transition using nanopore direct RNA sequencing

BackgroundRiccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition.ResultsA significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant’s resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions.ConclusionsThese insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant’s dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

Transfer learning enables identification of multiple types of RNA modifications using nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

PCR-based genome walking methods (review)

The review discusses a range of classical and modern methods used to determine the nucleotide sequence of unknown DNA regions flanking known ones. These methods are applied to decipher the regulatory regions of genes, identify integration sites of T-DNA or viruses, and so on, in cases where the use of whole-genome sequencing is not justified. To amplify a DNA segment, a binding site for a primer must be added to the end of the unknown sequence. This can be achieved either by ligating an adapter or by annealing a degenerate primer under gentle conditions, or by looping the DNA fragment so that the target region is surrounded by known sequences. The second important task is to eliminate the inevitable products of nonspecific binding of adapters or degenerate primers, which is often resolved through multiple rounds of nested PCR. Different methods vary significantly in terms of complexity, prevalence, and the availability of required reagents.

Preimplantation genetic testing for a Chinese pedigree affected with Rett syndrome

To carry out preimplantation genetic testing (PGT) for a Chinese pedigree affected with Rett syndrome (RTT). A pedigree affected with RTT who had presented at the First Hospital of Jilin University on June 4, 2021 was selected as the study subject. Variant of the MECP2 gene was analyzed by next generation sequencing (NGS) and Sanger sequencing. Direct sequencing was also used to determine the carrier status for the c.925C>T variant of the MECP2 gene in the blastocysts, and Sanger sequencing was used to validate the results. The MECP2 gene and 168 effective single nucleotide polymorphism (SNP) loci within 2 Mb ranges up- and downstream of the gene were used to construct a haplotype for analyzing the variant site in the embryos, and embryos without the variant were subjected to the analysis for chromosomal aneuploidies. PGT analysis revealed that five out of seven blastocysts did not harbor the pathogenic variant. The results of aneuploidy analysis indicated that two out of five blastocysts without the variant were euploid. Following genetic counselling, the couple had opted to transplant the optimal blastocyst. Following clinical pregnancy, prenatal diagnosis showed that the fetus has a normal chromosomal karyotype, and the c.925C>T variant was not detected in the amniotic fluid sample. A healthy girl was born by Cesarean section at full term. NGS can attain efficient PGT detection and reduce the risk of disease recurrence in families affected with RTT.

On abelian closures of infinite non-binary words

Two finite words u and v are called abelian equivalent if each letter occurs equally many times in both u and v. The abelian closure A(x) of an infinite word x is the set of infinite words y such that, for each factor u of y, there exists a factor v of x which is abelian equivalent to u. The notion of an abelian closure gives a characterization of Sturmian words: among uniformly recurrent binary words, periodic and aperiodic Sturmian words are exactly those words for which A(x) equals the shift orbit closure Ω(x). In this paper, we investigate how this property extends to non-binary words. We consider the abelian closures of most natural generalizations of Sturmian words to non-binary alphabets, such as balanced words and minimal complexity words. We characterize the abelian closures of words in these families and show that in both families, there exist both words which satisfy the property A(x)=Ω(x) and which do not. We observe that for Arnoux-Rauzy words, we always have a strict inclusion Ω(x)⊂A(x). We also consider abelian closures of general subshifts and make some initial observations of their abelian closures and pose some related open questions.