The DNA base modification N6-methyladenine (m
A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method ...to detect base modifications. Here, we show that a neural network can improve m
A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.
Highlights ► Adaptive laboratory evolution (ALE) as a tool for metabolic engineering. ► Latent pathway activation by ALE. ► ALE has successfully been used to optimize the phenotype of a ...microorganism. ► Adaptation to changing environments by ALE.
The antimicrobial resistance (AMR) phenotypic properties, multiple drug resistance (MDR) gene profiles, and genes related to potential virulence and pathogenic properties of five Enterobacter ...bugandensis strains isolated from the International Space Station (ISS) were carried out and compared with genomes of three clinical strains. Whole genome sequences of ISS strains were characterized using the hybrid de novo assembly of Nanopore and Illumina reads. In addition to traditional microbial taxonomic approaches, multilocus sequence typing (MLST) analysis was performed to classify the phylogenetic lineage. Agar diffusion discs assay was performed to test antibiotics susceptibility. The draft genomes after assembly and scaffolding were annotated with the Rapid Annotations using Subsystems Technology and RNAmmer servers for downstream analysis.
Molecular phylogeny and whole genome analysis of the ISS strains with all publicly available Enterobacter genomes revealed that ISS strains were E. bugandensis and similar to the type strain EB-247
and two clinical isolates (153_ECLO and MBRL 1077). Comparative genomic analyses of all eight E. bungandensis strains showed, a total of 4733 genes were associated with carbohydrate metabolism (635 genes), amino acid and derivatives (496 genes), protein metabolism (291 genes), cofactors, vitamins, prosthetic groups, pigments (275 genes), membrane transport (247 genes), and RNA metabolism (239 genes). In addition, 112 genes identified in the ISS strains were involved in virulence, disease, and defense. Genes associated with resistance to antibiotics and toxic compounds, including the MDR tripartite system were also identified in the ISS strains. A multiple antibiotic resistance (MAR) locus or MAR operon encoding MarA, MarB, MarC, and MarR, which regulate more than 60 genes, including upregulation of drug efflux systems that have been reported in Escherichia coli K12, was also observed in the ISS strains.
Given the MDR results for these ISS Enterobacter genomes and increased chance of pathogenicity (PathogenFinder algorithm with > 79% probability), these species pose important health considerations for future missions. Thorough genomic characterization of the strains isolated from ISS can help to understand the pathogenic potential, and inform future missions, but analyzing them in in-vivo systems is required to discern the influence of microgravity on their pathogenicity.
Telomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, and whose lengths can be correlated with age and adverse health ...risk factors. Yet, given their length and repetitive nature, telomeric regions are not easily reconstructed from short-read sequencing, thus making telomere sequencing, mapping, and variant resolution challenging problems. Recently, long-read sequencing, with read lengths measuring in hundreds of kilobase pairs, has made it possible to routinely read into telomeric regions and inspect their sequence structure. Here, we describe a framework for extracting telomeric reads from whole-genome single-molecule sequencing experiments, including de novo identification of telomere repeat motifs and repeat types, and also describe their sequence variation. We find that long, complex telomeric stretches and repeats can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize noncanonical telomere sequence motifs (both previously reported, as well as novel), and validate them in short-read sequence data. These data reveal extensive intra- and inter-population diversity of repeats in telomeric haplotypes, reveal higher paternal inheritance of telomeric variants, and represent the first motif composition maps of multi-kilobase-pair human telomeric haplotypes across three distinct ancestries (Ashkenazi, Chinese, and Utah), which can aid in future studies of genetic variation, aging, and genome biology.
Pseudomonas putida is a Gram-negative, non-fermenting bacterium frequently encountered in various environmental niches. P. putida rarely causes disease in humans, though serious infections and ...outbreaks have been reported from time to time. Some have suggested that P. putida functions as an exchange platform for antibiotic resistance genes (ARG), and thus represents a serious concern in the spread of ARGs to more pathogenic organisms within a hospital. Though poorly understood, the frequency of ARG exchange between P. putida and the more virulent Pseudomonas aeruginosa and its clinical relevance are particularly important for designing efficient infection control strategies, such as deciding whether high-risk patients colonized with a multidrug resistant but typically low pathogenic P. putida strain should be contact isolated or not.
In this study, 21,373 screening samples (stool, rectal and throat swab) were examined to determine the presence of P. putida in a high-risk group of haemato-oncology patients during a 28-month period. A total of 89 P. putida group strains were isolated from 85 patients, with 41 of 89 (46.1%) strains harbouring the metallo-beta-lactamase gene bla
. These 41 clinical isolates, plus 18 bla
positive environmental P. putida isolates, and 17 bla
positive P. aeruginosa isolates, were characterized by whole genome sequencing (WGS). We constructed a maximum-likelihood tree to separate the 59 bla
positive P. putida group strains into eight distinct phylogenetic clusters. Bla
was present in 6 clusters while bla
was detected in 4 clusters. Five P. putida group strains contained both, bla
and bla
genes. In contrast, all P. aeruginosa strains belonged to a single genetic cluster and contained the same ARGs. Apart from bla
and sul genes, no other ARGs were shared between P. aeruginosa and P. putida. Furthermore, the bla
gene in P. aeruginosa was predicted to be only chromosomally located.
These data provide evidence that no exchange of comprehensive ARG harbouring mobile genetic elements had occurred between P. aeruginosa and P. putida group strains during the study period, thus eliminating the need to implement enhanced infection control measures for high-risk patients colonized with a bla
positiv P. putida group strains in our clinical setting.
Telomeres, repetitive terminal features of chromosomes essential for maintaining genome integrity, shorten with cell division, lifestyle factors and stresses, and environmental exposures, and so they ...provide a robust biomarker of health, aging, and age-related diseases. We assessed telomere length dynamics (changes over time) in three unrelated astronauts before, during, and after 1-year or 6-month missions aboard the International Space Station (ISS). Similar to our results for National Aeronautics and Space Administration’s (NASA’s) One-Year Mission twin astronaut (Garrett-Bakelman et al., 2019), significantly longer telomeres were observed during spaceflight for two 6-month mission astronauts. Furthermore, telomere length shortened rapidly after return to Earth for all three crewmembers and, overall, telomere length tended to be shorter after spaceflight than before spaceflight. Consistent with chronic exposure to the space radiation environment, signatures of persistent DNA damage responses were also detected, including mitochondrial and oxidative stress, inflammation, and telomeric and chromosomal aberrations, which together provide potential mechanistic insight into spaceflight-specific telomere elongation.
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•Telomere elongation during spaceflight was observed for two 6-month mission astronauts•Telomere elongation was not dependent on mission duration, sample, or measurement type•Chronic space radiation exposure was associated with persistent DNA damage responses•Telomeres shortened rapidly after return to Earth, and long-term individual differences were observed
Consistent with findings first observed for NASA’s One-Year Mission twin astronaut, Luxton et al. report spaceflight-specific telomere elongation in ISS crewmembers on shorter duration missions. Signatures of persistent DNA damage responses in the space radiation environment are also observed, providing potential mechanistic insight into telomere maintenance pathways during spaceflight.
Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, ...a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.
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•MapToCleave method allows simultaneous screening of 12,472 RNA structures in cells•The biogenesis of ∼15% of human miRNAs is influenced by cell-dependent factors•We perform a systematic comparison of the importance of miRNA biogenesis features•Stability of first seven base pairs of the stem tunes processing in cells and tissues
Numerous miRNA features that facilitate biogenesis are known, but most have been identified in vitro. Kang et al. re-evaluate miRNA biogenesis in living cells and in animal tissues, and they find that stability of the first seven base pairs of the stem is particularly important for processing in cells.
Future multi-year crewed planetary missions will motivate advances in aerospace nutrition and telehealth. On Earth, the Human Cell Atlas project aims to spatially map all cell types in the human ...body. Here, we propose that a parallel Human Cell Space Atlas could serve as an openly available, global resource for space life science research. As humanity becomes increasingly spacefaring, high-resolution omics on orbit could permit an advent of precision spaceflight healthcare. Alongside the scientific potential, we consider the complex ethical, cultural, and legal challenges intrinsic to the human space omics discipline, and how philosophical frameworks may benefit from international perspectives.High-resolution omics data have facilitated the ongoing Human Cell Atlas project. In this Perspective, Rutter and colleagues propose that a parallel Human Cell Space Atlas initiative would provide a platform for spaceflight-associated research and healthcare.
The National Aeronautics and Space Administration (NASA) Twins Study created an integrative molecular profile of an astronaut during NASA’s first 1-year mission on the International Space Station ...(ISS) and included comparisons to an identical Earth-bound twin. The unique biochemical profiles observed when landing on Earth after such a long mission (e.g., spikes in interleukin-1 IL-1/6/10, c-reactive protein CRP, C-C motif chemokine ligand 2 CCL2, IL-1 receptor antagonist IL-1ra, and tumor necrosis factor alpha TNF-α) opened new questions about the human body’s response to gravity and how to plan for future astronauts, particularly around initiation or resolution of inflammation. Here, single-cell, multi-omic (100-plex epitope profile and gene expression) profiling of peripheral blood mononuclear cells (PBMCs) showed changes to blood cell composition and gene expression post-flight, specifically for monocytes and dendritic cell precursors. These were consistent with flight-induced cytokine and immune system stress, followed by skeletal muscle regeneration in response to gravity. Finally, we examined these profiles relative to 6-month missions in 28 other astronauts and detail potential pharmacological interventions for returning to gravity in future missions.
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•IL-6, IL-10, IL-1ra, CCL2, and CRP rose significantly on landing after a year in space•IL-6 and IL-1ra are potential targets for pharmacological intervention in astronauts•These cytokines are also associated with muscle regeneration in response to gravity•Muscle-generated IL-6 drives classic signaling cascades in an anti-inflammatory role
Gertz et al. present a re-analysis of the landing data from the NASA Twins Study, suggesting that the biochemical signature reflects muscle regeneration after atrophy rather than a detrimental inflammatory response. This is mediated through muscle-derived IL-6 anti-inflammatory cascades. Single-cell analysis supports this role. Potential pharmacological interventions are also discussed.
DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the ...nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase.
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•The protein kinase DYRK1A is recruited to proximal promoters of growth-related genes•DYRK1A targets contain a TCTCGCGAGA motif, which drives DYRK1A-dependent activation•DYRK1A phosphorylates the CTD of RNAPII at Ser2 and Ser5•DYRK1A depletion leads to reduced Ser2/Ser5 phosphorylation at its target promoters
Di Vona et al. show that the Down syndrome-associated kinase DYRK1A regulates transcription of a subset of genes by associating to their proximal promoter regions and phosphorylating the CTD of the RNA polymerase II.
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