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Abstract Environmental DNA (eDNA) monitoring of species distribution has become a critical tool in ecology, conservation biology and fisheries for identifying the presence and distribution of diverse organisms, including important, threatened and invasive species. However, eDNA detection still has room for improvement in sensitivity, requiring time‐consuming real‐time polymerase chain reaction (qPCR) steps and costly machines. In this study, we report a CRISPR‐Cas13‐based method for rapid, easy, sensitive, low‐cost and on‐site detection of eDNA. The assay for the detection of common carp (Cyprinus carpio) and medaka (Oryzias latipes) nucleic acid employs a two‐step process, starting with recombinase polymerase amplification (RPA) and followed by cleavage using Cas13 nuclease, to effectively identify mitochondrial DNA or RNA. Our results showed that the Cas13‐based method has a higher sensitivity than the qPCR‐based method in detecting tiny amounts of eDNA. When combined with reverse transcription of environmental RNA (eRNA), our method increased detection sensitivity by approximately one order of magnitude. Cas13‐based detection could achieve on‐site detection of eDNA using a quick nucleic acid extraction solution and lateral flow strips. Cas13‐based detection of eDNA and eRNA requires minimal training efforts and can be performed in 1 h, without the need for centrifugation and qPCR machines. The portability of the Cas13‐based method, along with its accuracy and relative ease in designing primers and CRISPR RNA (crRNA), underscores its potential to broaden the application of eDNA and eRNA in various fields, including biodiversity conservation.

Abstract Accurate inference of orthologous genes constitutes a prerequisite for comparative and evolutionary genomics. SonicParanoid is one of the fastest tools for orthology inference; however, its scalability and accuracy have been hampered by time-consuming all-versus-all alignments and the existence of proteins with complex domain architectures. Here, we present a substantial update of SonicParanoid, where a gradient boosting predictor halves the execution time and a language model doubles the recall. Application to empirical large-scale and standardized benchmark datasets shows that SonicParanoid2 is much faster than comparable methods and also the most accurate. SonicParanoid2 is available at https://gitlab.com/salvo981/sonicparanoid2 and https://zenodo.org/doi/10.5281/zenodo.11371108 .

Abstract Convergence provides clues to unveil the non-random nature of evolution. Intermediate paths toward convergence inform us of the stochasticity and the constraint of evolutionary processes. Although previous studies have suggested that substantial constraints exist in microevolutionary paths, it remains unclear whether macroevolutionary convergence follows stochastic or constrained paths. Here, we performed comparative genomics for hundreds of lactic acid bacteria (LAB) species, including clades showing a convergent gene repertoire and sharing fructose-rich habitats. By adopting phylogenetic comparative methods we showed that the genomic convergence of distinct fructophilic LAB (FLAB) lineages was caused by parallel losses of more than a hundred orthologs and the gene losses followed significantly similar orders. Our results further suggested that the loss of adhE, a key gene for phenotypic convergence to FLAB, follows a specific evolutionary path of domain architecture decay and amino acid substitutions in multiple LAB lineages sharing fructose-rich habitats. These findings unveiled the constrained evolutionary paths toward the convergence of free-living bacterial clades at the genomic and molecular levels.

ABSTRACT Marine biofilms were newly revealed as a giant microbial diversity pool for global oceans. However, the cyanobacterial diversity in marine biofilms within the upper seawater column and its ecological and evolutionary implications remains undetermined. Here, we reconstructed a full picture of modern marine cyanobacteria habitats by re-analyzing 9.3 terabyte metagenomic data sets and 2,648 metagenome-assembled genomes (MAGs). The abundances of cyanobacteria lineages exclusively detected in marine biofilms were up to ninefold higher than those in seawater at similar sample size. Analyses revealed that cyanobacteria in marine biofilms are specialists with strong geographical and environmental constraints on their genome and functional adaption, which is in stark contrast to the generalistic features of seawater-derived cyanobacteria. Molecular dating suggests that the important diversifications in biofilm-forming cyanobacteria appear to coincide with the Great Oxidation Event (GOE), “boring billion” middle Proterozoic, and the Neoproterozoic Oxidation Event (NOE). These new insights suggest that marine biofilms are large and important cyanobacterial factories for the global oceans.IMPORTANCECyanobacteria, highly diverse microbial organisms, play a crucial role in Earth’s oxygenation and biogeochemical cycling. However, their connection to these processes remains unclear, partly due to incomplete surveys of oceanic niches. Our study uncovered significant cyanobacterial diversity in marine biofilms, showing distinct niche differentiation compared to seawater counterparts. These patterns reflect three key stages of marine cyanobacterial diversification, coinciding with major geological events in the Earth's history.

Abstract The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

Abstract This study integrated bacterial community and soil chemicals to characterize the soil ecosystem in an open upland field managed by six controlled fertilizer programs using the minimum amount of pesticides. Amplicon sequencing the 16S rRNA gene revealed that inorganic nitrogen fertilizer and compost altered the diversity and structure of the soil bacterial community throughout buckwheat (Fagopyrum esculentum Moench ‘Hitachiakisoba’) cultivation. The bacterial community comprised three clusters that contained bacteria that are prevalent in soils fertilized with nitrogen (cluster 1, 340 taxa), without nitrogen and compost (cluster 2, 234 taxa), and with compost-fertilized (cluster 3, 296 taxa). Cluster 2 contained more taxa in Actinobacteriota and less in Acidobacteriota, and cluster 3 contained more taxa in Gemmatimonadota compared with the other clusters. The most frequent taxa in cluster 1 were within the Chloroflexi phylum. The bacterial community structure correlated with soil chemical properties including pH, total organic carbon, SO4 2−, soluble Ca2+. A co-occurrence network of bacterial taxa and chemicals identified key bacterial groups comprising the center of a community network that determined topology and dynamics of the network. Temporal dynamics of the bacterial community structure indicated that Burkholderiales were associated with buckwheat ripening, indicating plant-bacteria interaction in the ecosystem.

Abstract The plant microbiome is crucial for plant growth, yet many important questions remain, such as the identification of specific bacterial species in plants, their genetic content, and location of these genes on chromosomes or plasmids. To gain insights into the genetic makeup of the rice-phyllosphere, we perform a metagenomic analysis using long-read sequences. Here, 1.8 Gb reads are assembled into 26,067 contigs including 142 circular sequences. Within these contigs, 669 complete 16S rRNA genes are clustered into 166 bacterial species, 121 of which show low identity (

Abstract It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

Abstract Background Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. Results The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. Conclusions Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

Abstract Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. Here we describe a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid pathways via a fungal secondary metabolism gene cluster related to the biosynthesis of sesquiterpene metabolites, including botrydial. Cluster activation during root infection suppresses host nutrient uptake-related genes and changes mineral contents, suggesting a role in manipulating host nutrition state. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic–mutualistic continuum in fluctuating environments.

Abstract Chelidonichthys spinosus, a secondary economic fish, is increasingly being exploited and valued in China. However, overfishing has led to it being recognized as one of the most depleted marine species in China. In this study, we generated a chromosome-level genome of C. spinosus using PacBio, Illumina, and Hi-C sequencing data. Ultimately, we assembled a 624.7 Mb genome of C. spinosus, with a contig N50 of 13.77 Mb and scaffold N50 of 28.11 Mb. We further anchored and oriented the assembled sequences onto 24 pseudo-chromosomes using Hi-C techniques. In total, 25,358 protein-coding genes were predicted, of which 24,072 (94.93%) genes were functionally annotated. The dot plot reveals a prominent co-linearity between C. spinosus and Cyclopterus lumpus, indicating a remarkably close phylogenetic relationship between these two species. The assembled genome sequences provide valuable information for elucidating the genetic adaptation and potential molecular basis of C. spinosus. They also have the potential to provide insight into the evolutionary investigation of teleost fish and vertebrates.

Electrochemical measurements, which exhibit high accuracy and sensitivity under low contamination, controlled electrolyte concentration, and pH conditions, have been used in determining various compounds. The electrochemical quantification capability decreases with an increase in the complexity of the measurement object. Therefore, solvent pretreatment and electrolyte addition are crucial in performing electrochemical measurements of specific compounds directly from beverages owing to the poor measurement quality caused by unspecified noise signals from foreign substances and unstable electrolyte concentrations. To prevent such signal disturbances from affecting quantitative analysis, spectral data of voltage-current values from electrochemical measurements must be used for principal component analysis (PCA). Moreover, this method enables highly accurate quantification even though numerical data alone are challenging to analyze. This study utilized boron-doped diamond (BDD) single-chip electrochemical detection to quantify caffeine content in commercial beverages without dilution. By applying PCA, we integrated electrochemical signals with known caffeine contents and subsequently utilized principal component regression to predict the caffeine content in unknown beverages. Consequently, we addressed existing research problems, such as the high quantification cost and the long measurement time required to obtain results after quantification. The average prediction accuracy was 93.8% compared to the actual content values. Electrochemical measurements are helpful in medical care and indirectly support our lives.

Soil plays a crucial role in human health through its impact on food and habitation. However, it often contains toxic heavy metals, with mercury being particularly hazardous when methylated. Currently, high-sensitivity, rapid detection of mercury is achievable only through electrochemical measurements. These measurements require pretreatment of the soil sample and the preparation of a calibration curve tailored to the sample’s condition. In this study, we developed a method to determine the environmental standard value of mercury content in soil by significantly reducing the pretreatment process. Our approach involves analyzing current peaks from electrodeposition times using specific electrodes and solvent settings. This method demonstrates low error rates under low concentration conditions and can detect mercury levels as low as 0.5 ppb in soil leachate and reagent dilution series. This research facilitates the determination of low mercury concentrations in solutions containing various soil micro-compounds without the need for calibration curves.

Abstract Metagenome-assembled genomes (MAGs) have revealed the hidden diversity and functions of uncultivated microbes, but their reconstruction from metagenomes remains a computationally difficult task. Repetitive or exogenous sequences, such as ribosomal RNA and horizontally transferred genes, are frequently absent from MAGs because of misassembly and binning errors. Here, we report that ribosomal protein genes are also often absent from MAGs, although they are neither repetitive nor exogenous. Comprehensive analyses of more than 190,000 MAGs revealed that these genes could be missing in more than 20–40% of near-complete (i.e., with completeness of 90% or higher) MAGs. While some uncultivated environmental microbes intrinsically lack some ribosomal protein genes, we found that this unexpected absence is largely due to special evolutionary patterns of codon usage bias in ribosomal protein genes and algorithmic characteristics of metagenomic binning, which is dependent on tetranucleotide frequencies of contigs. This problem reflects the microbial life-history strategy. Fast-growing microbes tend to have this difficulty, likely because of strong evolutionary pressures on ribosomal protein genes toward the efficient assembly of ribosomes. Our observations caution those who study genomics and phylogeny of uncultivated microbes, the diversity and evolution of microbial genes in the central dogma, and bioinformatics in metagenomics.

Abstract The use of metal deposition has been limited to a limited number of applicable samples due to the increased temperature caused by accelerated electron impact on the substrate surface. The surfaces of various biological samples have a nanoscale structure with specific properties, which have been simulated in numerous studies. However, no examples of nano/microscale reproductions of biological surface features have used moulds. In this study, a mould that imitates the surface shape of a cellular-level biological material was fabricated, for the first time, and the shape was successfully reproduced using the mould. Al thin films were deposited on bovine sperm using magnetron sputtering without thermal denaturation with a cathode operating at a biological temperature. It is difficult to deposit films used as metal coatings on pre-treated biological materials at temperatures below 40 °C during evaporation. The Al thin film was peeled off and used as a mould to reproduce the shape of the sperm with high accuracy using a polymer. The results of this study represent a major innovation in reproducible biomimetic moulding technology, demonstrating biological temperature sputtering. We expect our non-destructive metal deposition and metal nano-moulding methods for biological samples to be the basis for the effective utilization of various biological structures.

The authors report the landscape of circulating platelet aggregates in COVID-19 obtained by massive single-cell image-based profiling and temporal monitoring to elucidate the underlying process of COVID-19-assocaited microvascular thrombosis.

Summary: Raman microspectroscopy is a powerful tool for obtaining biomolecular information from single microbial cells in a nondestructive manner. Here, we detail steps to discriminate prokaryotic species using single-cell Raman spectra acquisitions followed by data preprocessing and random forest model tuning. In addition, we describe the steps required to evaluate the model. This protocol requires minimal preprocessing of Raman spectral data, making it accessible to non-spectroscopists, yet allows intuitive visualization of feature importance.For complete details on the use and execution of this protocol, please refer to Kanno et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Optical sensing offers several advantages owing to its non-invasiveness and high sensitivity. The miniaturization of optical sensors will mitigate spatial and weight constraints, expanding their applications and extending the principal advantages of optical sensing to different fields, such as healthcare, Internet of Things, artificial intelligence, and other aspects of society. In this study, we present the development of a miniature optical sensor for monitoring thrombi in extracorporeal membrane oxygenation (ECMO). The sensor, based on a complementary metal-oxide semiconductor integrated circuit (CMOS-IC), also serves as a photodiode, amplifier, and light-emitting diode (LED)-mounting substrate. It is sized 3.8 × 4.8 × 0.75 mm3 and provides reflectance spectroscopy at three wavelengths. Based on semiconductor and microelectromechanical system (MEMS) processes, the design of the sensor achieves ultra-compact millimeter size, customizability, prototyping, and scalability for mass production, facilitating the development of miniature optical sensors for a variety of applications.

Ferns, with about 12,000 species, are the second most diverse lineage of vascular plants after angiosperms. They have been the subject of numerous molecular phylogenetic studies, resulting in the publication of trees for every major clade and DNA sequences from nearly half of all species. Global fern phylogenies have been published periodically, but as molecular systematics research continues at a rapid pace, these become quickly outdated. Here, we develop a mostly automated, reproducible, open pipeline to generate a continuously updated fern tree of life (FTOL) from DNA sequence data available in GenBank. Our tailored sampling strategy combines whole plastomes (few taxa, many loci) with commonly sequenced plastid regions (many taxa, few loci) to obtain a global, species-level fern phylogeny with high resolution along the backbone and maximal sampling across the tips. We use a curated reference taxonomy to resolve synonyms in general compliance with the community-driven Pteridophyte Phylogeny Group I classification. The current FTOL includes 5,582 species, an increase of ca. 40% relative to the most recently published global fern phylogeny. Using an updated and expanded list of 51 fern fossil constraints, we find estimated ages for most families and deeper clades to be considerably older than earlier studies. FTOL and its accompanying datasets, including the fossil list and taxonomic database, will be updated on a regular basis and are available via a web portal (https://fernphy.github.io) and R packages, enabling immediate access to the most up-to-date, comprehensively sampled fern phylogeny. FTOL will be useful for anyone studying this important group of plants over a wide range of taxonomic scales, from smaller clades to the entire tree. We anticipate FTOL will be particularly relevant for macroecological studies at regional to global scales and will inform future taxonomic systems with the most recent hypothesis of fern phylogeny.

Abstract Environmental DNA (eDNA)‐based assessments of macro‐organisms have now become an essential approach for biomonitoring. eDNA survey methods have a number of advantages over conventional survey methods. However, the value of the data that will accumulate would be greatly enhanced by standardizing the analysis methods, which would allow us to compare data from multiple monitoring sites at different points in time. The eDNA Society (http://ednasociety.org/en/about), whose founding members consist of Japanese researchers conducting eDNA studies on macro‐organisms, was established in 2018, with the aim of expanding eDNA technology and science. Here, we introduce our key publication, “Environmental DNA Sampling and Experiment Manual” (http://ednasociety.org/en/manual), which was published under the initiative of the eDNA Society. Detailed methods for the surveys and experiments are described in the manual, including the selection of sampling sites, sampling methods, filtration methods, DNA extraction, species‐specific detection by real‐time polymerase chain reaction, and fish eDNA metabarcoding. The manual assists users in conducting standardized surveys and quality experiments, and provides a basis for collecting comparable data. Given that the efficacy of methods can be context dependent and variable, and that procedures may sometimes conflict with standardization, it is difficult to ensure that all processes are equally effective. However, even in such cases, it is important to maintain sufficiently high data quality by setting the minimum standards to be followed. Implementation of such standardized methodologies will enable the systematic and frequent collection of flawless, comparable eDNA data from around the world; this will provide important fundamental information for biodiversity conservation, as well as the sustainable use of fisheries resources.

Summary: Accessing enormous uncultivated microorganisms (microbial dark matter) in various Earth environments requires accurate, nondestructive classification, and molecular understanding of the microorganisms in in situ and at the single-cell level. Here we demonstrate a combined approach of random forest (RF) machine learning and single-cell Raman microspectroscopy for accurate classification of phylogenetically diverse prokaryotes (three bacterial and three archaeal species from different phyla). Our RF classifier achieved a 98.8 ± 1.9% classification accuracy among the six species in pure populations and 98.4% for three species in an artificially mixed population. Feature importance scores against each wavenumber reveal that the presence of carotenoids and structure of membrane lipids play key roles in distinguishing the prokaryotic species. We also find unique Raman markers for an ammonia-oxidizing archaeon. Our approach with moderate data pretreatment and intuitive visualization of feature importance is easy to use for non-spectroscopists, and thus offers microbiologists a new single-cell tool for shedding light on microbial dark matter.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene. [ABSTRACT FROM AUTHOR]

In this study, the microreactor system was investigated and compared with the batch-wise system as rapid and effective extractive Pb(II) separation over Fe(III), Cu(II) and Zn(II) with tetraacetic acid calix[4]arene. By using a microreactor system, the Pb(II) extraction percentages reached the maximum of 73, 89 and 100% in 8 sec residence time at equilibrium pH of 2.00, 2.25 and 2.50, respectively. The stripping percentage was 92% at 8 sec residence time by using a microreactor system with 2.0 M HNO3 as a stripping reagent. Complete separation of Pb(II) over Fe(III), Cu(II) and Zn(II) ions with the tetraacetic acid calix[4]arene in a competitive metal system was achieved at pH 2.00. However, the batch system required 24 h to reach the equilibrium for both extraction and stripping processes. The results suggested that the microreactor system enhanced the Pb(II) extraction and stripping rate up to 104 times faster than the batch-wise system.

Our knowledge of DNA methylation systems in prokaryotes is mostly limited to those of culturable microbes. Here, Hiraoka et al. analyse DNA methylation patterns in metagenomic data from a microbial community, revealing new methylated motifs and experimentally validating the methyltransferases’ specificities.

Abstract Short peptides with sequences of alternating l‐ and d‐residues are known to form antiparallel double β‐helical structures, but their equilibrium structures have not been characterized in detail. Here, we use metal coordination of a simple octapeptide, ‐(l‐Val‐d‐Val)4‐, modified with two coordinating side chains at the (i, j)‐th residues to uncover these elusive structures. When (i, j) = (3, 5), complexation with ZnI2 induces a parallel double β‐helix, which is not commonly seen. In contrast, when (i, j) = (5, 7), a commonly occurring antiparallel double β‐helix (Type I) is formed. Interestingly, complexation of the peptide with (i, j) = (3, 7) gives another antiparallel double β‐helix, the unknown Type II structure, which has an inverted orientation of the two strands. Complexation of a monotopic peptide (i = 3) with trans‐PdCl2 yields a Pd(II)‐linked dimeric bundle of two antiparallel β‐helices. These results demonstrate that metal coordination can induce even as‐yet unrecognized structures in the folding and assembly pathways of short peptides. Key points Structural elucidation of elusive peptide nanostructures Precise structural control of double helical molecules Fusion of peptide folding and metal‐directed self‐assembly

[This corrects the article DOI: 10.1371/journal.pone.0213535.].

Owing to its simplicity and sensitivity, electrochemical analysis is of high significance in the detection of pollutants and highly toxic substances in the environment. In electrochemical analysis, the sensitivity of the sensor and reliability of the obtained signal are especially dependent on the electrode characteristics. Electrodes with a high density of nanomaterials, which exhibit excellent activity, are useful as sensor substrates for pollutant detection. However, the effective placement of high-density nanomaterials requires a high degree of control over the particle size, particle shape, and distance between the particles on the substrate. In this study, we exploited the properties of boron-doped diamond (BDD) electrodes, which have a wide potential window, and succeeded in coating a highly dense layer of gold nanoparticles (AuNPs) at high potential. The AuNP-modified BDD (AuNP-BDD) electrodes comprising less than 100 nm AuNPs at a density of 125 particles/µm were electrochemically synthesized over a short period of 30–60 s. The AuNP-BDD electrodes were applied for detecting arsenic, which is one of the most abundant elements, and exhibited a limit of detection of 0.473 ppb in solution.

Fluid control on a paper channel is necessary for analysis with multiple reagents, such as enzyme-linked immunosorbent assay (ELISA) in microfluidic paper-based analytical devices (µPADs). In this study, a thermo-responsive valve was fabricated by polymerizing N-isopropylacrylamide on a PVDF porous membrane by plasma-induced graft polymerization. The polymerized membrane was observed by scanning electron microscopy (SEM), and it was confirmed that more pores were closed at temperatures below 32 °C and more pores were opened at temperatures above 32 °C. Valve permeability tests confirmed that the proposed polymerized membrane was impermeable to water and proteins at temperatures below 32 °C and permeable to water at temperatures above 32 °C. The valve could also be reversibly and repeatedly opened and closed by changing the temperature near 32 °C. These results suggest that plasma-induced graft polymerization may be used to produce thermo-responsive valves that can be opened and closed without subsequent loss of performance. These results indicate that the thermo-responsive valve fabricated by plasma-induced graft polymerization could potentially be applied to ELISA with µPADs.

ABSTRACT Shinkaia crosnieri is an invertebrate that inhabits an area around deep-sea hydrothermal vents in the Okinawa Trough in Japan by harboring episymbiotic microbes as the primary nutrition. To reveal physiology and phylogenetic composition of the active episymbiotic populations, metatranscriptomics is expected to be a powerful approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. Here, we conducted direct metatranscriptomic analysis of S. crosnieri episymbionts by applying in situ RNA stabilization equipment. As expected, we obtained RNA expression profiles that were substantially different from those obtained by conventional metatranscriptomics (i.e., stabilization after retrieval). The episymbiotic community members were dominated by three orders, namely, Thiotrichales, Methylococcales, and Campylobacterales, and the Campylobacterales members were mostly dominated by the Sulfurovum genus. At a finer phylogenetic scale, the episymbiotic communities on different host individuals shared many species, indicating that the episymbionts on each host individual are not descendants of a few founder cells but are horizontally exchanged. Furthermore, our analysis revealed the key metabolisms of the community: two carbon fixation pathways, a formaldehyde assimilation pathway, and utilization of five electron donors (sulfide, thiosulfate, sulfur, methane, and ammonia) and two electron accepters (oxygen and nitrate/nitrite). Importantly, it was suggested that Thiotrichales episymbionts can utilize intercellular sulfur globules even when sulfur compounds are not usable, possibly also in a detached and free-living state. IMPORTANCE Deep-sea hydrothermal vent ecosystems remain mysterious. To depict in detail the enigmatic life of chemosynthetic microbes, which are key primary producers in these ecosystems, metatranscriptomic analysis is expected to be a promising approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. In this study, we conducted direct metatranscriptome analysis of microbial episymbionts of deep-sea squat lobsters (Shinkaia crosnieri) by applying in situ RNA stabilization equipment. Compared to conventional metatranscriptomics (i.e., RNA stabilization after retrieval), our method provided substantially different RNA expression profiles. Moreover, we discovered that S. crosnieri and its episymbiotic microbes constitute complex and resilient ecosystems, where closely related but various episymbionts are stably maintained by horizontal exchange and partly by their sulfur storage ability for survival even when sulfur compounds are not usable, likely also in a detached and free-living state.

Summary: The recent prevalence of high-throughput sequencing has been producing numerous prokaryotic community structure datasets. Although the trait-based approach is useful to interpret those datasets from ecological perspectives, available trait information is biased toward culturable prokaryotes, especially those of clinical and public health relevance, and thus may not represent the breadth of microbiota found across many of Earth's environments. To facilitate habitat-based analysis free of such bias, here we report a ready-to-use prokaryotic habitat database, ProkAtlas. ProkAtlas comprehensively links 16S rRNA gene sequences to prokaryotic habitats, using public shotgun metagenome datasets. We also developed a computational pipeline for habitat-based analysis of given prokaryotic community structures. After confirmation of the method effectiveness using 16S rRNA gene sequence datasets from individual genomes and the Earth Microbiome Project, we showed its validness and effectiveness in drawing ecological insights by applying it to six empirical prokaryotic community datasets from soil, aquatic, and human gut samples.

Logic relationship analysis is a data mining method that comprehensively detects item triplets that satisfy logic relationships from a binary matrix dataset, such as an ortholog table in comparative genomics. Thanks to recent technological advancements, many binary matrix datasets are now being produced in genomics, transcriptomics, epigenomics, metagenomics, and many other fields for comparative purposes. However, regardless of presumed interpretability and importance of logic relationships, existing data mining methods are not based on the framework of statistical hypothesis testing. That means, the type-1 and type-2 error rates are neither controlled nor estimated. Here, we developed Logicome Profiler, which exhaustively detects statistically significant triplet logic relationships from a binary matrix dataset (Logicome means ome of logics). To test all item triplets in a dataset while avoiding false positives, Logicome Profiler adjusts a significance level by the Bonferroni or Benjamini-Yekutieli method for the multiple testing correction. Its application to an ocean metagenomic dataset showed that Logicome Profiler can effectively detect statistically significant triplet logic relationships among environmental microbes and genes, which include those among urea transporter, urease, and photosynthesis-related genes. Beyond omics data analysis, Logicome Profiler is applicable to various binary matrix datasets in general for finding significant triplet logic relationships. The source code is available at https://github.com/fukunagatsu/LogicomeProfiler.

Abstract Background Teleosts transiting from freshwater (FW) to seawater (SW) environments face an immediate osmotic stress from ion influxes and water loss, but some euryhaline species such as eels can maintain a stable plasma osmolality during early SW exposure. The time course changes in the gene expression, protein abundance, and localization of key ion transporters suggested that the reversal of the ion transport systems was gradual, and we investigate how eels utilize a Na-binding strategy to slow down the ion invasion and complement the transporter-mediated osmoregulation. Results Using an electron probe micro-analyzer, we localized bound Na in various eel tissues in response to SW transfer, suggesting that the Na-binding molecules were produced to sequester excess ionic Na+ to negate its osmotic potential, thus preventing acute cellular dehydration. Mucus cells were acutely activated in digestive tract, gill, and skin after SW transfer, producing Na-binding molecule-containing mucus layers that fence off high osmolality of SW. Using gel filtration HPLC, some molecules at 18 kDa were found to bind Na in the luminal secretion of esophagus and intestine, and higher binding was associated with SW transfer. Transcriptome and protein interaction results indicated that downregulation of Notch and β-catenin pathways, and dynamic changes in TGFβ pathways in intestine were involved during early SW transition, supporting the observed histological changes on epithelial desquamation and increased mucus production. Conclusions The timing for the activation of the Na-binding mechanism to alleviate the adverse osmotic gradient was temporally complementary to the subsequent remodeling of branchial ionocytes and transporting epithelia of the digestive tract. The strategy to manipulate the osmotic potential of Na+ by specific binding molecules is similar to the osmotically inactive Na described in human skin and muscle. The Na-binding molecules provide a buffer to tolerate the salinity changes, which is advantageous to the estuary and migrating fishes. Our data pave the way to identify this unknown class of molecules and open a new area of vertebrate osmoregulation research.

Microbes adapting to broad and specialized ranges of environments (generalists and specialists) have distinct ecological roles and properties. Via meta-analysis of community sequencing datasets, Sriswasdi et al. show that generalists have higher speciation rates and persistence advantage over specialists.

The fisheries resources of the Yangtze Estuary and its adjacent waters have undergone dramatic declines as a consequence of environmental changes and human activities, with traditional ecological investigations demonstrating progressive decreases in species diversity and annual numbers in the fisheries resource. Environmental DNA (eDNA) technology has been demonstrated as an effective tool by many studies for detecting fish species, monitoring fish biodiversity, and indicating the abundance of fish. In the present study, we chose the Yangtze Estuary and its adjacent waters as a primary research area to investigate fish assemblage structure using eDNA technology. A total of 50 eDNA samples were collected in the estuary in 2018. The results showed that 41 operational taxonomic units were identified from three seasons, with 18, 12, and 33 fish species associated specifically with spring, summer and autumn, respectively. The fish assemblage differed significantly among seasons. Canonical correspondence analysis showed that water temperature, salinity, and dissolved oxygen were the main environmental factors affecting structure of the seasonal assemblages. Results of the present study indicate that eDNA technology can be an effective tool not only for fisheries monitoring, but might also importantly assist marine resources conservation, sustainable exploitation of fisheries, the aquatic products processing industry, eco-friendly development, and socioeconomic stability.

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 μM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.

Lithium extraction remains a challenge in the hydrometallurgy process due to its economic value and maldistribution sources. Employing calix[4]arene derivatives in solvent extraction techniques results in high selectivity and extraction capability, but a slow extraction rate. The slow kinetics of batch-wise extraction can be drastically accelerated by using a T-type microreactor system. Therefore, a combination of calix[4]arene and a microreactor system serves as an ideal platform for efficient lithium extraction. In this work, the fluid dynamics of lithium extraction using a monoacetic acid calix[4]arene derivative in a T-type microreactor system were studied. Increasing the O/A ratio increases the average length, surface area, and volume of the organic droplets, but decreases the specific surface area. In contrast, increasing the Reynolds number decreases the average length, surface area, and volume of the organic droplets, but increases the specific surface area. It was found that shorter diffusion distance, larger specific surface area, and faster vortex velocity were the factors that play the most pivotal roles in achieving great extraction rate enhancement in T-type microreactor systems compared to batch-wise systems. These findings represent an important new concept in the study of the fluid dynamics of lithium extraction using monoacetic acid calix[4]arene derivatives in T-type microreactor systems.