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FeGenie: a comprehensive tool for the identification of iron genes and iron gene neighborhoods in genomes and metagenome assemblies

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earth’s crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and surge in publicly available sequence data has made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition, iron redox cycling, iron storage, and magnetosome formation in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) based on genes related to iron acquisition, storage, and reduction/oxidation in Bacteria and Archaea. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate provided datasets with respect to iron-related genes and gene neighborhood. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. We validated FeGenie against a selected set of 28 isolate genomes and showcase its utility in exploring iron genes present in 27 metagenomes, 4 isolate genomes from human oral biofilms, and 17 genomes from candidate organisms, including members of the candidate phyla radiation. We show that FeGenie accurately identifies iron genes in isolates. Furthermore, analysis of metagenomes using FeGenie demonstrates that the iron gene repertoire and abundance of each environment is correlated with iron richness. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenie’s database will be maintained and continually updated as new genes are discovered.

59 BASIC BIOLOGICAL SCIENCES↗

A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

16S rDNA↗

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.

59 BASIC BIOLOGICAL SCIENCES↗

Eukaryotic genomes from a global metagenomic data set illuminate trophic modes and biogeography of ocean plankton

ABSTRACT Metagenomics is a powerful method for interpreting the ecological roles and physiological capabilities of mixed microbial communities. Yet, many tools for processing metagenomic data are neither designed to consider eukaryotes nor are they built for an increasing amount of sequence data. EukHeist is an automated pipeline to retrieve eukaryotic and prokaryotic metagenome-assembled genomes (MAGs) from large-scale metagenomic sequence data sets. We developed the EukHeist workflow to specifically process large amounts of both metagenomic and/or metatranscriptomic sequence data in an automated and reproducible fashion. Here, we applied EukHeist to the large-size fraction data (0.8–2,000 µm) from Tara Oceans to recover both eukaryotic and prokaryotic MAGs, which we refer to as TOPAZ (Tara Oceans Particle-Associated MAGs). The TOPAZ MAGs consisted of >900 environmentally relevant eukaryotic MAGs and >4,000 bacterial and archaeal MAGs. The bacterial and archaeal TOPAZ MAGs expand upon the phylogenetic diversity of likely particle- and host-associated taxa. We use these MAGs to demonstrate an approach to infer the putative trophic mode of the recovered eukaryotic MAGs. We also identify ecological cohorts of co-occurring MAGs, which are driven by specific environmental factors and putative host-microbe associations. These data together add to a number of growing resources of environmentally relevant eukaryotic genomic information. Complementary and expanded databases of MAGs, such as those provided through scalable pipelines like EukHeist, stand to advance our understanding of eukaryotic diversity through increased coverage of genomic representatives across the tree of life. IMPORTANCE Single-celled eukaryotes play ecologically significant roles in the marine environment, yet fundamental questions about their biodiversity, ecological function, and interactions remain. Environmental sequencing enables researchers to document naturally occurring protistan communities, without culturing bias, yet metagenomic and metatranscriptomic sequencing approaches cannot separate individual species from communities. To more completely capture the genomic content of mixed protistan populations, we can create bins of sequences that represent the same organism (metagenome-assembled genomes [MAGs]). We developed the EukHeist pipeline, which automates the binning of population-level eukaryotic and prokaryotic genomes from metagenomic reads. We show exciting insight into what protistan communities are present and their trophic roles in the ocean. Scalable computational tools, like EukHeist, may accelerate the identification of meaningful genetic signatures from large data sets and complement researchers’ efforts to leverage MAG databases for addressing ecological questions, resolving evolutionary relationships, and discovering potentially novel biodiversity.

59 BASIC BIOLOGICAL SCIENCES↗

Leptothrix ochracea genomes reveal potential for mixotrophic growth on Fe(II) and organic carbon

ABSTRACT Leptothrix ochracea creates distinctive iron-mineralized mats that carpet streams and wetlands. Easily recognized by its iron-mineralized sheaths, L. ochracea was one of the first microorganisms described in the 1800s. Yet it has never been isolated and does not have a complete genome sequence available, so key questions about its physiology remain unresolved. It is debated whether iron oxidation can be used for energy or growth and if L. ochracea is an autotroph, heterotroph, or mixotroph. To address these issues, we sampled L. ochracea -rich mats from three of its typical environments (a stream, wetlands, and a drainage channel) and reconstructed nine high-quality genomes of L. ochracea from metagenomes. These genomes contain iron oxidase genes cyc2 and mtoA, showing that L. ochracea has the potential to conserve energy from iron oxidation. Sox genes confer potential to oxidize sulfur for energy. There are genes for both carbon fixation (RuBisCO) and utilization of sugars and organic acids (acetate, lactate, and formate). In silico stoichiometric metabolic models further demonstrated the potential for growth using sugars and organic acids. Metatranscriptomes showed a high expression of genes for iron oxidation; aerobic respiration; and utilization of lactate, acetate, and sugars, as well as RuBisCO, supporting mixotrophic growth in the environment. In summary, our results suggest that L. ochracea has substantial metabolic flexibility. It is adapted to iron-rich, organic carbon-containing wetland niches, where it can thrive as a mixotrophic iron oxidizer by utilizing both iron oxidation and organics for energy generation and both inorganic and organic carbon for cell and sheath production. IMPORTANCE Winogradsky's observations of L. ochracea led him to propose autotrophic iron oxidation as a new microbial metabolism, following his work on autotrophic sulfur-oxidizers. While much culture-based research has ensued, isolation proved elusive, so most work on L. ochracea has been based in the environment and in microcosms. Meanwhile, the autotrophic Gallionella became the model for freshwater microbial iron oxidation, while heterotrophic and mixotrophic iron oxidation is not well-studied. Ecological studies have shown that Leptothrix overtakes Gallionella when dissolved organic carbon content increases, demonstrating distinct niches. This study presents the first near-complete genomes of L. ochracea , which share some features with autotrophic iron oxidizers, while also incorporating heterotrophic metabolisms. These genome, metabolic modeling, and transcriptome results give us a detailed metabolic picture of how the organism may combine lithoautotrophy with organoheterotrophy to promote Fe oxidation and C cycling and drive many biogeochemical processes resulting from microbial growth and iron oxyhydroxide formation in wetlands.

59 BASIC BIOLOGICAL SCIENCES↗

Integration of evidence across human and model organism studies: A meeting report

The National Institute on Drug Abuse and Joint Institute for Biological Sciences at the Oak Ridge National Laboratory hosted a meeting attended by a diverse group of scientists with expertise in substance use disorders (SUDs), computational biology, and FAIR (Findability, Accessibility, Interoperability, and Reusability) data sharing. The meeting's objective was to discuss and evaluate better strategies to integrate genetic, epigenetic, and 'omics data across human and model organisms to achieve deeper mechanistic insight into SUDs. Specific topics were to (a) evaluate the current state of substance use genetics and genomics research and fundamental gaps, (b) identify opportunities and challenges of integration and sharing across species and data types, (c) identify current tools and resources for integration of genetic, epigenetic, and phenotypic data, (d) discuss steps and impediment related to data integration, and (e) outline future steps to support more effective collaboration—particularly between animal model research communities and human genetics and clinical research teams. This review summarizes key facets of this catalytic discussion with a focus on new opportunities and gaps in resources and knowledge on SUDs.

59 BASIC BIOLOGICAL SCIENCES↗

Genome Analysis of a Verrucomicrobial Endosymbiont With a Tiny Genome Discovered in an Antarctic Lake

Organic Lake in Antarctica is a marine-derived, cold (–13°C), stratified (oxic-anoxic), hypersaline (>200 gl –1 ) system with unusual chemistry (very high levels of dimethylsulfide) that supports the growth of phylogenetically and metabolically diverse microorganisms. Symbionts are not well characterized in Antarctica. However, unicellular eukaryotes are often present in Antarctic lakes and theoretically could harbor endosymbionts. Here, we describe Candidatus Organicella extenuata, a member of the Verrucomicrobia with a highly reduced genome, recovered as a metagenome-assembled genome with genetic code 4 (UGA-to-Trp recoding) from Organic Lake. It is closely related to Candidatus Pinguicocccus supinus (163,218 bp, 205 genes), a newly described cytoplasmic endosymbiont of the freshwater ciliate Euplotes vanleeuwenhoeki (Serra et al., 2020). At 158,228 bp (encoding 194 genes), the genome of Ca. Organicella extenuata is among the smallest known bacterial genomes and similar to the genome of Ca. Pinguicoccus supinus (163,218 bp, 205 genes). Ca. Organicella extenuata retains a capacity for replication, transcription, translation, and protein-folding while lacking any capacity for the biosynthesis of amino acids or vitamins. Notably, the endosymbiont retains a capacity for fatty acid synthesis (type II) and iron–sulfur (Fe-S) cluster assembly. Metagenomic analysis of 150 new metagenomes from Organic Lake and more than 70 other Antarctic aquatic locations revealed a strong correlation in abundance between Ca. Organicella extenuata and a novel ciliate of the genus Euplotes. Like Ca. Pinguicoccus supinus, we infer that Ca. Organicella extenuata is an endosymbiont of Euplotes and hypothesize that both Ca. Organicella extenuata and Ca. Pinguicocccus supinus provide fatty acids and Fe-S clusters to their Euplotes host as the foundation of a mutualistic symbiosis. The discovery of Ca. Organicella extenuata as possessing genetic code 4 illustrates that in addition to identifying endosymbionts by sequencing known symbiotic communities and searching metagenome data using reference endosymbiont genomes, the potential exists to identify novel endosymbionts by searching for unusual coding parameters.

59 BASIC BIOLOGICAL SCIENCES↗

DRAM example narrative

DRAM example narrative DRAM on KBase let's anyone run annotations using DRAM in the cloud. DRAM is an annotation tool that can annotate bacterial, archaeal and viral genomes and distills those annotatios into represetations of the functional genomic potential of those organisms. If you want to read more about DRAM you can check out the GitHub, wiki and journal article. DRAM annotate assemblies In KBase Assembly objects contain nucleotide sequences from genomes or metagenomes. DRAM can predict genes and annotate their function from KBase Assembly objects which may be microbial isolate genomes, metagenome assembled genomes or metagenomes. This is done with the Annotate and Distill Assemblies with DRAM app. This app can also anntoate AssemblySet objects which contain collection of Assembly objects. It also generates a Genome object and a GenomeSet object which can be used for further analysis with other KBase apps. The full annotations and other DRAM files are also available for download in the app.

59 BASIC BIOLOGICAL SCIENCES↗

UnigeneFinder: An Automated Pipeline for Gene Calling From Transcriptome Assemblies Without a Reference Genome

ABSTRACT For most species, transcriptome data are much more readily available than genome data. Without a reference genome, gene calling is cumbersome and inaccurate because of the high degree of redundancy in de novo transcriptome assemblies. To simplify and increase the accuracy of de novo transcriptome assembly in the absence of a reference genome, we developed UnigeneFinder. Combining several clustering methods, UnigeneFinder substantially reduces the redundancy typical of raw transcriptome assemblies. This pipeline offers an effective solution to the problem of inflated transcript numbers, achieving a closer representation of the actual underlying genome. UnigeneFinder performs comparably or better, compared with existing tools, on plant species with varying genome complexities. UnigeneFinder is the only available transcriptome redundancy solution that fully automates the generation of primary transcript, coding region, and protein sequences, analogous to those available for high‐quality reference genomes. These features, coupled with the pipeline’s cross‐platform implementation, focus on automation, and an accessible, user‐friendly interface, make UnigeneFinder a useful tool for many downstream sequence‐based analyses in nonmodel organisms lacking a reference genome, including differential gene expression analysis, accurate ortholog identification, functional enrichments, and evolutionary analyses. UnigeneFinder also runs efficiently both on high‐performance computing (HPC) systems and personal computers, further reducing barriers to use.

Xue, Bo [Plant Resilience Institute Michigan State↗

Extreme dimensions — how big (or small) can tailed phages be?

Bacteriophages (phages), that is, viruses that infect bacteria, represent an extremely diverse, yet under-characterized, group of viruses. Although most known phages harbour genomes that are shorter than 200 kb packaged into capsids with a diameter under 100 Å, more and more ‘extremely large’ phages are being discovered. Early reports of phages with genomes larger than 200 kb defined them as ‘jumbo phages’1. Phylogenetic analysis of these jumbo phages revealed that they form distinct and cohesive groups with multiple independent origins. This suggested that jumbo phages are not merely mistakes or aberrations of smaller phages that got too big, but that large genome size is generally a stable trait. In fact, a larger genome can be advantageous; almost all jumbo phages encode their own specific transcription factors, at least some parts of the replication machinery, and many also have their own tRNA genes, which enables increased independence from their host. Here, large genomes also provide more flexibility in terms of transcription strategy; smaller phage genomes tend to have a strict modular genome structure to increase efficiency, whereas jumbo phage genomes may be much less organized in the absence of a strong selection pressure to maintain a compact genome.

59 BASIC BIOLOGICAL SCIENCES↗

Transforming the study of organisms: Phenomic data models and knowledge bases

The rapidly decreasing cost of gene sequencing has resulted in a deluge of genomic data from across the tree of life; however, outside a few model organism databases, genomic data are limited in their scientific impact because they are not accompanied by computable phenomic data. The majority of phenomic data are contained in countless small, heterogeneous phenotypic data sets that are very difficult or impossible to integrate at scale because of variable formats, lack of digitization, and linguistic problems. One powerful solution is to represent phenotypic data using data models with precise, computable semantics, but adoption of semantic standards for representing phenotypic data has been slow, especially in biodiversity and ecology. Some phenotypic and trait data are available in a semantic language from knowledge bases, but these are often not interoperable. In this review, we will compare and contrast existing ontology and data models, focusing on nonhuman phenotypes and traits. We discuss barriers to integration of phenotypic data and make recommendations for developing an operationally useful, semantically interoperable phenotypic data ecosystem.

59 BASIC BIOLOGICAL SCIENCES↗

RNA nanotechnology to build a dodecahedral genome of single-stranded RNA virus

The quest for artificial RNA viral complexes with authentic structure while being non-replicative is on its way for the development of viral vaccines. RNA viruses contain capsid proteins that interact with the genome during morphogenesis. The sequence and properties of the protein and genome determine the structure of the virus. For example, the Pariacoto virus ssRNA genome assembles into a dodecahedron. Virus-inspired nanotechnology has progressed remarkably due to the unique structural and functional properties of viruses, which can inspire the design of novel nanomaterials. RNA is a programmable biopolymer able to self-assemble sophisticated 3D structures with rich functionalities. RNA dodecahedrons mimicking the Pariacoto virus quasi-icosahedral genome structures were constructed from both native and 2'-F modified RNA oligos. The RNA dodecahedron easily self-assembled using the stable pRNA three-way junction of bacteriophage phi29 as building blocks. The RNA dodecahedron cage was further characterized by cryo-electron microscopy and atomic force microscopy, confirming the spontaneous and homogenous formation of the RNA cage. The reported RNA dodecahedron cage will likely provide further studies on the mechanisms of interaction of the capsid protein with the viral genome while providing a template for further construction of the viral RNA scaffold to add capsid proteins for the assembly of the viral nucleocapsid as a model. Understanding the self-assembly and RNA folding of this RNA cage may offer new insights into the 3D organization of viral RNA genomes. Finally, the reported RNA cage also has the potential to be explored as a novel virus-inspired nanocarrier.

59 BASIC BIOLOGICAL SCIENCES↗

KBase Narrative - Comparing Proteomes of Cupriavidus basilensis 4G11 and Cupriavidus necator H16

Species of Cupriavidus are among the best bioplastic producing microorganisms known, though intracellular poly-hydroxybutyrate (PHB) is made exclusively during unbalanced growth (e.g. nitrogen limitation), a state characterized by poor growth and cellular stress. Much work has been done studying bioplastic production in the model/type strain C. necator H16. We use an RB-TnSeq library of the closely related C. basilensis 4G11 to assess for genes which decouple PHB production from growth state. In this narrative we compute genome-wide protein homology between these two organisms, and generate a synteny plot across the genomes as well.

Sander, Kyle↗

Draft genome of Rosenbergiella nectarea strain 8N4 T provides insights into the potential role of this species in its plant host

Background: Rosenbergiella nectarea strain 8N4 T , the type species of the genus Rosenbergiella, was isolated from Amygdalus communis (almond) floral nectar. Other strains of this species were isolated from the floral nectar of Citrus paradisi (grapefruit), Nicotiana glauca (tobacco tree) and from Asphodelus aestivus. R. nectarea strain 8N4 T is a Gram-negative, oxidase-negative, facultatively anaerobic bacterium in the family Enterobacteriaceae. Results: Here we describe features of this organism, together with its genome sequence and annotation. The DNA GC content is 47.38%, the assembly size is 3,294,717 bp, and the total number of genes are 3,346. The genome discloses the possible role that this species may play in the plant. The genome contains both virulence genes, like pectin lyase and hemolysin, that may harm plant cells and genes that are predicted to produce volatile compounds that may impact the visitation rates by nectar consumers, such as pollinators and nectar thieves. Conclusions: The genome of R. nectarea strain 8N4T reveals a mutualistic interaction with the plant host and a possible effect on plant pollination and fitness.

59 BASIC BIOLOGICAL SCIENCES↗

Machine learning analysis of RB-TnSeq fitness data predicts functional gene modules in Pseudomonas putida KT2440

ABSTRACT There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida . Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

09 BIOMASS FUELS↗

Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus

Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.

59 BASIC BIOLOGICAL SCIENCES↗

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades—primarily plant—associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

59 BASIC BIOLOGICAL SCIENCES↗