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SM007

Omics-Lethal Human Virus, SARS - SM007

131 BASIC BIOLOGICAL SCIENCES↗

Regulation of Absorption and Emission in a Protein/Fluorophore Complex

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore’s aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Quantifying Antibiotic Distribution in Solid and Liquid Fractions of Manure Using a Two-Step, Multi-Residue Antibiotic Extraction

Antibiotic distribution and analysis within liquid and solid fractions of manure are highly variable due to each compound’s respective physiochemical properties. This study developed and evaluated a uniform method extracting 10 antibiotics from 4 antibiotic classes (tetracycline, sulfonamides, macrolides, and β-lactam) from unprocessed manure, solid–liquid separated manure, and composted solids. Through systematic manipulation of previously published liquid chromatography tandem mass spectrometry methods; this study developed an extraction protocol with optimized recovery efficiencies for varied manure substrates. The method includes a two-step, liquid-solid extraction using 10 mL of 0.1 M EDTA-McIlviane buffer followed by 10 mL of methanol. Antibiotics recoveries from unprocessed manure, separated liquids, separated solids, and heat-treated solids using the two-step extraction method had relative standard deviations < 30% for all but ceftiofur. Total antibiotic recoveries were 67–131% for tetracyclines, 56% for sulfonamide, 49–53% for macrolides, and 1.3–66% for β-lactams. This is the first study to use one protocol to assess four classes of antibiotics in liquid and solid manure fractions. This study allowed for more precise risk assessment of antibiotic transport in manure waste stream applied to fields as a liquid or solid compost.

59 BASIC BIOLOGICAL SCIENCES↗

Glacier ice archives nearly 15,000-year-old microbes and phages

Background Glacier ice archives information, including microbiology, that helps reveal paleoclimate histories and predict future climate change. Though glacier-ice microbes are studied using culture or amplicon approaches, more challenging metagenomic approaches, which provide access to functional, genome-resolved information and viruses, are under-utilized, partly due to low biomass and potential contamination. Results We expand existing clean sampling procedures using controlled artificial ice-core experiments and adapted previously established low-biomass metagenomic approaches to study glacier-ice viruses. Controlled sampling experiments drastically reduced mock contaminants including bacteria, viruses, and free DNA to background levels. Amplicon sequencing from eight depths of two Tibetan Plateau ice cores revealed common glacier-ice lineages including Janthinobacterium, Polaromonas, Herminiimonas, Flavobacterium, Sphingomonas, and Methylobacterium as the dominant genera, while microbial communities were significantly different between two ice cores, associating with different climate conditions during deposition. Separately, ~355- and ~14,400-year-old ice were subject to viral enrichment and low-input quantitative sequencing, yielding genomic sequences for 33 vOTUs. These were virtually all unique to this study, representing 28 novel genera and not a single species shared with 225 environmentally diverse viromes. Further, 42.4% of the vOTUs were identifiable temperate, which is significantly higher than that in gut, soil, and marine viromes, and indicates that temperate phages are possibly favored in glacier-ice environments before being frozen. In silico host predictions linked 18 vOTUs to co-occurring abundant bacteria (Methylobacterium, Sphingomonas, and Janthinobacterium), indicating that these phages infected ice-abundant bacterial groups before being archived. Functional genome annotation revealed four virus-encoded auxiliary metabolic genes, particularly two motility genes suggest viruses potentially facilitate nutrient acquisition for their hosts. Finally, given their possible importance to methane cycling in ice, we focused on Methylobacterium viruses by contextualizing our ice-observed viruses against 123 viromes and prophages extracted from 131 Methylobacterium genomes, revealing that the archived viruses might originate from soil or plants. Conclusions Together, these efforts further microbial and viral sampling procedures for glacier ice and provide a first window into viral communities and functions in ancient glacier environments. Such methods and datasets can potentially enable researchers to contextualize new discoveries and begin to incorporate glacier-ice microbes and their viruses relative to past and present climate change in geographically diverse regions globally.

59 BASIC BIOLOGICAL SCIENCES↗

Mining metatranscriptomes reveals a vast world of viroid-like circular RNAs

Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗

Safe passage of American Eels through a novel hydropower turbine

Abstract Objective Study the effects of downstream passage through a novel turbine designed for fish safety, the Restoration Hydro Turbine (RHT), on American Eels Anguilla rostrata in a recirculating turbine test facility. Methods A 55‐cm‐diameter RHT was operated under 10 m of hydraulic head and 667 revolutions/min. In total, 131 eels were passed through the turbine and 43 eels were used as experimental controls (length = 33.9–65.5 cm). High‐speed video of passage through the runner region was captured for 89% of turbine‐passed eels, and injury and behavioral effects were recorded immediately before and after passage, as well as after a 48‐h holding period. A subset of 37 eels was additionally examined with X‐ray imaging for internal injuries. Result The 48‐h survival rate for both treatment and control groups was 100%, with no major internal or external injuries present after the holding period. Conclusion This is a substantial improvement over eel survival rates through conventional Kaplan and Francis turbines, which may range from 40% to 95%, and suggests that hydropower turbines designed for safe downstream fish passage could be implemented without major impacts to eels.

59 BASIC BIOLOGICAL SCIENCES↗

MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

59 BASIC BIOLOGICAL SCIENCES↗