Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nanolattice-Forming Hybrid Collagens in Protective Shark Egg Cases.

Nanoscopic structural control with long-range ordering remains a profound challenge in nanomaterial fabrication. The nanoarchitectured egg cases of elasmobranchs rely on a hierarchically ordered latticework for their protective function─serving as an exemplary system for nanoscale self-assembly. Although the proteinaceous precursors are known to undergo intermediate liquid crystalline phase transitions before being structurally arrested in the final nanolattice architecture, their sequences have so far remained unknown. By leveraging RNA-seq and proteomic techniques, we identified a cohort of nanolattice-forming proteins comprising a collagenous midblock flanked by domains typically associated with innate immunity and network-forming collagens. Structurally homologous proteins were found in the genomes of other egg-case-producing cartilaginous fishes, suggesting a conserved molecular self-assembly strategy. The identity and stabilizing role of cross-links were subsequently elucidated using mass spectrometry and in situ small-angle X-ray scattering. Our findings provide a new design approach for protein-based liquid crystalline elastomers and the self-assembly of nanolattices.

TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae.

BACKGROUND: Target of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear. RESULTS: We demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-responsive genes mainly via the Tap42-Sit4-Rrd1/2 pathway. To confirm TORC1's function in glucose signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination. CONCLUSIONS: Our studies indicate that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

Elephant shark genome provides unique insights into gnathostome evolution.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

Functional VEGFA knockdown with artificial 3'-tailed mirtrons defined by 5' splice site and branch point.

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons - 3'-tailed mirtrons - has hairpins precisely defined on the 5' end by the 5' splice site and 3' end by the branch point. Here, we present design principles for artificial 3'-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.

Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined ß-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to ß-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in ß-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

Diethyl phthalate (DEP) perturbs nitrogen metabolism in Saccharomyces cerevisiae.

Phthalates are ubiquitously used as plasticizers in various consumer care products. Diethyl phthalate (DEP), one of the main phthalates, elicits developmental and reproductive toxicities but the underlying mechanisms are not fully understood. Chemogenomic profiling of DEP in S. cerevisiae revealed that two transcription factors Stp1 and Dal81 involved in the Ssy1-Ptr5-Ssy5 (SPS) amino acid-sensing pathway provide resistance to DEP. Growth inhibition of yeast cells by DEP was stronger in poor nitrogen medium in comparison to nitrogen-rich medium. Addition of amino acids to nitrogen-poor medium suppressed DEP toxicity. Catabolism of amino acids via the Ehrlich pathway is required for suppressing DEP toxicity. Targeted metabolite analyses showed that DEP treatment alters the amino acid profile of yeast cells. We propose that DEP inhibits the growth of yeast cells by affecting nitrogen metabolism and discuss the implications of our findings on DEP-mediated toxic effects in humans.

A Multiplex Thyroid-Specific Assay for Quantification of Circulating Thyroid Cell-Free RNA in Plasma of Thyroid Cancer Patients.

BACKGROUND: The standard of care for thyroid cancer management is thyroidectomy and adjuvant radioactive iodine (RAI). There is a paucity of clinical tool that quantifies residual thyroid volume reliably for precise adjuvant RAI dosing. Serum thyroglobulin (TG), tumour marker for thyroid cancer, takes 4 weeks for complete clearance due to its long half-life, and might be undetectable in 12% of structural disease patients. It detects recurrence with a sensitivity of 19-40%, mainly attributed to issue of TG antibody interference with TG immunometric assay. We hypothesise that the quantity of thyroid-specific cell-free RNA (cfRNA) is indicative of amount of thyroid tissues, and that during thyroid surgery, cfRNA levels decrease accordingly. METHODS: We identified 11 biologically significant and highly expressed thyroid-specific targets from Human Protein Atlas and literature. To assess for a fall in thyroid-specific cfRNA level, we recruited 16 patients undergoing thyroid surgery or RAI for malignant or benign thyroid disease, and tracked longitudinal trend of cfRNA. To assess the utility of cfRNA in detecting metastatic thyroid cancer, cfRNA of 11 patients at intermediate to high risk of recurrence was measured during surveillance and at time of clinical recurrence. RESULTS: The multiplex assay was capable of amplifying and quantifying multiple thyroid-specific genes in a single reaction. The selected targets were amplified successfully from RNA extracted directly from the thyroid (positive control), indicating that they were highly expressed within thyroid tissue. These cfRNAs were present in plasma, in amounts quantifiable using qRT-PCR. Four cfRNA transcripts (TPO, GFRA2, IVD, TG) fell post-treatment in more than 50% of cohort. The thyroid peroxidase (TPO) cfRNA fell post-therapy in 63% of cohort by 80%, as early as 1 day post-treatment, supporting the potential role as early indicator of remnant thyroid tissue volume. We demonstrated the clinical relevance of circulating TPO cfRNA by tracking temporal changes in setting of peri-treatment, recurrence, and TG Ab positive state. CONCLUSION: Using a multiplex pre-amplification approach, the TPO cfRNA was a potential biomarker that can track residual thyroid mass. It can be further optimised for quantification of thyroid volume to guide RAI doses and for detection of thyroid cancer recurrence.

Smad­binding decoy reduces extracellular matrix expression in human hypertrophic scar fibroblasts.

The exact mechanisms underlying hypertrophic scarring is yet to be fully understood. However, excessive collagen deposition by fibroblasts has been demonstrated to result in hypertrophic scar formation, and collagen synthesis in dermal fibroblasts is regulated by the transforming growth factor­ß1/Smad signaling pathway. In view of this, a Smad­binding decoy was designed and its effects on hypertrophic scar­derived human skin fibroblasts was evaluated. The results of the present study revealed that the Smad decoy attenuates the total amount of collagen, collagen I and Smad2/3 expression in scar fibroblasts. Data from RNA sequencing indicated that the Smad decoy induced more than 4­fold change in 178 genes, primarily associated with to the extracellular matrix, compared with the untreated control. In addition, results from quantitative real­time polymerase chain reaction further confirmed that the Smad decoy significantly attenuated the expression of extracellular matrix­related genes, including COL1A1, COL1A2 and COL3A1. Furthermore, the Smad decoy reduced transforming growth factor­ß1­induced collagen deposition in scar fibroblasts. Data generated from the present study provide evidence supporting the use of the Smad decoy as a potential hypertrophic scar treatment.

Chemical-genetic interaction landscape of mono-(2-ethylhexyl)-phthalate using chemogenomic profiling in yeast.

Integration of chemical-genetic interaction data with biological functions provides a mechanistic understanding of how toxic compounds affect cells. Mono-(2-ethylhexyl)-phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl)-phthalate (DEHP), a commonly used plasticizer. MEHP adversely affects human health causing hepatotoxicity and reproductive toxicity. How MEHP affects cellular physiology is not fully understood. We utilized a genome-wide competitive fitness-based assay called 'chemogenomic profiling' to determine the genetic interaction map of MEHP in Saccharomyces cerevisiae. Gene Ontology enrichment analysis of 218 genes that provide resistance to MEHP indicated that MEHP affects seven cellular processes namely: (1) cellular amino acid biosynthetic process, (2) sterol biosynthetic process, (3) cellular transport, (4) transcriptional and translational regulation, (5) protein glycosylation, (6) cytokinesis and cell morphogenesis and (7) ionic homeostasis. We show that MEHP protects yeast cells from membrane perturbing agents such as amphotericin B, dihydrosphingosine and phytosphingosine. Moreover, we also demonstrate that MEHP compromises the integrity of the yeast plasma membrane and cell wall. Our work provides a basis for further investigation of MEHP toxicity in humans.

Correction: Chemogenomic profiling in yeast reveals antifungal mode-of-action of polyene macrolactam auroramycin.

[This corrects the article DOI: 10.1371/journal.pone.0218189.].

Chemogenomic profiling in yeast reveals antifungal mode-of-action of polyene macrolactam auroramycin.

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization. This was verified by disruption of vacuolar structure and membrane integrity of yeast cells with auroramycin treatment. Addition of salt but not sorbitol to the medium rescued the growth of auroramycin-treated yeast cells suggesting that auroramycin causes ionic stress. Furthermore, auroramycin caused hyperpolarization of the yeast plasma membrane and displayed a synergistic interaction with cationic hygromycin. Our data strongly suggest that auroramycin inhibits yeast cells by causing leakage of cations from the cytoplasm. Thus, auroramycin's mode-of-action is distinct from known antifungal polyenes, reinforcing the importance of natural products in the discovery of new anti-infectives.

Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells.

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.

Supramolecular propensity of suckerin proteins is driven by ß-sheets and aromatic interactions as revealed by solution NMR.

Suckerin proteins are a family of block co-polymer-like structural proteins that self-assemble into robust supramolecular structures - the sucker ring teeth (SRT) - which are located on the arms and tentacles of cephalopods and used to firmly capture preys. Suckerins are promising biomimetic protein-based biopolymers, but the supramolecular interactions stabilizing SRT remain unknown. Here, we report multi-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy structural studies of an engineered suckerin protein composed of two main sequence modules. The protein adopts a dynamic structure with regions in both module 1 (M1: residues A42-A52) and module 2 (M2: residues G30-Y37 and G58-Y65) folding into anti-parallel ß-sheets and displaying ß-strand propensity, respectively. The obtained structure highlights that aromatic residues present in glycine (Gly)-rich M2 modules are involved in π-π stacking interactions, leading to the stabilization of the structural core. In addition, hydrogen/deuterium (H/D) exchange studies demonstrate a high protection of residues involved in intra-molecular ß-sheets. Gaining a better understanding of the molecular structure of suckerin provides key molecular lessons that may be mimicked in the de novo design of peptide- and protein-based biomaterials with applications in medicine, tissue engineering and nanotechnology.

Structure-activity relationship studies of ultra-short peptides with potent activities against fluconazole-resistant Candida albicans.

Vulvovaginal candidiasis (VVC) is a genital fungal infection afflicting approximately 75% of women globally and is primarily caused by the yeast Candida albicans. The extensive use of fluconazole, the first-line antifungal drug of choice, has led to the emergence of fluconazole-resistant C. albicans, creating a global clinical concern. This, coupled to the lack of new antifungal drugs entering the market over the past decade, has made it imperative for the introduction of new antifungal drug classes. Peptides with antifungal properties are deemed potential drug candidates due to their rapid membrane-disrupting mechanism of action. By specifically targeting and rapidly disrupting fungal membranes, they reduce the chances of resistance development and treatment duration. In a previous screening campaign involving an antimicrobial peptide library, we identified an octapeptide (IKIKIKIK-NH2) with potent activity against C. albicans. Herein, we report a structure-activity relationship study on this peptide with the aim of designing a more potent peptide for further development. The lead peptide was then tested against a panel of fluconazole-resistant C. albicans, subjected to a fungicidal/static determination assay, a human dermal fibroblast viability assay and a homozygous profiling assay to gain insights into its mechanism of action and potential for further development as a topical antifungal agent.

Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing.

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science.

Efforts to engineer new materials inspired by biological structures are hampered by the lack of genomic data from many model organisms studied in biomimetic research. Here we show that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization. This approach can be applied to a broad range of systems, as we illustrate by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation. In one example, we rapidly engineer recombinant squid sucker ring teeth proteins into a range of structural and functional materials, including nanopatterned surfaces and photo-cross-linked films that exceed the mechanical properties of most natural and synthetic polymers. Integrating RNA-seq with proteomics and materials science facilitates the molecular characterization of natural materials and the effective translation of their molecular designs into a wide range of bio-inspired materials.