Cinnamosyn, a Cinnamoylated Synthetic-Bioinformatic Natural Product with Cytotoxic Activity.

Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids. Here we increase the bioinformatic and synthetic complexity of the synBNP approach by targeting BGCs that encode N-cinnamoyl lipids. This led to our synthesis of cinnamosyn, a 10-mer N-cinnamoyl-containing peptide that is cytotoxic to human cells.

Identification of an Optimized Clinical Development Candidate from Cilagicin, an Antibiotic That Evades Resistance by Dual Polyprenyl Phosphate Binding.

Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens. Here, we manipulated the acyl tail and the peptide core of cilagicin to identify an optimized collection of structural features that maintain potent antibiotic activity against a wide range of pathogens in the presence of serum. This led to the identification of the optimized antibiotic dodecacilagicin, which contains an N-terminal dodecanoic acid. Dodecacilagicin exhibits low MICs against clinically relevant pathogens in the presence of serum, retains polyprenyl phosphate binding, and evades resistance development even after long-term antibiotic exposure, making dodecacilagicin an appealing candidate for further therapeutic development.

DNA glycosylases provide antiviral defence in prokaryotes.

Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

The Chalcidoidea bush of life: evolutionary history of a massive radiation of minute wasps.

Chalcidoidea are mostly parasitoid wasps that include as many as 500 000 estimated species. Capturing phylogenetic signal from such a massive radiation can be daunting. Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 ultra-conserved elements (UCEs) for 433 taxa including all extant families, >95% of all subfamilies, and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between the molecular results and our collective knowledge of morphology and biology, we detected bias in the analyses that was driven by the saturation of nucleotide data. Our final results are based on a concatenated analysis of the least saturated exons and UCE datasets (2054 loci, 284 106 sites). Our analyses support an expected sister relationship with Mymarommatoidea. Seven previously recognized families were not monophyletic, so support for a new classification is discussed. Natural history in some cases would appear to be more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a transition from smaller soft-bodied wasps to larger and more heavily sclerotized wasps, with egg parasitism as potentially ancestral for the entire superfamily. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the "Angiosperm Terrestrial Revolution". Our dating analyses suggest a middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9-169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea may have undergone a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about the host taxa of chalcid wasps, their fossil record and Earth's palaeogeographic history.

Out of the desert: Paleoclimatic changes drove the diversification of arid-adapted Ocymyrmex ants in southern Africa.

A highly endemic ant fauna is found in the arid regions of southern Africa, including species in the genus Ocymyrmex. This genus of ants has higher species richness in the western arid regions of southern Africa compared to tropical and subtropical parts of the continent. The processes that have produced these patterns of diversity and distribution of arid adapted ants in southern Africa have never been investigated. The diversification of many other taxa in the region has been associated with past climate fluctuations that occurred during the Miocene epoch. In this study, the nature and timing of historical processes that may have led to the diversification within Ocymyrmex were assessed. We hypothesized that past climate oscillations, characterized by long periods of aridification, have driven the current distribution of Ocymyrmex species that resulted in the highest species richness of the genus in the Deserts & xeric shrublands biome in southern Africa. Ninety-four Ocymyrmex worker specimens from Botswana, Kenya, Namibia, South Africa, Tanzania and Zimbabwe, representing 21 currently described species and six morphospecies, were included in a phylogenomic analysis. Phylogenies for the genus, based on next generation sequencing data from ultraconserved elements, were inferred using Maximum Likelihood, and a dating analysis was performed using secondary age estimates as calibration points. A distribution database of Ocymyrmex records was used to assign species ranges, which were then coded according to major biomes in southern Africa and used as input for biogeographical analysis. We explored the phylogenomic relationships of Ocymyrmex and analysed these within a biogeographical and paleoclimatic framework to disentangle the potential processes responsible for diversification in this group. Dating analyses estimated that the crown age of Ocymyrmex dates to the Oligocene, around 32 Ma. Diversification within this group occurred between the mid-Miocene (∼12.5 Ma) and Pleistocene (∼2 Ma). Our biogeographic analyses suggest that Ocymyrmex species originated in the south-western region of southern Africa, which is now part of the Deserts & xeric shrublands biome and diversified into eastern subtropical areas during the Pliocene. Paleoclimatic changes resulting in increased aridity during the Miocene likely drove the diversification of the genus Ocymyrmex. It is most likely that the diversification of grasslands, because of historical climate change, facilitated the diversification of these ants to the eastern parts of southern Africa when open grasslands replaced forests during the early Miocene.

A Family of Antibiotics That Evades Resistance by Binding Polyprenyl Phosphates.

Cilagicin is a Gram-positive active antibiotic that has a dual polyprenyl phosphate binding mechanism that impedes resistance development. Here we bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to search for antibiotics that might similarly avoid resistance development. Synthesis and bioactivity screening of the predicted structures that we identified led to three antibiotics that are active against multidrug-resistant Gram-positive pathogens, two of which, paenilagicin and virgilagicin, did not lead to resistance even after prolonged antibiotic exposure.

Bacterial cGAS senses a viral RNA to initiate immunity.

Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.

The evolutionary history of bees in time and space.

Bees are the most significant pollinators of flowering plants. This partnership began ca. 120 million years ago, but the uncertainty of how and when bees spread across the planet has greatly obscured investigations of this key mutualism. We present a novel analysis of bee biogeography using extensive new genomic and fossil data to demonstrate that bees originated in Western Gondwana (Africa and South America). Bees likely originated in the Early Cretaceous, shortly before the breakup of Western Gondwana, and the early evolution of any major bee lineage is associated with either the South American or African land masses. Subsequently, bees colonized northern continents via a complex history of vicariance and dispersal. The notable early absences from large landmasses, particularly in Australia and India, have important implications for understanding the assembly of local floras and diverse modes of pollination. How bees spread around the world from their hypothesized Southern Hemisphere origin parallels the histories of numerous flowering plant clades, providing an essential step to studying the evolution of angiosperm pollination syndromes in space and time.

Present and future outlooks on environmental DNA-based methods for antibiotic discovery.

Novel antibiotics are in constant demand to combat a global increase in antibiotic-resistant infections. Bacterial natural products have been a long-standing source of antibiotic compounds, and metagenomic mining of environmental DNA (eDNA) has increasingly provided new antibiotic leads. The metagenomic small-molecule discovery pipeline can be divided into three main steps: surveying eDNA, retrieving a sequence of interest, and accessing the encoded natural product. Improvements in sequencing technology, bioinformatic algorithms, and methods for converting biosynthetic gene clusters into small molecules are steadily increasing our ability to discover metagenomically encoded antibiotics. We predict that, over the next decade, ongoing technological improvements will dramatically increase the rate at which antibiotics are discovered from metagenomes.

Key innovations and the diversification of Hymenoptera.

The order Hymenoptera (wasps, ants, sawflies, and bees) represents one of the most diverse animal lineages, but whether specific key innovations have contributed to its diversification is still unknown. We assembled the largest time-calibrated phylogeny of Hymenoptera to date and investigated the origin and possible correlation of particular morphological and behavioral innovations with diversification in the order: the wasp waist of Apocrita; the stinger of Aculeata; parasitoidism, a specialized form of carnivory; and secondary phytophagy, a reversal to plant-feeding. Here, we show that parasitoidism has been the dominant strategy since the Late Triassic in Hymenoptera, but was not an immediate driver of diversification. Instead, transitions to secondary phytophagy (from parasitoidism) had a major influence on diversification rate in Hymenoptera. Support for the stinger and the wasp waist as key innovations remains equivocal, but these traits may have laid the anatomical and behavioral foundations for adaptations more directly associated with diversification.

High-throughput retrieval of target sequences from complex clone libraries using CRISPRi.

The capture of metagenomic DNA in large clone libraries provides the opportunity to study microbial diversity that is inaccessible using culture-dependent methods. In this study, we harnessed nuclease-deficient Cas9 to establish a CRISPR counter-selection interruption circuit (CCIC) that can be used to retrieve target clones from complex libraries. Combining modern sequencing methods with CCIC cloning allows for rapid physical access to the genetic diversity present in natural ecosystems.

Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History.

The use of DNA barcoding has revolutionised biodiversity science, but its application depends on the existence of comprehensive and reliable reference libraries. For many poorly known taxa, such reference sequences are missing even at higher-level taxonomic scales. We harvested the collections of the Smithsonian's National Museum of Natural History (USNM) to generate DNA barcoding sequences for genera of terrestrial arthropods previously not recorded in one or more major public sequence databases. Our workflow used a mix of Sanger and Next-Generation Sequencing (NGS) approaches to maximise sequence recovery while ensuring affordable cost. In total, COI sequences were obtained for 5,686 specimens belonging to 3,737 determined species in 3,886 genera and 205 families distributed in 137 countries. Success rates varied widely according to collection data and focal taxon. NGS helped recover sequences of specimens that failed a previous run of Sanger sequencing. Success rates and the optimal balance between Sanger and NGS are the most important drivers to maximise output and minimise cost in future projects. The corresponding sequence and taxonomic data can be accessed through the Barcode of Life Data System, GenBank, the Global Biodiversity Information Facility, the Global Genome Biodiversity Network Data Portal and the NMNH data portal.

Multiplexed mobilization and expression of biosynthetic gene clusters.

Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.

Discovery of Paenibacillaceae Family Gram-Negative-Active Cationic Lipopeptide Antibiotics Using Evolution-Guided Chemical Synthesis.

Cationic nonribosomal lipopeptides (CNRLPs) from Paenibacillus spp. have been a rewarding source of Gram-negative-active antibiotics. Here we systematically screened sequenced bacterial genomes for CNRLP biosynthetic gene clusters (BGCs) that we predicted might encode additional Gram-negative-active antibiotics. Total chemical synthesis of the bioinformatically predicted products of seven such BGCs led to our identification of new laterocidine, tridecaptin, and paenibacterin-like antibiotics with potent activity against both multiple-drug-resistant Gram-negative and Gram-positive pathogens.

Automated diagnosis and prognosis of COVID-19 pneumonia from initial ER chest X-rays using deep learning.

BACKGROUND: Airspace disease as seen on chest X-rays is an important point in triage for patients initially presenting to the emergency department with suspected COVID-19 infection. The purpose of this study is to evaluate a previously trained interpretable deep learning algorithm for the diagnosis and prognosis of COVID-19 pneumonia from chest X-rays obtained in the ED. METHODS: This retrospective study included 2456 (50% RT-PCR positive for COVID-19) adult patients who received both a chest X-ray and SARS-CoV-2 RT-PCR test from January 2020 to March of 2021 in the emergency department at a single U.S. INSTITUTION: A total of 2000 patients were included as an additional training cohort and 456 patients in the randomized internal holdout testing cohort for a previously trained Siemens AI-Radiology Companion deep learning convolutional neural network algorithm. Three cardiothoracic fellowship-trained radiologists systematically evaluated each chest X-ray and generated an airspace disease area-based severity score which was compared against the same score produced by artificial intelligence. The interobserver agreement, diagnostic accuracy, and predictive capability for inpatient outcomes were assessed. Principal statistical tests used in this study include both univariate and multivariate logistic regression. RESULTS: Overall ICC was 0.820 (95% CI 0.790-0.840). The diagnostic AUC for SARS-CoV-2 RT-PCR positivity was 0.890 (95% CI 0.861-0.920) for the neural network and 0.936 (95% CI 0.918-0.960) for radiologists. Airspace opacities score by AI alone predicted ICU admission (AUC = 0.870) and mortality (0.829) in all patients. Addition of age and BMI into a multivariate log model improved mortality prediction (AUC = 0.906). CONCLUSION: The deep learning algorithm provides an accurate and interpretable assessment of the disease burden in COVID-19 pneumonia on chest radiographs. The reported severity scores correlate with expert assessment and accurately predicts important clinical outcomes. The algorithm contributes additional prognostic information not currently incorporated into patient management.

Unraveling function and diversity of bacterial lectins in the human microbiome.

The mechanisms by which commensal organisms affect human physiology remain poorly understood. Lectins are non-enzymatic carbohydrate binding proteins that all organisms employ as part of establishing a niche, evading host-defenses and protecting against pathogens. Although lectins have been extensively studied in plants, bacterial pathogens and human immune cells for their role in disease pathophysiology and as therapeutics, the role of bacterial lectins in the human microbiome is largely unexplored. Here we report on the characterization of a lectin produced by a common human associated bacterium that interacts with myeloid cells in the blood and intestine. In mouse and cell-based models, we demonstrate that this lectin induces distinct immunologic responses in peripheral and intestinal leukocytes and that these responses are specific to monocytes, macrophages and dendritic cells. Our analysis of human microbiota sequencing data reveal thousands of unique sequences that are predicted to encode lectins, many of which are highly prevalent in the human microbiome yet completely uncharacterized. Based on the varied domain architectures of these lectins we predict they will have diverse effects on the human host. The systematic investigation of lectins in the human microbiome should improve our understanding of human health and provide new therapeutic opportunities.

Bioinformatic prospecting and synthesis of a bifunctional lipopeptide antibiotic that evades resistance.

Emerging resistance to currently used antibiotics is a global public health crisis. Because most of the biosynthetic capacity within the bacterial kingdom has remained silent in previous antibiotic discovery efforts, uncharacterized biosynthetic gene clusters found in bacterial genome-sequencing studies remain an appealing source of antibiotics with distinctive modes of action. Here, we report the discovery of a naturally inspired lipopeptide antibiotic called cilagicin, which we chemically synthesized on the basis of a detailed bioinformatic analysis of the cil biosynthetic gene cluster. Cilagicin's ability to sequester two distinct, indispensable undecaprenyl phosphates used in cell wall biosynthesis, together with the absence of detectable resistance in laboratory tests and among multidrug-resistant clinical isolates, makes it an appealing candidate for combating antibiotic-resistant pathogens.

Phylogenomics of braconid wasps (Hymenoptera, Braconidae) sheds light on classification and the evolution of parasitoid life history traits.

The parasitoid lifestyle is largely regarded as a key innovation that contributed to the evolutionary success and extreme species richness of the order Hymenoptera. Understanding the phylogenetic history of hyperdiverse parasitoid groups is a fundamental step in elucidating the evolution of biological traits linked to parasitoidism. We used a genomic-scale dataset based on ultra-conserved elements and the most comprehensive taxon sampling to date to estimate the evolutionary relationships of Braconidae, the second largest family of Hymenoptera. Based on our results, we propose Braconidae to comprise 41 extant subfamilies, confirmed a number of subfamilial placements and proposed subfamily-level taxonomic changes, notably the restoration of Trachypetinae stat. rev. and Masoninae stat. rev. as subfamilies of Braconidae, confirmation that Apozyx penyai Mason belongs in Braconidae placed in the subfamily Apozyginae and the recognition of Ichneutinae sensu stricto and Proteropinae as non-cyclostome subfamilies robustly supported in a phylogenetic context. The correlation between koinobiosis with endoparasitoidism and idiobiosis with ectoparasitoidism, long thought to be an important aspect in parasitoid life history, was formally tested and confirmed in a phylogenetic framework. Using ancestral reconstruction methods based on both parsimony and maximum likelihood, we suggest that the ancestor of the braconoid complex was a koinobiont endoparasitoid, as was that of the cyclostome sensu lato clade. Our results also provide strong evidence for one transition from endo- to ectoparasitoidism and three reversals back to endoparasitoidism within the cyclostome sensu stricto lineage. Transitions of koino- and idiobiosis were identical to those inferred for endo- versus ectoparasitoidism, except with one additional reversal back to koinobiosis in the small subfamily Rhysipolinae.