Critical Assessment of Metagenome Interpretation: the second round of challenges.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

Liver-specific adiponectin gene therapy suppresses microglial NLRP3-inflammasome activation for treating Alzheimer's disease.

Adiponectin (APN) is an adipokine which predominantly expresses in adipocytes with neuroprotective and anti-inflammatory effects. We have recently indicated that circulatory trimeric APN can enter the brain by crossing the blood-brain barrier (BBB) and modulate microglia-mediated neuroinflammation. Here, we found that the microglial NLR family pyrin domain containing 3 (NLRP3)-inflammasome activation was exacerbated in APN-/-5xFAD mice in age-dependent manner. The focus of this study was to develop a new and tractable therapeutic approach for treating Alzheimer's disease (AD)-related pathology in 5xFAD mice using peripheral APN gene therapy. We have generated and transduced adeno-associated virus (AAV2/8) expressing the mouse mutated APN gene (APNC39S) into the liver of 5xFAD mice that generated only low-molecular-weight trimeric APN (APNTri). Single dose of AAV2/8-APNC39S in the liver increased circulatory and cerebral APN levels indicating the overexpressed APNTri was able to cross the BBB. Overexpression of APNTri decreased both the soluble and fibrillar Aß in the brains of 5xFAD mice. AAV2/8-APNTri treatment reduced Aß-induced IL-1ß and IL-18 secretion by suppressing microglial NLRP3-inflammasome activation. The memory functions improved significantly in AAV-APNTri-treated 5xFAD mice with reduction of dystrophic neurites. These findings demonstrate that peripheral gene delivery to overexpress trimeric APN can be a potential therapy for AD.

Chronic oral administration of adipoRon reverses cognitive impairments and ameliorates neuropathology in an Alzheimer's disease mouse model.

Circulating adiponectin (APN) levels decrease with age and obesity. On the other hand, a reduction in APN levels is associated with neurodegeneration and neuroinflammation. We previously showed that aged adiponectin knockout (APN-/-) mice developed Alzheimer's like pathologies, cerebral insulin resistance, and cognitive impairments. More recently, we also demonstrated that APN deficiency increased Aß-induced microglia activation and neuroinflammatory responses in 5xFAD mice. There is compelling evidence that deregulated insulin activities or cerebral insulin resistance contributes to neuroinflammation and Alzheimer's disease (AD) pathogenesis. Here, we demonstrated that APN levels were reduced in the brain of AD patients and 5xFAD mice. We crossbred 5xFAD mice with APN-/- mice to generate APN-deficient 5xFAD (5xFAD;APN-/-). APN deficiency in 5xFAD mice accelerated amyloid loading, increased cerebral amyloid angiopathy, and reduced insulin-signaling activities. Pharmacokinetics study demonstrated adipoRon (APN receptor agonist) was a blood-brain barrier penetrant. AdipoRon improved neuronal insulin-signaling activities and insulin sensitivity in vitro and in vivo. Chronic adipoRon treatment improved spatial memory functions and significantly rescued neuronal and synaptic loss in 5xFAD and 5xFAD;APN-/- mice. AdipoRon lowered plaque and Aß levels in AD mice. AdipoRon also exerted anti-inflammatory effects by reducing microglial and astrocytes activation as well as suppressing cerebral cytokines levels. The microglial phagocytic activity toward Aß was restored after adipoRon treatment. Our results indicated that adipoRon exerts multiple beneficial effects providing important therapeutic implications. We propose chronic adipoRon administration as a potential treatment for AD.

Adapting medical education during crisis: Student-Faculty partnerships as an enabler of success.

Restrictions imposed by the COVID-19 pandemic have required medical educators to reimagine almost every aspect of undergraduate medical training, including curriculum delivery and assessments in a short timeline. In this personal view article, executive members of the University of Toronto medical student government and Faculty leads of pre-clerkship and clerkship education highlight five practical ways in which a student-Faculty partnership enabled the rapid and smooth adaptation of curricula during the COVID-19 pandemic. These included involving students as partners in decision making to contribute learner perspectives early, agile and collaborative meeting structures, frequent and consistent communication with the student body, providing learners with Faculty perspectives from the frontlines, and striking a balance in the level of feedback collected from students. These strategies may be of utility to medical administrators, educators, and student leaders in future crises affecting medical learners.

Bifunctional Doscadenamides Activate Quorum Sensing in Gram-Negative Bacteria and Synergize with TRAIL to Induce Apoptosis in Cancer Cells.

New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa, characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi, indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.

Autometa: automated extraction of microbial genomes from individual shotgun metagenomes.

Shotgun metagenomics is a powerful, high-resolution technique enabling the study of microbial communities in situ. However, species-level resolution is only achieved after a process of 'binning' where contigs predicted to originate from the same genome are clustered. Such culture-independent sequencing frequently unearths novel microbes, and so various methods have been devised for reference-free binning. As novel microbiomes of increasing complexity are explored, sometimes associated with non-model hosts, robust automated binning methods are required. Existing methods struggle with eukaryotic contamination and cannot handle highly complex single metagenomes. We therefore developed an automated binning pipeline, termed 'Autometa', to address these issues. This command-line application integrates sequence homology, nucleotide composition, coverage and the presence of single-copy marker genes to separate microbial genomes from non-model host genomes and other eukaryotic contaminants, before deconvoluting individual genomes from single metagenomes. The method is able to effectively separate over 1000 genomes from a metagenome, allowing the study of previously intractably complex environments at the level of single species. Autometa is freely available at https://bitbucket.org/jason_c_kwan/autometa and as a docker image at https://hub.docker.com/r/jasonkwan/autometa under the GNU Affero General Public License 3 (AGPL 3).

Memantine ameliorates motor impairments and pathologies in a mouse model of neuromyelitis optica spectrum disorders.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) are central nervous system (CNS) autoimmune inflammatory demyelinating diseases characterized by recurrent episodes of acute optic neuritis and transverse myelitis. Aquaporin-4 immunoglobulin G (AQP4-IgG) autoantibodies, which target the water channel aquaporin-4 (AQP4) on astrocytic membrane, are pathogenic in NMOSD. Glutamate excitotoxicity, which is triggered by internalization of AQP4-glutamate transporter complex after AQP4-IgG binding to astrocytes, is involved in early NMOSD pathophysiologies. We studied the effects of memantine, a N-methyl-D-aspartate (NMDA) receptor antagonist, on motor impairments and spinal cord pathologies in mice which received human AQP4-IgG. METHODS: Purified IgG from AQP4-IgG-seropositive NMOSD patients were passively transferred to adult C57BL/6 mice with disrupted blood-brain barrier. Memantine was administered by oral gavage. Motor impairments of the mice were assessed by beam walking test. Spinal cords of the mice were assessed by immunofluorescence and ELISA. RESULTS: Oral administration of memantine ameliorated the motor impairments induced by AQP4-IgG, no matter the treatment was initiated before (preventive) or after (therapeutic) disease flare. Memantine profoundly reduced AQP4 and astrocyte loss, and attenuated demyelination and axonal loss in the spinal cord of mice which had received AQP4-IgG. The protective effects of memantine were associated with inhibition of apoptosis and suppression of neuroinflammation, with decrease in microglia activation and neutrophil infiltration and reduction of increase in levels of proinflammatory cytokines including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). In addition, memantine elevated growth factors including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) in the spinal cord. CONCLUSIONS: Our findings support that glutamate excitotoxicity and neuroinflammation play important roles in complement-independent pathophysiology during early development of NMOSD lesions, and highlight the potential of oral memantine as a therapeutic agent in NMOSD acute attacks.

Accessing chemical diversity from the uncultivated symbionts of small marine animals.

Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for new bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context wherein biological interactions take place.

Adiponectin suppresses amyloid-ß oligomer (AßO)-induced inflammatory response of microglia via AdipoR1-AMPK-NF-κB signaling pathway.

BACKGROUND: Microglia-mediated neuroinflammation is important in Alzheimer's disease (AD) pathogenesis. Extracellular deposition of ß-amyloid (Aß), a major pathological hallmark of AD, can induce microglia activation. Adiponectin (APN), an adipocyte-derived adipokine, exerts anti-inflammatory effects in the periphery and brain. Chronic APN deficiency leads to cognitive impairment and AD-like pathologies in aged mice. Here, we aim to study the role of APN in regulating microglia-mediated neuroinflammation in AD. METHODS: Inflammatory response of cultured microglia (BV2 cells) to AßO and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor α [TNFα] and interleukin-1ß [IL-1ß]) in cultured medium before and after exposure to AßO, with and without APN pretreatment. Adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were targeted by small interference RNA. To study the neuroprotective effect of APN, cultured HT-22 hippocampal cells were treated with conditioned medium of AßO-exposed BV2 cells or were co-cultured with BV2 cells in transwells. The cytotoxicity of HT-22 hippocampal cells was assessed by MTT reduction. We generated APN-deficient AD mice (APN-/-5xFAD) by crossing APN-knockout mice with 5xFAD mice to determine the effects of APN deficiency on microglia-mediated neuroinflammation in AD. RESULTS: AdipoR1 and AdipoR2 were expressed in BV2 cells and microglia of mice. Pretreatment with APN for 2 h suppressed TNFα and IL-1ß release induced by AßO in BV2 cells. Additionally, APN rescued the decrease of AMPK phosphorylation and suppressed nuclear translocation of nuclear factor kappa B (NF-κB) induced by AßO. Compound C, an inhibitor of AMPK, abolished these effects of APN. Knockdown of AdipoR1, but not AdipoR2 in BV2 cells, inhibited the ability of APN to suppress proinflammatory cytokine release induced by AßO. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with AßO-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APN-/-5xFAD mice associated with upregulation of TNFα and IL-1ß in the cortex and hippocampus. CONCLUSIONS: Our findings demonstrate that APN inhibits inflammatory response of microglia to AßO via AdipoR1-AMPK-NF-κB signaling, and APN deficiency aggravates microglia activation and neuroinflammation in AD mice. APN may be a novel therapeutic agent for inhibiting neuroinflammation in AD.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Stenotrophomonas-Like Bacteria Are Widespread Symbionts in Cone Snail Venom Ducts.

Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized.IMPORTANCE In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world.

Interpreting Microbial Biosynthesis in the Genomic Age: Biological and Practical Considerations.

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo, we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam (slm) BGC.

The Stability Challenges of Oxygen Evolving Catalysts: Towards a Common Fundamental Understanding and Mitigation of Catalyst Degradation.

This Review addresses the technical challenges, scientific basis, recent progress, and outlook with respect to the stability and degradation of catalysts for the oxygen evolution reaction (OER) operating at electrolyzer anodes in acidic environments with an emphasis on ion exchange membrane applications. First, the term "catalyst stability" is clarified, as well as current performance targets, major catalyst degradation mechanisms, and their mitigation strategies. Suitable in situ experimental methods are then evaluated to give insight into catalyst degradation and possible pathways to tune OER catalyst stability. Finally, the importance of identifying universal figures of merit for stability is highlighted, leading to a comprehensive accelerated lifetime test that could yield comparable performance data across different laboratories and catalyst types. The aim of this Review is to help disseminate and stress the important relationships between structure, composition, and stability of OER catalysts under different operating conditions.

Origin of Chemical Diversity in Prochloron-Tunicate Symbiosis.

UNLABELLED: Diversity-generating metabolism leads to the evolution of many different chemicals in living organisms. Here, by examining a marine symbiosis, we provide a precise evolutionary model of how nature generates a family of novel chemicals, the cyanobactins. We show that tunicates and their symbiotic Prochloron cyanobacteria share congruent phylogenies, indicating that Prochloron phylogeny is related to host phylogeny and not to external habitat or geography. We observe that Prochloron exchanges discrete functional genetic modules for cyanobactin secondary metabolite biosynthesis in an otherwise conserved genetic background. The module exchange leads to gain or loss of discrete chemical functional groups. Because the underlying enzymes exhibit broad substrate tolerance, discrete exchange of substrates and enzymes between Prochloron strains leads to the rapid generation of chemical novelty. These results have implications in choosing biochemical pathways and enzymes for engineered or combinatorial biosynthesis. IMPORTANCE: While most biosynthetic pathways lead to one or a few products, a subset of pathways are diversity generating and are capable of producing thousands to millions of derivatives. This property is highly useful in biotechnology since it enables biochemical or synthetic biological methods to create desired chemicals. A fundamental question has been how nature itself creates this chemical diversity. Here, by examining the symbiosis between coral reef animals and bacteria, we describe the genetic basis of chemical variation with unprecedented precision. New compounds from the cyanobactin family are created by either varying the substrate or importing needed enzymatic functions from other organisms or via both mechanisms. This natural process matches successful laboratory strategies to engineer the biosynthesis of new chemicals and teaches a new strategy to direct biosynthesis.

Lack of Overt Genome Reduction in the Bryostatin-Producing Bryozoan Symbiont "Candidatus Endobugula sertula".

The uncultured bacterial symbiont "Candidatus Endobugula sertula" is known to produce cytotoxic compounds called bryostatins, which protect the larvae of its host, Bugula neritina The symbiont has never been successfully cultured, and it was thought that its genome might be significantly reduced. Here, we took a shotgun metagenomics and metatranscriptomics approach to assemble and characterize the genome of "Ca Endobugula sertula." We found that it had specific metabolic deficiencies in the biosynthesis of certain amino acids but few other signs of genome degradation, such as small size, abundant pseudogenes, and low coding density. We also identified homologs to genes associated with insect pathogenesis in other gammaproteobacteria, and these genes may be involved in host-symbiont interactions and vertical transmission. Metatranscriptomics revealed that these genes were highly expressed in a reproductive host, along with bry genes for the biosynthesis of bryostatins. We identified two new putative bry genes fragmented from the main bry operon, accounting for previously missing enzymatic functions in the pathway. We also determined that a gene previously assigned to the pathway, bryS, is not expressed in reproductive tissue, suggesting that it is not involved in the production of bryostatins. Our findings suggest that "Ca Endobugula sertula" may be able to live outside the host if its metabolic deficiencies are alleviated by medium components, which is consistent with recent findings that it may be possible for "Ca Endobugula sertula" to be transmitted horizontally. IMPORTANCE: The bryostatins are potent protein kinase C activators that have been evaluated in clinical trials for a number of indications, including cancer and Alzheimer's disease. There is, therefore, considerable interest in securing a renewable supply of these compounds, which is currently only possible through aquaculture of Bugula neritina and total chemical synthesis. However, these approaches are labor-intensive and low-yielding and thus preclude the use of bryostatins as a viable therapeutic agent. Our genome assembly and transcriptome analysis for "Ca Endobugula sertula" shed light on the metabolism of this symbiont, potentially aiding isolation and culturing efforts. Our identification of additional bry genes may also facilitate efforts to express the complete pathway heterologously.

Genome streamlining and chemical defense in a coral reef symbiosis.

Secondary metabolites are ubiquitous in bacteria, but by definition, they are thought to be nonessential. Highly toxic secondary metabolites such as patellazoles have been isolated from marine tunicates, where their exceptional potency and abundance implies a role in chemical defense, but their biological source is unknown. Here, we describe the association of the tunicate Lissoclinum patella with a symbiotic α-proteobacterium, Candidatus Endolissoclinum faulkneri, and present chemical and biological evidence that the bacterium synthesizes patellazoles. We sequenced and assembled the complete Ca. E. faulkneri genome, directly from metagenomic DNA obtained from the tunicate, where it accounted for 0.6% of sequence data. We show that the large patellazoles biosynthetic pathway is maintained, whereas the remainder of the genome is undergoing extensive streamlining to eliminate unneeded genes. The preservation of this pathway in streamlined bacteria demonstrates that secondary metabolism is an essential component of the symbiotic interaction.

Grassypeptolides as natural inhibitors of dipeptidyl peptidase 8 and T-cell activation.

Natural products made by marine cyanobacteria are often highly modified peptides and depsipeptides that have the potential to act as inhibitors for proteases. In the interests of finding new protease inhibition activity and selectivity, grassypeptolide A (1) was screened against a panel of proteases and found to inhibit DPP8 selectively over DPP4. Grassypeptolides were also found to inhibit IL-2 production and proliferation in activated T-cells, consistent with a putative role of DPP8 in the immune system. These effects were also observed in Jurkat cells, and DPP activity in Jurkat cell cytosol was shown to be inhibited by grassypeptolides. In silico docking suggests two possible binding modes of grassypeptolides-at the active site of DPP8 and at one of the entrances to the internal cavity. Collectively these results suggest that grassypeptolides might be useful tool compounds in the study of DPP8 function.

Evaluation of class I HDAC isoform selectivity of largazole analogues.

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.

Discovery of a lagriamide polyketide by integrated genome mining, isotopic labeling, and untargeted metabolomics.

Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia. Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger, while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells.

Environmental metabolomics characterization of modern stromatolites and annotation of ibhayipeptolides.

Lithified layers of complex microbial mats known as microbialites are ubiquitous in the fossil record, and modern forms are increasingly identified globally. A key challenge to developing an understanding of microbialite formation and environmental role is how to investigate complex and diverse communities in situ. We selected living, layered microbialites (stromatolites) in a peritidal environment near Schoenmakerskop, Eastern Cape, South Africa to conduct a spatial survey mapping the composition and small molecule production of the microbial communities from environmental samples. Substrate core samples were collected from nine sampling stations ranging from the upper point of the freshwater inflow to the lower marine interface where tidal overtopping takes place. Substrate cores provided material for parallel analyses of microbial community diversity by 16S rRNA gene amplicon sequencing and metabolomics using LC-MS2. Species and metabolite diversities were correlated, and prominent specialized metabolites were targeted for preliminary characterization. A new series of cyclic hexadepsipeptides, named ibhayipeptolides, was most abundant in substrate cores of submerged microbialites. These results demonstrate the detection and identification of metabolites from mass-limited environmental samples and contribute knowledge about microbialite chemistry and biology, which facilitates future targeted studies of specialized metabolite function and biosynthesis.