Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. Bacillus subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after Escherichia coli and Thermus thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are a significant cause of both epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system for HuNoVs was a major obstacle in studying virus replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We previously optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present additional advancements to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs made from human embryonic stem cell-derived human intestinal organoids that were transplanted into mice (H9tHIEs), genetically-engineered (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ KO ]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4 FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. Importance: Human noroviruses (HuNoVs) are very contagious and cause significant acute gastroenteritis globally, but studying them has been hindered by the lack of a reproducible culture system for nearly 50 years. This barrier was overcome by successfully cultivating multiple HuNoV strains in human intestinal enteroids (HIEs), advancing HuNoV research. We previously optimized culture conditions and developed genetically modified HIEs to enhance HuNoV replication. In this study, we tested different media, unique HIE lines, and additional virus strains, evaluating HuNoV infectivity in new HIE models. These models include HIEs from various intestinal segments of adult donors, human embryonic stem cell-derived HIEs transplanted into mice (H9tHIEs), genetically-engineered HIEs (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ KO ]), HIEs from a common variable immunodeficiency (CVID) patient, and from infants. Our findings show that adult small intestinal HIEs, H9tHIEs, CVID patient HIEs, and infant HIEs support HuNoV replication with segment and strain-specific differences. J4 FUT2-KI HIEs exhibited the highest susceptibility, allowing cultivation of a broader range of HuNoV strains. These results enhance the understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.

A complementary metaproteomic approach to interrogate microbiome cultivated from clinical colon biopsies.

The human gut microbiome plays a vital role in preserving individual health and is intricately involved in essential functions. Imbalances or dysbiosis within the microbiome can significantly impact human health and are associated with many diseases. Several metaproteomics platforms are currently available to study microbial proteins within complex microbial communities. In this study, we attempted to develop an integrated pipeline to provide deeper insights into both the taxonomic and functional aspects of the cultivated human gut microbiomes derived from clinical colon biopsies. We combined a rapid peptide search by MSFragger against the Unified Human Gastrointestinal Protein database and the taxonomic and functional analyses with Unipept Desktop and MetaLab-MAG. Across seven samples, we identified and matched nearly 36,000 unique peptides to approximately 300 species and 11 phyla. Unipept Desktop provided gene ontology, InterPro entries, and enzyme commission number annotations, facilitating the identification of relevant metabolic pathways. MetaLab-MAG contributed functional annotations through Clusters of Orthologous Genes and Non-supervised Orthologous Groups categories. These results unveiled functional similarities and differences among the samples. This integrated pipeline holds the potential to provide deeper insights into the taxonomy and functions of the human gut microbiome for interrogating the intricate connections between microbiome balance and diseases.

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar KD.

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer-ligand complex with a remarkable set of properties: Its unprecedented KD of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

Artificial RNA tags that tightly bind fluorogenic ligands have many RNA imaging and RNA-protein biomolecular purification applications. Here, we report and structurally characterize a very small (20-nt) biologically compatible G-quadruplex based aptamer that can be inserted into commonly found GNRA tetraloops. This aptamer binds its fluorogenic ligand with an unprecedented picomolar binding affinity and is very stable against thermal and chemical insults. As the ligand can be modified to include biotin, this RNA tag can also be bound to streptavidin magnetic beads. After washing, tagged RNA can be cleanly eluted by exposing the beads to intense green light, which photobleaches the bound fluorogenic ligand, triggering the release of the bound RNA complex.

A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies.

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe ß-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. ß-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. ß-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that ß-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that ß-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Targeted sampling of natural product space to identify bioactive natural product-like polyketide macrolides.

Polyketide or polyketide-like macrolides (pMLs) continue to serve as a source of inspiration for drug discovery. However, their inherent structural and stereochemical complexity challenges efforts to explore related regions of chemical space more broadly. Here, we report a strategy termed the Targeted Sampling of Natural Product space (TSNaP) that is designed to identify and assess regions of chemical space bounded by this important class of molecules. Using TSNaP, a family of tetrahydrofuran-containing pMLs are computationally assembled from pML inspired building blocks to provide a large collection of natural product-like virtual pMLs. By scoring functional group and volumetric overlap against their natural counterparts, a collection of compounds are prioritized for targeted synthesis. Using a modular and stereoselective synthetic approach, a library of polyketide-like macrolides are prepared to sample these unpopulated regions of pML chemical space. Validation of this TSNaP approach by screening this library against a panel of whole-cell biological assays, reveals hit rates exceeding those typically encountered in small molecule libraries. This study suggests that the TSNaP approach may be more broadly useful for the design of improved chemical libraries for drug discovery.

Enantioselective Total Synthesis of the Marine Macrolides Salarins A and C.

Here we report the first total synthesis of the marine macrolide salarin C, a potent anticancer agent, and demonstrate the biomimetic oxidation-Wasserman rearrangement to access salarin A. This synthesis relies on L-proline catalysis to install a chlorohydrin function that masks the sensitive C16-C17 epoxide and potentially mimics the biosynthesis of these compounds where a related chlorohydrin may yield both THF- and epoxide-containing salarins. Additional and key features of the synthesis include (i) macrocycle formation via ring-closing metathesis, (ii) macrocyclic substrate-controlled epoxidation of the C12-C13 allylic alcohol, and (iii) a late-stage Julia-Kocienski olefination to install the side chain. Importantly, this work provides a platform for the synthesis of other salarins and analogues of these potentially important anticancer natural products.

sp3 -Rich Heterocycle Synthesis on DNA: Application to DNA-Encoded Library Production.

DNA encoded library (DEL) synthesis represents a convenient means to produce, annotate and store large collections of compounds in a small volume. While DELs are well suited for drug discovery campaigns, the chemistry used in their production must be compatible with the DNA tag, which can limit compound class accessibility. As a result, most DELs are heavily populated with peptidomimetic and sp2 -rich molecules. Herein, we show that sp3 -rich mono- and bicyclic heterocycles can be made on DNA from ketochlorohydrin aldol products through a reductive amination and cyclization process. The resulting hydroxypyrrolidines possess structural features that are desirable for DELs and target a distinct region of pharmaceutically relevant chemical space.

Resveratrol for the Management of Human Health: How Far Have We Come? A Systematic Review of Resveratrol Clinical Trials to Highlight Gaps and Opportunities.

Resveratrol has long been proposed as being beneficial to human health across multiple morbidities, yet there is currently no conclusive clinical evidence to advocate its recommendation in any healthcare setting. A large cohort with high-quality clinical data and clearly defined biomarkers or endpoints are required to draw meaningful conclusions. This systematic review compiles every clinical trial conducted using a defined dose of resveratrol in a purified form across multiple morbidities to highlight the current 'state-of-play' and knowledge gaps, informing future trial designs to facilitate the realisation of resveratrol's potential benefits to human health. Over the last 20 years, there have been almost 200 studies evaluating resveratrol across at least 24 indications, including cancer, menopause symptoms, diabetes, metabolic syndrome, and cardiovascular disease. There are currently no consensus treatment regimens for any given condition or endpoint, beyond the fact that resveratrol is generally well-tolerated at a dose of up to 1 g/day. Additionally, resveratrol consistently reduces inflammatory markers and improves aspects of a dysregulated metabolism. In conclusion, over the last 20 years, the increasing weight of clinical evidence suggests resveratrol can benefit human health, but more large, high-quality clinical trials are required to transition this intriguing compound from health food shops to the clinic.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Vaginal microbial dynamics and pathogen colonization in a humanized microbiota mouse model.

Vaginal microbial composition is associated with differential risk of urogenital infection. Although Lactobacillus spp. are thought to confer protection against infection, the lack of in vivo models resembling the human vaginal microbiota remains a prominent barrier to mechanistic discovery. Using 16S rRNA amplicon sequencing of C57BL/6J female mice, we found that vaginal microbial composition varies within and between colonies across three vivaria. Noting vaginal microbial plasticity in conventional mice, we assessed the vaginal microbiome of humanized microbiota mice (HMbmice). Like the community structure in conventional mice, HMbmice vaginal microbiota clustered into community state types but, uniquely, HMbmice communities were frequently dominated by Lactobacillus or Enterobacteriaceae. Compared to conventional mice, HMbmice were less susceptible to uterine ascension by urogenital pathobionts group B Streptococcus (GBS) and Prevotella bivia. Although Escherichia and Lactobacillus both correlated with the absence of uterine GBS, vaginal pre-inoculation with exogenous HMbmouse-derived E. coli, but not Ligilactobacillus murinus, reduced vaginal GBS burden. Overall, HMbmice serve as a useful model to elucidate the role of endogenous microbes in conferring protection against urogenital pathogens.

Development and application of decatungstate catalyzed C-H 18F- and 19F-fluorination, fluoroalkylation and beyond.

Over the past few decades, photocatalytic C-H functionalization reactions have received increasing attention due to the often mild reaction conditions and complementary selectivities to conventional functionalization processes. Now, photocatalytic C-H functionalization is a widely employed tool, supporting activities ranging from complex molecule synthesis to late-stage structure-activity relationship studies. In this perspective, we will discuss our efforts in developing a photocatalytic decatungstate catalyzed C-H fluorination reaction as well as its practical application realized through collaborations with industry partners at Hoffmann-La Roche and Merck, and extension to radiofluorination with radiopharmaceutical chemists and imaging experts at TRIUMF and the BC Cancer Agency. Importantly, we feel that our efforts address a question of utility posed by Professor Tobias Ritter in "Late-Stage Fluorination: Fancy Novelty or Useful Tool?" (ACIE, 2015, 54, 3216). In addition, we will discuss decatungstate catalyzed C-H fluoroalkylation and the interesting electrostatic effects observed in decatungstate-catalyzed C-H functionalization. We hope this perspective will inspire other researchers to explore the use of decatungstate for the purposes of photocatalytic C-H functionalization and further advance the exploitation of electrostatic effects for both rate acceleration and directing effects in these reactions.

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling.

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit, the anti-inflammatory immune system, wound healing, normal social behavior in mice, and prevents bone reabsorption. Oxytocin impacts these functions and oxytocin signaling is required for L. reuteri-mediated wound healing and social behavior; however, the events in the gut leading to oxytocin stimulation and beneficial effects are unknown. Here we report evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We find that L. reuteri facilitates oxytocin secretion from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

Potent and Selective Cell-Active Iminosugar Inhibitors of Human α-N-Acetylgalactosaminidase (α-NAGAL).

Glycoside hydrolases (GHs) are a class of enzymes with emerging roles in a range of disease. Selective GH inhibitors are sought to better understand their functions and assess the therapeutic potential of modulating their activities. Iminosugars are a promising class of GH inhibitors but typically lack the selectivity required to accurately perturb biological systems. Here, we describe a concise synthesis of iminosugar inhibitors of N-acetyl-α-galactosaminidase (α-NAGAL), the GH responsible for cleaving terminal α-N-acetylgalactosamine residues from glycoproteins and other glycoconjugates. Starting from non-carbohydrate precursors, this modular synthesis supported the identification of a potent (490 nM) and α-NAGAL selective (∼200-fold) guanidino-containing derivative DGJNGuan. To illustrate the cellular activity of this new inhibitor, we developed a quantitative fluorescence image-based method to measure levels of the Tn-antigen, a cellular glycoprotein substrate of α-NAGAL. Using this assay, we show that DGJNGuan exhibits excellent inhibition of α-NAGAL within cells using patient derived fibroblasts (EC50 =150 nM). Moreover, in vitro and in cell assays to assess levels of lysosomal ß-hexosaminidase substrate ganglioside GM2 show that DGJNGuan is selective whereas DGJNAc exhibits off-target inhibition both in vitro and within cells. DGJNGuan is a readily produced and selective tool compound that should prove useful for investigating the physiological roles of α-NAGAL.

Low microplastic loads in riverine European eel (Anguilla anguilla) from southwest England during their marine-freshwater transition.

The microplastic loads in elvers of the critically endangered European eel Anguilla anguilla, sampled in the lower reaches of three English rivers, were very low (incidence: 3.3%, mean ± s.d.: 0.03 ± 0.18 particles) and did not vary with body length or between rivers. Particles were mostly black, polyolefins, fibres and fragments of size 101-200 µm. Current levels indicate a low contamination pressure locally and, consequently, management efforts might prioritise mitigating the effects of other stressors affecting the species.

An α-chloroaldehyde-based formal synthesis of eribulin.

Eribulin (Halaven) is the most structurally complex non-peptidic drug made by total synthesis and has challenged preconceptions of synthetic feasibility in drug discovery and development. However, despite decades of research, the synthesis and manufacture of eribulin remains a daunting task. Here, we report syntheses of the most complex fragment of eribulin (C14-C35) used in two distinct industrial routes to this important anticancer drug. Our convergent strategy relies on a doubly diastereoselective Corey-Chaykovsky reaction to affect the union of two tetrahydrofuran-containing subunits. Notably, this process relies exclusively on enantiomerically enriched α-chloroaldehydes as building blocks for constructing the three densely functionalized oxygen heterocycles found in the C14-C35 fragment and all associated stereocenters. Overall, eribulin can now be produced in a total of 52 steps, which is a significant reduction from that reported in both academic and industrial syntheses.