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Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians' diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli-compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs-e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer-as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.

The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.

IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors on the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing identified IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells.

Increased extracellular Ca 2+ concentrations ([Ca 2+ ] ex ) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca 2+ ] ex . Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1β release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1β release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca 2+ ] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA. How extracellular calcium can trigger Nlrp3 inflammasome activation has been somewhat controversial and unclear. Here the authors show calciprotein particles are taken up by myeloid cells via calcium-sensing receptor-dependent macropinocytosis in response to high levels of extracellular Ca2+ and this pathway might be critical to inflammatory conditions.

Background In Australia, methamphetamine use is a significant public health concern, and is common among people involved with the criminal justice system. This study aimed to investigate the prevalence and correlates of adult imprisonment history among adults who primarily smoke methamphetamine. Methods A cross-sectional study was conducted using baseline data from ‘VMAX’, a cohort of adults who regularly use methamphetamine. Data were collected between June 2016 and March 2020 from 718 participants. Sampling methods included convenience and respondent-driven sampling. Prison exposure was measured by asking if participants had ever been imprisoned due to a conviction (and was distinguished from juvenile detention). Logistic regression was used to examine how this correlated with socio-demographics, drug use, mental health, and criminogenic characteristics. Results Nearly one-third (30%) of 718 participants reported having been imprisoned. Increased odds of reporting a history of imprisonment were found for participants reporting older age, male gender, non-metropolitan residential location, past-year homelessness, not being currently employed, schooling  ≤  Year 9, ≥ weekly methamphetamine use, past-year illicit opioid use, injecting drug use history, and juvenile detention history. In contrast, participants reporting past-year other illicit stimulant (cocaine, ecstasy, illicit pharmaceutical stimulant) use were less likely to report a history of imprisonment. Conclusions Social characteristics, patterns of drug use, and juvenile detention history were found to be correlated with imprisonment history. These findings point to the importance of providing targeted services to address characteristics of social disadvantage and drug use behaviours among people who use drugs, including among people who primarily smoke methamphetamine.

The German twin family study ‘TwinLife’ was designed to enhance our understanding of the development of social inequalities over the life course. The interdisciplinary project investigates mechanisms of social inequalities across the lifespan by taking into account psychological as well as social mechanisms, and their genetic origin as well as the interaction and covariation between these factors. Main characteristics of the study are: (1) a multidimensional perspective on social inequalities, (2) the assessment of developmental trajectories in childhood, adolescence, and young adulthood in a longitudinal design by using (3) a combination of a multi-cohort cross-sequential and an extended twin family design, while (4) capturing a large variation of behavioral and environmental factors in a representative sample of about 4,000 German twin families. In the present article, we first introduce the theoretical and empirical background of the TwinLife study, and second, describe the design, content, and implementation of TwinLife. Since the data will be made available as scientific use file, we also illustrate research possibilities provided by this project to the scientific community.

Typhoid persists as a major cause of global morbidity. While several licensed vaccines to prevent typhoid are available, they are of only moderate efficacy and unsuitable for use in children less than two years of age. Development of new efficacious vaccines is complicated by the human host-restriction of Salmonella enterica serovar Typhi (S. Typhi) and lack of clear correlates of protection. In this study, we aimed to evaluate the protective efficacy of a single dose of the oral vaccine candidate, M01ZH09, in susceptible volunteers by direct typhoid challenge. We performed a randomised, double-blind, placebo-controlled trial in healthy adult participants at a single centre in Oxford (UK). Participants were allocated to receive one dose of double-blinded M01ZH09 or placebo or 3-doses of open-label Ty21a. Twenty-eight days after vaccination, participants were challenged with 104CFU S. Typhi Quailes strain. The efficacy of M01ZH09 compared with placebo (primary outcome) was assessed as the percentage of participants reaching pre-defined endpoints constituting typhoid diagnosis (fever and/or bacteraemia) during the 14 days after challenge. Ninety-nine participants were randomised to receive M01ZH09 (n = 33), placebo (n = 33) or 3-doses of Ty21a (n = 33). After challenge, typhoid was diagnosed in 18/31 (58.1% [95% CI 39.1 to 75.5]) M01ZH09, 20/30 (66.7% [47.2 to 87.2]) placebo, and 13/30 (43.3% [25.5 to 62.6]) Ty21a vaccine recipients. Vaccine efficacy (VE) for one dose of M01ZH09 was 13% [95% CI -29 to 41] and 35% [-5 to 60] for 3-doses of Ty21a. Retrospective multivariable analyses demonstrated that pre-existing anti-Vi antibody significantly reduced susceptibility to infection after challenge; a 1 log increase in anti-Vi IgG resulting in a 71% decrease in the hazard ratio of typhoid diagnosis ([95% CI 30 to 88%], p = 0.006) during the 14 day challenge period. Limitations to the study included the requirement to limit the challenge period prior to treatment to 2 weeks, the intensity of the study procedures and the high challenge dose used resulting in a stringent model. Despite successfully demonstrating the use of a human challenge study to directly evaluate vaccine efficacy, a single-dose M01ZH09 failed to demonstrate significant protection after challenge with virulent Salmonella Typhi in this model. Anti-Vi antibody detected prior to vaccination played a major role in outcome after challenge. ClinicalTrials.gov (NCT01405521) and EudraCT (number 2011-000381-35).

Background Medium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria. They activate G protein-coupled receptors, including GPR84 and HCA 3 , which regulate metabolism and immune functions. Although both receptors are coupled to G i proteins, share at least one agonist and show overlapping tissue expression, GPR84 exerts pro-inflammatory effects whereas HCA 3 is involved in anti-inflammatory responses. Here, we analyzed signaling kinetics of both HCA 3 and GPR84, to unravel signal transduction components that may explain their physiological differences. Methods To study the signaling kinetics and components involved in signal transduction of both receptors we applied the label-free dynamic mass redistribution technology in combination with classical cAMP, ERK signaling and β-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models. Results We present strong evidence for a natural biased signaling of structurally highly similar agonists at HCA 3 and GPR84. We show that HCA 3 signaling and trafficking depends on dynamin-2 function. Activation of HCA 3 by 3-hydroxyoctanoic acid but not 3-hydroxydecanoic acid leads to β-arrestin-2 recruitment, which is relevant for cell-cell adhesion. GPR84 stimulation with 3-hydroxydecanoic acid causes a sustained ERK activation but activation of GPR84 is not followed by β-arrestin-2 recruitment. Conclusions In summary, our results highlight that biased agonism is a physiological property of HCA 3 and GPR84 with relevance for innate immune functions potentially to differentiate between endogenous, non-pathogenic compounds and compounds originating from e.g. pathogenic bacteria. 9ZbwrhpX5Yj3XBGuPG7oW- Video Abstract. Graphical abstract

Background The adaptation of cellular metabolism is considered a hallmark of cancer. Oncogenic signaling pathways support tumorigenesis and cancer progression through the induction of certain metabolic phenotypes associated with altered regulation of key metabolic enzymes. Hydroxycarboxylic acid receptor 2 (HCA 2 ) is a G protein-coupled receptor previously shown to act as a tumor suppressor. Here, we aimed to unveil the connection between cellular metabolism and HCA 2 in BT-474 cells. Moreover, we intend to clarify how well this metabolic phenotype is reflected in transcriptional changes and metabolite levels as determined by global metabolomics analyses. Methods We performed both, siRNA mediated knockdown of HCA 2 and stimulation with the HCA 2 -specific agonist monomethyl fumarate. Seahorse technology was used to determine the role of HCA 2 in BT-474 breast cancer cell metabolism and its potential to induce a switch in the metabolic phenotype in the presence of different energy substrates. Changes in the mRNA expression of metabolic enzymes were detected with real-time quantitative PCR (RT-qPCR). Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolic profiling was used to determine changes in metabolite levels. Results Knockdown or stimulation of HCA 2 induced changes in the metabolic phenotype of BT474 cells dependent on the availability of energy substrates. The presence of HCA 2 was associated with increased glycolytic flux with no fatty acids available. This was reflected in the increased mRNA expression of the glycolytic enzymes PFKFB4 and PKM2, which are known to promote the Warburg effect and have been described as prognostic markers in different types of cancer. With exogenous palmitate present, HCA 2 caused elevated fatty acid oxidation and likely lipolysis. The increase in lipolysis was also detectable at the transcriptional level of ATGL and the metabolite levels of palmitic and stearic acid. Conclusions We combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA 2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA 2 , for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.