Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers.

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2'-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose-response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

Intestinal carcinogenicity screening of environmental pollutants using organoid-based cell transformation assay.

The high incidence of colorectal cancer (CRC) is closely associated with environmental pollutant exposure. To identify potential intestinal carcinogens, we developed a cell transformation assay (CTA) using mouse adult stem cell-derived intestinal organoids (mASC-IOs) and assessed the transformation potential on 14 representative chemicals, including Cd, iPb, Cr-VI, iAs-III, Zn, Cu, PFOS, BPA, MEHP, AOM, DMH, MNNG, aspirin, and metformin. We optimized the experimental protocol based on cytotoxicity, amplification, and colony formation of chemical-treated mASC-IOs. In addition, we assessed the accuracy of in vitro study and the human tumor relevance through characterizing interdependence between cell-cell and cell-matrix adhesions, tumorigenicity, pathological feature of subcutaneous tumors, and CRC-related molecular signatures. Remarkably, the results of cell transformation in 14 chemicals showed a strong concordance with epidemiological findings (8/10) and in vivo mouse studies (12/14). In addition, we found that the increase in anchorage-independent growth was positively correlated with the tumorigenicity of tested chemicals. Through analyzing the dose-response relationship of anchorage-independent growth by benchmark dose (BMD) modeling, the potent intestinal carcinogens were identified, with their carcinogenic potency ranked from high to low as AOM, Cd, MEHP, Cr-VI, iAs-III, and DMH. Importantly, the activity of chemical-transformed mASC-IOs was associated with the degree of cellular differentiation of subcutaneous tumors, altered transcription of oncogenic genes, and activated pathways related to CRC development, including Apc, Trp53, Kras, Pik3ca, Smad4 genes, as well as WNT and BMP signaling pathways. Taken together, we successfully developed a mASC-IO-based CTA, which might serve as a potential alternative for intestinal carcinogenicity screening of chemicals.

Moderate body lipid accumulation in mice attenuated benzene-induced hematotoxicity via acceleration of benzene metabolism and clearance.

Recent population and animal studies have revealed a correlation between fat content and the severity of benzene-induced hematologic toxicity. However, the precise impact of lipid deposition on benzene-induced hematotoxicity and the underlying mechanisms remain unclear. In this study, we established a mouse model with moderate lipid accumulation by subjecting the mice to an 8-week high-fat diet (45% kcal from fat, HFD), followed by 28-day inhalation of benzene at doses of 0, 1, 10, and 100 ppm. The results showed that benzene exposure caused a dose-dependent reduction of peripheral white blood cell (WBC) counts in both diet groups. Notably, this reduction was less pronounced in the HFD-fed mice, suggesting that moderate lipid accumulation mitigates benzene-related hematotoxicity. To investigate the molecular basis for this effect, we performed bioinformatics analysis of high-throughput transcriptome sequencing data, which revealed that moderate lipid deposition alters mouse metabolism and stress tolerance towards xenobiotics. Consistently, the expression of key metabolic enzymes, such as Cyp2e1 and Gsta1, were upregulated in the HFD-fed mice upon benzene exposure. Furthermore, we utilized a real-time exhaled breath detection technique to monitor exhaled benzene metabolites, and the results indicated that moderate lipid deposition enhanced metabolic activation and increased the elimination of benzene metabolites. Collectively, these findings demonstrate that moderate lipid deposition confers reduced susceptibility to benzene-induced hematotoxicity in mice, at least in part, by accelerating benzene metabolism and clearance.

Assessing the Effects of Nicotinamide Mononucleotide Supplementation on Pulmonary Inflammation in Male Mice Subchronically Exposed to Ambient Particulate Matter.

BACKGROUND: Chronic lung injury and dysregulated cellular homeostasis in response to particulate matter (PM) exposure are closely associated with adverse health effects. However, an effective intervention for preventing the adverse health effects has not been developed. OBJECTIVES: This study aimed to evaluate the protective effects of nicotinamide mononucleotide (NMN) supplementation on lung injury and elucidate the mechanism by which NMN improved immune function following subchronic PM exposure. METHODS: Six-week-old male C57BL/6J mice were placed in a real-ambient PM exposure system or filtered air-equipped chambers (control) for 16 wk with or without NMN supplementation in drinking water (regarded as Con-H2O, Exp-H2O, Con-NMN and Exp-NMN groups, respectively) in Shijiazhuang City, China (n=20/group). The effects of NMN supplementation (500mg/kg) on PM-induced chronic pulmonary inflammation were assessed, and its mechanism was characterized using single-cell transcriptomic sequencing (scRNA-seq) analysis of whole lung cells. RESULTS: The NMN-treated mice exhibited higher NAD+ levels in multiple tissues. Following 16-wk PM exposure, slightly less pulmonary inflammation and less collagen deposition were noted in mice with NMN supplementation in response to real-ambient PM exposure (Exp-NMN group) compared with the Exp-H2O group (all p<0.05). Mouse lung tissue isolated from the Exp-NMN group was characterized by fewer neutrophils, monocyte-derived cells, fibroblasts, and myeloid-derived suppressor cells induced by subchronic PM exposure as detected by scRNA-seq transcriptomic analysis. The improved immune functions were further characterized by interleukin-17 signaling pathway inhibition and lower secretion of profibrotic cytokines in the Exp-NMN group compared with the Exp-H2O group. In addition, reduced proportions of differentiated myofibroblasts and profibrotic interstitial macrophages were identified in the NMN-supplemented mice in response to PM exposure. Furthermore, less immune function suppression and altered differentiation of pathological cell phenotypes NMN was related to intracellular lipid metabolism activation. DISCUSSION: Our novel findings suggest that NMN supplementation mitigated PM-induced lung injury by regulating immune functions and improving lipid metabolism in male mice, providing a putative intervention method for prevention of human health effects associated with PM exposure. https://doi.org/10.1289/EHP12259.

An integrative analysis of lipidomics and transcriptomics in various mouse brain regions in response to real-ambient PM2.5 exposure.

Exposure to Fine particulate matter (PM2.5) has been associated with various neurological disorders. However, the underlying mechanisms of PM2.5-induced adverse effects on the brain are still not fully defined. Multi-omics analyses could offer novel insights into the mechanisms of PM2.5-induced brain dysfunction. In this study, a real-ambient PM2.5 exposure system was applied to male C57BL/6 mice for 16 weeks, and lipidomics and transcriptomics analysis were performed in four brain regions. The findings revealed that PM2.5 exposure led to 548, 283, 304, and 174 differentially expressed genes (DEGs), as well as 184, 89, 228, and 49 distinctive lipids in the hippocampus, striatum, cerebellum, and olfactory bulb, respectively. Additionally, in most brain regions, PM2.5-induced DEGs were mainly involved in neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, and calcium signaling pathway, while PM2.5-altered lipidomic profile were primarily enriched in retrograde endocannabinoid signaling and biosynthesis of unsaturated fatty acids. Importantly, mRNA-lipid correlation networks revealed that PM2.5-altered lipids and DEGs were obviously enriched in pathways involving in bile acid biosynthesis, De novo fatty acid biosynthesis, and saturated fatty acids beta-oxidation in brain regions. Furthermore, multi-omics analyses revealed that the hippocampus was the most sensitive part to PM2.5 exposure. Specifically, dysregulation of Pla2g1b, Pla2g, Alox12, Alox15, and Gpx4 induced by PM2.5 were closely correlated to the disruption of alpha-linolenic acid, arachidonic acid and linoleic acid metabolism in the hippocampus. In summary, our findings highlight differential lipidomic and transcriptional signatures of various brain regions by real-ambient PM2.5 exposure, which will advance our understanding of potential mechanisms of PM2.5-induecd neurotoxicity.

Type 1 diabetes and diet-induced obesity predispose C57BL/6J mice to PM2.5-induced lung injury: a comparative study.

BACKGROUND: Pre-existing metabolic diseases may predispose individuals to particulate matter (PM)-induced adverse health effects. However, the differences in susceptibility of various metabolic diseases to PM-induced lung injury and their underlying mechanisms have yet to be fully elucidated. RESULTS: Type 1 diabetes (T1D) murine models were constructed by streptozotocin injection, while diet-induced obesity (DIO) models were generated by feeding 45% high-fat diet 6 weeks prior to and throughout the experiment. Mice were subjected to real-ambient PM exposure in Shijiazhuang City, China for 4 weeks at a mean PM2.5 concentration of 95.77 µg/m3. Lung and systemic injury were assessed, and the underlying mechanisms were explored through transcriptomics analysis. Compared with normal diet (ND)-fed mice, T1D mice exhibited severe hyperglycemia with a blood glucose of 350 mg/dL, while DIO mice displayed moderate obesity and marked dyslipidemia with a slightly elevated blood glucose of 180 mg/dL. T1D and DIO mice were susceptible to PM-induced lung injury, manifested by inflammatory changes such as interstitial neutrophil infiltration and alveolar septal thickening. Notably, the acute lung injury scores of T1D and DIO mice were higher by 79.57% and 48.47%, respectively, than that of ND-fed mice. Lung transcriptome analysis revealed that increased susceptibility to PM exposure was associated with perturbations in multiple pathways including glucose and lipid metabolism, inflammatory responses, oxidative stress, cellular senescence, and tissue remodeling. Functional experiments confirmed that changes in biomarkers of macrophage (F4/80), lipid peroxidation (4-HNE), cellular senescence (SA-ß-gal), and airway repair (CCSP) were most pronounced in the lungs of PM-exposed T1D mice. Furthermore, pathways associated with xenobiotic metabolism showed metabolic state- and tissue-specific perturbation patterns. Upon PM exposure, activation of nuclear receptor (NR) pathways and inhibition of the glutathione (GSH)-mediated detoxification pathway were evident in the lungs of T1D mice, and a significant upregulation of NR pathways was present in the livers of T1D mice. CONCLUSIONS: These differences might contribute to differential susceptibility to PM exposure between T1D and DIO mice. These findings provide new insights into the health risk assessment of PM exposure in populations with metabolic diseases.

Morphological alterations in C57BL/6 mouse intestinal organoids as a tool for predicting chemical-induced toxicity.

Intestinal organoid may serve as an alternative model for toxicity testing. However, the linkage between specific morphological alterations in organoids and chemical-induced toxicity has yet to be defined. Here, we generated C57BL/6 mouse intestinal organoids and conducted a morphology-based analysis on chemical-induced toxicity. Alterations in morphology were characterized by large spheroids, hyperplastic organoids, small spheroids, and protrusion-loss organoids, which responded in a concentration-dependent manner to the treatment of four metal(loid)s including cadmium (Cd), lead (Pb), hexavalent chromium (Cr-VI), and inorganic trivalent arsenic (iAs-III). Notably, alterations in organoid morphology characterized by abnormal morphology rate were correlated with specific intestinal toxic effects, including reduction in cell viability and differentiation, induction of apoptosis, dysfunction of mucus production, and damage to epithelial barrier upon repeated administration. The benchmark dose (BMDL10) values of morphological alterations (0.007-0.195 µM) were lower than those of conventional bioassays (0.010-0.907 µM). We also established that the morphologic features of organoids upon Cd, Pb, Cr-VI, or iAs-III treatment were metal specific, and mediated by Wnt, bone morphogenetic protein, apoptosis induction, and Notch signaling pathways, respectively. Collectively, these findings provide novel insights into the relevance of morphological alterations in organoids to specific toxic endpoints and identify specific morphological alterations as potential indicators of enterotoxicity.

Single-cell transcriptomics reveals immune dysregulation mediated by IL-17A in initiation of chronic lung injuries upon real-ambient particulate matter exposure.

BACKGROUND: Long-term exposure to fine particulate matter (PM2.5) increases susceptibility to chronic respiratory diseases, including inflammation and interstitial fibrosis. However, the regulatory mechanisms by which the immune response mediates the initiation of pulmonary fibrosis has yet to be fully characterized. This study aimed to illustrate the interplay between different cell clusters and key pathways in triggering chronic lung injuries in mice following PM exposure. RESULTS: Six-week-old C57BL/6J male mice were exposed to PM or filtered air for 16 weeks in a real-ambient PM exposure system in Shijiazhuang, China. The transcriptional profiles of whole lung cells following sub-chronic PM exposure were characterized by analysis of single-cell transcriptomics. The IL-17A knockout (IL-17A-/-) mouse model was utilized to determine whether the IL-17 signaling pathway mediated immune dysregulation in PM-induced chronic lung injuries. After 16-week PM exposure, chronic lung injuries with excessive collagen deposition and increased fibroblasts, neutrophils, and monocytes were noted concurrent with a decreased number of major classes of immune cells. Single-cell analysis showed that activation of the IL-17 signaling pathway was involved in the progression of pulmonary fibrosis upon sub-chronic PM exposure. Depletion of IL-17A led to significant decline in chronic lung injuries, which was mainly triggered by reduced recruitment of myeloid-derived suppressor cells (MDSCs) and downregulation of TGF-ß. CONCLUSION: These novel findings demonstrate that immunosuppression via the IL-17A pathway plays a critical role in the initiation of chronic lung injuries upon sub-chronic PM exposure.

Protein phosphatase 2A regulates cytotoxicity and drug resistance by dephosphorylating AHR and MDR1.

Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and metabolism. However, its specific actions in many of these critical pathways are unclear. To explore mechanisms underlying metabolic enzyme regulation in the liver, we investigated the key pathways involved in regulation of xenobiotic-metabolizing enzymes in a mouse model with hepatocyte-specific deletion of Ppp2r1a, encoding the Aα subunit of PP2A. We performed transcriptome and phosphoproteome analysis in mouse livers at the age of 3 months and identified 2695 differentially expressed genes and 549 upregulated phosphoproteins in homozygous knockout mouse livers compared with WT littermates. In particular, the expression of metabolic enzymes Cyp2e1, Cyp1a1, Cyp1a2, Mdr1a, and Abcg2 was dramatically altered in homozygous knockout mouse livers. We also demonstrated that activation of PP2A reversed the decline of metabolic enzyme expression in primary mouse hepatocytes. We found that specific PP2A holoenzymes were involved in metabolic enzyme induction through dephosphorylation of transcription factors, nuclear receptors, or the target enzymes themselves, leading to dysregulation of xenobiotic metabolism or drug-induced hepatotoxicity. Notably, we confirmed that a regulatory axis, PP2A B56α-aryl hydrocarbon receptor-Cyp1a1, was involved in benzo(a)pyrene-induced cytotoxicity through dephosphorylation of the metabolic nuclear receptor, aryl hydrocarbon receptor, at serine 36. In addition, we showed that PP2A B56δ complexes directly dephosphorylated the multidrug efflux pump MDR1 (encoded by multi-drug resistance gene 1), contributing to drug resistance against the chemotherapeutic 5-fluorouracil. Taken together, these novel findings demonstrate the involvement of PP2A in the regulation of liver metabolism.

Identification of miRNAs involved in liver injury induced by chronic exposure to cadmium.

To elaborate the molecular mechanism underlying the hepatotoxicity induced by chronic exposure to cadmium (Cd), a mouse model with hepatocyte-specific deletion of Ppp2r1a (encoding protein phosphatase 2 A Aα subunit, PP2A Aα) gene was used to investigate the effect of cadmium exposure on liver injury. The wild type littermates (WT) and PP2A Aα-/- mice (KO) were treated with cadmium chloride (CdCl2) at concentrations of 0 mg/L, 10 mg/L, 100 mg/L in drinking water for 3, 6 and 9 months (KO mice only for 9 months), respectively. The pathological findings were characterized by progressive inflammation, steatosis, and liver fibrosis upon treatment of CdCl2 in a dose-response and time-dependent manner. Notably, PP2A Aα depletion leads to a more profound liver injury induced by CdCl2 treatment. The transcriptome analysis in livers of KO mice revealed 20 differentially expressed microRNAs (miRNAs) appeared in both 3- and 9-month. Particularly, the alterations of miR-34a-5p, miR-345-5p, and miR-30e-5p expressions were implicated in the development of liver disease and correlated with the degree of liver injury induced by cadmium treatment. Further analysis indicated that miR-34a-5p, miR-345-5p, and miR-30e-5p might be involved in CdCl2-induced liver injury, in part by dysregulation of lipid metabolism and inflammation. The in vitro studies showed that miR-34a-5p was involved in regulation of CdCl2-induced cytotoxicity through directly targeted adiponectin receptor 2 (AdipoR2) mRNA. Taken together, we identified that specific miRNAs were implicated in hepatotoxicity induced by chronic exposure to CdCl2. These findings also provide new insight into the role of PP2A in regulation of miRNAs-mediated liver injury.

Assessment of intestinal injury of hexavalent chromium using a modified in vitro gastrointestinal digestion model.

Intestinal injury assessment of hexavalent chromium (Cr-VI) in humans is crucial for quantifying assessment of adverse health risk posed by the intake of Cr (VI)-contaminated water. To overcome the deficiency in simulating human gastric reduction and intestinal absorption, we modified the constituents of simulated gastric fluid in in vitro digestion method by adding reductants glutathione (18 µM) and ascorbic acid (180 µM), which incorporated with human intestinal epithelial model to construct an in vitro gastrointestinal digestion (IVGD) model for intestinal injury assessment. Cr-VI bioaccessibility results from IVGD model showed that weak gastric acidity significantly increased the intestinal accessible Cr-VI dose by 22.41-38.43 folds. The time-course intestinal absorption indicated prolongation of intestinal exposure destroyed the intestinal epithelium, and 24 h after Cr-VI treatment was a good time point to perform intestinal absorption and toxicity assessment. A series of cell-based bioassays provided initial warning of adverse effect, suggesting that epithelial integrity exhibited greatest sensitivity to Cr-VI exposure and might be used as a sensitive marker for the toxicity assessment of oral exposure to Cr-VI. Notably, this study provides a feasible strategy for delineation of Cr-VI biotransformation and intestinal injury following ingestion exposure, which contributes to address the toxicity data gap of low-dose exposure in humans and puts forward a reference for intestinal toxicity assessment of other chemicals.

Discovery of a Potential Liver Fibrosis Inhibitor from a Mushroom Endophytic Fungus by Genome Mining of a Silent Biosynthetic Gene Cluster.

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-ß (TGF-ß) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.

Photo-induced energy transfer relay of N-heterocyclic carbene catalysis: an asymmetric α-fluorination/isomerization cascade.

The geometric configuration of olefin products is often driven by thermodynamic control in synthesis. Methods enabling switching of cis/trans selectivity are rare. Recently, photosensitized approaches have emerged as a powerful tool for accomplishing this task. In this report, we report an in situ isomerization of an N-heterocyclic carbene (NHC)-bound intermediate by a photo-induced energy transfer process that leads to selective access of chiral allylic fluorides with a cis-olefin geometry. In the absence of a photocatalyst or light, the reaction proceeds smoothly to give (E)-olefin products, while the (Z)-isomer can be obtained under photosensitizing conditions. Preliminary mechanistic experiments suggest that an energy transfer process might be operative.

Characterization of cGAS homologs in innate and adaptive mucosal immunities in zebrafish gives evolutionary insights into cGAS-STING pathway.

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution.

Promiscuous Enzymes Cause Biosynthesis of Diverse Siderophores in Shewanella oneidensis.

The siderophore synthetic system in Shewanella species is able to synthesize dozens of macrocyclic siderophores in vitro with synthetic precursors. In vivo, however, although three siderophores are produced naturally in Shewanella algae B516, which carries a lysine decarboxylase (AvbA) specific for siderophore synthesis, only one siderophore can be detected from many other Shewanella species. In this study, we examined a siderophore-overproducing mutant of Shewanella oneidensis which lacks an AvbA counterpart, and we found that it can also produce these three siderophores. We identified both SpeC and SpeF as promiscuous decarboxylases for both lysine and ornithine to synthesize the siderophore precursors cadaverine and putrescine, respectively. Intriguingly, putrescine is mainly synthesized from arginine through an arginine decarboxylation pathway in a constitutive manner, not liable to the concentrations of iron and siderophores. Our results provide further evidence that the substrate availability plays a determining role in siderophore production. Furthermore, we provide evidence to suggest that under iron starvation conditions, cells allocate more putrescine for siderophore biosynthesis by downregulating the expression of the enzyme that transforms putrescine into spermidine. Overall, this study provides another example of the great flexibility of bacterial metabolism that is honed by evolution to better fit living environments of these bacteria.IMPORTANCE The simultaneous production of multiple siderophores is considered a general strategy for microorganisms to rapidly adapt to their ever-changing environments. In this study, we show that some Shewanella spp. may downscale their capability for siderophore synthesis to facilitate adaptation. Although S. oneidensis lacks an enzyme specifically synthesizing cadaverine, it can produce it by using promiscuous ornithine decarboxylases. Despite this ability, this bacterium predominately produces the primary siderophore while restraining the production of secondary siderophores by regulating substrate availability. In addition to using the arginine decarboxylase (ADC) pathway for putrescine synthesis, cells optimize the putrescine pool for siderophore production. Our work provides an insight into the coordinated synthesis of multiple siderophores by harnessing promiscuous enzymes in bacteria and underscores the importance of substrate pools for the biosynthesis of natural products.

Enantio- and Diastereoselective Hydrofluorination of Enals by N-Heterocyclic Carbene Catalysis.

In contrast to well-established asymmetric hydrogenation reactions, enantioselective protonation is an orthogonal approach for creating highly valuable methine chiral centers under redox-neutral conditions. Reported here is the highly enantio- and diastereoselective hydrofluorination of enals by an asymmetric ß-protonation/α-fluorination cascade catalyzed by N-heterocyclic carbenes (NHCs). The two nucleophilic sites of a homoenolate intermediate, generated from enals and an NHC, are sequentially protonated and fluorinated. The results show that controlling the relative rates of protonation, fluorination, and esterification is crucial for this transformation, and can be accomplished using a dual shuttling strategy. Structurally diverse carboxylic acid derivatives with two contiguous chiral centers are prepared in a single step with excellent d.r. and ee values.

The profiles and networks of miRNA, lncRNA, mRNA, and circRNA in benzo(a)pyrene-transformed bronchial epithelial cells.

Our aim was to demonstrate the significance of miRNA, lncRNA, and circRNA in the transformation of human bronchial epithelial cells induced by benzo(a)pyrene (BaP), and to investigate their regulatory networks. Hierarchical clustering, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and network regulation analysis were used to analyze the high-throughput sequencing results of human bronchial epithelial cell line BEAS-2B and BaP-transformed BEAS-2B cells (BEAS-2B-T). 76,191,786 and 3,431differentially-expressed miRNA, lncRNA, mRNA and circRNA were detected, respectively; 43 miRNA, 48 lncRNA, 438 mRNA and 2,079 circRNA were up-regulated; 33 miRNA, 143 lncRNA, 348 mRNA and 1,352 circRNA were down-regulated. Through GO analysis, 257 biological process (BP) terms, 12 cell composition (CC) terms and 49 molecular function (MF) terms were found in differentially-expressed lncRNA; 143 BP terms, 32 CC terms, and 48 MF terms were found in differentially-expressed circRNA. Pathways of KEGG analysis of lncRNA and circRNA could be classified into the categories "human diseases" and "organism systems". From miRNA-circRNA, circRNA-mRNA, and lncRNA-circRNA networks analysis, we found that mir-137, circ-RPS5, circ-ZNF292, circ-ERBB2IP, circ-SEMA3C, circ-IGF1R, circ-RTN4, APOC1, and CDKN2A may be of great significance for cell transformation. From the analysis of miRNA, lncRNA, mRNA, and circRNA networks, we found that PDGFRB, lncRNA RGMB-AS1, circ-ZNF292 are associated with miR-138-5p. Our study shows that miRNA, lncRNA, and circRNA have a significant regulatory role in the transformation of human bronchial epithelial cell induced by BaP.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10.

Antibiotic-producing microorganisms have evolved several self-resistance mechanisms to prevent auto-toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP-binding cassette (ABC) transporter-encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5-de-epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up-regulated in response to intracellular accumulation of natamycin and 4,5-de-epoxynatamycin, suggesting a key role in the stress response for self-resistance. This article discusses a novel antibiotic-related efflux and response system in Streptomyces, as well as a self-resistance mechanism in antibiotic-producing strains.

FkbN and Tcs7 are pathway-specific regulators of the FK506 biosynthetic gene cluster in Streptomyces tsukubaensis L19.

FK506 (tacrolimus), which is produced by many Streptomyces strains, is clinically used as an immunosuppressive agent and for treatment of inflammatory skin diseases. Here, we identified that the FK506 biosynthetic gene cluster in an industrial FK506-producing strain Streptomyces tsukubaensis L19 is organized as eight transcription units. Two pathway-specific regulators, FkbN and Tcs7, involved in FK506 biosynthesis from S. tsukubaensis L19 were characterized in vivo and in vitro. FkbN activates the transcription of six transcription units in FK506 biosynthetic gene cluster, and Tcs7 activates the transcription of fkbN. In addition, the DNA-binding specificity of FkbN was determined. Finally, a high FK506-producing strain was constructed by overexpression of both fkbN and tcs7 in S. tsukubaensis L19, which improved FK506 production by 89 % compared to the parental strain.