CXCL12+ Tumor-associated Endothelial Cells Promote Immune Resistance in Hepatocellular Carcinoma.

BACKGROUND: The tumor microenvironment (TME) plays a crucial role in the limited efficacy of existing treatments for hepatocellular carcinoma (HCC), with tumor-associated endothelial cells (TECs) serving as fundamental TME components that substantially influence tumor progression and treatment efficacy. However, the precise roles and mechanisms of TECs in HCC remain inadequately understood. METHODS: We employed a multi-omics profiling strategy to investigate the single-cell and spatiotemporal evolution of TECs within the microenvironment of HCC tumors showcasing varied responses to immunotherapy. Through an analysis of a clinical cohort of HCC patients, we explored the correlation between TEC subpopulations and immunotherapy outcomes. The influence of TEC subsets on the immune microenvironment was confirmed through comprehensive in vitro and in vivo studies. To further explore the mechanisms of distinct TEC subpopulations in microenvironmental modulation and their impact on immunotherapy, we utilized TEC subset-specific knockout mouse models as well as humanized mouse models. RESULTS: In this research, we identified a new subset of CXCL12+ TECs that exert a crucial role in immune suppression within the HCC TME. Functionally, CXCL12+ TECs impede the differentiation of CD8+ naïve T cells into CD8+ cytotoxic T cells by secreting CXCL12. Furthermore, they attract myeloid-derived suppressor cells (MDSCs). A bispecific antibody was developed to target both CXCL12 and PD1 specifically, showing significant promise in bolstering anti-tumor immune responses and advancing HCC therapy. CONCLUSIONS: CXCL12+ TECs are pivotal in mediating immunosuppression within HCC microenvironment and targeting CXCL12+ TECs presents a promising approach to augment the efficacy of immunotherapies in HCC patients. IMPACT AND IMPLICATION: This investigation reveals a pivotal mechanism in the HCC TME, where CXCL12+ TECs emerge as crucial modulators of immune suppression. The discovery of CXCL12+ TECs as inhibitors of CD8+ naïve T cell activation and recruiters of MDSCs significantly advances our grasp of the dynamic between HCC and immune regulation. Moreover, the development and application of a bispecific antibody precisely targeting CXCL12 and PD1 has proven to enhance immune responses in a humanized mouse HCC model. This finding underscores a promising therapeutic direction for HCC, offering the potential to amplify the impact of current immunotherapies.

Regulating structural stability and photoelectrical properties of FAPbI3via formamidine cation orientation.

Organic cations play a significant role in the structural stability and photoelectrical properties of organic-inorganic hybrid perovskites. The orientation of organic cations impacts its interaction with inorganic octahedrons [PbI]6-, subsequently modifying the atomic structure and electronic and optical properties of perovskite materials. However, it is still challenging to regulate the stability of perovskites with different orientations. In this work, density functional theory calculations were performed to investigate the effects of the formamidine cation (FA+) located at the angles of 0°, 45°, 90° and 180° (relative to the normal of the crystal plane) along the typical crystal directions ([001], [010], [110] and [111]) on the structural stability and photoelectrical properties of formamidine lead iodide (FAPbI3). The results show that when FA+ is located at 45° along the [111] direction, FAPbI3 achieves the highest stability and excellent photoelectrical properties. The energy evolution curves display that the system with the orientation of [111] has the minimum energy value, signifying stronger stability than the other orientations. Especially, when FA+ is located at 45° along the [111] direction. it exhibits a stronger hydrogen bond between H and I atoms, shorter Pb-I bond length and smaller [PbI]6- octahedral tilt bond angle. The band gap in the [110] direction changes from direct to indirect while FAPbI3 with other FA+ orientations still maintains the direct band structure located at the high symmetric R point. Furthermore, FA+ orientation drives the redshift of FAPbI3 towards the long wavelength region in the [111] crystal direction, which enhances the light absorption coefficient. This work can offer guidance in employing molecular regulation technology for the development of stable perovskite solar cells.

Exosome-transmitted circCABIN1 promotes temozolomide resistance in glioblastoma via sustaining ErbB downstream signaling.

Although temozolomide (TMZ) provides significant clinical benefit for glioblastoma (GBM), responses are limited by the emergence of acquired resistance. Here, we demonstrate that exosomal circCABIN1 secreted from TMZ-resistant cells was packaged into exosomes and then disseminated TMZ resistance of receipt cells. CircCABIN1 could be cyclized by eukaryotic translation initiation factor 4A3 (EIF4A3) and is highly expressed in GBM tissues and glioma stem cells (GSCs). CircCABIN1 is required for the self-renewal maintenance of GSCs to initiate acquired resistance. Mechanistically, circCABIN1 regulated the expression of olfactomedin-like 3 (OLFML3) by sponging miR-637. Moreover, upregulation of OLFML3 activating the ErbB signaling pathway and ultimately contributing to stemness reprogramming and TMZ resistance. Treatment of GBM orthotopic mice xenografts with engineered exosomes targeting circCABIN1 and OLFML3 provided prominent targetability and had significantly improved antitumor activity of TMZ. In summary, our work proposed a novel mechanism for drug resistance transmission in GBM and provided evidence that engineered exosomes are a promising clinical tool for cancer prevention and therapy.

Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood; what is known is that it starts at a high speed followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate that is toxic with regard to the metabolism is produced in the reaction. We found that a Rid protein (designated Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate, imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumed imine intermediate produced in the Pno reaction and that, in the presence of Rid-NC, Pno maintained a high level of activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene led to slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins.IMPORTANCE Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumed reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to the RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5'-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.

Smoking Affects the Patterns of Metabolic Disorders and Metabolic Syndrome in Patients With First-Episode Drug-Naive Schizophrenia: A Large Sample Study Based on the Chinese Han Population.

OBJECTIVE: Although metabolic disorders and smoking are common in schizophrenia, few studies have investigated the effects of smoking on metabolic disorders or metabolic syndrome (MetS) in schizophrenia patients, especially in first-episode drug-naïve (FEDN) patients. We sought to investigate the differences in metabolic disorders and MetS between smoking and nonsmoking FEDN schizophrenia patients. METHODS: A total of 428 FEDN schizophrenia patients and 435 controls were recruited. Blood pressure, waist circumference, body mass index (BMI), lipid profiles, and glucose metabolism were measured. The psychopathology was evaluated by Positive and Negative Syndrome Scale. RESULTS: FEDN schizophrenia patients had a higher smoking rate than controls (23.8% vs 14.0%, P < .001). After adjusting for confounding variables, the prevalence of MetS, overweight, hypertension, hypertriglyceridemia, elevated insulin, and insulin resistance in smoking patients was higher than those in nonsmoking patients, while overweight and hypertension were higher in the smoking controls than in nonsmoking controls (all P < .05). In smoking patients, triglyceridemia, high-density lipoprotein cholesterol, and fasting blood glucose were the main contributing components to MetS, while in nonsmoking patients, waist circumference, systolic blood pressure, triglyceridemia, high-density lipoprotein cholesterol, and fasting blood glucose were the main contributing components to MetS. In smoking patients, BMI and homeostatic model assessment for insulin resistance were associated factors of MetS (both P < .05). In nonsmoking patients, sex, BMI, insulin, and homeostatic model assessment for insulin resistance were associated factors of MetS (all P < .05). CONCLUSIONS: Our study indicates that smoking schizophrenia patients have a higher prevalence of MetS and metabolic disorders than nonsmoking patients. Moreover, smoking and nonsmoking patients have different contributing components and associated factors for MetS.

The protective role of protocatechuic acid against chemically induced liver fibrosis in vitro and in vivo.

Liver fibrosis is the result of long-term liver injury and has a high incidence worldwide. Protocatechuic acid (PCA) is ubiquitous in vegetables, nuts, brown rice and herbal medicines, which is reported to possess anti-asthmatic, anti-cancer, and anti-oxidation properties. Our research aimed to investigate the effect of PCA on liver fibrosis. In vitro, TNF-α-induced hepatic stellate cell (HSC) model was used to assess the anti-fibrosis effects of PCA. In vivo, mice were treated with thioacetamide (TAA) to develop liver fibrosis. Body weight, organ index, histological changes, and proteins alteration of factors associated with TGF-ß signaling pathway were used to assess the anti-fibrosis effects of PCA. Our results showed that PCA not only inhibited cell viability in TNF-α activated HSC-T6 cells in vitro, but also efficiently mitigated TAA-induced liver damage and fibrosis in vivo. Further experiments indicated that PCA played a protective role in liver fibrosis through regulation of the TGF-ß signaling pathway downregulating the protein expression of p-Smad2, p-ERK, c-Jun. In summary, our findings provide a pharmacological justification for the clinical application of PCA in preventing or treating liver fibrosis.

A long-acting isomer of Ac-SDKP attenuates pulmonary fibrosis through SRPK1-mediated PI3K/AKT and Smad2 pathway inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening lung disease with a poor prognosis. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a critical negative regulator of fibrosis development. However, it's extremely short half-life greatly limits its applications. Previously, we reported an Ac-SDKP analog peptide in which Asp and Lys residues were replaced with D-amino acids (Ac-SDD KD P). Ac-SDD KD P exhibits better resistance to angiotensin-1-converting enzyme (ACE)-mediated degradation and a longer half-life than Ac-SDKP in rat and human sera. The objective of this study was to explore the potential application of Ac-SDD KD P for the treatment of IPF and to clarify the underlying mechanisms. We found that Ac-SDD KD P exerted similar antifibrotic effects as Ac-SDKP on human fetal lung fibroblast-1 (HFL-1) proliferation, α-smooth muscle actin (α-SMA), collagen I and collagen III expression, and Smad-2 phosphorylation in vitro. In vivo, Ac-SDD KD P exhibited significantly greater protective effects against bleomycin-induced pulmonary fibrosis than Ac-SDKP in mice. α-SMA, CD45, collagen I and collagen III expression, and Smad-2 phosphorylation were significantly decreased in the lungs of Ac-SDD KD P-treated but not Ac-SDKP-treated mice. Furthermore, a pull-down experiment was used to screen for molecules that interact with Ac-SDKP. Co-immunoprecipitation (Co-IP) and computer-based molecular docking experiments demonstrated an interaction between Ac-SDKP or Ac-SDD KD P (Ac-SDKP/Ac-SDD KD P) and serine/arginine-rich protein-specific kinase 1 (SRPK1) that caused inhibition SRPK1-mediated phosphatidylinositol-3 kinase/ serine/threonine kinase (PIK3/AKT) signaling pathway activation and Smad2 phosphorylation and thereby attenuated lung fibrosis. Our data suggest that long-acting Ac-SDD KD P may potentially be an effective drug for the treatment of pulmonary fibrosis. The interacting molecule and antifibrotic mechanism of Ac-SDKP/Ac-SDD KD P were also identified, providing an experimental and theoretical foundation for the clinical application of the drug.

The influence of linkages between 1-hydroxy-2(1H)-pyridinone coordinating groups and a tris(2-aminoethyl)amine core in a novel series of synthetic hexadentate iron(III) chelators on antimicrobial activity.

Resistance of pathogens to antimicrobials is a major current healthcare concern. In a series of linked studies, we have investigated synthetic iron chelators based on hydroxy-pyridinone ligands as novel bacteriostatic agents. Herein we describe our synthesis of several useful building blocks based on the 1-hydroxy-2(1H)-pyridinone moiety, including a novel formyl derivative, which were combined with a tris(2-aminoethyl)amine core to obtain a series of new high-affinity hexadentate Fe(III) chelators. The design principle examined by this series is the size and flexibility of the linker between the core and the metal ligands. Measurement of the pKa and stability constants (Fe3+ and Cu2+) of representative coordinating groups was performed to help rationalise the biological activity of the chelators. The novel chelators were tested on a panel of representative microorganisms with some effectively inhibiting microbial growth. We demonstrate that the nature and position of the linker between the hydroxypyridinone and the tris(2-aminoethyl)amine core has considerable impact upon microbial growth inhibition and that both amide or amine linkages can give efficacious chelators.

Preclinical safety evaluation of body protective compound-157, a potential drug for treating various wounds.

BPC157 displays protective activity in various organs and tissues. This report presents preclinical toxicity studies with BPC157 in mice, rats, rabbits and dogs. The single-dose toxicity study did not show any test-related effects that could be attributed to the test article. In repeated-dose toxicity evaluations, BPC157 was well tolerated in dogs, with no abnormal changes between the BPC157-treated groups and the solvent control group, with the exception of a decrease in creatinine level at a dose of 2 mg/kg but not at lower doses. The animals recovered spontaneously after 2 weeks of withdrawal. This may be due to the pharmacological activity of BPC157. A local tolerance test showed that the irritation caused by BPC157 was mild. BPC157 also showed no genetic or embryo-fetal toxicity. In summary, BPC157 was well tolerated and did not cause any serious toxicity in mice, rats, rabbits and dogs. These preclinical safety data contribute to the initiation of an ongoing clinical study. Based on the stability and protective effect of BPC157, which has been widely reported, BPC157 may have a better application prospect than the widely used cytokine drugs in wound therapy.

6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Synergistic tumoricidal effect of combined hPD-L1 vaccine and HER2 gene vaccine.

Immunotherapy is gathering momentum as a kind of important therapy for cancer patients. However, monotherapies have limited efficacy in improving outcomes and benefit only in a small subset of patients. Combination therapies targeting multiple pathways often can augment an immune response to improve survival further. Here, the tumoricidal effects of the dual hPD-L1(human programmed cell death ligand 1) vaccination/HER2(human epidermal growth factor receptor 2) gene vaccination immunotherapy against the established HER2-expressed cancers were observed. Animals treated with combination therapy using hPD-L1 vaccine and HER2 gene vaccine had significantly improved survival in a mammary carcinoma model. We observed an increase in tumor growth inhibition following treatment. The percentage of the tumor-free mice (%) was much higher in the combined PD-L1/HER2 group. Furthermore, under the tumor-burden condition, hPD-L1 vaccine enhanced humoral immunity of HER2 gene vaccine. And the combination treatment increased the IFN-γ-producing effector T cells. Additionally, splenocytes from the combined PD-L1/HER2 group immunized mice possessed higher CTL activity. Notably, vaccination with combination therapy induced a significant decrease in the percentage of CD4+CD25+ Treg cells. Collectively, these data demonstrate that PD-L1/HER2 gene vaccine combination therapy synergistically generates marked tumoricidal effects against established HER2-expressing cancers.

Acetylation and Amination Protect Angiotensin 1-7 from Physiological Hydrolyzation and Therefore Increases Its Antitumor Effects on Lung Cancer.

The recently reported inhibitory effects of angiotensin 1-7 (Ang-(1-7)) on various cancers indicate its potential use as a therapeutic agent for primary and metastatic cancers. However, its extremely short half-life in the circulation greatly compromises its potential applications. Here, we reported an Ang-(1-7) analogue peptide with the amino and carboxy termini protected by acetylation and amination. The in vitro and in vivo degradation of the resulting analogue, Ang-AA, were determined using high-performance liquid chromatography (HPLC). At the same time, small RNA interference and competition studies were performed to evaluate the specific capacity of Ang-AA to bind to the cell surface Mas receptor. Cell Counting Kit-8 (CCK8), wound-healing, and Boyden chamber assays were performed to investigate the inhibitory effects of Ang-AA on A549 cells. Finally, the synergistic inhibitory effects of Ang-AA and paclitaxel (PTX) on A549 xenografts in mice were observed using animal imaging systems and survival observations. The toxicity of Ang-AA in mice was evaluated. Our results showed that acetylation and amination significantly inhibited the hydrolyzation of Ang-(1-7) in vitro and in vivo. The half-life of Ang-(1-7) in rats was prolonged from 2.4 ± 0.6 min to 238.7 ± 61.3 min ( p < 0.001). The specific binding of Ang-AA to the Mas receptor was well preserved, and Ang-AA exerted significantly greater inhibitory effects on the proliferation, migration, and invasion of A549 cells than Ang-(1-7). The combination of Ang-AA and PTX exhibited a significantly greater synergistic inhibitory effect on A549 xenografts than the combination of Ang-(1-7) and PTX. Ang-AA did not display obvious toxicity in mice. Our findings indicate acetylation and amination is a simple and effective method for producing Ang-(1-7) as a bioactive peptide.

Genome-Wide Investigation of Genes Regulated by ERα in Breast Cancer Cells.

Estrogen receptor alpha (ERα), which has been detected in over 70% of breast cancer cases, is a driving factor for breast cancer growth. For investigating the underlying genes and networks regulated by ERα in breast cancer, RNA-seq was performed between ERα transgenic MDA-MB-231 cells and wild type MDA-MB-231 cells. A total of 267 differentially expressed genes (DEGs) were identified. Then bioinformatics analyses were performed to illustrate the mechanism of ERα. Besides, by comparison of RNA-seq data obtained from MDA-MB-231 cells and microarray dataset obtained from estrogen (E2) stimulated MCF-7 cells, an overlap of 126 DEGs was screened. The expression level of ERα was negatively associated with metastasis and EMT in breast cancer. We further verified that ERα might inhibit metastasis by regulating of VCL and TNFRSF12A, and suppress EMT by the regulating of JUNB and ID3. And the relationship between ERα and these genes were validated by RT-PCR and correlation analysis based on TCGA database. By PPI network analysis, we identified TOP5 hub genes, FOS, SP1, CDKN1A, CALCR and JUNB, which were involved in cell proliferation and invasion. Taken together, the whole-genome insights carried in this work can help fully understanding biological roles of ERα in breast cancer.

Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 µmol · min-1 · mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 µM and 3.61 ± 0.23 µM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.

Performance and mechanism of triclosan removal in simultaneous nitrification and denitrification (SND) process under low-oxygen condition.

Wastewater treatment under low dissolved oxygen (DO) conditions is promising for its low energy consumption. However, the removal process of some organic micropollutants, such as triclosan (TCS), could be inhibited under anaerobic conditions. So, it is worth investigating the TCS removal performance at low-oxygen condition. In this study, simultaneous nitrification and denitrification (SND) process, with DO ranging from 0.30 to 0.80 mg L-1, was chosen to investigate. Results showed that the water quality of the effluent was deteriorated after TCS addition at the beginning, with removal efficiency of NH4+-N dropped from almost 100 ± 0.70 to 88.30 ± 0.98% and COD decreased from 95.15 ± 1.55 to 65.81 ± 2.42 %. However, the performance recovered from the 3rd day and almost stabilized on the 14th day with the removal efficiencies of NH4+-N were over 98.00 ± 0.60 %, and COD was above 94.00 ± 1.70 % in effluent. Besides, TCS removal efficiencies were more than 93.00 %, and the contributions for TCS removal by the water effluent, sludge sorption, and other effects including biodegradation were 6.46 ± 2.25, 16.27 ± 3.30, and 77.27 ± 4.45 %, respectively. Although the results of absolute abundances of related genes showed no difference (P > 0.05), Illumina MiSeq sequencing analysis presented the variation of microbial community after TCS addition, in which T-45 had the highest Shannon and Simpson diversity index, followed by T-0 and T-2. Relative abundances of alpha and beta-Proteobacteria, which were related to TCS biodegradation, were increased. Compared with Bacteroidetes in T-0, the abundance of Bacteroidetes took up more than 15.6 % in T-45, which should play a more important role under low-oxygen conditions with TCS addition.

Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure.

NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD(+). Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg(187) is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH(•)/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed.

Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33.

Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.

Heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin.

Ferredoxins are small, acidic proteins containing iron-sulfur clusters that are widespread in living organisms. They play key roles as electron carriers in various metabolic processes, including respiration, photosynthesis, fermentation, nitrogen fixation, carbon dioxide fixation, and hydrogen production. However, only several kinds of ferredoxins are commercially available now, greatly limiting the investigation of ferredoxin-related enzymes and metabolic processes. Here we describe the heterologous overproduction of 2[4Fe4S]- and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin. Adding extra iron and sulfur sources to the medium in combination with using Escherichia coli C41(DE3) harboring pCodonplus and pRKISC plasmids as host greatly enhanced iron-sulfur cluster synthesis in the three ferredoxins. After induction for 12 h in terrific broth and purification by affinity chromatography and anion exchange chromatography, approximately 3.4 mg of streptavidin (Strep)-tagged and 3.7 mg of polyhistidine (His)-tagged clostridial 2[4Fe4S] ferredoxins were obtained from 1 l of culture. Excitingly, after induction for 24 h in terrific broth, around 40 mg of His-tagged clostridial [2Fe2S] and 23 mg of His-tagged agrobacterial [2Fe2S] ferredoxins were purified from 1 l of culture. The recombinant ferredoxins apparently exhibited identical properties and physiological function to native ferredoxins. No negative impact of two different affinity tags on ferredoxin activity was found. In conclusion, we successfully developed a convenient method for heterologous overproduction of the three kinds of ferredoxins with satisfactory yields and activities, which would be very helpful for the ferredoxin-related researches.

FOXP3 suppresses breast cancer metastasis through downregulation of CD44.

Forkhead box protein 3 (FOXP3) plays an important role in breast cancer as an X-linked tumor suppressor gene. However, the biological functions and significance of FOXP3 in breast cancer metastasis remain unclear. Here, we find that, clinically, nuclear FOXP3 expression is inversely correlated with breast cancer metastasis. Moreover, we demonstrate that FOXP3 significantly inhibits adhesion, invasion and metastasis of breast cancer cells in vivo and in vitro. In addition, the adhesion molecule CD44 is found to be suppressed by FOXP3 through transcriptome sequence analysis (RNA-seq). A luciferase reporter assay, chromatin immunoprecipitation and electrophoretic mobility shift assay identify CD44 as a direct target of FOXP3. The expression of CD44 is downregulated by FOXP3 in breast cancer cells. Importantly, anti-CD44 antibody reverses the FOXP3 siRNA-induced effects on the breast cancer cells in vitro and FOXP3 expression level in the nucleus of breast cancer cells is inversely correlated with CD44 expression level in clinic breast cancer tissues. Taken together, the results from the present study suggest that FOXP3 is a suppressor of breast cancer metastasis. FOXP3 directly binds to the promoter of CD44 and inhibits its protein expression, thereby suppressing adhesion and invasion of human breast cancer cells. This finding highlights the therapeutic potential of FOXP3-CD44 signaling to inhibit breast cancer metastasis.

Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.