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Gut microbiota and their metabolic products might play important roles in regulating the pathogenesis of choledocholithiasis concurrent with cholangitis (CC). The aim of this study was to explore the characteristic gut dysbiosis, metabolite profiles and the possible roles in patients with CC.A case-control study was carried out to analyze the alterations in the intestinal microbiota and their metabolites in patients with CC (n = 25) compared with healthy controls (HCs) (n = 25) by metagenomic sequencing to define the gut microbiota community and liquid chromatography/mass spectrometry (LC/MS) analysis to characterize the metabolite profiles.Significantly reduced Shannon diversity index (p = 0.043) and differential overall fecal microbiota community in CCs were observed. Twelve dominant altered species were identified and analyzed (LDA score 3.0, p 0.05) (Q value 0.05), including unclassified_f_Enterobacteriaceae, Escherichia_coli, Roseburia_faecis and Eubacterium rectale. Moreover, the levels of KEGG pathways related to biofilm formation of Escherichia coli, lipopolysaccharide (LPS) biosynthesis, and the metabolism of propanoate and glutathione in CCs were significantly altered. Finally, 47 markedly changed metabolites (VIP 1.0 and p 0.05), including low level of kynurenic acid (KYNA) and high concentration of N-palmitoylsphingosine involving tryptophan metabolism and sphingolipid signaling pathways, were identified to validate aberrant metabolic patterns in CCs, and multiple correlated metabolic modules involving bile inflammation were altered in CCs.Our study provides novel insights into compositional and functional alterations in the gut microbiome and metabolite profiles in CC and the underlying mechanisms between gut microbiota and bile inflammation.

The presence of pesticide residues in Dendrobium officinale (D. officinale), a commonly used herbal medicine, has attracted much attention in recent years. Therefore, this study presents the levels of 141 pesticide residues in forty D. officinale samples, which were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). And we used a deterministic estimate model to assess chronic and acute dietary exposure risk, as well as the cumulative risks for adults, children, and specific groups of consumers. Furthermore, the residual risk of individual pesticides was sorted by adapting the matrix-ranking scheme. In 92.5% of the samples, 43 pesticides were detected, of which difenoconazole had the highest detection frequency. Multiple residues were detected in 85.0% of the samples, and one sample contained even up to 17 pesticides. The chronic hazard quotient (HQc) and the acute hazard quotient (HQa) were far below 100%, and both cumulative chronic and acute hazard indices (HI) did not exceed 100%. The risk scoring scheme showed that four pesticides were considered to pose a comparatively potential high risk, including difenoconazole, carbofuran, fipronil, and emamectin benzoate. The results indicated that the occurrence of pesticide residues in D. officinale could not pose a serious health problem to the public.

Two Gram-stain-negative bacterial strains, SM1969T and SM1979T, were isolated from coastal surface seawater of Qingdao, China. They were taxonomically characterized by the phylogenetic, genomic, chemotaxonomic and phenotypic analyses. The two strains shared 97.0% 16S rRNA gene sequence similarity with each other and the highest similarity (96.8–97.5%) with type strains of six species in the genera Shimia, Tritonibacter and Tropicibacter in the Roseobacter group of the family Rhodobacteraceae. In the phylogenetic tree based on single-copy orthologous clusters (OCs), both strains clustered with known species of the genus Tritonibacter and together formed a separate branch adjacent to Tritonibacter ulvae. Although sharing many chemotaxonomic and phenotypic characteristics, the two strains could be differentiated from each other and closely related species by numerous traits. Particularly, strain SM1969T was found to have a DMSP lyase coding gene dddW in its genome and have the ability to produce DMS from DMSP while strain SM1979T was not. The average nucleotide identity and in silico DNA-DNA hybridization values between strains SM1969T and SM1979T and type strains of closely related species were all below the thresholds to discriminate bacterial species, demonstrating that they constitute two new species in the genus Tritonibacter. The names Tritonibacter aquimaris sp. nov. and Tritonibacter litoralis sp. nov. are proposed for the two new species, with type strains being SM1969T (= MCCC 1K04320T = KCTC 72843T) and SM1979T (= MCCC 1K04321T = KCTC 72842T), respectively.

Rhinocerebral mucormycosis caused by Rhizopus oryzae in a patient with acute myeloid leukemia: A case report

Microeunicellols A (1) and B (2), two undescribed eunicellin diterpenoids, were isolated from the culture of a bacterial symbiont, Streptomyces albogriseolus SY67903. Their structures, including ab...

Ovarian cancer has the highest mortality in gynecologic malignancies. LncRNA BLACAT1 serves crucial functions in various cancers, but its role in ovarian cancer has not been investigated. In this article, our team explored the role and the potential regulatory mechanism of BLACAT1 in ovarian cancer. Quantitative RT-PCR showed that BLACAT1 was aberrantly up-regulated in ovarian cancer tissues compared with normal tissues. In vitro, BLACAT1 knockdown induced cell cycle arrest and inhibited the proliferation, migration and invasion of ovarian cancer cells using flow cytometry, MTT and EdU assays, wound healing assay and transwell assay, respectively. Luciferase assay verified the binding relationship between microRNA-519d-3p and lncRNA BLACAT1, and BLACAT1 negatively regulated the expression of miR-519d-3p. We also found that miR-519d-3p overexpression could inhibit ovarian cancer cells proliferation, migration and invasion. Further, Western blot demonstrated that the expression of RPS15A and nuclear β-catenin expression was markedly reduced by BLACAT1 knockdown, and cytoplasmic β-catenin level was not obviously affected. In vivo, BLACAT1 knockdown inhibited the tumor growth, and immunohistochemistry showed that ki67 expression was decreased by BLACAT1 suppression. Inhibition of BLACAT1 was sufficient to down-regulate the expression of RPS15A and nuclear β-catenin but did not cause an obvious change in cytoplasmic β-catenin expression. Taken together, BLACAT1 knockdown inhibited the progression of ovarian cancer by suppressing the Wnt/β-catenin signaling pathway via regulating miR-519d-3p. Our work provided a proper understanding of the critical roles of BLACAT1 in ovarian cancer.

A Gram-stain-negative, pink-pigmented, rod-shaped, non-flagellated, aerobic bacterium, designated strain SM1701T, was isolated from a rotten seaweed collected off Fildes Peninsula, King George Island, West Antarctica. The strain grew at 4–30 °C, pH 6.0–8.0 and with 0.5–5 % (w/v) NaCl. It hydrolysed gelatin and Tweens (40, 60 and 80), but did not reduce nitrates to nitrites. The major cellular fatty acids of strain SM1701T were iso-C15 : 0, iso-C15 : 1G, iso-C16 : 1G, C16 : 0 and iso-C17 : 0 3-OH. Polar lipids included phosphatidylethanolamine, one unidentified aminolipid, two unidentified glycolipids and one unidentified aminoglycolipid. The major respiratory quinone was MK-7. The genomic DNA G+C content of strain SM1701T was 34.1 mol%. It showed high 16S rRNA gene sequence similarities to Crocinitomix algicola (93.8 %) and Crocinitomix catalasitica (92.5 %) and less than 91 % sequence similarities to other known members in the family Crocinitomicaceae . Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1701T constituted a distinct lineage within the family Crocinitomicaceae . The phylogenetic trees based on concatenated 261 protein sequences from genome sequences showed that strain SM1701T occupied a branch separated from those of known genera in the family of Crocinitomicaceae , indicating it may belong to a new genus. On the basis of the polyphasic characterization of strain SM1701T in this study, it is considered to represent a novel species in a new genus in the family Crocinitomicaceae , for which the name Putridiphycobacter roseus gen. nov., sp. nov. is proposed. The type strain is SM1701T (=KCTC 62302T=NBRC 113201T=CGMCC 1.16510T).

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The N-terminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Purpose: The purpose of this study was to investigate the production of IL-27 p28 and EBI3 in the ocular inflammatory sites, and the role of IL-27 signaling in a model of HSV-1 induced herpetic stromal keratitis (HSK). Methods: The BALB/c mice were injected intraperitoneally (24 h before infection) with anti-IL-27 antibody or IgG antibody as control, infected with HSV-1 via corneal scarification, and then injected intraperitoneally with anti-IL-27 antibody or IgG antibody at 1, 3, and 5 days postinfection. Slit lamp and histopathology were used to assess disease outcome. The levels of IL-27 p28 and EBI3 in corneas were determined by western blotting and immunofluorescence. Furthermore, viral titers were determined, and immune cell infiltrates were collected and analyzed by flow cytometry. Results: We found that the levels of IL-27 p28 and EBI3 in corneas were elevated significantly at the peak of HSK, and both of them were expressed simultaneously in the epithelium, stroma, and endothelium of corneas. In the group of anti-IL-27 treatment, the severity of the corneal lesion and CD4+ T cells infiltration were significantly decreased, and the percentage of CD4+ Foxp3+ Tregs was upregulated markedly in the spleen, DLNs and cornea of HSK mice compared to IgG treatment. Conclusion: These results provided evidence that IL-27 as a pathogenic pro-inflammatory cytokine controlled CD4+ Foxp3+ Tregs production in HSK, which ultimately resulted in promoting the progression of HSK and poor prognosis.

Objective: The present study was designed to evaluate whether and how itraconazole affects the macrophage polarization and its reactivity to Candida albicans. Methods: Cell toxicity of itraconazole was measured using cell counting kit-8 assay in RAW264.7 cells. The cell models were induced by lipopolysaccharide (LPS), interleukin (IL)-4, or Candida albicans. Levels of cytokines secreted by RAW246.7 treated with itraconazole were detected by Luminex or Cytometric Bead Array compared to the controls without itraconazole treatment, and the expressions of inducible nitric oxide synthase and arginase (Arg) were determined by Western blot. Phagocytosis ability was measured by both flow cytometry and fluorescence microscope. The Student’s t test and one-way analysis of variance were used to calculate the differences between groups. Results: In comparison to the control, itraconazole inhibited the growth of the cells in both a time- and a dose-dependent manner. Increased secretion of IL-6 (0.25 μmol/L ITZ [538.03 ± 60.23 pg/mL, P < 0.05], 0.5 μmol/L [550.32 ± 47.87 pg/mL, P < 0.05] and 1 μmol/L [626.95 ± 75.24 pg/mL, P < 0.01] vs. control [370.43 ± 33.98 pg/mL]) and tumor necrosis factor-alpha (TNF-α) (1 μmol/L ITZ vs. control: 2521.51 ± 444.06 pg/mL vs. 1617.85 ± 94.57 pg/mL, P < 0.05) were detected in the LPS-induced cell model with itraconazole treatment. In the cells induced by IL-4, itraconazole increased the secretion of IL-6 (1 μmol/L ITZ vs. control: 528.33 ± 11.60 pg/mL vs. 466.99 ± 28.32 pg/mL, P < 0.05), TNF-α (1 μmol/L ITZ vs. control: 4.85 ± 0.32 pg/mL vs. 4.30 ± 0.19 pg/mL, P < 0.05), and IL-1β (0.25 μmol/L [325.95 ± 13.97 pg/mL, P < 0.05], 0.5 μmol/L [332.38 ± 11.97 pg/mL, P < 0.05] and 1 μmol/L [334.35 ± 16.23 pg/mL, P < 0.05] vs. control [291.62 ± 17.03 pg/mL]), and reduced the secretion of IL-10 (1 μmol/L ITZ vs. control: 7.21 ± 0.68 pg/mL vs. 9.11 ± 0.14 pg/mL, P < 0.05). The secretion of IL-6 (1 μmol/L ITZ vs. control: 38.34 ± 1.36 pg/mL vs. 32.32 ± 0.84 pg/mL, P < 0.05) and TNF-α (1 μmol/L ITZ vs. control: 1060.17 ± 80.16 pg/mL vs. 890.84 ± 52.82 pg/mL, P < 0.01) was improved in Candida albicans-stimulated RAW264.7 cells under the treatment of itraconazole, while the secretion of IL-4 (0.5 μmol/L [2.86 ± 0.20 pg/mL, P < 0.05] and 1 μmol/L [2.24 ± 0.33 pg/mL, P < 0.001] vs. control [3.91 ± 0.23 pg/mL]) and IL-10 (1 μmol/L ITZ vs. control: 19.46 ± 2.05 pg/mL vs. 25.67 ± 1.95pg/mL, P < 0.05) decreased. In all three activated patterns, itraconazole enhanced the expression of inducible nitric oxide synthase (P < 0.01) and slightly inhibited the Arg-1 expression (P < 0.05). Phagocytosis ability of RAW264.7 cells at 1 μmol/L ITZ treatment was increased by 7.53% ± 2.21 % (P < 0.01) and 9.73% ± 2.03% (P < 0.01) at the ratio of cells: yeast of 1:4 and 1:8, respectively, in comparison to the control group. Conclusion: Itraconazole improved M1 polarization of RAW264.7 cells and enhanced the phagocytic capacity of RAW264.7 to Candida albicans, indicating a significant immunological enhancement. The study improves the understanding of undergoing mechanisms related to the anti-tumor and anti-infection effects of itraconazole. Key words: itraconazole; macrophage; immunomodulatory; macrophage polarization; Candida albicans

Catalpol is the main active ingredient of an extract from Radix rehmanniae, which in a previous study showed a protective effect against various types of tissue injury. However, a protective effect of catalpol on uterine inflammation has not been reported. In this study, to investigate the protective mechanism of catalpol on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and mouse endometritis, in vitro and in vivo inflammation models were established. The Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB), and immunofluorescence techniques. The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) and CXCL5, both in bEECs and in uterine tissue. From the experimental results of WB, qRT-PCR, and immunofluorescence, the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group. The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase (MPO) activity. The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.

A Gram-stain-negative, aerobic, non-flagellated, rod-shaped bacterium, designated strain SM1703T, was isolated from Antarctic seawater collected near the Chinese Antarctic Great Wall Station, King George Island, West Antarctica. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1703T formed a distinct phylogenetic lineage within the family ‘Rhodobacteraceae’, sharing high 16S rRNA gene sequence similarity with Marivita litorea (95.5%). The strain grew at 10–37 °C (optimum, 25 °C) and with 0.5–13% (w/v) NaCl (optimum, 3–5%). The major cellular fatty acids were C19:0 cyclo ω8c, C18:1ω7c, C18:1 2-OH and C16:0 2-OH. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and an unidentified lipid. The genomic DNA G+C content of strain SM1703T was 64.6 mol%. Based on the results of the polyphasic characterization for strain SM1703T, it is classified as the representative of a novel species in a new genus of the family ‘Rhodobacteraceae’, for which the name Chachezhania antarctica gen. nov., sp. nov. is proposed. The type strain of Chachezhania antarctica is SM1703T (= MCCC 1K03470T = KCTC 62794T = CCTCC AB 2018351T).

Background Syphilis is a common sexually transmitted disease caused by the Treponema pallidum (T. pallidum). Malignant syphilis is a rare presentation of secondary syphilis. Here, we present a case diagnosed with malignant syphilis accompanied with neurosyphilis. Case summary A 56-year-old man present with a 2-mo history of spreading ulcerous and necrotic papules and nodules covered with thick crusts over the face, trunk, extremities, and genitalia. The patient was diagnosed with malignant syphilis accompanied by neurosyphilis based on the characteristic morphology of the lesions, positive serological and cerebrospinal fluid tests for syphilis, brain magnetic resonance imaging, and histopathology, along with resolution of the lesions following the institution of penicillin therapy. The lesions and neurological condition successfully resolved after a course of treatment with penicillin. Conclusion We suggest that neurosyphilis should be considered whenever people have psychiatric symptoms without cutaneous lesions or human immunodeficiency virus.

Nest survival is a vital component of breeding success, and affects population dynamics, as the loss of nests is the main cause of reproductive failure in birds. To identify key factors for the conservation of Chinese grouse Tetrastes sewerzowi, we tested the effects of nest concealment, nest age, nesting season, and habitat edge on nest daily survival rate (DSR) of Chinese grouse using 54 nests found at Lianhuashan Nature Reserve, Gansu, China, 2009–2012. Moreover, we controlled for the effect of research activity by testing the effect of nest checks on DSR. Overall, mammal predation caused 93% of nest failures. DSR was 0.986 ± 0.0038 in the constant model and the probability of a nest with a full clutch of 6 eggs surviving the entire 40-day nesting period was 0.526 ± 0.090. DSR decreased with nest age and nesting season (from 19 May to 3 July). Mammals instead of avian predators being responsible for most nest failures suggest that nest sites might be selected to avoid visual avian predators, but not olfactory mammalian predators, and the decreasing trend of DSR with nest age and nesting season could attribute to an additive exposure effect. Moreover, nest checks conducted by investigators significantly lowered nest DSR, especially during the late period of nesting season and for older nests. Mammalian predators might locate the nest site by following the investigator’s odor. Based on our results, we suggest that the late incubation stage is a particularly vulnerable period for nest survival of Chinese grouse and those researchers should adjust their activities around nests to balance the need of acquiring accurate data and decreasing nest predation risk.

Collagens from marine animals are an important component of marine organic nitrogen. Collagenase-producing bacteria and their collagenases play important roles in collagen degradation and organic nitrogen recycling in the ocean. However, only a few collagenase-producing marine bacteria have been so far discovered. Here, we reported the isolation and characterization of a collagenase-secreting bacterium, designated strain SM1988T, isolated from a green alga Codium fragile sample. Strain SM1988T is a Gram-negative, aerobic, oxidase-, and catalase-positive, unipolar flagellated, and rod-shaped bacterium capable of hydrolyzing casein, gelatin and collagens. Phylogenetic analysis revealed that strain SM1988T formed a distinct phylogenetic lineage along with known genera within the family Pseudoalteromonadaceae, with 16S rRNA gene sequence similarity being less than 93.3% to all known species in the family. Based on the phylogenetic, genomic, chemotaxonomic and phenotypic data, strain SM1988T was considered to represent a novel species in a novel genus in the family Pseudoalteromonadaceae, for which the name Flocculibacter collagenilyticus gen. nov., sp. nov. is proposed, with the type strain being SM1988T (= MCCC 1K04279T = KCTC 72761T). Strain SM1988T showed a high production of extracellular collagenases, which had high activity against both bovine collagen and codfish collagen. Biochemical tests combined with genome and secretome analyses indicated that the collagenases secreted by strain SM1988T are serine proteases from the MEROPS S8 family. These data suggest that strain SM1988T acts as an important player in marine collagen degradation and recycling and may have a promising potential in collagen resource utilization.

PurposeGlioblastoma multiforme (GBM) is one of the deadliest tumors, which is involved in numerous dysregulated microRNAs including miR-137. However, the mechanism of how miR-137 suppression associated with cancer progression and chemoresistance still remains to be elucidated.MethodsQuantitative reverse transcriptase-PCR (qRT-PCR), DNA methylation analysis, cell proliferation assay, flow cytometric analysis, invasion assay, in situ tumor formation experiment were performed to test the expression levels and functions of miR-137 in GBM. Bioinformatics analysis, luciferase reporter assay, qRT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry assay were used to identify and verify the target of miR-137.ResultsWe found that miR-137 was downregulated in primary and recurrent GBM compared with normal brain tissues. Overexpression of miR-137 inhibited cell invasion and enhanced cell chemosensitivity to temozolomide (TMZ) by directly targeting low-density lipoprotein receptor-related protein 6 (LRP6) in GBM. Forced expression of LRP6 cDNA without its 3’-UTR region partly restored the effects of miR-137 in vitro and in vivo. Hypoxia-induced miR-137 methylation was responsible for the miR-137 suppression, leading to the cell chemoresistance and poor prognosis of GBM.ConclusionsThese findings demonstrated the detailed molecular mechanism of miR-137 in regulating GBM growth and chemoresistance in hypoxia microenvironment, suggesting the potentiality of miR-137 as a therapeutic target for GBM.

Initial malarial infection mostly causes symptomatic illness in humans. Infection that is not fatal induces complete protection from severe illness and death, and thus complete protection from severe illness or death is granted with sufficient exposure. However, malaria parasite immunity necessitates constant exposure. Therefore, it is important to evaluate lowered immunity and recurrent susceptibility to symptomatic disease in lower transmission areas. We aimed to investigate selection pressure based on transmission levels, antimalarial drug use, and environmental factors. We whole genome sequenced (WGS) P. falciparum clinical samples from Chinese hosts working in Ghana and compared the results with the WGS data of isolates from native Ghanaians downloaded from pf3k. The P. falciparum samples were generally clustered according to their geographic origin, and Chinese imported samples showed a clear African origin with a slightly different distribution from the native Ghanaian samples. Moreover, samples collected from two host populations showed evidence of differences in the intensity of selection. Compared with native Ghanaian samples, the China-imported isolates exhibited a higher proportion of monoclonal infections, and many genes associated with RBC invasion and immune evasion were found to be under less selection pressure. There was no significant difference in the selection of drug-resistance genes due to a similar artemisinin-based combination therapy medication profile. Local selection of malarial parasites is considered to be a result of differences in the host immunity or disparity in the transmission opportunities of the host. In China, most P. falciparum infections were imported from Africa, and under these circumstances, distinct local selective pressures may be caused by varying acquired immunity and transmission intensity. This study revealed the impact of host switching on the immune system, and it may provide a better understanding of the mechanisms that enable clinical immunity to malaria.

Whole plants of Gentianella turkestanorum are commonly used as a traditional Uighur medicine. A phytochemical investigation led to the isolation of eight undescribed gentianellane-type sesterterpenoids (18-epi-nitidasin, gentianelloids D−F, and 18-epi-gentianelloids C–F), one undescribed 11,12-seco-gentianellane (18-epi-alborosin), and three known analogs (nitidasin, gentianelloid C and alborosin) among which gentianelloid C was found for the first time from a natural source. The structures of these compounds were elucidated by extensive spectroscopic analyses (including 1D and 2D NMR, HRMS, IR, and specific rotation) and in the case of 18-epi-gentianelloid C by the single-crystal X-ray diffraction analysis. A putative biosynthetic route for these sesterterpenoids was proposed. The immunosuppressive activity of the isolated compounds was also evaluated by their ability to inhibit the proliferation of T cells and T cell cytokine IFN-γ production. Nitidasin suppressed IFN-γ production with an IC50 value of 16.50 μM, while gentianelloid F and alborosin inhibited the proliferation of and IFN-γ production in T cells with IC50 values of 12.40–14.66 μM.

Phthalate esters (PAEs), one class of the most frequently detected endocrine-disrupting chemicals (EDCs) in marine environment, have aroused wide public concerns because of their carcinogenicity, teratogenicity, and mutagenicity. However, the environmental fate of PAEs in the occurrence of harmful algal blooms remains unclear. In this research, four PAEs with different alkyl chains, i.e., dimethyl phthalate (DMP), diethyl phthalate (DEP), diallyl phthalate (DAP), and dipropyl phtalate (DPrP) were selected as models to investigate toxicity, uptake, and degradation of PAEs in seawater grown with K. brevis, one of the common harmful red tide species. The 96-h median effective concentration (96h-EC50) values followed the order of DMP (over 0.257 mmol L−1) > DEP (0.178 mmol L−1) > DAP (0.136 mmol L−1) > DPrP (0.095 mmol L−1), and the bio-concentration factors (BCFs) were positively correlated to the alkyl chain length. These results indicate that the toxicity of PAEs and their accumulation in K. brevis increased with increasing alkyl chains, due to the higher lipophicity of the longer chain PAEs. With growth of K. brevis for 96 h, the content of DMP, DEP, DAP, and DPrP decreased by 93.3%, 68.2%, 57.4% and 46.7%, respectively, mainly attributed to their biodegradation by K. brevis, accounting for 87.1%, 61%, 46%, 40% of their initial contents, respectively. It was noticed that abiotic degradation had little contribution to the total reduction of PAEs in the algal cultivation systems. Moreover, five metabolites were detected in the K. brevis when exposed to DEP including dimethyl phthalate (DMP), monoethyl phthalate (MEP), mono-methyl phthalate (MMP), phthalic acid (PA), and protocatechuic acid (PrA). While when exposed with to DPrP, one additional intermediate compound diethyl phthalate (DEP) was detected in the cells of K. brevis in addition to the five metabolites mentioned above. These results confirm that the main biodegradation pathways of DEP and DPrP by K. brevis included de-esterification, demethylation or transesterification. These findings will provide valuable evidences for predicting the environmental fate and assessing potential risk of PAEs in the occurrence of harmful algal blooms in marine environment.

Despite advancements in diagnosis and control, Aeromonas infections are considered the leading cause of economic aquaculture loss. In this study, to enhance DNA vaccine efficacy against Aeromonas infections, a fused DNA fragment (1504 bp) of the OmpAI gene from Aeromonas veronii (A. veronii) combined with the C5–I gene from the common carp was generated with splicing by overlapping PCR (SOE-PCR) and expressed in Lactobacillus casei strain CC16. Protein C5–I served as a molecular adjuvant for the antigen OmpAI. Two types of fusion antigens were developed (anchored and secretory). Generally, anchored-type antigens are more effective in inducing immune responses in fish than secretory antigens. Western blot analysis showed that the bands of both antigens were present at 58 kDa. After oral immunization, both DNA vaccines enhanced the serum levels of AKP, ACP, SOD and LZM in immunized carp; the genes IL-10, IL-1β, TNF-α, and IFN-γ in the heart, liver, spleen, head kidney, and intestinal tract were upregulated; and a stronger phagocytic response was triggered in immunized fish. In addition, common carp administered the fused antigens were more protected from Aeromonas challenge (60–73.3% protection). Recombinant Lactobacillus bacteria expressing the fused protein showed a greater propensity for colonization in the intestinal tract in immunized fish than in controls. Here, we provide a promising approach to improve DNA vaccine immunogenicity for protecting common carp from A. veronii infections.

To investigate the dietary exposure risk of flonicamid application on Lonicerae Japonicae Flos and the effect of flonicamid on constituents of Lonicerae Japonicae Flos, field experiments were conducted in Fengqiu, Henan province, and flonicamid residue in samples collected was detected by gas chromatography equipped with electron capture detector (GC-ECD). And chlorogenic acid and luteoloside were determined by HPLC. Dietary exposure risk assessment was conducted through comparing the estimated daily intake (EDI) which was calculated by using the consumed residual level along with the acceptable daily intake (ADI). The effect of flonicamid on chlorogenic acid and luteoloside were obtained by ANOVA statistical analysis and least significant difference (LSD)-t test. The results showed that the terminal-residue contents of flonicamid were under 1.6 mg kg-1. And risk quotient ranged from 0.0011 to 0.0028, indicating the long-term exposure to flonicamid residual through consumption of Lonicerae Japonicae Flos in consumers was relatively low. Flonicamid could suppress the generation of luteoloside, so it was not advised to be used in L. japonica flowering phase. The study aims at providing the useful suggestion on the reasonable flonicamid usage and the reference for the establishment of maximum residue limits (MRLs) of flonicamid in Lonicerae Japonicae Flos.

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h1DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h1DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h2DNA and h3DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels.

Engineered fluorescent indicators for visualizing mercury ion (Hg2+) are powerful tools to illustrate the intracellular distribution and serious toxicity of the ion. However, the sensitive and specific detection of Hg2+ in living cells and in vivo is challenging. This paper reported the development of fluorescent indicators for Hg2+ in green or red color by inserting a circularly permuted fluorescent protein into a highly mercury-specific repressor. These sensors provided a rapid, sensitive, specific, and real-time read-out of Hg2+ dynamics in solutions, bacteria, subcellular organelles of mammalian cells, and zebrafish, thereby providing a useful new method for Hg2+ detection and bioimaging. In conjunction with the hydrogen peroxide sensor HyPer, we found mercury uptake would trigger subcellular oxidative events at the single-cell level, and provided visual evidence of the causality of mercury and oxidative damage. These sensors would paint the landscape of mercury toxicity to cell functions.

The aim of this work was to investigate the application of the nested PCR assay for the detection of Treponema pallidum (TP) DNA from the blood of patients with different stages of syphilis. In this study, a nested PCR method targeting the Tpp47 and polA genes (Tpp47-Tp-PCR and polA-Tp-PCR) was developed to detect TP-DNA in whole blood samples collected from 262 patients with different stages of syphilis (84 primary syphilis, 97 secondary syphilis, and 81 latent syphilis patients). The PCR assay detected T. pallidum DNA in 53.6% and 62.9% of the patients with primary and secondary syphilis, respectively, which was much higher than the detection levels in patients with latent syphilis (7.4%) (both p

A Gram-negative, aerobic, yellow pigmented, non-flagellated, non-gliding, rod-shaped bacterial strain, designated SM1355T, was isolated from Antarctic intertidal sediment collected near the Chinese Antarctic Great Wall Station. The strain grew at 4–35 °C and with 0.5–7.0 % (w/v) NaCl. It hydrolysed aesculin but didn’t reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1355T formed a distinct phylogenetic lineage within the family Flavobacteriaceae , sharing the highest 16S rRNA gene sequence similarity with Flaviramulus ichthyoenteri (96.3 %) and fairly high sequence similarities (95.0–96.0 %) with over 20 recognized species in eight genera of the family Flavobacteriaceae . The predominant fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C15 : 1 G. The major polar lipids were phosphatidylethanolamine and one unidentified lipid. The genomic DNA G+C content of strain SM1355T was 36.2 mol%. Based on the results of the polyphasic characterization for strain SM1355T, it is identified as the representative of a novel species in a new genus of the family Flavobacteriaceae , for which the name Changchengzhania lutea gen. nov., sp. nov. is proposed. The type strain of Changchengzhania lutea is SM1355T (=JCM 30336T=CCTCC AB 2014246T).

This study was aimed to investigate the relationship between the methylation status of serotonin transporter (5-HTT) and major depressive disorder (MDD) in Chinese Han population. A total of 96 patients with MDD and 55 healthy volunteers were recruited, and the methylation index (MtI) at six positions in the cytosine-phosphate-guanosine island of 5-HTT gene was measured for each subject using bisulfite pyrosequencing. MtIs at positions 5 and 6 were higher in patients with MDD than those in controls. According to the multivariable logistic regression analysis, MtIs at positions 4 and 5 were significantly associated with MDD. Besides, depression education was an independent risk factor, whereas higher educational levels were protective factors for MDD. In addition, positions 1 and 4 were negatively correlated with weight and diurnal variation. Therefore, 5-HTT methylation might be closely related with MDD in Chinese Han population because of the correlation with diurnal variation and weight.

The insecticide cypermethrin has been considered as an endocrine-disrupting chemicals (EDCs) with anti-androgenic activity by interfering with interleukin-6 (IL-6) – induced ligand-independent AR signaling. The purpose of this study was to clarify whether the signal transducer and activator of transcription 3 (STAT3) was involved in the antagonism effect of cypermethrin. In this study, the Western blot was to test the level of STAT3 phosphorylation and the mammalian two-hybrid assay was developed to assess the AR–STAT3 interaction. The date showed that IL-6 increased the phosphorylation level of STAT3 and enhanced the AR–STAT3 interaction. Cypermethrin did not affect the phosphorylation level of STAT3 induced by IL-6, while suppressed the AR-STAT3 interaction induced by IL-6 significantly at the concentration of 10−5 M (p

Background Previous studies documented that humoral immune responses participated in neurological damage in neurosyphilis patients. However, the mechanisms that trigger and maintain humoral immunity involved in neurosyphilis remain unknown. Methods Using flow cytometry, expression of B cells was measured in neurosyphilis and non-neurosyphilis. Expression of immunoglobulin indices and chemokine ligand CXCL13 was detected by enzyme-linked immunosorbent assay. The migration and inhibition assays were evaluated by modified chamber assays. The presence of CXCL13+ cells, cluster of differentiation (CD)20+ B cells, CD3+ T cells, CD138+ plasma cells and CD35+ follicular dendritic cells was studied by immunohistochemistry. Results Enrichment of B cells was observed and activated in the cerebrospinal fluid (CSF) of neurosyphilis patients. Immunoglobulin indices were increased and associated with the progress to neurosyphilis. High expression of CSF CXCL13 mediated B cell migration both in vitro and in vivo. There was a positive correlation among the CSF B cells, immunoglobulin indices, and CSF CXCL13 levels. Ectopic germinal centers (EGCs), important structures for humoral immunity, were observed in the intracranial syphilitic gumma. Conclusions CXCL13/CXCR5 mediated the aggregation of B cells, that directed the aberrant humoral immune responses via the formation of EGCs, which suggests a molecular mechanism of neurological damage in neurosyphilis.

The photoelectrochemical (PEC) biosensor, in which light is utilized to excite the photoactive species and current is employed as the detection signal, is a newly appeared yet dynamically developing technique for biological analysis. Based on the assay of DNA binding proteins upon visible light irradiation, a PEC biosensor is constructed for successfully probing a DNA-protein interaction.

Monoacylglycerol lipases (MGLs) are present in all domains of life. However, reports on bacterial MGLs are still limited. Until now, reported bacterial MGLs are all thermophilic/mesophilic enzymes from warm terrestrial environments or deep-sea hydrothermal vent, and none of them originates from marine environments vastly subject to low temperature, high salts, and oligotrophy. Here, we characterized a novel MGL, GnMgl, from the marine cold-adapted and halophilic bacterium Glaciecola nitratireducens FR1064T. GnMgl shares quite low sequence similarities with characterized MGLs (lower than 31%). GnMgl and most of its bacterial homologs harbor a catalytic Ser residue located in the conserved C(A/S)HSMG motif rather than in the typical GxSxG motif reported on other MGLs, suggesting that GnMgl-like enzymes might be different from reported MGLs in catalysis. Phylogenetic analysis suggested that GnMgl and its bacterial homologs are clustered as a separate group in the monoglyceridelipase_lysophospholipase family of the Hydrolase_4 superfamily. Recombinant GnMgl has no lysophospholipase activity but could hydrolyze saturated (C12:0-C16:0) and unsaturated (C18:1 and C18:2) MGs and short-chain triacylglycerols, displaying distinct substrate selectivity from those of reported bacterial MGLs. The substrate preference of GnMgl, predicted to be a membrane protein, correlates to the most abundant fatty acids within the strain FR1064T, suggesting the role of GnMgl in the lipid catabolism in this marine bacterium. In addition, different from known bacterial MGLs that are all thermostable enzymes, GnMgl is a cold-adapted enzyme, with the maximum activity at 30°C and retaining 30% activity at 0°C. GnMgl is also a halotolerant enzyme with full activity in 3.5M NaCl. The cold-adapted and salt-tolerant characteristics of GnMgl may help its source strain FR1064T adapt to the cold and saline marine environment. Moreover, homologs to GnMgl are found to be abundant in various marine bacteria, implying their important physiological role in these marine bacteria. Our results on GnMgl shed light on marine MGLs.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is highly resistant to current treatments. ESCC harbors a subpopulation of cells exhibiting cancer stem-like cell (CSC) properties that contribute to therapeutic resistance including radioresistance, but the molecular mechanisms in ESCC CSCs are currently unknown. Here, we report that ribosomal S6 protein kinase 4 (RSK4) plays a pivotal role in promoting CSC properties and radioresistance in ESCC. RSK4 was highly expressed in ESCC CSCs and associated with radioresistance and poor survival in patients with ESCC. RSK4 was found to be a direct downstream transcriptional target of ΔNp63α, the main p63 isoform, which is frequently amplified in ESCC. RSK4 activated the β-catenin signaling pathway through direct phosphorylation of GSK-3β at Ser9. Pharmacologic inhibition of RSK4 effectively reduced CSC properties and improved radiosensitivity in both nude mouse and patient-derived xenograft models. Collectively, our results strongly suggest that the ΔNp63α/RSK4/GSK-3β axis plays a key role in driving CSC properties and radioresistance in ESCC, indicating that RSK4 is a promising therapeutic target for ESCC treatment.

Background Syphilis caused by the spirochetes Treponema pallidum, which transmitted mainly through sexual contact and blood transmission. The spirochetes spread from the damaged skin to bloodstream, at which point any organ may be affected included bone, central nervous system, and visceral organs-the stomach. Therefore, when treating patients with syphilis, the clinician must rule out the involvement of other organs. Gastric syphilis is a rare presentation of infection with Treponema pallidum, which has no-specific symptoms. Gastric syphilis may occur in any phase of syphilis, predominantly in the secondary (50%), but rarely in the third(6%). There are no standard recommendations specifically address the treatment of gastric syphilis. The general approach is to treat according to the stage of the disease. However, if the clinician does not have experience the disease will be misdiagnosed. Here, we will describe a case of a patient with gastric involvement, whose symptom repeated after conventional treatment. Methods The patent’s history, clinical examination, biology, cerebrospinal fluid,blood nest PCR findings and reatment are reported. We also discuss the profit treatment options. Results we described a case of a patient with the complaint of epigastralgia, pyrosis, and regurgitation. The gastroscopy revealed multiple ulcerative lesions at the antrum, gastric angle and duodenal ampulla, in which suspected cancer. Mucosal biopsy revealed an inflammatory infiltrate with multiple mature plasma cells. Immunohistochemistry and a nested PCR showed the presence of Treponema pallidum in the gastric tissues. But, with conventional treatment, the symptom repeated. Conclusion The patient was diagnosed as having neurosyphilis with gastric syphilis.Her epigastric pain improved on the seventh day of the treatment. Follow-up gastroscopic findings tree months after diagnosis showed improved ulcerative lesions on gastric antrum and duodenal ampulla. The CSF examination was negative. Disclosure No significant relationships.

Background Incubating birds must balance the conflict between thermal needs of the developing embryos and their self-maintenance needs for energy. The Chinese Grouse (Tetrastes sewerzowi) lives in high mountain conifer forests and faces energy stress, cold environment, and predation pressure. Females might adjust incubation rhythm to adapt to these constraints. Methods Two methods were used to investigate egg laying and incubation pattern of the Chinese Grouse; 25 nests were monitored by data loggers and 12 nests by infrared video cameras. Results Female Chinese Grouses usually laid an egg every 2 days. The incubation period was 28–31 days. Overall incubation constancy for Chinese Grouse was 93%. The females took 5.0 recesses per day and 34% of all 1696 recesses were taken in the crepuscular period. The average recess duration was 20.3 min. Females took more and shorter recesses in the latter part of incubation. The females who allocated more time to foraging had a higher reproductive success. Conclusions Probably due to its high egg/body mass ratio, the Chinese Grouse has a long laying interval of 49 h. We suggest that, due to energy stress, females have relatively more recesses and they increase the number of recesses as incubation progresses. To compensate for the embryos’ thermal needs, they extend the incubation period and shorten the recess duration in this cold environment.

Two new spliceostatin analogs, designed as spliceostatins J and K (1 and 2), were isolated and identified from the culture of Pseudomonas sp., along with two known ones, FR901464 (3) and spliceostatin E (4). Their structures were elucidated by detailed interpretation of their spectroscopic data, especially 2D-NMR and HR-ESI-MS. Spliceostatin J (1) represented the first example of spliceostatins bearing an unusual hexahydrofuro[3,4-b]furan moiety. Biological assay showed all the isolated compounds except 1 displayed potent cytotoxic activities against two cancer cell lines (MDA-MB-231 and A-549). Structure-activity-relationship studies revealed that the tetrahydropyran ring in spliceostatin analogs was necessary for their bioactive retention.

A Gram-stain-negative, aerobic, oxidase- and catalase-positive, non-flagellated, non-gliding, yellow-pigmented, and rod-shaped bacterium with appendages, designated strain SM1704T, was isolated from surface seawater collected from the South China Sea. The strain grew at 15–42 °C and with 1–10 % NaCl. It hydrolysed aesculin, but did not hydrolyse gelatin and Tween 80 nor reduce nitrate to nitrite. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SM1704T was affiliated with the genus Muricauda , sharing 94.1–95.9 % sequence similarities with type strains of recognized Muricauda species. The major fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH and the main polar lipids were phosphatidylethanolamine, three unidentified lipids and three unidentified aminolipids. The major respiratory quinone was menaquinone-6. The genomic DNA G+C content of strain SM1704T was 40.7 mol%. On the basis of results from polyphasic analysis of strain SM1704T, it is considered to represent a novel species within the genus Muricauda , for which the name Muricauda nanhaiensis sp. nov. is proposed. The type strain is SM1704T (=KCTC 62797T=MCCC 1K03557T).

Trimethylamine (TMA) and trimethylamine N-oxide (TMAO) are widespread in the ocean and are important nitrogen source for bacteria. TMA monooxygenase (Tmm), a bacterial flavin-containing monooxygenase (FMO), is found widespread in marine bacteria and is responsible for converting TMA to TMAO. However, the molecular mechanism of TMA oxygenation by Tmm has not been explained. Here, we determined the crystal structures of two reaction intermediates of a marine bacterial Tmm (RnTmm) and elucidated the catalytic mechanism of TMA oxidation by RnTmm. The catalytic process of Tmm consists of a reductive half-reaction and an oxidative half-reaction. In the reductive half-reaction, FAD is reduced and a C4a-hydroperoxyflavin intermediate forms. In the oxidative half-reaction, this intermediate attracts TMA through electronic interactions. After TMA binding, NADP+ bends and interacts with D317, shutting off the entrance to create a protected micro-environment for catalysis and exposing C4a-hydroperoxyflavin to TMA for oxidation. Sequence analysis suggests that the proposed catalytic mechanism is common for bacterial Tmms. These findings reveal the catalytic process of TMA oxidation by marine bacterial Tmm and first show that NADP+ undergoes a conformational change in the oxidative half-reaction of FMOs.

Sensitive and accurate analysis of DNA is crucial to better understanding of DNA functions and early diagnosis of fatal disease. Herein, an enhanced photoelectrochemical (PEC) DNA biosensor was proposed based on dual-signal amplification via coupling inorganic-organic nanocomposites sensitization with λ-exonuclease (λ-Exo)-assisted target recycling. The short DNA sequence about chronic myelogenous leukemia (CML, type b3a2) was selected as target DNA (tDNA). ZnO nanoplates were deposited with CdS nanocrystals to form ZnO/CdS hetero-nanostructure, and it was used as PEC substrate for immobilizing hairpin DNA (hDNA). CdTe quantum dots (QDs) covalently linked with meso-tetra(4-carboxyphenyl)porphine (TCPP) to form CdTe/TCPP inorganic-organic nanocomposites, which were utilized as sensitization agents labeling at the terminal of probe DNA (pDNA). When the hDNA-modified sensing electrode was incubated with tDNA and λ-Exo, hDNA hybridized with tDNA, and meanwhile it could be recognized and cleaved by λ-Exo, resulting in the release of tDNA. The rest of nonhybridized hDNA would continuously hybridize with the released tDNA, cleave by λ-Exo, and set free the tDNA again. After λ-Exo-assisted tDNA recycling, more amounts of short DNA (sDNA) fragments coming from digestion of hDNA produced on the electrode and hybridized with CdTe/TCPP-labeled pDNA (pDNA-CdTe/TCPP conjugates). In this case, the sensitization of CdTe/TCPP inorganic-organic nanocomposites occurred, which evidently extend the absorption range and strengthened the absorption intensity of light energy, and accordingly the photocurrent signal significantly promoted. Through introducing the dual-signal amplification tactics, the developed PEC assay allowed a low calculated detection limit of 25.6 aM with a wide detection scope from 0.1 fM to 5 pM for sensitive and selective determination of tDNA.

Self-incompatibility (SI) is an important mating system to prevent inbreeding and promote outcrossing. ARC1 and Exo70A1 function as the downstream targets of the S-locus receptor kinase and play conservative roles in Brassica SI signaling. Based on the sequence homology, Exo70A1 is divided into four subdomains: leucine zipper (Leu(128)-Leu(149)), hypervariable region (Ser(172)-Leu(197)), SUMO modification motif (Glu(260)-Ile(275)), and pfamExo70 domain (His(271)-Phe(627)). ARC1 contains four domains as follows: leucine zipper (Leu(116)-Leu(137)), coiled-coil domain (Thr(210)-Val(236)), U-box (Asp(282)-Trp(347)) motif, and ARM (Ala(415)-Thr(611)) domain. Bioinformatics analysis, yeast two-hybrid screening and pull-down assays show that leucine zipper and coiled-coil motifs of ARC1116-236 are required for the interaction with Exo70A1, while the addition of ARM motif results in loss of the interaction with Exo70A1. Meanwhile, the N-terminal of Exo70A1 without any domains shows a weak interaction with ARC1, and the level of LacZ expression increases with addition of leucine zipper and reaches the maximum value with hypervariable region and SUMO modification motif, indicating that hypervariable region and SUMO modification motif of Exo70A1172-275 is mainly responsible for the binding with ARC1, whereas pfamExo70 domain has little affinity for ARC1. Lys(181) located in the Exo70A1 hypervariable region may be the ubiquitination site mediating the interaction between ARC1 and Exo70A1. Therefore, both the leucine zipper with coiled-coil structure of ARC1116-236, and the hypervariable region and SUMO modification motif of Exo70A1172-275 are the core interaction domains between ARC1 and Exo70A1. Any factors affecting these core domains would be the regulators of ARC1 mediating ubiquitin degradation in self-incompatible system.

It is hypothesized that the pesticide cypermethrin may induce androgen receptor (AR) antagonism via ligand-independent mechanisms. The Real-Time Cell Analysis (RTCA) iCELLigence system was used to investigate the inhibitory effect of cypermethrin on interleukin-6 (IL-6)-induced ligand-independent LNCaP cell growth. Then, the mammalian two-hybrid assays were applied to clarify whether the mechanism of IL-6-induced AR antagonism of cypermethrin was associated with the interactions of the AR and co-activator steroid receptor co-activator-1 (SRC-1) and co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT). Cypermethrin inhibited the LNCaP cell growth induced by IL-6. The interactions of AR-SRC-1 and AR-SMRT mediated by IL-6 were suppressed by cypermethrin. The results indicate that the IL-6-mediated AR antagonism induced by cypermethrin is related to repress the recruitment of co-regulators SRC-1 and SMRT to the AR in a ligand-independent manner. Inhibition of the interactions of AR-SRC-1 and AR-SMRT mediated by IL-6 contributes to the AR antagonism induced by cypermethrin.

Four kinds of 2',3'-cNMPs have been identified in animals and plants, and their physiological roles are also suggested. However, in prokaryotes, while 2',3'-cCMP and 2',3'-cUMP are reported, 2',3'-cGMP or 2',3'-cAMP have never been identified from bacteria or archaea. In addition, there has been no biological method to prepare these cyclic nucleotides. During the study of a novel gene LfliZ from deep-sea bacterium Pseudoalteromonas sp. SM9913, we found that the recombinant LfliZ oligomers contained endogenous substrates, which were then identified to be 2',3'-cCMP, 2',3'-cUMP, 2',3'-cGMP, and 2',3'-cAMP, showing the first evidence for the biological existence of four kinds of 2',3'-cNMPs in prokaryotes. In addition, we further developed a biological method to simultaneously prepare four kinds of 2',3'-cNMPs from Escherichia coli through expressing LfliZ.

Undecylenic acid can effectively control skin fungal infection, but the mechanism of its fungal inhibition is unclear. Hyphal growth of Candida albicans (C. albicans) and biofilm formation have been well recognized as important virulence factors for the initiation of skin infection and late development of disseminated infection. In this study, we seek to investigate antifungal mechanisms of undecylenic acid by evaluating the virulence factors of C. albicans during biofilm formation. We found that undecylenic acid inhibits biofilm formation of C. albicans effectively with optimal concentration above 3 mM. In the presence of this compound, the morphological transition from yeast to filamentous phase is abolished ultimately when the concentration of undecylenic acid is above 4 mM. Meanwhile, the cell surface is crumpled, and cells display an atrophic appearance under scanning electron microscopy even with low concentration of drug treatment. On the other hand, the drug treatment decreases the transcriptions of hydrolytic enzymes such as secreted aspartic protease, lipase, and phospholipase. Hyphal formation related genes, like HWP1, are significantly reduced in transcriptional level in drug-treated biofilm condition as well. The down-regulated profile of these genes leads to a poorly organized biofilm in undecylenic acid treated environment.

As with most annotated two-component systems (TCSs) of Streptomyces coelicolor , the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using a S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNaseI foot-printing of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNaseI foot-printing and electrophoretic mobility shift assays in vitro and by ChIP-qPCR in vivo , and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728 , sco4924 , and sco4011 , markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein MmpA (SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae , respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in Streptomyces . IMPORTANCE Two-component systems (TCS) help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces .

A new demethyl abietane diterpenoid, Triptotin K (3) together with three known compounds, friedelin (1), canophyllal (2), and triptonoterpene (4) were isolated from the roots of Tripterygium wilfordii Hook. f. by silica gel column and preparative high performance liquid chromatography. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Triptotin K showed cytotoxic activities against KB, KBv200, HepG2, and MCF-7/ADM cells lines with IC50 values of 29.88, 36.50, 39.55, and 41.38 μM, respectively.

Summary Understanding of NAD+ metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD+ metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD+ dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD+ concentrations and sensitively respond to increases and decreases in NAD+. Utilizing FiNad, we performed a head-to-head comparison study of common NAD+ precursors in various organisms and mapped their biochemical roles in enhancing NAD+ levels. Moreover, we showed that increased NAD+ synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD+ declines during aging in situ. The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD+-associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.

Na+/K+ ATPase (NKA) is important in maintaining cellular functions. We found that loss of NKA activities in NKAα1+/− mice is associated with increased susceptibility to ischemic injuries following transient middle cerebral artery occlusion (tMCAO). This is corroborated by the neuroprotective effects of an antibody raised against an extracellular DR region (897DVEDSYGQQWTYEQR911, sequence number as in rat) of NKAα subunit (DR-Ab) in both preventive and therapeutic settings. DR-Ab protects cortical neurons against glutamate-induced toxicity by stimulating activities of NKA and Na+/Ca2+ exchanger (NCX), which resulted in accelerated Ca2+ extrusion. DR-Ab also enhanced the association between NKA and GluR2 and therefore reduced the internalization of both proteins from membrane induced by glutamate toxicity. The mechanism appears to involve suppression of GluR2 phosphorylation through PKCα/PICK pathway. Our data indicate that DR-region of NKA may be a novel therapeutic target for drug development for the treatment of ischemic stroke.

Cellular oxidation–reduction reactions are mainly regulated by pyridine nucleotides (NADPH/NADP+ and NADH/NAD+), thiols, and reactive oxygen species (ROS) and play central roles in cell metabolism, cellular signaling, and cell-fate decisions. A comprehensive evaluation or multiplex analysis of redox landscapes and dynamics in intact living cells is important for interrogating cell functions in both healthy and disease states; however, until recently, this goal has been limited by the lack of a complete set of redox sensors. We recently reported the development of a series of highly responsive, genetically encoded fluorescent sensors for NADPH that substantially strengthen the existing toolset of genetically encoded sensors for thiols, H2O2, and NADH redox states. By combining sensors with unique spectral properties and specific subcellular targeting domains, our approach allows simultaneous imaging of up to four different sensors. In this protocol, we first describe strategies for multiplex fluorescence imaging of these sensors in single cells; then we demonstrate how to apply these sensors to study changes in redox landscapes during the cell cycle, after macrophage activation, and in living zebrafish. This approach can be adapted to different genetically encoded fluorescent sensors and various analytical platforms such as fluorescence microscopy, high-content imaging systems, flow cytometry, and microplate readers. A typical preparation of cells or zebrafish expressing different sensors takes 2–3 d; microscopy imaging or flow-cytometry analysis can be performed within 5–60 min. This protocol describes how to combine up to four genetically encoded fluorescent sensors to image redox landscapes. The procedure describes applications in live imaging and flow cytometry of cultured cells, and in vivo imaging in zebrafish larvae.

A yellow-pigmented, rod-shaped, non-flagellated, aerobic and Gram-reaction-negative bacterium, designated strain SM1212T, was isolated from intertidal sand of Kongsfjorden, Svalbard. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1212T constituted a distinct lineage within the family Flavobacteriaceae. It shared highest 16S rRNA gene sequence similarities with the type strains of Bizionia echini (96.0 %), Lacinutrix jangbogonensis (95.8 %) and Psychroserpens damuponensis (95.7 %) and

Trimethylamine N -oxide (TMAO) is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter , has an ATP-binding cassette transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural, and mutational analyses. A Ca 2+ ion chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between “open” and “closed” states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Trp are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO. IMPORTANCE Trimethylamine N -oxide (TMAO) is an important nitrogen source for marine bacteria. The products of TMAO metabolized by bacteria are part of the precursors of the greenhouse gas nitrous oxide. It is unclear how TMAO is recognized and imported by bacteria. TmoX is the substrate-binding protein of a TMAO-specific transporter. Here, the substrate specificity of TmoX of Ruegeria pomeroyi DSS-3 was characterized. The TMAO-binding mechanism of TmoX was studied by biochemical, structural, and mutational analyses. Moreover, our results suggest that the TMAO-binding mechanism may have universal significance in marine bacteria. This study sheds light on how marine microorganisms utilize TMAO and should lead to a better understanding of marine nitrogen cycling.

Brown adipose tissue (BAT) functions to dissipate energy in response to cold exposure or overfeeding. Counteracting obesity has been extensively considered as a promising target. Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. However, the potential biological functions of lncRNAs during mouse brown fat cell differentiation have not been fully understood. Here, we performed lncRNA and mRNA expression profile analysis using microarray technology and identified 1064 lncRNAs with differential expression (fold change| ≥4, p ≤ 0.01) on day 0 and day 8 during differentiation. Furthermore, candidate lncRNAs were characterized by comprehensive examination of their genomic context, gene ontology (GO) enrichment of their associated protein-coding genes and pathway analysis. We identified three lncRNAs (Gm15051, Tmem189 and Cebpd) associated with their flanking coding genes (Hoxa1, C/EBPβ and C/EBPδ), which participated in adipose commitment. Collectively, our findings indicated lncRNAs are involved in mouse BAT development and provide potential targets for obesity therapy.