Isolation and genomic characterization of Klebsiella Lw3 with polychlorinated biphenyl degradability.

Bioremediation of sediment organic pollution has been intensely investigated, but the degradation of complex organic compounds, pesticide residues, and polychlorinated biphenyls (PCBs) remains poorly studied. In this study, sediments were collected from Zhanjiang Mangrove Reserve and inoculated in an inorganic salt medium using only biphenyl (BP) and PCBs as the carbon sources to obtain a PCB-degrading strain. A gram-negative bacterium that metabolized PCBs was isolated and identified as Klebsiella Lw3 by 16S rDNA phylogenetic analysis. Genomic sequencing showed that this bacterium possessed genes related to BP/PCB degradation, and its GC content was 58.2%; we identified 3326 cellular pathways. Gas chromatography-mass spectrometry was employed to test the PCB degrading ability; the results showed that the strain had a good degradation effect on PCB3 at concentrations of 5, 10, 20, 40, and 60 mg/L and that the final degradation rate was higher than 97% after 96 h. Interestingly, this strain showed good biodegradability of PCBs despite having no classical PCB degradation pathway, providing a new direction for Klebsiella research with practical significance for in situ bioremediation of PCB contamination. Overall, this study provides valuable insights into the genetic structure of PCB-degrading strains as well as eco-friendly and low-cost PCB degradation and lays a foundation for the discovery of new degradation pathways.

Effects of Aroclor 1254 on Intestinal Immunity, Metabolism, and Microflora in Zebrafish.

Polychlorinated biphenyls (PCBs) are widely distributed environmental toxicants, whose biological toxicity is magnified step by step through the transmission of the food chain. However, there is little research about the effect of PCBs on intestinal epithelial barrier function. In this experiment, the effects of PCB exposure on the intestines of zebrafish were evaluated. Animals were exposed to Aroclor 1254 (5 µg/L, 10 µg/L, 15 µg/L). After 21 days, the changes in histology, enzyme biomarkers, intestinal microorganisms, and metabolomics were detected. The inflammation and oxidative stress in the intestines of zebrafish were observed. Additionally, there were significant changes in intestinal microbiota and tissue metabolism, most of which were associated with oxidative stress, inflammation, and lipid metabolism. The results showed that PCBs exposure resulted in intestinal inflammation and oxidative stress in zebrafish.Moreover, intestinal metabolites and intestinal microflora of zebrafish were also disturbed. This study verified that exposure can lead to intestinal damage and changes in intestinal metabolic capacity and microorganisms, enlightening the consequences of PCB exposure.

Enhanced M-CSF/CSF1R Signaling Closely Associates with PrPSc Accumulation in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Activation and proliferation of microglia are one of the hallmarks of prion disease and is usually accompanied by increased levels of various cytokines and chemokines. Our previous study demonstrated that the level of brain macrophage colony-stimulating factor (M-CSF) was abnormally elevated during prion infection, but its association with PrPSc is not completely clear. In this study, colocalization of the increased M-CSF with accumulated PrPSc was observed by IHC with serial brain sections. Reliable molecular interaction between total PrP and M-CSF was observed in the brain of 263 K-infected hamsters and in cultured prion-infected cell line. Immunofluorescent assays showed that morphological colocalization of M-CSF with neurons and microglia, but not with astrocytes in brains of scrapie-infected animals. The transcriptional and expressing levels of CSF1R were also significantly increased in prion-infected cell line and mice, and colocalization of CSF1R with neurons and microglia was observed in the brains of prion-infected mouse models. Removal of PrPSc replication by resveratrol in SMB-S15 cells induced limited reductions of cellular levels of M-CSF and CSF1R. In addition, we found that the level of IL-34, another ligand of CSF1R, did not change significantly after prion infection, but its distribution on the cell types in the brains shifted from neurons in healthy mice to the proliferated astrocytes and microglia in scrapie-infected mice. Our data demonstrate activation of M-CSF/IL-34/CSF1R signaling in the microenvironment of prion infection, strongly indicating its vital role in the pathophysiology of prions. It provides solid scientific evidence for the therapeutic potential of inhibiting M-CSF/CSF1R signaling in prion diseases.

Different Aberrant Changes of mGluR5 and Its Downstream Signaling Pathways in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a G-protein-coupled receptor found widely in the central nervous system. It has been involved in the development and progression of some neurodegenerative diseases, but its role in prion diseases is rarely described. In this study, the changes of mGluR5 and its downstream signaling pathways in prion-infected cell line SMB-S15 and the brains of scrapie-infected experimental rodents were evaluated by various methodologies. We found the levels of mGluR5 were significantly increased in a prion-infected cell line SMB-S15 and the cultured cells transiently express an abnormal form PrP (Cyto-PrP). Using immunoprecipitation tests and immunofluorescent assays (IFA), molecular interaction and morphological colocalization between PrP and mGluR5 were observed in the cultured cells. We identified that the (GPCRs)-IP3-IP3R-Ca2+ pathway was activated and the levels of the downstream kinases p38, ERK, and JNK were increased in SMB-S15 cells. After treated with mGluR5 antagonist (MTEP) or the removal of prion replication by resveratrol in SMB-S15 cells, the upregulations of mGluR5 and the downstream kinases were restored in a certain degree. Moreover, increased mGluR5 contributes to the cell damage in prion-infected cells. Contrarily, the levels of mGluR5 in the brains of several scrapie-infected rodent models were decreased at terminal stage. IFA of the brain sections of scrapie-infected rodents demonstrated that the signals of mGluR5 were preferentially colocalized with the NeuN-positive cells, accompanying with severe neuron losses in Nissl staining, which might be a reason for the decrease of mGluR5. Our data indicate the different aberrant alterations of mGluR5 and the downstream signaling pathways during prion infection in vivo and in vitro.

Triterpenoids as bivalent and dual inhibitors of acetylcholinesterase/butyrylcholinesterase from the fruiting bodies of Inonotus obliquus.

Inonotus obliquus, an edible and medicinal mushroom parasitic on birches, has been used in human diet and for traditional therapies in the high latitude regions of Europe and Asia for a long time. Our phytochemical study of this fungus led to the identification of fourteen triterpenoids including four undescribed ones, and two pairs of undescribed phenolic enantiomers. The undescribed compounds were elucidated by extensive spectroscopic analysis including 1D and 2D NMR and HRESIMS, quantum chemical NMR and ECD calculations, as well as single-crystal X-ray diffraction analysis. Bioassays revealed that eight compounds showed dual inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 values ranging from 2.40 ± 0.05 to 28.72 ± 0.46 µM, while 3ß-hydroxy-lanosra-8,24-dien-21-al and trametenolic acid only presented BuChE inhibitory activities with IC50 values of 22.21 ± 1.01 and 7.68 ± 0.13 µM, respectively. In the kinetic studies, the most active three compounds acted as non-competitive inhibitors for both cholinesterases. Furthermore, molecular docking simulations revealed that three compounds demonstrated dual-sites bounding to AChE/BuChE. These triterpenoids emerged as bivalent and dual inhibitors of AChE/BuChE and could be effective drug candidates to prevent and treat Alzheimer's disease in the future.

PrPSc Inhibition and Cellular Protection of DBL on a Prion-Infected Cultured Cell via Multiple Pathways.

Prion diseases are kinds of fatal neurodegenerative diseases without effective therapeutic and prophylactic tools currently. In this study, the inhibition of PrPSc propagation and cellular protectivity of 3,4-dihydroxybenzalacetone (DBL), a small catechol-containing compound isolated and purified from the ethanol extract of Inonotus obliquus, upon a prion-infected cell line SMB-S15 were evaluated. Western blots showed that after incubation with 10 µM of DBL for 14 days, the level of PrPSc in SMB-S15 cells was significantly decreased. Meanwhile, the levels of ROS and hydrogen peroxide were decreased with a dose-dependent manner, whereas the levels of some antioxidant factors, such as HO-1, GCLC and GCLM, were significantly increased. The activities of total glutathione and SOD were up-regulated. DBL-treated SMB-S15 cells also showed the up-regulation of UPR-related proteins, including PERK, IRE1α, ATF6 and GRP78, and activation of autophagy system. Furthermore, the SIRT3 abnormalities caused by prion infection were relieved by DBL treatment. On the contrary, these comprehensive changes were not significantly noticed in the normal partner cell line SMB-PS under the same experimental condition. Those data indicate that treatment of DBL on prion-infected cells can reduce PrPSc level, activate UPR and autophagy system and meanwhile relieve intracellular oxidative stress, endoplasmic reticulum stress and mitochondrial dysfunction by raising the levels of multiple antioxidant factors. The PrPSc inhibition and protective effectiveness of DBL upon the prion-infected cells in vitro make it worthy of further study.

The Status of Polychlorinated Biphenyls (PCBs) Extract from Zhanjiang Mangrove Sediments and the Effects on Tissue Structure and Inflammatory Cytokines in Zebrafish Liver.

Polychlorinated biphenyls (PCBs) are released into the environment from a wide range of sources. The aim of the present study was to investigate the effect of the PCBs extracted from the Zhanjiang mangrove sediments on the immune function of zebrafish. The sediments were collected from 3 mangrove forest points in Zhanjiang (Guangdong Province, China), and the results showed that PCB153 was detected in the sediments of the Guangdong Zhanjiang Mangrove National Nature Reserve (MNNR) and Gaoqiao Mangrove Reserve (GMR), while PCB101, PCB112, PCB155, and PCB198 were detected in the sediments of the Leizhou Peninsula (LP). The zebrafish were exposed to different concentrations of PCBs, i.e., control group, positive control group (Aroclor1254; 10 µg/L), low dose group (LD; 0.6 µg/L), medium-dose group (MD; 3.0 µg/L) and high dose group (HD; 15 µg/L) for 14 days. As compared to the control group, the liver index increased significantly in all PCB treated groups. The liver tissue structure was destroyed in all PCB-treated groups as compared to the control group. In addition, the relative mRNA expression of the target genes (IL-1ß, IL-8, and TNF-α) was significantly expressed in each concentration group. Therefore, these findings suggest that exposure of zebrafish to PCBs can destroy the liver histology and increase the liver index and mRNA expression of inflammatory cytokines in a dose and time-dependent manner.

Effects of Environmental and Spatial Variables on Bacteria in Zhanjiang Mangrove Sediments.

The bottom mud of mangroves contains numerous microbial groups that play an important role in the main ecological functions of the mangrove ecosystem. The diversity and functional and environmental factors related to microbial communities, in terms of the assembly process and in environmental adaptation of the abundance and rare bacterial communities in the mangrove ecosystem, have not been fully explored. We used 16S high-throughput sequencing and operational taxonomic unit analysis to compare the diversity and composition of bacterial communities in different tidal zones in the sediments of the Zhanjiang Gaoqiao Mangrove Nature Reserve, compare the ecological adaptation thresholds and phylogenetic signals of bacterial communities under different environmental gradients, and examine the factors affecting the composition of the bacterial community. The diversity of microbial species and structure and function of the mangrove sediments were affected by the environment, showing the trend: mid tide zone > climax zone > low tide zone. Organic matter content, oxygen content, pH, and total phosphorus were identified as important environmental factors determining the functional diversity of bacterial communities and survival, while pH influences species evolution. The abundant taxa showed a wider response threshold and stronger phylogenetic signals of ecological preference across environmental gradients compared to rare taxa. The abundant bacterial groups have broader environmental adaptability than rare bacterial groups, and different environmental factors affect different communities and functions in the mangrove ecological environment. These results elucidate the mechanism underlying the generation and maintenance of bacterial diversity in response to global environmental changes.

Effects of Microplastics on Microbial Community in Zhanjiang Mangrove Sediments.

Microplastics are easily consumed by marine animals, thereby entering the food chain and endangering animal health. However, there are few studies focusing on the effects of microplastics in mangrove sediments on microbial communities. In order to study the influence of microplastics on microorganisms, microplastics and microorganisms were extracted from Zhanjiang (Guangdong Province, China) mangrove sediments and analyzed. The results showed that there were differences in Shannon and Simpson indices of the microbial community in microplastics (p < 0.05), and there were also differences between JG30_KF_CM45 and Natranaerovirga at the genus level, indicating that microplastics may affect the diversity and composition of microorganisms in sediments. In addition, FAPROTAX function prediction analysis showed that microplastics may affect the nitrification of microbial communities. The results from this study indicate that microplastics affected the diversity and richness of microorganisms in mangrove sediments, which provides an experimental basis for the relationship between microplastics and microorganisms.

Phenolic compounds from the sclerotia of Inonotus obliquus.

Three phenolic compounds (±1 and 2) including a pair of new enantiomers were isolated from the sclerotia of Inonotus obliquus. Their structures were assigned by extensive spectroscopic analyses. All the compounds were evaluated for the neuroprotective activity against oxidative damage on human neuroblastoma SH-SY5Y cells induced by H2O2. Compound 2 showed remarkable neuroprotective effect and significantly improved the cell viability of SH-SY5Y cells treated by H2O2.

Genome-wide identification and expression analysis of terpene synthase gene family in Aquilaria sinensis.

Agarwood is the resinous portion of Aquilaria trees, and has been widely used as medicine and incense. Sesquiterpenes are the main chemical characteristic constituents of agarwood. Terpene synthase (TPS) is a critical enzyme responsible for biosynthesis of sesquiterpene compounds. However, limited information is available on genome-wide identification and characterization of the TPS family in Aquilaria trees. In this study, TPS gene family was identified and characterized in Aquilaria sinensis by bioinformatics methods. The expression of those genes was analyzed by RNA-seq and quantitative real-time PCR. Transcription factors regulating TPS gene expression were identified by yeast one-hybrid and dual-luciferase assay. In total, 26 AsTPS genes (AsTPS1-AsTPS26) were identified, which were classified into five subgroups. Many putative cis-elements putatively involved in stresses and phytohormones (especially jasmonic acid) were identified in the promoter regions of AsTPSs, suggesting that AsTPSs genes may be regulated by stresses and jasmonic acid. Expression analysis revealed seven TPS genes encoding sesquiterpene synthetases were induced by wounding and methyl jasmonic acid (MeJA), which may be related to sesquiterpene biosynthesis. By yeast one-hybrid screening, a ERF transcription factor AsERF1 was identified to interact with the AsTPS1 promoter. Subcellular localization analysis indicated AsERF1 was a nucleus-localized protein. Transient transfection of AsERF1 in leaves of Nicotiana benthamiana significantly enhanced the promoter activation of AsTPS1, suggesting AsERF1 may participate in sesquiterpene biosynthesis by regulating AsTPS1 expression. These data generated in this study provide a foundation for future studies on functional roles and regulation mechanisms of AsTPS in sesquiterpene biosynthesis and agarwood formation.

The low levels of nerve growth factor and its upstream regulatory kinases in prion infection is reversed by resveratrol.

Resveratrol shows ability to eliminate prion replication, but the exact mechanism for prion eradication was not clear yet. Our previous studies demonstrate a downregulation of brain-derived nerve growth factor (BDNF) during prion infection, meanwhile recovery of cerebral nerve growth factor (NGF) level by resveratrol treatment has been reported in other neurodegenerative models. To obtain the possible changes of brain NGF and its upstream regulatory cascade during prion infection and after removal of prion propagation, the levels of NGF and its upstream regulatory factors in various prion-infected and prion-eradicated SMB cell lines and mice brains inoculated with various SMB cellular lysates were assessed with various methodologies. The levels of NGF were significantly decreased during prion replication, while recovered after removal of PrPSc by resveratrol in vitro. Morphological assays revealed that the NGF signals mainly colocalized within neurons, but not in the proliferative astrocytes and microglia. The upstream positive regulatory kinases, such as p-CREB, p-CaMKIV, CaMKK2 were decreased in the prion infected cells and mice brains, whereas the negative regulatory one, p-CaMKK2, was increased. The aberrant situations of those kinases in prion infected cell lines or mice brains could be also partially reversed by removal of prion agent. Moreover, we demonstrated that the signals of CaMKK2 and p-CaMKK2 were also distributed predominately in neurons in the brain tissues. The data illustrate a direct linkage of abnormally repressive NGF and its upstream regulatory kinases with prion infection. Resveratrol has not only the ability to inhibit prion replication, but also to improve the expression of NGF via CaMKK2/CaMKIV cascade, which might benefit the microenvironment in brains.

IP10, KC and M-CSF Are Remarkably Increased in the Brains from the Various Strains of Experimental Mice Infected with Different Scrapie Agents.

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system (CNS) of patients with prion diseases are frequently observed. To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection, the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were firstly screened with Luminex assay. Significant upregulations of interferon gamma-induced protein 10 (IP10), keratinocyte chemoattractant (KC) and macrophage colony stimulating factor (M-CSF) were frequently detected in the brain lysates of many strains of scrapie infected mice. The upregulations of those three cytokines in the brains of scrapie infected mice were further validated by the individual specific ELISA and immunohistochemical assay. Increased specific mRNAs of IP10, M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR. Dynamic analyses of the brain samples collected at different time points post infection revealed the time-dependent increases of those three cytokines, particularly IP10 during the incubation period of scrapie infection. In addition, we also found that the levels of IP10 in cerebral spinal fluid (CSF) of 45 sporadic Creutzfeldt-Jakob disease (sCJD) patients were slightly but significantly higher than those of the cases who were excluded the diagnosis of prion diseases. These data give us a better understanding of inflammatory reaction during prion infection and progression of prion disease.

Dimeric sesquiterpenoid-4H-chromone derivatives from agarwood of Aquilaria crassna and their cytotoxicity.

Six previously undescribed uncommon ester-bonded dimeric compounds (aquilacrassnins A-F) containing a sesquiterpenoid and a 5,6,7,8-tetrahydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydro -4H-chromone units were isolated from the EtOAc extract of agarwood originating from Aquilaria crassna. Their structures were elucidated by spectroscopic (NMR, UV, IR, MS, and ECD) methods. All the compounds were tested for AChE inhibitory activity and cytotoxicity against K562, BEL-7402, SGC-7901, Hela, and A549 tumor cell lines. The results showed that aquilacrassnin A, B, and E exhibited weak cytotoxicity against the five tested cell lines, whereas all the compounds were inactive against AChE.

Aberrant alterations of the expressions and S-nitrosylation of calmodulin and the downstream factors in the brains of the rodents during scrapie infection.

The aberrant alterations of calmodulin (CaM) and its downstream substrates have been reported in some neurodegenerative diseases, but rarely described in prion disease. In this study, the potential changes of Ca2+/CaM and its associated agents in the brains of scrapie agent 263K-infected hamsters and the prion infected cell line SMB-S15 were evaluated by various methodologies. We found that the level of CaM in the brains of 263K-infected hamsters started to increase at early stage and maintained at high level till terminal stage. The increased CaM mainly accumulated in the regions of cortex, thalamus and cerebellum of 263K-infected hamsters and well localization of CaM with NeuN positive cells. However, the related kinases such as total and phosphorylated forms of CaMKII and CaMKIV, as well as the downstream proteins such as CREB and BDNF in the brain of 263K-infected hamsters were decreased. Further analysis showed a remarkable increase of S-nitrosylated (SNO) form of CaM in the brains of 263K-infected hamsters. Dynamic analysis of S-nitrosylated CaM showed the SNO form of CaM abnormally increases in a time-dependent manner during prion infection. Compared with that of the normal partner cell line SMB-PS, the CaM level in SMB-S15 cells was increased, meanwhile, the downstream proteins, such as CaMKII, p-CaMKII, CREB, as well as BDNF, were also increased, especially in the nucleic fraction. No SNO-CaM was detected in the cell lines SMB-S15 and SMB-PS. Our data indicate an aberrant increase of CaM during prion infection in vivo and in vitro.

Four new bi-2-(2-phenylethyl)chromone derivatives of agarwood from Aquilaria crassna.

Four new bi-2-(2-phenylethyl)chromone derivatives, crassins A-D (1-4), were isolated from the EtOAc extract of agarwood originating from Aquilaria crassna. The structures including the absolute configurations of compounds were unambiguously elucidated by extensive spectroscopic methods (1D and 2D NMR, UV, ECD, IR, MS), and by comparison with the literature. The isolated compounds were tested for their acetylcholinesterase (AChE) and α-glucosidase inhibitory activities, as well as cytotoxic activities. All the compounds showed weak inhibitory activity against AChE, while compounds 3 and 4 also displayed weak cytotoxicity against human myeloid leukemia cell line (K562).

Nectar properties and the role of sunbirds as pollinators of the golden-flowered tea (Camellia petelotii).

PREMISE OF THE STUDY: Properties of floral nectar have been used to predict if a plant species is pollinated by birds. To see whether winter-flowering plants evolve nectar properties corresponding to bird pollinators, nectar properties of several Camellia species (including the golden-flowered tea), as well as the role of floral visitors as effective pollinators, were examined. METHODS: Potential pollinators of Camellia petelotii were identified at different times of day and under various weather conditions. A bird exclusion experiment was used to compare the pollination effectiveness of birds and insects. Nectar sugar components (fructose, glucose, and sucrose) from C. petelotii growing wild and another seven Camellia species and 22 additional cultivars (all in cultivation) were examined by high-performance liquid chromatography (HPLC). KEY RESULTS: The sunbird Aethopyga siparaja and honeybees were the most frequent floral visitors to C. petelotii. Honeybee visits were significantly reduced in cloudy/rainy weather. The fruit and seed set of flowers with birds excluded were reduced by 64%, indicating that bird pollination is significant. For the wild populations of C. petelotii, a bagged flower could secrete 157 µL nectar; this nectar has a low sugar concentration (19%) and is sucrose-dominant (87%). The eight Camellia species and 22 cultivars had an average sugar concentration of around 30% and a sucrose concentration of 80%, demonstrating sucrose-dominant nectar in Camellia species. CONCLUSIONS: The nectar sugar composition of Camellia species was characterized by sucrose dominance. In addition, the large reduction in seed set when birds are excluded in the golden-flowered tea also supports the suggestion that these winter-flowering plants may have evolved with birds as significant pollinators.