A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.

Archaeal community in a human-disturbed watershed in southeast China: diversity, distribution, and responses to environmental changes.

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of ß-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.

Berberine suppresses gero-conversion from cell cycle arrest to senescence.

Berberine (BRB), a natural alkaloid, has a long history of medicinal use in both Ayurvedic and old Chinese medicine. Recently, available as a dietary supplement, Berberine is reported to have application in treatment of variety diseases. Previously we observed that BRB inhibited mTOR/S6 signaling concurrently with reduction of the level of endogenous oxidants and constitutive DNA damage response. We currently tested whether Berberine can affect premature, stress-induced cellular senescence caused by mitoxantrone. The depth of senescence was quantitatively measured by morphometric parameters, senescence-associated ß-galactosidase, induction of p21WAF1, replication stress (γH2AX expression), and mTOR signaling; the latter revealed by ribosomal S6 protein (rpS6) phosphorylation. All these markers of senescence were distinctly diminished, in a concentration-dependent manner, by Berberine. In view of the evidence that BRB localizes in mitochondria, inhibits respiratory electron chain and activates AMPK, the observed attenuation of the replication stress-induced cellular senescence most likely is mediated by AMPK that leads to inhibition of mTOR signaling. In support of this mechanism is the observation that rhodamine123, the cationic probe targeting mitochondrial electron chain, also suppressed rpS6 phosphorylation. The present findings reveal that: (a) in cells induced to senescence BRB exhibits gero-suppressive properties by means of mTOR/S6 inhibition; (b) in parallel, BRB reduces the level of constitutive DNA damage response, previously shown to report oxidative DNA damage by endogenous ROS; (c) there appears to a causal linkage between the (a) and (b) activities; (d) the in vitro model of premature stress-induced senescence can be used to assess effectiveness of potential gero-suppressive agents targeting mTOR/S6 and ROS signaling; (e) since most of the reported beneficial effects of BRB are in age-relate diseases, it is likely that gero-suppression is the primary activity of this traditional medicine.

GeoChip-based analysis of the functional gene diversity and metabolic potential of soil microbial communities of mangroves.

Mangroves are unique and highly productive ecosystems and harbor very special microbial communities. Although the phylogenetic diversity of sediment microbial communities of mangrove habitats has been examined extensively, little is known regarding their functional gene diversity and metabolic potential. In this study, a high-throughput functional gene array (GeoChip 4.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of microbial communities in mangrove habitats from mangrove national nature reserves in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 4.0 were detected in the mangrove microbial communities, including carbon (C) fixation, C degradation, methane generation, nitrogen (N) fixation, nitrification, denitrification, ammonification, N reduction, sulfur (S) metabolism, metal resistance, antibiotic resistance, and organic contaminant degradation. Detrended correspondence analysis (DCA) of all detected genes showed that Spartina alterniflora (HH), an invasive species, did not harbor significantly different microbial communities from Aegiceras corniculatum (THY), a native species, but did differ from other species, Kenaelia candel (QQ), Aricennia marina (BGR), and mangrove-free mud flat (GT). Canonical correspondence analysis (CCA) results indicated the microbial community structure was largely shaped by surrounding environmental variables, such as total nitrogen (TN), total carbon (TC), pH, C/N ratio, and especially salinity. This study presents a comprehensive survey of functional gene diversity of soil microbial communities from different mangrove habitats/species and provides new insights into our understanding of the functional potential of microbial communities in mangrove ecosystems.