40S ribosomal protein S18 is a novel maternal peptidoglycan-binding protein that protects embryos of zebrafish from bacterial infections.

Previous studies have shown that ribosomal proteins play important roles in ribosome assembly and protein translation, but other biological functions remain ill-defined. Here it is clearly demonstrated that RPS18 is a newly identified PGN-binding protein which is present abundantly in the eggs/embryos of zebrafish. Recombinant RPS18 not only identifies the bacterial signature molecule PGN, LPS, and LTA, and binds the bacteria as a pattern recognition receptor, but also kills the Gram-positive and Gram-negative bacteria as an antibacterial effector molecule. What is important is that, we reveal that microinjection of rRPS18 into early embryos significantly improved the resistance of the embryos against pathogenic Aeromonas hydrophila challenge, and co-injection of anti-RPS18 antibody could markedly reduced this improved bacterial resistance. In summary, these results indicate that RPS18 is a maternal immune factor that can protect the early embryos of zebrafish against pathogenic attacks. This work also provides another angle for understanding the biological functions of ribosomal proteins.

ELAVL1a is an immunocompetent protein that protects zebrafish embryos from bacterial infection.

Previous studies have shown that ELAVL1 plays multiple roles, but its overall biological function remains ill-defined. Here we clearly demonstrated that zebrafish ELAVL1a was a lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs/embryos of zebrafish. ELAVL1a acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, as well as bacteria, but also as an effector molecule, capable of inhibiting the growth of Gram-positive and -negative bacteria. Furthermore, we reveal that the C-terminal 62 residues of ELAVL1a positioned at 181-242 were indispensable for ELAVL1a antibacterial activity. Additionally, site-directed mutagenesis revealed that the hydrophobic residues Val192/Ile193, as well as the positively charged residues Arg203/Arg204, were the functional determinants contributing to the antimicrobial activity of rELAVL1a. Importantly, microinjection of rELAVL1a into embryos markedly promoted their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was considerably reduced by co-injection of anti-ELAVL1a antibody or by knockdown with morpholino for elavl1a. Collectively, our results indicate that ELAVL1a is a maternal immune factor that can protect zebrafish embryos from bacterial infection. This work also provides another angle for understanding the biological roles of ELAVL1a.

Effects of late-onset dietary intake of salidroside on insulin/insulin-like growth factor-1 (IGF-1) signaling pathway of the annual fish Nothobranchius guentheri.

Salidroside (SDS) is the main active ingredient of Rhodiola which has many biological functions including anti-fatigue, anti-tumor, and immune regulation activities. Our last paper demonstrated that SDS prolonged longevity of the annual fish Nothobranchius guentheri, a promising vertebrate model for anti-aging research. However, little is known about its effect on insulin/insulin-like growth factor-1 (IGF-1) signaling pathway (IIS pathway). In this study, we show that SDS is able to decrease accumulation of SA-ß-Gal. We also show that SDS administraton could reduce the expression levels of Igf-1 and Igf-1R, downregulate the expressions of p-PI3K and p-Akt and upregulate the expression levels of Sirt1 and Foxo3a, both of which are the downstream regulators of the IIS pathway. We also find that SDS could alleviate DNA damage, which could result in increased expression of transcription factor Foxo3a. Collectively, these data indicate that SDS may take part in the IIS pathway.

Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.

Previous studies have shown that ATP synthase α subunit (ATP5A1) plays multiple roles, but our understanding of its biologic functions remains poor and incomprehensive. Here, we clearly demonstrated that zebrafish ATP5A1 was a newly characterized lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs and embryos of zebrafish. Zebrafish ATP5A1 acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, but also as an effector molecule, capable of inhibiting the growth of both gram-positive and -negative bacteria. ATP5A1 could disrupt the bacterial membranes by a combined action of membrane depolarization and permeabilization. We also found that the N-terminal 65 residues were critical for the antibacterial activity of zebrafish ATP5A1. In particular, we showed that microinjection of exogenous recombinant (r)ATP5A1 into early embryos could promote their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was markedly reduced by the coinjection of anti-ATP5A1 antibody or by the knockdown with morpholino for atp5a1 but not by the coinjection of anti-actin antibody. Moreover, each egg/embryo contains a sufficient amount of ATP5A1 in vivo to kill A. hydrophila. Furthermore, the N-terminal 65 residues 1-65 of ATP5A1 α subunit (rA1-65) with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the N-terminal 69 residues 66-134 (rA66-134) or C-terminal residues 135-551 (rA135-551) of ATP5A1 α subunit without in vitro antibacterial activity did not. Finally, we showed that the antibacterial activity of the N-terminal 65 residues of ATP5A1 α subunit was conserved throughout animal evolution. Collectively, these results indicate that ATP5A1 is a novel maternal immunocompetent factor that can protect the early embryos of zebrafish from bacterial infection. This work also provides a new viewpoint for understanding the biologic roles of ATP5A1, which is ubiquitously present in animals.-Ni, S., Zhou, Y., Chen, Y., Du, X., Zhang, S. Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.

Targeted gene therapy of the HSV-TK/hIL-12 fusion gene controlled by the hSLPI gene promoter of human non-small cell lung cancer in vitro.

The incidence of lung cancer and lung cancer-associated mortality have markedly increased worldwide, and gene-targeted therapy has emerged as a promising treatment strategy. The present study aimed to explore the targeted antitumor effect of the herpes simplex virus-thymidine kinase/human interleukin-12 (HSV-TK/hIL-12) fusion gene regulated by the human secretory leukocyte protease inhibitor (hSLPI) promoter of human non-small cell lung cancer (hNSCLC). There were four recombinant eukaryotic expression vectors: pcDNA3.1-CMV-TK, pcDNA3.1-CMV-TK/hIL-12, pcDNA3.1-phSLP-TK and pcDNA3.1-phSLP-TK/hIL-12. These were constructed and transfected into the A549, SPC-A1 and HepG2 cell lines in vitro. The expression of the HSV-TK/hIL-12 fusion gene was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the content of hIL-12 was measured using an ELISA. The antitumor effect of the fusion gene on the A549, SPC-A1 and HepG2 cell lines was determined using an MTT assay. Analysis of the experimental data demonstrated that genes regulated by the cytomegalovirus promoter were expressed at the same level in three different tumor cell lines. Genes regulated by the hSLPI promoter were expressed in the A549 and SPC-A1 cell lines, but not in the HepG2 cell line. Coincidentally, the hIL-12 expression levels were similar to those observed in previous RT-PCR findings. In the Pcmv-TK/Pcmv-TK-hIL-12 group for all three cell lines, as well as in the PSLPI-TK/PSLPI-TK-hIL-12 group for the A549 and SPC-A1 cell lines, the cell survival rate declined significantly and the fusion gene transfection group indicated a lower cell survival rate, when compared with single gene transfection group. The present study indicated that the fusion gene regulated by the hSLPI promoter had a targeted antitumor effect on hNSCLC, and that the combined suicide gene and immune gene therapy had a stronger antitumor effect, compared with single gene therapy.

Time-dependent effects of late-onset dietary intake of salidroside on lifespan and age-related biomarkers of the annual fish Nothobranchius guentheri.

One of the most studied and widely accepted conjectures of aging process is the oxidative stress theory. Previous studies have shown that salidroside can protect D-galactose-induced mouse model against aging and a formulation of Rhodiola rosea extracts (SHR-5) containing salidroside increases lifespan of fruit fly. However, direct evidence linking salidroside itself with the observed anti-aging effect in vivo and relevant molecular mechanisms are poorly defined. In this study, we first demonstrated that salidroside exhibited a time-dependent effect, and late-onset long-term salidroside dietary intake extended the lifespan in the annual fish Nothobranchius guentheri. We then showed that salidroside reduced the accumulation of lipofuscin in the gills as well as the levels of protein oxidation, lipid peroxidation and reactive oxygen species in the muscles; enhanced the activities of catalase, glutathione peroxidase, and superoxide dismutase in the fish; and decelerated the increase of P66shc, a critical factor for regulation of intracellular reactive oxygen species contents. Collectively, these data indicate that salidroside can prolong the lifespan and retard the onset of age-related biomarkers via the antioxidant system in aging fish. It also suggests that salidroside may have a potential usefulness in prolonging the lifespan of the elderly.

Zinc finger protein 365 is a new maternal LPS-binding protein that defends zebrafish embryos against gram-negative bacterial infections.

Zinc finger protein 365 (ZNF365) is widespread in animals, but its function and mechanism remains poorly defined. Here we clearly demonstrate that zebrafish ZNF365 is a newly identified LPS-binding protein capable of interacting with the gram-negative bacteria Escherichia coli, Vibrio anguillarum, and Aeromonas hydrophila, and functions as an antibacterial effector molecule capable of directly killing the bacteria. We also reveal that N-terminal residues 30-55 consisting of the ZnF_C2H2 domain are indispensable for ZNF365 antimicrobial activity. Importantly, microinjection of recombinant ZNF365 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, whereas down-regulation of ZNF365 by injection of znf365 morpholino into embryos considerably lowered their resistance against A. hydrophila challenge. Furthermore, the N-terminal peptide Z30-55 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z56-345 without in vitro antibacterial activity did not. Collectively, these results indicate that ZNF365 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role to be assigned to ZNF365 proteins. This work also provides new insights into the immunologic function of the zinc finger proteins that are widely distributed in various animals.-Du, X., Zhou, Y., Song, L., Wang, X., Zhang, S. Zinc finger protein 365 is a new maternal LPS-binding protein that defends zebrafish embryos against gram-negative bacterial infections.

Identification of neuroglobin as a novel player in anti-bacterial responses in amphioxus.

Theoretical considerations support various functions of neuroglobin (Ngb), but further studies are required for full characterization of these functions. In this study, we identified the presence of a single Ngb gene, BjNgb, in the amphioxus Branchiostoma japonicum. BjNgb was expressed in various tissues including the notochord, gonads (ovary and testis) and gill, and up-regulated significantly in response to the challenge with LPS and LTA, suggesting involvement in immune response of amphioxus against bacterial infection. In accord, we demonstrated for the first time that recombinant BjNgb (rBjNgb) not only interacted with the Gram-positive and negative bacteria as well as their conserved surface components LPS and LTA, but also enhanced the phagocytosis of bacteria by macrophages. Collectively, these data suggest that BjNgb is a novel player in amphioxus, via functioning as a pattern recognition molecule and an opsonin.

Association between tea consumption and risk of cognitive disorders: A dose-response meta-analysis of observational studies.

BACKGROUND: The epidemiological evidence for a dose-response relationship between tea consumption and risk of cognitive disorders is sparse. The aim of the study was to summarize the evidence for the association of tea consumption with risk of cognitive disorders and assess the dose-response relationship. METHODS: We searched electronic databases of Pubmed, Embase, and Cochrane Library (from 1965 to Jan 19, 2017) for eligible studies that published in the international journals. A random-effects model was used to pool the most adjusted odds ratios (ORs) and the corresponding 95% confidence intervals (CIs). RESULTS: Seventeen studies involving 48,435 participants were included in our study. The meta-analysis showed that a higher tea consumption was associated with a significant reduction in the risk of cognitive disorders (OR=0.73, 95% CI: 0.65-0.82). When considering the specific types of tea consumption, the significantly inverse association is only found in green tea consumption (OR=0.64, 95% CI: 0.53-0.77) but not in black/oolong tea consumption (OR=0.75, 95% CI: 0.55-1.01). Dose-response meta-analysis indicated that tea consumption is linearly associated with a reduced risk of cognitive disorders. An increment of 100 ml/day, 300 ml/day, and 500 ml/day of tea consumption was associated with a 6% (OR=0.94, 95% CI: 0.92-0.96), 19% (OR=0.81, 95% CI: 0.74-0.88), and 29% (OR=0.71, 95% CI: 0.62-0.82) lower risk of cognitive disorders. CONCLUSIONS: Tea consumption is inversely and linearly related to the risk of cognitive disorders. More studies are needed to further confirm our findings.

Intermittent food restriction initiated late in life prolongs lifespan and retards the onset of age-related markers in the annual fish Nothobranchius guentheri.

Two of the most studied and widely accepted conjectures on possible aging mechanisms are the oxidative stress hypothesis and the insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway. Intermittent fasting (IF) is known to modulate aging and to prolong lifespan in a variety of organisms, but the mechanisms are still under debate. In this study, we first demonstrated that late-onset two consecutive days a week fasting, a form of IF, termed intermittent food restriction (IFR), exhibited a time-dependent effect, and long-term late-onset IFR extended the mean lifespan and maximum lifespan by approximately 3.5 and 3 weeks, respectively, in the annual fish Nothobranchius guentheri. We also showed that IFR reduced the accumulation of lipofuscin in the gills and the protein oxidation and lipid peroxidation levels in the muscles. Moreover, IFR was able to enhance the activities of antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase in the fish. Finally, IFR was also able to decelerate the decrease of SirT1 and Foxo3A, but accelerate the decrease of IGF-1. Collectively, our findings suggest that late-onset IFR can retard the onset of age-related markers, and prolong the lifespan of the aging fish, via a synergistic action of an anti-oxidant system and the IIS pathway. It also proposes that the combined assessment of anti-oxidant system and IIS pathway will contribute to providing a more comprehensive view of anti-aging process.

Functional characterization of avidins in amphioxus Branchiostoma japonicum: Evidence for a dual role in biotin-binding and immune response.

Avidin is well known for its high affinity to biotin and has been found in many egg-laying vertebrate species. However, little is known about avidin in invertebrate species to date. Here we clearly showed the presence of two avidin genes, Bjavidin1 and Bjavidin2, in the amphioxus Branchiostoma japonicum, the first ones in non-vertebrate animals. We also showed that the expression of both Bjavidin1 and Bjavidin2 were inducible by progesterone, LTA and LPS. Moreover, we demonstrated for the first time that in addition to biotin-binding, the recombinant proteins rBjAVIDIN1 and rBjAVIDIN2 were not only able to interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also to enhance phagocytosis of bacteria by macrophages, suggesting that BjAVIDIN1 and BjAVIDIN2 both function as pattern recognition receptors and opsonins. It is thus clear that avidin may play a dual role in biotin-binding and immune response.

Functional characterization of Vitellogenin_N domain, domain of unknown function 1943, and von Willebrand factor type D domain in vitellogenin of the non-bilaterian coral Euphyllia ancora: Implications for emergence of immune activity of vitellogenin in basal metazoan.

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. However, the time when Vg was endowed with immunological activities during animal evolution remains elusive. Here we demonstrate for the first time that the recombinant proteins rVitellogenin_N, rDUF1943, and rVWD from Vg of the basal metazoan coral Euphyllia ancora not only interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also enhance phagocytosis of bacteria by macrophages. Moreover, challenge with LPS results in a marked up-regulation of vg in the coral E. ancora. These data suggest that E. ancora Vg, like that described in the bilaterian oviparous animals fish and amphioxus, is a molecule related to antibacterial defense, indicating that the timing of the emergence of immune role of Vg predates the divergence of the cnidarian (non-bilaterian) and bilaterian lineages.

Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections.

Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11-37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals.

Identification of a novel antimicrobial peptide from amphioxus Branchiostoma japonicum by in silico and functional analyses.

The emergence of multi-drug resistant (MDR) microbes leads to urgent demands for novel antibiotics exploration. We demonstrated a cDNA from amphioxus Branchiostoma japonicum, designated Bjamp1, encoded a protein with features typical of antimicrobial peptides (AMPs), which is not homologous to any AMPs currently discovered. It was found that Bjamp1 was expressed in distinct tissues, and its expression was remarkably up-regulated following challenge with LPS and LTA. Moreover, the synthesized putative mature AMP, mBjAMP1, underwent a coil-to-helix transition in the presence of TFE or SDS, agreeing well with the expectation that BjAMP1 was a potential AMP. Functional assays showed that mBjAMP1 inhibited the growth of all the bacteria tested, and induced membrane/cytoplasmic damage. ELISA indicated that mBjAMP1 was a pattern recognition molecule capable of identifying LPS and LTA. Importantly, mBjAMP1 disrupted the bacterial membranes by a membranolytic mechanism. Additionally, mBjAMP1 was non-cytotoxic to mammalian cells. Collectively, these data indicate that mBjAMP1 is a new AMP with a high bacterial membrane selectivity, rendering it a promising template for the design of novel peptide antibiotics against MDR microbes. It also shows for the first time that use of signal conserved sequence of AMPs is effective identifying potential AMPs across different animal classes.

De Novo Transcriptome Assembly and Differential Gene Expression Profiling of Three Capra hircus Skin Types during Anagen of the Hair Growth Cycle.

Despite that goat is one of the best nonmodel systems for villus growth studies and hair biology, limited gene resources associated with skin or hair follicles are available. In the present study, using Illumina/Solexa sequencing technology, we de novo assembled 130 million mRNA-Seq reads into a total of 49,115 contigs. Searching public databases revealed that about 45% of the total contigs can be annotated as known proteins, indicating that some of the assembled contigs may have previously uncharacterized functions. Functional classification by KOG and GO showed that activities associated with metabolism are predominant in goat skin during anagen phase. Many signaling pathways was also created based on the mapping of assembled contigs to the KEGG pathway database, some of which have been previously demonstrated to have diverse roles in hair follicle and hair shaft formation. Furthermore, gene expression profiling of three skin types identified ~6,300 transcript-derived contigs that are differentially expressed. These genes mainly enriched in the functional cluster associated with cell cycle and cell division. The large contig catalogue as well as the genes which were differentially expressed in different skin types provide valuable candidates for further characterization of gene functions.

Differential gene expression analysis between anagen and telogen of Capra hircus skin based on the de novo assembled transcriptome sequence.

Capra hircus, an economically important livestock, plays an indispensable role in the world animal fiber industry. In the present study, using Illumina/Solexa high throughput sequencing technology, we sequenced and de novo assembled the goat skin transcriptome corresponding to the anagen and telogen of the hair growth cycle. Approximately 53Mb of transcriptome sequences consisting of 57,040 high quality contigs was obtained. More than 8300 contigs were predicted to contain a full length coding sequence. Approximately 43% of the total contigs were identified as harboring homologs of sequences from other organisms in the public database. Based on the assembled transcript-derived contigs, we identified about 7000 transcripts that were differentially expressed between the anagen and telogen libraries. These differentially expressed genes were mainly enriched in signal transduction mechanisms, extracellular structures and cytoskeleton from the KOG database and in ECM receptor interaction, focal adhesion and gap junction from the KEGG pathway database, indicating the essential roles of these genes may play in cell-to-cell and cell-to-matrix communications during the active hair growth phase. In addition, many signaling pathway associated ligands and/or receptors were also identified as up-regulated genes during the anagen phase compared with the telogen stage, suggesting that enhanced cross-talk among signaling transduction pathways may be required for anagen of the hair cycle. These differentially expressed genes, especially those that were over-represented in each of the functional clusters and biochemical pathways, provide valuable resources and opportunities for characterizing the gene functions associated with hair fiber growth as well as for breeding elite Cashmere goat species.

[Experimental studies on the antitumor immunity induced by total laryngeal carcinoma RNA- transfected dendritic cells].

OBJECTIVE: The efficiency of antitumor immunity induced by dendritic cells (DCs) transfected with total RNA of laryngeal carcinoma cells was explored. METHOD: DCs, induced from human peripheral blood mononuclear cells, were transfected with total RNA of laryngeal carcinoma cells. The specific antitumor immunity of cytotoxic T Lymphocytes (CTLs) that were actived by RNA-transfected DCs were detected by MTT methods in vitro. In vivo, antitumor-specific CTLs were subcutaneously injected into the nude mice previously. After 7 days, the laryngeal carcinoma cells were seeded and the tumor occurrence rate was observed. Tumor-loaded nude mice were treated by specific CTLs once (the treated group) or twice (the retreated group). The growth of the implanted tumor was observed too. RESULT: DCs that transfected with tumor RNA can significantly active CTLs which induced antitumor-specific immune response against laryngeal carcinoma in vitro. In vivo, the tumor occurrence rate of the treatment group was predominantly reduced compared with that of the control (P < 0.01). The implanted tumor size of the treated and retreated groups were both significantly reduced (P < 0.05, P < 0.01) compared with the control too, especially the retreated ones (P < 0.05). CONCLUSION: The tumor RNA loaded DCs can significantly active CTLs and the antitumor specific CTLs can both induce antitumor specific immune response against laryngeal carcinoma in vitro and inhibit the growth of the implanted tumor in vivo.