The functional logic of odor information processing in the Drosophila antennal lobe.

Recent advances in molecular transduction of odorants in the Olfactory Sensory Neurons (OSNs) of the Drosophila Antenna have shown that the odorant object identity is multiplicatively coupled with the odorant concentration waveform. The resulting combinatorial neural code is a confounding representation of odorant semantic information (identity) and syntactic information (concentration). To distill the functional logic of odor information processing in the Antennal Lobe (AL) a number of challenges need to be addressed including 1) how is the odorant semantic information decoupled from the syntactic information at the level of the AL, 2) how are these two information streams processed by the diverse AL Local Neurons (LNs) and 3) what is the end-to-end functional logic of the AL? By analyzing single-channel physiology recordings at the output of the AL, we found that the Projection Neuron responses can be decomposed into a concentration-invariant component, and two transient components boosting the positive/negative concentration contrast that indicate onset/offset timing information of the odorant object. We hypothesized that the concentration-invariant component, in the multi-channel context, is the recovered odorant identity vector presented between onset/offset timing events. We developed a model of LN pathways in the Antennal Lobe termed the differential Divisive Normalization Processors (DNPs), which robustly extract the semantics (the identity of the odorant object) and the ON/OFF semantic timing events indicating the presence/absence of an odorant object. For real-time processing with spiking PN models, we showed that the phase-space of the biological spike generator of the PN offers an intuit perspective for the representation of recovered odorant semantics and examined the dynamics induced by the odorant semantic timing events. Finally, we provided theoretical and computational evidence for the functional logic of the AL as a robust ON-OFF odorant object identity recovery processor across odorant identities, concentration amplitudes and waveform profiles.

Fluorescent sensor based on quantum dots and nano-porphyrin for highly sensitive and specific determination of ethyl carbamate in fermented food.

BACKGROUND: Ethyl carbamate (EC) is a potentially toxic carcinogen produced during fermentation and storage of fermented foods, and many countries have set thresholds for its content in food. Therefore, sensitive, rapid and accurate detection of EC is meaningful to ensure the quality of fermented food. RESULTS: This study introduces a CdTe quantum dots/nano-5,10,15,20-tetrakis (4-methoxyphenyl)-porphyrin (nano TPP-OCH3 ) fluorescence sensor system detection of EC. The specificity of this sensing mainly relies on a photo-induced electron transfer and electrostatic force interaction between EC and nano TPP-OCH3 . This sensor presented a linear range of 10 to 1000 µg L-1 (R2  = 0.9903) with a low detection limit of 7.14 µg L-1 . Meanwhile, the recovery (91.19-101.09%) and precision [relative standard deviation (RSD) = 0.64-3.05%] of the sensor for the analysis of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples were good and could meet the requirements of practical detection. Moreover, the detection results of fermented food (yellow rice wine, soy sauce, Chinese spirits, Pu-erh tea) samples by this sensor are basically consistent with those of high-performance liquid chromatography with fluorescence detector (HPLC-FLD). CONCLUSION: This method was expected to provide a potential platform for sensitive and accurate detection of EC in food safety monitoring, which would provide knowledge of the flavor and quality related to fermented food. © 2021 Society of Chemical Industry.

A 3D screening approach identifies the compound epitajixanthone hydrate as a new inhibitor of cancer cell growth and invasion.

With unique advantages, the small-molecule anticancer drugs have recently gained growing attention. Particular strategies, exemplified by high-throughput screening, fragment-based drug discovery, virtual screening and knowledge-based design, have been developed to identify active compounds. However, such screens generally rely on sophisticated and expensive instrumentations. Herein, we developed a simple spheroids 3D culture system to enable direct screening of small molecules with reliable results. Using this system, we screened 27 fungal natural products and three fungal crude extracts for their inhibitory effects on cancer cell growth, and invasion. We identified that the compound M23 (epitajixanthone hydrate, a derivative of prenylxanthone) and the crude extracts (MPT-191) from the fungi Taxus chinensis showed potential anticancer activity. The effect of epitajixanthone hydrate on cancer cell growth and invasion were further confirmed by the assays of cells viability, trans-well migration and invasion, colony formation and cells reattachment. Overall, Epitajixanthone hydrate was identified as an effective inhibitor of cancer cell growth and invasion by our simple and fast screening platform.

Emerones A-C: three novel merosesquiterpenoids with unprecedented skeletons from Emericella sp. XL029.

Three novel highly oxygenated α-pyrone merosesquiterpenoids, emerones A-C (1-3), have been obtained from the fungus, Emericella sp. XL029, which was isolated from the leaves of the traditional Chinese medicinal plant, Panax notoginseng. The structures and absolute configurations of 1-3 were determined by NMR experiments, X-ray diffraction analysis, and computational methods. Structurally, compound 1 possessed an unprecedented 5/7 bicyclic ring architecture, compound 2 had an unusual substituted 10-membered ring, and compound 3 had an undescribed norsesquiterpene skeleton. In addition, compounds 1 and 2 showed moderate activity towards several types of bacteria and fungi, exhibiting MIC values ranging from 12.5 to 50 µg mL-1, whereas compound 3 showed no antimicrobial activity.

Aureonitols A and B, Two New C13 -Polyketides from Chaetomium globosum, an Endophytic Fungus in Salvia miltiorrhiza.

Two new C13 -polyketides, aureonitols A and B (1 and 2), along with five known compounds (3-7), were isolated from the solid fermentation culture of the plant endophytic fungus Chaetomium globosum from the aerial parts of Salvia miltiorrhiza. The structures and absolute configurations of 1 and 2 were determined by comprehensive spectroscopic data analysis and computed methods. Compound 5 was found to display the remarkable antimicrobial activities against four multidrug-resistant bacteria (Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Staphylococcus epidermidis) with MIC values of 3.13-6.25 µg/mL (ciprofloxacin: 0.78-1.56 µg/mL), and also against all tested fungal strains with MIC values of 3.13-25 µg/mL (ketoconazole: 0.78-12.50 µg/mL).

Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation.

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a "core complex" that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

Sequence analysis and subcellular localization of crucian carp Carassius auratus viperin.

Human viperin is known as an interferon (IFN)-inducible antiviral protein and localizes to endoplasmic reticulum (ER) via its N-terminal amphipathic α-helix. Little is known about subcellular localization of fish viperin. Herein, we characterized subcellular localization of a fish viperin from crucian carp Carassius auratus. Crucian carp viperin is nearly identical to the other viperin proteins in sequence, with the exception of the first N-terminal 70 amino acids that are defined as N-terminal variable domain including an amphipathic α-helix. In addition to N-terminal variable domain, crucian carp viperin protein harbors a conserved middle radical SAM domain and a conserved C-terminal domain. Subcellular localization analyses indicate that crucian carp viperin is a cytoplasmic protein associated with ER. Sequence analyses reveal that amino acids 1-74 forms an amphipathic α-helix domain that drives ER-localization of crucian carp viperin. In addition, Coimmunoprecipitation assays show that crucian carp viperin proteins are able to self-associate. These results together indicate that similar to mammalian homologs, fish viperins likely play important roles in IFN response.

Fish MITA serves as a mediator for distinct fish IFN gene activation dependent on IRF3 or IRF7.

In mammals, cytosolic sensors retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) activate multiple signaling cascades initiating IFN-α/ß expression. IFN regulatory factor 3 (IRF3) is required for the activation of IFN-ß, which, in turn, primes the expression of most IFN-α genes by IFN-induced IRF7 through the STAT1 pathway. In fish, RIG-I overexpression inhibits virus infection by induction of IFN response; however, the subtle signaling cascade mechanism remains to be identified. In this study, we clone an ortholog of MITA, a recently identified adaptor responsible for RLR pathway, from crucian carp (Carassius auratus L.), and demonstrate its ability to suppress viral replication through IRF3/7-dependent IFN response. The pivotal signaling molecules of RLR pathway, including RIG-I, melanoma differentiation-associated gene 5, laboratory of genetics and physiology 2, and TANK-binding kinase 1, are also cloned and characterized, confirming that the RLR-mediated IFN activation is conserved from fish to mammals. Further characterization of distinct IFN gene activation reveals that zebrafish IFN1 and IFN3 are induced by the MITA pathway but are dependent on distinct transcription factors. Whereas fish IFN genes cannot be classified into IFN-α or IFN-ß, zebrafish IFN1 is primarily regulated by IRF3, thereby resembling that of IFN-ß, and zebrafish IFN3 is regulated by IRF7, thereby resembling of those of IFN-αs. In contrast with mammalian IFN-α/ß, zebrafish IFN1 and IFN3 are induced by the basally expressed IRF3 or IRF7, both of which are upregulated by IFN and virus infection. Collectively, these data suggest that IFN genes in fish and mammals have evolved independently to acquire a similar mechanism triggering their expression.

A sensitive fluorescence sensor based on a glutathione modified quantum dot for visual detection of copper ions in real samples.

Copper (Cu2+), as a heavy metal, accumulates in the human body to a certain extent, which can induce various diseases and endanger human health. Rapid and sensitive detection of Cu2+ is highly desired. In present work, a glutathione modified quantum dot (GSH-CdTe QDs) was synthesized and applied in a "turn-off" fluorescence probe to detect Cu2+. The fluorescence of GSH-CdTe QDs could be rapidly quenched in the presence of Cu2+ through aggregation-caused quenching (ACQ), resulting from the interaction between the surface functional groups of GSH-CdTe QDs and Cu2+ and the electrostatic attraction. In the range of 20-1100 nM, the Cu2+ concentration showed a good linear relationship with the fluorescence decline of the sensor, and the LOD is 10.12 nM, which was lower than the U.S. Environmental Protection Agency (EPA) defined limit (20 µM). Moreover, aiming to attain visual analysis, colorimetric method was also used for rapidly detecting Cu2+ by capturing the change in fluorescence color. Interestingly, the proposed approach has successfully been applied for the detection of Cu2+ in real samples (i.e., environment water, food and traditional Chinese medicine) with satisfactory results, which provides a promising strategy for the detection of Cu2+ in practical application with the merits of being rapid, simple and sensitive.

Cooperative roles of fish protein kinase containing Z-DNA binding domains and double-stranded RNA-dependent protein kinase in interferon-mediated antiviral response.

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus.

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.

Characterization of fish IRF3 as an IFN-inducible protein reveals evolving regulation of IFN response in vertebrates.

In mammals, IFN regulatory factor (IRF) 3 is a critical player in modulating transcription of type I IFN and IFN-stimulated genes (ISGs). In this study, we describe the roles of crucian carp (Carassius auratus L.) IRF3 in activating fish IFN and ISGs. Fish IRF3 exhibits a large sequence divergence from mammalian orthologs. Whereas mammalian IRF3 is constitutively expressed, fish IRF3 protein is significantly upregulated by IFN, poly-IC, and other stimuli known as IFN inducers in mammals. The IFN-inducible property of fish IRF3 is consistent with the comparative analysis of 5' flanking regulatory region of vertebrate IRF3 genes, which reveals the presence of typical IFN-stimulated response elements in fish and amphibians, but an absence in tetrapods. Furthermore, either IFN or poly-IC induces phosphorylation and cytoplasmic-to-nuclear translocation of IRF3, which seems essential for its function in that phosphomimic active IRF3 exhibits stronger transactivation than wild type IRF3. Finally, overexpression of fish IRF3 activates production of IFN that in turn triggers ISG transcription through Stat1 pathway, whereas transfection of dominant negative mutant IRF3-DN abrogates poly-IC induction of ISGs, probably owing to blockade of IFN production. Therefore, regulation of IFN response by vertebrate IRF3 is another ancient trait. These data provide evidence of the evolving function of vertebrate IRF3 on regulating IFN response.

Accurate identification of the geographical origins of lily using near-infrared spectroscopy combined with carbon dot-tetramethoxyporphyrin nanocomposite and chemometrics.

Near-infrared spectroscopy technique is a prevailing tool for quality control of foods and traditional Chinese medicines. However, it usually faced the problems of severe peak overlap, low classification accuracy and poor specificity. In this work, the potential of carbon dot-tetramethoxyporphyrin nanocomposite-based nano-effect near-infrared spectroscopy sensor combined with chemometric method was investigated for the accurate identification lily from different geographical origins. Partial least squares-discriminant analysis (PLS-DA) was used for differentiating geographical origins of lily based on the collected traditional and nano-effect near-infrared spectroscopy. Compared with traditional near-infrared spectroscopy, the nano-effect near-infrared spectroscopy obtains superior classification performance with 100% accuracy on the training and test set. The results showed that the proposed method based on near-infrared spectroscopy combined with nanocomposites and chemometrics could be considered as a promising tool for rapid discrimination of the authenticity of food and traditional Chinese medicine in the future.

Rapid identification of the geographical origin of Eucommia ulmoides by using excitation-emission matrix fluorescence combined with chemometric methods.

Eucommia ulmoides is an important and valuable traditional Chinese medicine with various medical functions, and has been widely used as health food in China, Japan, South Korea and other Asian countries for many years. The efficacy and quality of E. ulmoides are closely associated with the geographical origin. In this work, the potential of excitation-emission matrix (EEMs) fluorescence coupled with chemometric methods was investigated for simple, rapid and accurate for identification E. ulmoides from different geographical origins. Parallel factor analysis (PARAFAC) was applied for characterizing the fluorescence fingerprints of E. ulmoides samples. Moreover, k-nearest neighbor (kNN), principal component analysis-linear discriminant analysis (PCA-LDA) and partial least squares discriminant analysis (PLS-DA) models were used for the classification of E. ulmoides samples according to their geographical origins. The results showed that kNN model was more suitable for identification of E. ulmoides samples from different provinces. The kNN model could identify E. ulmoides samples from eight different geographical origins with 100% accuracy on the training and test sets. Therefore, the proposed method was available for conveniently and accurately determining the geographical origin of E. ulmoides, which can expect to be an attractive alternative method for identifying the geographic origin of other traditional Chinese medicines.

Accelerating with FlyBrainLab the discovery of the functional logic of the Drosophila brain in the connectomic and synaptomic era.

In recent years, a wealth of Drosophila neuroscience data have become available including cell type and connectome/synaptome datasets for both the larva and adult fly. To facilitate integration across data modalities and to accelerate the understanding of the functional logic of the fruit fly brain, we have developed FlyBrainLab, a unique open-source computing platform that integrates 3D exploration and visualization of diverse datasets with interactive exploration of the functional logic of modeled executable brain circuits. FlyBrainLab's User Interface, Utilities Libraries and Circuit Libraries bring together neuroanatomical, neurogenetic and electrophysiological datasets with computational models of different researchers for validation and comparison within the same platform. Seeking to transcend the limitations of the connectome/synaptome, FlyBrainLab also provides libraries for molecular transduction arising in sensory coding in vision/olfaction. Together with sensory neuron activity data, these libraries serve as entry points for the exploration, analysis, comparison, and evaluation of circuit functions of the fruit fly brain.

Sesquiterpenoids and diterpenes with antimicrobial activity from Leptosphaeria sp. XL026, an endophytic fungus in Panax notoginseng.

Two new sesquiterpenoids, leptosphins A (1) and B (2), and a new cyclopiane diterpene, leptosphin C (3), along with four known diterpenes (4-7) were isolated from the solid fermentation cultures of an endophytic fungus Leptosphaeria sp. XL026 isolated from the leaves of Panax notoginseng. Their structures were elucidated by extensive spectroscopic methods and single-crystal X-ray diffraction (data). Compound 1 represents the first sulfur-containing eremophilane sesquiterpene. Compounds 5 and 7 displayed medium antifungal activity against Rhizoctonia cerealis, as well as 6 against Verticillium dahliae Kleb with an MIC value of 12.5 µg/mL. Furthermore, compounds 2, 5, 6 and 7 showed medium antibacterial activity against Bacillus cereus with MIC values of 12.5-6.25 µg/mL, as well as 6 also against Pseudomonas aeruginosa with an MIC value of 12.5 µg/mL.

Characterization of protein arginine methyltransferase of TgPRMT5 in Toxoplasma gondii.

BACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.

Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development.

Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction-a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Fish viperin exerts a conserved antiviral function through RLR-triggered IFN signaling pathway.

Mammalian viperin is a typical interferon (IFN)-induced antiviral protein. Fish have viperin homologs; however, little is known about the expression regulation of fish viperins. In this study, we report the expression regulation and antiviral function of a fish viperin from crucian carp Carassius auratus during IFN response. Crucian carp viperin is induced at mRNA and protein levels by fish IFNs and IFN stimuli such as poly(I:C). Consistently, this gene promoter contains multiple transcription factor binding sites including IFN-stimulated response elements (ISRE) and IFN gamma activation sequences (GAS), and is activated by two types of fish IFNs and also by the intracellular and extracellular poly(I:C). Activation of crucian carp viperin promoter by the intracellular poly(I:C) is mediated by retinoic acid-inducing gene I (RIG-I)-like receptors (RLR)-triggered IFN signaling pathway, which is further verified by the findings that each signaling molecule of RLR pathway is able to induce the expression of crucian carp viperin at mRNA and protein levels. Finally, overexpression of crucian carp viperin in cultured fish cells confers significant protection against infection of grass carp reovirus (GCRV). These data suggest that similar to mammalian homologs, crucian carp viperin exerts a conserved function through RLR-triggered IFN signaling pathway.

Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.

BACKGROUND: Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii. METHODS: Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity. RESULTS: TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif. CONCLUSIONS: TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.