SnTe/SnSe Heterojunction Based Ammonia Sensors with Excellent Withstand to Ambient Humidities.

Non-invasive breath testing has gained increasing importance for early disease screening, spurring research into cheap sensors for detecting trace biomarkers such as ammonia. However, real-life deployment of ammonia sensors remains hindered by susceptibility to humidity-induced interference. The SnTe/SnSe heterojunction-based chemiresistive-type sensor demonstrates an excellent response/recovery to different concentrations of ammonia from 0.1 to 100 ppm at room temperature. The improved sensing properties of the heterojunctions-based sensors compared to single-phased SnTe or SnSe can be attributed to the stronger NH3 adsorptions, more Te vacancies, and hydrophobic surface induced by the formed SnTe/SnSe heterojunctions. The sensing mechanisms are investigated in detail by using in situ techniques such as diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS), Kelvin probe, and a.c. impedance spectroscopy together with the Density-Function-Theory calculations. The formed heterojunctions boost the overall charge transfer efficiency between the ammonia and the sensing materials, thus leading to the desirable sensing features as well, with excellent resistance to ambient humidities.

Molecular confirmation and functional study of signal transducer and activator of transcription genes in the Pacific oyster Crassostrea gigas.

The JAK (Janus kinase)-STAT (Signal transducer and activator of transcription) is a well-known functional signaling pathway that plays a key role in several important biological activities such as apoptosis, cell proliferation, differentiation, and immunity. However, limited studies have explored the functions of STAT genes in invertebrates. In the present study, the gene sequences of two STAT genes from the Pacific oyster (Crassostrea gigas), termed CgSTAT-Like-1 (CgSTAT-L1) and CgSTAT-Like-2 (CgSTAT-L2), were obtained using polymerase chain reaction (PCR) amplification and cloning. Multiple sequence comparisons revealed that the sequences of crucial domains of these proteins were conserved, and the similarity with the protein sequence of other molluscan STAT is close to 90 %. The phylogenetic analyses indicated that CgSTAT-L1 and CgSTAT-L2 are novel members of the mollusk STAT family. Quantitative real-time PCR results implied that CgSTAT-L1 and CgSTAT-L2 mRNA expression was found in all tissues, and significantly induced after challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), or poly(I:C). After that, dual-luciferase reporter assays denoted that overexpression of CgSTAT-L1 and CgSTAT-L2 significantly activated the NF-κB signaling, and, interestingly, the overexpressed CgSTAT proteins potentiated LPS-induced NF-κB activation. These results contributed a preliminary analysis of the immune-related function of STAT genes in oysters, laying the foundation for deeper understanding of the function of invertebrate STAT genes.

A newly identified scallop MyD88 interacts with TLR and functions in innate immunity.

Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-ß, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.

Mechanistic Study of the Hydride Migration-Induced Reversible Isomerization in Au22(SR)15H Isomers.

Unraveling the evolution mechanism of metal nanoclusters is of great importance in understanding the formation and evolution of metallic condensed matters. In this work, the specific evolution process between a pair of gold nanocluster (Au NC) isomers is completely revealed by introducing hydride ligands to simplify the research system. A hydride-containing Au NC, Au22(SR)15H, was synthesized by kinetic control, and the positions of the hydrides were then confirmed by combining X-ray diffraction, neutron diffraction, and DFT calculations. Importantly, a reversible structural isomerization was found to occur on this Au22(SR)15H. By combining the crystal structures and theoretical calculations, the focus was placed on the hydride-binding site, and a [Au-H] migration mechanism of this isomerization process is clearly shown. Furthermore, this [Au-H] migration mechanism is confirmed by the subsequent capture and structural determination of theoretically predicted intermediates. This work provides insight into the dynamic behavior of hydride ligands in nanoclusters and a strategy to study the evolution mechanism of nanoclusters by taking the hydride ligand as the breakthrough point.

Low-Dimensional High-Entropy Alloys for Advanced Electrocatalytic Reactions.

Low-dimensional high-entropy alloy (HEA) nanomaterials are widely employed as electrocatalysts for energy conversion reactions, due to their inherent advantages, including high electron mobility, rich catalytically active site, optimal electronic structure. Moreover, the high-entropy, lattice distortion, and sluggish diffusion effects also enable them to be promising electrocatalysts. A thorough understanding on the structure-activity relationships of low-dimensional HEA catalyst play a huge role in the future pursuit of more efficient electrocatalysts. In this review, we summarize the recent progress of low-dimensional HEA nanomaterials for efficient catalytic energy conversion. By systematically discussing the fundamentals of HEA and properties of low-dimensional nanostructures, we highlight the advantages of low-dimensional HEAs. Subsequently, we also present many low-dimensional HEA catalysts for electrocatalytic reactions, aiming to gain a better understanding on the structure-activity relationship. Finally, a series of upcoming challenges and issues are also thoroughly proposed as well as their future directions.

CfIRF8-like interacts with the TBK1/IKKε family protein and regulates host antiviral innate immunity.

The interferon regulatory factor (IRF) family, a class of transcription factors with key functions, are important in host innate immune defense and stress response. However, further research is required to determine the functions of IRFs in invertebrates. In this study, the coding sequence of an IRF gene was obtained from the Zhikong scallop (Chlamys farreri) and named CfIRF8-like. The open reading frame of CfIRF8-like was 1371 bp long and encoded 456 amino acids. Protein domain prediction revealed a typical IRF domain in the N-terminus of the CfIRF8-like protein and a typical IRF3 domain in the C-terminus. Multiple sequence alignment confirmed the conservation of the amino acid sequences of these two functional protein domains. Phylogenetic analysis showed that CfIRF8-like clustered with mollusk IRF8 proteins and then clustered with vertebrate IRF3, IRF4, and IRF5 subfamily proteins. Quantitative real-time PCR detected CfIRF8-like mRNA in all tested scallop tissues, with the highest expression in the gills. Simultaneously, the expression of CfIRF8-like transcripts in gills was significantly induced by polyinosinic-polycytidylic acid challenge. The results of protein interaction experiments showed that CfIRF8-like could directly bind the TBK1/IKKε family protein of scallop (CfIKK2) via its N-terminal IRF domain, revealing the presence of an ancient functional TBK1/IKKε-IRF signaling axis in scallops. Finally, dual-luciferase reporter assay results showed that the overexpression of CfIRF8-like in human embryonic kidney 293T cells could specifically activate the interferon ß promoter of mammals and the interferon-stimulated response element promoter in dose-dependent manners. The findings of this preliminary analysis of the signal transduction and immune functions of scallop CfIRF8-like protein lay a foundation for an in-depth understanding of the innate immune function of invertebrate IRFs and the development of comparative immunology. The experimental results also provide theoretical support for the breeding of scallop disease-resistant strains.

Molecular characterization and functional analysis of a mollusk Rel homologous gene.

Members of the nuclear factor-kappa B (NF-κB) family are crucial regulators of physiological processes such as apoptosis, inflammation, and the immune response, acting as vital transcription factors to perform their function. In this study, we identified a NF-κB homologous gene (CfRel1) in Zhikong scallops. The 3006-bp-long open reading frame encodes 1001 amino acids. The N-terminus of the CfRel1 protein harbors a conserved Rel homology domain (RHD) that contains a DNA-binding domain and a dimerization domain. According to the multiple sequence alignment results, both the DNA-binding and dimerization domains are highly conserved. Phylogenetic analysis indicated that CfRel1 is closely related to both the Dorsal protein of Pinctada fucata and the Rel2 protein of Crassostrea gigas. CfRel1 mRNA was expressed in all tissues tested in the quantitative reverse transcription PCR experiments, with hepatopancreatic tissue expressing the highest levels. Furthermore, after stimulation with lipopolysaccharide, peptidoglycan, or polyinosinic:polycytidylic acid, the mRNA expression level of CfRel1 was markedly increased. The co-immunoprecipitation test results showed that CfRel1 interacted with scallop IκB protein through its RHD DNA-binding domain, suggesting that IκB may regulate the activity of Rel1 by binding to this domain. Dual-luciferase reporter gene assays revealed that CfRel1 overexpression in HEK293T cells activated the activator protein 1 (AP-1), NF-κB, interferon (IFN)α, IFNß, and IFNγ reporter genes, indicating the diverse functions of the protein. In summary, CfRel1 is capable of responding to attacks from pathogen-associated molecular patterns, participating in immune signaling, and activating NF-κB and IFN reporter genes. Our findings contribute to the advancement of invertebrate innate immunity theory, enrich the theory of comparative immunology, and serve as a reference for the future screening of disease-resistant strains in scallops.

Effect of Substituent Groups on the Strength of Intramolecular Hydrogen Bonds in 2,4-Dihydroxybenzophenone UV Absorbers.

2,4-Dihydroxybenzophenone is the most widely used molecule in the benzophenone group of UV absorbers. It is known that the UV absorption ability is dependent on the substituents. Numerous studies have shown that the strength of intramolecular hydrogen bonds is the main factor affecting this type of UV absorber. However, the effect of substituents on the formation and nature of the hydrogen bonds has not been well studied. In this work, the effect of the type of substituent and the substitution position on the absorption intensity of 2,4-dihydroxybenzophenone molecules is verified both experimentally and theoretically. The effect of substituents on the intramolecular hydrogen bonding of 2,4-dihydroxybenzophenone was investigated by DFT calculations. The results indicate that the addition of different substituents leads to various changes in the strength of the hydrogen bonding in 2,4-dihydroxybenzophenone. On the X-substitution site or the Y-substitution site, halogen groups and electron-absorbing groups such as -CN and -NO2 increase the strength of the hydrogen bond, while electron-giving groups such as -N(CH3)2 and -OCH3 decrease the strength of the bond. For the same substituent, the one at the Y site has a higher effect on hydrogen bonding than that at the X site. By NBO analysis, it was found that the substituents would cause charge redistribution of the individual atoms of 2,4-dihydroxybenzophenones, thus affecting the formation and strength of the hydrogen bonds. Moreover, when the substituent is at the Y substitution site, the oxygen atom of the carbonyl group is less able to absorb electrons and more charge is attracted to the oxygen atom of the hydroxyl group, resulting in a larger charge difference between the two oxygen atoms and an increase of bond energy. Finally, a multiple linear regression analysis of the NPA charge number of the atoms involved in the formation of the hydrogen-bonded chelated six-membered ring was performed with the energy of the hydrogen bond and the percentage of influencing factors estimated, which were found to jointly affect the strength of hydrogen bonding. The aim of this study is to provide theoretical guidance for the design of benzophenone-based UV absorbers that absorb UV light of specific wavelength bands.

Scallop RIG-I-like receptor 1 responses to polyinosinic:polycytidylic acid challenge and its interactions with the mitochondrial antiviral signaling protein.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a class of pattern recognition receptors located in the cytoplasm that play a key role in antiviral innate immunity in animals. However, few studies have been conducted on the function of RLR proteins in invertebrates. In this study, the complete coding sequence of the RLR gene of the Zhikong scallop, Chlamys farreri, was obtained and named CfRLR1 with an aim to study the response of CfRLR1 to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and the interaction between the CfRLR1 and C. farreri mitochondrial antiviral signaling (MAVS) protein. Sequence analysis revealed that CfRLR1 encodes 1161 amino acids, and the encoded protein covers two tandem caspase activation and recruitment domains (CARDs), a helicase domain, and a C-terminal regulatory domain. Phylogenetic analysis revealed that CfRLR1 belongs to the RLR family of mollusks. Quantitative real-time polymerase chain reaction showed that CfRLR1 mRNA was expressed in all tested tissues, with its highest expression observed in feet and gill tissues. Furthermore, CfRLR1 expression in the gill tissues was significantly induced after the poly (I:C) challenge. Finally, the results of co-immunoprecipitation and yeast two-hybrid assays revealed that CfRLR1 can bind to the CfMAVS protein via CARD-CARD interactions. Overall, our results elucidate the immune function of invertebrate RLR proteins and provide valuable information on viral disease control for scallop farming.

Transcriptional expression analysis reveals multiple effects of nonylphenol exposure on scallop immune system.

Nonylphenol (NP) is an endocrine disruptor and environmental hormone representing alkylphenol compounds. Marine mollusks are an important source of protein for people worldwide. Many researchers have begun to study the effect of NP on marine mollusks immune system in view of its toxicity; however, the underlying molecular mechanisms require in-depth analysis. In this study, we focused on the transcriptional expression change of immune-related genes and antioxidant enzymes activities variation after NP exposure in a marine bivalve mollusk, Chlamys farreri, to explore the immunomodulatory capacity of NP in marine mollusks. We identified MAVS (Mitochondrial antiviral signaling protein), a key adaptor molecule in the RLR (RIG-I like receptor) pathway, and studied the expression of multiple immune-related genes in response to different concentrations of NP. The key genes involved in RLR/TLR (Toll like receptor) innate immune pathway, apoptosis, and cellular antioxidation mechanism were investigated. Changes in the enzymatic activities of scallop antioxidant enzymes after NP exposure were also examined. The results revealed that the genes expression and the antioxidant enzymes activities show significant changes, thus proving that NP stimulation affects the scallop immune system. Our research results demonstrate the immunomodulatory capacity of NP in marine bivalve mollusks and lay the foundation for further in-depth analysis of the molecular mechanism of NP toxicity.

The first identified invertebrate LGP2-like homolog gene in the Pacific oyster Crassostrea gigas.

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology.

On the Mechanism of Anti-galvanic Metal Displacement Reaction between [Au25(SR)18]- and Metal-Thiolate Complex.

Metal displacement reaction is widely used for preparing alloy nanomaterials. In this study, the mechanism of anti-galvanic metal displacement reaction between the atomic precision [Au25(SC2H4Ph)18]- cluster and the metal-thiolate complexes SR-M-SR (M = Ag, Cd, and Hg) is studied based on dispersion correction density functional theory (DFT-D) calculations. The present study reveals that the metal displacement reaction of the Au25 cluster is carried out through two-stage metal diffusion including the rapid diffusion of the metal heteroatom from metal thiolate to the ligand layer of Au25 cluster and then gradual diffusion of the metal heteroatom into the icosahedral 13-atom core. The atomic charge analysis confirms that the SR group plays a crucial role. Due to the partial reducibility of SR group, it can nucleophilic attack Au atom to result in the fracture of the Au-S bond in the ligand layer and the formation of atomic vacancy on the surface of the metal core, which facilitates the metal heteroatom diffusion from the metal-SR complex to the ligand layer of gold cluster and then to the surface of gold core.

Regulation of Tyrosinase Gene Expression by Retinoic Acid Pathway in the Pacific Oyster Crassostrea gigas.

Retinoic acid (RA) plays important roles in various biological processes in animals. RA signaling is mediated by two types of nuclear receptors, namely retinoic acid receptor (RAR) and retinoid x receptor (RXR), which regulate gene expression by binding to retinoic acid response elements (RAREs) in the promoters of target genes. Here, we explored the effect of all-trans retinoic acid (ATRA) on the Pacific oyster Crassostera gigas at the transcriptome level. A total of 586 differentially expressed genes (DEGs) were identified in C. gigas upon ATRA treatment, with 309 upregulated and 277 downregulated genes. Bioinformatic analysis revealed that ATRA affects the development, metabolism, reproduction, and immunity of C. gigas. Four tyrosinase genes, including Tyr-6 (LOC105331209), Tyr-9 (LOC105346503), Tyr-20 (LOC105330910), and Tyr-12 (LOC105320007), were upregulated by ATRA according to the transcriptome data and these results were verified by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, increased expression of Tyr (a melanin-related TYR gene in C. gigas) and Tyr-2 were detected after ATRA treatment. The yeast one-hybrid assay revealed the DNA-binding activity of the RA receptors CgRAR and CgRXR, and the interaction of CgRAR with RARE present in the Tyr-2 promoter. These results provide evidence for the further studies on the role of ATRA and the mechanism of RA receptors in mollusks.

Unraveling the Nucleation Process from a Au(I)-SR Complex to Transition-Size Nanoclusters.

Atomically precise noble metal nanoclusters provide a critical benchmark for the fundamental research of the origin of condensed matter because they retain the original state of the metal bonds. Also, knowledge about the transition from organometallic complexes to a nanoclusters is important for understanding the structural evolution of the nanoclusters, particularly their nucleation mechanism. Herein, three transition-size gold nanoclusters are prepared via a controlled diphosphine-mediated top-down routine. Starting from small-size nanoclusters, three new nanoclusters including Au13(SAdm)8(L4)2(BPh4) (Au13), Au14(S-c-C6H11)10L4 (Au14), and Au16(S-c-C6H11)11LPh* (Au16) are obtained by controlled clipping on the surface and kernel of initial nanoclusters. Combining their atomically precise structures with DFT theoretical calculations, the overall atom-by-atom structural evolution process from Au12(SR)12 (0 e-) to Au18(SR)14 (4 e-) is mapped out. In addition, studies on their electronic structures show that the evolution from an organometallic complex to nanoclusters is accompanied by a dramatic decrease in the HOMO-LUMO gaps. Most importantly, the formation of the first Au-Au bond is captured in the "Au4S4 to Au5" nucleation process from Au12(SR)12 complex to the Au13 nanocluster. This work provides a deep insight into the origin of inner core in Au NCs and their structural transition relationship with metal complexes.

Ambient ultraviolet B radiation induced valve behavioral acclimation of Pacific oyster which resulted from the different response strategies of smooth and striated adductor muscles.

Light not only conveys image-forming vision but also has an impact on various physiological functions. In particular, ultraviolet B (UVB) radiation has the closest relationship with living organisms. For Pacific oysters (Crassostrea gigas), alteration of valve behavior is one of the most important ways responding to ambient UVB. In the present study, the response of adult C. gigas to sunlight (especially UVB) was evaluated by monitoring valve activity and further elucidated at the physiological and metabolomic levels. After exposure, the valve activity of C. gigas demonstrated flexible acclimation to the ambient conditions. The potential adjustment of osmoregulation and oxidative stress might be related to ambient UVB radiation. Mycosporine-like amino acids might contribute to the protection of C. gigas against UVB, while precursors of ß-alanine and degradation products of 5-hydroxytryptamine might adjust the contraction of the adductor muscles. The different responses of the adductor muscles (smooth and striated) were manifested in signal transduction and metabolisms of energy and nucleotide. This study not only indicated the correlation between the valve behavioral changes in oysters and light radiation, especially UVB, but illustrated the acclimation strategies of oysters to ambient light (UVB) environment.

Functional relationship between CgMyD88-1 and CgMyD88-2 in the Pacific oyster.

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2, have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity.

Molecular characterization and functional analysis of a Rel gene in the Pacific oyster.

The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in regulating many physiological processes such as development, inflammation, apoptosis, cell proliferation, differentiation and immune responses. And the NF-κB/Rel family members were considered as the most important transcription factors in the NF-κB signaling pathway. In this study, we cloned a Rel homolog gene (named as CgRel2) from the Pacific oyster, Crassostrea gigas. The 2115-bp open reading frame (ORF) encodes 704 amino acids and CgRel2 possesses a conserved Rel Homology Domain (RHD) at the N-terminus. Phylogenetic analysis revealed that CgRel2 is most closely related to Pinctada fucata dorsal protein. CgRel2 transcripts are widely expressed in all tested tissues, with the highest expression observed in the labial palp and the gill. Moreover, the expression of CgRel2 is significantly upregulated after lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)] challenge. CgRel2 transfection into human cell lines activated NF-κB, TNFα and oyster IL-17 (CgIL-17) reporter genes in a dose-dependent manner, while CgRel2 overexpression cannot induce ISRE (Interferon stimulation response element) reporter gene's transcriptional activity. Additionally, the results of co-immunoprecipitation showed that CgRel2 or CgRel1 could interact with oyster IκB1, IκB2 and IκB3 proteins strongly, which may be critical for the immune signaling transduction and the regulation of its immune functions. Together, these results suggest that CgRel2 could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB, TNFα and CgIL-17 reporter genes. Thus, CgRel2 could play an important role in the oyster immune system.

RNAi based transcriptome suggests genes potentially regulated by HSF1 in the Pacific oyster Crassostrea gigas under thermal stress.

BACKGROUND: The Pacific oyster Crassostrea gigas is an important fishery resource that is sensitive to temperature fluctuations. Thus, it has evolved a protection mechanism against heat stress by increasing the expression of the gene coding for heat shock protein (HSP) 70 under elevated temperatures. In other animals, heat shock response is a transcriptional response driven by the heat shock transcription factor 1 (HSF1) and thermal stress can trigger HSP70 expression to protect the organism via HSF1. However, the regulatory relationship between HSF1 and HSP remains unclear in Pacific oyster. Therefore, in the present study, we examined the transcriptomic response of several to thermal stress following HSF1 interference. RESULTS: We identified 150 genes responsive to heat shock including seven HSP genes, six of which belonging to the group of 17 HSP genes enriched in response to heat shock, according to weighted gene co-expression network analysis (WGCNA). The other gene was enriched in the module correlated with HSF1 interference. In addition, we found 48 and 47 genes that were upregulated and downregulated by HSF1 in response to heat shock, respectively. In the upregulated genes, we identified one HSP70 potentially regulated by HSF1 in response to heat shock. Furthermore, based on differentially expressed genes and WGCNA analyses, we found that the hypoxia signaling pathway was enriched under heat shock conditions. Five genes were then selected to detect dynamic changes through time. The results suggested that gene expression was correlated with HSF1 expression. The regulation of HSP70 by HSF1 was preliminarily confirmed by binding site predictions and by a dual luciferase assay. CONCLUSIONS: Our results revealed that the expression of HSP70 and HSP20 was initially triggered after 2 h of heat shock, and one of the HSP70 genes was potentially regulated by HSF1. From these results, it is evident that not all heat-inducible genes were triggered simultaneously in response to heat shock stress. Overall, the results revealed a possible HSF1-HSP regulatory relationship in Pacific oyster, providing valuable information on the mechanisms of thermal tolerance in this commercially important oyster.

IKKε-like plays an important role in the innate immune signaling of the Pacific oyster (Crassostrea gigas).

IκB-related kinase ε (IKKε) plays a crucial role in the activation of nuclear factor κB (NF-κB) by phosphorylating inhibitor of NF-κB (IκB) and in the regulation of interferon (IFN) gene expression by phosphorylating IFN regulatory factors (IRFs). In this study, we cloned an IKKε homologue cDNA (designated as CgIKKε-like) from the Pacific oyster, Crassostrea gigas. The full 2896-bp cDNA sequence comprised a 2163-bp open reading frame (ORF) encoding 720 amino acids. CgIKKε-like is ubiquitously expressed, and its mRNA levels in hemocytes after poly I:C, V. alginolyticus, or OsHV-1 µVar challenge were analyzed by real-time PCR. Compared to that in the control, CgIKKε-like mRNA expression levels were significantly increased at 3 h and peaked at 6 h after OsHV-1 µVar challenge; no obvious changes were observed in expression levels until 24 h after either V. alginolyticus or poly I:C challenge, reaching a maximum at 24 h (p < 0.01) and then rapidly decreasing. CgIKKε-like transfection into human cell lines induced NF-κB and ISRE activation, while transfection with CgIKKε-like deletion mutants abolished NF-κB and ISRE reporter gene activation. Additionally, CgIKKε-like could interact with CgTBK1 and could form homodimers strongly, which may be critical for the immune signaling transduction. Last but not least, we found that CgIKKε-like may increase CgIκBs phosphorylation and could interact with CgIRF8. Together, these results suggest that CgIKKε-like could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB and ISRE reporter genes. Thus, CgIKKε-like could play an important role in the oyster immune system.

TANK-binding kinase-1 broadly affects oyster immune response to bacteria and viruses.

As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas. Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus, ostreid herpesvirus 1 (OsHV-1 reference strain and µvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C. gigas and demonstrated its role in the innate immune response to pathogen infections.