Breaking the vicious cycle: Targeting the NLRP3 inflammasome for treating sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a collection of clinical syndromes resulting from sepsis and characterized by widespread brain dysfunction. The high prevalence of SAE has adverse outcomes on the clinical management and prognosis of sepsis patients. However, currently, there are no effective treatments to ameliorate SAE. The pathogenesis of SAE is complex, including neuroinflammation and microglia activation, destruction of the blood-brain barrier (BBB), neurotransmitter dysfunction, cerebral metabolism and mitochondrial impairment, accumulation of amyloid beta and tauopathy, complement activation, among others. Furthermore, these mechanisms intertwine with each other, further complicating the comprehension of SAE. Among them, neuroinflammation mediated by hyperactivated microglia is considered the primary etiology of SAE. This instigates a detrimental cycle wherein BBB permeability escalates, facilitating direct damage to the central nervous system (CNS) by various neurotoxic substances. Activation of the NLRP3 inflammasome, situated within microglia, can be triggered by diverse danger signals, leading to cell pyroptosis, apoptosis, and tauopathy. These complex processes intricately regulate the onset and progression of neuroinflammation. In this review, we focus on elucidating the inhibitory regulatory mechanism of the NLRP3 inflammasome in microglia, which ultimately manifests as suppression of the inflammatory response. Our ultimate objective is to augment comprehension regarding the role of microglial NLRP3 inflammasome as we explore potential targets for therapeutic interventions against SAE.

OsMYB14, an R2R3-MYB Transcription Factor, Regulates Plant Height through the Control of Hormone Metabolism in Rice.

Plant growth must be regulated throughout the plant life cycle. The MYB TF family is one of the largest TF families and is involved in metabolism, lignin biosynthesis and developmental processes. Here, we showed that OsMYB14, a rice R2R3-MYB TF, was expressed in leaves and roots, especially in rice culm and panicles, and that it localized to the nucleus. Overexpression of OsMYB14 (OsMYB14-ox) in rice resulted in a 30% reduction in plant height compared to that of the wild type (WT), while the height of the osmyb14-ko mutant generated using the CRISPR/Cas9 system was not significantly different. Microscopic observations of the first internode revealed that the cell size did not differ significantly among the lines. RNA-seq analysis revealed that genes associated with plant development, regulation, lipid metabolism, carbohydrate metabolism, and gibberellin and auxin metabolic processes were downregulated in the OsMYB14-ox line. Hormone quantitation revealed that inactive GA19 accumulated in OsMYB14-ox but not in the WT or knockout plants, suggesting that GA20 generation was repressed. IAA and IAA-Asp accumulated in OsMYB14-ox and osmyb14-ko, respectively. Indeed, real-time PCR analysis revealed that the expression of OsGA20ox1, encoding Gibberellin20 oxidase 1, and OsGH3-2, encoding IAA-amido synthetase, was downregulated in OsMYB14-ox and upregulated in osmyb14-ko. A protein binding microarray (PBM) revealed the presence of a consensus DNA-binding sequence, the ACCTACC-like motif, in the promoters of the OsGA20ox1 and GA20ox2 genes. These results suggest that OsMYB14 may act as a negative regulator of biological processes affecting plant height in rice by regulating GA biosynthesis and auxin metabolism.

The dexd/H-box helicase DHX40 is a novel negative regulator during GCRV infection via targeting the host RLR helicases and viral nonstructural protein NS38.

RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.

Potential therapies for HCC involving targeting the ferroptosis pathway.

Liver cancer ranks as the third leading cause of cancer-related mortality worldwide, predominantly in the form of hepatocellular carcinoma (HCC). Conventional detection and treatment approaches have proven inadequate for addressing the elevated incidence and mortality rates associated with HCC. However, a significant body of research suggests that combating HCC through the induction of ferroptosis is possible. Ferroptosis is a regulated cell death process characterized by elevated levels of reactive oxygen species (ROS) and lipid peroxide accumulation, both of which are dependent on iron levels. In recent years, there has been an increasing focus on investigating ferroptosis, revealing its potential as an inhibitory mechanism against various diseases, including tumors. Therefore, ferroptosis induction holds great promise for treating multiple types of cancers, including HCC. This article provides a review of the key mechanisms involved in ferroptosis and explores the potential application of multiple targets and pathways associated with ferroptosis in HCC treatment to improve therapeutic outcomes.

Planar Cell Polarity Signaling: Coordinated Crosstalk for Cell Orientation.

The planar cell polarity (PCP) system is essential for positioning cells in 3D networks to establish the proper morphogenesis, structure, and function of organs during embryonic development. The PCP system uses inter- and intracellular feedback interactions between components of the core PCP, characterized by coordinated planar polarization and asymmetric distribution of cell populations inside the cells. PCP signaling connects the anterior-posterior to left-right embryonic plane polarity through the polarization of cilia in the Kupffer's vesicle/node in vertebrates. Experimental investigations on various genetic ablation-based models demonstrated the functions of PCP in planar polarization and associated genetic disorders. This review paper aims to provide a comprehensive overview of PCP signaling history, core components of the PCP signaling pathway, molecular mechanisms underlying PCP signaling, interactions with other signaling pathways, and the role of PCP in organ and embryonic development. Moreover, we will delve into the negative feedback regulation of PCP to maintain polarity, human genetic disorders associated with PCP defects, as well as challenges associated with PCP.

Effect of Pulsatilla decoction on vulvovaginal candidiasis in mice. Evidences for its mechanisms of action.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common infection that affects the female reproductive tract. Pulsatilla decoction (PD), a traditional Chinese herbal medicine, is a classic and effective prescription for VVC. However, its mechanism of action remains unclear. PURPOSE: This study aimed to evaluate the efficacy and potential mechanism of action of the n-butanol extract of Pulsatilla decoction (BEPD) in VVC treatment. METHODS: High performance liquid chromatography (HPLC) was used to detect the main active ingredients in BEPD. A VVC-mouse model was constructed using an estrogen-dependent method to evaluate the efficacy of BEPD in VVC treatment. Fungal burden and morphology in the vaginal cavity were comprehensively assessed. Candida albicans-induced inflammation was examined in vivo and in vitro. The effects of BEPD on the Protein kinase Cδ (PKCδ) /NLR family CARD domain-containing protein 4 (NLRC4)/Interleukin-1 receptor antagonist (IL-1Ra) axis were analyzed using by immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and reverse transcription-quantitative polymerase chain reaction (qRT-PCR). RESULTS: BEPD inhibited fungal growth in the vagina of VVC mice, preserved the integrity of the vaginal mucosa, and suppressed inflammatory responses. Most importantly, BEPD activated the "silent" PKCδ/NLRC4/IL-1Ra axis and negatively regulated NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thereby exerting a therapeutic efficacy on VVC. CONCLUSIONS: BEPD effects on mice with VVC were dose-dependent. BEPD protects against VVC by inhibiting inflammatory response and NLRP3 inflammasome via the activation of the PKCδ/NLRC4/IL-1Ra axis. This study revealed the pharmacological mechanism of BEPD in VVC treatment and provided further evidence for the application of BEPD in VVC treatment.

TrichomeLess Regulator 3 is required for trichome initial and cuticle biosynthesis in Artemisia annua.

Artemisinin is primarily synthesized and stored in the subepidermal space of the glandular trichomes of Artemisia annua. The augmentation of trichome density has been demonstrated to enhance artemisinin yield. However, existing literature lacks insights into the correlation between the stratum corneum and trichomes. This study aims to unravel the involvement of TrichomeLess Regulator 3 (TLR3), which encodes the transcription factor, in artemisinin biosynthesis and its potential association with the stratum corneum. TLR3 was identified as a candidate gene through transcriptome analysis. The role of TLR3 in trichome development and morphology was investigated using yeast two-hybrid, pull-down analysis, and RNA electrophoresis mobility assay. Our research revealed that TLR3 negatively regulates trichome development. It modulates the morphology of Arabidopsis thaliana trichomes by inhibiting branching and inducing the formation of abnormal trichomes in Artemisia annua. Overexpression of the TLR3 gene disrupts the arrangement of the stratum corneum and reduces artemisinin content. Simultaneously, TLR3 possesses the capacity to regulate stratum corneum development and trichome follicle morphology by interacting with TRICHOME AND ARTEMISININ REGULATOR 1, and CycTL. Consequently, our findings underscore the pivotal role of TLR3 in the development of glandular trichomes and stratum corneum biosynthesis, thereby influencing the morphology of Artemisia annua trichomes.

Structure, cooperativity and inhibition of the inosine 5'-monophosphate-specific phosphatase from Saccharomyces cerevisiae.

The nucleoside inosine is a main intermediate of purine nucleotide catabolism in Saccharomyces cerevisiae and is produced via the dephosphorylation of inosine monophosphate (IMP) by IMP-specific 5'-nucleotidase 1 (ISN1), which is present in many eukaryotic organisms. Upon transition of yeast from oxidative to fermentative growth, ISN1 is important for intermediate inosine accumulation as purine storage, but details of ISN1 regulation are unknown. We characterized structural and kinetic behavior of ISN1 from S. cerevisiae (ScISN1) and showed that tetrameric ScISN1 is negatively regulated by inosine and adenosine triphosphate (ATP). Regulation involves an inosine-binding allosteric site along with IMP-induced local and global conformational changes in the monomer and a tetrameric re-arrangement, respectively. A proposed interaction network propagates local conformational changes in the active site to the intersubunit interface, modulating the allosteric features of ScISN1. Via ATP and inosine, ScISN1 activity is likely fine-tuned to regulate IMP and inosine homeostasis. These regulatory and catalytic features of ScISN1 contrast with those of the structurally homologous ISN1 from Plasmodium falciparum, indicating that ISN1 enzymes may serve different biological purposes in different organisms.

A zebrafish NLRX1 isoform downregulates fish IFN responses by targeting the adaptor STING.

In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.

Stilbenes: a promising small molecule modulator for epigenetic regulation in human diseases.

Stilbenes are characterized by a vinyl group connecting two benzene rings to form the basic parent nucleus. Hydrogen atoms on different positions of the benzene rings can be substituted with hydroQxyl groups. These unique structural features confer anti-inflammatory, antibacterial, antiviral, antioxidant, anticancer, cardiovascular protective, and neuroprotective pharmacological effects upon these compounds. Numerous small molecule compounds have demonstrated these pharmacological activities in recent years, including Resveratrol, and Pterostilbene, etc. Tamoxifen and Raloxifene are FDA-approved commonly prescribed synthetic stilbene derivatives. The emphasis is on the potential of these small molecules and their structural derivatives as epigenetic regulators in various diseases. Stilbenes have been shown to modulate epigenetic marks, such as DNA methylation and histone modification, which can alter gene expression patterns and contribute to disease development. This review will discuss the mechanisms by which stilbenes regulate epigenetic marks in various diseases, as well as clinical trials, with a focus on the potential of small molecule and their derivatives such as Resveratrol, Pterostilbene, and Tamoxifen.

SlMYB7, an AtMYB4-Like R2R3-MYB Transcription Factor, Inhibits Anthocyanin Accumulation in Solanum lycopersicum Fruits.

Tomato is a horticultural crop with an incomplete flavonoid metabolic pathway that does not typically accumulate anthocyanins in the fruit. In recent years, intensive studies of the loci Anthocyanin fruit (Aft) and atroviolacium (atv) have clarified the functions of positive regulators (R2R3-MYBs) and a negative regulator (CPC-MYB) in anthocyanin biosynthesis in the fruits. However, little is known about the R2R3-MYB repressors. Here, we used transient overexpression analysis to show that SlMYB7, a subgroup 4 AtMYB4-like R2R3-MYB, inhibited anthocyanin accumulation and reduced expression of anthocyanin synthase genes in the 'black pearl' tomato fruits, which usually accumulate high concentrations of anthocyanins. These findings revealed that SlMYB7 served as a repressor of anthocyanin production. Furthermore, SlMYB7 actively repressed SlANS expression by binding its promoter and passively inhibited anthocyanin synthesis by interacting with the basic helix-loop-helix (bHLH) proteins SlJAF13 and SlAN1, which are involved in the formation of MBW complexes. Thus, SlMYB7 and the MBW complex may coregulate the anthocyanin content of 'black pearl' tomato fruits via a negative feedback loop. These findings provide a theoretical basis for the future enhancement of tomato anthocyanin contents through genetic manipulation of the biosynthetic regulatory network.

miR2118 Negatively Regulates Bacterial Blight Resistance through Targeting Several Disease Resistance Genes in Rice.

Plant miRNAs are a class of noncoding RNA with a length of 21-24 nt that play an important role in plant responses to biotic and abiotic stresses. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases in rice. Our previous work showed that osa-miR2118b/n was induced by Xoo infection. However, the biological function of miR2118 has not yet been characterized in experiments. Herein, we constructed MIR2118b OE, as well as single and double mutants of MIR2118b/n using CRISPR/Cas9. Further results showed that osa-MIR2118b OE plants exhibited longer lesion lengths than the wild type after Xoo inoculation, while MIR2118 CRISPR plants exhibited shorter lesion lengths than the wild type after Xoo inoculation. Co-transformation experiments in rice protoplasts indicated that osa-miR2118 negatively regulated the transcripts of three nucleotide-binding sites and leucine-rich repeat (NLR) genes (LOC_Os08g42700.1, LOC_Os01g05600.1, and LOC_Os12g37290.1) which are predicted target genes of miR2118, but not the mutated NLR genes with a 3 bp insertion at the center of the binding sites. The transcriptional level of the three NLR genes was reversed relative to osa-miR2118 in the MIR2118b OE and MIR2118b CRISPR plants. The above results demonstrate that osa-miR2118b/n negatively regulates the resistance to bacterial blight through negatively regulating several NLR genes.

Regulation of type I IFN responses by deubiquitinating enzyme A in inflammatory bowel diseases.

The development of Inflammatory bowel disease (IBD) is driven by excessive production of pro-inflammatory cytokines including TNF-α, IL-12, and IL-23. This notion is supported by the remarkable clinical success of biologics targeting these cytokines. Recognition of cell wall components derived from intestinal bacteria by Toll-like receptors (TLRs) induces the production of these pro-inflammatory cytokines by macrophages and dendritic cells in human IBD and experimental colitis model. Although sensing of bacterial nucleic acids by endosomal TLRs, specifically TLR3, TLR7, and TLR9 leads to robust production of type I IFNs, it remains debatable whether TLR-mediated type I IFN responses are pathogenic or protective in IBD patients. Additionally, recent studies identified deubiquitinating enzyme A (DUBA) as a novel negative regulator of TLR-mediated type I IFN responses. In light of these observations and their potential applications, in this review, we summarize recent findings on the roles of type I IFN responses and DUBA-mediated negative regulation of these responses in human IBD and experimental colitis model.

Molecular characterization of a novel mitochondrial NOD-like receptor X1 in chicken that negatively regulates IFN-ß expression via STING.

NOD-like receptor X1 (NLRX1) is known for its unique mitochondrial localization and plays a negative role in innate immunity. The initial characterization and function of chicken NLRX1 remain unclear. Here, chicken mitochondrial-targeted NLRX1 (chNLRX1) protein was identified. It had relatively conserved domains, a unique N-terminal "X" mitochondrial-targeting domain (MT) and 2 highly conserved motifs at positions 510-520 and 412-421. Furthermore, chNLRX1 had a unique 53aa N-terminus-MT consistent with its localization to mitochondria. Additionally, chNLRX1 was observed to reduce the DNA sensing adaptor stimulator of interferon genes (STING)-induced IFN-ß by attenuating the STING-TANK-binding kinase 1 (TBK1) interaction, which is a requisite for the STING-TBK1-IFN-ß signaling pathway. These results suggested that chNLRX1 negatively regulated type-I interferon production via STING in host innate immunity.

STING negatively regulates antifungal immunity.

During viral infections, stimulator of interferon genes (STING) exerts a positive protective immune response. Chen et al. now shed light on the distinct role of STING in fungal infections. STING translocates to the phagosome to negatively regulate the immune response against Candida albicans infection through the inhibition of Src-involved Syk phosphorylation.

TLR5S negatively regulates the TLR5M-mediated NF-κB signaling pathway in Epinephelus coioides.

Nuclear factor kappa-B (NF-κB) pathway is a key mediator of inflammation response that plays a role in host defense for pathogen elimination, but excessive activation may lead to tissue damage or pathogen transmission. The negative regulation of NF-κB in lower vertebrates is largely unknown, hindering further understanding of immune signaling evolution. Here, we provided evidence that Epinephelus coioides soluble toll-like receptor 5 (TLR5S), a member of the TLR5 subfamily, has been newly identified as a negative regulator of NF-κB signaling. EcTLR5S was a cytoplasmic protein consisting of 17 leucine-rich repeat domains, which specifically responded to Vibrio flagellin and suppressed flagellin-induced NF-κB signaling activation and cytokine expression. The amino-terminal LRR 1-5 region was necessary for its negative regulatory function. Dual-luciferase reporter assay showed that EcTLR5S significantly inhibited the NF-κB-luc activity induced by inhibitor of NF-κB kinase α (IKKα) and IKKß. Subsequently, the functional relationship between EcTLR5M and EcTLR5S was analyzed, revealing that the negative regulatory function of EcTLR5S targeted the activation of the NF-κB pathway mediated by EcTLR5M. The above results reveal that EcTLR5S negatively regulates the flagellin-induced EcTLR5M-NF-κB pathway activation, which may prevent over-activation of immune signaling and restore homeostasis.

[Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis].

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1ß, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1ß, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1ß, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

The apple 14-3-3 gene MdGRF6 negatively regulates salt tolerance.

The 14-3-3 (GRF, general regulatory factor) regulatory proteins are highly conserved and are widely distributed throughout the eukaryotes. They are involved in the growth and development of organisms via target protein interactions. Although many plant 14-3-3 proteins were identified in response to stresses, little is known about their involvement in salt tolerance in apples. In our study, nineteen apple 14-3-3 proteins were cloned and identified. The transcript levels of Md14-3-3 genes were either up or down-regulated in response to salinity treatments. Specifically, the transcript level of MdGRF6 (a member of the Md14-3-3 genes family) decreased due to salt stress treatment. The phenotypes of transgenic tobacco lines and wild-type (WT) did not affect plant growth under normal conditions. However, the germination rate and salt tolerance of transgenic tobacco was lower compared to the WT. Transgenic tobacco demonstrated decreased salt tolerance. The transgenic apple calli overexpressing MdGRF6 exhibited greater sensitivity to salt stress compared to the WT plants, whereas the MdGRF6-RNAi transgenic apple calli improved salt stress tolerance. Moreover, the salt stress-related genes (MdSOS2, MdSOS3, MdNHX1, MdATK2/3, MdCBL-1, MdMYB46, MdWRKY30, and MdHB-7) were more strongly down-regulated in MdGRF6-OE transgenic apple calli lines than in the WT when subjected to salt stress treatment. Taken together, these results provide new insights into the roles of 14-3-3 protein MdGRF6 in modulating salt responses in plants.

Zebrafish HERC7c Acts as an Inhibitor of Fish IFN Response.

In humans, four small HERCs (HERC3-6) exhibit differential degrees of antiviral activity toward HIV-1. Recently we revealed a novel member HERC7 of small HERCs exclusively in non-mammalian vertebrates and varied copies of herc7 genes in distinct fish species, raising a question of what is the exact role for a certain fish herc7 gene. Here, a total of four herc7 genes (named HERC7a-d sequentially) are identified in the zebrafish genome. They are transcriptionally induced by a viral infection, and detailed promoter analyses indicate that zebrafish herc7c is a typical interferon (IFN)-stimulated gene. Overexpression of zebrafish HERC7c promotes SVCV (spring viremia of carp virus) replication in fish cells and concomitantly downregulates cellular IFN response. Mechanistically, zebrafish HERC7c targets STING, MAVS, and IRF7 for protein degradation, thus impairing cellular IFN response. Whereas the recently-identified crucian carp HERC7 has an E3 ligase activity for the conjugation of both ubiquitin and ISG15, zebrafish HERC7c only displays the potential to transfer ubiquitin. Considering the necessity for timely regulation of IFN expression during viral infection, these results together suggest that zebrafish HERC7c is a negative regulator of fish IFN antiviral response.

A novel miRNA, Cse-miR-33, functions as an immune regulator by targeting CsTRAF6 in Chinese tongue sole (Cynoglossus semilaevis).

The tumor necrosis factor receptor-associated factor 6 (TRAF6) can act as a fundamental adaptor protein in a chain reaction of signal transduction and cascade events to finish off immune defenses. However, immunomodulatory research on TRAF6 gene is still limited in fish. In this study, a novel miRNA, Cse-miR-33 was identified from the whole genome of Chinese tongue sole (Cynoglossus semilaevis). After separate infections with three different Vibrio strains (V. harveyi, V. anguillarum, V. parahemolyticus) and one virus (nervous necrosis virus, NNV), the expressions of CsTRAF6 and Cse-miR-33 displayed significant time-dependent changes in immune related tissues and the trends were opposite in general. Through target gene prediction and dual luciferase reporter assay, Cse-miR-33 was proven to regulate CsTRAF6 by combining with 3'-UTR sequence of the gene. The results of qRT-PCR and western blotting (WB) analyses showed that Cse-miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level, rather than degrading the target mRNA. Further experiment indicated that Cse-miR-33 inhibitor largely reduced the death rate of Chinese tongue sole caused by V. harveyi and NNV. The expressions of CsTRAF6-associated immune genes (such as CsIL-1R, CsMYD88, CsIRAK1, CsTNFα, CsIL6 and CsIL8) were also significantly changed in response to Cse-miR-33 agomir and inhibitor. The study suggested that Cse-miR-33 affected the immune response via targeting CsTRAF6 in C. semilaevis, which would provide us deep insights into miRNA-mediated regulatory network and help improve the immunity in fish.