Tumor-associated macrophages as a potential therapeutic target in thyroid cancers.

Macrophages are important precursor cell types of the innate immune system and bridge adaptive immune responses through the antigen presentation system. Meanwhile, macrophages constitute substantial portion of the stromal cells in the tumor microenvironment (TME) (referred to as tumor-associated macrophages, or TAMs) and exhibit conflicting roles in the development, invasion, and metastasis of thyroid cancer (TC). Moreover, TAMs play a crucial role to the behavior of TC due to their high degree of infiltration and prognostic relevance. Generally, TAMs can be divided into two subgroups; M1-like TAMs are capable of directly kill tumor cells, and recruiting and activating other immune cells in the early stages of cancer. However, due to changes in the TME, M2-like TAMs gradually increase and promote tumor progression. This review aims to discuss the impact of TAMs on TC, including their role in tumor promotion, gene mutation, and other factors related to the polarization of TAMs. Finally, we will explore the M2-like TAM-centered therapeutic strategies, including chemotherapy, clinical trials, and combinatorial immunotherapy.

Arterial Baroreflex Dysfunction Promotes Neuroinflammation by Activating the Platelet CD40L/Nuclear Factor Kappa B Signaling Pathway in Microglia and Astrocytes.

Arterial baroreflex (ABR) dysfunction has previously been associated with neuroinflammation, the most common pathological feature of neurological disorders. However, the mechanisms mediating ABR dysfunction-induced neuroinflammation are not fully understood. In the present study, we investigated the role of platelet CD40 ligand (CD40L) in neuroinflammation in an in vivo model of ABR dysfunction, and microglia and astrocyte activation in vitro. ABR dysfunction was induced in Sprague‒Dawley rats by sinoaortic denervation (SAD). We used ELSA and immunofluorescence to assess the effect of platelet CD40L on glial cell polarization and the secretion of inflammatory factors. By flow cytometry, we found that rats subjected to SAD showed a high level of platelet microaggregation and upregulation of CD40L on the platelet surface. The promotion of platelet invasion and accumulation was also observed in the brain tissues of rats subjected to SAD. In the animal model and cultured N9 microglia/C6 astrocytoma cells, platelet CD40L overexpression promoted neuroinflammation and activated M1 microglia, A1 astrocytes, and the nuclear factor kappa B (NFκB) signaling pathway. These effects were partially blocked by inhibiting platelet activity with clopidogrel or inhibiting CD40L-mediated signaling. Our results suggest that during ABR dysfunction, CD40L signaling in platelets converts microglia to the M1 phenotype and astrocytes to the A1 phenotype, activating NFκB and resulting in neuroinflammation. Thus, our study provides a novel understanding of the pathogenesis of ABR dysfunction-induced neuroinflammation and indicates that targeting platelet CD40L is beneficial for treating central nervous system (CNS) disorders associated with ABR dysfunction.

Identification and characterisation of a glycine-rich RNA-binding protein as an endogenous suppressor of RNA silencing from Nicotiana glutinosa.

MAIN CONCLUSION: This study shows that NgRBP suppresses both local and systemic RNA silencing induced by sense- or double-stranded RNA, and the RNA binding activity is essential for its function. To counteract host defence, many plant viruses encode viral suppressors of RNA silencing targeting various stages of RNA silencing. There is increasing evidence that the plants also encode endogenous suppressors of RNA silencing (ESR) to regulate this pathway. In this study, using Agrobacterium infiltration assays, we characterized NgRBP, a glycine-rich RNA-binding protein from Nicotiana glutinosa, as an ESR. Our results indicated that NgRBP suppressed both local and systemic RNA silencing induced by sense- or double-stranded RNA. We also demonstrated that NgRBP could promote Potato Virus X (PVX) infection in N. benthamiana. NgRBP knockdown by virus-induced gene silencing enhanced PVX and Cucumber mosaic virus resistance in N. glutinosa. RNA immunoprecipitation and electrophoretic mobility shift assays showed that NgRBP bound to GFP mRNA, dsRNA rather than siRNA. These findings provide the evidence that NgRBP acts as an ESR and the RNA affinity of NgRBP plays the key role in its ESR activity. NgRBP responds to multiple signals such as ABA, MeJA, SA, and Tobacco mosaic virus infection. Therefore, it could participate in the regulation of gene expression under specific conditions.

Non-structural protein 1 from avian influenza virus H9N2 is an efficient RNA silencing suppressor with characteristics that differ from those of Tomato bushy stunt virus p19.

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional dimeric protein that contains a conserved N-terminal RNA binding domain. Studies have shown that NS1 suppresses RNA silencing and the NS1 proteins encoded by different influenza A virus strains exhibit differential RNA silencing suppression activities. In this study, we showed that the NS1 protein from avian influenza virus (AIV) H9N2 suppressed systemic RNA silencing induced by sense RNA or dsRNA. It resulted in more severe Potato virus X symptom, but could not reverse established systemic green fluorescent protein silencing in Nicotiana benthamiana. In addition, its systemic silencing suppression activity was much weaker than that of p19. The local silencing suppression activity of AIV H9N2 NS1 was most powerful at 7 dpi and was even stronger than that of p19. And the inhibition ability to RNA silencing of NS1 is stronger than that of p19 in human cells. Collectively, these results indicate that AIV H9N2 NS1 is an effective RNA silencing suppressor that likely targets downstream step(s) of dsRNA formation at an early stage in RNA silencing. Although NS1 and p19 both bind siRNA, their suppression mechanisms seem to differ because of differences in their suppression activities at various times post-infiltration and because p19 can reverse established systemic RNA silencing, but NS1 cannot.

Mutation analysis of the RNA silencing suppressor NS1 encoded by avian influenza virus H9N2.

Non-structural protein 1 (NS1) binds small interfering RNA and suppresses RNA silencing in plants, but the underlying mechanism of this suppression is not well understood. Therefore, here we characterized NS1 encoded by the avian influenza virus H9N2. The NS1 protein was able to suppress RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). Using deletion and point mutants, we discovered that the first 70 residues of NS1 could suppress RNA silencing triggered by sense transgene, but this sequence was not sufficient to block dsRNA-induced silencing. Any mutations of two arginine residues (35R and 46R) of NS1, which contribute to its homodimeric structure, caused the loss of its silencing suppression activity. These results indicate that the region after residue 70 of NS1 is essential for the repression activity on dsRNA-induced RNA silencing, and that the dimeric structure of NS1 plays a critical role in its RNA silencing suppression function.

Enhanced photosynthetic efficiency by nitrogen-doped carbon dots via plastoquinone-involved electron transfer in apple.

Artificially enhancing photosynthesis is critical for improving crop yields and fruit qualities. Nanomaterials have demonstrated great potential to enhance photosynthetic efficiency; however, the mechanisms underlying their effects are poorly understood. This study revealed that the electron transfer pathway participated in nitrogen-doped carbon dots (N-CDs)-induced photosynthetic efficiency enhancement (24.29%), resulting in the improvements of apple fruit qualities (soluble sugar content: 11.43%) in the orchard. We also found that N-CDs alleviated mterf5 mutant-modulated photosystem II (PSII) defects, but not psa3 mutant-modulated photosystem I (PSI) defects, suggesting that the N-CDs-targeting sites were located between PSII and PSI. Measurements of chlorophyll fluorescence parameters suggested that plastoquinone (PQ), the mobile electron carrier in the photosynthesis electron transfer chain (PETC), was the photosynthesis component that N-CDs targeted. In vitro experiments demonstrated that plastoquinone-9 (PQ-9) could accept electrons from light-excited N-CDs to produce the reduced plastoquinone 9 (PQH2-9). These findings suggested that N-CDs, as electron donors, offer a PQ-9-involved complement of PETC to improve photosynthesis and thereby fruit quality. Our study uncovered a mechanism by which nanomaterials enhanced plant photosynthesis and provided some insights that will be useful in the design of efficient nanomaterials for agricultural/horticultural applications.

Nitrogen-doped carbon dots enhanced seedling growth and salt tolerance with distinct requirements of excitation light.

Numerous nanomaterials with optical properties have demonstrated excellent capacities to enhance plant growth and stress tolerance. However, the corresponding mechanisms have only been partially characterized, especially the excitation-light dependencies of different actions. Here, nitrogen-doped carbon dots (N-CDs) were developed to explore the excitation-light dependence in N-CD-induced growth enhancement and salt tolerance. Compared to the control, N-CDs induced significant enhancements in Arabidopsis thaliana growth under excitation light, including fresh/dry weight of shoot (21.07% and 16.87%), chlorophyll content (9.17%), soluble sugar content (23.41%), leaf area (28.68%), total root length (34.07%) and root tip number (46.69%). In the absence of excitation light, N-CD-treated seedlings exhibited little differences in these parameters, except the enhancements in root length (24.51%) and root tip number (10.24%). On the other hand, N-CD-treatment could improve seedling salt tolerance with or without excitation light. Under salt stress (150 mM NaCl), in the presence of excitation light, the N-CDs treatment significantly increased shoot/root fresh weight and chlorophyll content by 43.29%, 50.66% and 22.59%, and reduced malondialdehyde (MDA) content and relative conductivity by 17.59% and 32.58% compared to the control group. In the absence of excitation light, significant enhancements in shoot/root fresh weight (34.22%, 32.60%) and chlorophyll content (10.45%), and obvious decreases in MDA content (28.84%) and relative conductivity (16.13%) were also found. These results indicated that N-CDs only induced growth enhancement under excitation light, but they improved salt tolerance with and without excitation light, suggesting that the two effects occurred via distinct signaling pathways. This study revealed the excitation-light dependencies of nanomaterial-involved agriculture applications, providing insight into designing more efficient nanomaterials in the future.

The NAC transcription factor MdNAC29 negatively regulates drought tolerance in apple.

Drought stress is an adverse stimulus that affects agricultural production worldwide. NAC transcription factors are involved in plant development and growth but also play different roles in the abiotic stress response. Here, we isolated the apple MdNAC29 gene and investigated its role in regulating drought tolerance. Subcellular localization experiments showed that MdNAC29 was localized to the nucleus and transcription was induced by the PEG treatment. Over-expression of MdNAC29 reduced drought tolerance in apple plants, calli, and tobacco, and exhibited higher relative conductivity, malondialdehyde (MDA) content, and lower chlorophyll content under drought stress. The transcriptomic analyses revealed that MdNAC29 reduced drought resistance by modulating the expression of photosynthesis and leaf senescence-related genes. The qRT-PCR results showed that overexpression of MdNAC29 repressed the expression of drought-resistance genes. Yeast one-hybrid and dual-luciferase assays demonstrated that MdNAC29 directly repressed MdDREB2A expression. Moreover, the yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that MdNAC29 interacted with the MdPP2-B10 (F-box protein), which responded to drought stress, and MdPP2-B10 enhanced the repressive effect of MdNAC29 on the transcriptional activity of the MdDREB2A. Taken together, our results indicate that MdNAC29 is a negative regulator of drought resistance, and provide a theoretical basis for further molecular mechanism research.

Oxidative post-translational modification of catalase confers salt stress acclimatization by regulating H2O2 homeostasis in Malus hupehensis.

Reactive oxygen species (ROS) play an essential role as both signaling molecule and damage agent during salt stress. As a signaling molecule, proper accumulation of H2O2 is crucial to trigger stress response and enhance stress tolerance. However, the dynamic regulation mechanism of H2O2 remains unclear. Here, we show that MhCAT2 (catalase 2 in Malus hupehensis) undergoes oxidative modification in an O2•--dependent manner and that oxidation at His225 residue reduces the MhCAT2 activity. Furthermore, the substitution of His225 with Tyr weakens the activity of MhCAT2. The oxidation modification provides a post-translational brake mechanism for the excessive scavenging of H2O2 caused by salt stress-induced catalase (CAT) over-expression. Overall, this finding provides mechanistic insights on stress tolerance augmentation by an O2•--mediated switch that regulates H2O2 homeostasis in Malus hupehensis.

A multifunctional protein encoded by turkey herpesvirus suppresses RNA silencing in Nicotiana benthamiana.

Many plant and animal viruses counteract RNA silencing-mediated defense by encoding diverse RNA silencing suppressors. We characterized HVT063, a multifunctional protein encoded by turkey herpesvirus (HVT), as a silencing suppressor in coinfiltration assays with green fluorescent protein transgenic Nicotiana benthamiana line 16c. Our results indicated that HVT063 could strongly suppress both local and systemic RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). HVT063 could reverse local silencing, but not systemic silencing, in newly emerging leaves. The local silencing suppression activity of HVT063 was also verified using the heterologous vector PVX. Further, single alanine substitution of arginine or lysine residues of the HVT063 protein showed that each selected single amino acid contributed to the suppression activity of HVT063 and region 1 (residues 138 to 141) was more important, because three of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed to induce a cell death phenotype in the infiltrated leaf region, and the HVT063 dilutions could decrease the silencing suppressor activity and alleviate the cell death phenotype. Collectively, these results suggest that HVT063 functions as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in the RNA silencing process. Positively charged amino acids in HVT063, such as arginine and lysine, might contribute to the suppressor activity by boosting the interaction between HVT063 and RNA, since HVT063 has been demonstrated to be an RNA binding protein.

New strategy of light quality regulation with leaf-spraying fluorescent coatings for enhancing photosynthesis efficiency.

Fluorescent coatings are a kind of emerging light quality regulation material that can improve plant light utilization efficiency through easy manipulation at a low price. Compared with the scheme of fluorescent nanomaterials alone or those physically dispersed in polymeric materials for photosynthesis enhancement, fluorescent polymeric coatings (FPCs) originating from the covalent copolymerization of nanomaterial monomers can function stably and continuously, circumventing the high-cost manipulation of continuous leaf-spraying or hydroponics of the previous scheme in practical applications. Herein, we developed a kind of FPCs consisting of UV-to-blue light-converting nitrogen-doped carbon dots (N-CDs) as the fluorescent monomer to induce the copolymerization of N-CDs and tannic acid (TA). In the FPCs, N-CDs and TA are covalently cross-linked together. The fluorescent ability of N-CDs and the strong adhesion of TA are integrated organically to the whole to endow FPCs with excellent properties of prolonged fluorescence capacity, rain-erosion resistance and stability. After spraying FPCs on tomato leaves grown under the full spectrum, both the chlorophyll content of the leaves and effective photochemical efficiency were increased significantly, and the growth rate was promoted with 38.3% and 43.2% enhancement in the dry and fresh weight. We also analyzed the human cytotoxicity of the coating and the toxicological experiments showed that the coating did not affect the proliferation of human cells.

Endodormancy Release Can Be Modulated by the GA4-GID1c-DELLA2 Module in Peach Leaf Buds.

Gibberellin (GA) plays a key role in the release of bud dormancy and the GA receptor GID1 (GIBBERELLIN INSENSITIVE DWARF1) and DELLA protein are the GA signaling parts, but the molecular mechanism of GA-GID1-DELLA module regulating leaf bud dormancy in peach (Prunus persica) is still not very clear. In this study, we isolated and characterized the GID1 gene PpGID1c from the peach cultivar "Zhong you No.4." Overexpressing PpGID1c in Arabidopsis promoted seed germination, which indicated that PpGID1c has an important function in dormancy. The expression level of PpGID1c in peach leaf buds during endodormancy release was higher than that during ecodormancy and was positively correlated with GA4 levels. Our study also found that GA4 had the most obvious effect on promoting the bud break, indicating that GA4 may be the key gibberellin to promoting peach leaf bud endodormancy release. Moreover, a quantitative real-time PCR (qRT-PCR) found that GA4 could increase the expression of the gibberellin signaling gene PpDELLA2. A yeast two-hybrid (Y2H) assay suggested that the PpGID1c interaction with the PpDELLA1 protein was not dependent on gibberellin, while the PpGID1c interaction with PpDELLA2 required GA4 or another gibberellin. These findings suggested that the GA4-GID1c-DELLA2 module regulates peach leaf bud endodormancy release, with this finding significantly enhancing our comprehensive understanding of bud endodormancy release and revealing a new mechanism for regulating leaf bud endodormancy release in peach.

Eosinophil recruitment is dynamically regulated by interplay among lung dendritic cell subsets after allergen challenge.

Eosinophil infiltration, a hallmark of allergic asthma, is essential for type 2 immune responses. How the initial eosinophil recruitment is regulated by lung dendritic cell (DC) subsets during the memory stage after allergen challenge is unclear. Here, we show that the initial eosinophil infiltration is dependent on lung cDC1s, which require nitric oxide (NO) produced by inducible NO synthase from lung CD24-CD11b+ DC2s for inducing CCL17 and CCL22 to attract eosinophils. During late phase responses after allergen challenge, lung CD24+ cDC2s inhibit eosinophil recruitment through secretion of TGF-ß1, which impairs the expression of CCL17 and CCL22. Our data suggest that different lung antigen-presenting cells modulate lung cDC1-mediated eosinophil recruitment dynamically, through secreting distinct soluble factors during the memory stage of chronic asthma after allergen challenge in the mouse.

Programmed Death Ligand-1 on Microglia Regulates Th1 Differentiation via Nitric Oxide in Experimental Autoimmune Encephalomyelitis.

Microglia are considered to be potential antigen-presenting cells and have the ability to present antigen under pathological conditions. Nevertheless, whether and how microglia are involved in immune regulation are largely unknown. Here, we investigated the suppressive activity of microglia during experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein, with the goal of understanding their role in regulating the T cell reaction. Using flow cytometric analysis, we found that microglia were characterized by increased cell number and up-regulated programmed death ligand-1 (PD-L1) at the peak phase of EAE. Meanwhile, both the CD4(+) T cells and microglia that infiltrated the central nervous system expressed higher levels of PD1, the receptor for PD-L1, accompanied by a decline of Th1 cells. In an ex vivo co-culture system, microglia from EAE mice inhibited the proliferation of antigen-specific CD4(+) T cells and the differentiation of Th1 cells, and this was significantly inhibited by PD-L1 blockade. Further, microglia suppressed Th1 cells via nitric oxide (NO), the production of which was dependent on PD-L1. Thus, these data suggest a scenario in which microglia are involved in the regulation of EAE by suppressing Th1-cell differentiation via the PD-L1-NO pathway.