Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture and causes huge economic loss annually. The roles of N6-methyladenosine (m6A) modification in silkworms following BmNPV infection are currently unclear. Here, methylated RNA immunoprecipitation with next-generation sequencing were applied to investigate the m6A profiles in silkworm midgut following BmNPV infection. A total of 9144 and 7384 m6A peaks were identified from the BmNPV-infected (TEST) and uninfected silkworm midguts (CON), respectively, which were distributed predominantly near stop codons. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common m6A peaks in nuclear genes revealed that these m6A-related transcripts were associated with crucial signaling pathways. Comparative transcriptome analysis showed that 1221 differential expressed m6A peaks were identified between TEST and CON, indicating that m6A modification is regulated following BmNPV infection. GO and KEGG pathway analysis of the differentially expressed m6A peaks showed their association with signal transduction, translation, and degradation. To understand further the effect of the m6A machinery on virus infection, expression levels of m6A-related genes were altered in silencing and overexpression experiments. Expression of viral structural protein VP39 was increased in BmN cells by siRNA-mediated depletion of methyltransferase-like (METTL) enzyme genes (BmMETTL3, BmMETTL14) and cytoplasmic YTH-domain family 3 (BmYTHDF3), while the reverse results were found after overexpression of the m6A-related enzymes in BmN cells. Overall, m6A modification might be a novel epigenetic mechanism that regulation BmNPV infection and interference with this mechanism may provide a novel antiviral strategy for preventing BmNPV disease.

To date, some DNA viruses and single-stranded RNA viruses have been found to generate circRNAs. However, the reports on circRNAs produced by double-stranded RNA viruses are very limited. In this study, Bombyx mori cypovirus (BmCPV), a typical double-stranded RNA virus belonging to the Reoviridae, was demonstrated to generate viral circRNAs (vcircRNAs) and a vcircRNA_000048 whose sequence corresponds with the region 164-1245 nt on the BmCPV genomic dsRNA S5 segment (GQ294468.1) was validated by PCR, Sanger sequencing, reverse transcription-rolling circle amplification, and Northern blotting. Furthermore, we verified that vcircRNA_000048 translates a micropeptide vsp21 with 21 amino acid residues in an IRES-dependent manner, and vsp21 attenuates the viral replication. These findings provided a novel clue to understanding the regulation of viral multiplication and interaction of reovirus with the host.

The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei.

Calcitonin gene-related peptide (CGRP) is a bioactive peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP is largely distributed in the cardiovascular and nervous systems, where it acts as a regulatory factor. CGRP is also expressed in organs and tissues involved in metabolic regulation, including white adipose tissue (WAT), where its function is largely unknown. In this study, we examined the effects of endogenous CGRP on metabolic function. When we administered a high-fat diet to CGRP-specific knockout (CGRP-/-) and wild-type (WT) mice for 10 weeks, we observed that food intake did not differ between the two groups, but body weight and visceral fat weight were significantly lower in CGRP-/- mice. Fatty liver changes were less severe in CGRP-/- mice, which also showed lower serum insulin and leptin levels. Glucose tolerance and insulin sensitivity were better in CGRP-/- than WT mice, and expired gas analysis revealed greater oxygen consumption by CGRP-/- mice. Adipocyte hypertrophy was suppressed in CGRP-/- mice, while expression of β-3-adrenergic receptor, hormone-sensitive lipase and adiponectin was enhanced. Isoproterenol-induced glycerol release from WAT was higher in CGRP-/- than WT mice, and CGRP-/- mice showed elevated sympathetic nervous activity. β-receptor-blockade canceled the beneficial effects of CGRP deletion on obesity. These results suggest that, in addition to its actions in the cardiovascular system, endogenous CGRP is a key regulator of metabolism and energy homeostasis in vivo.

Neointimal hyperplasia is the primary lesion underlying atherosclerosis and restenosis after coronary intervention. We previously described the essential angiogenic function of the adrenomedullin (AM)-receptor activity-modifying protein (RAMP) 2 system. In the present study, we assessed the vasoprotective actions of the endogenous AM-RAMP2 system using a wire-induced vascular injury model. We found that neointima formation and vascular smooth muscle cell proliferation were enhanced in RAMP2+/- male mice. The injured vessels from RAMP2+/- mice showed greater macrophage infiltration, inflammatory cytokine expression, and oxidative stress than vessels from wild-type mice and less re-endothelialization. After endothelial cell-specific RAMP2 deletion in drug-inducible endothelial cell-specific RAMP2-/- (DI-E-RAMP2-/-) male mice, we observed markedly greater neointima formation than in control mice. In addition, neointima formation after vessel injury was enhanced in mice receiving bone marrow transplants from RAMP2+/- or DI-E-RAMP2-/- mice, indicating that bone marrow-derived cells contributed to the enhanced neointima formation. Finally, we found that the AM-RAMP2 system augmented proliferation and migration of endothelial progenitor cells. These results demonstrate that the AM-RAMP2 system exerts crucial vasoprotective effects after vascular injury and could be a therapeutic target for the treatment of vascular diseases.

Thinned peach polyphenols (TPPs) were extracted by ultrasonic disruption and purified using macroporous resin. Optimized extraction conditions resulted in a TPPs yield of 1.59 ± 0.02 mg GAE/g FW, and optimized purification conditions resulted in a purity of 43.86% with NKA-9 resin. TPPs composition was analyzed by UPLC-ESI-QTOF-MS/MS; chlorogenic acid, catechin, and neochlorogenic acid were the most abundant compounds in thinned peaches. Purified TPPs exhibited scavenging activity on DPPH, ABTS, hydroxyl radical, and FRAP. TPPs inhibited α-amylase and α-glucosidase by competitive and noncompetitive reversible inhibition, respectively. TPPs also exhibited a higher binding capacity for bile acids than cholestyramine. In summary, TPPs from thinned peaches are potentially valuable because of their high antioxidant, hypoglycemic, and hypolipidemic capacities, and present a new incentive for the comprehensive utilization of thinned peach fruit.

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 ± 0.06, 1.0 ± 0.13 and 0.4 ± 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation.

The phylogenetic relationships of species in the Phasianidae, Order Galliformes, are the object of intensive study. However, convergent morphological evolution and rapid species radiation result in much ambiguity in the group. Further, matrilineal (mtDNA) genealogies conflict with trees based on nuclear DNA retrotransposable elements. Herein, we analyze 39 nearly complete mitochondrial genomes (three new) and up to seven nuclear DNA segments. We combine these multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids. The nuclear DNA tree is largely congruent with the tree derived from mt genomes. However, branching orders of mt/nuclear trees largely conflict with those based on retrotransposons. For example, Gallus/Bambusicola/Francolinus forms the sister-group of Coturnix/Alectoris in the nuclear/mtDNA trees, yet the tree based on retrotransposable elements roots the former at the base of the tree and not with the latter. Further, while peafowls cluster with Gallus/Coturnix in the mt tree, they root at the base of the phasianids following Gallus in the tree based on retrotransposable elements. The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in our study reveal the complex topology of the Phasianidae.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)that belongs to the genus Cypovirus in the family of Reoviridae is one of the problematic pathogens in sericulture. In our previous study, we have found that lipid-related constituents in the host cellular membrane are associated with the BmCPV life cycle. It is important to note that the lipids not only affect the cellular biological processes, they also impact the virus life cycle. However, the intracellular lipid homeostasis in BmN cells after BmCPV infection remains unclear. Here, the lipid metabolism in BmCPV-infected BmN cells was studied by lipidomics analysis. Our results revealed that the intracellular lipid homeostasis was disturbed in BmN cells upon BmCPV infection. Major lipids constituents in cellular membrane were found to be significantly induced upon BmCPV infection, which included triglycerides, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospholipids, glucoside ceramide, monoetherphosphatidylcholin, ceramide, ceramide phosphoethanolamine and cardiolipin. Further analysis of the pathways related to these altered lipids (such as PE and PC) showed that glycerophospholipid metabolism was one of the most enriched pathways. These results suggested that BmCPV may manipulate the lipid metabolism of cells for their own interest. The findings may facilitate a better understanding of the roles of lipid metabolic changes during virus infection in future studies.

Acetylcholinesterase (AChE) hydrolyses acetylcholine to choline and acetate, playing an important role in terminating the neurotransmission in brain and muscle. Recently, the non-neuronal functions of AChE have been proposed in different tissues, in which there are various factors to regulate the expression of AChE. In mammalian skin, AChE was identified in melanocytes and keratinocytes. Our previous study has indicated that AChE in keratinocyte affects the process of solar light-induced skin pigmentation; however, the expression of AChE in keratinocytes in responding to sunlight remains unknown. Here, we provided several lines of evidence to support a notion that AChE could be upregulated at transcriptional and translational levels in keratinocytes when exposed to solar light. The light-mediated AChE expression was triggered by Ca2+, supported by an induction of Ca2+ ionophore A23187 and a blockage by Ca2+ chelator BAPTA-AM. In addition, this increase on AChE transcriptional expression was eliminated by mutagenesis on the activating protein 1 (AP1) site in ACHE gene. Hence, the solar light-induced AChE expression is mediated by Ca2+ signalling through AP1 site. This finding supports the role of solar light in affecting the cholinergic system in skin cells, and which may further influence the dermatological function.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

Brain metastasis is a significant unmet clinical problem in breast cancer treatment. It is always associated with poor prognosis and high morbidity. Recently, Slit2/Robo1 pathway has been demonstrated to be involved in the progression of breast carcinoma. However, until present, there are no convincing reports that suggest whether the Slit2/Robo1 axis has any role in brain metastasis of breast cancer. In this study, we investigated the correlation between Slit2/Robo1 signaling and breast cancer brain metastasis for the first time. Our results demonstrated that (1) Invasive ductal carcinoma patients with low expression of Slit2 or Robo1 exhibited worse prognosis and brain-specific metastasis, but not liver, bone or lung. (2) Lower expression of Slit2 and Robo1 were observed in patients with brain metastasis, especially in their brain metastasis tumors, compared with patients without brain metastasis. (3) The interval from diagnosis of breast cancer to brain metastasis and brain metastasis to death were both much shorter in patients with low expression of Slit2 or Robo1 compared with the high expression group. Overall, our findings indicated that Slit2/Robo1 axis possibly be regarded as a significant clinical parameter for predicting brain metastasis in breast cancer patients.

Placenta-specific protein 1 (Plac1) has critical functions in multiple human malignancies, but its role in nasopharyngeal carcinoma (NPC) was unclear. Clinical samples of NPC and adjacent normal tissue were collected. Plac1 expressions in both tissues and cells were measured. After cell transfection, NPC cell viability, proliferation, migration and invasion were detected using cell counting kit-8 (CCK-8) assay, colony formation assay, scratch assay and Transwell assay. Relative expressions of Plac1 and proteins related to migration and invasion (E-Cadherin, N-cadherin, Matrix metalloproteinase2 (MMP2), and MMP9), Furin/Notch1 intracellular domain (NICD)/phosphate and tension homology (PTEN) pathway (NICD, PTEN, phosphorylated-Akt (p-Akt), Akt) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. The interaction between Plac1 and Furin, a member of Furin/NICD/PTEN Pathway, was analyzed using co-Immunoprecipitation (co-IP) assay. Plac1 expression was upregulated in both NPC tissue and cells. Overexpressed Plac1 promoted Plac1 and Furin expressions and increased cell viability, proliferation, migration and invasion of NPC cells, while silencing Plac1 showed the opposite effects. Plac1 interacted with Furin, overexpression of Furin reversed the inhibitory effects of silencing Plac1 on NPC cell proliferation, migration, and invasion, and also reversed the effects of silencing Plac1 on Furin/NICD/PTEN pathway-, cell migration-, and invasion-related protein expressions. Plac1 promoted NPC cell proliferation, migration and invasion via Furin/NICD/PTEN Pathway. The findings of this study provide a possible therapeutic method for NPC treatment.

Long non-coding RNAs (lncRNAs) play crucial roles in the progression and pathogenesis of cancer. Right now, less is known about the association between the expression of lncRNAs and the pathogenesis of pulmonary tuberculosis (PTB).

Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.

The grass carp accounts for a large proportion of aquacultural production in China, but the hemorrhagic disease caused by grass carp reovirus (GCRV) infection often causes huge economic losses to the industry. Interleukin 17 (IL-17) is an important cytokine that plays a critical role in the inflammatory and immune responses. Although IL-17 family members have been extensively studied in mammals, our knowledge of the activity of IL-17 proteins in teleosts in response to viral infection is still limited. In this study, the role of IL-17 in GCRV infection and its mechanism were investigated. The expression levels of IL-17AF1, IL-17AF2, and IL-17AF3 in Ctenopharyngodon idella kidney (CIK) cells gradually increased from 6 h after infection with GCRV. The nuclear translocation of p65, which acts in the NF-κB signaling pathway, was also increased by GCRV infection. The overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 also promoted the nuclear translocation of p65 and the levels of phospho-IκBα in CIK cells, and reduced the expression of the viral structural protein VP7. An NF-κB signal inhibitor abolished the inhibition of GCRV infection by IL-17 proteins. These results suggested that the NF-κB signaling pathway was activated by the overexpression of IL-17 proteins, resulting in the inhibition of viral infection. In conclusion, in this study, we demonstrated that IL-17AF1, IL-17AF2, and IL-17AF3 acted as immune cytokines, exerting an antiviral effect by activating the NF-κB signaling pathway.

Chimeric antigen receptor (CAR)-expressing macrophages (CAR-M) have a great potential to improve cancer therapy, as shown from several recent preclinical studies. However, unlike CAR-T cell therapy, which has been widely studied, the efficacy and limitations of CAR-M cells remain to be established. To address this issue, in the present study, we compared three intracellular signaling domains (derived from common γ subunit of Fc receptors (FcRγ), multiple EGF-like-domains protein 10 (Megf10), and the CD19 cytoplasmic domain that recruits the p85 subunit of phosphoinositide-3 kinase (PI3K), respectively) for their ability to promote primary CAR-M functions, and investigated the potential synergistic effect between CAR-M and CAR-T cells in their ability to kill tumor cells. We found that CAR-MFcRγ exerted more potent phagocytic and tumor-killing capacity than CAR-MMegf10 and CAR-MPI3K. CAR-M and CAR-T demonstrated synergistic cytotoxicity against tumor cells in vitro. Mechanistically, the inflammatory factors secreted by CAR-T increased the expression of costimulatory ligands (CD86 and CD80) on CAR-M and augmented the cytotoxicity of CAR-M by inducing macrophage M1 polarization. The upregulated costimulatory ligands may promote the fitness and activation of CAR-T cells in turn, achieving significantly enhanced cytotoxicity. Taken together, our study demonstrated for the first time that CAR-M could synergize with CAR-T cells to kill tumor cells, which provides proof-of-concept for a novel combinational immunotherapy.

β-Arrestin 2 is known to be a widely distributed adaptor protein in mammals but its function has never been reported in Lepidoptera insects. Herein, the β-Arrestin 2 (BmArrestin 2) gene from silkworm was cloned and characterized. The spatiotemporal expression level of BmArrestin 2 was highest in the gonads at the 3rd day of 5th instar, whereas the highest and lowest abundance of BmArrestin 2 were identified in the tracheal and testis, respectively. BmArrestin 2 is mainly distributed in the cytoplasm. Furthermore, in BmN cells，overexpression of BmArrestin 2 promoted Bombyx mori nucleopolyhedrovirus (BmNPV) and B. mori cytoplasmic polyhedrosis virus (BmCPV) replication as the increment of the concentration of plasmid transfection, whereas silencing the gene with specific siRNA inhibited viral replication. Replication of BmNPV and BmCPV also was weakened using BmArrestin 2 antiserum as the increment of the concentration. Immunofluorescent staining revealed the invasion of recombinant BmNPV or BmCPV was decreased after blocking endogenous BmArrestin 2. On the other hand, BmArrestin 2 co-localizes with recombinant BmNPV and BmCPV virions in BmN cells. These results suggest that BmArrestin 2 may represent a novel target for antiviral strategies, as it is an adaptor protein that plays a key role in virus replication.