Novel lncRNA 803 related to Marek’s disease inhibits apoptosis of DF-1 cells

Marek’s disease (MD) is a neoplastic disease that significantly affects the poultry industry. Long non-coding RNAs (lncRNAs) are crucial regulatory factors in various biological processes, including tumourigenesis. However, the involvement of novel lncRNAs in the course of MD virus (MDV) infection is still underexplored. Here, we present the first comprehensive characterization of differentially expressed lncRNAs in chicken spleen at different stages of MDV infection. A series of differentially expressed lncRNAs was identified at each stage of MDV infection through screening. Notably, our investigation revealed a novel lncRNA, lncRNA 803, which exhibited significant differential expression at different stages of MDV infection and was likely to be associated with the p53 pathway. Further analyses demonstrated that the overexpression of lncRNA 803 positively regulated the expression of p53 and TP53BP1 in DF-1 cells, leading to the inhibition of apoptosis. This is the first study to focus on the lncRNA expression profiles in chicken spleens during MDV pathogenesis. Our findings highlight the potential role of the p53-related novel lncRNA 803 in MD pathogenesis and provide valuable insights for decoding the molecular mechanism of MD pathogenesis involving non-coding RNA. RESEARCH HIGHLIGHTSDifferentially expressed lncRNAs in spleens of chickens infected with Marek’s disease virus at different stages were identified for the first time.The effects of novel lncRNA 803 on p53 pathway and apoptosis of DF-1 cells were reported for the first time. Differentially expressed lncRNAs in spleens of chickens infected with Marek’s disease virus at different stages were identified for the first time. The effects of novel lncRNA 803 on p53 pathway and apoptosis of DF-1 cells were reported for the first time.</p

PEGylated Self-Assembled Nano-Bacitracin A: Probing the Antibacterial Mechanism and Real-Time Tracing of Target Delivery in Vivo

Although nano-self-assemblies of hydrophobic-modified bacitracin A with poly­(d,l-lactic-<i>co</i>-glycolic acid) (PLGA) (nano-BA_PLGA) have demonstrated promising antibacterial activities, the application of nano-BA_PLGA was severely compromised by low water solubility. In this study, a series of PEGylated PLGA copolymers were selected to conjugate with the N-terminus of bacitracin A to construct PEGylated self-assembled nano-BAs and to further develop nano-self-assemblies of bacitracin A with strong antibacterial potency and high solubility. Compared with nano-BA_PLGA, all PEGylated nano-BAs, except nano-BA_5k, exhibited strong antibacterial efficiency against both Gram-positive and Gram-negative bacteria by inducing loss of cytoplasmic membrane potential, membrane permeabilization, and leakage of calcein from artificial cell membranes. Studies elucidating the underlying mechanism of PEGylated nano-BAs against Gram-negative bacteria indicated that the strong hydrophobic and van der Waals interactions between PLGA and lipopolysaccharide (LPS) could bind, neutralize, and disassociate LPS, facilitating cellular uptake of the nanoparticles, which could destabilize the membrane, resulting in cell death. Moreover, PEGylated nano-BAs (nano-BA_12k) with a longer PLGA block were expected to occupy a higher local density of BA mass on the surface and result in stronger hydrophobic and van der Waals interactions with LPS, which were responsible for the enhanced antibacterial activity against Gram-positive and emerging antibacterial activity against Gram-negative bacteria, respectively. In vivo imaging verified that PEGylated nano-BAs exhibited higher inflammatory tissue distribution and longer circulation time than nano-BA_PLGA. Therefore, although PEGylation did not affect antibacterial activity, it is necessary for target delivery and resistance to clearance of the observed PEGylated nano-BAs. In vivo, nano-BA_12k also showed the highest therapeutic index against infection burden in a mouse thigh infection model among the tested formulations, which showed good correlation with the in vitro results. In conclusion, nano-BA_12k showed high efficacy in the treatment of invasive infections. This new approach of constructing nanoantibiotics by modification of commercially available antibiotics with PEGylated copolymers is safe, cost-effective, and environmentally friendly

Auxin and ethylene responsiveness of <i>SlARF2A</i> and <i>SlARF2B</i> genes.

<p>(A) qPCR analysis of <i>SlARF2A</i> and <i>SlARF2B</i> transcripts in total RNA samples extracted from wild-type mature green fruits treated with 50 ml.L^-1 ethylene for 5 hours. (B) qPCR analysis of <i>SlARF2A</i> and <i>SlARF2B</i> transcripts in total RNA samples extracted from wild-type breaker fruits treated with 1-MCP (1.0 mg.L^-1) for 16 hours. (C) qPCR analysis of <i>SlARF2A</i> and <i>SlARF2B</i> transcripts in total RNA samples extracted from wild-type mature green fruits treated with 20 μM IAA or buffer (control) for 6 hours. The relative mRNA levels of <i>SlARF2A</i>/<i>SlARF2B</i> genes were normalized against actin. The results were expressed using control untreated fruit as reference with relative mRNA level set to 1. Error bars mean ±SD of three biological replicates. Stars indicate the statistical significance using Student’s t-test: *0.01 < p-value < 0.05, ** 0.001< p-value <0.01, *** p-value < 0.001. <i>E4</i>, <i>E8</i>: ethylene response genes; <i>GH3</i>, <i>SAUR</i>: auxin response genes.</p

Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.

<p>(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 <i>in vivo</i>. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called <i>DR5</i> fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.</p

Structural features and expression patterns of tomato <i>SlARF2A</i> and <i>SlARF2B</i> genes.

<p>(A) Genomic structure analysis of <i>SlARF2A</i> and <i>SlARF2B</i> genes were drawn using Fancy gene V1.4 software (<a href="http://bio.ieo.eu/fancygene/" target="_blank">http://bio.ieo.eu/fancygene/</a>) and <i>SlARF2A SlARF2B</i> iTAG2.40 gene model data. The pink portion represents the promoter region; the strandlines represent intron parts; the gray boxes indicate exon parts; the yellow boxes region responsible for dimerization with Aux/IAA proteins (domain III and IV); the red boxes correspond to the DNA binding domain (DBD); ERE and AuxRE correspond to the ethylene and auxin responsive <i>cis</i>-elements. (B) Expression pattern of <i>SlARF2A/2B</i> monitored by quantitative real-time RT-PCR (qPCR) in total RNA samples extracted from root (Rt), stem (St), leaf (Le), flower (Fl), fruit (Fr), mature green fruit (MG), breaker fruit (Br) and red fruit (Re). Relative mRNA levels corresponding to <i>SlARF2A</i>/<i>SlARF2B</i> genes were normalized against actin in each RNA sample. The relative mRNA levels of <i>SlARF2B</i> in root and at mature green (MG) stage were used as reference (relative mRNA level 1). Error bars mean ±SD of three biological replicates.</p

Ethylene production of <i>SlARF2AB-RNAi</i> fruits.

<p>(A) Ethylene production of wild-type and <i>SlARF2AB-RNAi</i> fruits picked at different ripening stages and assessed for ethylene production. MG = mature green stage; Br = breaker stage; Br+1 = 1 day post breaker stage; Br+2 = 2 days post breaker stage; Br+3, 3 days post breaker stage. (B) Ethylene production of wild-type and <i>SlARF2AB-RNAi</i> fruits picked at MG stage and left on the bench. Ethylene was measured at different days post mature green stage. Values represent means of at least 10 individual fruits. Vertical bars represent SD. AB1 = <i>SlARF2AB-RNAi</i> line 311; AB2 = <i>SlARF2AB-RNAi</i> line 223.</p

The expression of a number of ripening-related genes is altered in <i>SlARF2AB-RNAi</i> plants.

<p>Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and <i>SlARF2AB-RNAi</i> lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the <i>SlActin</i> gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.</p

DataSheet1_Two novel bombesin-like neuropeptides from the skin secretion of Pelophylax kl. esculentus: Ex vivo pharmacological characterization on rat smooth muscle types.docx

Mammalian bombesin-like neuropeptides (BLPs) play an important role in regulation of physiological and pathophysiological processes. Frog skin-derived BLPs, of smaller size and diverse lengths and sequences at their N-terminus, have attracted the attention of many researchers. However, these N-terminal variants and the receptors modulating their pharmacological actions are poorly studied and less understood. In this study, two BLPs, namely, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin with primary structures NLGKQWATGHFM and NLGKQWAVGHFM were isolated from the skin secretion of hybrid Pelophylax kl. esculentus. Both BLPs share a similar primary structure with only a single amino acid substitution at the eighth position (threonine to valine), while they have quite different myotropic potencies with EC50 values in the range of 22.64 ± 9.7 nM (N = 8) to 83.93 ± 46.9 nM (N = 8). The potency of [Asn3, Lys6, Thr10, Phe13]3–14-bombesin was approximately 3-fold higher than that of [Asn3, Lys6, Phe13]3–14-bombesin. Through the investigation of receptor selectivity using a canonical bombesin receptor antagonist, it was found that [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin had an affinity to both BB1 and BB2 receptors. Their contractile functions are mainly modulated by both BB1 and BB2 receptors on rat urinary bladder and BB2 alone on rat uterus smooth muscle preparations. These data may provide new insights into the design of potent and selective ligands for bombesin receptors. Moreover, [Asn3, Lys6, Thr10, Phe13]3–14-bombesin and [Asn3, Lys6, Phe13]3–14-bombesin did not induce significant hemolysis and toxicity in normal human cells, suggesting that these two natural novel BLPs have great potential for development into new drug candidates.</p

Altered pigment accumulation in <i>SlARF2AB-RNAi</i> fruits.

<p>(A) Changes in hue angle in WT and two independent <i>SlARF2AB-RNAi</i> lines during different ripening stages (breaker, Br; Br+1 to 41 days post-breaker;). AB1 = <i>SlARF2AB-RNAi</i> line 311; AB2 = <i>SlARF2AB-RNAi</i> line 223. (B) Quantitative RT-PCR relative expression of carotenoid biosynthesis genes in wild-type (WT) and <i>SlARF2AB-RNAi</i> tomato lines. Total RNA was extracted from different developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at breaker (Br) stage were standardized to 1.0, referring to the <i>SlActin</i> gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate a statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. ABL1 is <i>SlARF2AB-RNAi</i> line 311. PSY1 phytoene synthase; PDS phytoene desaturase; ZDS, carotenoid desaturase; ß-LCY1, ß-LCY2, CYC-ß lycopene b-cyclases.</p

Altered ripening phenotypes of <i>SlARF2</i> down-regulated lines.

<p>(A) Ripening phenotypes of <i>SlARF2A-RNAi; SlARF2B-RNAi</i> and <i>SlARF2AB-RNAi</i> fruits at mature green (upper panel) and ripe (lower panel) stages. The <i>SlARF2A/SlARF2B-RNAi</i> fruits show spiky phenotype at mature green stage and ripe stage fruits, <i>SlARF2AB-RNAi</i> mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent <i>SlARF2AB-RNAi</i> lines. (C) Ripening phenotypes of wild-type (WT) and <i>SlARF2AB-RNAi</i> fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and <i>SlARF2AB-RNAi</i> fruit. Mature green fruits from WT and <i>SlARF2AB-RNAi</i> lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while <i>SlARF2AB-RNAi</i> fruits treated or untreated displayed orange sectors on the fruit surface and never get red.</p