BRD4 inhibition exerts anti-viral activity through DNA damage-dependent innate immune responses.

Chromatin dynamics regulated by epigenetic modification is crucial in genome stability and gene expression. Various epigenetic mechanisms have been identified in the pathogenesis of human diseases. Here, we examined the effects of ten epigenetic agents on pseudorabies virus (PRV) infection by using GFP-reporter assays. Inhibitors of bromodomain protein 4 (BRD4), which receives much more attention in cancer than viral infection, was found to exhibit substantial anti-viral activity against PRV as well as a range of DNA and RNA viruses. We further demonstrated that BRD4 inhibition boosted a robust innate immune response. BRD4 inhibition also de-compacted chromatin structure and induced the DNA damage response, thereby triggering the activation of cGAS-mediated innate immunity and increasing host resistance to viral infection both in vitro and in vivo. Mechanistically, the inhibitory effect of BRD4 inhibition on viral infection was mainly attributed to the attenuation of viral attachment. Our findings reveal a unique mechanism through which BRD4 inhibition restrains viral infection and points to its potent therapeutic value for viral infectious diseases.

A novel ratiometric fluorescence probe for highly sensitive and specific detection of chlorotetracycline among tetracycline antibiotics.

It is of great importance to detect chlorotetracycline (CTC) in a highly sensitive and specific way because of its wide distribution in aquaculture and animal husbandry. Herein, we propose a novel ratiometric fluorescence strategy to assay CTC by using bovine serum albumin stabilized gold nanoclusters (BSA-AuNCs). The BSA-AuNCs consisting of 25 gold atoms (Au25NCs) display a red emission at 640 nm (λex = 370 nm). In the presence of CTC, a new blue emission at 425 nm is emerged and its intensity dramatically increases with the addition of more the analyte; meanwhile the red emission at 640 nm shows a linear decrease reversely. However, at identical conditions neither the analogues of CTC as tetracycline (TC), oxytetracycline (OTC) or doxycycline (DC) induces similar response of BSA-AuNCs. Such interesting phenomenon is proven related to the conversion from large Au25NCs to smaller nanoclusters composing 8 gold atoms (Au8NCs), which intrinsically originate from the interaction between CTC and the ligand BSA. Therefore, a ratiometric probe is established to sensitively detect CTC in the wide range (0.2-10 µM) with a low limit of detection (LOD) at 65 nM. In addition, this strategy can also be applied to assay CTC in human serum, showing great promise for practical applications in future.

Porcine Reproductive and Respiratory Syndrome Virus Activates Lipophagy To Facilitate Viral Replication through Downregulation of NDRG1 Expression.

Autophagy maintains cellular homeostasis by degrading organelles, proteins, and lipids in lysosomes. Autophagy is involved in the innate and adaptive immune responses to a variety of pathogens. Some viruses can hijack host autophagy to enhance their replication. However, the role of autophagy in porcine reproductive and respiratory syndrome virus (PRRSV) infection is unclear. Here, we show that N-Myc downstream-regulated gene 1 (NDRG1) deficiency induced autophagy, which facilitated PRRSV replication by regulating lipid metabolism. NDRG1 mRNA is expressed ubiquitously in most porcine tissues and most strongly in white adipose tissue. PRRSV infection downregulated the expression of NDRG1 mRNA and protein, while NDRG1 deficiency contributed to PRRSV RNA replication and progeny virus assembly. NDRG1 deficiency reduced the number of intracellular lipid droplets (LDs), but the expression levels of key genes in lipogenesis and lipolysis were not altered. Our results also show that NDRG1 deficiency promoted autophagy and increased the subsequent yields of hydrolyzed free fatty acids (FFAs). The reduced LD numbers, increased FFA levels, and enhanced PRRSV replication were abrogated in the presence of an autophagy inhibitor. Overall, our findings suggest that NDRG1 plays a negative role in PRRSV replication by suppressing autophagy and LD degradation.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-positive-stranded RNA virus, causes acute respiratory distress in piglets and reproductive failure in sows. It has led to tremendous economic losses in the swine industry worldwide since it was first documented in the late 1980s. Vaccination is currently the major strategy used to control the disease. However, conventional vaccines and other strategies do not provide satisfactory or sustainable prevention. Therefore, safe and effective strategies to control PRRSV are urgently required. The significance of our research is that we demonstrate a previously unreported relationship between PRRSV, NDRG1, and lipophagy in the context of viral infection. Furthermore, our data point to a new role for NDRG1 in autophagy and lipid metabolism. Thus, NDRG1 and lipophagy will have significant implications for understanding PRRSV pathogenesis for developing new therapeutics.

Nitrogen-terminated silicon nanoparticles obtained via chemical etching and passivation are specific fluorescent probes for creatinine.

A method is described here to prepare water-dispersible nitrogen-functionalized silicon nanoparticles (N-SiNPs). It consists of two steps, viz. etching of the oxidized shell of SiNPs and nitrogen-passivation of the exposed silicon. The resulting N-SiNPs have an average diameter of 2.6±0.7 nm and show blue fluorescence (with excitation/emission peaks at 340/420 nm). The fluorescence quantum yield is 23% and the decay time is in the nanosecond regime. Compared to etching methods using a plasma or hydrofluoric acid, the process described here (etching and passivation) is mild, continuous, fast, and air-compatible. The N-SiNPs modified with chlorotetracycline are shown to be a viable fluorescent probe for creatinine. Fluorescence drops in the 0 to 20 µM creatinine concentration range, and the limit of detection is 0.14 µM.

De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing.

Common carp (Cyprinus carpio) is one of the most important aquacultured species of the family Cyprinidae, and breeding this species for disease resistance is becoming more and more important. However, at the genome or transcriptome levels, study of the immunogenetics of disease resistance in the common carp is lacking. In this study, 60,316,906 and 75,200,328 paired-end clean reads were obtained from two cDNA libraries of the common carp spleen by Illumina paired-end sequencing technology. Totally, 130,293 unique transcript fragments (unigenes) were assembled, with an average length of 1400.57 bp. Approximately 105,612 (81.06%) unigenes could be annotated according to their homology with matches in the Nr, Nt, Swiss-Prot, COG, GO, or KEGG databases, and they were found to represent 46,747 non-redundant genes. Comparative analysis showed that 59.82% of the unigenes have significant similarity to zebrafish Refseq proteins. Gene expression comparison revealed that 10,432 and 6889 annotated unigenes were, respectively, up- and down-regulated with at least twofold changes between two developmental stages of the common carp spleen. Gene ontology and KEGG analysis were performed to classify all unigenes into functional categories for understanding gene functions and regulation pathways. In addition, 46,847 simple sequence repeats (SSRs) were detected from 35,618 unigenes, and a large number of single nucleotide polymorphism (SNP) and insertion/deletion (INDEL) sites were identified in the spleen transcriptome of common carp. This study has characterized the spleen transcriptome of the common carp for the first time, providing a valuable resource for a better understanding of the common carp immune system and defense mechanisms. This knowledge will also facilitate future functional studies on common carp immunogenetics that may eventually be applied in breeding programs.

Identification and characterization of microRNAs in the spleen of common carp immune organ.

MicroRNAs (miRNAs) play an important role in the regulation of many fundamental biological processes in eukaryotes; however, miRNAs associated with immune functions in the common carp have not been reported. In this study, a small-RNA cDNA library was constructed from the spleen of the common carp. A total of 10,603,456 high-quality clean reads, representing 293,603 unique sequences, were obtained from the small-cDNA library using the Solexa sequencing. By the bioinformatic analysis, 194 conserved miRNAs and 12 novel miRNAs were identified in the carp spleen. The abundant miRNAs principally belong to 30 miRNA gene families such as let-7, mir-10, mir-15, mir-30, and so on. The conservation analysis showed that 23 families were present both in protostomes and deuterostomes, 46 families were conserved only in vertebrates, and 5 families (mir-430, mir-722, mir-724, mir-734, and mir-738) were identified only in fish species. Furthermore, GO enrichment analysis and KEGG pathway analysis suggested that miRNAs expressed in the spleen of common carp are involved in immune system development, lymphoid organ development, lymphocyte activation, immune response, B cell receptor signaling pathway, T cell receptor signaling pathway, Fc gamma R-mediated phagocytosis, Toll-like receptor signaling pathway, and so on. This study described the miRNA transcriptome in spleen tissue for the first time in the common carp. The results expand the number of known common carp miRNAs and provides a meaningful framework to understand the common carp immune system and defense mechanisms.

Allicin induces apoptosis in EL-4 cells in vitro by activation of expression of caspase-3 and -12 and up-regulation of the ratio of Bax/Bcl-2.

Garlic (Allium sativum L.; Liliaceae) has been widely demonstrated in the role of cancer prevention, but the specific compound in garlic corresponding to this effect and its mechanisms are not clearly known. Allicin is one of the organic sulphur compounds derived from garlic. In the present study we investigated the anti-proliferative and pro-apoptotic activities of allicin in murine T-lymphocytes (EL-4) and the mechanism of inducing apoptosis in vitro. The results showed that allicin was effective in inhibiting the proliferation of EL-4 cells in vitro in a concentration-dependent manner. Further, allicin could induce the formation of apoptotic bodies, nuclear condensation, DNA spallation, and even activated the expression of caspase-3, -12 and cytochrome C (cyt C). Finally, allicin up-regulated the ratio of Bax/Bcl-2 and induced a mitochondrion membrane potential (MMP) decrease. Allicin induced apoptosis in EL-4 cells in a time- and concentration-dependent manner, in which the mitochondrial pathway might play a central role.

[Simultaneous determination of 6 phenolic acids in coffee beans by reversed-phase high performance liquid chromatography].

A method of reversed-phase high performance liquid chromatography coupled with diode array detection (HPLC-DAD) was established for the simultaneous determination of 6 phenolic acids in green coffee beans. These phenolic acids are caffeic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid. The separation was achieved using a Kromasil C18 column (200 mm x 4.6 mm, 5 microm) under the gradient elution with the mobile phases of acetonitrile and 0.1% (v/v) formic acid/water. The 6 phenolic acids were well separated within 45 min. The recoveries were from 90.76% to 104.73% with the relative standard deviations between 0.7% and 3.9%. The method is simple, rapid and highly sensitive, suitable for the simultaneous determination of 6 phenolic acids and quality control of coffee beans.

[Isolation and preparation of flavonoids from the leaves of Nelumbo nucifera Gaertn by preparative reversed-phase high performance liquid chromatography].

It has been confirmed that flavonoids in the leaves of Nelumbo nucifera Gaertn (lotus leaves) have many pharmacological activities. Currently, total flavones in the leaves of Nelumbo nucifera Gaertn have been studied extensively, however, only a few researches were able to investigate the individual components in it. At first, crude extract was obtained from lotus leaves by reflux extraction using 60% ethanol for three times. Then, the concentrated crude extract was separated on a D-101 column (eluted with 70% ethanol) and a polyamide column (step gradient 15% to 90% ethanol). The Fr-1 fraction was obtained from the eluate of 45% ethanol and was subjected to a preparative reversed-phase high performance liquid chromatograph (RP-HPLC) for the isolation of target components. The preparation of the individual flavonoids was carried out on an RP-HPLC with a Symmetry Prep C18 column, and the mobile phase was water-acetonitrile at a flow rate of 5.0 mL/min. Three compounds were identified with ultra violet absorbance (UV), infrared (IR), nuclear magnetic resonance (NMR) and mass spectrometry (MS). They were hyperin, isoquercetin and astragalin. To our knowledge that astragalin was the first time successfully isolated from this plant. The purity of the three compounds was all over 97%.

The role of prostaglandins and the hypothalamus in thermoregulation in the lizard, Phrynocephalus przewalskii (Agamidae).

Typically, small lizards rely heavily on behavioral thermoregulation rather than physiological mechanisms to control their rates of warming and cooling. We tested the hypothesis that prostaglandins participate in mediating the cardiovascular response to heating and cooling and temperature regulating neurons in the hypothalamus of the small lizard Phrynocephalus przewalskii. In vivo and in vitro treatments, heart rates (HRs) were all found to be higher during heating than during cooling, hysteresis was distinct below 30 and 26 degrees Celsius, respectively. In vivo, as administration of COX inhibitor, there were no differences in HR between heating and cooling at any body temperature and administration of agonist prostaglandins only produced a significant effect on HR below 25 degrees Celsius. Single-unit activity was recorded extracellularly in vitro with microelectrodes, found the firing rate of the continuous unit increased 23% when the temperature of the artificial cerebrospinal fluid dropped from 30-20 degrees Celsius. We conclude that prostaglandins appear to play only a limited role in modulating heart activity in Phrynocephalus przewalskii and suggest that cold-sensitive neurons in the preoptic and anterior hypothalamus (PO/AH) are involved in thermoregulatory control during heating or cooling.