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Objectives: Triterpenoid saponins of Ilex pubescens (IPTS), the main active components of Ilex pubescens, has a therapeutic effect on atherosclerosis (AS). The ingredients in IPTS that could be intracellularly transported by human umbilical vein endothelial cells (HUVECs) may play an essential role in AS. This study attempted to explore its mechanism from the perspectives of HUVECs' inflammation, apoptosis, and autophagy. Methods: By using a tumour necrosis factor-α (TNF-α)-induced HUVECs injury model, cell viability and the expression of intercellular adhesion molecule 1 (ICAM1), matrix metalloproteinase 9 (MMP9), cleave-caspase-3 and cleave-caspase-9, in combination with the results of flow cytometry, JC-1 and Hoechst 33258 staining were investigated to evaluate the anti-inflammatory and anti-apoptotic impact effects of IPTS on HUVECs. Afterwards, the expression of microtubule-associated proteins light chain 3II (LC3II) and sequestosome 1 (p62) was determined to test the effect of IPTS on autophagy. Finally, by adding an autophagy inhibitor 3-methyladenine (3-MA), we investigated whether IPTS exerts anti-inflammatory and anti-apoptotic effects through the autophagy pathway. Key findings: We firstly demonstrated that pretreatment with IPTS could increase the cell viability, maintain the cell morphology and reduce TNF-α-induced inflammation and apoptosis of HUVECs. Moreover, IPTS pretreatment was proved to raise the expression of LC3II /LC3I while decreasing the expression of p62, which indicated that IPTS could activate HUVECs' autophagy. IPTS has been shown for the first time to exert anti-inflammatory and anti-apoptotic effects through autophagy and thereby resisting TNF-α-induced inflammatory injury of HUVECs. Conclusions: This study preliminarily confirmed that IPTS ameliorated HUVECs' inflammation and apoptosis by increasing autophagy. [ABSTRACT FROM AUTHOR]

Elevated expression and genetic aberration of IRTKS, also named as BAIAP2L1, have been observed in many tumors, especially in tumor progression. however, the molecular and cellular mechanisms involved in the IRTKS-enhanced tumor progression are obscure. Here we show that higher IRTKS level specifically increases histone H3 lysine 9 trimethylation (H3K9me3) by promoting accumulation of the histone methyltransferase SETDB1. Furthermore, we reveal that IRTKS recruits the deubiquitinase OTUD4 to remove Lys48-linked polyubiquitination at K182/K1050 sites of SETDB1, thus blocking SETDB1 degradation via the ubiquitin-proteasome pathway. Interestingly, the enhanced IRTKS–OTUD4–SETDB1–H3K9me3 axis leads to a general decrease in chromatin accessibility, which inhibits transcription of CDH1 encoding E-cadherin, a key molecule essential for maintaining epithelial cell phenotype, and therefore results in epithelial-mesenchymal transition (EMT) and malignant cell metastasis. Clinically, the elevated IRTKS levels in tumor specimens correlate with SETDB1 levels, but negatively associate with survival time. Our data reveal a novel mechanism for the IRTKS-enhanced tumor progression, where IRTKS cooperates with OTUD4 to enhance SETDB1-mediated H3K9 trimethylation that promotes tumor metastasis via suppressing E-cadherin expression. This study also provides a potential approach to reduce the activity and stability of the known therapeutic target SETDB1 possibly through regulating IRTKS or deubiquitinase OTUD4. • Higher levels of IRTKS increase H3K9me3 by promoting SETDB1 accuumulation. • IRTKS recruits the deubiquitinase OTUD4 to blocking SETDB1 degradation. • The enhanced IRTKS–OTUD4–SETDB1–H3K9me3 axis decreases chromatin accessibility, and inhibits E-cadherin transcription, resulting in EMT and tumor metastasis. • Clinically, elevated IRTKS levels in tumor correlate with SETDB1 levels, and negatively associate with survival time. [ABSTRACT FROM AUTHOR]

Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721–1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene I (RIG-I), a crucial member of cytoplasmic pattern recognition receptors (PRRs), is initially characterized as a dsRNA-binding protein triggering interferon (IFN) induction in response to virus invasion. While the antiviral regulatory mechanism of RIG-I remains largely unclear. In this study, the mechanism of CiRIG-I ( Ctenopharyngodon idella RIG-I ) against grass carp reovirus (GCRV) would be revealed from the perspective of epigenetics. By prediction, three CpG islands (CGIs) were located in 5′-flanking region and the first exon. The first CGI and the second one, both located in the 5′-flanking region, were 109 base pairs (bp) and 134 bp in length, involving five CpG sites and four loci, respectively. The third CGI was of 386 bp spanning the 5′-flanking region and the first exon, densely possessing 24 CpG sites. DNA methylation statuses of CpG sites were identified by virtue of the bisulfite sequencing PCR (BSP) in spleen of all susceptible and resistant individuals post the challenge experiment. The resistance association analysis was performed with Chi-square test. And the relationship between DNA methylation and gene expression in CiRIG-I was investigated by quantitative real-time RT-PCR (qRT-PCR). Results indicated only the methylation of -534 CpG site in the second CGI possessing tight association with the resistance against GCRV, which was significantly higher in the susceptible individuals than that in the resistant individuals. In addition, the average expression of CiRIG-I was down-regulated in the susceptible group compared with the resistant one demonstrating gene transcription may be negatively-regulated by DNA methylation in CiRIG-I . Collectively, the methylation statuses of CiRIG-I were extremely related to the resistance against GCRV and maybe serve as a negative modulator on antiviral transcription of CiRIG-I . This study revealed the underlying antiviral regulatory mechanism of CiRIG-I and laid a theoretical foundation for the nosogenesis of hemorragic diseases in C. idella . [ABSTRACT FROM AUTHOR]

Melanoma differentiation-associated gene 5 (MDA5) plays a crucial role in recognizing intracellular viral infection, activating the interferon regulatory factor pathways as well as inducing antiviral response. While the antiviral regulatory mechanism of MDA5 remains unclear. In the present study, CiMDA5 ( Ctenopharyngodon idella MDA5 ) against grass carp reovirus (GCRV) would be initially revealed from the perspective of DNA methylation, a pivotal epigenetic modification. Two CpG islands (CGIs) were predicted located in the first exon of CiMDA5 , of which the first CpG island was 427 bp in length possessed 29 candidate CpG loci and 34 CpA loci, and the second one was 130 bp in length involving 7 CpG loci as well as 10 CpA loci. By bisulfite sequencing PCR (BSP), the methylation statuses were detected in spleen of 70 individuals divided into resistant/susceptible groups post challenge experiment, and the resistance-association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was carried out to explore the relationship between DNA methylation and gene expression in CiMDA5 . Results indicated that the methylation levels of CpA/CpG sites at +200, +202, +204, +207 nt, which consisted of a putative densely methylated element (DME), were significantly higher in the susceptible group than those in the resistant group. Meanwhile, the average transcription of CiMDA5 was down-regulated in the susceptible individuals compared with the resistant individuals. Evidently, the DNA methylation may be the negative modulator of CiMDA5 antiviral expression. Collectively, the methylation levels of CiMDA5 demonstrated the tight association with the resistance against GCRV and the negative-regulated roles in mRNA expression. This study first discovered the resistance-associated gene modulated by DNA methylation in teleost, preliminary revealed the underlying regulatory mechanism of CiMDA5 transcription against GCRV as well as laid a theoretical foundation on molecular nosogenesis of hemorrhagic diseases in C. idella . [ABSTRACT FROM AUTHOR]

As an intracellular pattern recognition receptor (PRR), laboratory of genetics and physiology 2 (LGP2) plays a pivotal role in detecting nucleic acids of invading pathogens and simultaneously modulating signaling by retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in type I interferon (IFN–I) pathway. Nevertheless, the underlying antiviral transcription mechanism of LGP2 remains obscure. The present study attempted to reveal the methylation levels of CiLGP2 ( Ctenopharyngodon idella LGP2 ) in muscle and spleen of grass carp and their association with the resistance against grass carp reovirus (GCRV). By prediction, the CpG island was 133 bp in length in 5′-flanking region, containing six candidate CpG loci, whose methylation statuses were investigated by virtue of the bisulfite sequencing PCR (BSP) among muscle and spleen tissues in 120 individuals that were divided into resistant/susceptible groups after a challenge experiment, and the association analysis was performed with Chi-square test. Quantitative real-time RT-PCR (qRT-PCR) was employed to ascertain the interrelation between methylation status and transcription of CiLGP2 . The CpG sites at −1394, −1366, −1331 and −1314 nt were identified as hypermethylated, inversely unmethylated at −1350 CpG site. The −1411 CpG site presented six methylation patterns as well as one mentionable type of mutation triggered by spontaneous deamination. Although there was no statistically significant difference on DNA methylation with resistance against GCRV at −1411 CpG site, the methylation levels were significantly lower in spleen than those in muscle, accompanied by higher mRNA expression of CiLGP2 in spleen. Notably, DNA methylation may be conceivably serve as an essential regulatory factor for CiLGP2 antiviral transcription in spleen. This research first demonstrated the relationship between DNA methylation and LGP2 gene expression, preliminary revealed the underlying transcription mechanism of CiLGP2 against GCRV as well as provided potential references and laid a theoretical foundation for viral recognition and regulation research of LGP2 in vertebrates. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that vitamin D (VD) has a therapeutic effect on non-alcoholic fatty liver disease (NAFLD). Pyroptosis and gut microbiota have been recognized as critical factors of the progression of NAFLD. However, the effect of VD on the pyroptosis and gut microbiota in NAFLD remains inconclusive. Herein, rats were fed high fat diet (HFD) for 12 weeks and concurrently treated with 5 μ g/kg 1,25(OH) 2 D 3 twice a week. BRL-3A cells were stimulated with 0.4 mmol/L palmitic acid (PA) and 1 μ g/ml lipopolysaccharide (LPS) for 16 h and treated with 10−6 mol/L 1,25(OH) 2 D 3. Effect of VD on the hepatic injury, lipid accumulation, activation of NLRP3 inflammasome and pyroptosis was determined in vivo and in vitro. Next, gasdermin D N -terminal (GSDMD-N) fragment was overexpressed in BRL-3A cells to investigate the role of pyroptosis in the therapeutic effect of VD on NAFLD. In addition, gut microbiota in NAFLD rats was also analyzed. Results showed that VD attenuated HFD-induced hepatic injury in vivo and PA-LPS-induced impairment of cell viability in vitro , and inhibited lipid accumulation, activation of NLRP3 inflammasome and pyroptosis in vivo and in vitro. GSDMD-N fragment overexpression suppressed the protective effect of VD on PA-LPS-induced activation of NLRP3 inflammasome, impairment of cell viability and lipid accumulation, indicating that VD might attenuate NAFLD through inhibiting pyroptosis. Additionally, VD also restored HFD-induced gut microbiota dysbiosis by increasing the relative abundance of Lactobacillus and reducing that of Acetatifactor , Oscillibacter and Flavonifractor. This study provides a novel mechanism underlying VD therapy against NAFLD. • Vitamin D inhibited activation of NLRP3 inflammasome and pyroptosis in NAFLD rat. • Vitamin D might attenuate HFD-induced NAFLD by inhibiting pyroptosis in NAFLD rat. • Vitamin D restored HFD-induced gut microbiota dysbiosis in NAFLD rat. [ABSTRACT FROM AUTHOR]

In this study, the pathogenic fungus of stem spot disease of pitaya (Hylocereus spp.) stems was identified by isolation and purification, pathogenicity test, morphological characteristics, and analysis of rDNA-ITS sequences. The results turned out that the rDNA-ITS sequences of the H1 strain showed 100% of identity with Botryosphaeria dothidea, indicating that the pathogenic fungus of stem spot disease of pitaya stems was Botryosphaeria dothidea. Meanwhile, the H1 strain was then used as a reference strain to screen some commercial fungicides. The bioassay test results indicated that prochloraz had an obvious inhibitory effect on Botryosphaeria dothidea with the EC50 value of 0.0798 μg/mL. Our study could provide a theoretical basis for the effective control method of stem spot disease of pitaya. [ABSTRACT FROM AUTHOR]

The Drosophila wing was proposed to be a taste organ more than 35 years ago, but there has been remarkably little study of its role in chemoreception. We carry out a differential RNA-seq analysis of a row of sensilla on the anterior wing margin and find expression of many genes associated with pheromone and chemical perception. To ask whether these sensilla might receive pheromonal input, we devised a dye-transfer paradigm and found that large, hydrophobic molecules comparable to pheromones can be transferred from one fly to the wing margin of another. One gene, Ionotropic receptor (IR)52a, is coexpressed in neurons of these sensilla with fruitless, a marker of sexual circuitry; IR52a is also expressed in legs. Mutation of IR52a and optogenetic silencing of IR52a+ neurons decrease levels of male sexual behavior. Optogenetic activation of IR52a+ neurons induces males to show courtship toward other males and, remarkably, toward females of another species. Surprisingly, IR52a is also required in females for normal sexual behavior. Optogenetic activation of IR52a+ neurons in mated females induces copulation, which normally occurs at very low levels. Unlike other chemoreceptors that act in males to inhibit male–male interactions and promote male–female interactions, IR52a acts in both males and females, and can promote male–male as well as male–female interactions. Moreover, IR52a+ neurons can override the circuitry that normally suppresses sexual behavior toward unproductive targets. Circuit mapping and Ca2+ imaging using the trans-Tango system reveals second-order projections of IR52a+ neurons in the subesophageal zone (SEZ), some of which are sexually dimorphic. Optogenetic activation of IR52a+ neurons in the wing activates second-order projections in the SEZ. Taken together, this study provides a molecular description of the chemosensory sensilla of a greatly understudied taste organ and defines a gene that regulates the sexual circuitry of the fly. [ABSTRACT FROM AUTHOR]

Context:Baicalin (BL) and baicalein (B) as the major flavonoids ofScutellaria baicalensisGeorgi (Lamiaceae) have been investigated intensively, and shown to possess a multitude of pharmacological activities. Objective:This study systematically evaluates the stability of BL and B in monomer and total flavonoid fraction (FSR) formin vitro, and further studies whether the protective measures are effective to make B and BL stable enough to meet the requirement of quantitative analysis in various biological samples. Materials and methods:The stability of BL and B was evaluated by investigating the influence factors such as pH (2.0, 3.0, 4.5, 6.8, 7.4 and 9.0), temperature (4, 25 and 40 °C), antioxidant (vitamin C and Na2SO3) and sunlight. After the protective measures were taken, stability of BL and B in plasma, urine and tissue homogenates was evaluated through post-preparative stability (stored at 4 °C for 24 h), three freeze–thaw cycles stability and long-term stability test (stored in refrigerator at −20 °C for 15 days). In addition, by comparing the degradation parameters of BL and B obtained from the monomer administration group with those of the FSR administration group, drug–drug interaction of coexistent components in FSR on the stability of BL and B was discussed. Results:The degradation of BL and B was both pH- and temperature-dependent with their correlation coefficents for first-order kinetics equation larger than 0.99, and acidic environment (pH 2–4.5), lower temperature (<4 °C) and acidic antioxidant (e.g. vitamin C) were conducive to stabilize B and BL. Furthermore, coexistent components in FSR were proved to have function on inhibiting the degradation of BL and B in our study for the first time, which was characteristic of prolonging their biological half-life (t1/2) significantly, e.g., from 2.89 h to indefinite for BL (pH 6.8, 25 °C), from 2.63 h to 4.48 h for B (pH 6.8, 25 °C) and so on. Antioxidant of Na2SO3could inhibit the degradation of BL witht1/2increasing from 1.8 h to 3.5 h, but aggravate the bio-transformation of B witht1/2decreasing from 0.92 h to 0.29 h. Our research proved that BL monomer, and BL and B in FSR form could be stabilized enough to meet the requirement of biological quantitative analysis under the protection of coexistent components in FSR. Discussion and conclusion:The results obtained indicated that the stability of BL and B was affected not only by its environmental parameters, but also by the coexistent components in the effective total flavonoids fractions. [ABSTRACT FROM PUBLISHER]

Toll-like receptor 8 (TLR8), a prototypical intracellular member of TLR family, is generally linked closely to antiviral innate immune through recognizing viral nucleic acid. In this study, 5′-flanking region of Ctenopharyngodon idella TLR8 ( CiTLR8 ), 671 bp in length, was amplified and eight SNPs containing one SNP in the intron, three SNPs in the coding region (CDS) and four SNPs in the 3′-untranslated region (UTR) were identified and characterized. Of which 4062 A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele ( P < 0.05), while 4168 C/T was extremely significantly associated with that ( P < 0.01) according to the case (susceptibility)–control (resistance) analysis. Following the verification experiment, further analyses of mRNA expression, linkage disequilibrium (LD), haplotype and microRNA (miRNA) target site indicated that 4062 A/T and 4168 C/T in 3′-UTR might affect the miRNA regulation, while the exertion of antiviral effects of 4062 A/T might rely on its interaction with other SNPs. Additionally, the high-density of SNPs in 3′-UTR might reflect the specific biological functions of 3′-UTR. And also, the mutation of 747 A/G in intron changing the potential transcriptional factor-binding sites (TFBS) nearby might affect the expression of CiTLR8 transcriptionally or post-transcriptionally. Moreover, as predicted, the A/G transition of the only non-synonymous SNP (3846 A/G) in CDS causing threonine/alanine variation, could shorten the length of the α-helix and ultimately affect the integrity of the Toll-IL-1 receptor (TIR) domain. The functional mechanism of 3846 A/G might also involve a threonine phosphorylation signaling. This study may broaden the knowledge of TLR polymorphisms, lay the foundation for further functional research of CiTLR8 and provide potential markers as well as theoretical basis for resistance molecular breeding of grass carp against GCRV. [ABSTRACT FROM AUTHOR]

The quantitative analysis of multiple components with a single marker (QAMS) method was firstly established for simultaneous determination of 18 active components in Ilex kudingcha C. J. Tseng by HPLC. Using rutin, isochlorogenic acid A and kudinoside A as internal refererence substances (IRS), compatibility results showed that the relative correction factors (RCFs) of all compounds showed good reproducibility under different chromatographic conditions. On the basis of previous studies, the accuracy of the QAMS method was systematically evaluated by investigating the influences of curve intercept, analytes and IRS concentration. The results showed that the concentration (especially at low level) of analytes and curve intercept were the major influencing parameters for the LRG-QAMS method (LRG = linear regression), whereas the influence of IRS concentration seemed more apparent in terms of the AVG-QAMS method (AVG = average). The two approaches were complementary with each other. In addition, hierarchical clustering analysis (HCA), principal components analysis (PCA) and similarity analysis (SA) were performed to differentiate and classify the samples based on the contents of 18 marker compounds. The results of the different chemometric analyses were completely consistent with each other, and could be supported by the quantification results. [ABSTRACT FROM AUTHOR]

The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches. [ABSTRACT FROM AUTHOR]

Background: High-throughput sequencing technology has yielded reliable and ultra-fast sequencing for DNA and RNA. For tumor cells of cancer patients, when combining the results of DNA and RNA sequencing, one can identify potential neoantigens that stimulate the immune response of the T cell. However, when the somatic mutations are abundant, it is computationally challenging to efficiently prioritize the identified neoantigen candidates according to their ability of activating the T cell immuno-response. Methods: Numerous prioritization or prediction approaches have been proposed to address this issue but none of them considers the original DNA loci of the neoantigens from the perspective of 3D genome. Based on our previous discoveries, we propose to investigate the distribution of neoantigens with different immunogenicity abilities in 3D genome and propose to adopt this important information into neoantigen prediction. Results: We retrospect the DNA origins of the immuno-positive and immuno-negative neoantigens in the context of 3D genome and discovered that DNA loci of the immuno-positive neoantigens and immuno-negative neoantigens have very different distribution pattern. Specifically, comparing to the background 3D genome, DNA loci of the immuno-positive neoantigens tend to locate at specific regions in the 3D genome. We thus used this information into neoantigen prediction and demonstrated the effectiveness of this approach. Conclusion: We believe that the 3D genome information will help to increase the precision of neoantigen prioritization and discovery and eventually benefit precision and personalized medicine in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Insulin signaling is mediated by a highly integrated network that controls glucose metabolism, protein synthesis, cell growth, and differentiation. Our previous work indicates that the insulin receptor tyrosine kinase substrate (IRTKS), also known as BAI1-associated protein 2-like 1 (BAIAP2L1), is a novel regulator of insulin network, but the mechanism has not been fully studied. In this work we reveal that IRTKS co-localizes with Src homology (SH2) containing inositol polyphosphate 5-phosphatase-2 (SHIP2), and the SH3 domain of IRTKS directly binds to SHIP2's catalytic domain INPP5c. IRTKS suppresses SHIP2 phosphatase to convert phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3, PIP3) to phosphatidylinositol (3,4) bisphosphate (PI(3,4)P2). IRTKS-knockout significantly increases PI(3,4)P2 level and decreases cellular PI(3,4,5)P3 content. Interestingly, the interaction between IRTKS and SHIP2 is dynamically regulated by insulin, which feeds back and affects the tyrosine phosphorylation of IRTKS. Furthermore, IRTKS overexpression elevates PIP3, activates the AKT–mTOR signaling pathway, and increases cell proliferation. Thereby, IRTKS not only associates with insulin receptors to activate PI3K but also interacts with SHIP2 to suppress its activity, leading to PIP3 accumulation and the activation of the AKT–mTOR signaling pathway to modulate cell proliferation. [ABSTRACT FROM AUTHOR]