Downregulation Of Transcript Levels Research Articles

Effects of Proteasome 20S Subunit Beta 8 on Proliferation,Migration,and Invasion of Clear Cell Renal Cell Carcinoma Cells via Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling Pathway

Objective To explore the effects of proteasome 20S subunit beta 8 (PSMB8) on the proliferation,migration,and invasion of clear cell renal cell carcinoma (ccRCC) cells and whether PSMB8 promotes tumor progression by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway. Methods The Cancer Genome Atlas was employed to analyze the mRNA levels of PSMB8 in ccRCC and normal tissue,and the expression levels of PSMB8 in ccRCC tissue and cells were determined by real-time quantitative PCR,Western blotting,and immunohistochemistry.Furthermore,the cell lines with stable overexpression and knockdown of PSMB8 were constructed.The CCK-8 assay and colony formation assay were employed to examine the cell proliferation,and the wound healing assay and Transwell assay were employed to examine the invasion and migration of cells.Kyoto Encyclopedia of Genes and Genomes pathway enrichment was performed to analyze the co-expressed genes of PSMB8.Western blotting was used to measure the phosphorylation levels of the proteins in the MEK/ERK signaling pathway.Finally,the rescue experiment was carried out with the ERK agonist C16-PAF. Results Compared with the normal tissue,the ccRCC tissue showed up-regulated mRNA and protein levels of PSMB8 (both P<0.001),which were associated with the TNM stage of patients with ccRCC (P<0.001).Compared with the negative control group,overexpression of PSMB8 promoted the proliferation (P=0.021,P=0.039),migration and invasion (all P<0.001) of 786-O and ACHN cells,and the knockdown of PSMB8 inhibited the proliferation (P=0.022,P=0.005),migration and invasion (all P<0.001) of 786-O and ACHN cells.The pathway enrichment analysis of co-expressed genes of PSMB8 predicted the mitogen-activated protein kinase signaling pathway (P<0.001).After the knockdown of PSMB8,786-O and ACHN cells showed lowered phosphorylation levels of MEK1/2 (P=0.017,P=0.016) and ERK1/2 (P=0.010,P=0.040) and down-regulated transcription levels of ERK downstream factors c-Myc (P=0.043,P=0.038),c-Fos (P=0.025,P=0.008),and CyclinD1 (P=0.006,P=0.047).Compared with the ERK agonist C16-PAF group,the PSMB8 knockdown + C16-PAF group showed inhibited proliferation (P=0.003,P=0.002),migration and invasion (all P<0.001) of 786-O and ACHN cells. Conclusion PSMB8 may promote the proliferation,migration,and invasion of ccRCC cells by activating the MEK/ERK signaling pathway.

WALL-ASSOCIATED KINASE Like 14 regulates vascular tissue development in Arabidopsis and tomato

Initiation of plant vascular tissue is regulated by transcriptional networks during development and in response to environmental stimuli. The WALL-ASSOCIATED KINASES (WAKs) and WAK-likes (WAKLs) are cell surface receptors involved in cell expansion and defence in cells with primary walls, yet their roles in regulation of vascular tissue development that contain secondary walls remains unclear. In this study, we showed tomato (Solanum lycopersicum) SlWAKL2 and the orthologous gene in Arabidopsis thaliana, AtWAKL14, were specifically expressed in vascular tissues. SlWAKL2-RNAi tomato plants displayed smaller fruit size with fewer seeds and vascular bundles compared to wild-type (WT) and over-expression (OE) lines. RNA-seq data showed that SlWAKL2-RNAi fruits down-regulated transcript levels of genes related to vascular tissue development compared to WT. Histological analysis showed T-DNA insertion mutant wakl14–1 had reduced plant stem length with fewer number of xylem vessels and interfascicular fibres compared to WT, with no significant differences in cellulose and lignin content. Mutant wakl14–1 also showed reduced number of vascular bundles in fruit. A proWAKL14::mCherry-WAKL14 fusion protein was able to complement wakl14–1 phenotypes and showed mCherry-WAKL14 associated with the plasma membrane. In vitro binding assays showed both SlWAKL2 and AtWAKL14 can interact with pectin and oligogalacturonides. Our results reveal novel roles of WAKLs in regulating vascular tissue development.

PRRSV degrades MDA5 via dual autophagy receptors P62 and CCT2 to evade antiviral innate immunity

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically devastating pathogen that has evolved various strategies to evade innate immunity. Downregulation of antiviral interferon largely promotes PRRSV immunoevasion by utilizing cytoplasmic melanoma differentiation-associated gene 5 (MDA5), a receptor that senses viral RNA. In this study, the downregulated transcription and expression levels of porcine MDA5 in PRRSV infection were observed, and the detailed mechanisms were explored. We found that the interaction between P62 and MDA5 is enhanced due to two factors: the phosphorylation modification of the autophagic receptor P62 by the upregulated kinase CK2α and the K63 ubiquitination of porcine MDA5 catalyzed by the E3 ubiquitinase TRIM21 in PRRSV-infected cells. As a result of these modifications, the classic P62-mediated autophagy is triggered. Additionally, porcine MDA5 interacts with the chaperonin containing TCP1 subunit 2 (CCT2), which is enhanced by PRRSV nsp3. This interaction promotes the aggregate formation and autophagic clearance of MDA5-CCT2-nsp3 independently of ubiquitination. In summary, enhanced MDA5 degradation occurs in PRRSV infection via two autophagic pathways: the binding of MDA5 with the autophagy receptor P62 and the aggrephagy receptor CCT2, leading to intense innate immune suppression. The research reveals a novel mechanism of immune evasion in PRRSV infection and provides fundamental insights for the development of new vaccines or therapeutic strategies.

Anti-Biofilm Activity of Laurel Essential Oil against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a primary seafood-associated pathogen that could cause gastroenteritis. It can attach to various surfaces and form a biofilm, which poses serious threats to food safety. Hence, an effective strategy is urgently needed to control the biofilm formation of V. parahaemolyticus. Laurel essential oil (LEO) is used in food, pharmaceutical and other industries, and is commonly used as a flavoring agent and valuable spice in food industries. The potential antibiofilm effects of LEO against V. parahaemolyticus were examined in this study. LEO obviously reduced biofilm biomass at subinhibitory concentrations (SICs). It decreased the metabolic activity and viability of biofilm cells. Microscopic images and Raman spectrum indicted that LEO interfered with the structure and biochemical compositions of biofilms. Moreover, it also impaired swimming motility, decreased hydrophobicity, inhibited auto-aggregation and reduced attachment to different food-contact surfaces. RT-qPCR revealed that LEO significantly downregulated transcription levels of biofilm-associated genes of V. parahaemolyticus. These findings demonstrate that LEO could be potentially developed as an antibiofilm strategy to control V. parahaemolyticus biofilms in food industries.

Taurochenodeoxycholic acid exerts anti-viral activities upon SGIV infection via anti-inflammatory response

Taurine chenodeoxycholic acid (TCDCA), a combination of taurine and chenodeoxycholic acid, plays anti-inflammatory roles in many diseases. However, little is known about its antiviral activity in aquaculture. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, is an important viral pathogen in grouper aquaculture. Here, the effects of TCDCA on SGIV replication in vitro and in vivo were investigated. TCDCA treatment inhibited SGIV infection in grouper spleen (GS) cells, evidenced by the delayed CPE progression and decreased viral gene transcription, protein expression and viral progeny production. TCDCA treatment suppressed the mRNA level of pro-inflammatory factors and NF-κB promoter activities in SGIV-infected cells compared with mock-infected cells. Inhibition of NF-κB activity enhanced the inhibitory effects of TCDCA on SGIV replication, indicating that TCDCA inhibited SGIV replication in vitro via regulating host inflammatory response. Furthermore, TCDCA treatment protected grouper from SGIV infection, evidenced by the decreased mortality, the down-regulated transcription levels of viral genes and the improved pathological symptoms. In addition, the expression levels of SGIV induced pro-inflammation factors were decreased, while those of the antioxidant genes were increased after the supplement with TCDCA. Thus, our findings will not only contribute greatly to understanding the mechanism of SGIV pathogenesis, but also demonstrate that TCDCA might be a potential antiviral agent against SGIV infection.

Life-cycle exposure to tris (2-chloroethyl) phosphate (TCEP) causes alterations in antioxidative status, ion regulation and histology of zebrafish gills

Tris (2-chloroethyl) phosphate (TCEP) has been receiving great concerns owing to its ubiquitous occurrence in various environmental compartments and potential risks to wildlife and humans. Gill is structural basis for ion regulation and homeostasis in fish and susceptible to xenobiotics. However, current knowledge on the impacts of long-term exposure to TCEP on the structure and physiological function of fish gills are insufficient. In this work, zebrafish were exposed to environmental realistic concentrations (0.8, 4, 20 and 100 μg/L) of TCEP from 3 h post ferterlization (hpf) till 120 days post ferterlization (dpf). Our results demonstrated that life-cycle exposure to TCEP significantly decreased the activity of glutathione S-transferase (GST), but elevated the activities of antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and increased malondialdehyde (MDA) content in zebrafish gills. Gene transcription analysis implied that the mRNA expressions of antioxidant-related genes (nrf2, cat and nqo1) were induced, while the transcription of gstα1, hmox1, keap1 were down-regulated, indicating that Nrf2-Keap1 pathway might be activated to defend the oxidative stress induced by TCEP. Additionally, the ion homeostasis was disrupted by TCEP exposure, evidenced by reduced activities of Na+/K+-ATPase (NKA), Ca2+-ATPase and Mg2+-ATPase and downregulated transcription levels of ncc, nkcc, cftr and clc-3. Besides, whole-life exposure to TCEP resulted in a series of structural damages to gills, including epithelial lifting, epithelial rupture, telangiectasis, vacuolation, edema and shortened gill lamellae. Overall, our results demonstrated that long-term TCEP exposure could induce oxidative stress, affect ion regulation and cause histological changes in zebrafish gills.

Design, Synthesis, and Bioassay for the Thiadiazole-Bridged Thioacetamide Compound as Cy-FBP/SBPase Inhibitors Based on Catalytic Mechanism Virtual Screening.

Cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase (Cy-FBP/SBPase) was an important regulatory enzyme in cyanobacterial photosynthesis and was a potential target enzyme for screening to obtain novel inhibitors against cyanobacterial blooms. In this study, we developed a novel pharmacophore screening model based on the catalytic mechanism and substrate structure of Cy-FBP/SBPase and screened 26 S series compounds with different structures and pharmacophore characteristics from the Specs database by computer-assisted drug screening. These compounds exhibited moderate inhibitory activity against Cy-FBP/SBPase, with 9 compounds inhibiting >50% at 100 μM. Among them, compound S5 showed excellent inhibitory activity against both Cy-FBP/SBPase and Synechocystis sp. PCC6803 (IC50 = 6.7 ± 0.7 μM and EC50 = 7.7 ± 1.4 μM). The binding mode of compound S5 to Cy-FBP/SBPase was predicted using the molecular docking theory and validated by sentinel mutation and enzyme activity analysis. Physiochemical, gene transcription level, and metabolomic analyses showed that compound S5 significantly reduced the quantum yield of photosystem II and the maximum electron transfer rate, downregulated transcript levels of related genes encoding the Calvin cycle and photosystem, reduced the photosynthetic efficiency of cyanobacteria, thus inhibited metabolic pathways, such as the Calvin cycle and tricarboxylic acid cycle, and eventually achieved an efficient algicide. In addition, compound S5 had a high safety profile for human-derived cells and zebrafish. In summary, the novel pharmacophore screening model obtained from the current work provides an effective solution to the cyanobacterial bloom problem.

OsPEX1, an extensin-like protein, negatively regulates root growth in a gibberellin-mediated manner in rice

Leucine-rich repeat extensins (LRXs) are required for plant growth and development through affecting cell growth and cell wall formation. LRX gene family can be classified into two categories: predominantly vegetative-expressed LRX and reproductive-expressed PEX. In contrast to the tissue specificity of Arabidopsis PEX genes in reproductive organs, rice OsPEX1 is also highly expressed in roots in addition to reproductive tissue. However, whether and how OsPEX1 affects root growth is unclear. Here, we found that overexpression of OsPEX1 retarded root growth by reducing cell elongation likely caused by an increase of lignin deposition, whereas knockdown of OsPEX1 had an opposite effect on root growth, indicating that OsPEX1 negatively regulated root growth in rice. Further investigation uncovered the existence of a feedback loop between OsPEX1 expression level and GA biosynthesis for proper root growth. This was supported by the facts that exogenous GA3 application downregulated transcript levels of OsPEX1 and lignin-related genes and rescued the root developmental defects of the OsPEX1 overexpression mutant, whereas OsPEX1 overexpression reduced GA level and the expression of GA biosynthesis genes. Moreover, OsPEX1 and GA showed antagonistic action on the lignin biosynthesis in root. OsPEX1 overexpression upregulated transcript levels of lignin-related genes, whereas exogenous GA3 application downregulated their expression. Taken together, this study reveals a possible molecular pathway of OsPEX1mediated regulation of root growth through coordinate modulation of lignin deposition via a negative feedback regulation between OsPEX1 expression and GA biosynthesis.

Amino Acid Conjugates of 2-Mercaptobenzimidazole Ameliorates High-Fat Diet-Induced Hyperlipidemia in Rats via Attenuation of HMGCR, APOB, and PCSK9

This study was designed to explore the antihyperlipidemic effects of amino acid derivatives of 2-mercaptobenzimidazole (4J and 4K) in high-fat diet (HFD)-fed rats. Male Sprague-Dawley rats were divided into nine groups which received either standard diet or HFD for 28 days. Blood samples were taken on 27th day from HFD-fed rats to ensure hyperlipidemia. HFD-induced hyperlipidemic rats later received daily dosing of either vehicle or simvastatin (SIM; 20 mg/kg) or 4J/4K compounds (10, 20, and 30 mg/kg) for 12 consecutive days. On 40th day, animals were sacrificed, and blood samples were collected for the determination of serum lipid profile and liver function parameters. Liver samples were harvested for histopathological, antioxidant, and qPCR analyses. Molecular docking of tested compounds with HMGCR was also performed to assess the binding affinities. 4J and 4K dose dependently decreased serum total cholesterol, triglycerides, low-density lipoprotein, very low-density lipoproteins, alanine transaminase (ALT), and aspartate aminotransferase (AST) levels while significantly alleviated high-density lipoproteins. However, SIM failed to reduce AST and ALT levels. Moreover, tested compounds displayed antioxidant effects by inducing superoxide dismutase and glutathione levels. Histopathology data also displayed protective effects of 4J and 4K against HFD-induced fatty changes and hepatic damage. In addition, 4J and 4K downregulated transcript levels of HMGCR, APOB, PCSK9, and VCAM1, and molecular docking analysis also supported the experimental data. It is conceivable from this study that 4J and 4K exert their antihyperlipidemic effects by modulating multiple targets regulating lipid levels.

Insight into the regulatory networks underlying the high lipid perennial ryegrass growth under different irradiances.

Under favourable conditions, perennial ryegrass (Lolium perenne) engineered to accumulated high lipid (HL) carbon sink in their leaves was previously shown to also enhance photosynthesis and growth. The greater aboveground biomass was found to be diminished in a dense canopy compared to spaced pots. Besides, the underlying genetic regulatory network linking between leaf lipid sinks and these physiological changes remains unknown. In this study, we demonstrated that the growth advantage was not displayed in HL Lolium grown in spaced pots under low lights. Under standard lights, analysis of differentiating transcripts in HL Lolium reveals that the plants had elevated transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling while reduced expression of genes participating in sugar biosynthesis and transportation. The plants also had altered several transcripts involved in mitochondrial oxidative respiration and redox potential. Many of the above upregulated or downregulated transcript levels were found to be complemented by growing the plants under low light. Overall, this study emphasizes the importance of carbon and energy homeostatic regulatory mechanisms to overall productivity of the HL Lolium through photosynthesis, most of which are significantly impacted by low irradiances.

Transcript-Targeted Therapy Based on RNA Interference and Antisense Oligonucleotides: Current Applications and Novel Molecular Targets

The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA (e.g., Inclisiran targets mRNA of protein PCSK9, permitting a longer half-life of LDL receptors in heterozygous familial hypercholesteremia), by arresting mRNA translation (i.e., Fomivirsen that binds to UL123-RNA and blocks the translation into IE2 protein in CMV-retinitis), or by reactivating modified functional protein (e.g., Eteplirsen able to restore a functional shorter dystrophin by skipping the exon 51 in Duchenne muscular dystrophy) or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs (e.g., Nusinersen acts on the splicing of exon 7 in SMN2 mRNA normally not expressed; it is used for spinal muscular atrophy) or by downregulation of transcript levels (e.g., Inotersen acts on the transthryretin mRNA to reduce its expression; it is prescribed for the treatment of hereditary transthyretin amyloidosis) in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed.

C2H2-type zinc finger OsZFP15 accelerates seed germination and confers salinity and drought tolerance of rice seedling through ABA catabolism

Environmental factors and plant hormones are major players in seed germination. Abscisic acid (ABA) not only delays seed germination but also acts as a central regulator in plant growth, development and response to stress conditions. In this study, a rice C2H2-type zinc finger protein, OsZFP15, was screened by microarray and characterized. RT-qPCR and GUS staining assays revealed that OsZFP15 was significantly induced by ABA and multiple abiotic stresses. Subcellular localization assay indicated that OsZFP15 protein was located in the nucleus of rice protoplast. Overexpression of OsZFP15 reduced the sensitivity to ABA and down-regulated transcript levels of ABA-responsive genes including LEA3 and LIP9, but increased transcript level of OsABA8ox2, suggesting that OsZFP15 is a positive regulator of ABA catabolism. Additionally, overexpression of OsZFP15 in rice enhanced ABA catabolism and thus accelerated seed germination. We further investigated the role of OsZFP15 in abiotic stress response. Overexpression of OsZFP15 promoted reactive oxygen species (ROS) production and accordingly reduced the tolerance to oxidative stress. Conversely, improvements in salinity and drought tolerance were observed in OsZFP15 overexpression plants.

Inhibitory effects of gallic acid on the activity of exosomal secretory pathway in breast cancer cell lines: A possible anticancer impact.

Introduction: Breast cancer cells produce exosomes that promote tumorigenesis. The anticancer properties of gallic acid have been reported. However, the mechanism underlying its anticancer effect on the exosomal secretory pathway is still unclear. We investigated the effect of gallic acid on exosome biogenesis in breast cancer cell lines. Methods: The cytotoxic effect of gallic acid on MCF-10a, MCF-7, and MDA-MD-231 cells was measured by MTT assay after 48 hours treatment. Expression of miRNAs including miRNA-21, -155, and 182 as well as exosomal genes such as Rab27a, b, Rab11, Alix, and CD63; along with HSP-70 (autophagy gene), was determined using Q-PCR. The subcellular distribution of it was monitored by flow cytometry analysis. Isolated exosomes were characterized by transmission and scanning electron microscopes and flow cytometry. Acetylcholinesterase activity is used to measure the number of exosomes in supernatants. In addition, autophagy markers including LC3 and P62 were measured by ELISA. Results: Data showed that gallic acid was cytotoxic to cells (P < 0.05). Gallic acid modulated expression of miRNAs and down-regulated transcript levels of exosomal genes and up-regulated the HSP-70 gene in three cell lines (P < 0.05). The surface CD63/total CD63 ratio as well as acetylcholinesterase activity decreased in treated cells (P < 0.05). The protein level of LC3 was increased in three cell lines, while the expression of P62 increased in MCF-7 and MDA-MB-231 cancer cell lines. Conclusion: Together, gallic acid decreased the activity of the exosomal secretory pathway in breast cancer cell lines, providing evidence for its anti-cancer effects.

Protein Elicitor EsxA Induces Resistance to Seedling Blight and PR Genes Differential Transcription in Rice

Protein elicitors can induce plant systemic resistance to pathogens. In an earlier study, we cloned an EsxA gene from the plant growth-promoting rhizobacterium Paenibacillus terrae NK3-4 and expressed it in Pichia pastoris. In addition to being important for the pathogenicity of animal pathogens, EsxA can also induce an immune response in animals. While, we found the exogenously expressed EsxA has the activity of elicitor, which can trigger hypersensitive response and reactive oxygen species burst in leaves as well as enhanced rice plant growth. The effects of EsxA on seedling blight (Fusarium oxysporum) resistance and gene transcription, including pathogenesis-related (PR) genes in rice were evaluated. The germination rate was 95.0% for seeds treated with EsxA and then inoculated with F. oxysporum, which was 2.8-times higher than that of F. oxysporum-infected control seeds that were not treated with EsxA (Con). The buds and roots of EsxA-treated seedlings were 2.4- and 15.9-times longer than those of Con seedlings. The plants and roots of seedlings dipped in an EsxA solution and then inoculated with F. oxysporum were longer than those of the Con seedlings. Theplant length, number of total roots, and number of white roots were respectively 23.2%, 1.74-times, and 7.42-times greater for the seedlings sprayed with EsxA and then inoculated with F. oxysporum than for the Con seedlings. The EsxA induction efficiency (spray treatment) on seedling blight resistance was 60.9%. The transcriptome analysis revealed 1137 and 239 rice genes with EsxA-induced up-regulated and down-regulated transcription levels, respectively. At 48 h after the EsxA treatment, the transcription of 611 and 160 genes was up-regulated and down-regulated, respectively, compared with the transcription levels for the untreated control at the same time-point. Many disease resistance-related PR genes had up-regulated transcription levels. The qPCR data were consistent with the transcriptome sequencing results. EsxA triggered rice ISR to seedling blight and gene differential transcription, including the up-regulated transcription of rice PR genes. These findings may be relevant for the use of EsxA as a protein elicitor to control plant diseases.

Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae

In insects, 20-hydroxyecdysone (20E) mediates developmental transitions and regulates molting processes through activation of a series of transcription factors. Broad-Complex (Br-C), a vital gene in the 20E signalling pathway, plays crucial roles during insect growth processes. However, whether Br-C affects chitin synthesis in insects remains unclear. In the present study, the Br-C gene from Lymantria dispar, a notorious defoliator of forestry, was identified based on transcriptome data, and subjected to bioinformatic analysis. The regulatory functions of LdBr-C in chitin synthesis and metabolism in L. dispar larvae were analysed by RNA interference (RNAi). The full-length LdBr-C gene (1431 bp) encodes a 477 amino acid (aa) polypeptide containing a common BRcore region (391 aa) at the N-terminus and a C-terminal Zinc finger domain (56 aa) harbouring two characteristic C2H2 motifs (CXXC and HXXXXH). Phylogenetic analyses showed that LdBr-C shares highest homology and identity with Br-C isoform 7 (83.12%) of Helicoverpa armigera. Expression profiles indicate that LdBr-C was expressed throughout larval and pupal stages, and highly expressed in prepupal and pupal stages. Furthermore, LdBr-C expression was strongly induced by exogenous 20E, and suppressed dramatically after application of dsLdBr-C. Bioassay results showed that knockdown of LdBr-C caused larval developmental deformity, significant weight loss, and a mortality rate of 67.18%. Knockdown of LdBr-C significantly down-regulated transcription levels of eight critical genes (LdTre1, LdTre2, LdG6PI, LdUAP, LdCHS1, LdCHS2, LdTPS and LdCHT) related to chitin synthesis and metabolism, thereby lowering the chitin content in the midgut and epidermis. Our findings demonstrate that Br-C knockdown impairs larval development and chitin synthesis in L. dispar.

Intron 1–Mediated Regulation of EGFR Expression in EGFR-Dependent Malignancies Is Mediated by AP-1 and BET Proteins

The epidermal growth factor receptor (EGFR) is overexpressed in numerous solid tumors and is the subject of extensive therapeutic efforts. Much of the research on EGFR is focused on protein dynamics and downstream signaling; however, few studies have explored its transcriptional regulation. Here, we identified two enhancers (CE1 and CE2) present within the first intron of the EGFR gene in models of glioblastoma (GBM) and head and neck squamous cell carcinoma (HNSCC). CE1 and CE2 contain open chromatin and H3K27Ac histone marks, enhance transcription in reporter assays, and interact with the EGFR promoter. Enhancer genetic deletion by CRISPR/Cas9 significantly reduces EGFR transcript levels, with double deletion exercising an additive effect. Targeted repression of CE1 and CE2 by dCas9-KRAB demonstrates repression of transcription similar to that of genomic deletion. We identify AP-1 transcription factor family members in concert with BET bromodomain proteins as modulators of CE1 and CE2 activity in HNSCC and GBM through de novo motif identification and validate their presence. Genetic inhibition of AP-1 or pharmacologic disruption of BET/AP-1 binding results in downregulated EGFR protein and transcript levels, confirming a role for these factors in CE1 and CE2. Our results identify and characterize these novel enhancers, shedding light on the role that epigenetic mechanisms play in regulating EGFR transcription in EGFR-dependent cancers. IMPLICATIONS: We identify critical constituent enhancers present in the first intron of the EGFR gene, and provide a rationale for therapeutic targeting of EGFR intron 1 enhancers through perturbation of AP-1 and BET in EGFR-positive malignancies.

Erythromycin Prevents Elastin Peptide-Induced Emphysema and Modulates CD4+T Cell Responses in Mice.

PurposeElastin peptides (EP) can induce lung inflammation and emphysema. Erythromycin has been shown to decrease acute exacerbation frequency and delay lung function decline in chronic obstructive pulmonary disease patients and ameliorate emphysema in murine models; however, the mechanism remains unclear. We aimed to observe the preventive and immunomodulatory effects of erythromycin in a mouse model of EP-induced emphysema.MethodsIn the in vivo study, Balb/c mice were treated with EP intranasally on day 0, and then administered erythromycin (100 mg/kg) or vehicle orally on day 1, which was continued every other day. Mice exposed to cigarette smoke were used as an emphysema positive control. The severity of emphysema and inflammation in the lungs of EP-exposed mice with or without erythromycin treatment were observed on day 40 after EP administration. In the in vitro study, naïve CD4+T cells were isolated from healthy mice spleens and stimulated by EP with or without erythromycin incubation. Flow cytometry was used to measure the proportions of Th1, Th17, and Treg cells. ELISA was used to detect cytokine levels of IFN-γ, IL-17, IL-6, and TGF-β. Transcript levels of Ifnγ, IL17a, and Foxp3 were evaluated by qRT-PCR.ResultsAfter exposure to EP, Th1 and Th17 cell percentages and the levels of inflammatory cytokines increased in vivo and in vitro, while Treg cells decreased in vivo. Erythromycin reduced IFN-γ, IL-17, IL-6 inflammatory cytokines, MLI, and the inflammation score in the lungs of EP-exposed mice. In vitro, erythromycin also limited Th17 and Th1 cell differentiation and downregulated transcript levels of Ifnγ and IL17a in the EP-stimulated CD4+T cells.ConclusionThe Th1 and Th17 cell responses were increased in EP-induced emphysema. Prophylactic use of erythromycin effectively ameliorated emphysema and modulated CD4+T cells responses in EP-induced lung inflammation in mice.

Transcriptomic profiling of effects of emamectin benzoate on the pine wood nematode Bursaphelenchus xylophilus.

Emamectin benzoate (EB) has recently been successfully applied as a trunk injection for preventative control of the pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Here, a whole-organism transcriptomic analysis provides comprehensive insights into the adverse effects of EB on B. xylophilus. A large set of differentially expressed genes (DEGs) were found, demonstrating the antagonistic effects of EB on B. xylophilus embryonic and larval development, reproduction, nervous and motor systems, and pathogenesis. In toxicity assays with EB, the number of eggs laid, hatching rate, thrashing frequency, and developmental rate of B. xylophilus were significantly suppressed at low concentrations (0.1μg mL-1 ). Moreover, the transcriptional changes validated by real-time quantitative PCR showed downregulated transcript levels of the genes encoding pectate lyases, β-1,4-endoglucanases, and upregulated the genes encoding glutamate-gated chloride channel, γ-aminobutyric acid type β receptor, uridine 5'-diphospho-glucuronosyl transferase, ATP-binding cassette transporter. The potential responses of B. xylophilus to EB included the upregulation of several genes putatively contributing to oocyte protection, stem cell renewal, and xenobiotic degradation, implying the potential for drug resistance to develop. Our findings further our understanding of the effects of EB for managing the PWD and may help to improve the pesticide-use strategies for controlling B. xylophilus. © 2019 Society of Chemical Industry.

Genomic and transcriptional changes in response to pinene tolerance and overproduction in evolved Escherichia coli

α-Pinene is an important monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals and biofuels. Although an evolved strain Escherichia coli YZFP, which had higher tolerance to pinene and titer, has been successfully used to produce high levels of pinene, the pinene titer is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. Moreover, the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear. To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer, a comparative genomics and transcriptional level analyses combining with CRISPR activation (CRISPRa) and interference (CRISPRi) were carried out. The results show that the tolerance to pinene and the overproduction of pinene in E. coli may be associated with: 1) the mutations of the DXP pathway genes, the rpoA and some membrane protein genes, and their upregulations of transcription levels; and 2) the mutations of some genes and their downregulation of transcriptional levels. These comparative omics analyses provided some genetic modification strategies to further improve pinene production. Overexpression of the mutated cbpA, tabA, pitA, rpoA, sufBCDS, mutS, ispH, oppF, dusB, dnaK, dxs, dxr and flgFGH genes further improved pinene production. This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy.

Effects of NaCl stress on growth of Portulaca oleracea and underlying mechanisms

The purslane Portulaca oleracea L. is a promising crop species for agriculture in saline–alkali soils. Up to date, biochemical and molecular changes in purslane in response to salinity were seldom reported. To investigate biochemical effects of salinity on purslane, seedlings were treated with different concentrations of NaCl for up to 14 days. The results showed that treatments with 150 mM and 200 mM NaCl significantly decreased dry and fresh weight of seedlings, shoot and root length as well as leaflet number on main stem, demonstrating inhibition on purslane growth. Treatments with 100–200 mM NaCl significantly decreased net photosynthetic rate, increased intercellular CO2 concentration, MDA content and production rate of O2−, induced activities of SOD, POD and CAT, suggesting inhibition on photosynthesis and induction of oxidant stress. With longer exposure time, activities of antioxidant enzymes showed decreasing tendency, probably due to denaturation of proteins and damages of cell membrane. In treatments with 150 mM and 200 mM NaCl, Na+ content increased in purslane leaf, stem and root, which then decreased K+ content, probably due to membrane depolarization and regulation of ion channels. Moreover, transcriptome profiles in leaves were compared among treatments with 200 mM for 0, 2, 6, 12 and 24 h. The results suggested that saline treatments down-regulated transcription levels of genes involved in photosynthesis, energy metabolism, lignin biosynthesis and signaling transduction. Overall, the present study uncovered biochemical and molecular responses of purslane to salinity, which should be useful for agriculture of purslane on saline–alkali soils.