Transient Transfection Assays Research Articles

Two mutations on S2 subunit were critical for Vero cell tropism expansion of infectious bronchitis virus HV80.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2′ cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2′ cleavage site induced low-level cell–cell fusion, while H855Q substitution hindered cell–cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

Induction of the human CDC45 gene promoter activity by natural compound trans‑resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans‑resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'‑upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT‑PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'‑upstream 556‑bp of the CDC45 gene was cloned and inserted into a multi‑cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation‑introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia

BackgroundIn recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold‑inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC).MethodsCCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot.ResultsOur results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)‑like population. Moreover, hyperthermia substantially improved the sensitivity of radiation‑resistant NPC cells and CSC‑like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti‑tumor‑killing activity of hyperthermia against NPC cells and CSC‑like cells, whereas ectopic expression of Cirbp compromised tumor‑killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC‑like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance.ConclusionTaken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

The Disruption of a Nuclear Export Signal in the C-Terminus of the Herpes Simplex Virus 1 Determinant of Pathogenicity UL24 Protein Leads to a Syncytial Plaque Phenotype.

UL24 of herpes simplex virus 1 (HSV-1) has been shown to be a determinant of pathogenesis in mouse models of infection. The N-terminus of UL24 localizes to the nucleus and drives the redistribution of nucleolin and B23. In contrast, when expressed alone, the C-terminal domain of UL24 accumulates in the Golgi apparatus; its importance during infection is unknown. We generated a series of mammalian expression vectors encoding UL24 with nested deletions in the C-terminal domain. Interestingly, enhanced nuclear staining was observed for several UL24-deleted forms in transient transfection assays. The substitution of a threonine phosphorylation site had no effect on UL24 localization or viral titers in cell culture. In contrast, mutations targeting a predicted nuclear export signal (NES) significantly enhanced nuclear localization, indicating that UL24 is able to shuttle between the nucleus and the cytoplasm. Recombinant viruses that encode UL24-harboring substitutions in the NES led to the accumulation of UL24 in the nucleus. Treatment with the CRM-1-specific inhibitor leptomycin B blocked the nuclear export of UL24 in transfected cells but not in the context of infection. Viruses encoding UL24 with NES mutations resulted in a syncytial phenotype, but viral yield was unaffected. These results are consistent with a role for HSV-1 UL24 in late cytoplasmic events in HSV-1 replication.

A streamlined guide RNA screening system for genome editing in Sorghum bicolor

BackgroundGenome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been developed for generating targeted mutations in plants. Although these tools hold promise for rapid crop improvement, target-specific guide RNAs exhibit variable activity. To improve genome editing, a rapid and precise method for evaluating their efficiency is necessary.ResultsHere we report an efficient system for screening single guide RNAs (sgRNAs) for genome editing in sorghum using a transient protoplast transfection assay. Protoplasts were isolated from leaves from sorghum plants cultivated under three different conditions. Cultivation for three days of continuous darkness following seven days with a 16-h light and 8-h dark photoperiod resulted in the highest yield of viable protoplasts and the highest protoplast transfection efficiency. We tested both plasmid-mediated and ribonucleoprotein-based delivery to protoplasts, via polyethylene glycol-mediated transfection, of CRISPR components targeting the sorghum genome. The frequencies of small insertions and deletions induced by a set of sgRNAs targeting four endogenous sorghum genes were analyzed via targeted deep sequencing. Our screening system induced indels in sorghum protoplasts at frequencies of up to 77.8% (plasmid) and 18.5% (RNP). The entire screening system was completed within 16 days.ConclusionsThe screening system optimized in this study for predicting sgRNA activity for genome editing in sorghum is efficient and straightforward. This system will reduce the time and effort needed for sorghum genome editing.

Antagonistic Regulation of ABA Responses by Duplicated Tandemly Repeated DUF538 Protein Genes in Arabidopsis.

The plant hormone ABA (abscisic acid) regulates plant responses to abiotic stresses by regulating the expression of ABA response genes. However, the functions of a large portion of ABA response genes have remained unclear. We report in this study the identification of ASDs (ABA-inducible signal peptide-containing DUF538 proteins), a subgroup of DUF538 proteins with a signal peptide, as the regulators of plant responses to ABA in Arabidopsis. ASDs are encoded by four closely related DUF538 genes, with ASD1/ASD2 and ASD3/ASD4 being two pairs of duplicated tandemly repeated genes. The quantitative RT-PCR (qRT-PCR) results showed that the expression levels of ASDs increased significantly in response to ABA as well as NaCl and mannitol treatments, with the exception that the expression level of ASD2 remained largely unchanged in response to NaCl treatment. The results of Arabidopsis protoplast transient transfection assays showed that ASDs were localized on the plasma membrane and in the cytosol and nucleus. When recruited to the promoter of the reporter gene via a fused GD domain, ASDs were able to slightly repress the expression of the co-transfected reporter gene. Seed germination and cotyledon greening assays showed that ABA sensitivity was increased in the transgenic plants that were over-expressing ASD1 or ASD3 but decreased in the transgenic plants that were over-expressing ASD2 or ASD4. On the other hand, ABA sensitivity was increased in the CRISPR/Cas9 gene-edited asd2 single mutants but decreased in the asd3 single mutants. A transcriptome analysis showed that differentially expressed genes in the 35S:ASD2 transgenic plant seedlings were enriched in several different processes, including in plant growth and development, the secondary metabolism, and plant hormone signaling. In summary, our results show that ASDs are ABA response genes and that ASDs are involved in the regulation of plant responses to ABA in Arabidopsis; however, ASD1/ASD3 and ASD2/ASD4 have opposite functions.

FZKA reverses gefitinib resistance by regulating EZH2/Snail/EGFR signaling pathway in lung adenocarcinoma

Ethnopharmacological relevanceFuzheng Kang-Ai (FZKA) decoction is mainly composed of 12 components with different types of herbs. In the last decade, FZKA has been used as an adjuvant treatment for lung cancer in clinical practice. Our previous studies have confirmed that FZKA shows a strong anti-cancer activity, significantly increases the clinical efficacy of gefitinib and reverses gefitinib resistance in non-small cell lung cancer (NSCLC). However, the molecular mechanism still needs to be further elucidated. Aim of the studyThe aim of this study was to investigate the role and mechanism by which FZKA inhibited the cell growth, proliferation and invasion of lung adenocarcinoma(LUAD) and reversed the acquired resistance of gefitinib for the therapy in LUAD. Materials and methodsCell viability assay and EDU assay were used for detecting of cell viability and cell proliferation. Transwell assay was performed to measure cell invasion. Western Blot and qRT-PCR were used for protein and gene expression test. The gene promoter activity was determined by dul-luciferase reporter assay. The in situ expression of protein was measured by cell immunofluorescence. Stabilized cell lines were established for stable overexpression of EZH2. Transient transfection assay was used for gene silence and overexpression. Xenograft tumors and bioluminescent imaging were used for in vivo experiments. ResultsFZKA significantly inhibited the cell viability, proliferation and cell invasion of LUAD, the combination of FZKA and gefitinib had a great synergy on the above processes. Moreover, FZKA significantly decreased EZH2 mRNA and protein expression, FZKA reversed the resistance of gefitinib by down-regulation of EZH2 protein. ERK1/2 kinase mediated the down-regulation of EZH2 reduced by FZKA. In addition, FZKA decreased the expression of Snail and EGFR by decreasing EZH2. Overexpression of Snail and EGFR significantly reversed the effect of FZKA-inhibited cell invasion and cell proliferation. More important, the combination of FZKA and gefitinib enhanced the inhibitory effect on EZH2, Snail and EGFR proteins. Furthermore, the growth inhibition and reversal of gefitinib resistance induced by FZKA were further validated in vivo. Finally, the expression and clinical correlation of EZH2,EGFR and Snail in cancer patients were further validated using bioinformatics analysis. ConclusionsFZKA significantly suppressed tumor progression and reversed gefitinib resistance by regulating the p-ERK1/2-EZH2-Snail/EGFR signaling pathway in LUAD.

TLR3 recognition of viral double-stranded RNA in human dental pulp cells is important for the innate immunity

Dental caries or trauma can expose human dental pulp cells (DPCs) to various oral microorganisms, which play an important role in the development of an innate immune response. In the present study, we examined the expression of Toll-like receptors (TLRs) for sensing microbe-associated molecular patterns in human DPCs. Interestingly, real-time PCR analysis demonstrated that TLR3 is the most highly expressed among 10 different TLRs in human DPCs. Poly(I:C), a representative TLR3 ligand mimicking viral double-stranded RNA, potently induced IL-8 expression in a time- and dose-dependent manner. Concordantly, poly(I:C) treatment substantially increased the expression of pro-inflammatory cytokines and chemokines such as IL-6, CCL2, and CXCL10. Human DPCs transfected with TLR3 siRNA exhibited decreased IL-8 production compared with non-targeting siRNA-transfected cells, suggesting that the expression of poly(I:C)-induced inflammatory cytokines is dependent on TLR3. IL-8 secretion induced by poly(I:C) was down-regulated by MAP kinase inhibitors, indicating that the MAP kinase pathway contributes to IL-8 production. Furthermore, C/EBPβ and NF-κB were essential transcriptional factors for poly(I:C)-induced IL-8 expression, as demonstrated by the transient transfection and reporter gene assay. Since lipoproteins are known as major immunostimulatory components of bacteria, human DPCs were treated with poly(I:C) together with Pam2CSK4, a synthetic lipopeptide mimicking bacterial lipoproteins. Pam2CSK4 and poly(I:C) co-treatment synergistically increased IL-8 production in comparison to Pam2CSK4 or poly(I:C) alone, implying that co-infection of viruses and bacteria can synergistically induce inflammatory responses in the dental pulp. Taken together, these results suggest that human DPCs potentially sense and respond to viral double-stranded RNAs, leading to effective induction of innate immune responses.

Fuziline Ameliorates Glucose and Lipid Metabolism by Activating Beta Adrenergic Receptors to Stimulate Thermogenesis.

Radix aconiti carmichaeli is a widely used traditional Chinese medicine that has been found to be effective in treating cardiovascular diseases and metabolic disorders. Patients with these diseases often experience a heat generation disorder, which is characterized by chilliness and can worsen the progression of the disease. This study established an in vitro screening model combining the examination of cellular mitochondrial membrane potential and mitochondrial temperature to screen drugs with thermogenic activity. After differentiation and determination of the content of characteristic metabolites of the drug-containing serum blood components, it was found that Fuziline (FZL) is the key thermogenic property in Radix aconiti carmichaeli, responsible for its thermogenic effects with a high relative importance of 33%. Experiments were conducted to evaluate the thermogenic activity of Radix aconiti carmichaeli and FZL in vivo by assessing temperature changes in various organs, including the rectum, liver, and brown adipose tissue. Moreover, the effects of intracellular β3-adrenergic receptor (β3-AR) agonistic effects were evaluated using transient β3-AR transfection and dual-luciferase assay systems. The molecular mechanism by which FZL promotes thermogenesis and improves mitochondrial function was investigated by verifying the β-adrenergic receptors (β-AR) downstream signaling pathway. The results suggest that FZL activates β-AR nonselectively, which in turn activates the downstream cAMP-PKA signaling pathway and leads to an increase in liver glycogenolysis and triglyceride hydrolysis, accompanied by enhancing mitochondrial energy metabolism. Consequently, the liver and brown adipose tissue receive energy to generate heat. In summary, these findings provide insight into the therapeutic application of Radix aconiti carmichaeli for metabolic disorders associated with heat generation disorders.

Chemico-Physical Properties of Some 1,1'-Bis-alkyl-2,2'-hexane-1,6-diyl-bispyridinium Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery.

The development of very efficient and safe non-viral vectors, constituted mainly by cationic lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the effect of the hydrophobic chain's length, we here report the synthesis, and the chemico-physical and biological characterization, of a new term of the homologous series of hydrogenated gemini bispyridinium surfactants, the 1,1'-bis-dodecyl-2,2'-hexane-1,6-diyl-bispyridinium chloride (GP12_6). Moreover, we have collected and compared the thermodynamic micellization parameters (cmc, changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA, MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence of a "tail" in the 288-320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data about the requirement of a proper length of the spacer to allow the molecule to form a sort of molecular tong able to intercalate DNA.

Transcription factors Dmrt1a, Foxl2, and Nr5a1a potentially interact to regulate cyp19a1a transcription in ovarian follicles of ricefield eel (Monopterus albus)

Aromatase (encoded by Cyp19a1) in the ovarian follicular cells catalyzes the production of estradiol from testosterone, which plays important roles in the ovarian development of vertebrates. In the present study, the interaction of Dmrt1, Foxl2, and Nr5a1a on the regulation of cyp19a1a transcription in ovarian follicles was examined in a teleost, the ricefield eel Monopterus albus. The expression of dmrt1a, foxl2, and nr5a1a was detected in ovarian follicular cells together with cyp19a1a at the mRNA and/or protein levels. Sequence analysis identified one conserved Foxo binding site in the proximal promoter region of ricefield eel cyp19a1a. Transient transfection assay showed that Foxl2 may bind to the conserved Foxo site to activate cyp19a1a transcription and act synergistically with Nr5a1a. Mutation of either the conserved Nr5a1 site or Foxo site abolished or significantly decreased the synergistic effects of Nr5a1a and Foxl2 on cyp19a1a transcription. The sequence between Region III and I-box of Nr5a1a was critical to this synergistic effect. Dmrt1a modulated the Foxl2- and Nr5a1a-induced activation of cyp19a1a transcription and their synergistic effects in a biphasic manner, with inhibitory roles observed at lower doses (10–50 ng) but release of the inhibition or even potentiating effects observed at higher doses (100–200 ng). Collectively, data of the present study suggest that the interaction of Dmrt1a, Foxl2, and Nr5a1a in the ovarian follicular cells may facilitate the adequate expression of cyp19a1a and the production of estradiol, and contribute to the development and maturation of ovarian follicles in ricefield eels and other vertebrates as well.

Low-dose phthalates promote breast cancer stem cell properties via the oncogene ΔNp63α and the Sonic hedgehog pathway

BackgroundThe omnipresence of human phthalate (PAE) exposure is linked to various adverse health issues, including breast cancer. However, the effects of low-dose PAE exposure on breast cancer stem cells (BCSCs) and the underlying mechanism remain unexplored. MethodsBCSCs from breast cancer cell lines (MDA-MB-231 and MCF-7) were enriched using a tumorsphere formation assay. Gene and protein expression was detected by measurement of quantitative real-time reverse transcription PCR, western blot, and immunofluorescence assays. Transient transfection assays were used to evaluate the involvement of Gli1, a signaling pathway molecule and ΔNp63α, an oncogene in influencing the PAE-induced characteristics of BCSCs. ResultsPAE (butylbenzyl phthalate, BBP; di-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) exposure of 10−9 M significantly promoted the tumorsphere formation ability in BCSCs. Breast cancer spheroids with a 10−9 M PAE exposure had higher levels of BCSC marker mRNA and protein expression, activated sonic hedgehog (SHH) pathway, and increased mRNA and protein levels of an oncogene, ΔNp63α. Furthermore, suppression of the SHH pathway attenuated the effects of PAEs on BCSCs. And the overexpression of ΔNp63α enhanced PAE-induced characteristics of BCSCs, while low expression of ΔNp63α inhibited the promotion effects of PAEs on BCSCs and the SHH pathway. ConclusionLow-dose PAE exposure promoted the stem cell properties of BCSCs in a ΔNp63α- and SHH-dependent manner. The influence of low-dose exposure of PAEs and its relevance for the lowest observed effect concentrations requires further investigation, and the precise underlying mechanism needs to be further explored.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 μg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

Genetic Diversity of the LTR Region of Polish SRLVs and Its Impact on the Transcriptional Activity of Viral Promoters.

A long terminal repeat (LTR) plays an indispensable role in small ruminant lentivirus (SRLV) gene expression. In this study, we present the LTR sequence of Polish SRLVs representing different subtypes, and analyzed their impact on SRLV promoter activity, as measured in transient transfection assays. Although certain nucleotide motifs (AML(vis), TATA box and the polyadenylation site (AATAAA)) were conserved across sequences, numerous mutations within the LTR sequences have been identified. Single nucleotide polymorphisms (SNPs) were detected in both regulatory (AP-1, AP-4, Stat and Gas) and non-regulatory sequences, and subtype-specific genetic diversity in the LTR region of Polish SRLVs was observed. In vitro assays demonstrated subtype-specific functional differences between the LTR regions of distinct SRLV subtypes. Our results revealed that the promoter activity of Polish strains was lower (1.64-10.8-fold) than that noted for the K1514 reference strain; however, the differences in most cases were not statistically significant. The lowest promoter activity was observed for strains representing subtype A5 (mean 69.067) while the highest promoter activity was observed for strain K1514 representing subtype A1 (mean 373.48). The mean LTR activities of strains representing subtypes A12, A17, A23, A18 and A24 were 91.22, 137.21, 178.41, 187.05 and 236.836, respectively. The results of the inter-subtype difference analysis showed that the promoter activity of strains belonging to subtype A5 was significantly lower than that for subtype A12 strains (1.32-fold; p < 0.00). The promoter activities of the A5 strain were 1.98-fold and 2.58-fold less active than that of the A17 and A23 strains, and the promoter activities of A12 strains were 1.955 and 1.5 times lower than the promoter activity of A23 and A17 strains, respectively. Furthermore, the promoter activity of A17 strains was 1.3 lower than the promoter activity of A23 strains. Our findings suggest that subtype-specific genetic diversity, mainly in the transcription factor's binding sites, has an impact on their transcriptional activity, producing a distinct activity pattern for the subtypes. This study provides new information that is important for better understanding the function of the SRLV LTR. However, further research including more strains and subtypes as well as other cell lines is needed to confirm these findings.

Characterization of the human &lt;i&gt;IDH1&lt;/i&gt; gene promoter

&lt;abstract&gt; &lt;p&gt;In cancer, the production of ATP depends mainly on glycolysis, usually accompanied by the dysfunction of the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Nicotinamide adenine dinucleotide (NAD&lt;sup&gt;+&lt;/sup&gt;) is a coenzyme for various biological enzymatic reactions such as those involved in the TCA cycle. To investigate the molecular mechanisms involved in carcinogenesis, the transcription system of genes associated with mitochondrial function should be elucidated. In this study, we isolated several mitochondrial function-associated bidirectional promoters and tested whether they responded to NAD&lt;sup&gt;+&lt;/sup&gt;-metabolism regulating compounds, namely, &lt;italic&gt;trans&lt;/italic&gt;-resveratrol (Rsv), 2-deoxy-D-glucose (2DG), 3-amino benzamide (3AB), and olaparib (OLA), in HeLa S3 cells. Transient transfection and luciferase (Luc) reporter assay showed that the &lt;italic&gt;IDH1&lt;/italic&gt; promoter was prominently activated by these compounds. The &lt;italic&gt;IDH1&lt;/italic&gt; gene, which encodes a nicotinamide adenine dinucleotide phosphate (NADP&lt;sup&gt;+&lt;/sup&gt;) dependent isocitrate dehydrogenase, is frequently mutated in glioma and leukemia cells. In this study, RT-PCR showed that &lt;italic&gt;IDH1&lt;/italic&gt; gene and protein expression was induced in response to the NAD&lt;sup&gt;+&lt;/sup&gt;-regulating drugs Rsv and 3AB. However, IDH1 protein amount was rather stable at control level. The result suggested that a post-transcriptional controlling system works to keep IDH1 at a stable level.&lt;/p&gt; &lt;/abstract&gt;

Effects of Caffeine, a DNA Damage Response Inhibitor, on Papillomavirus Genome Replication.

Epidemiological studies have revealed that caffeinated coffee imparts a reduced risk of oropharyngeal cancer, of which human papillomavirus (HPV) is one of the causative agents. Caffeine is a known inhibitor of the DNA damage response (DDR) pathway. We sought to test the effects of caffeine on the early replication of the HPV31 virus. It has been reported that the inhibition of several factors necessary for the DDR during the differentiation-dependent stage of HPV block genome amplification, while the HPV genome maintenance replication was unaffected. We first studied the effects of caffeine in the earliest stages of viral infection. Using pseudo-virions (PsV) expressing an m-Cherry reporter gene and quasi-virions (QsV) containing HPV31 genomes to mediate the infection, we found no evidence that caffeine impeded the viral entry; however, the infected cells displayed a reduced HPV copy number. In contrast, caffeine exposure increased the copy number of HPV31 episomes in the transient transfection assays and in the CIN612E cells that stably maintain viral episomes. There was a concomitant increase in the steady state levels of the HPV31 E1 and E2 transcripts, along with increased E2 loading at the viral origin of replication (ori). These results suggest that the caffeine-mediated inhibition of the DDR reduces viral genome replication in the early stage of infection, in contrast to the maintenance stage, in which the inhibition of the DDR may lead to an increase in viral amplicon replication.

New insights into the influence of NHX-type Cation/H+ antiporter on flower color in Phalaenopsis orchids

Vacuolar sodium/proton Na+(K+)/H+ exchanger (NHX) influence color formation because of their effects on cellular pH and Na+/K+ homeostasis. Research regarding NHXs has mainly focused on the vacuolar NHX family members. However, the NHX functions related to Phalaenopsis flower coloration remain relatively uncharacterized. In this study, we cloned and characterized PeNHX1, a vacuolar cation/H+ antiporter-encoding gene that is highly expressed in the Phalaenopsis equestris (orchid) flower lip. Phylogenetic and sequence analyses showed that PeNHX1 is a vacuolar NHX protein family member that is similar to other known vacuolar antiporters. The PeNHX1-GFP fusion protein was clearly localized to the vacuolar membrane in a transient transfection assay. A quantitative real-time PCR analysis revealed the increased expression of PeNHX1 in different flower developmental stages. Moreover, it was more highly expressed in the lip than in the other flower organs. On the basis of virus-induced gene silencing, we determined that decreased PeNHX1 expression significantly reduces P. equestris petal coloration. Furthermore, the overexpression of PeNHX1 in Phalaenopsis Big Chili caused the pH to increase and the petal color to change from red to blue. The results indicate that NHX1 may mediates the Na + or K+/H+ exchange, thereby regulating the vacuolar pH to promote blue coloration. This research provides a theoretical basis for the development of orchid varieties with blue flowers.

Molecular Pathogenesis of Colorectal Cancer: Impact of Oncogenic Targets Regulated by Tumor Suppressive miR-139-3p.

We recently determined the RNA sequencing-based microRNA (miRNA) expression signature of colorectal cancer (CRC). Analysis of the signature showed that the expression of both strands of pre-miR-139 (miR-139-5p, the guide strand, and miR-139-3p, the passenger strand) was significantly reduced in CRC tissues. Transient transfection assays revealed that expression of miR-139-3p blocked cancer cell malignant transformation (e.g., cell proliferation, migration, and invasion). Notably, expression of miR-139-3p markedly blocked RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation in CRC cells. A combination of in silico database and gene expression analyses of miR-139-3p-transfected cells revealed 29 putative targets regulated by miR-139-3p in CRC cells. RNA immunoprecipitation analysis using an Argonaute2 (AGO2) antibody revealed that KRT80 was efficiently incorporated into the RNA-induced silencing complex. Aberrant expression of Keratin 80 (KRT80) was detected in CRC clinical specimens by immunostaining. A knockdown assay using small interfering RNA (siRNA) targeting KRT80 showed that reducing KRT80 expression suppressed the malignant transformation (cancer cell migration and invasion) of CRC cells. Importantly, inhibiting KRT80 expression reduced AKT phosphorylation in CRC cells. Moreover, hexokinase-2 (HK2) expression was reduced in cells transfected with the KRT80 siRNAs or miR-139-3p. The involvement of miRNA passenger strands (e.g., miR-139-3p) in CRC cells is a new concept in miRNA studies. Our tumor-suppressive miRNA-based approach helps elucidate the molecular pathogenesis of CRC.

The secreted protein augurin is a novel modulator of canonical Wnt signalling involved in osteoblast differentiation

AbstractBackgroundECRG4/C2ORF40 is a tumour suppressor gene downregulated in several cancer types, which encodes the secreted protein augurin. A wide number of functions in health and disease have been assigned to augurin, but the signalling pathways it regulates are still poorly characterized. Augurin expression is strongly upregulated during in vitro differentiation of neonatal mouse osteoblasts.MethodsIn vitro differentiation assays of calvarial osteoblasts isolated from Ecrg4 ‐/‐ and wild‐type mice; transient transfection assays using reporters activated by Wnt signalling and other signal transduction pathways; Real‐time quantitative polymerase chain reaction for measurement of gene expression; protein expression in Chinese hamster ovary cells and Escherichia coli; in situ binding assays of proteins expressed as fusions to alkaline phosphatase with cells expressing various membrane receptors.ResultsOsteoblasts from Ecrg4 ‐/‐ mice have an accelerated differentiation compared to wild‐type and upregulation of Wnt markers. Augurin is a specific repressor of Wnt‐stimulated transcriptional activity, both when coexpressed together with the reporter and when added to the culture medium as a soluble protein. We confirmed the previously described binding of augurin to LOX1, a scavenger receptor, but an inhibitor of this molecule did not impair augurin repression of Wnt‐stimulated transcription specifically. Genome‐wide association studies showed an association of ECRG4 genomic variation with body height and osteoarthritis.ConclusionsOur study sheds new light on the wide spectrum of functions previously ascribed to augurin in brain function, stem cell biology, inflammation/immunity and cancer. Furthermore, our discovery paves the way to further characterization of the mechanisms involved in augurin repression of Wnt signalling and the development of agonists and antagonists for this protein, which have a wide array of potential applications in the clinic.

Regulator of G protein signaling 2 is inhibited by hypoxia-inducible factor-1α/E1A binding protein P300 complex upon hypoxia in human preeclampsia.

Preeclampsia is a pregnancy-related complication that causes maternal and fetal mortality. Despite extensive studies showing the role of hypoxia in preeclampsia progression, the specific mechanism remains unclear. The purpose of this study was to explore the possible mechanism underlying hypoxia in preeclampsia. Human trophoblast-like JEG-3 cell line was used to investigate the molecular mechanisms underlying hypoxia contribution to preeclampsia and the expression correlation of key molecules was examined in human placental tissues. Methods include JEG-3 cell culture and hypoxia induction, RNA isolation and quantitative real-time PCR, transient transfection and dual-luciferase assay, western blot, immunoprecipitation, immunofluorescence staining, cell proliferation assay, chromatin immunoprecipitation assay, obtainment of human placental tissue sample and immunohistochemistry staining. Hypoxia-Inducible Factor-1α is up-regulated in clinical preeclampsia samples, where Regulator of G Protein Signaling 2 is down-regulated. Mechanistically, Hypoxia-Inducible Factor-1α is induced in response to hypoxia, which up-regulates E1A binding protein P300 expression and thereby forms a Hypoxia-Inducible Factor-1α/E1A binding protein P300 protein-protein complex that binds to the promoter of gene Regulator of G Protein Signaling 2 and subsequently inhibits the transcription of Regulator of G Protein Signaling 2, possibly contributing to the preeclampsia development. In addition, the expression of E1A binding protein P300 is increased in preeclampsia samples, and the expression of Regulator of G Protein Signaling 2 in preeclamptic placentas inversely correlates with the levels of E1A binding protein P300. Our findings may provide novel insights into understanding the molecular pathogenesis of preeclampsia and may be a prognostic biomarker and therapeutic target for preeclampsia.