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Mosquito E-20-Monooxygenase Gene Knockout Increases Dengue Virus Replication in Aedes aegypti Cells.

E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E2 … (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieve …

EZH2 Promotes Corneal Endothelial Cell Apoptosis by Mediating H3K27me3 and Inhibiting HO-1 Transcription.

PURPOSE: This paper aims to explore the molecular mechanism of Enhancer of Zeste Homolog 2 (EZH2)-mediated H3K27me3 in human corneal endothelial cells (HCEC) apoptosis by inhibiting Heme oxygenase-1 (HO-1) transcription to provide a potential target for the treatment of corneal apoptosis. METHODS: HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved

The Long Noncoding RNA H19 Promotes Fibrotic Processes in Lens Epithelial Cells.

PURPOSE: The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular me … nd H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anteri …

A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells.

At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon

Silencing E6/E7 Oncoproteins in SiHa Cells Treated with siRNAs and Oroxylum indicum Extracts Induced Apoptosis by Upregulating p53/pRb Pathways.

E6 and E7 human papillomavirus (HPV) oncoproteins play a significant role in the malignant transformation of infected cervical cancer cells … ) were evaluated using western blot analysis before and after E6/E7 small interfering RNAs (siRNAs) transfection. In addition, the E6/E7 mRNA expression levels were assessed with real-time (RT)-PCR. The present study showed that the OI extract effectively hindered the proliferation of SiHa cells and instigated increments of cas-3 and cas-9 expressions but decr …

The miR-410-5p /ITGA6 axis participates in the pathogenesis of recurrent abortion by regulating the biological function of trophoblast.

The purpose of this study was to determine the regulation of the miR-410-5p /ITGA6 axis on the biological functions of trophoblast cells and … ectively. Meanwhile, the molecular mechanism of ITGA6 regulation on trophoblast cells was explored. Transfection with miR-410-5p mimic or si-ITGA6 attenuated the proliferation, migration and invasion and induced apoptosis of HTR-8/SVneo cells. Transfection of sh-miR-410-5p promoted proliferation, migration and invasion, and weakened apoptosis of HTR-8/SVneo ce …

Characterization of exonic variants of uncertain significance in very long-chain acyl-CoA dehydrogenase identified through newborn screening.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from mutations in the ACADVL gene and … ning control or variant ACADVL coding sequence were transfected into the ACADVL null HEK293T. While transfection of control ACADVL restored VLCAD protein and enzyme activity, cells expressing the VLCAD Val283Ala mutant had 18% VLCAD enzyme activity and reduced protein compared to control. VLCAD Ile420Leu, Gly179Arg, and Gln406Pro produced protein comparable to …

hsa-miR-199a-3p Inhibits Motility, Invasiveness, and Contractility of Ovarian Endometriotic Stromal Cells.

It is suggested that aberrantly expressed microRNAs are involved in the pathogenesis of endometriosis. Our previous study demonstrated that … at in NESCs, whereas the expression of p21-activated kinase 4 (PAK4) mRNA was significantly higher. Transfection of hsa-miR-199a-3p inhibited the migration, invasion, and contractility of ECSCs via inhibition of PAK4 mRNA expression. PAK4 was confirmed to be the direct target of hsa-miR-199a-3p. Transfection of PAK4 small interfering RNA and the PAK4 inhibitor …

LncRNA GACAT1 targeting miRNA-149 regulates the molecular mechanism of proliferation, apoptosis and autophagy of oral squamous cell carcinoma cells.

To explore the effects of lncRNA GACAT1/miR-149 molecular axis on the proliferation, apoptosis, migration and autophagy of oral squamous cel … the proliferation and migration of OSCC cells and promoted apoptosis and autophagy (P < 0.01). The transfection of miR-149 inhibitor had the opposite effect. Knockdown of lncRNA GACAT1 and transfection with miR-149 inhibitor reversed the effect of GACAT1 silencing on OSCC cells. Inhibition of lncRNA GACAT1 can inhibit the proliferation and migration of OSCC c …

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lin … promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expressio …

A rough Brucella mutant induced macrophage death depends on secretion activity of T4SS, but not on cellular Txnip- and Caspase-2-mediated signaling pathway.

Brucella is a facultative intracellular bacterium, dividing into smooth- and rough-type Brucella. Smooth-type Brucella can dissociate into r … y, 15 reported T4SS effectors were evaluated in inducing macrophage death using over-expression and transfection methods, the results showed that 15 recombinant strains with over-expression of respective effector were not cytotoxicity. In addition, 10 effectors transfected individually, or co-transfected with five effectors barely induced macrophage death, sug …

MiR-103 inhibiting cardiac hypertrophy through inactivation of myocardial cell autophagy via targeting TRPV3 channel in rat hearts.

Cardiac hypertrophy is a common pathological change frequently accompanied by chronic hypertension and myocardial infarction. Nevertheless, … ecreased in neonatal rat ventricular myocyte transfected with miR-103, but increased by AMO-103. Co-transfection of the miR-103 with the luciferase reporter vector into HEK293 cells caused a sharp decrease in luciferase activity compared with transfection of the luciferase vector alone. The miR-103-induced depression of luciferase activity was rescued by an AM …

Hypoxia-inducible factor 1α from a high-altitude fish enhances cytoprotection and elevates nitric oxide production in hypoxic environment.

Hypoxia-inducible factors (HIFs) are master transcription factor regulating hypoxic responses in vertebrates. Species of Schizothoracine, a sub-family of cyprinidae, are highly endemic to the hypoxic Qinghai-Tibetan Plateau (QTP). What roles the HIFs play in hypoxic adaptation in the Schizothoracine fish is little known. In this study, the HIF-1α/B gene from Gymnocypris dobula (Gd) was characterized. The predicted protein for Gd-HIF-1α/B contains the main domains (bHLH, PAS, PAC, ODD, N-TAD, and C-TAD). Moreover, a specific mutation that the proline hydroxylation motif (LXXLAP) mutated into Px

Targeted Inhibitory Effect of Nasopharyngeal Carcinoma Cells by Hre2.Grp78 Chimeric Promoter Regulating Fusion Gene TK/VP3.

OBJECTIVE: To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells. METHODS: Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression.

Role of PERK-eIF2α signaling pathway in fetal male rats with hypospadias induced by di-n-butyl phthalate.

This study aims to explore the role of PERK-eIF2α signaling pathway in fetal male rats with hypospadias induced by maternal exposure to di-n … tly decreased (all P < 0.05). In addition, the apoptosis rate was increased in GT fibroblasts after transfection of PERK-siRNA with the increased P62 and reduced LC3-II/LC3-I ratio (all P < 0.05). Activation of PERK-eIF2α signaling pathway can influence the GT development of fetal male rats with hypospadias induced by DBP through activation of autophagy and in …

A Novel PSEN1 K311R Mutation Discovered in Chinese Families with Late-Onset Alzheimer's Disease Affects Amyloid-β Production and Tau Phosphorylation.

BACKGROUND: Presenilin-1 (PSEN1) is the most frequently mutated gene in familial Alzheimer's disease (AD), whereas only several novel mutations have been reported in China and functional studies were seldom conducted. OBJECTIVE: We describe a novel PSEN1 K311R mutation in two Chinese families with late-onset AD and its functional impact on amyloid-β protein precursor (AβPP) processing and tau phosphorylation. METHODS: The mutation was detected by direct sequencing of PSEN1 exon 9. HEK293 cells stably expressing wild-type APP695 (HEK293-APP695wt) were transfected with plasmids containing huma

The role of miR-320a and IL-1β in human chondrocyte degradation.

ObjectivesThis study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA).MethodsHuman cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furth

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… Cell transfection and treatment. … These vectors were co-transfected with miR-320a, ASO-miR-320a or their negative controls into cells using Lipofectamine 2000. After 48 hours of transfection, luciferase assays were carried out using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin). …

MicroRNA-1825 induces proliferation of adult cardiomyocytes and promotes cardiac regeneration post ischemic injury.

In mammals, proliferative capacity of cardiomyocytes is lost soon after birth, while zebrafish and other lower organisms like newts are know … d the direct targets of miR-199a, namely p16, Rb1, and Meis2 to be downregulated following miR-1825 transfection. However, miR-199a alone did not have similar proliferation inducing effects as miR-1825, indicating that miR-1825 works through multiple pathways and is a master regulator of cardiomyocyte proliferation. In addition, our in-vivo analysis in animal …

AMP010014A09 in Sus Scrofa Encodes an Analog of G Protein-Coupled Receptor 109A, Which Mediates the Anti-Inflammatory Effects of Beta-Hydroxybutyric Acid.

BACKGROUND: Hydroxy-carboxylic acid receptor 2 (HCA2, also called GPR109A) belongs to the G protein-coupled receptor (GPCR) family and is fo … T) and IPEC-J2 cells were detected with or without BHBA treatment and following silencing or stable transfection of the AMP+ gene. RESULTS: AMP+ in swine exhibited a high level of homology with HM74A in humans and PUMA-G in mice. BHBA inhibited the LPS-induced secretion of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and the inflammatory protein COX-2 …