Gene Expression In Cultured Cells Research Articles

DNA origami drives gene expression in a human cell culture system

Self-assembling DNA nanoparticles have the potential to significantly advance the targeted delivery of molecular cargo owing to their chemical and architectural flexibility. Recently, it has been demonstrated that the genetic code embedded in DNA nanoparticles produced by the method of DNA origami or related techniques can be recognized and copied by RNA polymerase in vitro. Further, sculpted DNA nanoparticles can serve as a substrate for Cas9-mediated gene modification and gene expression in cell culture. In the present study, we further investigate the ability of DNA origami nanoparticles to be expressed in a human cell line with emphasis on the impact of single-stranded DNA (ssDNA) domains and the contributions of the architectural disposition of genetic control elements, namely promoter and enhancer sequences. Our findings suggest that while cells possess the remarkable capability to express genes within highly folded architectures, the presence and relative density and location of ssDNA domains appears to influence overall levels of gene expression. These results suggest that it may be possible to nuance folded DNA nanoparticle architecture to regulate the rate and/or level of gene expression. Considering the highly malleable architecture and chemistry of self-assembling DNA nanoparticles, these findings motivate further exploration of their potential as an economic nanotechnology platform for targeted gene editing, nucleic acid-based vaccines, and related biotherapeutic applications.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

Activation of Nrf2 by sulfuretin stimulates chondrocyte differentiation and increases bone lengths in zebrafish

Elongation of most bones occur at the growth plate through endochondral ossification in postnatal mammals. The maturation of chondrocyte is a crucial factor in longitudinal bone growth, which is regulated by a complex network of paracrine and endocrine signaling pathways. Here, we show that a phytochemical sulfuretin can stimulate hypertrophic chondrocyte differentiation in vitro and in vivo. We found that sulfuretin stabilized nuclear factor (erythroid-derived 2)-like 2 (Nrf2), stimulated its transcriptional activity, and induced expression of its target genes. Sulfuretin treatment resulted in an increase in body length of zebrafish larvae and induced the expression of chondrocyte markers. Consistently, a clinically available Nrf2 activator, dimethyl fumarate (DMF), induced the expression of hypertrophic chondrocyte markers and increased the body length of zebrafish. Importantly, we found that chondrocyte gene expression in cell culture and skeletal growth in zebrafish stimulated by sulfuretin were significantly abrogated by Nrf2 depletion, suggesting that such stimulatory effects of sulfuretin were dependent on Nrf2, at least in part. Taken together, these data show that sulfuretin has a potential use as supporting ingredients for enhancing bone growth.

US3 Serine/Threonine Protein Kinase from MDV-1, MDV-2, and HVT Differentially Regulate Viral Gene Expression and Replication.

Gallid alphaherpesvirus 2 (GaHV-2), commonly known as Marek’s disease virus type 1 (MDV-1), is an oncogenic avian alphaherpesvirus, and along with its close relatives—Gallid alphaherpesvirus 3 (GaHV-3) or MDV-2 and Meleagrid alphaherpesvirus 1 (MeHV-1) or turkey herpesvirus (HVT)—belongs to the Mardivirus genus. We and others previously showed that MDV-1 US3 protein kinase plays an important role in viral replication and pathogenesis, which could be partially compensated by MDV-2 and HVT US3. In this study, we further studied the differential roles of MDV-1, MDV-2 and HVT US3 in regulating viral gene expression and replication. Our results showed that MDV-2 and HVT US3 could differentially compensate MDV-1 US3 regulation of viral gene expression in vitro. MDV-2 and HVT US3 could also partially rescue the replication deficiency of MDV-1 US3 null virus in the spleen and thymus, as determined by immunohistochemistry analysis of MDV-1 pp38 protein. Importantly, using immunohistochemistry and dual immunofluorescence assays, we found that MDV-2 US3, but not HVT US3, fully compensated MDV-1 US3 regulation of MDV-1 replication in bursal B lymphocytes. In conclusion, our study provides the first comparative analysis of US3 from MDV-1, MDV-2 and HVT in regulating viral gene expression in cell culture and MDV-1 replication in lymphocytes.

Effects of Oxygen on Renal Function and Oxidative Stress During Hypothermic Machine Perfusion in an Experimental Porcine Model of Kidney Donation after Cardiac Death

The efficacy and safety of using high concentrations of oxygen during hypothermic machine perfusion (HMP) have not been fully elucidated to date. This study investigated the impact of administering high concentrations of oxygen on renal function during HMP in a porcine donation after circulatory death (DCD), as well as the metabolic and biochemical effects of this method. A randomized nonblinded cohort study was established in a porcine transplant (KT) model mimicking Maastricht type III DCD under oxygen-supplemented HMP (Ox-HMP) compared to non-supplemented (nOx-HMP) (LifePort® kidney transporter) conditions. The primary endpoint was evolution of renal function post-KT, whereas secondary endpoints included changes in perfusion dynamics, miRNA expression and cellular lesion measured by LDH and lactate levels in perfusate, lipid peroxidation in kidney biopsies, ATP generation, epithelial mesenchymal transition (EMT) and oxidative gene expression in cell cultures and histology evaluation. ATP generation and oxidative stress, as measured by lipid peroxidation, increased simultaneously after warm ischemia in the Ox-HMP group. Ox-HMP did not exhibit a significant effect on kidney function or animal survival. A significant increase in lipid peroxidation was observed in the Ox-HMP group. This resulted in a greater expression of the genes responsible for producing superoxide dismutase 1 (SOD-1) and catalase oxygenation enzymes, although only SOD-1 showed statistical significance. Respiratory chain dysfunction was maintained in the Ox-HMP group with a non-significant decrease in ATP production, increased proton leakage, and a decrease in respiratory reserve. Regarding epithelial-mesenchymal transition, an upward trend in the expression of vimentin, fibronectin, and collagen genes was observed only in the Ox-HMP group. Finally, the expression levels of miR-101 and miR-126, related to characteristic functions of the tubular epithelium, were significantly modified (miRNA levels expressed as DCT. A brief bubble Ox-HMP treatment did not show a clear positive effect on renal function and oxidative stress markers. The role and safety of adding oxygen during HMP still need to be elucidated. Currently, this Ox-HMP method cannot be considered standard practice.

Interleukin-8 gene expression and apoptosis induced by Salmonella Typhimurium in the presence of Bacillus probiotics in the epithelial cell.

This study aimed to evaluate the effects of three Bacillus probiotics on Salmonella Typhimurium, and interleukin-8 (IL-8) gene expression in the co-culture of the Bacillus and the pathogen in vitro. Bacillus subtilis, Bacillus indicus and Bacillus coagulans were initially turned to spore and heat-inactivated forms. The cellular damages of the probiotics on the HT-29 cells were investigated individually and in combination with S. Typhimurium using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorescence assays. To extract cell free supernatants (CFS) of the probiotics, they were cultured in selective media. The inhibitory activity of CFSs were then assayed against the pathogen. The gene expression of IL-8 of the HT-29 cells was evaluated by real-time PCR in all the groups. The results showed that the CFSs of three probiotics could inhibit the growth of S. Typhimurium by more than 50%. Inhibitory effects of B. indicus and B. subtilis CFSs were related to the production of pepsin-sensitive compounds, except B. coagulans in which the high inhibitory effect was due to organic acids. The spores of the three probiotics and the heat-inactivated forms of B. subtilis and B. coagulans could reduce the cytotoxicity of S. Typhimurium. The cell viability also increased applying both forms probiotics against the pathogen. In all co-culture groups, the IL-8 gene expression induced by S. Typhimurium was reduced. The three Bacillus probiotics can be considered as proper candidates for the prevention and treatment of S. Typhimurium food poisoning. Applying probiotics as live bacteria is universally noted in foods. This study tried to discover the effects of Bacillus probiotics in the form of spore or even heat-killed bacteria against S. Typhimurium and evaluate ratio of IL-8 gene expression in cell culture. The most effective Bacillus probiotic will be recommended. This approach will help to use probiotics as nonvegetative cells in foods to fight gastrointestinal pathogens.

CreLite: An optogenetically controlled Cre/loxP system using red light.

Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The cyclic recombinase (Cre)-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here, we present CreLite, an optogenetically controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multicolor fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multispectral cell labeling in several different tissues. Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models.

P3482Leptin, receptor to leptin and expression in adipocytes isolated from different adipose depots in patients with ischemic heart disease

Abstract Purpose To study the peculiarities of the content of leptin and the leptin receptor in adipocyte culture of subcutaneous (SAT), epicardial (EAT) and perivascular (PVAT) adipose tissue and the expression of their genes. Methods 29 patients with coronary heart disease were examined. Adipocytes were isolated from SAT, EAT and PVAT samples obtained during coronary artery bypass grafting, followed by evaluation (after 24 hours) of the leptin concentration (Human resistin ELISA, R&D Systems, USA) and leptin gene expression in cell culture. Magnetic resonance imaging (MRI) was used to determine the area of the visceral (VAT) and subcutaneous adipose tissue (SAT) of the abdominal region, the thickness of epicardial adipose tissue (EAT). The calculation of the free leptin index (FLI) was determined by the formula: leptin/SOB-R * 100. Leptin resistance was defined as an FLI >0.25. All study was carried out in compliance with the Helsinki Declaration, and its protocol was approved by the Ethical Committee of Research Institute. Statistical analysis was performed using Statistica 10.0. Results In adipocytes of EAT, leptin secretion was 1.2 times higher than in adipocytes of PVAT, which is consistent with the expression of the leptin gene. The content of leptin soluble receptor was opposite in the adipocytes of PVAT higher than in EAT and SAT 2.3 and 3.1 times, respectively. At the same time, the index of free leptin was higher in SAT adipocytes 1.3 and 3.2 times in comparison with EAT and PVAT. The correlation analysis confirmed the relationship between EAT thickness and the serum concentrations of leptin. Thus, in patients with VO, the leptin concentration had a negative dependence on the thickness of the left and right ventricle (r=−0.28, p=0.02; r=−0.33, p=0.02, respectively). A negative dependence on the EAT thickness was also established for the FLI (r=−0.28; p=0.03). PVAT of the abdominal aorta is similar to the properties of visceral AT. An analysis of the possible relationship between perivascular adipocytes and adipokine exchange rates showed a direct relationship between the volume PVAT of the abdominal aortic ventricle and leptin (r=0.44, p=0.01), as well as the FLI (r=0.56; p=0.03). Conclusion Adipocytes SAT, EAT and PVAT differed in the level of secretion of leptin. In epicardial and, to a greater extent, in perivascular adipocytes, a protective mechanism against a high concentration of leptin was observed due to increased secretion of the soluble receptor to leptin. The high content of leptin and the low soluble receptor for leptin led to the development of leptin resistance, which was more pronounced in the culture of adipocytes SAT. Meanwhile, the EAT thickness was inversely related to the concentration of leptin and the FLI index (one of the main leptin-resistance markers).

Transcriptional Reprogramming and Inhibition of Tumor-propagating Stem-like Cells by EC-8042 in ERG-positive Prostate Cancer

BackgroundThe TMPRSS2-ERG gene fusion is the most frequent genetic rearrangement in prostate cancers and results in broad transcriptional reprogramming and major phenotypic changes. Interaction and cooperation of ERG and SP1 may be instrumental in sustaining the tumorigenic and metastatic phenotype and could represent a potential vulnerability in ERG fusion–positive tumors. ObjectiveTo test the activity of EC-8042, a compound able to block SP1, in cellular and mouse models of ERG-positive prostate cancer. Design, setting, and participantsWe evaluated the activity of EC-8042 in cell cultures and ERG/PTEN transgenic/knockout mice that provide reliable models for testing novel therapeutics in this specific disease context. Using a new protocol to generate tumor spheroids from ERG/PTEN mice, we also examined the effects of EC-8042 on tumor-propagating stem-like cancer cells with high self-renewal and tumorigenic capabilities. Outcome measurements and statistical analysisThe efficacy of EC-8042 was determined by measuring the proliferative capacity and target gene expression in cell cultures, invasive and metastatic capabilities in chick chorioallantoic membrane assays, and tumor development in mice. Significance was determined using statistical test. Results and limitationsEC-8042 blocked transcription of ERG-regulated genes and reverted the invasive and metastatic phenotype of VCaP cells. EC-8042 blocked the expansion of stem-like tumor cells in tumor spheroids from VCaP cells and mouse-derived tumors. In ERG/PTEN mice, systemic treatment with EC-8042 inhibited ERG-regulated gene transcription, tumor progression, and tumor-propagating stem-like tumor cells. ConclusionsOur data support clinical testing of EC-8042 for the treatment of ERG-positive prostate cancer in precision medicine approaches. Patient summaryIn this study, EC-8042, a novel compound with a favorable pharmacological and toxicological profile, exhibited relevant activity in cell cultures and in vivo in a genetically engineered mouse model that closely recapitulates the features of clinically aggressive ERG-positive prostate cancer. Our data indicate that further evaluation of EC-8042 in clinical trials is warranted.

Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency

ABSTRACTThe development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282–90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8+ T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

Effects of ultra-high dilutions of sodium butyrate on viability and gene expression in HEK 293 cells

Several recent studies reported the capability of high diluted homeopathic medicines to modulate gene expression in cell cultures. In line with these studies, we examined whether ultra-high dilutions (30C and 200C) of sodium butyrate (SB) can affect the expression levels of genes involved in acquisition of a senescence-associated secretory phenotype (SASP) in human embryonic kidney (HEK) 293 cells. Cell viability was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of TNF-α, interleukin (IL)-2, IL-4, IL-6 and IL-10 genes were determined by real-time PCR assay. Exposure to both 30C and 200C during 48h led to a significant decrease of the level of expression of TNF-α gene, while expression of IL-2 gene was increased when exposed to 30C, and expression of IL-10 gene was decreased when exposed to 200C. No changes in expression levels of all genes studied were observed in cells treated with both 30C and 200C remedies of SB during the 24h. Observed changes in gene expression levels after exposure to 30C and 200C remedies of SB during 48h suggest that extremely low concentrations of this agent can modulate the transcriptome of HEK 293 cells. These results are in line with findings from other studies confirming the ability of homeopathic remedies to modulate gene expression in cell cultures.

An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors.

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1: has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1: in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1: distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1: show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608-17. ©2015 AACR.

Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes.

The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. Cdk12 is, however, not essential for bulk mRNA transcription and cells lacking CDK12 function are viable and able to proliferate. Consistent with previous findings on the DNA damage and heat shock responses, it emerges that Cdk12 may be specifically required for stress activated gene expression. Transcriptome analysis revealed that antioxidant gene expression is compromised in flies with reduced Cdk12 function, which makes them oxidative stress sensitive. In addition to supporting Reactive Oxygen Species (ROS) induced gene activation, Cdk12 suppresses genes that support metabolic functions in stressed conditions. We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to “stress-defence mode” when challenged by external stress.

Fabrication and evaluation of Nb-containing amorphous calcium phosphate coating film on Ti

Event Abstract Back to Event Fabrication and evaluation of Nb-containing amorphous calcium phosphate coating film on Ti Kyosuke Ueda1, Takayuki Narushima1, Akiko Obata2, Toshihiro Kasuga2 and Kouetsu Ogasawara3 1 Tohoku University, Department of Materials Processing, Japan 2 Nagoya Institute of Technology, Department of Frontier Materials, Japan 3 Tohoku University, Institute of Development, Aging and Cancer, Japan Introduction: Calcium phosphate coating can potentially improve the bone compatibility of titanium (Ti). We previously reported an amorphous calcium phosphate (ACP) coating film fabricated using radiofrequency (RF) magnetron sputtering[1]. Evaluated in vivo, the ACP coating film showed high bioresorbability, and dissolution of this coating improved the bone biocompatibility of Ti[2]. Controlling the bioresorbability of the ACP coating film can extend its application to areas such as drug delivery systems (DDSs). It has been reported that niobium (Nb) stabilizes calcium phosphate glasses and decreases their resorption in simulated body fluids. Additionally, Nb ions enhance osteoblast differentiation and mineralization[3]. In this study, ACP coating films with various amounts of Nb were fabricated by RF magnetron sputtering. Subsequently, bonding strength between coating films and substrates, resorbability in simulated body fluid, and gene expression in cell culture were investigated. Materials and Methods: Coating films were fabricated by RF magnetron sputtering on mirror-polished commercially pure (CP) Ti and blasted Ti-6Al-4V substrates (10×10×1 mm) using sintered target consisting of calcium phosphate and Nb2O5. The coating films were analyzed by ICP, XRD, Raman spectroscopy, and XPS. The bonding strength between the coating film and substrate was measured by the pulling test. Amount of ions eluted from the coating film in Tris buffer solution was determined. Quantitative analysis of gene expression from SaOS-2 cells was conducted by real-time PCR. Results and Discussion: Nb content in coating films increased and Ca/P atomic ratio of coating films decreased with addition of Nb. It is speculated that Nb reached the substrate with higher probability compared with Ca and P during sputtering. Moreover, addition of Nb stabilized the ACP phase. Raman spectra of the Nb-containing ACP coating films showed that the addition of Nb caused an increase and decrease in NbO4 and (O-P-O)s(Q0) peak intensities, respectively. Thus, Nb probably acted as network former. Bonding strength between the coating film and blasted substrate was >60 MPa, which is greater than that recommended in ISO13779-2 (>15 MPa). Figure 1 shows the effect of Nb content in the coating film on amounts of eluted ions after immersion of coating films in Tris buffer solution for 86.4 ks. Total amount of eluted ions from the coating films decreased with an increase in Nb content of the coating films. It was considered that the formation of NbO4 structure was one of the reasons for suppressing ion elution. The amount of eluted Nb ion was constant, independent of Nb content in the coating films. Gene expressions such as ALP, COLI and TGF-β1 from Nb-containing ACP coating films were identical to that from non-coated substrate after a 7-day cell culture. Conclusion: Nb ions were act as network former in the ACP coating film and controlled the bioresorbability of ACP coating films. Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Effects of achilline on lipid metabolism gene expression in cell culture

Objective . Evaluation in vitro of the mechanisms of the hypolipidemic effect of sesquiterpene γ-lactone achilline in the hepatoma tissue culture (HTC). Materials and methods .The influence of sesquiterpene γ-lactone achilline and gemfibrozil (comparison drug) on the viability, lipid content and expression of key genes of lipid metabolism in the hepatoma tissue culture. The lipid content was assessed by fluorescent method with the vital dye Nile Red, the cell viability was assessed using MTT assay. Results. Cultivation of of cell cultures of rat’s hepatoma cell line HTC for 48 h with achilline in a concentration of from 0.25 to 1.0 mm and gemfibrozil from 0,25 to 0,5 mm did not change cell viability compared to control. In these same concentrations of the test substance reduced the lipid content in the cells, assessed by fluorescent method with the vital dye Nile Red. To study the mechanism of hypolipidemicaction of achillinedetermined the expression of key genes of lipid metabolism in cell culture lines HTC. The possible mechanism of hypolipidemic action of achilline can be attributed to the increased transport and oxidation of long-chain fatty acids in mitochondria, as evidenced by the increase in the gene expression of carnitine-palmitoyltransferase 2 (Cpt2). The decrease in cholesterol level may be due to increased synthesis of bile acids from cholesterol, due to increased gene expression of 7-alphahydroxylase (Cyp7a1). Conclusion. In cell cultures of rat’s hepatoma cell line HTC sesquiterpene γ-lactone achilline reduces the accumulation of lipids in cells, as evidenced by the decrease in the fluorescence of Nile Red, increased gene expression of the carnitine-palmitoyltransferase 2 (Cpt2) gene and 7-alpha-hydroxylase (Cyp7a1).

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

Clinical and immunological comparisons of therapeutic regimens for corneal infiltrates secondary to adenoviral keratoconjunctivitis

The problem with post-adenoviral corneal infiltrates is that they cause a significant and persistent decrease in visual function, while corticosteroids in monotherapy bring only temporary improvement. to perform a comparative evaluation of topical corticosteroids and 0.05% cyclosporine A efficacy in the treatment of post-adenoviral corneal infiltrates on the basis of clinical presentation and local cytokine status. The study involved two groups of patients after adenoviral keratoconjunctivitis: group 1 (25 patients, 45 eyes) were prescribed a diminishing regimen of corticosteroid eye drops for 12 weeks and corneal protectors; group 2 (24 patients, 42 eyes) received the same treatment as described above plus topical 0.05% cyclosporine A for 6 months. The follow-up period was from 6 to 12 months. Visual acuity measurements, biomicroscopy, and pneumatic tonometry were performed at months 1, 3, and 6. Local cytokine status was assessed by studying cytokine gene expression in cell culture from conjunctival scrapings and cytokines levels in the supernatant. The tests were done before the beginning of the treatment in both groups, at month 1 in group 1, at month 4 in group 2 (i.e. in a month after the cessation of dexamethasone) and also in a group of healthy volunteers (30 persons, 30 eyes). Long-term combined anti-inflammatory therapy with corticosteroids and 0.05% cyclosporine A in patients with post-adenoviral corneal infiltrates has yielded positive clinical results, including a persistent increase in visual acuity and complete resolution of corneal opacities (92.8%). In addition, we revealed a correlation between local cytokine status changes and clinical results. The proposed therapeutic regimen enabled complete suppression of residual interferon-α antiviral activity, an increase in interleukin-4 that regulates local humoral immunity, and a decrease (down to a complete suppression) in anti-inflammatory interleukin-2, which is responsible for activation of cell-mediated immunity, thus, resulting in resolution of the immune-mediated inflammation in the cornea.

MRNA Transfection of Mouse and Human Neural Stem Cell Cultures

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain

Manipulation of gene expression in the brain is fundamental for understanding the function of proteins involved in neuronal processes. In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection. We show that neurons accumulate these LNPs in an apolipoprotein E–dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs. Effective targeted knockdown was demonstrated by showing that intracortical delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor (NMDAR)) selectively reduced synaptic NMDAR currents in vivo as compared with synaptic AMPA receptor currents. Therefore, LNP delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling the development of gene therapies for neurological disorders.