Levels Of Green Fluorescent Protein Research Articles

A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency.

Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term "endocytosis" from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automatedMATrix LABoratory(MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.

Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications

BackgroundPichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities.ResultsTwo novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in β-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage.ConclusionsTwo novel P. pastoris chassis hosts with impaired β-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

Effects of signal peptides on the secreted green fluorescent protein (GFP) expression by recombinant Saccharomyces cerevisiae

Recombinant proteins secreted by Saccharomyces cerevisiae have been widely applied in the food industry and biopharmaceuticals because of their valuable features. However, low-secreted protein production in S. cerevisiae is a pending challenge that has hampered applications based on S. cerevisiae. To improve the secreted protein expression in S. cerevisiae, it is important to develop an efficient cassette for the secretory expression of recombinant protein by S. cerevisiae. In this study, three cassettes were constructed in which a gene of interest was fused with genes coding for secretory signal peptides from AGA2, MFα and α-glucoamylase. Green fluorescent protein (GFP) was employed as a model protein to investigate the secretion of recombinant protein mediated by these signal peptides in S. cerevisiae. The secreted expression of GFP was detected by Western Blot and dot-blot analyses using a specific antibody against GFP which showed the strongest GFP levels from MFα signal-related cassette. Therefore, their cassettes could be employed for following studies on the secretion of some other proteins.

Oxidative stress strongly restricts the effect of codon choice on the efficiency of protein synthesis in Escherichia coli.

The response of enterobacteria to oxidative stress is usually considered to be regulated by transcription factors such as OxyR and SoxR. Nevertheless, several reports have shown that under oxidative stress the levels, modification and aminoacylation of tRNAs may be altered suggesting a role of codon bias in regulation of gene expression under this condition. In order to characterize the effects of oxidative stress on translation elongation we constructed a library of 61 plasmids, each coding for the green fluorescent protein (GFP) translationally fused to a different set of four identical codons. Using these reporters, we observed that GFP production levels vary widely (~15 fold) when Escherichia coli K-12 is cultured in minimal media as a consequence of codon choice variations. When bacteria are cultured under oxidative stress caused by paraquat the levels of GFP produced by most clones is reduced and, in contrast to control conditions, the range of GFP levels is restricted to a ~2 fold range. Restricting elongation of particular sequences does not increase the range of GFP production under oxidative stress, but altering translation initiation rates leads to an increase in this range. Altogether, our results suggest that under normal conditions the speed of translation elongation is in the range of the speed of initiation and, consequently, codon choice impacts the speed of protein synthesis. In contrast, under oxidative stress translation initiation becomes much slower than elongation, limiting the speed of translation such that codon choice has at most only subtle effects on the overall output of translation.

Citrus sudden death-associated virus as a new expression vector for rapid in planta production of heterologous proteins, chimeric virions, and virus-like particles

The more we understand the strategies used by viruses for protein expression, the more possibilities we have to exploit viruses as expression vectors for heterologous protein production. Advances in the development of virus-based expression systems have been possible due to generation of many virus infectious clones, especially those derived from plant viruses, which have the capability for rapid and high-level transient expression of proteins in plant cells, a robust and low-cost bioreactor. In this work, we generated new replicative virus expression vectors based on a previously constructed citrus sudden death-associated virus (CSDaV) infectious cDNA clone. These vectors were generated to express the reporter green fluorescent protein (GFP) in Nicotiana benthamiana leaves by taking advantage of the expression strategies used by CSDaV to produce its structural proteins. We show that higher amounts of GFP can be produced from a coat protein (CP)-independent CSDaV-based vector, compared to levels of GFP expressed from a widely used non-replicative vector (pEAQ series); or GFP can be produced in fusion with the major CSDaV CP (CPp21) to be incorporated into chimeric virions. However, GFP-recombinant CSDaV virions do not appear uniformly assembled, but more likely as mosaic particles. Cryo-electron microscopy analysis from this work revealed the structures of the wild-type and the GFP-recombinant CSDaV virions, but it was not able to reveal where exactly the GFP is displayed in the chimeric virions. We show though that the incorporation of GFP-CPp21 fusion protein into virions occurs solely due to its interaction with free/non-fused CPp21, independent of other viral proteins. Therefore, individual co-expression of GFP-CPp21 and CPp21 in the same plant cells leads to the production of chimeric virus-like particles (VLPs), while GFP-CPp21 fusion protein itself is not able to self-assemble into VLPs. The new CSDaV-based expression vectors may provide an alternative platform for use in molecular farming, either for production of heterologous proteins or as scaffold for heterologous protein display.

Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor.

Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.

OFF-switching property of quorum sensor LuxR via As(III)-induced insoluble form

Whole-cell sensors for arsenite detection have been developed exclusively based on the natural arsenite (As(III)) sensory protein ArsR for arsenic metabolism. This study reports that the quorum-sensing LuxR/Plux system from Vibrio fischeri, which is completely unrelated to arsenic metabolism, responds to As(III) in a dose-dependent manner. Due to as many as 9 cysteine residues, which has a high binding affinity with As(III), LuxR underwent As(III)-induced insoluble form, thereby reducing its effective cellular concentration. Accordingly, the expression level of green fluorescent protein under the control of Plux gradually decreased with increasing As(III) concentration in the medium. This is a novel As(III)-detection system that has never been proposed before, with a unique ON-to-OFF transfer function.

744 Multi-armed myxoma virus has therapeutic potential for treatment of multiple myeloma

BackgroundDespite improvements with new therapeutics, multiple myeloma (MM) patients still relapse and become refractory. Myxoma virus (MYXV) selectively replicates in human tumor cells and stimulates the immune system. MYXV selectively kills human patient MM cells and spares normal progenitors. MYXV also eradicates growth of a disseminated mouse MM in vivo. MYXV is a large, double stranded DNA poxvirus, and has a genome size amenable to insertion of multiple transgenes. We generated MYXV carrying IL-12 and decorin. IL-12 is an immune modulator that activates T- and NK-cells. Cellular responses to decorin include tumor cell intrinsic signaling effects, tumor matrix modulation, and inhibition of the TGF-beta pathway. This represents a promising therapeutic option for MM patients that do not respond well to immunotherapy. The current work suggests MYXV armed with IL-12 and decorin could be an effective anti-MM therapy.MethodsCytotoxicity assays were performed using a cell viability assay. Transgene expression levels were analyzed by microscopy, flow cytometry, and ELISA.ResultsHuman MM cell line U266 infected with MYXV (vMYX-hIL-12/Dec) carrying human IL-12, decorin, and green fluorescent protein (GFP) produced transgenes in a dose and time responsive manner. A panel of human MM cell lines was infected with vMYX-hIL-12/Dec and transgene expression in supernatant, cell killing EC50, and GFP levels were evaluated. Sensitive and resistant human MM cell lines were identified. The comparison of replication, cell killing capacity, and transgene expression highlighted the independent importance of these mechanisms in overall activity.ConclusionsThe current work describes the oncolytic activity and transgene expression following exposure to vMYX-hIL-12/Dec in human MM cell lines in vitro. Our initial studies suggest there is significant value in pursuing vMYX-hIL-12/Dec and other armed MYXV as a new approach towards MM therapy.

Characterization of endogenous promoters of GapC1 and GS for recombinant protein expression in Phaeodactylum tricornutum.

Although diatoms have been utilized as a cellular factory to produce biopharmaceuticals, recombinant proteins, and biofuels, only a few numbers of gene promoters are available. Therefore, the development of novel endogenous promoters is essential for the production of a range of bioactive substances. Here, we characterized the activities of endogenous promoters glyceraldehyde‐3‐phosphate dehydrogenase (GapC1) and glutamine synthetase (GS) of Phaeodactylum tricornutum using green fluorescent protein (GFP) under different culture conditions. Compared with the widely used fucoxanthin chlorophyll‐binding protein A (fcpA) promoter, the GS promoter constitutively drove the expression of GFP throughout all growth phases of P. tricornutum, regardless of culture conditions. Additionally, the GFP level driven by the GapC1 promoter was the highest at the log phase, similar to the fcpA promoter, and increased light and nitrogen‐starvation conditions reduced GFP levels by inhibiting promoter activity. These results suggested that the GS promoter could be utilized as a strong endogenous promoter for the genetic engineering of P. tricornutum.

A New Geniposidic Acid Derivative Exerts Antiaging Effects through Antioxidative Stress and Autophagy Induction.

Two compounds that can prolong the replicative lifespan of yeast, geniposidic acid (Compound 1) and geniposide (Compound 2), were isolated from Gardenia jasminoides Ellis. Compared with Compound 1, Compound 2 was different at C11 and showed better bioactivity. On this basis, seven new geniposidic derivatives (3–9) were synthesized. Geniposidic 4-isoamyl ester (8, GENI), which remarkably prolonged the replicative and chronological lifespans of K6001 yeast at 1 µM, was used as the lead compound. Autophagy and antioxidative stress were examined to clarify the antiaging mechanism of GENI. GENI increased the enzymes activities and gene expression levels of superoxide dismutase (SOD) and reduced the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) to improve the survival rate of yeast under oxidative stress. In addition, GENI did not extend the replicative lifespan of ∆sod1, ∆sod2, ∆uth1, ∆skn7, ∆cat, and ∆gpx mutants with K6001 background. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 was increased by GENI. The protein level of free GFP showed a considerable increase and was time-dependent. Furthermore, GENI failed to extend the replicative lifespans of ∆atg32 and ∆atg2 yeast mutants. These results indicated that antioxidative stress and autophagy induction were involved in the antiaging effect of GENI.

Microscopy deep learning predicts virus infections and reveals mechanics of lytic-infected cells

SummaryImaging across scales reveals disease mechanisms in organisms, tissues, and cells. Yet, particular infection phenotypes, such as virus-induced cell lysis, have remained difficult to study. Here, we developed imaging modalities and deep learning procedures to identify herpesvirus and adenovirus (AdV) infected cells without virus-specific stainings. Fluorescence microscopy of vital DNA-dyes and live-cell imaging revealed learnable virus-specific nuclear patterns transferable to related viruses of the same family. Deep learning predicted two major AdV infection outcomes, non-lytic (nonspreading) and lytic (spreading) infections, up to about 20 hr prior to cell lysis. Using these predictive algorithms, lytic and non-lytic nuclei had the same levels of green fluorescent protein (GFP)-tagged virion proteins but lytic nuclei enriched the virion proteins faster, and collapsed more extensively upon laser-rupture than non-lytic nuclei, revealing impaired mechanical properties of lytic nuclei. Our algorithms may be used to infer infection phenotypes of emerging viruses, enhance single cell biology, and facilitate differential diagnosis of non-lytic and lytic infections.

Involvement of the endocannabinoid system in the inhibition of Sindbis virus replication: a preliminary study

BackgroundSindbis virus (Alphaviridae) is a plus-strand RNA virus that is dependent on the host cell for replication. Cannabinoid (CB) receptors are found on most human cells, including virally infected cells. Activation of cannabinoid receptors has been shown to alter normal cellular physiology. This study aimed to assess how agonist (ACEA) or antagonists/inverse agonist (AM251) of the cannabinoid receptors would alter the cellular environment and impact Sindbis virus replication.MethodsHuman hepatoma (Huh7) cells were used as our model for viral replication. Cells were infected with Sindbis virus (SINV) and then treated with CB agonist (ACEA) (10 μM) or antagonist/inverse agonist (AM-251) (10 μM) and virus replication was monitored. A double subgenomic Sindbis virus containing a green fluorescent protein (GFP) reporter gene inserted into a 3′ subgenomic promoter was utilized for these assays to quickly measure viral replication. GFP fluorescent cells were analyzed using flow cytometry to measure the percentage of cells expressing the viral reporter and also quantify the levels of GFP fluorescence.ResultTreatment of SINV-infected Huh7 cells with CB1 receptor antagonist/inverse agonist (AM251, 10 μM) resulted in a significant decrease in viral replication, while infected cells treated with a CB1 receptor agonist (ACEA, 10 μM) resulted in a significant increase of viral infection. The data indicates that activation of CB1 receptor by cannabinoids significantly influences the ability of Sindbis virus to replicate in the host cell.ConclusionBlocking CB1 receptor activity with 10 μM AM251 reduced viral replication, but activating the CB1 receptor with 10 μM ACEA resulted in an increase in viral infection. These results indicate cannabinoids may significantly impact a virus replicating in human liver cells. Future confirmation with other viruses and cell lines will be performed to better understand the impact of cannabinoids on viral infections.

A Model System to Explore the Detection Limits of Antibody-Based Immuno-SPECT Imaging of Exclusively Intranuclear Epitopes.

Imaging of intranuclear epitopes using antibodies tagged to cell-penetrating peptides has great potential given its versatility, specificity, and sensitivity. However, this process is technically challenging because of the location of the target. Previous research has demonstrated a variety of intranuclear epitopes that can be targeted with antibody-based radioimmunoconjugates. Here, we developed a controlled-expression model of nucleus-localized green fluorescent protein (GFP) to interrogate the technical limitations of intranuclear SPECT using radioimmunoconjugates, notably the lower target-abundance detection threshold. Methods: We stably transfected the lung adenocarcinoma cell line H1299 with an enhanced GFP (EGFP)-tagged histone 2B (H2B) and generated 4 cell lines expressing increasing levels of GFP. EGFP levels were quantified using Western blot, flow cytometry, and enzyme-linked immunosorbent assay. An anti-GFP antibody (GFP-G1) was modified using dibenzocyclooctyne-N3-based strain-promoted azide-alkyne cycloaddition with the cell-penetrating peptide TAT (GRKKRRQRRRPPQGYG), which also includes a nuclear localization sequence, and the metal ion chelator N3-Bn-diethylenetriamine pentaacetate (DTPA) to allow radiolabeling with 111In. Cell uptake of 111In-GFP-G1-TAT was evaluated across 5 cell clones expressing different levels of H2B-EGFP invitro. Tumor uptake in xenograft-bearing mice was quantified to determine the smallest amount of target epitope that could be detected using 111In-GFP-G1-TAT. Results: We generated 4 H1299 cell clones expressing different levels of H2B-EGFP (0-1 million copies per cell, including wild-type H1299 cells). GFP-G1 monoclonal antibody was produced and purified in house, and selective binding to H2B-EGFP was confirmed. The affinity (dissociation constant) of GFP-G1 was determined as 9.1 ± 3.0 nM. GFP-G1 was conjugated to TAT and DTPA. 111In-GFP-G1-TAT uptake in H2B-EGFP-expressing cell clones correlated linearly with H2B-EGFP expression (P < 0.001). In vivo xenograft studies demonstrated that 111In-GFP-G1-TAT uptake in tumor tissue correlated linearly with expression of H2B-EGFP (P = 0.004) and suggested a lower target-abundance detection threshold of approximately 240,000 copies per cell. Conclusion: Here, we present a proof-of-concept demonstration that antibody-based imaging of intranuclear targets is capable both of detecting the presence of an epitope of interest with a copy number above 240,000 copies per cell and of determining differences in expression level above this threshold.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2weeks of severe hypoxia (PO2=35mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

Sox9EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Background & AimsDefining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types.MethodsSox9EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence.ResultsBECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFPhigh BECs. Single Sox9EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation.ConclusionsOur data demonstrate that Sox9EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

Development of fibrin hydrogel-based in vitro bioassay system for assessment of skin permeability to and pro-inflammatory activity mediated by zinc ion released from nanoparticles.

Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel-based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.

Development of an experimental method of systematically estimating protein expression limits in HEK293 cells

Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein’s expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins’ expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.

CCL2 affects fat metabolism in liver regeneration by regulating ADRP expression

This study aimed to elucidate the effect of chemokine (c-c motif) ligand 2 (CCL2) on fat metabolism in liver regeneration. CCL2 shRNA expression plasmids were constructed and transfected into rats using hydraulic transgenic technology. Transfection efficiency was measured using a fluorescence microscope. We also measured serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to test liver functioning. The weights of regenerative livers were recorded and the liver index was calculated. We used immunohistochemistry to determine the expression of PCNA, and used western blot to measure expression levels of adipose differentiation related protein (ADRP). After transfection of pGenesil-1.0-ccl2 into the liver, expression levels of green fluorescent protein were 35% at 6 h, and the liver index as well as levels of ALT, AST, PCNA, and ADRP were all lower than those in the group that underwent partial hepatectomy. We conclude that CCL2 may affect fat metabolism in liver regeneration by inhibiting the expression of ADRP.