Green Fluorescent Protein Gene Research Articles

P-031 Presence of HPV 16 and HPV 18 in spermatozoa and embryos of mice

Abstract Study question The clarification of Human Pappiloma Virus (HPV) internalization in spermatozoa and early preimplantation embryos. Summary answer Spermatozoa are able to internalize constructs of cloned high risk HPV viruses either as integrated or as episomal DNA. What is known already Human Pappiloma Virus (HPV) is a non-enveloped, circular, double stranded DNA virus. Persistent infection with oncogenic HPV genotypes triggers HPV DNA integration into the host genome, eventually leading to chromosomal damage accumulation and genome destabilization in infected cells. Living spermatozoa of almost all species are able to take up spontaneously exogenous DNA and internalize a part of it into their nucleus. Study design, size, duration Taking into account the capability of sperm cells to integrate exogenous DNA into their genome, we sought to clarify HPV internalization into sperm genome. A total of 26 sperm samples were studied. The control group included 97 embryos. As for the HPV 16 and HPV 18 group, a total of 89 embryos were studied. Participants/materials, setting, methods Sperm was incubated with plasmid vectors containing the complete genome of human HPV 16 and HPV 18 tagged with the green fluorescent protein (GFP) gene, to investigate HPV 16 and HPV 18 integration in mouse spermatozoa. Oocytes were in vitro fertilized with preincubated spermatozoa to investigate HPV 16 and HPV 18 potential transfer to mouse embryos. Main results and the role of chance Spermatozoa were able to internalize constructs of cloned high-risk HPV either as integrated or as episomal DNA. Constructs of cloned HPV can also be transferred to mouse embryos, through in vitro fertilization of the oocytes by mouse spermatozoa. Limitations, reasons for caution Viral DNA integration into the sperm nucleus could not be directly demonstrated, but nevertheless integration is evident by GFP gene expression. Wider implications of the findings This study highlights the possibility of viral DNA transmission to the early embryo via sperm, opening new perspectives on the effect of HPV in reproductive cells. HPV persistence may impair sperm parameters, suggesting caution in the use of these cells for assisted reproduction techniques or sperm banking. Trial registration number Not applicable

Defining an optimal control for RNAi experiments with adult Schistosoma mansoni

In parasites such as Schistosoma mansoni, gene knockdown by RNA interference (RNAi) has become an indispensable tool for functional gene characterization. To distinguish target-specific RNAi effects versus off-target effects, controls are essential. To date, however, there is still no general agreement about suitable RNAi controls, which limits the comparability between studies. To address this point, we investigated three selected dsRNAs for their suitability as RNAi controls in experiments with adult S.mansoni in vitro. Two dsRNAs were of bacterial origin, the neomycin resistance gene (neoR) and the ampicillin resistance gene (ampR). The third one, the green fluorescent protein gene (gfp), originated from jellyfish. Following dsRNA application, we analyzed physiological parameters like pairing stability, motility, and egg production as well as morphological integrity. Furthermore, using RT-qPCR we evaluated the potential of the used dsRNAs to influence transcript patterns of off-target genes, which had been predicted by si-Fi (siRNA-Finder). At the physiological and morphological levels, we observed no obvious changes in the dsRNA treatment groups compared to an untreated control. However, we detected remarkable differences at the transcript level of gene expression. Amongst the three tested candidates, we suggest dsRNA of the E. coli ampR gene as the most suitable RNAi control.

NIR-II window absorbing graphene oxide-coated gold nanorods and graphene quantum dot-coupled gold nanorods for photothermal cancer therapy

Abstract The graphene-based materials have been used as a potential coating material for nanoparticles due to their excellent passivation. Herein, we report for the first time the colloidal stability, photothermal profile, thermal stability, cytotoxicity, and photo-cytotoxicity of graphene quantum dots (GQDs) coupled with the second infrared window (NIR-II) absorbing gold nanorods (AuNRs/GQDs) and compare it to graphene oxide (GO)-coated NIR-II absorbing AuNRs (AuNRs/GO). The composites were achieved by electrostatic interaction of the GO or GQDs with AuNRs. The results revealed that (i) AuNRs/GQDs were more stable in the aqueous phosphate buffer and cell culture media than AuNRs/GO and AuNRs; (ii) GO enhanced the photothermal efficiency of the AuNRs, whereas GQDs reduced it; (iii) GQDs enhanced the photothermal stability of AuNRs than GO; (iv) both AuNRs/GO and AuNRs/GQDs were biocompatible with mouse colon carcinoma (C26) cell lines and malignant fibrous histiocytoma‐like, expressing a fusion of the luciferase and enhanced green fluorescent protein genes (KM-Luc/GFP) cell lines; and (v) photo-cytotoxicity of AuNRs/GO and AuNRs/GQDs conducted against C26 cell lines showed significantly improved cell death compared to laser irradiation alone; however, AuNRs/GO exhibited high photo-toxicity than AuNRs/GQDs. This study shows that AuNRs/GO and AuNRs/GQDs composites possess unique properties to improve AuNRs and be utilised in photothermal applications.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

Food-grade expression system of Lactobacillus plantarum using β-galactosidase small subunit as selection marker and lactose as screening condition

Lactobacillus plantarum, one of the most important and widely studied lactic acid bacteria, has been used in many fields. In recent years, the construction and application of food-grade expression systems in L. plantarum has shown significant advantages in the fields of food and medicine. In this study, a food-grade expression system of L. plantarum with β-galactosidase as a screening marker was constructed by exploring the complementary properties of the gene lacLM of β-galactosidase in L. plantarum. Firstly, L. plantarum knockout strains ΔlacM and ΔlacAlacM were constructed, and it was determined that the knockout strain ΔlacAlacM was endowed with the phenotype of being unable to metabolize lactose. The knockout strain ΔlacAlacM was selected as the food-grade host for the food-grade expression system. The lacM gene was further expressed by plasmid pMG36e and transformed into L. plantarum ΔlacAlacM. The complementary strain restored lactose utilization ability, and its biomass reached 87.3% of that of L. plantarum LP01. Based on this phenomenon, a food-grade vector pMG36m with lacM gene as a selection marker was obtained by removing the antibiotic resistance gene. The genetic stability test showed that the plasmid existence rate was still close to 100% after 98 generations of L. plantarum ΔlacAlacM/pMG36m. Subsequently, an expression vector pMG36m-gfp was constructed with green fluorescent protein gene (gfp) as the reporter gene. These results indicated that a novel food-grade expression system of L. plantarum with lacM gene as a selection marker and lactose as a screening condition was successfully established, laying a foundation for the application of L. plantarum.

Comparison of In-Frame Deletion, Homology-Directed Repair, and Prime Editing-Based Correction of Duchenne Muscular Dystrophy Mutations.

Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing (PE, PE2, and PE3)-based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74-77%), followed by HDR (21-24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.

Infection tracing and organ tropism of Siniperca chuatsi rhabdovirus expressing enhanced green fluorescent protein

Siniperca chuatsi Rhabdovirus (SCRV) is one of the important pathogens that infect mandarin fish. Recovery of recombinant viruses expressing fluorescent protein is an important method for virology research. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted between the leader and the N gene of the SCRV genome. The recombinant SCRV of rS3-LeNEGFP expressing the EGFP was rescued, and the virus titer was as high as 2 × 106 PFU/mL in EPC cells. The insertion of the EGFP gene in the genome of SCRV was stable. EGFP was expressed efficiently after 10 passages in EPC cells. In vitro growth characterization, pathogenicity and organ tropism in vivo were studied. Compared to the parental rS3, rS3-LeNEGFP was defective in vitro, but not attenuated in vivo. The mortality of red zebrafish caused by rS3-LeNEGFP was the same as the parental SCRV rS3. The earliest expression of EGFP was detectable at 2 h, and clear fluorescence spots appeared at 8 h post-infection in EPC cells. The liver, heart, and brain were the major organ targets of rS3-LeNEGFP in vivo, and the kidney, gill, and spermatocyte were also invaded. The organ infection in vitro was different from that in vivo. This study is of great significance for revealing the pathogenesis of SCRV and developing vaccines and antiviral drugs for SCRV in the future.

Rapid selection of potyviral cross-protection effective mutants from the local lesion host after nitrous acid mutagenesis.

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, alocal lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%-100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.

Optimization of Biolistic Transformation Parameters for <i>Nicotiana tabacum</i>

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species.

PO-02-244 AAV6-MEDIATED GFP GENE TRANSFER INDUCES TACHYARRHYTHMIAS IN A PORCINE MODEL OF AV BLOCK

Green fluorescent protein (GFP) is a reporter widely utilized to evaluate gene transfer efficiency and used as control in therapeutic overexpression strategies. Although it is frequently considered to be an innocuous protein, there is also evidence indicating interference with contractile function and electrophysiological properties, potentially leading to cardiac dilation and functional deterioration. Cognizant of these possible limitations, we considered GFP gene transfer to provide a valuable starting point to evaluate local gene transfer and its potential deleterious effects. To evaluate gene transfer efficiency and potential pro-arrhythmic effects in a porcine model of complete atrioventricular block (CAVB). A total of eight pigs were implanted with a pacemaker, after which a radiofrequency ablation CAVB was generated. Four weeks after induction of the model the animals received catheter-based intramyocardial injections of saline, AAV6-CMV-GFP or were not injected at all. All animals were then observed for four more weeks. Monitoring included weekly pacemaker interrogations. Ventricular arrhythmias were assessed by means of increase in maximum heart rates and pacemaker dependency. Four weeks post-ablation all pigs displayed maximal heart rates of 75 ± 16,9 bpm which notably increased in AAV6-CMV-GFP-injected animals two weeks post-treatment (240 ± 10 bpm; Figure A), but not in the control animals (n = 5, 78 ± 29,5 bpm; Figure A). Electronically paced beats showed a remarkable reduction in transduced animals two weeks post-injection (40 ± 37,36 %; Figure B). Although GFP is commonly used in gene transfer studies, our experiments reveal the occurrence of ventricular arrhythmias after treatment. Mechanistically, these arrhythmias could be triggered by local tissue damage stemming from a possible inflammatory response exacerbated by a reaction to the capsid and transgene. As a result, locally injected AAV6-CMV-GFP should not be used as a control transgene in large animal studies to validate cardiac gene therapies.

Study on the Promotion of hADSCs Migration and Chemotaxis by SDF-1.

The purpose of this study was to investigate the chemotaxis effect of stromal cell-derived factor-1 (SDF-1) on human adipose-derived stem cells (hADSCs). A lentivirus vector with the enhanced green fluorescent protein gene was constructed and transfected to hADSCs. A control group and an SDF-1 induction group were set to estimate the efficacy of SDF-1 in promoting hADSCs chemotaxis and migration. After 7 days of infection with hADSCs by enhanced green fluorescent protein lentivirus, the positive rate of fluorescence expression detected by flow cytometry was 100%. After the addition of SDF-1 induction, the invasion ability of hADSCs was enhanced. SDF-1 can promote hADSCs migration and chemotaxis, which may play a role in stem cell transplantation.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

Establishment of reverse genetics for genotype VII Newcastle disease virus and altering the cell tropism by inserting TMPRSS2 into the viral genome.

Newcastle disease (ND) is the most important infectious disease in poultry, which is caused by avian orthoavulavirus type 1 (AOAV-1), previously known as Newcastle disease virus (NDV). In this study, an NDV strain SD19 (GenBank accession number OP797800) was isolated, and phylogenetic analysis suggested the virus belongs to the class II genotype VII. After generating wild-type rescued SD19 (rSD19), the attenuating strain (raSD19) was generated by mutating the F protein cleavage site. To explore the potential role of the transmembrane protease, serine S1 member 2 (TMPRSS2), the TMPRSS2 gene was inserted into the region between the P and M genes of raSD19 to generate raSD19-TMPRSS2. Besides, the coding sequence of the enhanced green fluorescent protein (EGFP) gene was inserted in the same region as a control (rSD19-EGFP and raSD19-EGFP). The Western blot, indirect immunofluorescence assay (IFA), and real-time quantitative PCR were employed to determine the replication activity of these constructs. The results reveal that all the rescued viruses can replicate in chicken embryo fibroblast (DF-1) cells; however, the proliferation of raSD19 and raSD19-EGFP needs additional trypsin. We next evaluated the virulence of these constructs, and our results reveal that the SD19, rSD19, and rSD19-EGFP are velogenic; the raSD19 and raSD19-EGFP are lentogenic; and the raSD19-TMPRSS2 are mesogenic. Moreover, due to the enzymatic hydrolysis of serine protease, the raSD19-TMPRSS2 can support itself to proliferate in the DF-1 cells without adding exogenous trypsin. These results may provide a new method for the NDV cell culture and contribute to ND's vaccine development.

Transfection Efficacy and Cellular Uptake of Lipid-Modified Polyethyleneimine Derivatives for Anionic Nanoparticles as Gene Delivery Vectors.

Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI (Mw ∼ 1200 Da) via two different linkers, gallic acid (GA) and p-hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). 1H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC50) of lipophilic polymers to pDNA revealed increased BC50 with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.

Endophytic Phthalate-degrading Bacillus subtilis N-1-gfp colonizing in soil-crop system shifted indigenous bacterial community to remove di-n-butyl phthalate

Endophytic bacteria can degrade toxic phthalate (PAEs). Nevertheless, the colonization and function of endophytic PAE-degrader in soil-crop system and their association mechanism with indigenous bacteria in PAE removal remain unknown. Here, endophytic PAE-degrader Bacillus subtilis N-1 was marked with green fluorescent protein gene. Inoculated strain N-1-gfp could well colonize in soil and rice plant exposed to di-n-butyl phthalate (DBP) as directly confirmed by confocal laser scanning microscopy and realtime PCR. Illumina high-throughput sequencing demonstrated that inoculated N-1-gfp shifted indigenous bacterial community in rhizosphere and endosphere of rice plants with significant increasing relative abundance of its affiliating genus Bacillus than non-inoculation. Strain N-1-gfp exhibited efficient DBP degradation with 99.7% removal in culture solutions, and significantly promoted DBP removal in soil-plant system. Strain N-1-gfp colonization help plant enrich specific functional bacteria (e.g., pollutant-degrading bacteria) with significant higher relative abundances and stimulated bacterial activities (e.g., pollutant degradation) compared with non-inoculation. Furthermore, strain N-1-gfp displayed strong interaction with indigenous bacteria for accelerating DBP degradation in soil, decreasing DBP accumulation in plants and promoting plant growth. This is the first report on well colonization of endophytic DBP-degrader Bacillus subtilis in soil-plant system and its bioaugmentation with indigenous bacteria for promoting DBP removal.

Investigating the molecular mechanisms behind the transcriptional response in pressure adaptation.

Despite 80% of Earth's microbial biomass existing in high pressure habitats in the Deep Ocean and within the continental crust, the mechanisms that allow for survival and growth in these thermodynamically unfavorable environments are poorly understood. Moreover, pressure resistance in pathogenic bacteria commonly found in food, such as Listeria monocytogenes and Escherichia coli, has emerged as more foods are sterilized by high pressure, rather than high temperature. To understand pressure adaptation, we have begun studying the molecular mechanisms of transcriptional adaptation to pressure. One important pathway in the pressure response is the heat shock pathway. To compare the heat shock response to pressure and temperature, we generated green fluorescent protein (GFP) promoter fusions of the central sigma factor genes’ transcriptional promoters and those of their downstream effectors. Activity of these promoter fusions was evaluated after heat shock and after pressure shock using scanning number and brightness (sN&B) fluorescence microscopy to determine the absolute number of GFP molecules produced in single cells and compared to the absolute number of GFP molecules produced in no shock conditions. Assuming transcriptional bursting at these promoters arises from release of supercoiling tension, we calculated the burst size and burst frequency from the distribution of the number of GFP molecules produced in each cell under each condition. These studies were carried out in the mesophilic E. coli MG1655 and in a moderately piezophilic (pressure-loving) strain adapted from MG1655, AN62. Studying both strains allowed us to quantify how the transcriptional response to changes in hydrostatic pressure occurs in both mesophiles and piezophiles, providing a more holistic model of the pressure response and adaptation phenomena.

Development of fluorescence-labeled antibody for immune checkpoint inhibitor using engineered probiotics

Here, we developed a genetically modified lactic acid bacteria (gmLAB) that produces green fluorescent protein (GFP)-conjugating, anti-programmed death-ligand 1 (PD-L1) single-chain variable fragments (scFv) for use as an anti-cancer device that targets immune checkpoint molecules. Since PD-L1 plays a key role as an immune checkpoint molecule in the tumor microenvironment, inhibition and detection of PD-L1 are important in cancer research. The anti-PD-L1 scFv was designed based on atezolizumab, a humanized IgG1 monoclonal antibody, and integrated into a lactococcal GFP gene expression vector. Gene expression from the constructed gmLAB was confirmed by western blotting and GFP fluorescence. The ability of GFP-conjugating anti-PD-L1 scFv against the target antigen, PD-L1 protein, was shown using an enzyme-linked immunosorbent assay. Finally, the ability to recognize PD-L1-expressing tumor-cell lines was confirmed using flow cytometry and fluorescence microscopy. Our results suggest that the gmLAB could be applied to in vivo imaging in cancer as an affordable diagnostic/treatment tool.Graphical

Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results: Bands on Western Blots with 6×His-tag monoclonal antibody (primary antibody) and Goat anti-mouse IgG HRP (secondary antibody) showed the EGFP polyarginine and EGFP proteins were expressed in Saccharomyces cerevisiae INVSc1 at relatively low levels. The lyticase incubation time modification and administration of 3-5 kDa microfilter to concentrate increased the yield of isolated protein. Conclusions: The sufficient amount of protein isolation in S. cerevisiae can be achieved by using lyticase and sonicators combination for the lysis process.

Oct-4 induces cisplatin resistance and tumor stem cell-like properties in endometrial carcinoma cells

ObjectiveResearch has suggested that tumor-initiating tumor stem cells are derived from normal stem cells and that tumor cells undergo progressive de-differentiation to achieve a stem cell-like state. Tumor stem cells are characterized by high proliferation ability, high plasticity, expression of multi-drug resistance proteins, and the ability to seed new tumors. Octamer-binding transcription factor 4 (Oct-4) and its activation targets are overexpressed in the tumor stem cells of various types of tumors, and this expression is associated with the pathogenesis, development, and poor prognosis of tumors. The primary objective of this study was to test if a stably transfected with Oct-4 gene cell line, RL95-2/Oct-4, has the characteristics of tumor stem cells. Materials and methodsHuman endometrial carcinoma cells (RL95-2) were transfected with a plasmid carrying genes for Oct-4 and green fluorescent protein (GFP). The stably transfected cells, RL95-2/Oct-4, were selected using G418 and observed to express the GFP reporter gene under the control of the Oct-4 promoter. GFP expression levels of RL95-2/Oct-4 cells were measured using flow cytometry. The proliferation potential of cells was determined according to cumulative population doubling and colony-formation efficiency. Gene expression was analyzed using reverse transcription-polymerase chain reaction. ResultsRL95-2/Oct-4 cells not only exhibited increased expression of the three most important stem cell genes, Oct-4, Nanog, and Sox2, but also had increased expression of the endometrial tumor stem cell genes CD133 and ALDH1. Furthermore, enhanced expression of these genes in the RL95-2/Oct-4 cells was associated with higher colony-forming ability and growth rate than in parental RL95-2 cells. We also observed that cisplatin induced less cell death in RL95-2/Oct-4 cells than in RL95-2 cells, indicating that RL95-2/Oct-4 cells were more resistant to chemotherapeutic agents. ConclusionThe study findings contribute to investigate the effects of Oct-4 on tumor stem cell origins.