Fluorescent Protein DsRed Research Articles

Endothelial Cell-Specific Prolyl Hydroxylase-2 Deficiency Augments Angiotensin II-Induced Arterial Stiffness and Cardiac Pericyte Recruitment in Mice.

Endothelial prolyl hydroxylase-2 (PHD2) is essential for pulmonary remodeling and hypertension. In the present study, we investigated the role of endothelial PHD2 in angiotensin II-mediated arterial stiffness, pericyte recruitment, and cardiac fibrosis. Chondroitin sulfate proteoglycan 4 tracing reporter chondroitin sulfate proteoglycan 4- red fluorescent protein (DsRed) transgenic mice were crossed with PHD2flox/flox (PHD2f/f) mice and endothelial-specific knockout of PHD2 (PHD2ECKO) mice. Transgenic PHD2f/f (TgPHD2f/f) mice and TgPHD2ECKO mice were infused with angiotensin II for 4 weeks. Arterial thickness, stiffness, and histological and immunofluorescence of pericytes and fibrosis were measured. Infusion of TgPHD2f/f mice with angiotensin II resulted in a time-dependent increase in pulse-wave velocity. Angiotensin II-induced pulse-wave velocity was further elevated in the TgPHD2ECKO mice. TgPHD2ECKO also reduced coronary flow reserve compared with TgPHD2f/f mice infused with angiotensin II. Mechanistically, knockout of endothelial PHD2 promoted aortic arginase activity and angiotensin II-induced aortic thickness together with increased transforming growth factor-β1 and ICAM-1/VCAM-1 expression in coronary arteries. TgPHD2f/f mice infused with angiotensin II for 4 weeks exhibited a significant increase in cardiac fibrosis and hypertrophy, which was further developed in the TgPHD2ECKO mice. Chondroitin sulfate proteoglycan 4 pericyte was traced by DsRed+ staining and angiotensin II infusion displayed a significant increase of DsRed+ pericytes in the heart, as well as a deficiency of endothelial PHD2, which further promoted angiotensin II-induced pericyte increase. DsRed+ pericytes were costained with fibroblast-specific protein 1 and α-smooth muscle actin for measuring pericyte-myofibroblast cell transition. The knockout of endothelial PHD2 increased the amount of DsRed+/fibroblast-specific protein 1+ and DsRed+/α-smooth muscle actin+ cells induced by angiotensin II infusion. Knockout of endothelial PHD2 enhanced angiotensin II-induced cardiac fibrosis by mechanisms involving increasing arterial stiffness and pericyte-myofibroblast cell transitions.

Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

DNA Delivery to Intact Plant Cells by Casein Nanoparticles with Confirmed Gene Expression

AbstractThis research presents gene expression after DNA delivery into intact plant cells by protein nanoparticles. The DNA delivery is carried out by casein nanoparticles (CNPs). A plasmid harboring the red fluorescent protein DsRed sequence is absorbed to the CNPs surface by electrostatic interaction and served as a model DNA in this study, and its expression is monitored by the fluorescence of the DsRed protein. The zeta potential of the CNPs is tuned by altering the pH to obtain sufficient electrostatic interaction between the CNPs and the DsRed plasmid for successful DNA delivery into the cells of the model plant Nicotiana benthamiana. The CNPs are covalently modified with the green fluorescent dye 6‐Aminofluorescein (6‐AF) to determine their location in the plant. To assess the ability of the CNPs to deliver DNA into the cells, CNP/6‐AF/DsRed plasmid electrostatic conjugates are infiltrated into N. benthamiana leaves. Confocal fluorescence microscopy results showed successful intracellular and nucleus uptake of the conjugates at pH 4.5 and a concentration of 2 mg mL−1 at CNPs: DsRed plasmid ratio of 1:0.01. The successful gene expression is confirmed by RT‐PCR and qRT‐PCR. The first appearance of the emitted red signal of the DsRed protein is observed 24 h post‐infiltration.

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control Anopheles gambiae the mosquito vector of human malaria

BackgroundPopulation suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity.MethodsAmino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived.ResultsWhile Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible.ConclusionsBioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.

Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules

ABSTRACT In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

Expression of GFP and DsRed fluorescent proteins after gene electrotransfer of tumour cells in vitro

Fluorescent reporter genes are widely used to study the transfection of various types of primary cells and cell lines. The aim of our research was to investigate the expression dynamics of GFP and DsRed reporter genes individually and combined after gene electrotransfer of plasmids with two different electroporation protocols in B16F10 and CT26 cells in vitro. The cytotoxicity after gene electrotransfer of both plasmids was first determined. Second, the intensity of fluorescence and the percentage of cells transfected with both plasmids individually and in combination were monitored in real time. The results show that the percentage of viability after gene electrotransfer of plasmids using the EP2 pulses was significantly higher compared to the EP1 pulses. In contrast, the percentage of transfected cells and fluorescence intensity were higher after gene electrotransfer with the EP1 pulse protocol. Moreover, the percentage of transfected cells was higher and started earlier in the B16F10 cell line than in the CT26 cell line. However, fluorescence intensity was higher in CT26 cells. Co-expression of fluorescent proteins was achieved only in a small number of cells. In conclusion, this study elucidated some of the dynamics of reporter gene expression in cancer cell lines after gene electrotransfer.

Characterization of photosynthetic Bradyrhizobium sp. strain SSBR45 isolated from the root nodules of Aeschynomene indica

ABSTRACT We isolated a novel strain of Bradyrhizobium sp., SSBR45, from the nodulated roots of Aeschynomene indica and labeled it with Discosoma sp. red fluorescent protein (dsRED) or enhanced green fluorescent protein (eGFP) and determined its draft genomic sequence. The labeled SSBR45 stimulated the growth of A. indica markedly on a nitrogen-free medium, as observed by visualizing the fluorescent root nodules. The nodulated roots also exhibited high acetylene reduction activities. The SSBR45 genome included genes involved in nitrogen fixation, photosynthesis, and type IV secretion system; however, it did not consist of canonical nodABC genes and type III secretion system genes. SSBR45, a novel species of the genus Bradyrhizobium, consisted of an average nucleotide identity and average amino acid identity of 87% and 90%, respectively, with the closest strain B. oligotrophicum S58.

Intraplacental injection of AAV9-CMV-iCre results in the widespread transduction of multiple organs in double-reporter mouse embryos.

Adeno-associated virus serotype 9 (AAV9) has become a popular tool for gene transfer because of its ability to cross the blood-brain barrier and efficiently transduce genetic material into a variety of cell types. The study utilized GRR (Green-to-Red Reporter) mouse embryos, in which the expression of iCre results in the disappearance of Green Fluorescent Protein (GFP) expression and the detection of Discosoma sp. Red Fluorescent Protein (DsRed) expression by intraplacental injection. Our results demonstrate that AAV9-CMV-iCre can transduce multiple organs in embryos at developmental stages E9.5-E11.5, including the liver, heart, brain, thymus, and intestine. These findings suggest that intraplacental injection of AAV9-CMV-iCre is a viable method for the widespread transduction of GRR mouse embryos.

Development of a transgenic tissue visualization system in representatives of <i>Fabaceae</i>

Plant organisms are the objects of green biotechnology, which have been used by humans in various areas of life. The family of legumes (Fabaceae) that is being studied in this work is not an exception here. It is a very diverse family widespread throughout the globe.
 Barrel medic (Medicago truncatula) and common pea (Pisum sativum), members of the legume family, were selected in this study for the development of an vital imaging system for transgenic tissues. As a part of the work, we tested an imaging system with post-mortem staining based on the GUS reporter, as well as a system for vital detection of transgenic tissue with fluorescent proteins GFP and DsRed. At this stage, vital imaging systems based on betalain and anthocyanin staining of transgenic tissues are designed and currently being adapted to the model objects under study.
 The results of this study may be useful for subsequent attempts to solve the problem of the low efficiency of transformation of P. sativum. Moreover, such a system should be useful in the study of gene regulatory networks and factors that regulate the process of induction and subsequent development of somatic embryos. In addition, it can be used to track the dynamics of expression of genes of interest on living objects, while studying other fundamental and biotechnological processes.
 The study is supported by the Russian Foundation for Basic Research (20-016-00124).

Generating a construct harboring the rpb1 promoter for enhancing recombinant protein expression in the medicinal mushroom Cordyceps militaris

Cordyceps militaris is a valuable medicinal mushroom that contains numerous bioactive ingredients such as cordycepin, adenosine, pentostatin, polysaccharides, and carotenoids. However, the contents of these substances in wild-type strains of C. militaris are relatively low. Recently, the genome of C. militaris has been fully sequenced, and genes involved in the biosynthesis of beneficial constituents have been identified. Therefore, research on enhancing the biosynthesis of the bioactive compounds in this fungus by recombinant DNA technology has advantages. In the present study, the authors have successfully generated a binary vector with a T-DNA region carrying a construct regulated by the native rpb1 promoter that can enhance gene expression in C. militaris. The T-DNA structure from the binary vector was then transferred into the C. militaris genome by Agrobacterium tumefaciens-mediated transformation. Analysis of some fungal transformants by PCR using the specific primer pairs indicated that the T-DNA structure was successfully integrated into the fungal genome. Observation of the transformants under fluorescence microscopy confirmed the overexpression of the DsRed fluorescent protein under the regulation of the rpb1promoter. The binary vector carrying the rpb1 promoter constructed in this study can be employed to promote the expression of target genes in the medicinal mushroom C. militaris.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora

Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.

Simultaneous manipulation of multiple genes within a same regulatory stage for iterative evolution of Trichoderma reesei

BackgroundWhile there is growing interest in developing non-canonical filamentous fungi as hosts for producing secretory proteins, genetic engineering of filamentous fungi for improved expression often relies heavily on the understanding of regulatory mechanisms.ResultsIn this study, using the cellulase-producing filamentous fungus Trichoderma reesei as a model system, we designed a semi-rational strategy by arbitrarily dividing the regulation of cellulase production into three main stages-transcription, secretion, and cell metabolism. Selected regulatory or functional genes that had been experimentally verified or predicted to enhance cellulase production were overexpressed using strong inducible or constitutive promoters, while those that would inhibit cellulase production were repressed via RNAi-mediated gene silencing. A T. reesei strain expressing the surface-displayed DsRed fluorescent protein was used as the recipient strain. After three consecutive rounds of engineering, the cellulase activity increased to up to 4.35-fold and the protein concentration increased to up to 2.97-fold in the genetically modified strain.ConclusionsWe demonstrated that, as a proof-of-concept, selected regulatory or functional genes within an arbitrarily defined stage could be pooled to stimulate secretory cellulase production, and moreover, this method could be iteratively used for further improvement. This method is semi-rational and can essentially be used in filamentous fungi with little regulatory information.

Fluorescently labeled Pseudomonas syringae DC3000 and 1449b wild-type strains constitutively expressing either eGFP, eCFP, or dsRED.

Here we describe the generation of fluorescently labeled derivatives of the plant pathogen Pseudomonas syringae DC3000 and 1449b strains, with each derivative constitutively expressing either the enhanced green (eGFP), enhanced cyan (eCFP), or Discosoma sp. red (dsRED) fluorescent proteins. The fluorophore-expressing cassetes are stably located in a neutral locus in the chromosome, and its expression does not affect bacterial fitness, while allowing efficient detection by microscopy or flow cytometry. We have generated these strains as a complementary set of labeled strains to those previously generated in our laboratory, thus extending the range of applications.

Molecular Characterization and Functional Localization of a Novel SUMOylation Gene in Oryza sativa.

Simple SummaryThe small ubiquitin-related modifier genes regulate the function of the cellular proteins, which are associated with cell stress-tolerance. Identification and understanding the functional localization of these genes are very important to mitigate the stresses. In this study, we identified a novel small ubiquitin-related modifier gene and studied its functional localization in the cell. This new finding will be very valuable in increasing our understanding of the mechanism of stress-tolerance.Small ubiquitin-related modifier (SUMO) regulates the cellular function of diverse proteins through post-translational modifications. The current study defined a new homolog of SUMO genes in the rice genome and named it OsSUMO7. Putative protein analysis of OsSUMO7 detected SUMOylation features, including di-glycine (GG) and consensus motifs (ΨKXE/D) for the SUMOylation site. Phylogenetic analysis demonstrated the high homology of OsSUMO7 with identified rice SUMO genes, which indicates that the OsSUMO7 gene is an evolutionarily conserved SUMO member. RT-PCR analysis revealed that OsSUMO7 was constitutively expressed in all plant organs. Bioinformatic analysis defined the physicochemical properties and structural model prediction of OsSUMO7 proteins. A red fluorescent protein (DsRed), fused with the OsSUMO7 protein, was expressed and localized mainly in the nucleus and formed nuclear subdomain structures. The fusion proteins of SUMO-conjugating enzymes with the OsSUMO7 protein were co-expressed and co-localized in the nucleus and formed nuclear subdomains. This indicated that the OsSUMO7 precursor is processed, activated, and transported to the nucleus through the SUMOylation system of the plant cell.

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells.

A core challenge in the field of tissue engineering is the ability to establish pipeline workflows for the design and characterization of scaffold technologies with clinically translatable attributes. The parallel development of biomaterials and stem cell populations represents a self-sufficient and streamlined approach for establishing such a pipeline. In the current study, rat dental pulp stem cell (rDPSC) populations were established to assess functionalized polycaprolactone (PCL) constructs. Initial optimization and characterization of rDPSC extraction and culture conditions confirmed that cell populations were readily expandable and demonstrated surface markers associated with multi-potency. Subset populations were transduced to express DsRed fluorescent protein as a mechanism of tracking both cells and cell-derived extracellular matrix content on complex scaffold architecture. Thermoplastic constructs included reduced graphene oxide (rGO) as an additive to promote cellular attachment and were further modified by surface etching a weak acetic acid solution to roughen surface topographical features, which was observed to dramatically improve cell surface coverage in vitro. Based on these data, the modified rGO-functionalized PCL constructs represent a versatile platform for bone tissue engineering, capable of being applied as a standalone matrix or in conjunction with bio-active payloads such as DPSCs or other bio-inks.

Miltefosine enhances infectivity of a miltefosine-resistant Leishmania infantum strain by attenuating its innate immune recognition.

BackgroundMiltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency.Methodology/Principal findingsTo enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production.Conclusions/SignificanceDifferential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.

Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses’ furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases.

The hAT-family transposable element, hopper, from Bactrocera dorsalis is a functional vector for insect germline transformation

BackgroundThe hopper hAT-family transposable element isolated from the Oriental fruit fly, Bactrocera dorsalis, is distantly related to both the Drosophila hobo element and the Activator element from maize. The original 3120 bp hopperBd-Kah element isolated from the Kahuku wild-type strain was highly degenerate and appeared to have a mutated transposase and terminal sequences, while a second 3131 bp element, hopperBd-we, isolated from a white eye mutant strain had an intact transposase reading frame and terminal sequences consistent with function.ResultsThe hopperBd-we element was tested for function by its ability to mediate germline transformation in two dipteran species other than B. dorsalis. This was achieved by creating a binary vector/helper transformation system by linking the hopperBd-we transposase reading frame to a D. melanogaster hsp70 promoter for a heat-inducible transposase helper plasmid, and creating vectors marked with the D. melanogaster mini-white+ or polyubiquitin-regulated DsRed fluorescent protein markers.ConclusionsBoth vectors were successfully used to transform D. melanogaster, and the DsRed vector was also used to transform the Caribbean fruit fly, Anastrepha suspensa, indicating a wide range of hopper function in dipteran species and, potentially, non-dipteran species. This vector provides a new tool for insect genetic modification for both functional genomic analysis and the control of insect populations.

Modeling a synthetic aptamer-based riboswitch biosensor sensitive to low hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) concentrations.

RNA aptamers are relatively short nucleic acid sequences that bind targets with high affinity, and when combined with a riboswitch that initiates translation of a fluorescent reporter protein, can be used as a biosensor for chemical detection in various types of media. These processes span target binding at the molecular scale to fluorescence detection at the macroscale, which involves a number of intermediate rate-limiting physical (e.g., molecular conformation change) and biochemical changes (e.g., reaction velocity), which together complicate assay design. Here we describe a mathematical model developed to aid environmental detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using the DsRed fluorescent reporter protein, but is general enough to potentially predict fluorescence from a broad range of water-soluble chemicals given the values of just a few kinetic rate constants as input. If we expose a riboswitch test population of Escherichia coli bacteria to a chemical dissolved in media, then the model predicts an empirically distinct, power-law relationship between the exposure concentration and the elapsed time of exposure. This relationship can be used to deduce an exposure time that meets or exceeds the optical threshold of a fluorescence detection device and inform new biosensor designs.