Red Fluorescent Protein Expression Research Articles

Orthogonality of Redesigned tRNA Molecules with Three Stop Codons

Comprehensive SummaryThe ability to expand genetic code in living cells has emerged as a powerful method with diverse applications. Here, we design replacement of the anticodons of E. coli tRNAs that recognize codons for 20 natural amino acids, with three anti‐stop codons, resulting in a total of 60 engineered tRNA constructs. We test these constructs one by one in E. coli, and found that six tRNAsCUA (tyrV, serX, hisR, trpT, glnV and leuX), two tRNAsUCA (trpT and leuX) and one tRNAUUA (tyrV) allowed efficient expression of Red Fluorescence Protein (RFP) with the presence of a corresponding stop codon in frame. Furthermore, we exploit the mutual orthogonality of tRNASerCUA, tRNATrpUCA and tRNATyrUUA with corresponding stop codons and demonstrated that the tRNASerCUA and the tRNATrpUCA can provide dynamic range and low crosstalk. Finally, we show the TAG and TGA can not only be used as an “AND gate” circuit to control the translation of target gene, but also be used to control the translation of a prodeoxyviolacein (PDV) pathway and a reporter in parallel. Overall, this work provides flexible tools for translational control and holds great potential to promote synthetic biology studies.

Improvement of 2,3-butanediol production by dCas9 gene expression system in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been widely used in bioproduction. To produce a target product other than ethanol, ethanol production must be decreased to enhance target production. An ethanol non-producing yeast strain was previously constructed by knocking out pyruvate decarboxylase (PDC) genes in the ethanol synthetic pathway. However, glucose uptake by the ethanol-non-producing yeast strain was significantly decreased. In this study, dead Cas9 (dCas9) was used to reduce ethanol synthesis during 2,3-butanediol production without reduction of glucose. The binding site of guide RNA used to effectively suppress PDC1 promoter-driven red fluorescent protein expression by dCas9 was identified and applied to control PDC1 expression. The production of 2,3-butanediol rather than ethanol was improved in repetitive test tube culture. Additionally, ethanol production was decreased and 2,3-butanediol production was increased in the strain expressing dCas9 targeting the PDC1 promoter in the third round of cultivation, compared with the control strain.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, Prsp_7571 and Prsp_6124, showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a Prsp_7571-derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

Construction and Characterization of a Gradient Strength Promoter Library for Fine-Tuned Gene Expression in Bacillus licheniformis.

Bacillus licheniformis DW2 is an important industrial strain for bacitracin production, and it is also used for biochemical production, however, the lack of effective toolkit for precise regulation of gene expression hindered its application seriously. Here, a gradient strength promoter library was constructed based on bacitracin synthetase gene cluster promoter PbacA. First, different PbacA promoter variants were constructed via coupling PbacA with various 5'-UTRs, and expression ranges of 32.6-741.8% were attained among these promoters. Then, three promoters, PUbay (strong), PbacA (middle), and PUndh (weakest), were applied for red fluorescent protein (RFP) and keratinase expression assays, and these promoters were proven to have good universality for different proteins. Second, the promoter of bacitracin synthetase gene cluster was replaced by these three promoters, and bacitraicn titer was enhanced by 14.62% when PUbay was applied, which was decreased by 98.05% under the mediation of PUndh compared with that of the original strain DW2. Third, promoters PUbay, PUyvgO, and PUndh were selected to regulate the expression levels of critical genes that are responsible for pucheriminic acid synthesis, and pucheriminic acid yield was increased by 194.1% via manipulating synthetic and competitive pathways. Finally, promoters PUbay, PbacA, and PUndh were applied for green fluorescent protein (GFP) and RFP expression in Escherichia coli, and consistent effects were attained based on our results. Taken together, a gradient strength promoter library was constructed in this research, which provided an effective toolkit for fine-tuning gene expression and reprogramming metabolite metabolic flux in B. licheniformis.

Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair

Delivery of the CRISPR ribonucleoprotein (RNP) for homology-directed repair (HDR) has been challenging due to the low efficiency. Herein, we developed a Cas9 conjugate complex system which can induce efficient HDR editing with the use of a minimal amount of carrier material. Cas9 from Streptococcus pyogenes was purified and conjugated with low molecular weight polymer (LP). The Cas9-LP conjugates were complexed with single guide RNA (sgRNA) and donor DNA, which showed greatly enhanced internalization into cells compared to native Cas9 complexes, as well as a high extent of co-localization of Cas9 with sgRNA. The cytotoxicity of Cas9-LP complexes was evaluated, demonstrating low cytotoxicity compared to the conventional lipofectamine formulation. Finally, the treatment of Cas9-LP complexes to HEK293T reporter cell line expressing a mutant red fluorescent protein (RFP) results in efficient base correction of the RFP gene (up to 31%), leading to restoration of RFP expression and fluorescence. We anticipate that the current method can be widely used as a platform for efficient HDR editing via ‘minimal carrier-assisted’ delivery without the aid of any external physical stimuli, which can be potentially applied for in vivo and ex vivo editing of cellular targets.

Engineering a Bifunctional ComQXPA-PsrfA Quorum-Sensing Circuit for Dynamic Control of Gene Expression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for the production of amino acids and other chemicals. However, the engineering of C.glutamicum is inflexible due to the lack of dynamic regulation tools. In this study, a quorum sensing (QS) circuit and its modulated hfq-sRNA cassette were constructed, and the dynamic control of gene expression by these bifunctional circuits was researched. First, the ComQXPA-PsrfA QS system of Bacillus subtilis was harnessed and modified to create an upregulating QS circuit, in which the transcription of genes controlled by the PsrfA promoter may be promoted at high cell density. This QS circuit successfully activated the expression of green fluorescent protein (GFP) to 6.35-fold in a cell density-dependent manner in C.glutamicum. Next, the hfq-sRNA-mediated downregulating circuit under the control of the ComQXPA-PsrfA QS system was established, and the expression of GFP was autonomously repressed by 96.1%. Next, to fine-tune these two QS circuits, a library of synthetic PsrfA based promoters was constructed, and a series of mutant PsrfAM promoters with 0.4-1.5-fold strength of native PsrfA were selected. Subsequently, the ComQXPA-PsrfAM QS circuit was utilized to upregulate the expression of red fluorescent protein, and the same QS-based hfq-sRNA system was utilized to downregulate the expression of GFP simultaneously. Last, this bifunctional ComQXPA-PsrfAM QS circuit was verified again by fine-tuning the expression of α-amylase. Therefore, the engineered ComQXPA-PsrfAM QS cassette can be applied as a novel bifunctional QS circuit to flexibly control gene expression in C.glutamicum.

Construction of an Integrated mCherry Red Fluorescent Protein Expression System for Labeling and Tracing in Lactiplantibacillus plantarum WCFS1.

Thorough intestinal adhesion and colonization greatly promote the probiotic properties of lactic acid bacteria (LAB). Labeling and tracing with fluorescent proteins are effective and reliable for studying the in vivo physiological activities of LAB including localization, adhesion, and colonization. Lactiplantibacillus plantarum WCFS1 was successfully traced with a red fluorescent protein (RFP), which was expressed by the bacteria-carrying recombinant plasmids. In this study, we aimed to construct a stable RFP mCherry expression system, whose encoding gene was integrated into the bacterial chromosome via double-crossed homologous recombination, and use it for labeling WCFS1 with the goal of avoiding the potential loss of non-chromosomal plasmids along with intestinal growth. First, the constitutive expression of the mCherry protein was improved after adjusting the length of the spacer between the promoter and the gene start codon. Then, the optimized mCherry gene expression cassette was integrated into the chromosome of WCFS1. The resulting strain had normal unimpaired growth and strong fluorescent signals, even after 100 generations, indicating its stability. Furthermore, quantitative polymerase chain reaction (PCR) results revealed a strong positive correlation between the fluorescence intensity of the strain and the number of viable cells, demonstrating its potential usage for the quantification of in vivo WCFS1 cells. Finally, the increased adhesion ability of WCFS1 due to the recombinant expression of the bsh gene was visualized and evaluated using fluorescence intensity, the results of which were consistent with those obtained using the previously established quantification methods. These results suggest that the chromosomal-integrated mCherry labeling system can be extensively used to examine the distribution, colonization, and survival of LAB in vivo in order to determine the mechanism of its probiotic function.

Evolution of Reproductive Division of Labor - Lessons Learned From the Social Amoeba Dictyostelium discoideum During Its Multicellular Development.

The origin of multicellular life from unicellular beings is an epochal step in the evolution of eukaryotes. There are several factors influencing cell fate choices during differentiation and morphogenesis of an organism. Genetic make-up of two cells that unite and fertilize is the key factor to signal the formation of various cell-types in due course of development. Although ploidy of the cell-types determines the genetics of an individual, the role of ploidy in cell fate decisions remains unclear. Dictyostelium serves as a versatile model to study the emergence of multicellular life from unicellular life forms. In this work, we investigate the role played by ploidy status of a cell on cell fate commitments during Dictyostelium development. To answer this question, we created Dictyostelium cells of different ploidy: haploid parents and derived isogenic diploids, allowing them to undergo development. The diploid strains used in this study were generated using parasexual genetics. The ploidy status of the haploids and diploids were confirmed by microscopy, flow cytometry, and karyotyping. Prior to reconstitution, we labeled the cells by two methods. First, intragenic expression of red fluorescent protein (RFP) and second, staining the amoebae with a vital, fluorescent dye carboxyfluorescein succinimidyl ester (CFSE). RFP labeled haploid cells allowed us to track the haploids in the chimeric aggregates, slugs, and fruiting bodies. The CFSE labeling method allowed us to track both the haploids and the diploids in the chimeric developmental structures. Our findings illustrate that the haploids demonstrate sturdy cell fate commitment starting from the aggregation stage. The haploids remain crowded at the aggregation centers of the haploid–diploid chimeric aggregates. At the slug stage haploids are predominantly occupying the slug posterior, and are visible in the spore population in the fruiting bodies. Our findings show that cell fate decisions during D. discoideum development are highly influenced by the ploidy status of a cell, adding a new aspect to already known factors Here, we report that ploidy status of a cell could also play a crucial role in regulating the cell fate commitments.

N-Acetyl-D-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration.

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK–NudC–Lis1–dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein–NudC–Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography.

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli.

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.

Intron with transgenic marker (InTraM) facilitates high-throughput screening of endogenous gene reporter lines.

The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation.

Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

MCherry fusions enable the subcellular localization of periplasmic and cytoplasmic proteins in Xanthomonas sp.

Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.

Provision of rapid and specific ex vivo diagnosis of central nervous system lymphoma from rodent xenograft biopsies by a fluorescent aptamer.

Differentiating central nervous system (CNS) lymphoma from other intracranial malignancies remains a clinical challenge in surgical neuro-oncology. Advances in clinical fluorescence imaging contrast agents and devices may mitigate this challenge. Aptamers are a class of nanomolecules engineered to bind cellular targets with antibody-like specificity in a fraction of the staining time. Here, the authors determine if immediate ex vivo fluorescence imaging with a lymphoma-specific aptamer can rapidly and specifically diagnose xenografted orthotopic human CNS lymphoma at the time of biopsy. The authors synthesized a fluorescent CNS lymphoma-specific aptamer by conjugating a lymphoma-specific aptamer with Alexa Fluor 488 (TD05-488). They modified human U251 glioma cells and Ramos lymphoma cells with a lentivirus for constitutive expression of red fluorescent protein and implanted them intracranially into athymic nude mice. Three to 4 weeks postimplantation, acute slices (biopsies, n = 28) from the xenografts were collected, placed in aptamer solution, and imaged with a Zeiss fluorescence microscope. Three aptamer staining concentrations (0.3, 1.0, and 3.0 μM) and three staining times (5, 10, and 20 minutes) followed by a 1-minute wash were tested. A file of randomly selected images was distributed to neurosurgeons and neuropathologists, and their ability to distinguish CNS lymphoma from negative controls was assessed. The three staining times and concentrations of TD05-488 were tested to determine the diagnostic accuracy of CNS lymphoma within a frozen section time frame. An 11-minute staining protocol with 1.0-μM TD05-488 was most efficient, labeling 77% of positive control lymphoma cells and less than 1% of negative control glioma cells (p < 0.001). This protocol permitted clinicians to positively identify all positive control lymphoma images without misdiagnosing negative control images from astrocytoma and normal brain. Ex vivo fluorescence imaging is an emerging technique for generating rapid histopathological diagnoses. Ex vivo imaging with a novel aptamer-based fluorescent nanomolecule could provide an intraoperative tumor-specific diagnosis of CNS lymphoma within 11 minutes of biopsy. Neurosurgeons and neuropathologists interpreted images generated with this molecular probe with high sensitivity and specificity. Clinical application of TD05-488 may permit specific intraoperative diagnosis of CNS lymphoma in a fraction of the time required for antibody staining.

Study of the effects of 3.1 THz radiation on the expression of recombinant red fluorescent protein (RFP) in E. coli.

In recent years, many studies have been conducted to investigate the non-thermal effects of THz radiation on different organisms, but further studies are needed to fully elucidate the effects, especially on the molecular level. In this study, we explored the effects of at 3.1 THz radiation on protein expression in Escherichia coli (E. coli) using red fluorescent protein as a reporter molecule. After 8 hours of continuous THz irradiation of bacteria on LB (Luria-Bertani) solid plates at an average power of 33 mW/cm2 and 10 Hz pulse repetition frequency, we found that the plasmid copy number, protein expression and fluorescence intensity of bacteria from the irradiated area were 3.8-, 2.7-, and 3.3 times higher than in bacteria from the un-irradiated area, respectively. These findings suggest that plasmid replication changed significantly in bacteria exposed to 3.1 THz radiation, resulting in increased protein expression as evidenced by increased fluorescence intensity of the RFP reporter.

Detection of transformed endothelial cells associated with survived cardiomyocytes in sub‐endocardial region of infarct myocardium

Previous studies have shown that cardiomyocytes in the sub‐endocardial region of infarct myocardium survive from ischemic injury. The present study was undertaken to explore a possible mechanism by which these cardiomyocytes survive in response to myocardial ischemia. Wild type mice (C57BL/6J, male, 8–10 weeks), or endothelial cell‐specific red fluorescent protein (RFP) expression transgenic mice (Tie2CRETdTomato) were subjected to permanent ligation of left anterior descending of coronary artery along with sham‐operated controls in which the same surgery procedure was applied with an exception of the artery ligation. Pimonidazole was injected intravenously to define the hypoxic region, and lectin was injected to trace blood infiltration before sacrifice in all of the mice. The hearts were harvested on day one after the surgery and cut into serial sections for detection of cardiomyocytes and endothelial cells. The staining of pimonidazole showed that most of the infarct myocardium was under hypoxic condition except the sub‐endocardial region in which survived cardiomyocytes were detected. The RFP positive endothelial cells in the sub‐endocardial region also survived but displayed a suppressed expression of PECAM‐1 and VEGFR2. Correspondently, lectin infiltration analysis suggested blood leakage in the sub‐endocardial region although there was no blood infiltration in other regions of the infarct area. The results suggest that blood infiltration through the transformed endothelial cells in the sub‐endocardial region of infarcted myocardium would provide a non‐hypoxic microenvironment, contributing to the survival of cardiomyocytes in this region.Support or Funding InformationWest China Hospital, Sichuan UniversityThe changes of hypoxic microenvironment in the infarct myocardium region.Hypoxic condition (partial pressure of O2 was less than 10 mmHg) was marked with hpi (pimonidazole) at 1st day after ischemic injury. The blood infiltrations were reflected by FITC‐conjugated WGA through tail vein at 1st, 4th, 7th day after LAD ligation. Hypoxic region stained by pimonidazole (red), and nuclear labeled by DAPI (blue). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 1The change of blood supplement in the sub‐endocardial region.The blood infiltrations were completely reflected by Lectin (FITC‐conjugated WGA) through tail vein at 1st day after LAD ligation. Cardiomyocytes stained by TNNI3 (red), Blood infiltration labeled by FITC‐conjugated WGA (green), and nuclear labeled by DAPI (blue). Abbreviations: WGA, wheat germ agglutinin; DAPI, 4′, 6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 2

Expression and Characterization of a Bright Far-red Fluorescent Protein from the Pink-Pigmented Tissues of Porites lobata.

Members of the anthozoan green fluorescent protein (GFP) family display a diversity of photo-physical properties that can be associated with normal and damaged coral tissues. Poritid coral species often exhibit localized pink pigmentation in diseased or damaged tissues. Our spectral and histological analyses of pink-pigmented Porites lobata lesions show co-localization of bright red fluorescence with putative amoebocytes concentrating in the epidermis, suggesting an activated innate immune response. Here we report the cloning, expression, and characterization of a novel red fluorescent protein (plobRFP) from the pink-pigmented tissues associated with lesions on Porites lobata. In vitro, the recombinant plobRFP exhibits a distinct red emission signal of 614 nm (excitation maximum: 578 nm), making plobRFP the furthest red-shifted natural fluorescent protein isolated from a scleractinian coral. The recombinant protein has a high molar extinction coefficient (84,000 M-1 cm-1) and quantum yield (0.74), conferring a notable brightness to plobRFP. Sequence analysis suggests the distinct brightness and marked red shift may be inherent features of plobRFP's chromophore conformation. While plobRFP displays a tendency to aggregate, its high pH stability, photostability, and spectral properties make it a candidate for cell imaging applications and a potential template for engineering optimized RFPs. The association of plobRFP with a possible immune response furthers its potential use as a visual diagnostic and molecular biomarker for monitoring coral health.

Respiratory syncytial virus exhibits differential tropism for distinct human placental cell types with Hofbauer cells acting as a permissive reservoir for infection.

BackgroundRespiratory syncytial virus (RSV) is capable of transient viremia and extrapulmonary dissemination. Recently, this virus has been identified in fetal cord blood, suggesting the possibility of in utero acquisition in humans. Here, we assess permissivity and kinetics of RSV replication in primary human placental cells, examine their potential to transfer this infection to neighboring cells, and measure the inflammatory response evoked by the virus.Methods and findingsHuman placental villus tissue was collected immediately upon delivery and processed for isolation of placental cytotrophoblast, fibroblast, and macrophage (Hofbauer) cells. Isolated cells were infected with a recombinant RSV-A2 strain (rrRSV) expressing red fluorescent protein (RFP) and analyzed by fluorescence microscopy, Western blot, and quantitative PCR (qPCR). Based on RFP expression, rrRSV exhibited differential tropism for the three major placental cell types. Placental fibroblasts and Hofbauer cells were permissive and supported productive rrRSV replication. While infected cytotrophoblast cells expressed viral glycoprotein (G protein), only limited RSV replication was detected. Importantly, qPCR and fluorescence-focused unit assay revealed that the viral progeny remains trapped within infected Hofbauer cells for up to 30 days, with no release into surrounding media. Yet, Hofbauer cells passed the infection onto overlaid naïve 16HBE cells, suggesting contact-dependent trans-infection. Lastly, a significant increase in proinflammatory cytokines, particularly IL-6, TNF-alpha, and IFN-gamma was measured in the supernatant of infected Hofbauer cells by multiplex cytokine assay and conventional ELISA.ConclusionsThis study demonstrates that RSV can replicate in human placenta, exhibits differential tropism for distinct placental cell types, can be stored and transferred to neighboring cells by Hofbauer cells, and elicits an inflammatory response. It also supports the hypothesis that this respiratory virus can be vertically transferred to the fetus and potentially affect its development and the outcome of pregnancies.

Targeting transgene to seed resulted in high rate of morphological abnormalities of Camelina transformants

Abstract False flax (Camelina sativa L.) is currently under-exploited but highly promising oilseed crop. Combining Camelina’s attractive agronomic traits with its unprecedented ease for genetic engineering makes it an ideal plant chassis for biotechnology applications, in particular synthetic biology strategies. For targeted expression of transgene particularly to seeds requires identification and application of seed specific promoters. In the present study two cultivars of Camelina, namely Zuzana and Smilowska, were used for transformation at early flowering stage using the floral dip method. The plants were inoculated with Agrobacterium bearing a construct for expression of red fluorescent protein (RFP) under the control of the seed specific cruciferin promoter CRUC from Arabidopsis. Transgenic seeds and plants were identified on the basis of red fluorescence (RFP) and kanamycin resistance. Relatively high transformation efficiency of 8 % was achieved particularly for the cultivar Zuzana. However, many of regenerants exerted developmental deformations such as lack of shoot apical meristem, deformed or absent cotyledons, etc. Furthermore, the activity of the CRUC promoter was still active also in true leaves rendering this promoter as inappropriate for seed targeting of the transgene. Nevertheless, genetic transformation remains a tool for direct modulation of pathways for oil synthesis in oilseed crops.