Fluorescent Protein mCherry Research Articles

Application of the Fluorescent Dye BODIPY in the Study of Lipid Dynamics of the Rice Blast Fungus Magnaporthe oryzae.

Rice blast is one of the most serious diseases affecting rice yield which is caused by Magnaporthe oryzae, a model organism for studies on plant pathogenic fungi. Lipids stored in M. oryzae cells have been shown to be crucial for the development of appressorium turgor and the ability of the pathogen to cause infection. Nile red staining is a common method to study lipid dynamics in phytopathogenic fungi. However, the disadvantages of this dye include its wide spectrum, poor water solubility, and susceptibility to quenching. Boron dipyrromethene (BODIPY) is a new type of fluorescent dye that has a different emission wavelength to that of Nile red as well as many desirable spectral and chemical properties. In this study, we used BODIPY to stain the lipids in M. oryzae cells to seek a possible substitute to Nile red in the study of lipid dynamics in plant pathogenic fungi. Our data showed that through simple and routine procedures, BODIPY was able to distinctly label lipids in the cells of mycelia and conidia. The positions of lipids labeled by BODIPY were essentially identical to those labeled by Nile red, but with more clear fluorescence labelling, lower background, and higher specificity. The use of BODIPY to stain germinating M. oryzae conidia allowed the lipid dynamics to be clearly tracked during this process. We also achieved double and multiple fluorescent staining conidia by combining BODIPY with the red fluorescent protein mCherry and other fluorescent dyes, such as Calcofluor white and DAPI, in conidia, mycelia, and sexual structures of M. oryzae. These results indicate that BODIPY is an ideal fluorescent dye for staining fungal lipids and provide a method for the study of the lipid dynamics and lipid metabolism in plant pathogenic fungi.

Safety markers for rhabdomyosarcoma cells using an in�vivo imaging system

In vivo imaging system (IVIS) is a novel and rapidly expanding technology that is widely applied in life sciences, including cell tracing. IVIS is able to quantify biological events, including tumor proliferation, through counting the number of photons emitted from a specimen. PLA802-enhanced green fluorescent protein (EGFP), PLA802-monomeric cherry fluorescent protein (mCherry), RH30-EGFP and RH30-mCherry tumor cells were injected into 18 BALB/c female nude mice subcutaneously with 5×106 cells in 100 µl to quantitatively analyze EGFP and mCherry cells traced by IVIS. Inversion fluorescence microscopy revealed no transfection efficiency difference between PLA802-EGFP (95.3±1.2%) and PLA802-mCherry (95.8±1.7%), or between RH30-EGFP (94.7±2.1%) and RH30-mCherry (95.2±1.9%). Transfection did not influence the cell morphology of PLA802 or RH30. The cell migration, invasion and proliferation assay results of lentivirus-EGFP and lentivirus-mCherry revealed no significant difference prior to or following transfection. Therefore, lentivirus-EGFP and lentivirus-mCherry may serve as safety biological markers for PLA802 and RH30 cells. In vivo experiments demonstrated that lentivirus-EGFP and lentivirus-mCherry tumor luminescence signals were observed in all mice by IVIS. Hematoxylin-eosin staining and immunohistochemistry indicated that PLA802-EGFP, PLA802-mCherry, RH30-EGFP and RH30-mCherry cell lines exhibited rhabdomyosarcoma (RMS) characteristics like the maternal cells. In summary, mCherry and green fluorescent protein in human RMS PLA802 and RH30 cancer cells may be safely and stably expressed for a long time in vitro and in vivo.

Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy In Vitro.

Triple-negative breast cancer (TNBC) is one of the most aggressive types of cancer, and treatment is limited to chemotherapy and radiation. Oncolytic virotherapy may be a promising approach to treat TNBC. However, oncolytic adenovirus (OAd)-based mono-therapeutic clinical trials have resulted in modest outcomes. The OAd potency could be increased by chemotherapy-induced autophagy, an intracellular degradation system that delivers cytoplasmic constituents to the lysosome. In this study, the ability of alkylating agent temozolomide (TMZ)-induced autophagy to increase OAd replication and oncolysis in TNBC cells was evaluated. Human TNBC MDA-MB-231 and HCC1937 cells and mouse 4T1 cells were infected with an OAd expressing the red fluorescent protein mCherry on the virus capsid (OAdmCherry) alone or in combination with TMZ. TNBC cells treated with OAdmCherry/TMZ displayed greater mCherry and adenovirus (Ad) early region 1A (E1A) expression and enhanced cancer-cell killing compared to OAdmCherry or TMZ alone. The combined therapy-mediated cell death was associated with virus replication and accumulation of the autophagy marker light chain 3 (LC3)-II. Overall, this study provides experimental evidence of TMZ’s ability to increase oncolytic virotherapy in both human and murine TNBC cells.

PTRA - A reporter system for monitoring the intracellular dynamics of gene expression.

The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

BackgroundResuscitation promoting factor proteins (Rpfs) are peptidoglycan glycosidases capable of resuscitating dormant mycobacteria, and have been found to play a role in the pathogenesis of tuberculosis. However, the specific roles and localisation of each of the 5 Rpfs in Mycobacterium tuberculosis remain mostly unknown. In this work our aim was to construct fluorescent fusions of M. tuberculosis Rpf proteins as tools to investigate their function.ResultsWe found that Rpf-fusions to the fluorescent protein mCherry are functional and able to promote cell growth under different conditions. However, fusions to Enhanced Green Fluorescent Protein (EGFP) were non-functional in the assays used and none were secreted into the extracellular medium, which suggests Rpfs may be secreted via the Sec pathway. No specific cellular localization was observed for either set of fusions using time-lapse video microscopy.ConclusionsWe present the validation and testing of five M. tuberculosis Rpfs fused to mCherry, which are functional in resuscitation assays, but do not show any specific cellular localisation under the conditions tested. Our results suggest that Rpfs are likely to be secreted via the Sec pathway. We propose that such mCherry fusions will be useful tools for the further study of Rpf localisation, individual expression, and function.

Modified HeLa Cells and C.elegans Multicellular Model as Unique Systems for the Study of Purinosome Formation

Purines are essential molecules for synthesis of nucleic acids, universal carriers of chemical energy and parts of signaling molecules in all living organisms. Their cellular level is maintained by the de novo purine synthesis (DNPS), effective recycling by salvage pathway and eventual degradation. DNPS includes ten reactions catalyzed by six enzymes, four of them are multifunctional in high eukaryotes. Some DNPS intermediates are unstable and/or toxic, therefore the proximity of enzymes in multienzyme complex called purinosome would be essential to ensure the vital metabolic function. The first direct evidence of its formation was demonstrated by detecting spatial overlap of signals of transiently expressed fluorescently labeled DNPS proteins in HeLa cells grown in the purine depleted medium. However, this model and its eventual utility for further research was questioned and attributed to aggregation of overexpressed proteins and stress granules resulting from the exposure of the cells to the dialyzed, and therefore nutrient reduced growth media. Another possibility for detection of purinosome complexes is immunofluorescent labeling of endogenous proteins involved in the DNPS pathway, so the artificial protein overexpression is avoided. The disadvantage of this method is the inability to study purinosome formation in vivo.In this work we detected the purinosome by combining two previously used methods in prepared purinosome non‐forming cells deficient for particular DNPS steps (CR‐DNPS cells) and in cells deficient in salvage pathway which build purinosome regardless to the purine levels (CR‐HGPRT cells). Moreover, we have prepared the model organism C. elegans with fluorescently labeled DNPS enzymes for in vivo study of the purinosome formation.We transiently transfected the CR‐DNPS and CR‐HGPRT cells with relevant vectors coding BFP labeled wild‐type proteins and observed normalization (e.g. restoration or disruption) of purinosome formation in the cells.To control if the cells undergoing transient transfection and nutrition starvation formed purinosomes or stress bodies, we transfected one type of CR‐DNPS cells with vector coding enzyme with mutation resulting in zero enzyme activity. We did not find any purinosomes regardless of the quantity of purines in the growth media.We fused DNPS proteins in C. elegans with fluorescent protein GFP or mCherry and observed the intracellular compartmentalization of purinosome by fluorescencent confocal microscopy.In conclusion, both methods, transient transfection and immunofluorescence, are useful for the detection of purinosomes in HeLa cells. Moreover, prepared cell‐based models and multicellular model organism C. elegans with translational reporters represent a unique system for purinosome assembly studies. From these models, we expect a greater understanding of the complexity of purine metabolism.Support or Funding InformationThis work was supported by grants AZV 15‐28979A (Ministry of Health, CR); PRIMUS/17/MED/6, GAUK 1102217 and PROGRES Q26/LF1 (Charles University, CR); and LQ1604 NPU II (Ministry of Education, Youth and Sports, CR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization).

P.1.005 - Mutating the cholesterol binding site on amyloid precursor protein alters the processing and production of Aβ-amyloid peptides

Levels of cholesterol are elevated in the brains of Alzheimer’s disease (AD) patients [1,2]. While the precise pathophysiological consequences remain unclear, an increase in cholesterol at the plasma membrane promotes endocytosis of the amyloid precursor protein (APP) thereby leading to increased Aβ production [3]. An interaction between the Aβ fragment C99 (resulting from cleavage of APP by β-secretase), and cholesterol was recently proposed [4]. In the present work, we studied the nature of this putative cholesterol binding site (CBS) on APP by analyzing APP mutants within the CBS (located between 691-710 of APP770). We measured the influence of mutants on production of amyloid peptides, cleavage by secretases and the subcellular localization of APP mutants in HEK293T cells. A variety of mutants of the CBS of APP751-mCherry (APP isoform which contain 751 residues fused to the fluorescent protein mCherry) were constructed using site-directed mutagenesis: E693K, S697A, K699A, K699E, G700A, G704A, V710A, S697A/K699A and G700A/G704A (numbering according to APP770 sequence). The amounts of secreted Aβ40 and Aβ42 peptides were measured by ELISA (Meso Scale Discovery) and short peptides (28 to 42aa) by ELISA from IBL (Immuno-Biological Laboratories) in the culture medium of HEK293T cells transfected with various mutants. We studied the activity of secretases by analyzing the levels of C99 and AICD (APP Intracellular Domain) using western blot. In addition, we analyzed the subcellular localization of mutant APP-mCherry by confocal microscopy, using immunocytochemistry with anti-Early Endosome Antigen 1 antibodies. Levels of secreted Aβ40 and Aβ42 were significantly decreased in HEK293T cells transfected with all mutants (except for S697A) as compared to HEK293T cells transfected with APPwt (p In conclusion, these results suggest that the CBS is a key player in the processing of APP and generation of neurotoxic fragments like Aβ42. Further, a specific interaction between cholesterol and the APP CBS controls the size as well as amount of Aβ peptides. We hypothesize that CBS mutants are positioned more deeply in the membrane thus being processed differently leading to the formation of shorter Aβ peptides. These data demonstrate the functional importance of an interaction between cholesterol and APP, provide insights into the pathology of AD, and suggest potential routes to novel therapies.

P.2.036 - Somatostatin-expressing neurons in the bed nucleus of stria terminalis in anxiety- and addiction-related behaviours in mice

The bed nucleus of stria terminalis (BNST), part of the extended amygdala circuitry, is currently studied due to its role in the anxiety-, stress-, and fear-related behaviours, as well as in addiction [1]. The BNST is a highly heterogeneous area constituting of set of subnuclei and a variety of neuron populations, properties of which have only partially been revealed so far. One of the neuron populations, on which only a very little research has been conducted, is the somatostatin (Sst) -expressing neurons, highly abundant in the anterodorsal part of the BNST (adBNST), especially in oval and juxtacapsular nuclei of the BNST [2]. We aimed to elucidate the role of the Sst-neurons in behaviours such as anxiety and reward. To study the projections of the Sst-neurons in the adBNST, we injected Cre-inducible myristoylated green fluorescent protein (myr-GFP) to the adBNST of Sst-Cre mice (n=2). Following a whole-brain screening for fluorescent neurites, an abundancy of neurites were observed in the lateral hypothalamus (LH) and amygdaloidal nuclei, mostly central (CeA) and medial nucleus of amygdala (MeA). Neurites were also observed nucleus accumbens (NAc) as well as in midbrain structures like ventral tegmental area (VTA) and substantia nigra (SN). To follow up the tracing results we ran tests on amygdala and VTA associated behaviours. We used Sst-IRES-Cre mice (n=21) and targeted the Cre-expressing neurons using stereotaxic delivery of AAV-vector encoding Cre-inducible hM3Dq-DREADDs with Cre-inducible mCherry fluorescent protein as the control (n=20). The mice were treated with 1.0 mg/kg i.p. clozapine-N-oxide (CNO), to activate the Sst-neuron firing, 30 min prior to the behavioural tests. To assess acute anxiety-like behaviour, we used the elevated-plus maze paradigm and a modified open field test, in which a novel object is introduced to the arena in the middle of the trial [3]. To study the potential effect on reward-associated behaviours, we used the biased conditioned place preference (CPP) test. In the elevated-plus maze, the activation of the Sst-neurons did not affect the locomotor activity of the mice, the time spent in the open arms, nor the exploratory activities, like the frequency of the head dips or the stretch-attend postures, as compared to the controls. In the open field test, no effects on the movement were observed. The DREADD group spent less time in the centre of the field during the last 3 min of the test (t(39)=2.143 p=0.038), but no differences in the number of contacts with the novel object were observed. The CPP test failed to show any meaningful rewarding or aversive properties of CNO-induced activation of the Sst-neurons. During the conditioning trials, the animals in the DREADD and control groups moved similar distances after the activation. Chemogenetic activation of the adBNST Sst-neurons failed to show any significant behavioural effects in the chosen paradigm for the reward-related behaviour, but showed a minor change in anxiety-associated parameter in open field test. These data support the initial tracing results, but further research is needed to clarify the role of the Sst-subcircuitry of adBNST.

Oral Application of Recombinant Bacillus subtilis Spores to Dogs Results in a Humoral Response against Specific Echinococcus granulosus Paramyosin and Tropomyosin Antigens.

Bacillus subtilis is known as an endospore- and biofilm-forming bacterium with probiotic properties. We have recently developed a method for displaying heterologous proteins on the surface of B. subtilis biofilms by introducing the coding sequences of the protein of interest into the bacterial genome to generate a fusion protein linked to the C terminus of the biofilm matrix protein TasA. Although B. subtilis is a regular component of the gut microflora, we constructed a series of recombinant B. subtilis strains that were tested for their ability to be used to immunize dogs following oral application of the spores. Specifically, we tested recombinant spores of B. subtilis carrying either the fluorescent protein mCherry or else selected antigenic peptides (tropomyosin and paramyosin) from Echinococcus granulosus, a zoonotic intestinal tapeworm of dogs and other carnivores. The application of the recombinant B. subtilis spores led to the colonization of the gut with recombinant B. subtilis but did not cause any adverse effect on the health of the animals. As measured by enzyme-linked immunosorbent assay and immunoblotting, the dogs were able to develop a humoral immune response against mCherry as well as against E. granulosus antigenic peptides. Interestingly, the sera of dogs obtained after immunization with recombinant spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, which represent the infective form of the head of the tapeworms. These results represent an essential step toward the establishment of B. subtilis as an enteric vaccine agent.

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15min for the scanner, 500 cells and 2min for the plate fluorimeter, and 1000 cells and less than 1min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry.

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Characterization of the Sorbitol Utilization Cluster of the Probiotic Pediococcus parvulus 2.6: Genetic, Functional and Complementation Studies in Heterologous Hosts.

Pediococcus parvulus 2.6 secretes a 2-substituted (1,3)-β-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the P. parvulus 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (gutFRMCBA), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) P. parvulus 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-β-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of gutFRMCBA. The promoter-probe vector pRCR based on the mrfp gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (Pgut) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: Lactobacillus plantarum and Lactobacillus casei. Upon growth in the presence of sorbitol, but not of glucose, only the presence of Pgut was required to support expression of mrfp in L. plantarum. In L. casei the presence of sorbitol in the growth medium and the pediococcal gutR or gutR plus gutM in the genome was required for Pgut functionality. This demonstrates that: (i) Pgut is required for expression of the gut cluster, (ii) Pgut is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, trans-complementation for activation of Pgut exists in L. plantarum but not in L. casei.

Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis

Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β–catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β–catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe2O4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens.

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

An analysis of the effect of the internal ribosome entry site of the encephalomyocarditis virus on the expression of the second gene in the bicistronic matrix in neurons of primary hippocampal cultures

Molecular biological experiments sometimes require expression of two or more genes in a single cell with an accurate ratio between their expression levels. One of the methods to provide this control is the use of the internal ribosome entry site (IRES) from the encephalomyocarditis virus as a separating insert between two target genes in the expression vector. Previously, it was shown that the efficacy of translation of the gene after IRES varies considerably in a range from 6 to 100% depending on the cell type. In neurons, the exact ratio between the expression levels of genes that are located before and after the IRES in the expression vector is unknown. Here, we analyzed the ratio between the amounts of products of the first and second genes located before and after the IRES in a plasmid that was used to transfect neurons in a primary hippocampal culture. We created two plasmid vectors that contain genes of the yellow (Venus) and red (mCherry) fluorescent proteins in different orders, which are separated by the IRES. We found that the unmodified IRES sequence of the encephalomyocarditis virus decreases the expression in the second cistron by a factor of 2.7 in a primary culture of hippocampal neurons. These data will help us to use currently available libraries of mutant IRES sequences for accurate control of the relationships between the expression of different target genes in neurons.

Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor.

SummaryAlthough hundreds of genetic male sterility (GMS) mutants have been identified in maize, few are commercially used due to a lack of effective methods to produce large quantities of pure male‐sterile seeds. Here, we develop a multicontrol sterility (MCS) system based on the maize male sterility 7 (ms7) mutant and its wild‐type Zea mays Male sterility 7 (ZmMs7) gene via a transgenic strategy, leading to the utilization of GMS in hybrid seed production. ZmMs7 is isolated by a map‐based cloning approach and encodes a PHD‐finger transcription factor orthologous to rice PTC1 and Arabidopsis MS1. The MCS transgenic maintainer lines are developed based on the ms7‐6007 mutant transformed with MCS constructs containing the (i) ZmMs7 gene to restore fertility, (ii) α‐amylase gene ZmAA and/or (iii) DNA adenine methylase gene Dam to devitalize transgenic pollen, (iv) red fluorescence protein gene DsRed2 or mCherry to mark transgenic seeds and (v) herbicide‐resistant gene Bar for transgenic seed selection. Self‐pollination of the MCS transgenic maintainer line produces transgenic red fluorescent seeds and nontransgenic normal colour seeds at a 1:1 ratio. Among them, all the fluorescent seeds are male fertile, but the seeds with a normal colour are male sterile. Cross‐pollination of the transgenic plants to male‐sterile plants propagates male‐sterile seeds with high purity. Moreover, the transgene transmission rate through pollen of transgenic plants harbouring two pollen‐disrupted genes is lower than that containing one pollen‐disrupted gene. The MCS system has great potential to enhance the efficiency of maize male‐sterile line propagation and commercial hybrid seed production.

Chemical regulation of Fea1 driven transgene expression in Chlamydomonas reinhardtii

Inducible promoters can provide regulated gene expression allowing the biosynthesis of gene products at most suitable moments of cultivation. In this study, parameters of induction and deactivation of the iron-responsive Fea1 promoter (Allen et al., 2007) were investigated in the green alga Chlamydomonas reinhardtii. Our results indicate that the construct used, ble-2A-mCherry, can be expressed successfully by the Fea1 promoter under iron-deficient conditions. The fluorescence signals of the fluorescent protein mCherry obtained via flow cytometry were detectable at different intensities in response to concentrations of iron ranging from 0μM to 20μM in media. We also demonstrate that the addition of the iron chelator deferroxamine (DFO) to iron-replete media leads to promoter activation, resulting in the increase of mCherry fluorescence. Reversibility of promoter induction is detected already within 3h after transferring the cells to iron-replete chelator-free media. In this case, the progressive decrease in mCherry fluorescence can reach, within 48h, as low as 5% of the fluorescence observed in a 40h – treatment with DFO. Cell viability after DFO treatment is not affected up to a concentration of 100μM of the chelator, which enables the establishment of a cyclic process of induction and repression for the production of recombinant proteins under the control of the Fea1 inducible promoter.