Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Sensitive Methods to Detect Single-Stranded Nucleic Acids of Food Pathogens Based on Cell-Free Protein Synthesis and Retroreflection Signal Detection.

Cell-free protein synthesis (CFPS) has recently gained considerable attention as a new platform for developing methods to detect various molecules, ranging from small chemicals to biological macromolecules. Retroreflection has been used as an alternative signal to develop analytical methods because it can be detected by using a simple instrument comprising a white light source and a camera. Here, we report a novel reporter protein that couples the capability of CFPS and the simplicity of retroreflection signal detection. The design of the reporter was based on two pairs of protein-peptide interactions, SpyCatcher003-SpyTag003 and MDM2-PMI(N8A). MDM2-MDM2-SpyCatcher003 was decided as the reporter protein, and the two peptides, SpyTag003 and PMI(N8A), were immobilized on the surfaces of retroreflective Janus particles and microfluidic chips, respectively. The developed retroreflection signal detection system was combined with a previously reported CFPS reaction that can transduce the presence of a single-stranded nucleic acid into protein synthesis. The resulting methods were applied to detect 16S rRNAs of several foodborne pathogens. Concentration-dependent relationships were observed over a range of 10° fM to 102 pM, with the limits of detection being single-digit femtomolar concentrations. Considering the designability of the CFPS system for other targets, the retroreflection signal detection method will enable the development of novel methods to detect various molecules.

Development of recombinant West Nile virus expressing mCherry reporter protein.

West Nile virus (WNV) is transmitted to humans and animals by a mosquito and enters the central nervous system, leading to lethal encephalitis. Reporter viruses expressing fluorescent proteins enable detection of infected cells in vitro and in vivo, facilitating evaluation of the dynamics of viral infection, and the development of diagnostic or therapeutic methods. In this study, we developed a method for production of a recombinant replication-competent WNV expressing mCherry fluorescent protein. The expression of mCherry was observed in viral antigen-positive cells in vitro and in vivo, but the growth of the reporter WNV was reduced as compared to the parental WNV. The expression of mCherry was stable during 5 passages in reporter WNV-infected culture cells. Neurological symptoms were observed in mice inoculated intracranially with the reporter WNV. The reporter WNV expressing mCherry will facilitate research into WNV replication in mouse brains.

In vivo visualization of murine melanoma cells B16-derived exosomes through magnetic resonance imaging.

BACKGROUND: Numerous studies demonstrated that exosomes play a powerful role in mediating intercellular communication to induce a pro-tumoral environment to promote tumor progression, including pre-metastatic niche formation and metastasis. Noninvasive imaging could determine the in vivo kinetics of exosomes in real time to provide better understanding of the mechanisms of the tumor formation, progression and metastasis. Magnetic resonance imaging (MRI) is an ideal technique which provides excellent anatomical resolution, intrinsic soft tissue contrast, unlimited penetration depth and no radiation exposure. METHODS: A fusion protein composed of ferritin heavy chain (FTH1) and lactadherin was designed for visualizing exosomes through MRI. FTH1 was served as MRI reporter protein and lactadherin is a membrane-associated protein that is distributed on exosome surface. The characterizations of labeled exosomes were validated through transmission electron microscopy, western blot, nanoparticle tracking analysis and finally visualized in vitro and in vivo through MRI. RESULTS: MR imaging showed that the labeled exosomes are able to be visualized in vitro and in vivo. Verification of the characterizations of exosomes observed no significant difference between labeled and unlabeled exosomes. CONCLUSION: The proposed FTH1 labeling method was useful for visualizing exosomes through MRI. GENERAL SIGNIFICANCE: The present study first reported a novel self-label method for imaging labeled exosomes of tumor cells in vivo through MR with cell endogenous MRI reporter protein. It may be further used as a tool to enhance understanding the role of exosomes in various pathophysiological conditions.

A chimeric vector for dual use in cyanobacteria and Escherichia coli, tested with cystatin, a nonfluorescent reporter protein.

BACKGROUND: Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803. METHODS: We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target. RESULTS: With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.

Dual-fluorescent bacterial two-hybrid system for quantitative Protein-Protein interaction measurement via flow cytometry.

Characterization of protein-protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement of the binding strength remains challenging. Building upon the classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that the relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, is an intrinsic characteristic associated with the binding strength between the two interacting proteins. In this study, we inserted fluorescent protein tdTomato in the chromosome as the reporter protein by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) to tag one of the interacting proteins, which can be further labeled by a membrane-permeable biarsenical dye. The combined use of tdTomato and TC-tag offers rapid and high-throughput analysis of the expression levels of both the reporter protein and one of the interacting proteins at the single-cell level by multicolor flow cytometry, which simplifies the quantitative measurement of PPI. The use of the as-developed RRPE-tdTomato-TC-BACTH approach was demonstrated in three demanding applications. First, binding affinities could be correctly ranked for discriminating interaction strengths with a tenfold difference or of the same order of magnitude. We demonstrate that the method is sensitive enough to discriminate affinities with a small difference of 1.4-fold. Moreover, residues involved in PPI can be easily mapped and ranked. Lastly, protein interaction inhibitors can be rapidly screened.

Microbial whole-cell biosensors: Current applications, challenges, and future perspectives.

Microbial Whole-Cell Biosensors (MWCBs) have seen rapid development with the arrival of 21st century biological and technological capabilities. They consist of microbial species which produce, or limit the production of, a reporter protein in the presence of a target analyte. The quantifiable signal from the reporter protein can be used to determine the bioavailable levels of the target analyte in a variety of sample types at a significantly lower cost than most widely used and well-established analytical instrumentation. Furthermore, the versatile and robust nature of MWCBs shows great potential for their use in otherwise unavailable settings and environments. While MWCBs have been developed for use in biomedical, environmental, and agricultural monitoring, they still face various challenges before they can transition from the laboratory into industrialized settings like their enzyme-based counterparts. In this comprehensive and critical review, we describe the underlying working principles of MWCBs, highlight developments for their use in a variety of fields, detail challenges and current efforts to address them, and discuss exciting implementations of MWCBs helping redefine what is thought to be possible with this expeditiously evolving technology.

Plant Cell Wall Proteomes: Bioinformatics and Cell Biology Tools to Assess the Bona Fide Cell Wall Localization of Proteins.

The purification of plant cell walls is challenging because they constitute an open compartment which is not limited by a membrane like the cell organelles. Different strategies have been established to limit the contamination by proteins of other compartments in cell wall proteomics studies. Non-destructive methods rely on washing intact cells with various types of solutions without disrupting the plasma membrane in order to elute cell wall proteins. In contrast, destructive protocols involve the purification of cell walls prior to the extraction of proteins with salt solutions. In both cases, proteins known to be intracellular have been identified by mass spectrometry in cell wall proteomes. The aim of this chapter is to provide tools to assess the subcellular localization of the proteins identified in cell wall proteomics studies, including: (1) bioinformatic predictions, (2) immunocytolocalization of proteins of interest on tissue sections and (3) in muro observation of proteins of interest fused to reporter fluorescent proteins by confocal microscopy. Finally, a qualitative assessment of the work can be performed and the strategy used to prepare the samples can be optimized if necessary.

[Advances in the research and applications of orange fluorescent protein].

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Immunohistochemical amplification of mCherry fusion protein is necessary for proper visualization.

Fluorescent reporter proteins are a powerful tool being increasingly integrated into biological experiments. Their utility spans techniques such as live-cell imaging, validating transgene expression, and studying cell-type specific anatomy. As these reporters become more widely used, it is necessary to fully understand their benefits and limitations. One such recently developed red fluorescent protein, mCherry, has been well utilized due to its stability, brightness, and pH resistance. In the course of an experiment using the fluorescent reporter protein mCherry fused to a G-protein coupled receptor (mCherry fusion protein), our lab discovered a notable inability for the fusion protein to faithfully produce fluorescent signal representative of its expression in fixed tissue. Here, we demonstrate the importance of immunohistochemical amplification in tissue injected with various adeno-associated viruses (AAVs), containing mCherry fusion protein as a reporter. Our findings demonstrate that antibody amplification consistently provides a stronger signal when mCherry fusion protein is used as a reporter protein.

QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence.

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

Engineering microbial consortia by division of labor.

During microbial applications, metabolic burdens can lead to a significant drop in cell performance. Novel synthetic biology tools or multi-step bioprocessing (e.g., fermentation followed by chemical conversions) are therefore needed to avoid compromised biochemical productivity from over-burdened cells. A possible solution to address metabolic burden is Division of Labor (DoL) via natural and synthetic microbial consortia. In particular, consolidated bioprocesses and metabolic cooperation for detoxification or cross feeding (e.g., vitamin C fermentation) have shown numerous successes in industrial level applications. However, distributing a metabolic pathway among proper hosts remains an engineering conundrum due to several challenges: complex subpopulation dynamics/interactions with a short time-window for stable production, suboptimal cultivation of microbial communities, proliferation of cheaters or low-producers, intermediate metabolite dilution, transport barriers between species, and breaks in metabolite channeling through biosynthesis pathways. To develop stable consortia, optimization of strain inoculations, nutritional divergence and crossing feeding, evolution of mutualistic growth, cell immobilization, and biosensors may potentially be used to control cell populations. Another opportunity is direct integration of non-bioprocesses (e.g., microbial electrosynthesis) to power cell metabolism and improve carbon efficiency. Additionally, metabolic modeling and 13C-metabolic flux analysis of mixed culture metabolism and cross-feeding offers a computational approach to complement experimental research for improved consortia performance.

Induction of Cu/Zn Superoxide Dismutase (SOD1) Aggregation in Living Cells.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration and loss of motor neurons that appears to spread through the neuroaxis in a spatiotemporally restricted manner. In the familial form of ALS, the presence of any one of over 180 inherited mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1) leads to its eventual misfolding and aggregation. Once the pathological SOD1 seed is formed, it can continue growing into a larger aggregate through nucleation of other SOD1 substrate molecules. To date, there is no effective and rapid method to study the nucleation of SOD1 or to test therapeutics against this mechanism in living cells. In this chapter, we describe a series of protocols used to study induced aggregation of SOD1 in a simple but robust cell culture model. This assay can also be used to evaluate the potential therapeutic efficacy of small molecules targeting the induced aggregation mechanisms of SOD1.

The mKate fluorescent protein expressed by Leishmania mexicana modifies the parasite immunopathogenicity in BALB/c mice.

Parasites have been engineered to express fluorescent reporter proteins, yet the impact of red fluorescent proteins on Leishmania infections remains largely unknown. We analysed the infection outcome of Leishmania mexicana parasites engineered for the constitutive expression of mKate protein and evaluated their immunogenicity in BALB/c mice. Infection of BALB/c mice with mKate transfected L. mexicana (LmexmKate ) parasites caused enlarged lesion sizes, leading to ulceration, and containing more parasites, as compared to LmexWT . The mKate protein showed immunogenic properties inducing antibody production against the mKate protein, as well as enhancing antibody production against the parasite. The augmented lesion sizes and ulcers, together with the more elevated antibody production, were related to an enhanced number of TNF-α and IL-1ß producing cells in the infected tissues. We conclude that mKate red fluorescent protein is an immunogenic protein, capable of modifying disease evolution of L. mexicana.

Microencapsulation of Lactic Acid Bacteria Improves the Gastrointestinal Delivery and in situ Expression of Recombinant Fluorescent Protein.

The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both in vitro and in vivo, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (L. lactis ssp. cremoris MG1363) carrying the pExu vector encoding the fluorescence protein mCherry [L. lactis MG1363 (pExu:mCherry)] was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:mCherry vector, was demonstrated in vitro by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0-168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated L. lactis bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel.

Red fluorescent protein (DsRFP) optimization for Entamoeba histolytica expression.

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications.

Cell-Type Specific Metabolic Flux Analysis: A Challenge for Metabolic Phenotyping and a Potential Solution in Plants.

Stable isotope labelling experiments are used routinely in metabolic flux analysis (MFA) to determine the metabolic phenotype of cells and tissues. A complication arises in multicellular systems because single cell measurements of transcriptomes, proteomes and metabolomes in multicellular organisms suggest that the metabolic phenotype will differ between cell types. In silico analysis of simulated metabolite isotopomer datasets shows that cellular heterogeneity confounds conventional MFA because labelling data averaged over multiple cell types does not necessarily yield averaged flux values. A potential solution to this problem-the use of cell-type specific reporter proteins as a source of cell-type specific labelling data-is proposed and the practicality of implementing this strategy in the roots of Arabidopsis thaliana seedlings is explored. A protocol for the immunopurification of ectopically expressed green fluorescent protein (GFP) from Arabidopsis thaliana seedlings using a GFP-binding nanobody is developed, and through GC-MS analysis of protein hydrolysates it is established that constitutively expressed GFP reports accurately on the labelling of total protein in root tissues. It is also demonstrated that the constitutive expression of GFP does not perturb metabolism. The principal obstacle to the implementation of the method in tissues with cell-type specific GFP expression is the sensitivity of the GC-MS system.

Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate.

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Promoter, transgene, and cell line effects in the transfection of mammalian cells using PDMAEMA-based nano-stars.

Non-viral transfection protocols are typically optimized using standard cells and reporter proteins, potentially underestimating cellular or transgene effects. Here such effects were studied for two human (Jurkat, HEK-293) and two rodent (CHO-K1, L929) cell lines and three fluorescent reporter proteins. Expression of the enhanced green fluorescent protein (EGFP) was studied under the control of the human elongation factor 1 alpha promoter and three viral promoters (SV40, SV40/enhancer, CMV), that of ZsYellow1 (yellow fluorescence) and mCherry (red fluorescence) for the CMV promoter. Results varied with the cell line, in particular for the Jurkat cells. Pair-wise co-transfection of the CMV controlled transgenes resulted in a significant fraction of monochromatic cells (EGFP for EGFP/YFP and EGFP/RFP co-transfections, YFP in case of YFP/RFP co-transfections). Only Jurkat cells were almost incapable of expressing YFP. Dilution of the plasmid DNA with a non-expressed plasmid showed cell line dependent effects on transfection efficiency and/or expression levels.