Fluorescent Protein mCherry Research Articles

An endogenous mCherry-tagged COSA-1 as a crossover investigation tool in Caenorhabditis elegans.

The C. elegans cosa-1 gene encodes the crossover site-associated-1 (COSA-1) protein, a cyclin-related protein that functions in promoting crossovers (COs) during meiosis. Previous studies regarding CO dynamics in live C. elegans have mostly relied on the green fluorescent protein-tagged cosa-1 transgenic strain, which was generated by the microparticle bombardment method. Here, we insert the red fluorescence protein mCherry at the C-terminal of the cosa-1 gene to establish cosa-1::mCherry transgenic worm by the CRISPR/Cas9 technique. The COSA-1::mCherry was observed to appear from the early pachytene, and disappear in the diplotene zone of the germline, with 6 COSA-1:: mCherry foci in the late pachytene, which colocalized with GFP::COSA-1 from AV630 strain. Furthermore, the transgenic strain harboring a cosa-1::mCherry fusion shows no defect in the brood size, progeny viability and male frequency, which provides a useful tool for the meiotic analysis in C. elegans .

Preparing sequencing grade RNAs from a small number of FACS-sorted larvae macrophages isolated from enzyme free dissociated zebrafish larvae

Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages.•An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages.•A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality.•Production of high integrity RNAs from a small number of isolated macrophages.

Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers.

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Monitoring Virus Intercellular Movement from Primary Infected Cells to Neighboring Cells in Plants.

Viral cell-to-cell movement from the primary infected cells to neighboring cells is an essential step for viruses to establish systemic infection in plants. The classic experimental design for studying this process involves the application of a reporter protein such as β-glucuronidase (GUS), green fluorescent protein (GFP), or monomeric red fluorescent protein (mRFP or mCherry). However, such experimental settings are unable to unambiguously distinguish primary and secondary infected cells. In recent years, we have developed several double-labeling potyvirus infectious clones. Upon introduction of such vectors into plant leaf tissues, primary infected cells emit dual fluorescence (green and red) whereas secondary infected cells emit only green fluorescence. In this chapter, we provide detailed protocols on (1) construction of a GFP and mCherry-tagged turnip mosaic virus infectious clone, (2) delivery of the recombinant viral clones into plant cells by agroinfiltration, (3) confocal imaging of viral cell-to-cell movement, and (4) analysis of viral systemic infection. Using this dual-color imaging system, we have revealed coat protein (CP) is essential for TuMV cell-to-cell movement. This system provides a valuable and robust tool to study plant virus cell-to-cell movement.

Functionalized Protein Nanotubes Based on the Bacteriophage vB_KleM-RaK2 Tail Sheath Protein.

We report on the construction of functionalized nanotubes based on tail sheath protein 041 from vB_KleM-RaK2 bacteriophage. The truncated 041 protein (041Δ200) was fused with fluorescent proteins GFP and mCherry or amidohydrolase YqfB. The generated chimeric proteins were successfully synthesized in E. coli BL21 (DE3) cells and self-assembled into tubular structures. We detected the fluorescence of the structures, which was confirmed by stimulated emission depletion microscopy. When 041Δ200GFP and 041Δ200mCherry were coexpressed in E. coli BL21 (DE3) cells, the formed nanotubes generated Förster resonance energy transfer, indicating that both fluorescent proteins assemble into a single nanotube. Chimeric 041Δ200YqfB nanotubes possessed an enzymatic activity, which was confirmed by hydrolysis of N4-acetyl-2′-deoxycytidine. The enzymatic properties of 041Δ200YqfB were similar to those of a free wild-type YqfB. Hence, we conclude that 041-based chimeric nanotubes have the potential for the development of delivery vehicles and targeted imaging and are applicable as scaffolds for biocatalysts.

Latently KSHV-Infected Cells Promote Further Establishment of Latency upon Superinfection with KSHV.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related virus which engages in two forms of infection: latent and lytic. Latent infection allows the virus to establish long-term persistent infection, whereas the lytic cycle is needed for the maintenance of the viral reservoir and for virus spread. By using recombinant KSHV viruses encoding mNeonGreen and mCherry fluorescent proteins, we show that various cell types that are latently-infected with KSHV can be superinfected, and that the new incoming viruses establish latent infection. Moreover, we show that latency establishment is enhanced in superinfected cells compared to primary infected ones. Further analysis revealed that cells that ectopically express the major latency protein of KSHV, LANA-1, prior to and during infection exhibit enhanced establishment of latency, but not cells expressing LANA-1 fragments. This observation supports the notion that the expression level of LANA-1 following infection determines the efficiency of latency establishment and avoids loss of viral genomes. These findings imply that a host can be infected with more than a single viral genome and that superinfection may support the maintenance of long-term latency.

Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells.

Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the nonexpressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA-mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA-mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA-mediated nuclear localization.

Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria.

Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately.

Expanding the toolbox of exosome-based modulators of cell functions

Exosomes are cell-derived extracellular vesicles and play important roles in mediating intercellular communications. Due to their unique advantages in transporting a variety of biomolecules, exosomes have been emerging as a new class of nanocarriers with great potential for therapeutic applications. Despite advancements in loading chemotherapeutics and interfering RNAs into exosomes, active incorporation of protein molecules into exosomes remains challenging owing to their distinctive physicochemical properties and/or a lack of knowledge of cargo sorting during exosome biogenesis. Here we report the generation of a novel type of engineered exosomes with actively incorporated membrane proteins or soluble protein cargos, named genetically infused functionally tailored exosomes (GIFTed-Exos). Through genetic fusion with exosome-associated tetraspanin CD9, transmembrane protein CD70 and glucocorticoid‐induced tumor necrosis factor receptor family‐related ligand (GITRL) could be displayed on exosome surface, resulting in GIFTed-Exos with excellent T-cell co-stimulatory activities. By genetically linking to a CD9-photocleavable protein fusion, fluorescent protein mCherry, apoptosis-inducing protein apoptin, and antioxidant enzyme catalase could be effectively packed into exosomes for light-controlled release. The generated GIFTed-Exos display notable in vitro and in vivo activities for delivering distinct types of protein cargos to target cells. As a possibly general approach, GIFTed-Exos provide new opportunities to create exosomes with new functions and properties for biomedical research.

Photo-Switching of Protein Dynamical Collectivity

We examine changes in the picosecond structural dynamics with irreversible photobleaching of red fluorescent proteins (RFP) mCherry, mOrange2 and TagRFP-T. Measurements of the protein dynamical transition using terahertz time-domain spectroscopy show in all cases an increase in the turn-on temperature in the bleached state. The result is surprising given that there is little change in the protein surface, and thus, the solvent dynamics held responsible for the transition should not change. A spectral analysis of the measurements guided by quasiharmonic calculations of the protein absorbance reveals that indeed the solvent dynamical turn-on temperature is independent of the thermal stability/photostate however the protein dynamical turn-on temperature shifts to higher temperatures. This is the first demonstration of switching the protein dynamical turn-on temperature with protein functional state. The observed shift in protein dynamical turn-on temperature relative to the solvent indicates an increase in the required mobile waters necessary for the protein picosecond motions, that is, these motions are more collective. Melting-point measurements reveal that the photobleached state is more thermally stable, and structural analysis of related RFP’s shows that there is an increase in internal water channels as well as a more uniform atomic root mean squared displacement. These observations are consistent with previous suggestions that water channels form with extended light excitation providing O2 access to the chromophore and subsequent fluorescence loss. We report that these same channels increase internal coupling enhancing thermal stability and collectivity of the picosecond protein motions. The terahertz spectroscopic characterization of the protein and solvent dynamical onsets can be applied generally to measure changes in collectivity of protein motions.

Neuropeptide Y suppresses thermogenic and cardiovascular sympathetic nerve activity via Y1 receptors in the paraventricular nucleus and dorsomedial hypothalamus.

In hungry animals, neuropeptide Y (NPY) neurones in the arcuate nucleus (ArcN) are activated to suppress energy expenditure, in part by decreasing brown adipose tissue sympathetic nerve activity (BAT SNA); however, the NPY receptor subtype and brain neurocircuitry are unclear. In the present study, we investigated the inhibition of BAT SNA by exogenous and endogenous NPY via binding to Y1 receptors (NPY1R) in the hypothalamic paraventricular nucleus (PVN) and dorsomedial hypothalamus (DMH), in anaesthetised male rats. Downstream projections of PVN/DMH NPY1R-expressing neurones were identified using male Npy1r-cre mice and localised unilateral DMH or PVN injections of an adeno-associated virus, which allows for the cre-dependent expression of a fluorescent protein (mCherry) in the cell bodies, axon fibres and nerve terminals of NPY1R-containing neurones. Nanoinjections of NPY into the DMH of cooled rats decreased BAT SNA, as well as mean arterial pressure (MAP) and heart rate (HR), and these responses were reversed by subsequent injection of the selective NPY1R antagonist, BIBO3304. In warmed rats, with little to no BAT SNA, bilateral nanoinjections of BIBO3304 into the DMH or PVN increased BAT SNA, MAP and HR. DMH NPY1R-expressing neurones projected heavily to the raphe pallidus (RPa), which houses BAT presympathetic neurones, as well as the PVN. In anaesthetised mice, DMH BIBO3304 increased splanchnic SNA, MAP and HR, all of which were reversed by nonselective blockade of the PVN with muscimol, suggesting that DMH-to-PVN connections are involved in this DMH BIBO3304 disinhibition. PVN Y1R expressing neurones also projected to the RPa, as well as to the nucleus tractus solitarius. We conclude that NPY tonically released in the DMH and PVN suppresses BAT SNA, MAP and HR via Y1R. Downstream neuropathways for BAT SNA may utilise direct projections to the RPa. Release of tonic NPY inhibition of BAT SNA may contribute to feeding- and diet-induced thermogenesis.

Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame).

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

Abstract 1150: CARG-2020 artificial oncolytic virus delivering three immune-modulators prevents tumor recurrence in a syngeneic model of ovarian cancer

Abstract Background: Ovarian cancer is the most lethal of all gynecologic malignancies with a grim 5-year survival rate of ~47%. Mortality occurs in the setting of recurrent disease wherein the co-presentation of chemoresistance and carcinomatosis limits the value of standard of care. It is therefore imperative to develop approaches that can prevent recurrent ovarian cancer. Oncolytic viruses are genetically modified replication competent viruses that can selectively infect cancer cells to induce cell lysis and death. This approach has proven to be clinically safe making it a promising approach to complement standard of care. The objective of this study is to evaluate the efficacy of CARG-2020, a virus-like vesicles (VLV) delivering three immune-modulators including IL-12, IL-17 antagonist and shRNA-PD-L1 in preventing recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Materials and Methods: In vitro: Triple knockout (TKO; p53LSL-R172H/Dicerflox/flox/Ptenflox/flox) mouse ovarian cancer cells were seeded in 96-well plates and treated with different doses of CARG-2020. Cell death was determined using the Incucyte imaging system. In vivo: TKO ovarian cancer cells stably expressing the mCherry fluorescent protein were injected i.p. in C57bl/6 mice (day 0) to mimic the establishment of recurrent disease. Treatment commenced on day 3 with the treatment group receiving three doses of 1x10^8 PFU of CARG-2020 given every other day (n=5). Control group received PBS (n=5). Both treatments were given i.p. Tumor growth was monitored by live imaging using mCherry fluorescence ROI area. Progression-free survival (PFS) was defined as the day ROI area reached 2,000. Results: CARG-2020 (1x10^5 PFU) demonstrated cytolytic effect in vitro and induced 100% cell death within 24h. In vivo, CARG-2020 induced significant decrease in i.p. tumor growth (p = 0.0128) compared to PBS control. Treatment with CARG-2020 completely delayed the establishment of recurrent disease (PFS, p = 0.0003). Whereas PBS control group demonstrated median PFS of 20 days, mice in the CARG-2020 group remained disease-free until day 60. Conclusion: CARG-2020 is able to prevent the establishment of recurrent disease in a syngeneic mouse model of recurrent ovarian cancer. Our results provide rationale for the further development of this platform as a therapeutic modality for ovarian cancer patients. Citation Format: Ayesha B. Alvero, Alexandra Fox, Bhaskara Madina, Valerian Nakaar, Timur Yarovinsky, Marie Krady, Bijan Almassian, Gil Mor. CARG-2020 artificial oncolytic virus delivering three immune-modulators prevents tumor recurrence in a syngeneic model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1150.

Peroxisomes Implicated in the Biosynthesis of Siderophores and Biotin, Cell Wall Integrity, Autophagy, and Response to Hydrogen Peroxide in the Citrus Pathogenic Fungus Alternaria alternata.

Little is known about the roles of peroxisomes in the necrotrophic fungal plant pathogens. In the present study, a Pex6 gene encoding an ATPase-associated protein was characterized by analysis of functional mutations in the tangerine pathotype of Alternaria alternata, which produces a host-selective toxin. Peroxisomes were observed in fungal cells by expressing a mCherry fluorescent protein tagging with conserved tripeptides serine-lysing-leucine and transmission electron microscopy. The results indicated that Pex6 plays no roles in peroxisomal biogenesis but impacts protein import into peroxisomes. The number of peroxisomes was affected by nutritional conditions and H2O2, and their degradation was mediated by an autophagy-related machinery termed pexophagy. Pex6 was shown to be required for the formation of Woronin bodies, the biosynthesis of biotin, siderophores, and toxin, the uptake and accumulation of H2O2, growth, and virulence, as well as the Slt2 MAP kinase-mediated maintenance of cell wall integrity. Adding biotin, oleate, and iron in combination fully restored the growth of the pex6-deficient mutant (Δpex6), but failed to restore Δpex6 virulence to citrus. Adding purified toxin could only partially restore Δpex6 virulence even in the presence of biotin, oleate, and iron. Sensitivity assays revealed that Pex6 plays no roles in resistance to H2O2 and superoxide, but plays a negative role in resistance to 2-chloro-5-hydroxypyridine (a hydroxyl radical-generating compound), eosin Y and rose Bengal (singlet oxygen-generating compounds), and 2,3,5-triiodobenzoic acid (an auxin transport inhibitor). The diverse functions of Pex6 underscore the importance of peroxisomes in physiology, pathogenesis, and development in A. alternata.

LED control of gene expression in a nanobiosystem composed of metallic nanoparticles and a genetically modified E. coli strain

BackgroundWithin the last decade, genetic engineering and synthetic biology have revolutionized society´s ability to mass-produce complex biological products within genetically-modified microorganisms containing elegantly designed genetic circuitry. However, many challenges still exist in developing bioproduction processes involving genetically modified microorganisms with complex or multiple gene circuits. These challenges include the development of external gene expression regulation methods with the following characteristics: spatial–temporal control and scalability, while inducing minimal permanent or irreversible system-wide conditions. Different stimuli have been used to control gene expression and mitigate these challenges, and they can be characterized by the effect they produce in the culture media conditions. Invasive stimuli that cause permanent, irreversible changes (pH and chemical inducers), non-invasive stimuli that cause partially reversible changes (temperature), and non-invasive stimuli that cause reversible changes in the media conditions (ultrasound, magnetic fields, and light).MethodsOpto-control of gene expression is a non-invasive external trigger that complies with most of the desired characteristics of an external control system. However, the disadvantage relies on the design of the biological photoreceptors and the necessity to design them to respond to a different wavelength for every bioprocess needed to be controlled or regulated in the microorganism. Therefore, this work proposes using biocompatible metallic nanoparticles as external controllers of gene expression, based on their ability to convert light into heat and the capacity of nanotechnology to easily design a wide array of nanostructures capable of absorbing light at different wavelengths and inducing plasmonic photothermal heating.ResultsHere, we designed a nanobiosystem that can be opto-thermally triggered using LED light. The nanobiosystem is composed of biocompatible gold nanoparticles and a genetically modified E. coli with a plasmid that allows mCherry fluorescent protein production at 37 °C in response to an RNA thermometer.ConclusionsThe LED-triggered photothermal protein production system here designed offers a new, cheaper, scalable switchable method, non-destructive for living organisms, and contribute toward the evolution of bioprocess production systems.

An N-terminal fusion allele to study melanin concentrating hormone receptor 1.

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherry MCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

Localization of two interacting protein components of the conjugation machinery of Bacillus subtilis

Conjugation, or mating, is a major form of horizontal gene transfer, contributing to the spread of antibiotic resistance and virulence genes in bacterial populations. During conjugation, DNA is transferred from donor to recipient cell through a large multi-protein DNA translocation channel produced in the donor cell. We are interested in understanding the localization of the conjugation machinery of the conjugative element ICEBs1 of Bacillus subtilis. We previously identified several proteins that make up the ICEBs1 DNA translocation channel, including the peripheral membrane protein ConE and the transmembrane protein ConB. ConE fused to green fluorescent protein (GFP) localizes to the cell membrane, predominantly at the poles. ConE interacts with the bitopic membrane protein ConB. This interaction is functionally important as ConE-GFP mislocalizes to the cytoplasm in a strain deleted for conB. Here, we discovered that a fusion of ConB to the red fluorescent protein mCHERRY also localizes to the cell membrane at the poles, similarly to ConE-GFP. We also show that ConB-mCHERRY localization is independent of other conjugation proteins, but dependent on its own transmembrane segment. Finally, we discovered which domains of ConB are required for ConE-GFP localization. Our work provides new insights into the subcellular localization of two conserved interacting proteins that are critical for mediating horizontal gene transfer.

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System.

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

Replication-Competent ΔNS1 Influenza A Viruses Expressing Reporter Genes.

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.

Automated multi-scale cascade of parallel stirred-tank bioreactors for fast protein expression studies

Automation, parallelization and autonomous operation of standard lab equipment, usually applied for manual bioprocess development, is considered as the key for reduction of bioprocess development time and costs. An automated bioreactor system with 4 stirred-tank bioreactors on a L-scale was combined with a custom-made biomass transfer system to distribute the cell suspensions produced on the L-scale into 48 parallel stirred-tank bioreactors on a mL-scale. Afterwards parallel protein expression studies automated by a liquid handling system with integrated fluorescence reader were performed. Isopropyl β-D-1-thiogalactopyranoside-induced (IPTG) expression of the red fluorescence protein mCherry was studied as an example of using fed-batch processes with recombinant Escherichia coli. In a first automated study, IPTG concentrations were varied in 48 parallel fed-batch processes with E. coli cells produced at a growth rate of 0.1 h−1 on an L-scale and transferred automatically to the mL-scale. The mCherry expression rate increased with increasing inducer concentration until the highest protein expression rate was observed at > 9 μM IPTG. In a second automated study, the growth rate of E. coli was varied between 0.1−0.2 h-1 in parallelly-operated stirred-tank bioreactors on a L-scale. The cells were automatically transferred and distributed into the stirred-tank bioreactors on a mL-scale and the concentration of the inducer IPTG was varied as before in parallel fed-batch processes. An increased growth rate during the production of the recombinant E. coli cells and/or higher cell densities during protein expression resulted in the increased IPTG concentrations necessary to achieve identical expression rates compared to a growth rate of 0.1 h−1 with the exception of very low inducer concentrations and inducer concentrations in excess. The new automated multi-scale cascade of parallel stirred-tank bioreactors should easily be applicable for performing fast optimisation studies with other microbial production systems and will have the potential to reduce bioprocess development time and staff assignment considerably.