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One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4 , encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1 , a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP. [ABSTRACT FROM AUTHOR]

Background/Aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research. [ABSTRACT FROM AUTHOR]

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 ('Chicago') to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes. [ABSTRACT FROM AUTHOR]

Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.

The RFX-mod2 installation is planned to be completed by 2024 and the start of operations is expected in 2025. The high flexibility of the machine (already tested in the previous RFX-mod experiment) allows operation in Reversed Field Pinch and tokamak configuration as well as ultra-low q pulses. In this work we present predictive analysis on transport, performances and plasma control in RFX-mod2 in view of the first experimental campaigns.

Simple Summary: Prions are self-perpetuating protein aggregates that cause fatal neurodegenerative diseases in humans and other mammalian species. In yeast, in contrast to mammals, prions can be maintained in the cell population and confer adaptive traits. Fluorescence microscopy is commonly used to visualize prion aggregates in living cells, providing important information regarding the morphology and localization of prion particles in the cell. In most studies, various constructs with green fluorescent protein (GFP) are used to detect particles of the most-studied yeast prion, [PSI+]. In our work, we tried to substitute GFP with two different red fluorescent protein variants to expand the application of prion particle imaging. Surprisingly, we found that the processing of the fluorescently labeled prionogenic protein can prevent the detection of prion particles. This pattern was observed for one of the studied red fluorescent proteins (mCherry) and was not dependent on any tested protein degradation systems. The present work thus highlights the limitations of aggregate labeling with fluorescent proteins and suggests labeling with mCherry should be avoided. Yeast is a convenient model for studying protein aggregation as it is known to propagate amyloid prions. [PSI+] is the prion form of the release factor eRF3 (Sup35). Aggregated Sup35 causes defects in termination of translation, which results in nonsense suppression in strains carrying premature stop codons. N-terminal and middle (M) domains of Sup35 are necessary and sufficient for maintaining [PSI+] in cells while preserving the prion strain's properties. For this reason, Sup35NM fused to fluorescent proteins is often used for [PSI+] detection and investigation. However, we found that in such chimeric constructs, not all fluorescent proteins allow the reliable detection of Sup35 aggregates. Particularly, transient overproduction of Sup35NM-mCherry resulted in a diffuse fluorescent pattern in the [PSI+] cells, while no loss of prions and no effect on the Sup35NM prion properties could be observed. This effect was reproduced in various unrelated strain backgrounds and prion variants. In contrast, Sup35NM fused to another red fluorescent protein, TagRFP-T, allowed the detection of [PSI+] aggregates. Analysis of protein lysates showed that Sup35NM-mCherry is actively degraded in the cell. This degradation was not caused by vacuolar proteases and the ubiquitin-proteasomal system implicated in the Sup35 processing. Even though the intensity of this proteolysis was higher than that of Sup35NM-GFP, it was roughly the same as in the case of Sup35NM-TagRFP-T. Thus, it is possible that, in contrast to TagRFP-T, degradation products of Sup35NM-mCherry still preserve their fluorescent properties while losing the ability to decorate pre-existing Sup35 aggregates. This results in diffuse fluorescence despite the presence of the prion aggregates in the cell. Thus, tagging with fluorescent proteins should be used with caution, as such proteolysis may increase the rate of false-negative results when detecting prion-bearing cells. [ABSTRACT FROM AUTHOR]

We report the different properties of two types of red fluorescent proteins (RFP), undescribed species, extracted from two octocorals, Scleronephthya sp. 1 (S. sp. 1) and S. sp, 2 (Alcyonacea, Nephtheidae). S. sp. 1, named Alc‐Orange, emits strong green emission at 492 nm and weak red emission at 590 and 630 nm when excited at 449 and 574 nm, respectively. S. sp. 2, LS‐Red, emits strong deep red at 642 nm and weak green at 480 and 510 nm when excited at 574 nm and 434 nm, respectively. LS‐Red has a very large Stokes shift of about 208 nm emitting at 642 nm when excited at 434 nm. Interestingly, LS‐Red shows some emissions at 480 (blue emission), 514 (green emission), 563 (orange emission), and 642 nm (deep red emission) continuously at pH 7.5, which means multicolored fluorescence protein by one excitation at 434 nm. In pH dependence of fluorescence of Alc‐Orange (pH 13 to 3.5), no relation between 'green and red FPs' was observed, whereas LS‐Red showed the interconversion between 'green and red forms' depending on pH (11.5 to 4.5). [ABSTRACT FROM AUTHOR]

Key message: An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events. Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5′ or 3′ processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments. [ABSTRACT FROM AUTHOR]

Background: Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency. Methods and results: We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures. Conclusion: Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme. [ABSTRACT FROM AUTHOR]

The freshwater angelfish (Pterophyllum scalare Schultze, 1823) is a common aquarium species. Similar to many other cichlids, angelfish provide parental care for their embryos and larvae. In contrast to zebrafish and medaka, it is difficult to raise angelfish eggs in the lab. Therefore, obtaining angelfish embryos for transgenic purposes will also be challenging. Our aim in this study was to employ microinjection techniques to develop transgenic angelfish expressing red fluorescent protein (RFP). Using the zebrafish myosin light chain 2 (mylz2) promoter, the expression patterns of the RFP founder transgenic angelfish were analyzed. An open loop of plasmid pDsred2-1 was cloned with the 1999-bp Mylz2 promoter fragment at the SacI and AgeI sites to generate the pMylz2-RFP transgenic construct. After angelfish laid their eggs on the substrate, the embryos were carefully collected with a plastic pipette in preparation for the microinjection procedure. A single-cell-stage angelfish embryo was microinjected with pMylz2-RFP. Sixteen of 524 pMylz2-RFP microinjected embryos survived to five days post fertilization (5 dpf), with twelve displaying red fluorescence. Only two RFP-positive larvae survived to adulthood. In adult angelfish, red LED illumination clearly displayed RFP expression in trunk muscles, indicating successful transmission of the transgene. The pMylz2-RFP transgene, however, was not passed on to offspring, indicating that it was not incorporated into the germline. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.

Alternative splicing, a key regulatory process of gene expression, is controlled by trans-acting factors that recognize cis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise. Determining splicing regulators and/or upstream modulators has been difficult with low throughput, low sensitivity, and low specificity. IRAS (identifying regulators of alternative splicing) is a novel cell-based high-throughput screening strategy designed specifically to address these challenges and has achieved high throughput, high sensitivity, and high specificity. Here, we describe the IRAS method in detail with a pair of dual-fluorescence minigene reporters that produces GFP and RFP fluorescent signals to assay the two spliced isoforms exclusively. These two complementary mini-gene reporters alter GFP/RFP output ratios in the opposite direction in response to only a true splicing change. False positives from a signal screen do not stimulate opposite changes in GFP/RFP ratios. The reporter pair in conjunction with robotic liquid handlers and arrayed libraries allows IRAS to screen for both positive and negative splicing regulators with high sensitivity and specificity in a high-throughput manner.

Abstract Background Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. Result In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. Conclusions We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS’s potential molecular mechanism and will help breed B. napus hybrids.

Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.

Our laboratory previously developed a highly-invasive, triple-negative breast cancer (TNBC) variant using serial orthotopic implantation of the human MDA-MB-231 cell line in nude mice. The isolated variant was highly-invasive in the mammary gland and lymphatic channels and metastasized to lymph nodes in 10 of 12 mice compared to 2 of 12 of the parental cell line. In the present study, the tumor-selective telomerase dependent OBP-401 adenovirus was injected intratumorally (i.t.) (1 × 108 PFU) when the high-metastatic MDA-MB-231 primary tumor expressing red fluorescent protein (MDA-MB-231-RFP) reached approximately 500 mm3 (diameter; 10 mm). The mock-infected orthotopic primary tumor grew rapidly. After i.t. OBP-401 injection, the growth of the orthotopic tumors was arrested. Six weeks after implantation, the fluorescent area and fluorescence intensity showed no increase from the beginning of treatment. OBP-401 was then injected into high-metastatic MDA-MB-231-RFP primary orthotopic tumor growing in mice which already had developed metastasis within lymphatic ducts. All 7 of 7 control mice subsequently developed lymph node metastasis. In contrast, none of 7 mice which received OBP-401 had lymph node metastasis. Seven of 7 control mice also had gross lung metastasis. In contrast, none of the 7 mice which received OBP-401 had gross lung metastasis. Confocal laser microscopy imaging demonstrated that all control mice had diffuse lung metastases. In contrast, all 7 mice which received OBP-401 only had a few metastatic cells in the lung. OBP-401 treatment significantly extended survival of the treated mice.

Alternative splicing is a fundamental regulatory process of gene expression. Defects in alternative splicing can lead to various diseases, and modification of disease-causing splicing events presents great therapeutic promise. Splicing outcome is commonly affected by extracellular stimuli and signaling cascades that converge on RNA-binding splicing regulators. These trans-acting factors recognize cis-elements in pre-mRNA transcripts to affect spliceosome assembly and splice site choices. Identification of these splicing regulators and/or upstream modulators has been difficult and traditionally done by piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and low specificity of screening assays. Here we describe a unique screening strategy, IRAS (identifying regulators of alternative splicing), using a pair of dual-output minigene reporters to allow for sensitive detection of exon splicing changes. Each dual-output reporter produces green fluorescent protein (GFP) and red fluorescent protein (RFP) fluorescent signals to assay the two spliced isoforms exclusively. The two complementary minigene reporters alter GFP/RFP output ratios in the opposite direction in response to splicing change. Applying IRAS in cell-based high-throughput screens allows sensitive and specific identification of splicing regulators and modulators for any alternative exons of interest. In comparison to previous high-throughput screening methods, IRAS substantially enhances the specificity of the screening assay. This strategy significantly eliminates false positives without sacrificing sensitive identification of true regulators of splicing.

Light‐responsive proteins are widely employed in bioimaging, for example, fluorescent proteins (FPs), which are comprised of a chromophore centered within a barrel‐shaped protein. FPs exhibit remarkable one‐ and multi‐photon absorption (1PA and MPA, respectively) in addition to their emissive properties. Over the last two decades, many types of quantum mechanical, molecular dynamics, and combined quantum mechanical/molecular‐mechanical (QM/MM) approaches have been employed in the study of the photophysics of FPs. Among the latter, QM/MM approaches have proven to be capable of capturing the strong correlation between FPs' light‐responsive properties and their chromophore–environment interactions. In particular, polarizable embedding QM/MM methods are gaining attention by reason of their outstanding performance in the computation of MPA in FPs. Herein, we discuss the outcomes of some of the investigations performed on the 1PA, MPA, and emissive features of FPs using QM/MM approaches. In addition, critical aspects of the use of QM/MM approaches to study FPs' 1PA and MPA features are described. To those researchers interested in starting to perform MPA computations for FPs using QM/MM methods, this review aims to be a compass to navigate among the relevant available literature. This article is categorized under:Electronic Structure Theory > Combined QM/MM MethodsStructure and Mechanism > Computational Biochemistry and Biophysics [ABSTRACT FROM AUTHOR]

In higher eukaryotes, defects in regulated protein turnover are intimately linked to development of diseases and aging. Systematic investigation of proteostasis and protein degradation pathways is of high importance for understanding basic cellular events as well as developmental processes in higher organisms. Recently, novel fluorescent protein-based tools for monitoring protein degradation and mapping degradation pathways were described that facilitate this task. Here we give an overview of these tools and relate them to biophysical properties of fluorescent proteins. We focus on methods for the identification of degradation pathways, the discovery of novel degradation sequences, the investigation of proteome dynamics, and the characterization of protein stability. One can expect systematic application of these tools in the near future by systems biology approaches enhancing understanding of the ubiquitin-proteasome system from single protein degradation pathways to its influence on developmental processes.

En el año 2006 se identificó el primer movimiento transfronterizo de peces cebra fluorescentes al territorio peruano. Se tiene poco conocimiento del posible impacto ambiental ante la liberación descontrolada de estos peces transgénicos fluorescentes. Así como, los orígenes y flujo génico de estos organismos hidrobiológicos. Se analizó por PCR los genes de fluorescencia GFP (Proteína fluorescente verde) y RFP (Proteína fluorescente roja) en peces cebra transgénicos introducidos dentro del territorio peruano y de peces cebra procedentes de Colombia, Chile y de Taiwán como controles positivos. Todas las secuencias nucleotídicas correspondían a la proteína fluorescente roja (drFP583) de la anémona marina Discosoma sp. con un tamaño de 505pb (RFP) y de su variante verde (GFP) con un tamaño de 570pb. Asimismo, el análisis filogenético evidenció un origen asiático del movimiento transfronterizo de estos peces por una ruta sudamericana vía Colombia principalmente hacia territorio peruano.

The estimation of integral wall force using solely magnetic measurement in RFX-mod experiment is presented. The vertical and sideways forces are directly obtained from the magnetic field measured outside the vacuum vessel. Several theoretical predictions related to tokamak are also verified for the reversed field pinch configuration. The contribution of different modes to the force is also considered and analyzed. This method of calculation would be relevant for future nuclear fusion reactors where magnetic measurements will be located only outside the vacuum vessel.

Background/aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument., Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP., Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system., Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment., Competing Interests: AW, NC, and SG are employees of Analytik Jena. JR is an employee of Anticancer Inc. KM, SM, MS, BMK, and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research., (©2024 The Author(s). Published by the International Institute of Anticancer Research.)

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research., Competing Interests: The Authors declare no competing interests., (©2024 The Author(s). Published by the International Institute of Anticancer Research.)

Background/aim: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis., Materials and Methods: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer., Results: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer., Conclusion: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Colon cancer liver metastasis is often the lethal aspect of this disease. Well-isolated metastases are candidates for surgical resection, but recurrence is common. Better adjuvant treatment is therefore needed to reduce or prevent recurrence. In the present study, HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used to establish liver metastases in nude mice. Mice with a single liver metastasis were randomized into bright-light surgery (BLS) or the combination of BLS and adjuvant treatment with tumor-targeting S. typhimurium A1-R. Residual tumor fluorescence after BLS was clearly visualized at high magnification by fluorescence imaging. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase survival and disease-free survival after BLS of liver metastasis. The results suggest the future clinical potential of adjuvant S. typhimurium A1-R treatment after liver metastasis resection.

We report here a color-coded imaging model in which metastatic niches in the lung and liver of breast cancer can be identified. The transgenic green fluorescent protein (GFP)-expressing nude mouse was used as the host. The GFP nude mouse expresses GFP in all organs. However, GFP expression is dim in the liver parenchymal cells. Mouse mammary tumor cells (MMT 060562) (MMT), expressing red fluorescent protein (RFP), were injected in the tail vein of GFP nude mice to produce experimental lung metastasis and in the spleen of GFP nude mice to establish a liver metastasis model. Niche formation in the lung and liver metastasis was observed using very high resolution imaging systems. In the lung, GFP host-mouse cells accumulated around as few as a single MMT-RFP cell. In addition, GFP host cells were observed to form circle-shaped niches in the lung even without RFP cancer cells, which was possibly a niche in which future metastasis could be formed. In the liver, as with the lung, GFP host cells could form circle-shaped niches. Liver and lung metastases were removed surgically and cultured in vitro. MMT-RFP cells and GFP host cells resembling cancer-associated fibroblasts (CAFs) were observed interacting, suggesting that CAFs could serve as a metastatic niche.

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We previously reported monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor, intravitally in live mice, using a fluorescence ubiquitination-based cell-cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after cessation of chemotherapy. These results suggested why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. In the present report, we used FUCCI imaging and Gelfoam® collagen-sponge-gel histoculture, to demonstrate in real time, that the cell-cycle phase distribution of cancer cells in Gelfoam® and in vivo tumors is highly similar, whereby only the surface cells proliferate and interior cells are quiescent in G0/G1. This is in contrast to 2D culture where most cancer cells cycle. Similarly, the cancer cells responded similarly to toxic chemotherapy in Gelfoam® culture as in vivo, and very differently than cancer cells in 2D culture which were much more chemosensitive. Gelfoam® culture of FUCCI-expressing cancer cells offers the opportunity to image the cell cycle of cancer cells continuously and to screen for novel effective therapies to target quiescent cells, which are the majority in a tumor and which would have a strong probability to be effective in vivo.

Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G2/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G2/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G2/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.

Yeast is a convenient model for studying protein aggregation as it is known to propagate amyloid prions. [PSI+] is the prion form of the release factor eRF3 (Sup35). Aggregated Sup35 causes defects in termination of translation, which results in nonsense suppression in strains carrying premature stop codons. N-terminal and middle (M) domains of Sup35 are necessary and sufficient for maintaining [PSI+] in cells while preserving the prion strain’s properties. For this reason, Sup35NM fused to fluorescent proteins is often used for [PSI+] detection and investigation. However, we found that in such chimeric constructs, not all fluorescent proteins allow the reliable detection of Sup35 aggregates. Particularly, transient overproduction of Sup35NM-mCherry resulted in a diffuse fluorescent pattern in the [PSI+] cells, while no loss of prions and no effect on the Sup35NM prion properties could be observed. This effect was reproduced in various unrelated strain backgrounds and prion variants. In contrast, Sup35NM fused to another red fluorescent protein, TagRFP-T, allowed the detection of [PSI+] aggregates. Analysis of protein lysates showed that Sup35NM-mCherry is actively degraded in the cell. This degradation was not caused by vacuolar proteases and the ubiquitin-proteasomal system implicated in the Sup35 processing. Even though the intensity of this proteolysis was higher than that of Sup35NM-GFP, it was roughly the same as in the case of Sup35NM-TagRFP-T. Thus, it is possible that, in contrast to TagRFP-T, degradation products of Sup35NM-mCherry still preserve their fluorescent properties while losing the ability to decorate pre-existing Sup35 aggregates. This results in diffuse fluorescence despite the presence of the prion aggregates in the cell. Thus, tagging with fluorescent proteins should be used with caution, as such proteolysis may increase the rate of false-negative results when detecting prion-bearing cells.

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Time-lapse imaging demonstrated that tumor-targeting Salmonella typhimurium A1-R (A1-R) decoyed cancer cells in monolayer culture and in tumor spheres to cycle from G0/G1 to S/G2/M, as demonstrated by fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice also decoyed quiescent cancer cells, which were the majority of the cells in the tumors, to cycle from G0/G1 to S/G2/M, thereby making them sensitive to cytotoxic agents. The combination of A1-R and cisplatinum or paclitaxel reduced tumor size compared with A1-R monotherapy or cisplatinum or paclitaxel alone. The results of this study demonstrate that A1-R can decoy quiescent cancer cells to cycle to S/G2/M and sensitize them to cytotoxic chemotherapy. These results suggest a new paradigm of bacterial-decoy chemotherapy of cancer.

In order to develop a model for fluorescence-guided surgery (FGS), 143B human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the intramedullary cavity of the tibia in nude mice. The fluorescent areas of residual tumors after bright-light surgery (BLS) and FGS were 10.2 ± 2.4 mm(2) and 0.1 ± 0.1 mm(2) , respectively (p

Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.

CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC 50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC 50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.

Background: Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. Result: In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. Conclusions: We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS's potential molecular mechanism and will help breed B. napus hybrids. [ABSTRACT FROM AUTHOR]

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen‐derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens. [ABSTRACT FROM AUTHOR]

Currently, it is generally accepted that chromophore synthesis of red fluorescent proteins proceeds via an intermediate "blue" form with a maximum absorption at 400 nm, which directly turns into "red" form due to the formation of a double bond in the side chain of the chromophore-forming Tyr residue. In the present work, we show that the synthesis of the chromophore of a chromoprotein from Actinia equina (aeCP) proceeds via dehydration and two stages of oxidation, like in other red fluorescent proteins. However, the appearance of an additional intermediate "green" form with a maximum absorption around 530 nm is observed. [ABSTRACT FROM AUTHOR]

Axolotl limb regeneration is a fascinating characteristic that has attracted attention for several decades. Our previous studies on axolotl limb regeneration indicated that the satellite cells in the remnant muscles move distally into the blastema to regenerate new muscles that are separated by a gap from remnant muscles. Thereafter, the regenerative muscle fibers start to reconnect with remnant ones. In this study, the reconnection at the individual muscle fiber level was elucidated to test the hypothesis that this reconnection happens synchronously among involved muscles. Three pairs of EGFP+ mid-bud stage blastemas were transplanted onto freshly amputated stumps of RFP+ axolotls at the same thigh position to generate double fluorescence chimeric regenerative hindlimbs. These regenerative limbs were harvested very late far beyond they had reached the late differentiation stage. Fluorescence imaging of these limbs in cross sections revealed that in the proximal remnant part of the muscle fiber, reconnection occurred at a different pace among the muscles. In the major thigh muscle gracilis, the reconnection started from the periphery before it was completed. Furthermore, RFP+ muscle fibers contributed to muscle regeneration in the distal regenerative parts. Intriguingly, this red cell contribution was limited to ventral superficial muscles of the calf. This kind of double fluorescence chimeric limb regeneration model may help increase the understanding of the patterning of axolotl limb regeneration in late stages. [ABSTRACT FROM AUTHOR]

In higher eukaryotes, defects in regulated protein turnover are intimately linked to development of diseases and aging. Systematic investigation of proteostasis and protein degradation pathways is of high importance for understanding basic cellular events as well as developmental processes in higher organisms. Recently, novel fluorescent protein-based tools for monitoring protein degradation and mapping degradation pathways were described that facilitate this task. Here we give an overview of these tools and relate them to biophysical properties of fluorescent proteins. We focus on methods for the identification of degradation pathways, the discovery of novel degradation sequences, the investigation of proteome dynamics, and the characterization of protein stability. One can expect systematic application of these tools in the near future by systems biology approaches enhancing understanding of the ubiquitin-proteasome system from single protein degradation pathways to its influence on developmental processes. [ABSTRACT FROM AUTHOR]

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In 2006, the first transboundary movement of fluorescent zebrafish to the Peruvian territory was identified. Little is known about the possible environmental impact due to the uncontrolled release of these transgenic fluorescent fish. As well as, the origins and gene flow of these hydrobiological organisms. The fluorescence genes GFP (Green Fluorescent Protein) and RFP (Red Fluorescent Protein) in transgenic Zebrafish introduced into the Peruvian territory and Zebrafish from Colombia, Chile and Taiwan as positive controls were analyzed by PCR. All nucleotide sequences corresponded to the red fluorescent protein (drFP583) of the marine anemone Discosoma sp. with a size of 505pb (RFP) and its green variant (GFP) with a size of 570pb. Likewise, the phylogenetic analysis evidenced an Asian origin of the transboundary movement of these fish along a South American route via Colombia, mainly to Peruvian territory. [ABSTRACT FROM AUTHOR]