Photopharmacological Applications for Cherenkov Radiation Generated by Clinically Used Radionuclides.

Translational photopharmacological applications are limited through irradiation by light showing wavelengths within the bio-optical window. To achieve sufficient tissue penetration, using wavelengths >500 nm is mandatory. Nevertheless, the majority of photopharmacological compounds respond to irradiation with more energetic UV light, which shows only a minor depth of tissue penetration in the µm range. Thus, we became interested in UV light containing Cherenkov radiation (CR) induced as a by-product by clinically employed radionuclides labeling specific tissues. Therefore, CR may be applicable in novel photopharmacological approaches. To provide evidence for the hypothesis, we verified the clinically established radionuclides 68Ga and 90Y but not 18F in clinically used activities to be capable of generating CR in aqueous solutions. We then investigated whether the generated CR was able to photoactivate the caged kinase inhibitor cagedAZD5438 as a photoresponsive model system. Herein, 21% uncaging of the model system cagedAZD5438 occurred by incubation with 90Y, along with a non-specific compound decomposition for 68Ga and partly for 90Y. The findings suggest that the combination of a clinically employed radionuclide with an optimized photoresponsive agent could be beneficial for highly focused photopharmacological therapies.

Measurement of Exocytosis in Genetically Manipulated Mast Cells.

The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.

Genetically Encoded Red Photosensitizers with Enhanced Phototoxicity.

Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.

Efficient cellular uptake of click nucleic acid modified proteins.

Efficient intracellular delivery of biomacromolecules such as proteins continues to remain a challenge despite its potential for medicine. In this work, we show that mScarlet, a non cytotoxic red fluorescent protein (RFP) conjugated to Click Nucleic Acid (CNA), a synthetic analog of DNA, undergo cell uptake significantly more than either native proteins or proteins conjugated with similar amounts of DNA in MDA-MB-468 cells. We further demonstrate that the process of cell uptake is metabolically driven and that scavenger receptors and caveolae mediated endocytosis play a significant role. Co-localization studies using anti-scavenger receptor antibodies suggest that scavenger receptors are implicated in the mechanism of uptake of CNA modified proteins.

Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo.

Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, racemic mixtures, and addition of heavy metals hinders FDA approval in the United States. We developed a small ultra-red fluorescent protein, named smURFP, to have optical properties similar to the small-molecule Cy5, a heptamethine subclass of cyanine dyes (Ex/Em = 642/670 nm). smURFP has a fluorescence quantum yield of 18% and expresses so well in E. coli, that gram quantities of fluorescent protein are purified from cultures in the laboratory. In this research, the fluorescent protein smURFP was combined with bovine serum albumin into fluorescent protein nanoparticles. These nanoparticles are fluorescent with a quantum yield of 17% and 12-14 nm in diameter. The far-red fluorescent protein nanoparticles noninvasively image tumors in living mice via the enhanced permeation and retention (EPR) mechanism. This manuscript describes the use of a new fluorescent protein nanoparticle for in vivo fluorescent imaging. This protein nanoparticle core should prove useful as a biomacromolecular scaffold, which could bear extended chemical modifications for studies, such as the in vivo imaging of fluorescent protein nanoparticles targeted to primary and metastatic cancer, theranostic treatment, and/or dual-modality imaging with positron emission tomography for entire human imaging.

Herceptin: A First Assault on Oncogenes that Launched a Revolution.

This year's Lasker Clinical Research Award honors H. Michael Shepard, Dennis J. Slamon, and Axel Ullrich for their invention of Herceptin, the first monoclonal antibody that blocks a cancer-causing protein, and for its development as a life-saving therapy for women with breast cancer.

Photoprotective and Anti-Inflammatory Properties of Vina-Ginsenoside R7 Ameliorate Ultraviolet B-Induced Photodamage in Normal Human Dermal Fibroblasts.

Vina-ginsenoside R7 (R7) has been exhibited to engage in multiple pharmacological activities, such as antioxidant and anti-inflammatory activities. However, no photoaging-related studies have been performed on R7. Research is being conducted with the aim of assessing whether treatment with R7 has a protective effect on UVB-induced photoaging skin. Our results show that UVB exposure directly reduces matrix metalloproteinase (MMP) secretion through R7 by restraining the AP-1/MAPK pathway and blocks extracellular matrix (ECM) expression degradation. In addition, R7 improves the expression of transforming growth factor beta 1 (TGF-ß1), and type I procollagen also facilitates the synthesis of collagen by the TGF-ß/Smad signal transduction pathway. Finally, R7 valid blocks nuclear factor-κB (NF-κB) activation and enhances antioxidative stress capacity through activated nuclear factor (erythroid derived 2)-like 2 (Nrf2). In particular, the application of R7 restrains pro-inflammatory cytokines (TNF-α, IL-6, iNOS), which trigger ECM, degrade enzyme production, and suppress vascular endothelial growth factor (VEGF) secretion. In conclusion, R7 may constitute a promising cosmetic ingredient that can protect against skin photodamage resulting from detrimental UVB irradiation.

Death Mechanism of Breast Adenocarcinoma Cells Caused by BRET-Induced Cytotoxicity of miniSOG Depends on the Intracellular Localization of the NanoLuc-miniSOG Fusion Protein.

Photodynamic therapy (PDT) is widely used in clinical practice to influence neoplasms in the presence of a photosensitizer, oxygen, and light source. The main problem of PDT of deep tumors is the problem of delivering excitation light (without lost of its intensity) inside the body. An alternative to the external light sources can be the internal light sources based on luciferase-substrate bioluminescent systems. In our work, we used the NanoLuc-furimazine system as an internal light source. This system can be successfully used to excite the protein photosensitizer miniSOG and to induce the phototoxicity of this flavoprotein in cancer cells during bioluminescent resonance energy transfer (BRET). It was shown that the mechanism of cell death caused by BRET-induced phototoxicity of mimiSOG in the presence of furimazine depends on the intracellular localization of the NanoLuc-miniSOG fusion protein: BRET-mediated activation of miniSOG in mitochondrial localization causes apoptosis, while the membrane localization of PS causes necrosis of cancer cells.

Protein-based fluorescent bioassay for low-dose gamma radiation exposures.

The study suggests an application of a coelenteramide-containing fluorescent protein (CLM-CFP) as a simplest bioassay for gamma radiation exposures. "Discharged obelin," a product of the bioluminescence reaction of the marine coelenterate Obelia longissima, was used as a representative of the CLM-CFP group. The bioassay is based on a simple enzymatic reaction-photochemical proton transfer in the coelenteramide-apoprotein complex. Components of this reaction differ in fluorescence color, providing, by this, an evaluation of the proton transfer efficiency in the photochemical process. This efficiency depends on the microenvironment of the coelenteramide within the protein complex, and, hence, can evaluate a destructive ability of gamma radiation. The CLM-CFP samples were exposed to gamma radiation (137Cs, 2 mGy/h) for 7 and 16 days at 20 °C and 5 °C, respectively. As a result, two fluorescence characteristics (overall fluorescence intensity and contributions of color components to the fluorescence spectra) were identified as bioassay parameters. Both parameters demonstrated high sensitivity of the CLM-CFP-based bioassay to the low-dose gamma radiation exposure (up to 100 mGy). Higher temperature (20 °C) enhanced the response of CLM-CFP to gamma radiation. This new bioassay can provide fluorescent multicolor assessment of protein destruction in cells and physiological liquids under exposure to low doses of gamma radiation. Graphical abstract ᅟ.

Identification of In-Vitro Red Fluorescent Protein with Antipathogenic Activity from the Midgut of the Silkworm (Bombyx Mori L.).

BACKGROUND: The midgut of silkworm (Bombyx mori L.) plays an important role as a natural barrier and source of innate immunity. We had purified the novel red fluorescent protein (RFP) from the midgut of the silkworm Bombyx mori L. and bioassay studies confirmed RFPs possess antiviral, antifungal and antibacterial properties. N-terminal sequence of RFP analysis predicted chbp gene and it belongs to lipocalin gene family and is known to involve in anti-pathogenic activities. OBJECTIVE: The main objective of this study was to purify RFP from the midgut of Kolar Gold silkworm and confirm its antimicrobial activity. METHODS: For isolation of RFP, midgut juice was collected by brief exposure to chloroform vapours to fifth instar Kolar Gold silkworm larvae. Juice was purified by 40 % ammonium sulfate precipitation and purified by gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected. Molecular weight and purity of RFP was identified using PAGE, MALDI-TOF and HPLC. Antimicrobial property of purified RFP against BmNPV, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii was performed. N-terminal sequencing of RFP was performed using Edman degradation method. Using ten amino acid sequence, using default parameter BLAST search was performerd. From the fifth day old fifth instar silkworm midgut mRNA was isolated and cDNA was synthesized using oligo-dt primer and amplification of ChBP gene was carried out by using cDNA as the template and ChBP gene specific primers. chbp protein sequence as a input built the homology model by using SWISS-MODEL. RESULTS: RFP was purified by 40 % ammonium sulfate precipitation and gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected and SDS - PAGE revealed a size of 40 kDa. RFP purified by GFC was further reconfirmed by HPLC with a single peak with a retention time of 8.755 min. MALDI-TOF produced a peak at a molecular mass of 40 kDa. RFP from the midgut juice showed antiviral activity against the silkworm virus BmNPV, antibacterial activity against Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii. N-terminal sequencing of RFP by Edman degradation method sequenced TQTIETDYWV amino acids and BLAST analysis predicted the Chlorophyllide-a Binding Protein (chbp) with B. mori. PCR product was sequenced and obtained 911bp nucleotides encoding 302 amino acid residues and deposited with the accession number KX186723 in NCBI. Sequence analysis revealed Chbp belongs to lipocalin gene family and known to involve in antiviral, antifungal and anti-bacterial properties. Chbp gene homology model was predicted using crystal structure of insecticyanin A from the tobacco hornworm as a template. CONCLUSION: Our results indicated RFP present in midgut juice of 5th instar larvae of kolar gold silkworm. We have purified novel RFP with molecular mass of 40 kDa and showed its antipathogenic activities. Chbp gene synthesises RFP and further it could be utilized for agriculture and pharmaceutical industry.

The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo.

The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans-a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3-5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Nutritional supplements modulate fluorescent protein-bound advanced glycation endproducts and digestive enzymes related to type 2 diabetes mellitus.

BACKGROUND: Chronic hyperglycemia enhances the formation of advanced glycation endproducts (AGEs) and reactive oxygen species (ROS), contributing to diabetic complications. Thus, controlling blood glucose levels, inhibiting the formation of AGEs and reducing ROS are key therapeutic targets in early stage type 2 diabetes. METHODS: The inhibitory effects of seven commercial liquid nutritional supplements against carbohydrate hydrolysing enzymes, α-amylase and α-glucosidase, was determined by dinitrosalicylic (DNS) reagent and p-nitrophenyl-α-D-glucopyranoside solution, respectively. Antiglycation activity was determined using the formation of fluorescent protein-bound AGEs. Total phenolic and flavonoid content and antioxidant properties (1,1-diphenyl-2-picrylhydrazyl antioxidant activity (DPPH) and ferric reducing antioxidant power (FRAP)) were determined for correlation among these components and inhibitory activities. RESULTS: Samoan noni juice showed the greatest inhibitory effects against α-amylase, whereas chlorophyll extracts showed the greatest inhibitory effect against α-glucosidase. Inhibition of α-glucosidase correlated with TFC (r(2) = 0.766; p < 0.01) and FRAP (r(2) = 0.750; p < 0.01) whereas no correlation was observed for α-amylase inhibition. All supplements inhibited fluorescent protein-bound AGEs, with the greatest effect exerted by Olive Leaf Extract, Blood Sugar Support (IC50 = 0.5 mg/ml). The IC50 values negatively correlated with TPC (r(2) = -0.707; p < 0.001) and DPPH scavenging activities (r(2) = 0.515; p < 0.05). CONCLUSION: The findings of this study highlight the potential of liquid nutritional supplements in managing and treating type 2 diabetes mellitus.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.

Modeling and integral X-ray, optical, and MRI visualization of multiorgan metastases of orthotopic 4T1 breast carcinoma in BALB/c mice.

A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.

Halo-tag mediated self-labeling of fluorescent proteins to molecular beacons for nucleic acid detection.

We report here the generation of a fluorescent protein (FP)-based dual molecular beacon (MB) system for nucleic acid detection. Halo-tag mediated conjugation was used for the site-specific decoration of MBs with two different FP fusions, thereby enabling easy detection of target sequences by fluorescence resonance energy transfer or FRET. Enhanced intracellular delivery was demonstrated by simply tethering a well-known TAT peptide sequence to the N-terminus of the fusion proteins.

Multiresistant strains are as susceptible to photodynamic inactivation as their naïve counterparts: protoporphyrin IX-mediated photoinactivation reveals differences between methicillin-resistant and methicillin-sensitive Staphylococcus aureus strains.

OBJECTIVE: The current study was aimed at the investigation of differences in response to photoinactivation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. Moreover, we aimed to elucidate if the observed variation resulted from antimicrobial resistance mechanisms and strains' susceptibility to antibiotic therapy. BACKGROUND DATA: Because of the emergence of multidrug resistance, the development of alternative antimicrobial strategies seems to be required. The concept of photodynamic inactivation (PDI) involves cell exposure to appropriate wavelength light that leads to the excitation of photosensitizer molecules, resulting in the production of reactive oxygen species responsible for cell inactivation and death. Recently, we have demonstrated a strain-dependent response of S. aureus to photoinactivation, and observed elevated resistance to PDI among MRSA strains. Nevertheless, the mechanism underlying this phenomenon remains unexplained. METHODS: S. aureus response to protoporphyrin IX (PPIX)-mediated photoinactivation was studied for 424 MRSA/MSSA isolates. VITEK 2 Advanced Expert System was used to detect antimicrobial resistance mechanisms and strains' susceptibility to antibiotictherapy. RESULTS: Data obtained demonstrated that MRSA are significantly more resistant to photoinactivation than MSSA strains; however, the difference observed did not result from antimicrobial susceptibility or resistance mechanisms. Furthermore, regardless of the strains' origin, a similar effectiveness of PDI could be achieved. Moreover, it was determined that the ability to form biofilms in vitro, and the presence of mec element, does not explain the observed differences between MRSA and MSSA strains. CONCLUSIONS: PDI could be highly effective against multidrug resistant pathogens as well as their naïve counterparts. Nevertheless, regardless of the antimicrobial resistance mechanism, the difference in response to PDI between MRSA and MSSA exists.

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice.

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Intelligent design of nano-scale molecular imaging agents.

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on-off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.

Proteomic analysis of bovine nucleolus.

Nucleolus is the most prominent subnuclear structure, which performs a wide variety of functions in the eukaryotic cellular processes. In order to understand the structural and functional role of the nucleoli in bovine cells, we analyzed the proteomic composition of the bovine nucleoli. The nucleoli were isolated from Madin Darby bovine kidney cells and subjected to proteomic analysis by LC-MS/MS after fractionation by SDS-PAGE and strong cation exchange chromatography. Analysis of the data using the Mascot database search and the GPM database search identified 311 proteins in the bovine nucleoli, which contained 22 proteins previously not identified in the proteomic analysis of human nucleoli. Analysis of the identified proteins using the GoMiner software suggested that the bovine nucleoli contained proteins involved in ribosomal biogenesis, cell cycle control, transcriptional, translational and post-translational regulation, transport, and structural organization.