Universal membrane-labeling combined with expression of Katushka far-red fluorescent protein enables non-invasive dynamic and longitudinal quantitative 3D dual-color fluorescent imaging of multiple bacterial strains in mouse intestine.

BACKGROUND: The human gastrointestinal (GI) tract microbiota has been a subject of intense research throughout the 3rd Millennium. Now that a general picture about microbiota composition in health and disease is emerging, questions about factors determining development of microbiotas with specific community structures will be addressed. To this end, usage of murine models for colonization studies remains crucial. Optical in vivo imaging of either bioluminescent or fluorescent bacteria is the basis for non-invasive detection of intestinal colonization of bacteria. Although recent advances in in vivo fluorescence imaging have overcome many limitations encountered in bioluminescent imaging of intestinal bacteria, such as requirement for live cells, high signal attenuation and 2D imaging, the method is still restricted to bacteria for which molecular cloning tools are available. RESULTS: Here, we present usage of a lipophilic fluorescent dye together with Katushka far-red fluorescent protein to establish a dual-color in vivo imaging system to monitor GI transit of different bacterial strains, suitable also for strains resistant to genetic labeling. Using this system, we were able to distinguish two different E. coli strains simultaneously and show their unique transit patterns. Combined with fluorescence molecular tomography, these distinct strains could be spatially and temporally resolved and quantified in 3D. CONCLUSIONS: Developed novel method for labeling microbes and identify their passage both temporally and spatially in vivo makes now possible to monitor all culturable bacterial strains, also those that are resistant to conventional genetic labeling.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Circularly Permuted Far-Red Fluorescent Proteins.

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the "optical window" above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

Photoelectric Silk via Genetic Encoding and Bioassisted Plasmonics.

Genetically encoded photoelectric silk that can convert photons to electrons (light to electricity) over a wide visible range in a self-power mode is reported. As silk is a versatile host material with electrical conductivity, biocompatibility, and processability, a photoelectric protein is genetically fused with silk by silkworm transgenesis. Specifically, mKate2, which is conventionally known as a far-red fluorescent protein, is used as a photoelectric protein. Characterization of the electrochemical and optical properties of mKate2 silk allows designing a photoelectric measurement system. A series of in situ photocurrent experiments support the sensitive and stable performance of photoelectric conversion. In addition, as a plasmonic nanomaterial with a broad spectral resonance, titanium nitride (TiN) nanoparticles are biologically hybridized into the silk glands, taking full advantage of the silkworms' open circulatory system as well as the absorption band of mKate2 silk. This biological hybridization via direct feeding of TiN nanoparticles further enhances the overall photoelectric conversion ability of mKate2 silk. It is envisioned that the biologically derived photoelectric protein, its ecofriendly scalable production by transgenic silkworms, and the bioassisted plasmonic hybridization can potentially broaden the biomaterial choices for developing next-generation biosensing, retina prosthesis, and neurostimulation applications.

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins.

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the "window of tissue transparency". The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A.