Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.

We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

Immune cell imaging using multi-spectral optoacoustic tomography.

Multispectral optoacoustic tomography (MSOT) offers the potential to image in high-resolution cells tagged with optical labels. In contrast to single wavelength imaging, multispectral excitation and spectral unmixing can differentiate labeled moieties over tissue absorption in the absence of background measurements. This feature can enable longitudinal cellular biology studies well beyond the depths reached by optical microscopy. However, the relation between spectrally resolved fluorescently labeled cells and optoacoustic detection has not been systematically investigated. Herein, we measured titrations of fluorescently labeled cells and establish the optoacoustic signal generated by these cells as a function of cell number and across different cell types. We then assess the MSOT sensitivity to resolve cells implanted in animals.

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish.

The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.

Zwilling-A and -B, two related myelin proteins of teleosts, which originate from a single bicistronic transcript.

Myelination, the ensheathment of axons by membranes of highly specialized glial cells, has been a crucial innovation during early vertebrate evolution. It enables high nerve signal conduction velocities, while maintaining nervous system size and energy requirements at moderate levels. Consequently, myelination has been conserved in all extant gnathostome vertebrates. In a genomewide mRNA expression screen, we identified several novel neural crest and myelin-specific transcripts in the zebrafish (Danio rerio). Here, we describe the characterization of two proteins, Zwilling-A and -B (ZwiA and ZwiB), which are exclusively expressed in myelinating glia of teleosts. They are structurally homologous and are translated from a common, bicistronic transcript. No similarities to sequences or domains of other proteins were detected. Analysis of phylogeny, genomic organization, and genomic syntenies suggests that the zwi gene has appeared soon after the teleost-specific genome duplication event and evolved under conservative selective pressure. We hypothesize that ZwiA and ZwiB serve important physiological functions in teleost myelin.

External electric muscle stimulation improves burning sensations and sleeping disturbances in patients with type 2 diabetes and symptomatic neuropathy.

OBJECTIVE: External muscle stimulation (EMS) of the thighs was previously shown to have beneficial effects in a pilot study on painful diabetic neuropathy. However, differential effects on specific symptoms of neuropathy as well as determinants of treatment response have not been described. DESIGN: Ninety-two type 2 diabetes patients with different neuropathic symptoms were included in a prospective uncontrolled trial. Patients were treated twice a week for 4 weeks. Symptoms were graded on numeric scales at baseline, before the second and the eighth visit. RESULTS: Seventy-three percent of the participants reported marked improvement of symptoms. Subjective treatment response was positively and independently associated with symptom intensity but independent of disease extent, metabolic factors, age, or gender. Total symptoms graded by patients on numerical scales decreased significantly after 4 weeks of treatment. Patients in the upper tertile of symptom intensity showed significant improvement of paresthesia, pain, numbness and most pronounced for burning sensations and sleeping disturbances. CONCLUSIONS: In an uncontrolled setting, EMS seems to be an effective treatment for symptomatic neuropathy in patients with type 2 diabetes, especially in patients with strong symptoms.

Deep-tissue reporter-gene imaging with fluorescence and optoacoustic tomography: a performance overview.

PURPOSE: A primary enabling feature of near-infrared fluorescent proteins (FPs) and fluorescent probes is the ability to visualize deeper in tissues than in the visible. The purpose of this work is to find which is the optimal visualization method that can exploit the advantages of this novel class of FPs in full-scale pre-clinical molecular imaging studies. PROCEDURES: Nude mice were stereotactically implanted with near-infrared FP expressing glioma cells to from brain tumors. The feasibility and performance metrics of FPs were compared between planar epi-illumination and trans-illumination fluorescence imaging, as well as to hybrid Fluorescence Molecular Tomography (FMT) system combined with X-ray CT and Multispectral Optoacoustic (or Photoacoustic) Tomography (MSOT). RESULTS: It is shown that deep-seated glioma brain tumors are possible to visualize both with fluorescence and optoacoustic imaging. Fluorescence imaging is straightforward and has good sensitivity; however, it lacks resolution. FMT-XCT can provide an improved rough resolution of ∼1 mm in deep tissue, while MSOT achieves 0.1 mm resolution in deep tissue and has comparable sensitivity. CONCLUSIONS: We show imaging capacity that can shift the visualization paradigm in biological discovery. The results are relevant not only to reporter gene imaging, but stand as cross-platform comparison for all methods imaging near infrared fluorescent contrast agents.

Some comments on valence bond representations for the radical exchange reaction X*+R:Y-->X:R+Y*.

Qualitative valence bond formulations by Hiberty and co-workers (Hiberty, P. C.; Megret, C.; Song, L.; Wu, W.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 2836) of mechanisms for the radical exchange reactions H*+F:H-->H:F+H* and F*+H:F-->F:H+F* are compared to a previously published formulation of the generalized radical exchange reaction X*+R:Y-->X:R+Y*. The former formulation uses covalent-ionic VB complexes, and the latter formulation, which is more general, involves the formation of reactant-like and product-like complexes at intermediate stages along the reaction coordinate.