Single-Dose Intravenous Toxicity Study of IRDye 800CW in Sprague-Dawley Rats

Objective: Fluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate. Methods: Male and female Sprague–Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14 days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes. Results: Based on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity. Conclusion: A dose of 20 mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW

Vision 20/20: Molecular-guided surgical oncology based upon tumor metabolism or immunologic phenotype: Technological pathways for point of care imaging and intervention

Surgical guidance with fluorescence has been demonstrated in individual clinical trials for decades, but the scientific and commercial conditions exist today for a dramatic increase in clinical value. In the past decade, increased use of indocyanine green based visualization of vascular flow, biliary function, and tissue perfusion has spawned a robust growth in commercial systems that have near-infrared emission imaging and video display capabilities. This recent history combined with major preclinical innovations in fluorescent-labeled molecular probes, has the potential for a shift in surgical practice toward resection guidance based upon molecular information in addition to conventional visual and palpable cues. Most surgical subspecialties already have treatment management decisions partially based upon the immunohistochemical phenotype of the cancer, as assessed from molecular pathology of the biopsy tissue. This phenotyping can inform the surgical resection process by spatial mapping of these features. Further integration of the diagnostic and therapeutic value of tumor metabolism sensing molecules or immune binding agents directly into the surgical process can help this field mature. Maximal value to the patient would come from identifying the spatial patterns of molecular expression in vivo that are well known to exist. However, as each molecular agent is advanced into trials, the performance of the imaging system can have a critical impact on the success. For example, use of pre-existing commercial imaging systems are not well suited to image receptor targeted fluorophores because of the lower concentrations expected, requiring orders of magnitude more sensitivity. Additionally the imaging system needs the appropriate dynamic range and image processing features to view molecular probes or therapeutics that may have nonspecific uptake or pharmacokinetic issues which lead to limitations in contrast. Imaging systems need to be chosen based upon objective performance criteria, and issues around calibration, validation, and interpretation need to be established before a clinical trial starts. Finally, as early phase trials become more established, the costs associated with failures can be crippling to the field, and so judicious use of phase 0 trials with microdose levels of agents is one viable paradigm to help the field advance, but this places high sensitivity requirements on the imaging systems used. Molecular-guided surgery has truly transformative potential, and several key challenges are outlined here with the goal of seeing efficient advancement with ideal choices. The focus of this vision 20/20 paper is on the technological aspects that are needed to be paired with these agents

CARBON-13 NMR STUDIES OF CONFIGURATION AND CONFORMATION

Abstract not availabl

Genetic Architecture of Conspecific Sperm Precedence in Allonemobius fasciatus and A. socius

The evolution of barriers to gene exchange is centrally important to speciation. We used the crickets Allonemobius fasciatus and A. socius to investigate the genetic architecture of conspecific sperm precedence (CSP), a postinsemination prezygotic reproductive barrier. With amplified fragment-length polymorphism (AFLP) markers and controlled crosses we constructed linkage maps and estimated positions of QTL associated with CSP. The majority of QTL have low to moderate effects, although a few QTL exist in A. socius with large effects, and the numbers of QTL are comparable to numbers of genes accounting for species differences in other studies. The QTL are spread across many unlinked markers, yet QTL placed with linked markers are on a small number of linkage groups that could reflect the role of the large Allonemobius sex chromosome in prezygotic isolation. Although many QTL had positive effects on conspecific sperm utilization several QTL also exerted negative effects, which could be explained by intraspecific sexual conflict, sperm competition, or epistasis of introgressed genes on novel backgrounds. One unexpected outcome was that A. socius CSP alleles have a stronger effect than those from A. fasciatus in hybrid females, causing hybrids to behave like A. socius with regard to sperm utilization. Implications of this asymmetry in the Allonemobius hybrid zone are discussed

Near-Infrared Fluorescence Imaging of Mammalian Cells and Xenograft Tumors with SNAP-Tag

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP f is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAPf. This property makes SNAPf a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP f-Beta-2 adrenergic receptor (SNAP f-ADRb2) fusion protein were created. The ADRb2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAPf-ADRb2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAPf-ADRb2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAPf-ADRb2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP f-ADRb2 expressing tumor, possibly du

β-Hydroxybutyrate improves β-cell mitochondrial function and survival

Pharmacological interventions aimed at improving outcomes in type 2 diabetes and achieving normoglycaemia, including insulin therapy, are increasingly common, despite the potential for substantial side effects. Carbohydrate-restricted diets that result in increased ketogenesis have effectively been used to improve insulin resistance, a fundamental feature of type 2 diabetes. In addition, limited evidence suggests that states of ketogenesis may also improve β-cell function in type 2 diabetics. Considering how little is known regarding the effects of ketones on β-cell function, we sought to determine the specific effects of β-Hydroxybutyrate (βHB) on pancreatic β-cell physiology and mitochondrial function. βHB treatment increased β-cell survival and proliferation, while also increasing mitochondrial mass, respiration and adenosine triphosphate (ATP) production. Despite these improvements, were unable to detect an increase in β-cell or islet insulin production and secretion. Collectively, these findings have two implications. Firstly, they indicate that β-cells have improved survival and proliferation in the midst of βHB, the circulating form of ketones. Secondly, insulin secretion does not appear to be directly related to apparent improvements in mitochondrial function and cellular proliferation

Concentration dependent reaction of BG-800 with stable cells.

<p>293-SNAP_f (A), MDA-SNAP_f (B) and SKOV-SNAP_f (C) stable cells were reacted with different concentrations of BG-800. The fluorescence signals from the reaction were scanned on Odyssey Infrared Imaging System and quantified. TO-PRO-3 staining was used as the internal control. The insets represent scanned images of BG-800 (100 nM) stained stable cells (SNAP_f) and control cells (Ctrl). RFU, relative fluorescence unit.</p

Reaction of BG-800, CBG-800-QC1 and CBG-800-PEG-QC1 substrates with purified SNAP_f-EGF protein.

<p>(A) BG substrates (100 nM) were incubated with SNAP_f-EGF protein (15 ng/µl) in a 96-well plate. The plate was scanned after different time periods of reaction to measure the fluorescence signal. (B) The reaction mixtures of SNAP_f-EGF with different BG substrates were resolved on a SDS-PAGE gel and scanned to visualize the labeled protein.</p

Reaction of BG-800 with HEK293 cells expressing SNAP_f-ADRβ2 protein.

<p>HEK293 was transiently transfected with SNAP_f-ADRβ2 expression plasmid. After 2 d of culture, cells were reacted with BG-800. The fluorescence signal on the cells was measured by scanning on Odyssey Infrared Imaging System. HEK293 cells transfected with empty vector were used as a control. Shown in the inset is a representative microscopic image of BG-800 stained HEK293 cells expressing SNAP_f-ADRβ2.</p

<i>In vivo</i> imaging with BG-800 substrate.

<p>(A) Xenograft tumors were established using either SK-OV-3 parental cells as the control (Tm-C, left side) or SKOV-SNAP_f stable cells (Tm-S, right side). The mice were imaged 24 h after i.v. injection of 10 nmol of BG-800 substrate. The tumors were indicated with arrows. The tumors were dissected and imaged <i>ex vivo</i> after whole animal imaging (lower left corner inset). (B) Tissue distribution of BG-800. Tissues were dissected 24 h after BG-800 administration and imaged. Note that the tissues were from a different mouse as shown in (A). Ht, Heart; Int, intestine; Kd, kidney; Lg, lung; Lv, liver; Ms, muscle; Sk, skin; Spl, spleen; Tm-C, SK-OV-3 tumor; Tm-S, SKOV-SNAP_f tumor. (C) Gel analysis of tissue lysates from Ms, Tm-C, Tm-S and SKOV-SNAP_f cell lysate reacted with BG-800 (Cell-S).</p