Nerve Visualization using Phenoxazine-Based Near-Infrared Fluorophores to Guide Prostatectomy.

Fluorescence-guided surgery (FGS) is poised to revolutionize surgical medicine through near-infrared (NIR) fluorophores for tissue- and disease-specific contrast. Clinical open and laparoscopic FGS vision systems operate nearly exclusively at NIR wavelengths. However, tissue-specific NIR contrast agents compatible with clinically available imaging systems are lacking, leaving nerve tissue identification during prostatectomy a persistent challenge. Here, it is shown that combining drug-like molecular design concepts and fluorophore chemistry enabled the production of a library of NIR phenoxazine-based fluorophores for intraoperative nerve-specific imaging. The lead candidate readily delineated prostatic nerves in the canine and iliac plexus in the swine using the clinical da Vinci Surgical System that has been popularized for minimally invasive prostatectomy procedures. These results demonstrate the feasibility of molecular engineering of NIR nerve-binding fluorophores for ready integration into the existing surgical workflow, paving the path for clinical translation to reduce morbidity from nerve injury for prostate cancer patients.

Rhodium(II)-Catalyzed Hinsberg Dearomatization Using Trimethylsilyldiazomethane.

Rhodium(II) catalyzes carbene transfer from trimethylsilyldiazomethane to arylmethyl thioethers, generating sulfonium ylides that undergo [2,3]-sigmatropic rearrangement, punching quaternary centers into aromatic rings. The reaction works well with naphthalene, indole, and benzofuran ring systems, but the reaction is unsuccessful with the monocyclic benzene homologue. For aryl thioethers, Rh2(OAc)4 gives good results. For alkyl thioethers, the yields improve with Rh2(cap)4. Surprisingly, thioesters and thiocarbamates are also competent substrates for the reaction.

OregonFluor enables quantitative intracellular paired agent imaging to assess drug target availability in live cells and tissues.

Non-destructive fluorophore diffusion across cell membranes to provide an unbiased fluorescence intensity readout is critical for quantitative imaging applications in live cells and tissues. Commercially available small-molecule fluorophores have been engineered for biological compatibility, imparting high water solubility by modifying rhodamine and cyanine dye scaffolds with multiple sulfonate groups. The resulting net negative charge, however, often renders these fluorophores cell-membrane-impermeant. Here we report the design and development of our biologically compatible, water-soluble and cell-membrane-permeable fluorophores, termed OregonFluor (ORFluor). By adapting previously established ratiometric imaging methodology using bio-affinity agents, it is now possible to use small-molecule ORFluor-labelled therapeutic inhibitors to quantitatively visualize their intracellular distribution and protein target-specific binding, providing a chemical toolkit for quantifying drug target availability in live cells and tissues.

Tandem Insertion/[3,3]-Sigmatropic Rearrangement Involving the Formation of Silyl Ketene Acetals by Insertion of Rhodium Carbenes into S-Si Bonds.

Allyl 2-diazo-2-phenylacetates are shown to react with trimethylsilyl thioethers in the presence of rhodium(II) catalysts to generate α-allyl-α-thio silyl esters. The transformation involves a tandem process involving formal rhodium-catalyzed insertion of the carbene group into the S-Si bond to generate a silyl ketene acetal, followed by a spontaneous Ireland-Claisen rearrangement. The silyl ester products were isolated as the corresponding carboxylic acids after aqueous workup. Intramolecular cyclopropanation of the allyl fragment generally competes with addition of the heteroatom to the carbene center. The reaction occurs under mild conditions and in high yield, allowing for rapid entry into rearrangement tetrasubstituted products. Propargyl esters were shown to generate the corresponding α-allenyl products.

Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA.

Deoxyribonucleic acid (DNA) can be extracted from different tissue sources for genotyping of mice. Methods of collecting tissue vary with respect to their perceived invasiveness, and in some cases, tissue is collected multiple times in order to verify a genotype. The authors' goal was to refine and optimize tissue collection methods with quantitative measures for subsequent genotyping. To do this, the authors used real-time quantitative polymerase chain reaction (qPCR) analysis to quantify DNA extracted from fecal pellets, hair, buccal swab samples, ear punch samples and tail biopsy samples and then compared the quantities of DNA obtained by using either previously published protocols or commercial kits to extract DNA. They found that 2-mm tail biopsy samples yielded significantly more DNA than did the other sources and that commercial extraction kits generally yielded more DNA than did published protocols. The authors also assessed the stability of DNA extracted from the various tissue sources by repeating the qPCR analysis after the samples had been frozen and stored for 44 months. Although the quantities of DNA in the stored samples had decreased, all the samples could still be used for PCR-based genotyping. The authors' work supports the collection of a single minimal biopsy sample of 2 mm of tail or ear tissue, above other sources, for highest yield of DNA for genotyping. With appropriate storage, DNA remains usable for PCR-based genotyping for years without a need for repeated animal sampling.

Investigation of Oxazine and Rhodamine Derivatives as Peripheral Nerve Tissue Targeting Contrast Agent for In Vivo Fluorescence Imaging.

Accidental nerve transection or injury is a significant morbidity associated with many surgical interventions, resulting in persistent postsurgical numbness, chronic pain, and/or paralysis. Nerve-sparing can be a difficult task due to patient-to-patient variability and the difficulty of nerve visualization in the operating room. Fluorescence image-guided surgery to aid in the precise visualization of vital nerve structures in real time during surgery could greatly improve patient outcomes. To date, all nerve-specific contrast agents emit in the visible range. Developing a near-infrared (NIR) nerve-specific fluorophore is poised to be a challenging task, as a NIR fluorophore must have enough "double-bonds" to reach the NIR imaging window, contradicting the requirement that a nerve-specific agent must have a relatively low molecular weight to cross the blood-nerve-barrier (BNB). Herein we report our efforts to investigate the molecular characteristics for the nerve-specific oxazine fluorophores, as well as their structurally analogous rhodamine fluorophores. Specifically, optical properties, physicochemical properties and their in vivo nerve specificity were evaluated herein.

Thrombospondin-2 is an endogenous adipocyte inhibitor.

The matricellular protein thrombospondin-2 (TSP2) inhibits proliferation and enhances osteoblastogenesis of multipotent mesenchymal progenitor cells (MPC). Osteoblastogenesis and adipogenesis are reciprocally regulated, thus we hypothesized that TSP2 could be an inhibitor of adipogenesis. TSP2 gene expression is up-regulated during MPC osteoblast differentiation and down-regulated during adipogenic differentiation through a cAMP-PKA pathway, relative to control cells. Next, the importance of TSP2 in adipogenesis was studied by comparing gene-targeted knockout mice that lack TSP2 protein (TSP2-null) and control wild-type mice. TSP2-null marrow-derived MPC show 25% increased adipocytes. Similarly, TSP2-null adipose-derived MPC show increased adipocytes (25-50%) and proliferation (2-fold) relative to wild-type cells. However, the increase in TSP2-null adipocytes is not due to an increase in MPC number, because the percentage of adipocytes relative to total MPC is still significantly increased. Surprisingly, there are only minor alterations in the adipogenic transcriptional program (PPARγ and C/EBPα expression). Weight gain over time was evaluated in TSP2-null and control wild-type mice fed normal and high-fat diets. Female TSP2-null mice are 30% heavier than wild-type controls due to an increase in non-visceral adipose tissue, measured by dual-energy X-ray absorptiometry (DEXA) and direct weighing of fat pads. These results show that TSP2 is an endogenous matricellular protein produced by MPC that in addition to enhancing osteoblastogenesis, inhibits adipocytes and decreases subcutaneous adiposity.