Imaging Drug Uptake by Bioorthogonal Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy in tandem with bioorthogonal Raman labelling strategies is set to revolutionise the direct visualisation of intracellular drug uptake. Rational evaluation of a series of Raman-active labels has allowed the identification of highly active labels which have minimal perturbation on the biological efficacy of the parent drug. Drug uptake has been correlated with markers of cellular composition and cell cycle status, and mapped across intracellular structures using dual-colour and multi-modal imaging. The minimal phototoxicity and low photobleaching associated with SRS microscopy has enabled real-time imaging in live cells. These studies demonstrate the potential for SRS microscopy in the drug development process

Kinetic analysis of bioorthogonal reaction mechanisms using Raman microscopy

Raman spectroscopy is well-suited to the study of bioorthogonal reaction processes because it is a non-destructive technique, which employs relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum enabling live cell imaging. In addition, Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains. Combined with the rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), this technique has the potential to revolutionise our mechanistic understanding of the biochemical and medicinal chemistry applications of bioorthogonal reactions. Current approaches to the kinetic analysis of bioorthogonal reactions (including heat flow calorimetry, UV-vis spectroscopy, fluorescence, IR, NMR and MS) have a number of practical shortcomings for intracellular applications. We highlight the advantages offered by Raman microscopy for reaction analysis in the context of both established and emerging bioorthogonal reactions, including the copper(i) catalysed azide-alkyne cycloaddition (CuAAC) click reaction and Glaser-Hay coupling

Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells

Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug dis-tribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and speci-ficity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, na-nomolar concentrations; allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understanding of target engagement

The bovine paranasal sinuses: Bacterial flora, epithelial expression of nitric oxide and potential role in the in-herd persistence of respiratory disease pathogens

peer-reviewedThe bovine paranasal sinuses are a group of complex cavernous air-filled spaces, lined by respiratory epithelium, the exact function of which is unclear. While lesions affecting these sinuses are occasionally reported in cattle, their microbial flora has not been defined. Furthermore, given that the various bacterial and viral pathogens causing bovine respiratory disease (BRD) persist within herds, we speculated that the paranasal sinuses may serve as a refuge for such infectious agents. The paranasal sinuses of clinically normal cattle (n = 99) and of cattle submitted for post-mortem examination (PME: n = 34) were examined by microbial culture, PCR and serology to include bacterial and viral pathogens typically associated with BRD: Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica and Pasteurella multocida, bovine respiratory syncytial virus (BRSV) and bovine parainfluenza-3 virus (BPIV-3). Overall, the paranasal sinuses were either predominantly sterile or did not contain detectable microbes (83.5%: 94.9% of clinically normal and 50.0% of cattle submitted for PME). Bacteria, including BRD causing pathogens, were identified in relatively small numbers of cattle (<10%). While serology indicated widespread exposure of both clinically normal and cattle submitted for PME to BPIV-3 and BRSV (seroprevalences of 91.6% and 84.7%, respectively), PCR identified BPIV-3 in only one animal. To further explore these findings we investigated the potential role of the antimicrobial molecule nitric oxide (NO) within paranasal sinus epithelium using immunohistochemistry. Expression of the enzyme responsible for NO synthesis, inducible nitric oxide synthase (iNOS), was detected to varying degrees in 76.5% of a sub-sample of animals suggesting production of this compound plays a similar protective role in the bovine sinus as it does in humans

Robotic Testing of Proximal Tibio-Fibular Joint Kinematics for Measuring Instability Following Total Knee Arthroplasty

Pain secondary to instability in total knee arthroplasty (TKA) has been shown to be major cause of early failure. In this study, we focused on the effect of instability in TKA on the proximal tibio-fibular joint (PTFJ). We used a robotics model to compare the biomechanics of the PTFJ in the native knee, an appropriately balanced TKA, and an unbalanced TKA. The tibia (n = 5) was mounted to a six-degree-of-freedom force/torque sensor and the femur was moved by a robotic manipulator. Motion at the PTFJ was recorded with a high-resolution digital camera system. After establishing a neutral position, loading conditions were applied at varying flexion angles (0°, 30°, and 60°). These included: internal/external rotation (0 Nm, ±5 Nm), varus/valgus (0 Nm, ±10 Nm), compression (100 N, 700 N), and posterior drawer (0 N, 100 N). With respect to anterior displacement, external rotation had the largest effect (coefficient = 0.650; p \u3c 0.0001). Polyethylene size as well as the interaction between polyethylene size and flexion consistently showed substantial anterior motion. Flexion and mid-flexion instability in TKA have been difficult to quantify. While tibio-femoral kinematics is the main aspect of TKA performance, the effects on adjacent tissues should not be overlooked. Our data show that PTFJ kinematics are affected by the balancing of the TKA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:47–52, 201