Fluorescence Imaging and Spectroscopy of Biomaterials in Air and Liquid by Scanning Near-Field Optical/Atomic Force Microscopy

We have developed scanning near-field optical/atomic force microscopy (SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fiber cantilever is 15-40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/ AFM system contains photon counting system and polychrometer/intensified coupled charge devise (ICCD) system to observe fluorescence image and spectrograph of micro areas, respectively. Cultured cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-keratin antibody or FITC-labeled phalloidin after treatment with Triton X-100. Fluorescence and topographic images were obtained in air and water. The fluorescence images showed clear images of keratin and actin filaments. The SNOM/AFM is perfect to observe biomaterials in liquid with a liquid chamber while the topographic Images showed subcellular structures which correspond to keratin and actin filaments

The cartilage matrisome in adolescent idiopathic scoliosis

The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called "matrisome" and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility

Lipid phosphate phosphatase 3 enables efficient thymic egress

Lipid phosphate phosphatase 3 in endothelial and epithelial cells promotes efficient T cell emigration from the thymus to the periphery

In Vivo Imaging of HIF-Active Tumors by an Oxygen-Dependent Degradation Protein Probe with an Interchangeable Labeling System

Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases