Establishment of Clonal MIN-O Transplant Lines for Molecular Imaging via Lentiviral Transduction & In Vitro Culture

As the field of molecular imaging evolves and increasingly is asked to fill the discovery and validation space between basic science and clinical applications, careful consideration should be given to the models in which studies are conducted. The MIN-O mouse model series is an established in vivo model of human mammary precancer ductal carcinoma in situ with progression to invasive carcinoma. This series of transplant lines is propagated in vivo and experiments utilizing this model can be completed in non-engineered immune intact FVB/n wild type mice thereby modeling the tumor microenvironment with biological relevance superior to traditional tumor cell xenografts. Unfortunately, the same qualities that make this and many other transplant lines more biologically relevant than standard cell lines for molecular imaging studies present a significant obstacle as somatic genetic re-engineering modifications common to many imaging applications can be technically challenging. Here, we describe a protocol for the efficient lentiviral transduction of cell slurries derived from precancerous MIN-O lesions, in vitro culture of “MIN-O-spheres” derived from single cell clones, and the subsequent transplantation of these spheres to produce transduced sublines suitable for optical imaging applications. These lines retain the physiologic and pathologic properties, including multilineage differentiation, and complex microanatomic interaction with the host stroma characteristic of the MIN-O model. We also present the in vivo imaging and immunohistochemical analysis of serial transplantation of one such subline and detail the progressive multifocal loss of the transgene in successive generations

Automated segmentation and quantification of airway mucus with endobronchial optical coherence tomography

We propose a novel suite of algorithms for automatically segmenting the airway lumen and mucus in endobronchial optical coherence tomography (OCT) data sets, as well as a novel approach for quantifying the contents of the mucus. Mucus and lumen were segmented using a robust, multi-stage algorithm that requires only minimal input regarding sheath geometry. The algorithm performance was highly accurate in a wide range of airway and noise conditions. Mucus was classified using mean backscattering intensity and grey level co-occurrence matrix (GLCM) statistics. We evaluated our techniques in vivo in asthmatic and non-asthmatic volunteers

Citrullination of HP1γ chromodomain affects association with chromatin.

BACKGROUND: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance. RESULTS: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs. CONCLUSION: Citrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.Cancer Research UK (grant reference RG17001) Wellcome Trust (Core Grant reference WT203144) Cancer Research UK (grant reference C6946/A24843). Wellcome Trust (206291/Z/17/Z) Medical Research Council (MR/P019471/1 and MR/M010082/1). Royal Society Professorship (RP150066) Medical Research Council (MR/K015850/1

An Apical PDZ Protein Anchors the Cystic Fibrosis Transmembrane Conductance Regulator to the Cytoskeleton

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. In this study, we utilized segmental allergen challenge (SAC) of allergic asthmatics (AA) and allergic non-asthmatic controls (AC) to determine if there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin and increased MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-registered optical coherence tomography (OR-OCT). Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation

Acute Effects of Dry Needling on Myofascial Trigger Points in the Triceps Surae of Ballet Dancers: A Pilot Randomized Controlled Trial

# Background There is convincing evidence that dancers suffer injuries to the triceps surae musculature. Research on the immediate effects of dry needling (DN) is limited, and it is important to understand the acute effects of this treatment prior to performance. # Purpose The purpose of this pilot study was to assess the immediate effects of DN on myofascial trigger points in terms of skin surface temperature, pain, active and passive range of motion, and torque production in the triceps surae of ballet dancers. # Study Design Randomized, double-blinded pilot study # Methods Professional ballet dancers that fit inclusion and exclusion criteria (n=11) were randomly assigned to an experimental or control group. The dancers had three pre-determined standard point (SP) measurement spots that were used as a baseline for surface temperature comparisons. The dancers were also palpated for trigger point (TP) spots. Both SP and TP spots were marked for future measurements. The experimental group received DN, while the control group received sham DN (SHAM) to their bilateral calves at the TP spots. Immediately prior to and following treatment, both DN and SHAM groups were tested for skin surface temperature, pain, range of motion, and plantar flexion torque by blinded assessors. Paired t-tests and independent t-tests were performed to examine for differences between groups. # Results The surface temperature for the TP was higher than the SP measurements prior to intervention (Right calf p= .014; Left calf p= .031). There were no significant changes in VAS scale reported pain and ROM. The plantar flexion torque measurements showed an increase in the DN group of the left calf at the angular velocity of 60 degrees/sec. # Conclusion This was a unique pilot study examining the acute effects of DN on professional ballet dancers. The results were limited due to low sample size. However, the methodology for this study and surface temperature results invites future research. # Level of evidence Level 1