Injection, detection and repair of aesthetics in home movies

This paper details the design of an algorithm for automatically manipulating the important aesthetic element of video, visual tempo. Automatic injection, detection and repair of such aesthetic elements, it is argued, is vital to the next generation of amateur multimedia authoring tools. We evaluate the performance of the algorithm on a battery of synthetic data and demonstrate its ability to return the visual tempo of the final media a considerable degree closer to the target signal. The novelty of this work lies chiefly in the systematic manipulation of this high level aesthetic element of video.

Arthroscopic repair of humeral avulsion of glenohumeral ligament lesion : anterior and posterior techniques

Humeral avulsion of the inferior glenohumeral ligament complex is an unusual pathology, often implicated in traumatic shoulder instability. Traditional open techniques involve at least partial detachment of the subscapularis, and arthroscopic techniques are limited by neurovascular boundaries. The technique described here presents an anterior and posterior arthroscopic approach that can be used individually or in combination to treat different types of humeral avulsion of the inferior glenohumeral ligament lesions. The anterior approach is based on anatomic guidelines described in the literature. The posterior approach is based on the arthroscopic and cadaveric anatomic studies described by one of the authors (D.N.B.); use of the recently described axillary pouch portal (Bhatia portal) permits safe arthroscopic instrumentation access in the entire inferior glenohumeral recess and provides adequate access to the posteroinferior aspect of the humeral head. The differential mattress stitch technique ensures secure fixation of the avulsed ligaments and eliminates excessive capsular redundancy. Technical tips to avoid complications are discussed, and a detailed rehabilitation protocol is presented.

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-κB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Non-invasive collection and examination of human corneal epithelial cells

Purpose. To report the development of a new apparatus for non-invasive collection of human corneal epithelial cells.

Methods. Previous methods of non-invasive, irrigative corneal cell collection resulted in low cell yields limiting potential analysis. A new ocular surface cell collection apparatus (OSCCA) was designed to collect more epithelial cells from direct irrigation of the corneal surface to allow for clinical comparisons. Forty-five samples were obtained (unilateral or bilateral over seven visits) from five human participants. Cell yield, size, phenotype, and corneal staining (prior and post eye wash) were examined.

Results. On average 364 ± 230 epithelial cells were collected from the cornea per eye. Epithelial cell sizes ranged from 8.21 to 51.69 μm in diameter, and 67.30 to 2098.85 μm2 area. The proportion of corneal specific cells collected per sample was 75 ± 14% as determined by positive K3 expression with AE5. On average, 77 ± 0.2% of epithelial cells harvested were nucleated, the remainder were non-nucleated ghost cells. Corneal staining was reduced in the OSCCA-washed vs. contralateral non-washed eyes (p = 0.02).

Conclusions. The OSCCA allows collection of human corneal epithelial cells with significantly higher yields, and greater specificity than previously reported. Reduced corneal staining observed post eye-wash demonstrated the safety of the technique, and its ability to remove cells directly from the corneal surface. The OSCCA could provide an objective non-invasive method of investigating pathological changes, effects of topical therapeutics, and impact of contact lenses and care-solutions of the cells of the ocular surface.

Arthroscopic repair of a chondrolabral lesion associated with anterior glenohumeral dislocation

Chondrolabral lesions are uncommon after anteroinferior glenohumeral dislocations. This report describes a new dual-lesion complex that involved an avulsion of the anteroinferior glenoid labrum and a flap tear of the adjacent articular cartilage [glenoid labral tear and articular cartilage flap (GLAF) lesion]. The chondral component involved a large undermined region of the anterior half of the lower glenoid articular cartilage, and the labral component involved an avulsion from the 2.30–6 o’clock position on the glenoid. The labral tear was reconstructed with 3 suture anchors to form a neo-labrum in an attempt to overlap and stabilize the periphery of the chondral flap. A meniscal repair device was used to place a mattress stitch in the cartilage periphery to further stabilize the flap. This technique resulted in a secure repair without any chondral damage, and this remained intact on an MRI performed at a 3-month follow-up. A final 12-month follow-up showed complete recovery, as assessed by the Oxford shoulder instability score and Rowe score, and by a return to the pre-injury sporting level.

Target-specific delivery of doxorubicin to retinoblastoma using epithelial cell adhesion molecule aptamer

PURPOSE: To study target-specific delivery of doxorubicin (Dox) using an RNA aptamer against epithelial cell adhesion molecule (EpCAM) in retinoblastoma (RB) cells. METHODS: The binding affinity of the EpCAM aptamer to RB primary tumor cells, Y79 and WERI-Rb1 cells, and Müller glial cell lines were evaluated with flow cytometry. Formation of physical conjugates of aptamer and Dox was monitored with spectrofluorimetry. Cellular uptake of aptamer-Dox conjugates was monitored through fluorescent microscopy. Drug efficacy was monitored with cell proliferation assay. RESULTS: The EpCAM aptamer (EpDT3) but not the scrambled aptamer (Scr-EpDT3) bound to RB tumor cells, the Y79 and WERI-Rb1 cells. However, the EpCAM aptamer and the scrambled aptamer did not bind to the noncancerous Müller glial cells. The chimeric EpCAM aptamer Dox conjugate (EpDT3-Dox) and the scrambled aptamer Dox conjugate (Scr-EpDT3-Dox) were synthesized and tested on the Y79, WERI-Rb1, and Müller glial cells. The targeted uptake of the EpDT3-Dox aptamer caused cytotoxicity in the Y79 and WERI-Rb1 cells but not in the Müller glial cells. There was no significant binding or consequent cytotoxicity by the Scr-EpDT3-Dox in either cell line. The EpCAM aptamer alone did not cause cytotoxicity in either cell line. CONCLUSIONS: The results show that the EpCAM aptamer-Dox conjugate can selectively deliver the drug to the RB cells there by inhibiting cellular proliferation and not to the noncancerous Müller glial cells. As EpCAM is a cancer stem cell marker, this aptamer-based targeted drug delivery will prevent the undesired effects of non-specific drug activity and will kill cancer stem cells precisely in RB.

Airway clearance of apoptotic cells in COPD

Apoptosis of bronchial epithelial cells and the phagocytic clearance of these cells by alveolar macrophages (a process termed efferocytosis) are integral processes leading to repair of airway epithelial injury. Efferocytosis allows for the removal of apoptotic material with minimal inflammation and prevents the development of secondary necrosis and ongoing inflammation. Defective efferocytosis and the increased presence of apoptotic cells have been identified in the airways of subjects with chronic obstructive pulmonary disease (COPD). There are three major potential causes for this accumulation of apoptotic cells: (i) increased apoptosis per se as a result of an increase in apoptotic mediators, (ii) defects in the recognition of apoptotic cells by AM and (iii) failure to clear the unwanted cells by the process of efferocytosis. The implications of these processes in COPD and novel treatment strategies aimed at improving clearance of apoptotic cells form the focus of the present review.

hZip1 (hSLC39A1) regulates zinc homoeostasis in gut epithelial cells

Zinc is an essential trace element required for enzyme catalysis, gene regulation and signal transduction. Zinc absorption takes place in the small intestine, however, the mechanisms by which cells accumulate zinc are not entirely clear. Zip1 (SLC39A1) is a predicted transmembrane protein that is postulated, but not conclusively proven to mediate zinc influx in gut cells. The aim of this study was to investigate a role for hZip1 in mediating zinc uptake in human enterocytes. Both hZip1 mRNA and protein were detected in human intestinal tissue. In non-differentiated Caco-2 human gut cells, hZip1 was partially localised to the endoplasmic reticulum. In contrast, in differentiated Caco-2 cells cultured in extracellular matrix, the hZip1 protein was located in proximity to the apical microvilli. Lack of surface antibody binding and internalisation indicated that hZip1 was not present on the plasma membrane. Functional studies to establish a role for hZip1 in cellular zinc accumulation were carried out using 65Zn. In Caco-2 cells harbouring an hZip1 overexpression construct, cellular zinc accumulation was enhanced relative to the control. Conversely, Caco-2 cells with an hZip1 siRNA construct showed reduced zinc accumulation. In summary, we show that the Caco-2 cell differentiation endorses targeting of hZip1 to a region near the apical domain. Given the absence of hZip1 at the apical plasma membrane, we propose that hZip1 may act as an intracellular sensor to regulate zinc homoeostasis in human gut cells.

Copper homeostasis in human mammary epithelial cells

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. This thesis analysed copper transporting proteins in mammary epithelial cells and the impact of copper and lactational hormones upon the regulation these proteins was measured.

Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38±9 and 67±8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.