Organic metabolites produced by Vibrio parahaemolyticus strain An3 isolated from Goan mullet inhibit bacterial fish pathogens

Identification and action of several antibacterial metabolites produced by a fish pathogen Vibrio parahaemolyticus strain An3 from marine ecosystem of Goa has been demonstrated. Antibacterial activity of the crude cell extract of the test bacterium has been evaluated against indicator pathogenic bacterial strains such as Acinetobacter sp. An2, Aeromonas hydrophila strain An4, Staphylococcus arlettae strain An1 and Alteromonas aurentia strain SE3 by agar well diffusion method which clearly demonstrated comparatively more significant inhibitory effect on indicator bacteria as compared to several commonly used antibiotics. Gas chromatography mass spectrometry (GC-MS) analysis of crude cell extract of the test organism interestingly revealed presence of indole, phenyl acetic acid, n-(3- methyl-1, 2, 4-oxadiazol-5-yl) - 1- pyrrolidine carboximidamide, pyrrolopyrazines, tetramethyl pyrazine and other important phenolic compounds which may be responsible for antibacterial activity against indicator microorganisms tested. It has been clearly demonstrated that V. parahaemolyticus strain An3 produced several medically important organic metabolites during cultivation suggesting it as a potential candidate for production of several antibacterial metabolites to control pathogenic bacterial strains causing serious fish and human diseases.Key words: Antibacterial, gas chromatography mass spectrometry, metabolites, pathogenic bacteria, welldiffusion

Establishing Human Lacrimal Gland Cultures with Secretory Function

PURPOSE: Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in-vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures. METHODS: Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA. RESULTS: Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15-20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed 'spherules' with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons). CONCLUSION: The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future

Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis

The ophthalmology surgical competency assessment rubric for lateral tarsal strip surgery

PURPOSE: To produce an internationally valid tool to assess skill in performing lateral tarsal strip surgery. METHODS: A panel of 7 content experts adapted a previously published tool for assessing lateral tarsal strip surgery by using a modified Dreyfus scale of skill acquisition and providing behavioral descriptors for each level of skill in each category. The tools were then reviewed by 11 international content experts for their constructive comments. RESULTS: Experts\u27 comments were incorporated, establishing face and content validity. CONCLUSIONS: The tool International Council of Ophthalmology-Ophthalmology Surgical Competency Assessment Rubric for Lateral Tarsal Strip Surgery has face and content validity. It can be used globally to assess lateral tarsal strip surgical skill. Reliability and predictive validity still need to be determined

ELISA data: Secretion of tear proteins on various matrices post carbachol stimulation on day 6–7.

<p>ELISA data: Secretion of tear proteins on various matrices post carbachol stimulation on day 6–7.</p

List of antibodies and dilutions used for flow cytometry.

<p>List of antibodies and dilutions used for flow cytometry.</p

Other cell types in culture.

<p>a) Spindle shaped cells with slight granularity in their cytoplasm and distinct nucleus, are seen on uncoated culture dishes and these attain confluence within 5–7 days. b) Oval and plump cells that organize themselves in whorls are also seen. These may be myoepithelial in nature.</p

List of secondary antibodies and dilutions.

<p>List of secondary antibodies and dilutions.</p