Knockdown of S1PR2 attenuated S1PR2, p-PI3K, p-ERK, p-JNK, p-p38, and p-NF-kBp65 protein expressions in BMMs.

Murine BMMs were uninfected, infected with a S1PR2 shRNA lentivirus, or infected with a control shRNA lentivirus (moi 50) for 72 h. Then the cells were either unstimulated or stimulated with Aa (1.5 CFU/cell) for 1 to 4 h. (A) S1PR2, p-PI3K, p-ERK, p-JNK, p-p38, and p-NF-κBp65 protein expressions were evaluated by Western Blot. (B) S1PR2 protein density, (C) p-PI3K protein density, (D) p-ERK protein density, (E) p-JNK protein density, (F) p-p38 protein density, and (G) p-NF-κBp65 protein density were analyzed and normalized by GAPDH protein expression. The data are representatives from three separate experiments (n = 3, *PPP<0.001).</p

Secretory pathway of the filamentous fungus Trichoderma reesei

"Bottlenecks for overproduction of proteins in filamentous fungi possibly exist within the secretory pathway, therefore better understanding of this pathway is a key to achieving better yields. “Visible” data with high spatial and temporal resolution of morphology of the hyphal compartments, protein localisation, expression and secretion would need to be added to the existing knowledge to help understand protein secretion and to devise strategies for the improvement of protein production. A series of expression plasmids/cassettes containing a gene encoding the fluorescent protein(s) GFP2 and/or VenusYFP alone or fused to the ER-resident folding chaperone Bip1 and the main cellobiohydrolase I (CBHI) were constructed and introduced into a Trichoderma reesei strain Rut C-30. A transformant strain BV47 expressing the Bip1-Venus fusion protein was applied to visualise the endoplasmic reticulum and potential changes in the ER during Bip1-Venus overexpression. A transformant strain CV48 secreting the main cellobiohydrolase I of T. reesei fused with VenusYFP was used to monitor secretion of the CBHI-Venus fusion protein. In order to investigate the potential interaction between the Bip1 and the secretory protein CBHI, a GFP2/VenusYFP FRET pair system was developed. In the developed FRET system, the transformant strains BG29 expressing Bip1-GFP2, CV48 expressing CBHI-Venus, VG15 expressing Venus-GFP2 and BGCV101 coexpressing Bip1-GFP2 served as the donor, the acceptor, the positive FRET control and the FRET sample, respectively. The ER in the host strain T. reesei Rut C-30 was visualised as a typical network of parallel tubular membranes and some punctate-like bodies through the hyphae. The ER structure in the transformant BV47 expressing the Bip1-Venus fusion protein appeared unusual with an abundance of punctate structures and fewer tubular membranes demonstrating modified spatial organisation of the ER, different to what has been seen in other filamentous fungi studied so far. This type of modification of the ER may assist in forming an ER sub-domain to which overproduced and potentially misfolded proteins can be deposited to wait for further processing. The ER structural modifications appeared to have been caused by overproduction of the BiP1-Venus fusion protein. In addition to the changes in the ER morphology in T. reesei, it was also noted that BiP1 appeared to have escaped from the ER and become secreted into the culture medium, possibly due to overloading of the ER retention capability. Light microscopy and immunoelectron microscopy studies confirmed that the Golgi apparatus in T. reesei appeared in punctate bodies that were not surrounded with obvious membranes. The Golgi membrane invisibility could be associated with the chemical fixation method used in this study which failed to preserve the delicate Golgi membranes. The morphological characteristics of the Golgi apparatus in T. reesei observed in this study were different to those previously reported for T. reesei and yeast and were likely due to different methods for sample preparation and observation i.e. immunoelectron microscopy staining or ultrastructural observation without specific staining. Secretion of the CBHI-Venus fusion protein in the T. reesei transformant CV48 was tracked both intracellularly and extracellularly. Intracellular fluorescence of CBHI-Venus in CV48 was detected at the 12 h time point and was in line with the detection of cbh1-venus transcript at 12 h. There was a 6 h time lag between the first presence of intracellular fluorescence and the detectable level of CBHI-Venus in the culture supernatant at 12 h. Subcellular localisation of the fusion protein was studied by both light microscopy and immunoelectron microscopy. In addition to association with a typical ER network, the CBHI-Venus protein was found localised in distorted ER membrane structures assumed as ER-derived sub-domains similar to what seen in the BV47 overexpressing BiP1-Venus at both 24 and 48 h. The modification of the ER organisation and formation of ER sub-domains in the CV48 transformant with at least two copies of venus gene was assumed as a result of overexpression of the fusion protein. At the early culture stages of 24 and 48 h, CBHI-Venus also localised in the vesicles and Golgi bodies. After 24 h, the protein concentrated into the secretory vesicles for transport. Interestingly, the fusion protein was retained in the cell wall since 72 h, which was possibly the cause for reduction of secretion into the culture medium at the later stages of cultivation. Vacuolation of the hyphae occurred at 120 h. In general, secretion of the fusion protein CBHI-Venus follows the conventional secretory pathway through the ER to Golgi via secretory vesicles. In this study, a FRET system was developed to analyse the interaction between the ER-resident chaperone BiP1 and the highly secreted protein CBHI using GFP2 and VenusYFP as fusion partners to CBHI and BiP1. The positive control, transformant VG15 expressing venus-gfp2 demonstrated positive FRET signals although at a considerably low level. However the transformant BGCV101 coexpressing BiP-GFP2 and CBHI-Venus did not show significant FRET efficiency. This observation could be a result of multiple reasons as discussed in the thesis. The FRET work conducted here will aid in developing an efficient FRET tool for studying protein interactions in living filamentous fungi." -- Abstrac

Knockdown of S1PR2 suppressed osteoclastogenesis in BM cells induced by RANKL.

<p>BM cells were uninfected, infected with a S1PR2 shRNA lentivirus, or infected with a control shRNA lentivirus (moi 20), and co-cultured with M-CSF and RANKL as described in Methods. A control group of cells were cultured only with M-CSF. BM cells were either untreated or treated with <i>A</i>. <i>actinomycetemcomitans</i>-stimulated media (<i>Aa</i>-media) for 24 h. (A) Representative images show TRAP-stained cells with and without <i>Aa</i>-media stimulation. Pictures were taken at 100x magnification. (B) Number of TRAP⁺ multinucleated (more than 3 nuclei) osteoclasts/well (96-well) and (C) Total areas of osteoclasts/image were quantified. The data are representatives from three separate experiments (n = 3, ***<i>P</i><0.001).</p

Knockdown of S1PR2 inhibited bone resorption in BM cells induced by RANKL.

<p>BM cells were uninfected, infected with a S1PR2 shRNA lentivirus, or infected with a control shRNA lentivirus (moi 20), and co-cultured with M-CSF and RANKL, with or without <i>A</i>. <i>actinomycetemcomitans</i>-stimulated media (<i>Aa</i>-media) treatment as described in Methods. Control groups of cells were cultured only with M-CSF with or without <i>Aa</i>-media treatment. (A) Representative images show bone resorption pits. Pictures were taken at 100x magnification. (B) Total areas of bone resorption pits /image were quantified. The data are representatives from three separate experiments (n = 3, *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Knockdown of S1PR2 significantly attenuated Nfatc1, Ctsk, Acp5, Oscar, Dcstamp, and Ocstamp mRNA expressions in BM cells with or without treatment with <i>A</i>. <i>actinomycetemcomitans</i>-stimulated media (<i>Aa</i>-media).

<p>BM cells were uninfected, infected with a S1PR2 shRNA lentivirus, or infected with a control shRNA lentivirus (moi 20), and co-cultured with M-CSF and RANKL as described in Methods. A control group of cells were cultured only with M-CSF. BM cells were either unstimulated or stimulated with <i>Aa</i>- media for 4 h. (A) S1PR2 mRNA, (B) Nfatc1 mRNA, (C) Ctsk mRNA, (D) Acp5 mRNA, (E<b>)</b> Oscar mRNA, (F) Dcstamp mRNA, (G) Ocstamp mRNA, (H) RANKL mRNA, (I) RANK mRNA, (J) OPG mRNA, (K) CSF1 mRNA, and (L) CSF1R mRNA levels were quantified by real time PCR and normalized by GAPDH expression. The data are representatives from three separate experiments (n = 3, *<i>P</i><0.05, ***<i>P</i><0.001).</p

Knockdown of S1PR2 significantly decreased IL-1b, IL-6, and TNF-a protein expressions induced by <i>A</i>. <i>actinomycetemcomitans</i> (<i>Aa</i>) in BMMs.

<p>Murine BMMs were uninfected, infected with a S1PR2 shRNA lentivirus, or infected with a control shRNA lentivirus (moi 50) for 72 h. Then the cells were either unstimulated or stimulated with <i>Aa</i> (1.5 CFU/cell) for 4 h (S1PR2 mRNA assay) or 8 h (cytokine protein assays). (A) S1PR2 mRNA expression was determined by real time PCR, (B) IL-1β, (C) IL-6, and (D) TNF-α protein levels were quantified by ELISA and normalized by protein levels in cell lysates. The data are representatives from three separate experiments (n = 3, ***<i>P</i><0.001).</p

<i>IndividualAuthorCategory</i> testing sets.

<p><i>IndividualAuthorCategory</i> testing sets.</p

Results of text localization and up-sampling prior to the application of the OCR tool.

<p>Results of text localization and up-sampling prior to the application of the OCR tool.</p

Performance curve with each feature selection iteration.

<p>Performance curve with each feature selection iteration.</p

Sensitivity and specificity for all testing sets by logistic regression model.

<p>Sensitivity and specificity for all testing sets by logistic regression model.</p