A COMPARATIVE HISTOMORPHOLOGICAL AND MICRO CT STUDY OF THE PRIMARY STABILITY AND THE OSSEOINTEGRATION OF THE SYDNEY MINI-SCREW: AN ANIMAL STUDY USING NEW ZEALAND RABBITS

Introduction: Failure rate of orthodontic miniscrews (MSs) is 7-50%. To address this problem and to promote primary stability of the miniscrew (MS), we recently designed and developed The Sydney Mini Screw (SMS, Patent number: PCT2009014) which can be used with injectable bone graft substitutes (iBGS). The aim of this study was to assess in vivo dispersion of bone graft substitutes (BGS) and the integration of the SMS to the cortical and trabecular bone using New Zealand femur and tibia rabbit model. Method: Twenty-four MSs were randomly placed in each proximal tibia and femur of 6 New Zealand rabbits with an open surgery process. Aarhus MS was used as a control and the effect of injection of BGS was studied by implanting SMS with and without BGS injection. The dispersion and integration of the MS were studied by using micro CT (μCT) and histochemical analysis at two time points, 0 day and 8 weeks post-implantation. Results: BGS were successfully injected to the SMS and thereafter hardened in situ to fill the bone void. After 8 weeks, μCT results revealed that the iBGS were resorbed and bone tissue was formed around the MS and within its lateral exit holes. The osteointegration of the SMS samples showed similar histologic characteristics to that of Arhus controls, and initial drilling for injection of bone cements into SMS did not seem to affect adjacent bone quality. Conclusion: Results of this pilot animal study showed the high potential of SMS and the developed technique to promote the primary stability of MS. Keywords: Primary stability; orthodontic miniscrew; injectable bone graft substitute

Effects of partner proteins on BCA2 RING ligase activity

Abstract Background BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2. Methods Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays. Results Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = \u3c 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways. Conclusions The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression

Regulating BCA2: An Investigation into E3 Ligase Activity

The BCA2 E3 ligase is expressed in a majority of invasive breast cancers. BCA2 has inherent autoubiquitination activity which contributes to cell migration and proliferation processes. Here, ten novel BCA2 binding proteins were found using yeast and bacterial screening. Two of which were human homolog of Rad23 variant A (hHR23a) and 14-3-3σ. In vivo and in vitro assays confirmed that both hHR23a and 14-3-3σ bound BCA2 and were co-expressed with BCA2 in breast cancer cells. Interaction of BCA2 with hHR23a and 14-3-3σ affect the autoubiquitination and auto-degradation activity of BCA2. Multi-ubiquitination of hHR23a-bound BCA2 was dramatically lower than that of free BCA2, this corresponded to increased BCA2 expression and half-life. Furthermore, phosphorylated BCA2 protein was stabilized by interaction with 14-3-3σ, via substrate inhibition of BCA2 autoubiquitination. High expression of BCA2 is correlated with grade in breast cancer and regulation of this E3 ligase’s activity may be important to cancer progression.MAS

Cognitive heterogeneity among community-dwelling older adults with Cerebral Small Vessel Disease

Some degree of ischemic injury to white matter tracts occurs naturally with age and is visible on magnetic resonance imaging as focal or confluent white matter hyperintensities (WMHs). Its relationship to cognition, however, remains unclear. To explore this, community-dwelling adults between the ages 55-80 years old completed structural imaging, neuropsychological testing, and questionnaires to provide objective measures and subjective experience of executive functioning. Volumetric lesion burden derived from structural MRI identified those with significant WMH burden (~10 cubic cm). Half of those recruited met this criterion and were designated as the cerebral small vessel disease (CSVD) group. Subjective complaints but not objective test scores differentiated adults with and without CSVD. Hierarchical clustering revealed two CSVD subgroups that differentiated those with impaired versus preserved executive function relative to controls. Overall these results provide some explanation for behavioural heterogeneity often observed in studies of age-related white matter changes. They also support the use of questionnaires to assess subjective complaints that may be able to detect subtle effects of pathology not evident on standardized cognitive scores

Loss of <i>Igfbp7</i> Causes Precocious Involution in Lactating Mouse Mammary Gland

<div><p>Background</p><p>Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While <i>Igfbp7</i> has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated <i>Igfbp7</i>-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals.</p><p>Results</p><p>We report here that loss of <i>Igfbp7</i> significantly retards mammary gland development in the virgin animals. More significantly, the pregnant <i>Igfpb7</i>-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the <i>Igfbp7</i>-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating <i>Igfbp7</i>-null glands exhibit increased expression of <i>Stat3</i> and enhanced activation of (phosphorylated) <i>Stat3</i>, combined with decreased expression of <i>Stat5</i> suggesting that the absence of <i>Igfbp7</i> accelerates the onset of involution. We also found that in absence of <i>Igfpb7</i>, the lactating glands contain increased <i>Igfbp5</i> protein along with decreased expression of <i>IGF-1</i> Receptor and <i>Akt</i> activation. Finally, we show that during the normal course of involution, <i>Igfbp7</i> expression is significantly decreased in the mammary gland.</p><p>Conclusion</p><p>Our data suggest that loss of <i>Igfbp7</i> induces precocious involution possibly through diminished cell survival signals. Our findings identify <i>Igfbp7</i> as major regulator of involution in the mammary gland.</p></div

<i>Igfbp7</i> expression is substantially decreased during involution in the mammary gland.

<p>(A) To ascertain if expression of <i>Igfbp7</i> correlates with involution, protein extracts were prepared from the Wild Type glands on the lactation day 3 (WT LD3) or involuting WT glands (weaned for 5 days, WT Inv D5) and the expression of <i>Igfbp7</i> protein was examined using Western blots. A representative blot is shown and average <i>Igfbp7</i> expression was obtained from 3 independent protein extracts and depicted in the bar graph. As shown, <i>Igfbp7</i> expression is dramatically decreased during involution (*P<0.05). (B) <i>Igfbp7</i>^−/− transcript expression was examined using qPCR assays using RNA extracted from Wild Type 11 week-old virgin (WT 11Week), day 18.5 pregnant (WT D18.5 pregnant) or on lactation day 3 glands. The average transcript expressions obtained from 3 independent RNA samples are shown. While <i>Igfbp7</i> expression in the mammary gland does not show significant change during pregnancy, its expression is substantially increased during lactation. Statistical analysis was done through two-tailed t-test (***p<0.0005).</p