Comparative genomics reveals a functional thyroid-specific element in the far upstream region of the PAX8 gene

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the <it>Pax8 </it>gene.</p> <p>Results</p> <p>We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human <it>Pax8 </it>gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the <it>Pax8 </it>transcription start site. The <it>in silico </it>data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind <it>in vitro </it>and <it>in vivo </it>the Pax8 far upstream element, and is capable to activate transcription from it.</p> <p>Conclusions</p> <p>Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the <it>Pax8 </it>gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control <it>Pax8 </it>transcription during thyroid differentiation and are relevant to further studies on <it>Pax8 </it>as a candidate gene for thyroid dysgenesis.</p

Sustained release of decorin to the surface of the eye enables scarless corneal regeneration

Ophthalmology: novel eye drop brings sustained drug delivery to ocular surface An eye drop formulation that applies anti-scarring drugs to the surface of the eye helps reverse infection-induced corneal damage in mice. Hill et al. from the University of Birmingham, UK, formulated a fluid gel loaded with a wound-healing protein called decorin that conforms to the ocular surface and is cleared gradually through blinking. With colleagues in California, they applied the therapeutic eye drop to mice with bacterial eye infections that trigger sight-threatening corneal scarring. Within a matter of days, the team saw improvements in corneal transparency, with reductions in scar tissue and reconstitution of healthy cells. Such a drug delivery system, if successful in humans, could help save many people’s sight and reduce the need for corneal transplantation

Toward the understanding and treatment of human neurodegenerative disorders: a mouse model approach

Part 1 : Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by the deficiency of the lysosomal enzyme iduronate 2 sulphates (Ids). The inactivity of this enzyme results in the progressive accumulation of heparan and dermatan sulfates within the lysosomes of various tissues and organs, with consequent cellular degeneration. Patients affected by the severe form of MPS II are characterized, in addition, by a devastating involvement of the central nervous system. The MPSII mouse model reproduces the features of MPSII patients. The characterization of brain phenotype of mouse MPS II showed a progressive GAGs accumulation in almost all the regions of the brain, an increase in inflammation and severe neurodegeneration. Moreover, behavioral tests, showed a deficit in learning and motor coordination. Up to now, the only treatment for MPS II patients is the enzyme replacement therapy (ERT) with the systemic infusion of Ids enzyme; however, this treatment only obtains amelioration of visceral defects, but not of neurological ones. To this aim, we developed a new protocol of ERT to treat both, visceral and neurological defects. Importantly, we were successful in having the infused Ids enzyme reaching the brain with a good rescue of the CNS phenotype and behavioral performances. Among the ERT protocols tested, the one using low Ids concentration, was effective on young and adult MPS II mice. Part 2: Growing evidence suggest comorbidity between diabetes mellitus (DM) and Alzheimer's disease (AD); indeed, diabetic patients show increased risk of developing AD and cognitive deficits, while, AD patients show impaired insulin function and glucose metabolism. However, the molecular mechanisms linking these two disorders are still not fully understood. Here, we hypothesize that DM induces tau hyperphosphorylation generating the cognitive decline observed in AD. Interestingly, our results show that induction of type 1 DM by streptozotocin (STZ) administration in WT mice support our hypothesis showing, in these mice, tau hyperphosphorylation, decreased activity of the Insulin Receptor (IR)/PI3K/AKT pathway and increased phosphorylation of glycogen synthase kinase 3β (GSK3β), a kinase strongly involved in the pathogenesis of AD. At the behavioral levels, WT-STZ mice showed learning and memory deficits. Importantly, when type 1 DM was induced in knockout mice lacking tau proteins (here referred as mtauKO STZ), the behavioral and cellular phenotypes found in WT-STZ mice were not observed. Associated with these cognitive deficits, WT-STZ mice displayed a significant decrease of synaptic markers. Overall, our results indicate that tau phosphorylation is a critical and essential molecular mechanism underlying the link between DM and AD

Differential Roles for IL-1α and IL-1β in Pseudomonas aeruginosa Corneal Infection.

Pseudomonas aeruginosa is an important cause of dermal, pulmonary, and ocular disease. Our studies have focused on P. aeruginosa infections of the cornea (keratitis) as a major cause of blinding microbial infections. The infection leads to an influx of innate immune cells, with neutrophils making up to 90% of recruited cells during early stages. We previously reported that the proinflammatory cytokines IL-1α and IL-1β were elevated during infection. Compared with wild-type (WT), infected Il1b-/- mice developed more severe corneal disease that is associated with impaired bacterial killing as a result of defective neutrophil recruitment. We also reported that neutrophils are an important source of IL-1α and IL-1β, which peaked at 24 h postinfection. To examine the role of IL-1α compared with IL-1β in P. aeruginosa keratitis, we inoculated corneas of C57BL/6 (WT), Il1a-/-, Il1b-/-, and Il1a-/-Il1b-/- (double-knockout) mice with 5 × 104 ExoS-expressing P. aeruginosa. Il1b-/- and double-knockout mice have significantly higher bacterial burden that was consistent with delayed neutrophil and monocyte recruitment to the corneas. Surprisingly, Il1a-/- mice had the opposite phenotype with enhanced bacteria clearance compared with WT mice. Although there were no significant differences in neutrophil recruitment, Il1a-/- neutrophils displayed a more proinflammatory transcriptomic profile compared to WT with elevations in C1q expression that likely caused the phenotypic differences observed. To our knowledge, our findings identify a novel, non-redundant role for IL-1α in impairing bacterial clearance

Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3

Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18−/− and Dectin-1−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4−/− corneas; however, we found no difference between C57BL/6 and PAD4−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection