Faculty Bibliography

Endoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1alpha, ERO1beta, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Members of the vacuolar protein sorting 10 (Vps10) family of receptors (including sortilin, SorL1, SorCS1, SorCS2, and SorCS3) play pleiotropic functions in protein trafficking and intracellular and intercellular signaling in neuronal and non-neuronal cells. Interactions have been documented between Vps10 family members and the retromer coat complex, a key component of the intracellular trafficking apparatus that sorts cargo from the early endosome to the trans-Golgi network. In recent years, genes encoding several members of the Vps10 family of proteins, as well as components of the retromer coat complex, have been implicated as genetic risk factors for sporadic and autosomal dominant forms of neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, and Parkinson's disease, with risk for type 2 diabetes mellitus and atherosclerosis. In addition to their functions in protein trafficking, the Vps10 family proteins modulate neurotrophic signaling pathways. Sortilin can impact the intracellular response to brain-derived neurotrophic factor (BDNF) by regulating anterograde trafficking of Trk receptors to the synapse and direct control of BDNF levels, while both sortilin and SorCS2 function as cell surface receptors to mediate acute responses to proneurotrophins. This mini-review and symposium will highlight the emerging data from this rapidly growing area of research implicating the Vps10 family of receptors and the retromer in physiological intracellular trafficking signaling by neurotrophins and in the pathogenesis of neurodegeneration.

Sex estimation from the human skull is often a necessary step when constructing a biological profile from unidentified human remains. Traditional methods for determining the sex of a skull require observers to rank the expression of sexually dimorphic skeletal traits by subjectively assessing their qualitative differences. One of these traits is the prominence of the glabellar region above the browridge. In this paper, the volume of the browridge region was measured from digital 3D models of 128 dry crania (65 female, 63 male). The 3D models were created with a desktop laser scanner, and the browridge region of each 3D model was isolated using geometric planes defined by cranial landmarks. Statistical analysis of browridge-to-cranium volume ratios revealed significant differences between male and female crania. Differences were also observed between geographically distinct populations, and between temporally distinct populations from the same locale. The results suggest that in the future, sex determination of human crania may be assisted by quantitative computer-based volume calculations from 3D models, which can provide increased objectivity and repeatability when compared to traditional forensic techniques. The method presented in this paper can easily be extended to other volumetric regions of the human cranium.

Bone morphogenetic protein 2 (BMP2) is known to activate unfolded protein response (UPR) signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation have not yet been elucidated. In this study we demonstrate that BMP2 mediates mild ER stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation, and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5'-flanking regulatory region of XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II Collagen, type X Collagen and Runx2; however, knockdown of XBP1S via the RNA interference(RNAi) approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of Indian hedgehog (IHH) and parathyroid hormone-related peptide (PTHrP). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by (1) acting as a cofactor of Runx2 and (2) affecting IHH/PTHrP signaling.

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-gamma secreting CD4(+) T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-gamma/TNF-alpha/IL-2 secreting polyfunctional CD4(+) T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.

Elevated levels of circulating lipids are the major cause of cardiovascular disease, but beneficial outcomes might be realized by targeting lipid carriers. Two papers in this issue of Cell Metabolism (So et al., 2012; Wang et al., 2012) demonstrate how modulation of one arm of the unfolded protein response can decrease plasma levels of VLDL particles and their associated lipids.

BACKGROUND: Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. RESULTS: In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. CONCLUSIONS: The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.

The use of gadolinium-based contrast agents (GBCA) is integral to the field of diagnostic magnetic resonance imaging (MRI). Pharmacokinetic evaluation of the plasma clearance of GBCA is required for all new agents or improved formulations, to address concerns over toxicity or unforeseen side effects. Current methods to measure GBCA in plasma lack either a rapid readout or the sensitivity to measure small samples or require extensive processing of plasma, all obstacles in the development and characterization of new GBCA. Here, we quantify the plasma concentration of a labeled analogue of a common clinical GBCA by ligand triplet harvesting and energy transfer. The nonemittive GBCA becomes a "dark donor" to a fluorescent detector molecule, with a lower limit of detection of 10(-7) M in unprocessed plasma. On a time scale of minutes, we determine the plasma clearance rate in the wild-type mouse, using time-resolved fluorescence on a standard laboratory plate reader.