Faculty Bibliography

Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

The response of S. pombe, also known as fission yeast, to misfolded proteins involves mechanisms that have not been observed in other species.

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.