The role of domain I in laminin chain assembly

Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu et al., 1995). Using the yeast two-hybrid system, long arm fragments of the laminin chains β2, γ1, and αl were assayed for their ability to form dimers. This assay confirmed the strong specific interactions between the β and γ chains seen in other studies of recombinant laminin fragments (Nomizu et al., 1994 & 1996; Utani et al., 1994 & 1995). Interactions of the αl fragment with β2 or γ1 were weak or non existent in this assay. A region necessary for dimerization within the β2 chain was found between the C-terminal 75 and 38 amino acids, as the C-terminal 75 amino acids interacted strongly with γl domain I but the C-terminal 38 amino acids did not. Additionally, a domain I fragment of β2 containing a cysteine to serine substitution at amino acid 1765 (created to prevent disulfide bonding) was able to form dimers with y 1 domain I, indicating that noncovalent forces can mediate this interaction. To determine the ability of domain I alone to mediate specific dimerization of the β2 with the γl chain, the domain I regions of β2 and γl were switched to create two chimeric laminin chains. Epitope-tagged chimeras were tested for their ability to interact with the full-length wild-type β2 or γ1. In immunoprecipitation experiments wild type β2 associated only with the chimera containing domain I of γl and wild-type γ1 coprecipitated only with the chimera containing domain I of β2. These results indicate that the domain I of laminin chains as part of a full-length chain can impart specificity to chain assembly within a cell

Dyadic Speech-based Affect Recognition using DAMI-P2C Parent-child Multimodal Interaction Dataset

Automatic speech-based affect recognition of individuals in dyadic conversation is a challenging task, in part because of its heavy reliance on manual pre-processing. Traditional approaches frequently require hand-crafted speech features and segmentation of speaker turns. In this work, we design end-to-end deep learning methods to recognize each person's affective expression in an audio stream with two speakers, automatically discovering features and time regions relevant to the target speaker's affect. We integrate a local attention mechanism into the end-to-end architecture and compare the performance of three attention implementations -- one mean pooling and two weighted pooling methods. Our results show that the proposed weighted-pooling attention solutions are able to learn to focus on the regions containing target speaker's affective information and successfully extract the individual's valence and arousal intensity. Here we introduce and use a "dyadic affect in multimodal interaction - parent to child" (DAMI-P2C) dataset collected in a study of 34 families, where a parent and a child (3-7 years old) engage in reading storybooks together. In contrast to existing public datasets for affect recognition, each instance for both speakers in the DAMI-P2C dataset is annotated for the perceived affect by three labelers. To encourage more research on the challenging task of multi-speaker affect sensing, we make the annotated DAMI-P2C dataset publicly available, including acoustic features of the dyads' raw audios, affect annotations, and a diverse set of developmental, social, and demographic profiles of each dyad.Comment: Accepted by the 2020 International Conference on Multimodal Interaction (ICMI'20

A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.

Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc

Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by αvβ3 and α5β1 integrins

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as α3 chain of type IV collagen and α1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to αvβ3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind α5β1 integrin. The activity of human tumstatin is mediated by αvβ3 integrin, whereas the activity of human endostatin is mediated by α5β1 integrin. Additionally, although human tumstatin binding to αvβ3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to α5β1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis

Summary of PK parameters for SC rFVIIIFc and rFVIIIFc.

<p>Summary of PK parameters for SC rFVIIIFc and rFVIIIFc.</p