The Notebook: A Psychological Thriller

This was an entry into the 2007 Mashup Contest, in which students created trailers parodying well-known films.https://repository.upenn.edu/showcase_videos/1018/thumbnail.jp

A comparison of non-integrating reprogramming methods

Human induced pluripotent stem cells (hiPSCs1,2,3) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV)4, episomal (Epi)5 and mRNA transfection mRNA6 methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation

Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis

<div><p>The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies.</p></div

AR3 mAbs target overlapping but distinct epitopes on the neutralizing face of E2c.

<p><b>(A)</b> Data shown are the mutated residues for which binding for mAb AR3A was ≤25%, but >75% for at least one control mAb. Any mutations that resulted in global misfolding (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.g002" target="_blank">Fig 2B</a></b>) were excluded. Mutations leading to ≤25% binding for all four AR3 mAbs are highlighted in red. <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.s002" target="_blank">S2 Fig</a></b> summarizes data for AR3B-3D mAbs. The percent reactivity is the mean of two experiments. <b>(B)</b> Seven out of thirteen critical residues for the four AR3 mAbs (highlighted in red in <b>A</b>) are shown in red on the E2c structure [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>]. The remaining 6 residues are hidden in this orientation. Critical residues that are variable between AR3 mAbs are indicated in blue in the right panel. The exact location of C459 could not be visualized as aa 453–459 are disordered in the E2c structure. <b>(C)</b> The E2c-AR3C Fab crystal structure and hydrogen bonding analysis confirmed that several amino acids in the region identified by flow cytometry are likely involved in hydrogen bonding with the heavy chain CDR H3 and H1 of AR3C Fab as indicated. m.c -main chain; s.c—side chain.</p

The stalk, VR2, and a glycan-free face of E2 may play a role in the E1E2 interface.

<p><b>(A)</b> Classification of E1E2 residues based on the effects of mutations on antibody binding. <b>(B)</b> The location of <b>Class 2</b> residues (mutation affected E1E2 complex formation) are indicated in red on the structure of E2 with the transmembrane region removed (E2ΔTM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>]. Regions that are missing or disordered in the structure are represented by black dotted lines. The area containing HVR1 is highlighted in blue, VR2 is in purple, and the stalk and transmembrane regions are in light and dark green, respectively. The locations of glycans are indicated by green circles and are numbered beginning with the N-terminus. <b>(C)</b> A general model of the E1E2 interaction indicates that the stalk region, the base of VR2, and a portion of E2 opposite of the front layer and CD81bs (i.e. VR3, post-VR3 and back layer) possibly interacts with E1. Dots indicate region of E2 that may interact with E1.</p

Epitopes for AR1 monoclonal antibodies target the non-neutralizing face of E2c.

<p>Single residue mutations which resulted in ≤25% of mAb binding (relative to wild-type E2) but >75% for at least one control antibody are shown for mAbs AR1A <b>(A)</b> and AR1B <b>(B)</b>. Binding assays were performed twice with the range indicated. Black arrows indicate negative values. <b>(C)</b> The critical residues for AR1A and AR1B were visualized on the E2c structure [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>]. Residues in purple are required by both AR1A and AR1B. Residues specific for AR1A alone are in red. Dashed lines represent regions of E2c that are disordered or missing.</p

E2 back layer mutations modulate CD81 binding to the E2 front layer and CD81 binding loop.

<p><b>(A)</b> Based on flow cytometry and ELISA analysis, and previously published results [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>], residues in four distinct E2c regions were found to be important for CD81 binding: the front layer (cyan), Ig β-sandwich (red), CD81 loop (blue), and the back layer (green). The locations of mutations resulting in <25% CD81 binding in each of these regions are highlighted. <b>(B)</b> Hydrogen bond calculations indicate that two back layer residues in the α2-helix, Y613 and W616 (green), interact with L441 and W437 (cyan) of the front layer α1-helix, respectively. m.c—main chain; s.c—side chain. These interactions between front and back layer residues suggest that the back layer indirectly affects CD81 binding through structural interactions with the adjacent front layer. The residue colors follows those in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.g001" target="_blank">Fig 1C</a>. <b>(C)</b> Based on flow cytometry and ELISA, I622A, which was found to enhance CD81 binding, was analyzed further (along with F627A). Soluble E2c mutants harboring I622A, I622A/F627A and F627A were tested using ELISA for their ability to bind recombinant WT CD81-Fc (left panel) or a mutant that reduces CD81 dimerization, CD81-Fc (K124T) (middle panel). Binding signals to mAb HCV1 were used as an expression control for the mutants (right panel).</p

mAb AR2A binds the back layer of E2.

<p><b>(A)</b> Data shown are the mean reactivities determined by flow cytometry (FC) and ELISA of mAb AR2A, conformational mAbs AR1A and AR4A, and linear mAbs HCV1, AP33, and A4 to mutants T625A and K628A. The C-terminal V5 tag expression is also shown for the flow cytometry constructs. Each binding assay was performed twice with the range shown. <b>(B)</b> The mean binding value (percent relative to wild-type reactivity) of mAb AR2A to each mutant E1E2 library clone was plotted as a function of binding to conformational-dependent mAb AR4A (black circles). Clones with AR2A binding ≤25%, but at least 75% AR4A binding (red boxes), are considered to indicate crucial residues for AR2A. As shown, only K628A (red circle) fell within this threshold. <b>(C)</b> Back-layer residues T625 and K628 are highlighted in red on the E2c structure [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>]. The back layer is shown in green.</p

Overview of E1 and E2 glycoprotein structures.

<p><b>(A)</b> The approximately 3000 amino-acid HCV polyprotein generates 10 proteins following cleavage, of which E1 and E2 are two of the three structural proteins. <b>(B)</b> Spanning amino acids 192–383, the structure of E1 is poorly understood although crystallization of the first half of the protein (aa192-271) revealed secondary structures that could be present in native E1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref019" target="_blank">19</a>] including an α-helix flanked by several β-sheets. In contrast, E2 (aa 384–746) has several well-defined regions containing β-sheets, α-helices, and η (3₁₀) helices. Sequences for regions in which secondary structure is known (e.g. nE1, E2 front layer, β-sandwich core, back layer, etc.) are included (prototypic H77 sequence). Green-branched forks depict relative locations of N-linked glycans. <b>(C)</b> Crystal structure of E2c [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006735#ppat.1006735.ref015" target="_blank">15</a>] illustrated that E2 is characterized by a globular structure with a central Ig-like core flanked by front and back layers. The front layer, β-sandwich core, CD81 binding loop, and back layer are colored as depicted in panel <b>(B)</b>.</p