Rollins Alumni Record, October 1999

Rite of Passage: That bittersweet first day at Rollin

Morphology and Tensile Properties of Multigraft Copolymers with Regularly Spaced Tri-, Tetra-, and Hexafunctional Junction Points

The effect of chain architecture on the morphological and tensile properties of series of multigraft copolymers, with regularly spaced tri-, tetra-, and hexafunctional junction points, was investigated using transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and tensile testing. The materials were synthesized by coupling difunctional polyisoprene (PI) spacers and living polystyrene (PS) branches, made by anionic polymerization, with chlorosilanes of different functionalities. Since the coupling process is a step-growth polymerization, yielding polydisperse products, fractionation was utilized to separate each material into three fractions (high, middle, and low molecular weight), each of low polydispersity. All three fractions have the same chain architecture on a per junction point basis but differ in the number of junction point units per molecule. By applying the constituting block copolymer concept, the physical behavior of these molecules was compared with current theories. It was found that morphological behavior of these graft copolymers can be predicted using theoretical approaches and is independent on the number of junction points. The number of the junction points, however, greatly influences the long-range order of microphase separation. Additionally, two new parameters for adjusting mechanical properties of multigraft copolymers were found in this investigation: (1) functionality of the graft copolymerstri-, tetra-, or hexafunctionals and (2) number of junction points per molecule. An increase in functionality causes a change in morphology, resulting in a high level of tensile strength for tetrafunctional (cylindrical) and hexafunctional (lamellae) multigraft copolymers, leading to about the twice the strength of the spherical trifunctional multigrafts of similar overall composition. Tetrafunctional multigraft copolymers show a surprisingly high strain at break, far exceeding that of commercial block copolymer thermoplastic elastomers (TPEs). Strain at break and tensile strength increase linearly with the number of junction points per molecule. Hysteresis experiments at about 300-900% deformation demonstrate that multifunctional multigraft copolymers have improved high elasticity as compared to commercial TPEs like Kraton or Styroflex

The genomic and transcriptional landscape of primary central nervous system lymphoma

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.ISSN:0006-2960ISSN:1520-499

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

The heterotrimeric G-protein complex is minimally composed of Ga, Gb, and Gc subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification

Aza-SAHA Derivatives are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group (“aza-scan”) into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one atom replacement (C-->N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ-748, with potency and selectivity demonstrated by cellular and biochemical target-engagement, as well as thermal-shift, assays. Co-crystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling con-firmed cellular HDAC10-selectivity of DKFZ-748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, confirmed for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limited in vitro tumor model, DKFZ-748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ-748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings

Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group (“aza-scan”) into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ‑748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ‑748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ‑748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ‑748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ‑748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings