Vinculin-Arp2/3 interaction inhibits branched actin assembly to control migration and proliferation.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood.

High-affinity peptides developed against calprotectin and their application as synthetic ligands in diagnostic assays.

Common inflammatory disorders such as ulcerative colitis and Crohn's disease are non-invasively diagnosed or monitored by the biomarker calprotectin. However, current quantitative tests for calprotectin are antibody-based and vary depending on the type of antibody and assay used. Additionally, the binding epitopes of applied antibodies are not characterized by structures and for most antibodies it is unclear if they detect calprotectin dimer, tetramer, or both.

BRCA Mutations-The Achilles Heel of Breast, Ovarian and Other Epithelial Cancers.

Two related tumor suppressor genes, and , attract a lot of attention from both fundamental and clinical points of view. Oncogenic hereditary mutations in these genes are firmly linked to the early onset of breast and ovarian cancers. However, the molecular mechanisms that drive extensive mutagenesis in these genes are not known.

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells.

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific.

Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR).

High-concentration graphene dispersions with minimal stabilizer: a scaffold for enzyme immobilization for glucose oxidation.

Modified acrylate polymers are able to effectively exfoliate and stabilize pristine graphene nanosheets in aqueous media. Starting with pre-exfoliated graphite greatly promotes the exfoliation level. The graphene concentration is significantly increased up to 11 mg mL(-1) by vacuum evaporation of the solvent from the dispersions under ambient temperature.

Surface-Modified TiO Photocatalysts Prepared by a Photosynthetic Route: Mechanism, Enhancement, and Limits.

Surface-modified TiO photocatalysts were synthesized by a photosynthetic route involving visible-light-induced (λ>455 nm) activation of benzene and toluene at the surface of TiO leading to the formation of carbonaceous polymeric deposits. IR spectroscopic and photoelectrochemical experiments showed that the mechanism of the photosynthetic reactions involves intra-bandgap surface states at TiO related to surface OH groups interacting with adsorbed aromatic molecules. The photosynthesized surface-modified TiO materials exhibited enhanced activity, relative to pristine TiO , in photocatalytic degradation (and complete mineralization) of 4-chlorophenol.

Translating dosage compensation to trisomy 21.

Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells.

Optimization of a membraneless glucose/oxygen enzymatic fuel cell based on a bioanode with high coulombic efficiency and current density.

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite.

Mutual enhancement of the current density and the coulombic efficiency for a bioanode by entrapping bi-enzymes with Os-complex modified electrodeposition paints.

A bioanode with high current density and coulombic efficiency was developed by co-immobilization of pyranose dehydrogenase from Agaricus meleagris (AmPDH) with the dehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophiles (recDHCtCDH) expressed recombinantly in Escherichia coli. The two enzymes were entrapped in Os-complex modified electrodeposition polymers (Os-EDPs) with specifically adapted redox potential by means of chemical co-deposition. AmPDH oxidizes glucose at both the C2 and C3 positions whereas recDHCtCDH oxidizes glucose only at the C1 position.

A designed zinc-finger transcriptional repressor of phospholamban improves function of the failing heart.

Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure.

A new synthesis route for Os-complex modified redox polymers for potential biofuel cell applications.

A new synthesis route for Os-complex modified redox polymers was developed. Instead of ligand exchange reactions for coordinative binding of suitable precursor Os-complexes at the polymer, Os-complexes already exhibiting the final ligand shell containing a suitable functional group were bound to the polymer via an epoxide opening reaction. By separation of the polymer synthesis from the ligand exchange reaction at the Os-complex, the modification of the same polymer backbone with different Os-complexes or the binding of the same Os-complex to a number of different polymer backbones becomes feasible.

Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations.

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.

The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies.

Electron transfer between genetically modified Hansenula polymorpha yeast cells and electrode surfaces via Os-complex modified redox polymers.

Graphite electrodes modified with redox-polymer-entrapped yeast cells were investigated with respect to possible electron-transfer pathways between cytosolic redox enzymes and the electrode surface. Either wild-type or genetically modified Hansenula polymorpha yeast cells over-expressing flavocytochrome b2 (FC b(2) ) were integrated into Os-complex modified electrodeposition polymers. Upon increasing the L-lactate concentration, an increase in the current was only detected in the case of the genetically modified cells.

An engineered zinc finger protein activator of the endogenous glial cell line-derived neurotrophic factor gene provides functional neuroprotection in a rat model of Parkinson's disease.

Loss of dopaminergic neurons is primarily responsible for the onset and progression of Parkinson's disease (PD); thus, neuroprotective and/or neuroregenerative strategies remain critical to the treatment of this increasingly prevalent disease. Here we explore a novel approach to neurotrophic factor-based therapy by engineering zinc finger protein transcription factors (ZFP TFs) that activate the expression of the endogenous glial cell line-derived neurotrophic factor (GDNF) gene. We show that GDNF activation can be achieved with exquisite genome-wide specificity.

A rapid and general assay for monitoring endogenous gene modification.

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.

Redox electrodeposition polymers: adaptation of the redox potential of polymer-bound Os complexes for bioanalytical applications.

The design of polymers carrying suitable ligands for coordinating Os complexes in ligand exchange reactions against labile chloro ligands is a strategy for the synthesis of redox polymers with bound Os centers which exhibit a wide variation in their redox potential. This strategy is applied to polymers with an additional variation of the properties of the polymer backbone with respect to pH-dependent solubility, monomer composition, hydrophilicity etc. A library of Os-complex-modified electrodeposition polymers was synthesized and initially tested with respect to their electron-transfer ability in combination with enzymes such as glucose oxidase, cellobiose dehydrogenase, and PQQ-dependent glucose dehydrogenase entrapped during the pH-induced deposition process.

Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases.

Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem.

Electrodeposition polymers as immobilization matrices in amperometric biosensors: improved polymer synthesis and biosensor fabrication.

Electrodeposition polymers can be precipitated on electrode surfaces upon electrochemical-induced modulations of the pH value in the diffusion zone in front of the electrode. The formed polymer films can be used as immobilization matrices in amperometric biosensors. In order to rationally control the thus obtained biosensor properties, it is indispensable to develop strategies for the reproducible synthesis of electrodeposition polymers as well as methods for the non-manual and reproducible sensor fabrication.

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase.

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected.