Fluorescent sensors based on bacterial fusion proteins.

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker.

Biomaterial and cellular properties as examined through atomic force microscopy, fluorescence optical microscopies and spectroscopic techniques.

Colloids, polymers, gels, and biological materials are widely used for numerous technological applications. The design, fabrication, and understanding of the physico-chemical properties of such (bio)materials, however, represent a challenge for scientists and technologists. This review is a concise update of the latest achievements in surface and bulk analytical techniques applied to biomaterials and soft matter systems.

A fusion tag to fold on: the S-layer protein SgsE confers improved folding kinetics to translationally fused enhanced green fluorescent protein.

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP.

2D crystalline protein layers as immobilization matrices for the development of DNA microarrays.

There is a growing demand for functional layers for the immobilization of (bio)molecules on different kinds of substrates in the field of biosensors, microarrays, and lab-on-a-chip development. These functional coatings should have the ability to specifically bind (bio)molecules with a high binding efficiency, while showing low unspecific binding during the following assay. In this paper we present rSbpA surface layer proteins (S-layer proteins) as a versatile immobilization layer for the development of DNA microarrays.

Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma.

Background: G protein-coupled receptor 5D (GPRC5D) is a novel surface receptor. As this new subtype of G protein-coupled receptors was discovered, little is known about the role of this gene.

Materials And Methods: In this retrospective study, we investigated GPRC5D mRNA expression by real-time polymerase chain reaction (RT-PCR) in bone marrow (BM) of 48 patients with multiple myeloma (MM).

Fluorescence energy transfer in the bi-fluorescent S-layer tandem fusion protein ECFP-SgsE-YFP.

This work reports for the first time on the fabrication of a bi-functional S-layer tandem fusion protein which is able to self-assemble on solid supports without losing its functionality. Two variants of the green fluorescent protein (GFP) were genetically combined with a self-assembly system having the remarkable opportunity to interact with each other and act as functional nanopatterning biocoating. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the cyan ECFP donor protein at the SgsE N-terminus and with the yellow YFP acceptor protein at the C-terminus.

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei.

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids.

Absorption, steady-state fluorescence, fluorescence lifetime, and 2D self-assembly properties of engineered fluorescent S-layer fusion proteins of Geobacillus stearothermophilus NRS 2004/3a.

S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5.

Optical oxygen sensors based on Pt(II) porphyrin dye immobilized on S-layer protein matrices.

This paper describes the development of planar and fiber optic oxygen sensors utilizing surface layer (S-layer) proteins as immobilization matrix for oxygen sensitive dyes. S-layer proteins have the intrinsic capability to reassemble into two-dimensional arrays in suspension and at interfaces. Due to their crystalline character the distribution of functional groups, such as carboxylic groups, is repeated with the periodicity of the lattice and thus allows the reproducible and geometrically distinct binding of functional molecules.

Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays.

The mesophilic organism Lysinibacillus sphaericus CCM 2177 produces the surface (S)-layer protein SbpA, which after secretion completely covers the cell surface with a crystalline array exhibiting square lattice symmetry. Because of its excellent in vitro recrystallization properties on solid supports, SbpA represents a suitable candidate for genetically engineering to create a versatile self-assembly system for the development of a molecular construction kit for nanobiotechnological applications. The first goal of this study was to investigate the surface location of 3 different C-terminal amino acid positions within the S-layer lattice formed by SbpA.

Recombinant glycans on an S-layer self-assembly protein: a new dimension for nanopatterned biomaterials.

Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans.

Myeloid leukemias express a broad spectrum of VEGF receptors including neuropilin-1 (NRP-1) and NRP-2.

Vascular endothelial growth factor (VEGF) is produced in neoplastic cells in various myeloid neoplasms and may act as an autocrine growth-regulator. We have examined the expression of five VEGF receptors (VEGR1/Flt-1, VEGFR2/KDR, Flt-4, neuropilin-1 = NRP-1, NRP-2) in leukemic cells obtained from patients with acute myeloid leukemia (n = 28), chronic myeloid leukemia (n = 14), chronic eosinophilic leukemia (n = 3), chronic myelomonocytic leukemia (n = 9), or mast cell leukemia/systemic mastocytosis (n = 3) as well as in respective cell lines. Expression of VEGFR mRNA was analyzed by RT-PCR, and expression of VEGFR protein by immunocytochemistry.

Overexpression of the paternally expressed gene 10 (PEG10) from the imprinted locus on chromosome 7q21 in high-risk B-cell chronic lymphocytic leukemia.

We report high expression of the maternally imprinted gene PEG10 in high-risk B-CLL defined by high LPL mRNA expression. Differential expression was initially identified by microarray analysis and confirmed by real time PCR in 42 B-CLL patients. mRNA expression ranged from 0.

Upregulation of retinoic acid receptor-beta by the epidermal growth factor-receptor inhibitor PD153035 is not mediated by blockade of ErbB pathways.

Inhibiting epidermal growth factor-receptor (ErbB-1) represents a powerful anticancer strategy. Activation of retinoid pathways is also in development for cancer treatment. Retinoic acid receptor-beta-the tumor suppressor and main retinoid mediator--is silenced in many tumors.

Limited value of FLT3 mRNA expression in the bone marrow for prognosis and monitoring of patients with acute myeloid leukemia.

We studied wild-type FLT3 mRNA expression at diagnosis in bone marrow samples from 85 patients with acute myeloid leukemia (AML), 23 of whom were in complete remission, and determined its utility as a marker for minimal residual disease (MRD). We conclude that FLT3 expression is of limited value as a prognostic marker and for MRD monitoring.

An EGF receptor inhibitor induces RAR-beta expression in breast and ovarian cancer cells.

Inhibition of the epidermal growth factor (EGF)-receptor (EGFR) has become a promising anticancer treatment strategy. In addition, application of retinoids yields encouraging results for cancer prevention and therapy. Many tumors express no or low amounts of retinoic acid receptor-beta2 (RAR-beta2) due to epigenetic silencing via DNA hypermethylation.

Highly refractory acute myeloid leukemia.

In this study we evaluated 103 patients suffering from acute myeloid leukemia (AML) who did not respond to induction chemotherapy and defined a sub-group of patients with highly refractory disease characterized by a persistence of more than 1 G/L blast cells in the peripheral blood between days 12 and 16 of the first induction cycle. Only seven patients (one female, six males) met these criteria. Their median age was 65 years (range 41-82 years).

Relation of in vitro growth characteristics to cytogenetics and treatment outcome in acute myeloid leukemia: prognostic significance in patients with a normal karyotype.

We analyzed in vitro growth characteristics of bone marrow mononuclear cells (BMMCs) from 322 patients with acute myeloid leukemia (AML) in relation to cytogenetic abnormalities. Median colony growth was low in each of the cytogenetic changes associated with a favorable outcome. Most karyotypic abnormalities in the intermediate prognosis group were associated with low growth potential, but 11 q23 abnormalities exhibited 8 times higher in vitro growth.

Variable prognostic value of FLT3 internal tandem duplications in patients with de novo AML and a normal karyotype, t(15;17), t(8;21) or inv(16).

Introduction: Internal tandem duplication of the FLT3 gene (FLT3/ITD) has been linked to poor outcome in acute myeloid leukemia (AML). However, the prognostic value of FLT3/ITD in various cytogenetic risk groups is still a matter of debate. The aim of this study was to evaluate the prognostic significance in patients with de novo AML and a normal karyotype or a t(15;17), t(8;21) or inv(16) (good risk group).