Eugenia Pechkova

Immunosuppressive drug-free operational immune tolerance in human kidney transplant recipients: Part I. Blood gene expression statistical analysis

Survival of solid organ grafts depends on life‐long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous “operational tolerance” can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross‐validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis. J. Cell. Biochem. 103: 1681–1692, 2008.

Nanoproteomics enabling personalized nanomedicine.

Nucleic Acid Programmable Protein Arrays utilize a complex mammalian cell free expression system to produce proteins in situ. In alternative to fluorescent-labeled approaches a new label free method, emerging from the combined utilization of three independent and complementary nanotechnological approaches, appears capable to analyze protein function and protein-protein interaction in studies promising for personalized medicine. Quartz Micro Circuit nanogravimetry, based on frequency and dissipation factor, mass spectrometry and anodic porous alumina overcomes indeed the limits of correlated fluorescence detection plagued by the background still present after extensive washes. This could be further optimized by a homogeneous and well defined bacterial cell free expression system capable to realize the ambitious objective to quantify the regulatory protein networks in humans. Implications for personalized medicine of the above label free protein array using different test genes proteins are reported.

Protein nucleation and crystallization by homologous protein thin film template

A new method of protein nucleation and crystallization based on Langmuir–Blodgett technology is here utilized for the template stimulation of crystal growth of so far non‐crystallized proteins. Microcrystals (60–120 μm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth. J. Cell. Biochem. 85: 243–251, 2002.

Immunosuppressive drug‐free operational immune tolerance in human kidney transplants recipients. Part II. Non‐statistical gene microarray analysis

Kidney transplant is the reference treatment for patients with end‐stage renal disease, but patients may develop long‐term rejection of the graft. However, some patients do not reject the transplant, but instead are operationally tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray‐based identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney transplant in humans by utilizing a non/statistical bioinformatic approach based on the identification of “key genes,” either as those mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerges on the genes controlling the human transplant from the detailed comparison of these findings with the traditional statistical SAM (Tusher et al. 2001 Proc Natl Acad Sci USA 98:5116–5121) analysis of the microarrays and with the clinical study carried out in the accompanying part I article. J. Cell. Biochem. 103: 1693–1706, 2008.

Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2.

The three-dimensional crystal structure of the triple-point mutant of the catalytic subunit of human protein kinase CK2alpha has been determined at 2.4 A resolution. Microcrystals of mutant CK2 catalytic subunit were obtained by a protein-crystallization method based on thin-film nanotechnology. These microcrystals (of about 20 micro m in diameter) were used for diffraction data collection by means of the microfocus beamline at the ESRF synchrotron. A comparison between the human protein kinase CK2alpha and the corresponding enzyme from a lower organism (Zea mays) is made.

Prototypes of Newly Conceived Inorganic and Biological Sensors for Health and Environmental Applications

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.

Proteomics and nanocrystallography

1 The New Frontier at the Crossing of life and Physical Sciences.- 2 State of the Art in Proteomics, Crystallography and Nanobiotechnology.- 2.1 Methods of Protein Production.- 2.1.1 Recombinant DNA Technology: expression, purification and mutagenesis.- 2.2 Methods for Protein Identification.- 2.2.1 Two-dimensional electrophoresis.- 2.2.2 Mass spectrometry and its principles.- 2.3 Methods of Protein Crystallization.- 2.3.1. Importance of protein sample preparation.- 2.3.2 Protein solubility and supersaturation. Nucleation and crystal growth.- 2.3.3 Classical methods.- 2.3.4 Less common techniques.- 2.3.5 Advanced methods.- 2.4 Methods of Crystal Structure Determination.- 2.4.1 Protein X-ray crystallography.- 2.4.2 Crystal structure determination.- 2.4.3 Data collection and data processing.- 2.4.4 Traditional X-ray diffractometer.- 2.4.5 Electron microscopy.- 2.5 Other Methods of Protein Structure Investigation.- 2.5.1 Circular Dichroism measurements.- 2.5.2 High Resolution Nuclear Magnetic Resonance.- 2.6 Methods of Protein Immobilization In Solid Thin Films.- 2.6.1 Pressure-area isotherms.- 2.6.2 Surface potential.- 2.6.3 Methods of thin film deposition.- 2.6.4 Methods of thin film characterization.- 3 Nanocrystallography.- 3.1 Nanobiofilms Template.- 3.1.1 Substrate preparation.- 3.1.2 Protein thin film formation.- 3.1.3 Protein thin film characterization.- 3.2 Atomic Force Microscopy.- 3.2.1 Design of the chamber.- 3.2.2 The hardware and software apparatus.- 3.2.3 Performances of the system.- 3.3 Synchrotron Radiation.- 3.3.1 Principles of Synchrotron functioning.- 3.3.2 Microfocus beamline and ultramicrocrystal diffraction.- 3.3.3 Protein crystals synchrotron radiation analysis.- 3.3.4 Structure determination and refinement.- 3.4 From Art to Science With Protein Nanocrystallography.- 4 From Art to Science in Protein Crystallography.- 4.1 New Method Validation With Lysozyme as a Model Protein.- 4.1.1 Effect of the thin film template: kinetics of the lysozyme crystal growth.- 4.1.2 Thin film surface pressure influence on the template-induced crystallization.- 4.1.3 X-ray diffraction of lysozyme crystals.- 4.1.4 Synchrotron radiation analysis of lysozyme crystals.- 4.2 Yet Unresolved Proteins: Bovine Cytochrome P450scc.- 4.2.1 Cytochrome P450scc (Side-Chain Cleavage).- 4.2.2 Sample homogeneity.- 4.2.3 Pressure-area isotherm.- 4.2.4 Quartz crystal nanobalance.- 4.2.5 Cytochrome P450scc crystallization.- 4.2.6 Atomic Force Microscopy of cytochrome microcrystals.- 4.3 Yet unresolved proteins: Human kinase CK2? Catalytic Subunit.- 4.3.1 Protein kinase CK2? catalytic subunit.- 4.3.2 Sample homogenity.- 4.3.3 Pressure-area isotherm.- 4.3.4 Quartz crystal nanobalance.- 4.3.5 Human protein kinase CK2? crystallization.- 4.3.6 Synchrotron radiation: analysis, data processing and structure determination.- 4.4 Conclusions.- 5 From Science Technology in Proteomics.- 5.1 Automated Mass Spectrometry.- 5.2 Biomolecular Arrays.- 5.2.1 The surface patterning.- 5.2.2 The instrumental layout and data analysis.- 5.3 Unsolved Protein Structures of Scientific and Industrial Interest.- 6 Future Trends.- 6.1 Novel Composite Biomaterials and Nanodevices.- 6.2 nanocrystals.- References.

Conductometric monitoring of protein-protein interactions.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

µGISAXS and protein nanotemplate crystallization: methods and instrumentation

Microbeam grazing-incidence small-angle X-ray scattering (microGISAXS) has been used and the technique has been improved in order to investigate protein nucleation and crystal growth, assisted by a protein nanotemplate. The aim is to understand the protein nanotemplate method in detail, as this method has been proved capable of accelerating and increasing crystal size and quality as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing lysozyme crystals at three different stages of growth.

Radiation stability of proteinase K crystals grown by LB nanotemplate method

A detailed analysis of structural and intensity changes induced by X-ray radiation is presented for two types of proteinase K crystals: crystal grown by classical hanging drop method and those grown by Langmuir-Blodgett (LB) nanotemplate. The comparison of various parameters (e.g. intensity per sigma ratio, unit-cell volume, number of unique reflections, B-factors) and electron density maps as a function of radiation dose, demonstrates that crystals, grown by the LB nanotemplate method, appear to be more resistant against radiation damage than crystals grown by the classical hanging drop method.