Langmuir-Blodgett (LB)-based nanobiocrystallography at the frontiers of cancer proteomics.

BACKGROUND/AIM: Langmuir-Blodgett (LB) films used as templates for crystallization lead to marked changes in protein stability and water dehydration, despite slight changes in protein atomic structure. Herein, we discuss the importance of LB-based nanocrystallography at the frontiers of cancer proteomics focusing on two model proteins with important biological roles in cancer, namely CK2alpha and RNase A. MATERIALS AND METHODS: Computational mutagenesis using the KINARI Mutagen webserver exhibits different behaviors in terms of stability and robustness, as well as in terms of water dynamics. CONCLUSION: Introduction of LB film leads to the appearance of water molecules close to the protein surface with larger volume, causing changes in crystal stability against radiation and appearing replicated in mutant proteins. Implications for drug design, drug delivery and cancer-causing protein variants are herein presented, along with a review of the most recent findings in LB-based nanobiocrystallography.

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.

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.

Langmuir-Blodgett nanotemplate and radiation resistance in protein crystals: state of the art.

A state-of-the-art review of the role of the Langmuir-Blodgett nanotemplate on protein crystal structures is here presented. Crystals grown by nanostructured template appear more radiation resistant than the classical ones, even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The electron density maps and the changes in parameters such as total diffractive power, B-factor, and pairwise R-factor have been discussed. Protein crystals, grown by the Langmuir-Blodgett nanotemplate-based method, proved to be more radiation resistant compared to crystals grown by the classical hanging drop method in terms of both global and specific damage.

Real-time quantitative polymerase chain reaction analysis of patients with refractory chronic periodontitis.

BACKGROUND: Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients. METHODS: Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients >0.98 and efficiencies >96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/µL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value <0.01 was considered statistically significant. RESULTS: The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P <0.01), and it was not statistically significant for REL-A and NFKB1. CONCLUSION: This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.

Domain organization and properties of LB lysozyme crystals down to submicron size.

New topographic details appeared evident in protein crystal buffered with glycerol solution native on mica by atomic force microscopy and after laser irradiation on glass by light microscopy. This observation indicates the existence of distinct domains in the 3D crystal organisation that are quite different in size and number between the lysozyme crystals grown by Langmuir-Blodgett (LB) nanotemplate with respect to traditional hanging-drop vapour diffusion. Nanodiffraction by highly focused synchrotron radiation of laser cut submicron crystals confirmed the atomic structure of all residues of LB lysozyme crystals as being the most resistant to radiation damage. Crystals grown by LB nanotemplate still diffracted at good resolution after several steps of X-ray 'burning', while the classical crystals decayed very quickly at the same exposure.

An overview of nanotechnology-based functional proteomics for cancer and cell cycle progression.

Nanogenomics and nanoproteomics allow the study and comparison of the huge number of genes and proteins involved in the cell cycle progression of human T lymphocytes and in its transformation in lymphoma. Nanogenomics has, however, many pitfalls that only functional proteomics, called nucleic acid programmable protein array (NAPPA), is capable of overcoming by probing with unique sensitivity native in situ protein-protein interactions. This allows identification of the key proteins involved in the control of cancer and proliferation in the light of recent label-free NAPPA approaches based on nanotechnologies. Bioinformatics in combination with label free NAPPA, anodic porous alumina (APA) and DNA analyser (DNASER) microarrays appear capable of providing the long-range framework for the basic molecular understanding of cancer and cell cycle progression.

AKT1 leader gene and downstream targets are involved in a rat model of kidney allograft tolerance.

Tolerance is the so-called "Holy Grail" of transplantation but achieving this state is proving a major challenge, particularly in the clinical settings. This tolerance state can be induced in rodent models using a variety of maneuvers. This phenomenon is classically characterized by donor specificity (recipients accept a secondary donor-specific allograft but reject third-party allograft) as well as by the absence of chronic rejection lesion. We previously showed that administration and anti-donor anti-class II serum on the day of transplantation induce tolerance to a kidney allograft in the LEW-1W to LEW-1A strain combination. In this study, we used DNA microarrays to compare gene patterns involved in anti-donor anti-class II tolerated or untreated syngeneic kidney transplants in this strain combination. Statistical and non-statistical analyses were combined with ab initio analysis, using the recently developed leader gene approach, to shed new light on this phenomenon. Theoretical and experimental results suggest that tolerance and rejection outcome may be in large part determined by low expression variations of some genes, which can form a core gene network around specific genes such as Rac1, NFKB1, RelA, AKT1, IKBKB, BCL2, BCLX, and CHUK. Through this model, we showed that AKT1 gene, WNT pathway and NO synthesis are strictly connected to each other and may play an important role in kidney tolerance and rejection processes, with AKT1 gene being the center of this complex network of interactions.

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.

DNA bridging of yeast chromosomes VIII leads to near-reciprocal translocation and loss of heterozygosity with minor cellular defects.

Loss of heterozygosity (LOH) of tumor suppressor genes in somatic cells is a major process leading to several types of cancer; however, its underlying molecular mechanism is still poorly understood. In the present work, we demonstrate that a linear DNA molecule bridging two homologous chromosomes in diploid yeast cells via homologous recombination produce LOH-generating regions of hemizygosity by deletion. The result is a near-reciprocal translocation mutant that is characterized by slight cell cycle defects and increased expression of the multidrug-resistant gene VMR1. When the distance between target regions is approximately 40 kb, the specificity of gene targeting becomes less stringent and an ensemble of gross chromosomal rearrangements arises. These heterogeneous genomic events, together with the low frequency of specific translocation, confirm that several pathways contribute to the healing of a broken chromosome and suggest that uncontrolled recombination between parental homologs is actively avoided by the cell. Moreover, this work demonstrates that the common laboratory practice of making targeted gene deletions may result in a low, but not negligible, frequency of LOH due to the recombination events triggered between homologous chromosomes in mitosis.

Bioinformatic prediction of leader genes in human periodontitis.

BACKGROUND: Genes involved in different biologic processes form complex interaction networks. However, only a few have a high number of interactions with the other genes in the network. In previous bioinformatics and experimental studies concerning the T lymphocyte cell cycle, these genes were identified and termed "leader genes." In this work, genes involved in human periodontitis were tentatively identified and ranked according to their number of interactions to obtain a preliminary, broader view of molecular mechanisms of periodontitis and plan targeted experimentation. METHODS: Genes were identified with interrelated queries of several databases. The interactions among these genes were mapped and given a significance score. The weighted number of links (weighted sum of scores for every interaction in which the given gene is involved) was calculated for each gene. Genes were clustered according to this parameter. The genes in the highest cluster were termed leader genes. RESULTS: Sixty-one genes involved or potentially involved in periodontitis were identified. Only five were identified as leader genes, whereas 12 others were ranked in an immediately lower cluster. For 10 of 17 genes there is evidence of involvement in periodontitis; seven new genes that are potentially involved in this disease were identified. The involvement in periodontitis has been completely established for only two leader genes. CONCLUSIONS: We applied a validated bioinformatics algorithm to increase our knowledge of molecular mechanisms of periodontitis. Even with the limitations of this ab initio analysis, this theoretical study can suggest ad hoc experimentation targeted on significant genes and, therefore, simpler than mass-scale molecular genomics. Moreover, the identification of leader genes might suggest new potential risk factors and therapeutic targets.

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.

cAMP induced alterations of Chinese hamster ovary cells monitored by mass spectrometry.

Chinese Hamster Ovary fibroblasts (CHO-K1) have shown different protein contents when undergoing differentiation by 3',5'-cyclic adenosine monophosphate (cAMP), which is known to induce reverse transformation (RT) from malignancy to fibroblast-like characteristics. The mass spectrometry (MS) investigation here reported about the behavior of CHO-K1 cells before and after exposure to cAMP reveals a change in the composition of nuclear proteins associated to an inhibition of the protein expression. Possible implications of this finding on the control of cell reverse transformation are discussed.

Gene expression of human T lymphocytes cell cycle: experimental and bioinformatic analysis.

Human lymphocytes gene expression is monitored before and after PHA stimulation over 72 h, using DNA microarray technology. Results are then compared with our previous bioinformatics predictions, which identified six leader genes of highest importance in human T lymphocytes cell cycle. Experimental data are strikingly compatible with bioinformatic predictions of the specific role and interaction of PCNA, CDC2, and CCNA2 at all phases of the cell cycle and of CHEK1 in regulating DNA repair and preservation. It does not escape our notice that the conception and use of ad hoc arrays, based on a bioinformatics prediction which identifies the most important genes involved in a particular biological process, can really be an added value in cell biology and cancer research alternative to massive frequently misleading molecular genomics.

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. microGISAXS and microdiffraction of P450scc.

Ultrasmall P450scc cytochrome microcrystals are grown by classical hanging vapor diffusion and by its modification using homologous protein thin-film template displaying a long-range order. The nucleation and growth mechanisms of P450scc microcrystals are studied at the thin cytochrome film surface by a new microbeam grazing incidence small angle X-ray scattering (microGISAXS) technique developed at the microfocus beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. P450scc cytochrome crystals of about 5 microm are also investigated by synchrotron radiation diffraction in order to attempt a preliminary analysis of the atomic structure of this unique protein system yet unsolved.

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: II. microGISAX and microscopy of lysozyme.

The early steps of growth and nucleation of the lysozyme microcrystals by classical and nanotemplate-based hanging vapor diffusion methods are studied using microGISAXS at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Out-of-plane cuts in the Yoneda regions of the 2D scattering profiles point to the detection of ultrasmall lysozyme crystals by microGISAXS quite before than by light microscopy. Furthermore lysozyme crystal formation occurs quite earlier with the nanotemplate than with the classical method. Our data are compatible with two distinct modes of crystal nucleation and growth for P450sc and lysozyme.