Langmuir-Blodgett Protein Multilayer Nanofilms by XFEL

Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) has created many new opportunities for protein crystallography, including radiation damage mitigation and the study of dynamics at room temperature. This field is rapidly evolving, requiring new methods of macromolecule organization into diffracting arrays, since current methods of sample preparation and delivery are often the bottleneck which limits productivity. We propose here the LangmuirBlodgett (LB) protein nanofilm technology as a novel approach for direct \u201con chip\u201d protein molecules organization into the 3D ordered diffracting arrays. The combination of this advanced technology with the XFEL for fixed target SFX has the potential to become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins of life science interest and with pharmaceutical industry impac

Science and Technology for A Sustainable Human Development

At the end of the NanoWorld Conference in San Francisco 25 April 2018 it was decided to formalize the move of the NanoWorld Journal Headquarter from Texas (USA) at United Scientific Group to Bergamo (Europe) at Fondazione ELBA Nicolini, moving also to Paris the Nano World Conferences from March 4-6 2019, previously in San Francisco and Boston

Microarray-based Functional Nanoproteomics for an Industrial Approach to Cancer. II Mass Spectrometry and Nanoconductimetry

Using the New England BioLabs (NEBL) SNAP-based Genes Expression in conjunction with our “sub-micron arrays” (Anodic Porous Allumina and/ or Kapton based Nanopores), we exploit our proprietary microarrays scanner (DNASER, DNA analyzer) and Label Free Nanotechnologies to carry out the following tasks:\ud \ud 1) Construction of SNAP-based Genes Nanoarrays, using gold surface coated for 10 minutes with 2% solution of 3-Aminopropyltriethoxysilane (APTES) in acetone, rinsed in acetone and dried with filtered air. Full length complementary DNAs (cDNAs) for onco-suppressor 53 (p53), Cyclin-dependent kinase 2 (CDK2), SH2 (Src Homology 2) domain of the proto-oncogene tyrosine-protein kinase (Src) and tyrosine-protein phosphatase non-receptor type 11 (PTPN11) were amplified and cloned. Printing mix was prepared with 0.66 μg/μl DNA capture reagent BG-PEG-NH2 for the one-step synthesis of SNAP-tag substrates from esters on labels or surfaces;\ud \ud 2) Determination of Protein-Protein Interaction for the chosen cancer following the identification of leader genes (or hub genes, investigated with theoretical ab initio bioinformatics analysis using in-house software and algorithms, and then experimentally confirmed via DNASER). These genes are expressed by PURE (Protein synthesis Using Recombinant Elements) Express in spots less than 1 micron size piezo-microdispensed and then characterized via Label Free proprietary Autoflex Mass Spectrometry (MS) integrated with ad hoc software, namely the Spectrum Analyzer and Data Set manager (SpADS) and a proprietary Quartz Crystal Micro-balance with Dissipation factor monitoring (QCM_D) Nanoconductimetry, enabling to describe properties such as changes in frequency and conductance, viscoelasticity and dissipation factor. Solutions without DNA were prepared (called Master Mix, MM), as negative controls, in printing mix. Negative controls were prepared with a varying concentration range of SNAP capture reagent. As a positive control (for fluorescence analysis) mouse IgG or rabbit IgG (Pierce, IL, USA) were added in a printing mix instead of DNA

Protein Crystallization by Anodic Porous Alumina (APA) Template: The Example of Hen Egg White Lysozyme (HEWL)

In this communication, we report anodic porous alumina (APA) template induced crystallization. The APA nanotemplate was prepared on the glass substrate for the hen egg white lysozyme (HEWL) crystal growth. The changes in the lysozyme crystals morphology, namely in the a/c axis ratio, were observed in the crystal grown by APA nanotemplate, but not in the crystal obtained with classical hanging drop vapor diffusion method, under the same experimental conditions. The comparison of the diffraction data of the two crystals as well as bioinformatics and data mining approaches and molecular dynamics simulations suggest a possible explanation of the nanotemplate crystallization phenomenon and shed light on the APA-induced nanocrystallography

Synchrotron powder diffraction study of radiation damage in Langmuir Blodgett nanotemplate crystallised protein

Polycrystalline samples of lysozyme were prepared with and without a Langmuir-Blodgett (LB) thin film template via both the hanging drop method and batch crystallisation. Powder diffraction methods are used to compare these samples and to measure their resistance to radiation damage at room temperature. The X-ray induced amorphisation of the samples was followed as a function of time and it was shown that diffraction does not entirely disappear even at very long exposure times. Two distinct kinetic timescales are evident suggesting that early and late stage processes are quite different. Radiation damage was also shown to be localized in the sample in the region where the beam impinges

Langmuir-Blodgett Technology for Drugs Production and Delivery: Insights and Implications from an In Silico Study

Insulin dynameomics here reported as a database pertaining to a series of Molecular Dynamics (MD) ran for Protein Data Bank (PDB) entries consisting in bioinformatics and molecular dynamics simulation of Langmuir-Blodgett and classical insulin with and without insulin receptor, appears to have profound implications for drug design and endocrinology. The slight differences in conformation and dynamics here reported may indeed explain why LB-insulin is more stable when binds to its receptor (lower free energy) and this could be useful when designing new drugs and pharmaceuticals

Microarray-based Functional Nanoproteomics for an Industrial Approach to Cancer: I Bioinformatics and miRNAome

Oral Lichen Planus (OLP) is a common chronic inflammatory disease, which involves the mucous membranes of the oral cavity, with an overall age-standardized prevalence of 1.27% (0.96% in men and 1.57% in women) as reported in the literature by McCartan and Healy. In our previous work, we exploited a bioinformatics approach, namely the Leader Gene Algorithm (LGA), enabling to underpin the main hub genes (termed as Leader Genes) involved in biological processes. In the case of OLP, we found a complex network made up of 132 genes and, in particular, we found five Leader Genes (namely, JUN, EGFR, FOS, IL2, and ITGB4). Using a subsequent bioinformatics algorithm, we managed to find the 48.39% of the already established OLP-related microRNAs (miRNAs), suggesting that at least half of the OLP-related microRNAome (miRNAome) finely tunes few, highly interconnected hub genes. Now, we would use real clinical samples in order to validate our predicted biomarkers, using molecular biology techniques, mass-spectrometry (MS) and ad hoc in-house developed instruments, such as Nanoconductimetry via Quartz Crystal Microbalance with Dissipation factor monitoring (QCM_D). A unique combination of genomics and proteomics approaches can indeed represent a promising innovation for a personalized treatment of OLP and oral cancer

NAPPA-Based Vaccines for a New Proteogenomics Approach for Public Health

Vaccinology was developed for the first time in 1796 when Jenner empirically implemented a vaccine against smallpox using animal-to-human cowpox inoculation. Since then, it has become a very complex science due to the merging of disciplines ranging from structural and functional, cellular and molecular biology and immunology to bioinformatics and nanobiotechnology, as well as systems biology and synthetic biology and engineering. In the frame of evidence-based medicine (EBM), evidence-based vaccinology emerged as an important sub-field: vaccinology has nowadays become more and more predictive and personalized. With the discovery that many patients with cancer develop antibodies against p53 (the so-called oncoantibodies), it was evident that oncoprotein are immunogenic and can be used for immunotherapeutics purposes. In this manuscript, we report Nucleic Acid Programmable Protein Arrays (NAPPA)-based Quartz Crystal Microbalance (QCM) measurement of p53 immunogenicity and kinetics, in the perspective of developing an effective p53 therapy. NAPPA-based QCM_D can be a useful platform for proving the immunogenicity of oncoprotein-based vaccines. Recently, the field of vaccinology has extended from vaccines for infectious diseases to vaccines not only preventive but also therapeutic for chronic-degenerative diseases such as cancer. Peptide-based immunotherapeutics has been proven to be quite effective for cancer treatment and NAPPA-based QCM_D has the promise of providing clinicians with quick, rapid and cheap measurement of oncoprotein kinetics and bindings with immune cells. Moreover, it can be a precious tool for implementing personalized and predictive vaccinology

Determination of Protein-Protein Interaction for Cancer Control via Mass Spectrometry and Nanoconductimetry of NAPPA SNAP Arrays: An Overview

Background: Protein-protein interactions play a major role in Cancer Control and their detailed understanding by Label-Free Nanotechnology is essential especially within the framework of a personalized medicine-based approach. Material and Methods: We implemented an array of label-free nanobiotechnologies, including the Quartz Crystal Microbalance with Dissipation factor monitoring (QCM_D). We used it for the conductometric monitoring of an antiblastic (temozolomide) interacting with genes and proteins, such as MLH1, that represents a biomarker of the rate survival of patients suffering from brain tumors, outcome of chemotherapy and resistance to drug itself. We coupled the Nucleic Acid Programmable Protein Arrays (NAPPA) and the cell-free protein array with the quartz crystal microbalance technology. In another proof of principle, we coupled the NAPPA with the SNAP tag E. colicell-free expression system. The goal is to analyze the protein-protein interaction using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Bruker Ultraflex and \u201cProtein synthesis Using Recombinant Elements\u201d (PURE) system, thus avoiding the \u201cblack box\u201d nature of the cell extract. The E. coliin vitro transcription/translation system (IVTT) in respect to the reticulocyte lysate (RRL) or human lysate (HL) is totally characterized and represents an advantage for the subsequent mass spectrometry (MS) analysis. An R Script for Mass Spectrometry Data Preprocessing before Data Mining (SpADS) provides the user with peak recognition and amplitude independent subtraction functions. The MS samples are obtained from SNAP-NAPPA spots and printed on gold coated glass slides in higher density, in order to obtain an amount of protein appropriate for MS analysis. Conclusion: We developed a coherent approach that overcome the drawbacks and pitfalls of the traditional laborious and time-consuming labeled and fluorescence-based experimental procedures. This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography that can enable new strategies for drug design separately reported, defines our approach to cancer control