Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

Magnesium aminoclays were synthesized and used to transform non-competent Escherichia coli JM109 using the exogenous plasmid pUC19. The structure determined for the Mg aminoclays is analogous to 2:1 trioctahedral smectites such as talc, with an approximate composition R8Si8Mg6O16(OH)4, where R = CH2CH2NH2, morphologically arranged in layered sheets. Mg aminoclays were employed as a cationic vehicle that enabled the passage of plasmids across the cell envelope and led to genetic modification of the host. A stock solution of 10 mg/mL of Mg aminoclays was prepared, mixed with E. coli JM109 and pUC19 plasmid, and spread over Petri dishes containing lysogeny broth (LB), isopropyl ?-D-1-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-gal), ampicillin and various concentrations of agar (14%). The transformation efficiency obtained was higher for 1% and 2% agar even though transformation also occurred at agar concentrations of 3% and 4%. The optical density of E. coli JM109 and spreading time were also adjusted, favoring transformation when cells were used in their exponential growth phase (OD600 = 1.0) and spread for 90 s. Transformation was confirmed by the growth of blue colonies in LB/IPTG/X-gal/agar Petri dishes containing ampicillin, by regrowth of biomass in liquid media containing ampicillin and by agarose gel electrophoresis of the linearized pUC19 plasmid that followed plasmidic DNA extraction from 4 blue colonies. The maximum transformation efficiency achieved was 7.0 × 103 CFU/?g pUC19. This transformation approach proved to be suitable for a convenient, cost-effective, room-temperature, risk-free and rapid transformation of non-competent E. coli JM109.This study was supported by the Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects FCOMP-01- 0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/2013, UID/FIS/04650/2020, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioEnv - Biotech nology and Bioengineering for a sustainable world”. The authors acknowledge the fellowship SFRH/BD/71661/2010 awarded to Gabriel Mendes under the scope of the MIT-Portugal Program. The authors also thank Paul Brown and Takuya Harada for the help in obtaining TEM images.info:eu-repo/semantics/publishedVersio

Transformation of Escherichia coli JM109 using pUC19 by the Yoshida effect

Transformation of non-competent Escherichia coli JM109 was accomplished using pUC19 as donor plasmid and sepiolite as the acicular material to promote cell piercing via application of friction with a polystyrene stick or a magnetic bar on the surface of a hydrogel containing agar. An automatic spreading setup was built with a conventional stirring plate and compared to manual spreading. Several parameters were optimized, namely, the agar content of the hydrogel (2%), concentration of cells (OD=1.3 corresponding to 1.4x109 bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30 sec). Efficiency values up to 4.1 x 104 CFU/µg pUC19 were obtained. The method proved to be suitable for a rapid and low cost transformation of non-competent E. coli JM109, where higher values of efficiency do not need to be attained.Portuguese Foundation for Scienceand Technology (FCT) and the European Community fund FEDER,through Program COMPETE, under the scope of the Projects FCOMP-01-0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/ 2013, PEST-C/FIS/UI607/2013, RECI/BBB-EBI/0179/2012 (FCOMP-01- 0124-FEDER-027462) and the Projects “BioEnv — Biotechnology and Bioengineering for a sustainableworld” and “Matepro—Optimizing Materials and Processes”. NORTE-07-0124-FEDER-000048 was co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER. The authors also acknowledge the fellowship awarded to Gabriel Mendes SFRH/BD/71661/2010 under the scope of the MITPortugal Program

A flexible-docking approach for the design of novel cancer peptidomimetic drugs

Cancer is the second leading cause of death worldwide and the lack of alternative therapies has kept patients dependent on classic chemotherapy. The most occurring form of cancer amongst women is breast cancer and the triple negative cell subtype (TNBC) is responsible for a high metastatic and mortality rate, as it presents molecular and genetic shifts, lacks specific targeting and responds poorly to existing therapies. Recent studies have provided convincing evidence that the therapeutic outcome of chemotherapy may be affected by the expression and activity of receptor tyrosine kinases and their phosphatase pathways (MAPK/ERK, PI3/AKT, among others). These proteins are implicated in mechanisms of cell survival, drug resistance and Epithelial-Mesenchymal Transition (EMT), and are therefore key targets for TNBC cancer cell subtype. However, many inhibitors developed for these targets have not succeeded at a clinical level and present low solubility. As a result of the pronounced decline in productivity experienced by drug discovery efforts in the last years, novel approaches to the rational design of new drugs are now being pursued. A potential solution might be the use of natural or synthetic peptides and peptidomimetics targeting protein-protein interactions essential for signaling networks function. The combination of several bioinformatic approaches (docking, virtual screening, pharmacophore models, among others) allows the use of the vast amount of existing information on available compounds and protein-protein interactions in structural databases. In this study we designed a procedure for small peptidomimetics structure-based rational drug design capable of blocking the active sites of SNAiL1, a protein that has been suggested as a potent repressor of E-cadherin expression and consequently, as an inducer of EMT transition in TNBCs. A random library was created using a composite approach for drug-like compound identification from the PubChem and Development Therapeutics NCI/NIH compound databases, which combined structure-based virtual screening (known motifs of peptide structures within proteins and small molecules) and Z-score comparison. Docking studies were performed to map the polypeptides activity and stability: (1) point alteration studies using non-natural aminoacids for helical stability over a wider range (since linear peptides adopt many confirmations in aqueous solution) using Ramachandran plot dihedral angles estimation; (2) quantitative structure-activity relationships (QSAR) using radical modification chemical studies and (3) umbrella sampling for dissociations studies. The peptidomimetic SNAiL1 model created suggested at least two radical modifications for a strong inhibition

Identification of peptides targeting human osteoarthritic chondrocytes using phage display

Osteoarthritis (OA) is one of the most common degenerative joint disease and is characterized by a progressive degradation of articular cartilage extracellular matrix (ECM) leading to loss of joint mobility and function, accompanied by chronic pain. Currently used therapies for cartilage repair are still far from generating regenerated tissue with quality and stability comparable to native cartilage. We hypothesize that alterations of chondrocyte-ECM interactions in OA affect the expression of cell surface adhesion molecules and phage display can allow the identification of high affinity peptides by screening peptide libraries against these targets. Here, we report the use of phage display to identify novel peptides which specifically bind to human chondrocytes isolated from patients with OA. Chondrocytes were isolated from healthy and osteoarthritic cartilage obtained from patients undergoing partial knee arthroplasty and characterized, in terms of expression of cell surface proteins and expression of chondrocyte-specific genes, before the panning experiments to assess their phenotypic stage. A phage library displaying random 12-amino acid peptides was first incubated with chondrocytes from healthy donors (control cells) and then with osteoarthritic chondrocytes. A 12-amino acid peptide (GFQMISNNVYMR) was identified, showing high affinity to osteoarthritic chondrocyte cells (about 8-fold higher than the wild-type phage lacking recombinant peptides - control). Bioinformatics analysis was performed by creating a protein structure database of known and stereo-chemical validated OA-associated cell membrane proteins. Protein-peptide docking revealed, from the overall complex stability, solvent accessibility and binding site prediction that the membrane protein MMP28 is expected to be the putative receptor of the identified peptide ligand. Future work will be devoted to integrate the identified peptide sequence into nanocarrier systems to provide localization of therapeutic molecules into OA cartilage. If successful, these nanocarriers can offer important insights into the regenerative mechanisms of cartilage and could be applied for developing more efficient and less invasive therapies for treating OA

Targeted therapy using phage technology: a computational and experimental breast cancer study

During the past two decades cancer biology knowledge has widely increased and shifted the paradigm of cancer treatment from nonspecific cytotoxic agents to selective, mechanism-based therapeutics. Initially, cancer drug design was focused on compounds that rapidly killed dividing cells. Though still used as the backbone of current treatments, these highly unspecific targeting drugs lead to significant toxicity for patients, narrowing the therapeutic index, and frequently lead to drug resistance. Therefore, cancer therapies are now based on cancer immunotherapy and targeted agents, whereas novel treatments are strategically combining both to improve clinical outcomes. Despite the nanotechnology advances dictating the development of targeted therapies in diverse classes of nano-based carriers, virus-based vectors still remain highly used due to its biocompatibility and specificity for the target. Particularly, bacteriophages are an interesting alternative ‘nanomedicine’ that can combine biological and chemical components into the same drug delivery system. The great potential of this novel platform for cancer therapy is the ability to genetically manipulate the virus-vector to display specific targeting moieties. Phage display technology, a general technique used for detecting interfaces of various types of interacting proteins outside of the immunological context, allows the target agents to locate the target (with an increased selection process for the specific binding – termed biopanning) and play their essential role inhibiting molecular pathways crucial for tumour growth and maintenance. Phage display specificity core is related with the binding of small peptides displayed at their coat or capsid proteins, enriched during biopanning. Bioinformatics plays an important role in testing and improving phage display libraries by effective epitope mapping, selecting from a large set of random peptides those with a high binding affinity to a target of interest. In this work we demonstrate the screening of a manually constructed 7-mer peptide library of M13KE phage particles against MDA-MB-231 and -435 cancer cell lines. Two peptides – TLATVEV and PRLNVSP – with high affinity for the referred cells were identified, respectively. Based on computationally predicted epitopes based on the peptides extracted from this library the linear peptide sequence was docked onto known membrane proteins from the used cell lines and peptides-proteins interactions were mapped. Umbrella sampling studies were performed to predict the binding affinity and to improve future rational design of binding peptides to these cancer cells

Novel “nano-phage” interfaces for wireless biosensors

The prevention of food-borne illness has become a very important factor in public health. Meantime, the conventional microbiological detection techniques are time consuming, require proficiency and appropriate laboratory conditions. Recently, there has been an extensive work undertaken towards the development of diagnostic biosensor devices for on-site detection of biological threats that explore a diversity of transduction mechanisms and bio-recognition elements. In particular, the environmentally robust filamentous phages have been successfully used as an alternative to fragile antibodies in wireless biosensor system for real-time pathogen detection. However, when phages are used as interface, they can aggregate forming bundles of fibers that cannot cover completely the sensor’s interface leading to the decrease in sensor’s performance. In this work we developed novel wireless magnetoelastic biosensors with interface formed by biorecognition nanoparticles called “nano-phage”. “Nano-phage” comprises nanoparticles with diameter ~11 nm composed of self-assembled fusion major coat protein of landscape phages selected against the target analyte. For proof-of-concept, we investigated interfaces formed by three model phages selected from landscape libraries: streptavidin binders 7b1 and SAE10 and clone E2 highly specific and selective for S. typhimurium. Beside food borne pathogens, this new approach can be used to develop biosensors with increased performance for early detection of cancer diseases and other pathologies

M13 phage grafted with peptide motifs as a tool to detect amyloid- oligomers in brain tissue

Oligomeric clusters of amyloid- (A) are one of the major biomarkers for Alzheimers disease (AD). However, proficient methods to detect A-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying A-derived peptides on their surface preferentially interact with A-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized A-aggregates in hippocampus at an early age, prior to the occurrence of A-plaques. Similarly, the bacteriophages reveal the presence of such small A-aggregates in post-mortem hippocampus tissue of ADpatients. These results advocate bacteriophages displaying A-peptides as a convenient and low-cost tool to identify A-oligomers in post-mortem brain tissue of AD-model mice and AD patients.FCT -Fundação para a Ciência e a Tecnologia(SFRH/BD/101171/2014)info:eu-repo/semantics/publishedVersio

Combining self-assembly and phage display to develop a targeted nanodelivery system for cartilage therapies

The present work focuses on a specific challenge of great clinical importance: targeted therapy for osteoarthritis (OA). The identification of molecules expressed exclusively, or at elevated levels, by cartilage cells (chondrocytes) in OA conditions might provide a strategy for targeted OA therapy by enhancing drug specificity. Towards this goal, we report the identification of peptide ligands, that bind selectively and with affinity to OA chondrocytes, using phage display, a technology in which a library of phage particles expressing a wide diversity of peptides is screened to identify those that bind the desired target. A random 12-mer peptide library, displayed on the surface of a filamentous phage (M13), was screened by biopanning against the surface of OA chondrocytes to identify peptide ligands specific for these cells. Healthy and OA chondrocytes for the panning experiments were isolated from cartilage samples obtained in local hospitals under pre-established agreement and from patients after informed consent. Isolation and expansion of chondrocytes was performed according to published procedures and their phenotype was characterized by FACS (CD44, CD26, CD10 and CD95), RT-PCR (aggrecan, collagen I, II and X and Sox9), immunohistochemistry (collagen I, II and X), SDS-PAGE and western blot analyses. The identified peptide sequences are being integrated into nanocarrier systems formed by self-assembling approaches and the potential of these targeted delivery systems is currently being tested in vitro. This approach, if successful, will yield important insights into the regenerative mechanisms of cartilage and could be applied for developing more efficient and less invasive therapies for treating OA