Screening and characterization of novel specific peptides targeting MDA-MB-231 claudin-low breast carcinoma by computer-aided phage display methodologies

Background: Claudin-low breast carcinoma represents 19% of all breast cancer cases and is characterized by an aggressive progression with metastatic nature and high rates of relapse. Due to a lack of known specific molecular biomarkers for this breast cancer subtype, there are no targeted therapies available, which results in the worst prognosis of all breast cancer subtypes. Hence, the identification of novel biomarkers for this type of breast cancer is highly important for early diagnosis and targeted therapy. Methods: In this work, we propose the identification of peptides for the specific recognition of MDA-MB-231, a cell line representative of claudin-low breast cancers, using phage display (both conventional panning and BRASIL). Binding assays were performed to select the most interesting peptides and bioinformatics approaches were applied to putatively identify the biomarkers to which these peptides bind. Results: Two peptides were selected using this methodology specifically targeting MDA-MB-231 cells, as demonstrated by a 4 to 9 log higher affinity as compared to control cells. The use of bioinformatics approaches provided relevant insights into possible cell surface targets for each peptide identified. Conclusions: The peptides herein identified may contribute to an earlier detection of claudin-low breast carcinomas and possibly to develop more individualized therapies.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-FEDER021053 (PTDC/SAU-BMA/121028/2010), RECI/BBB-EBI/0179/2012 (FCOMP-01– 0124-FEDER-027462), the strategic funding of UID/BIO/04469/2013 unit, and the Projects “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, REF. NORTE-07–0124-FEDER-000027, and “BioInd – Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07–0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Franklin L. Nóbrega acknowledges FCT for the grant SFRH/BD/86462/2012

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

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

The national inventory of geological heritage: methodological approach and results

A existência de um inventário nacional de património geológico é fundamental para se poderem implementar estratégias de geoconservação. Este trabalho apresenta a metodologia usada no desenvolvimento do mais completo inventário de geossítios, realizado até ao momento em Portugal, assim como os principais resultados obtidos. O inventário vai integrar o Sistema de Informação do Património Natural e o Cadastro Nacional dos Valores Naturais Classificados, ambos geridos pelo Instituto de Conservação da Natureza e da Biodiversidade.The existence of a national inventory of the geological heritage is of paramount importance for the implementation of a geoconservation strategy. This paper presents the methodological approach used to produce the most complete geosites inventory in Portugal, so far, and the obtained results. This inventory will be uploaded into the National Database of Natural Heritage managed by the Portuguese authority for nature conservation.Este trabalho é apoiado pela Fundação para a Ciência e a Tecnologia, através do financiamento plurianual do CGUP e do projecto de investigação “Identificação, caracterização e conservação do património geológico: uma estratégia de geoconservação para Portugal” (PTDC/CTE-GEX/64966/2006).info:eu-repo/semantics/publishedVersio

Examination of potential virulence factors of Candida tropicalis clinical isolates from hospitalized patients

Candida tropicalis has been reported to be one of the Candida species which is most likely to cause bloodstream and urinary tract infections in hospitalized patients. Accordingly, the aim of this study was to characterize the virulence of C. tropicalis by assessing antifungal susceptibility and comparing the expression of several virulence factors. This study was conducted with seven isolates of C. tropicalis from urine and blood cultures and from central venous catheter. C. tropicalis ATCC 750 was used as reference strain. Yeasts adhered (2 h) to epithelial cells and silicone and 24 h biofilm biomass were determined by crystal violet staining. Pseudohyphae formation ability was determined after growth in fetal bovine serum. Enzymes production (hemolysins, proteases, phospholipases) was assessed by halo formation on agar plates. Susceptibility to antifungal agents was determined by E-test. Regarding adhesion, it can be highlighted that C. tropicalis strains adhered significantly more to epithelium than to silicone. Furthermore, all C. tropicalis strains were able to form biofilms and to express total hemolytic activity. However, protease was only produced by two isolates from urine and by the isolates from catheter and blood. Moreover, only one C. tropicalis (from catheter) was phospholipase positive. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. Four strains were susceptible-dose dependent to itraconazole and one clinical isolate was found to be resistant

Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Author Summary: Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is a necrotizing disease of the skin, subcutaneous tissue and bone. Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin for 8 weeks. However, in advanced stages of the disease, surgical resection of the destroyed skin is still required. The use of bacterial viruses (bacteriophages) for the control of bacterial infections has been considered as an alternative or a supplement to antibiotic chemotherapy. By using a mouse model of M. ulcerans footpad infection, we show that mice treated with a single subcutaneous injection of the mycobacteriophage D29 present decreased footpad pathology associated with a reduction of the bacterial burden. In addition, D29 treatment induced increased levels of IFN-γ and TNF in M. ulcerans -infected footpads, correlating with a predominance of a mononuclear infiltrate. These findings suggest the potential use of phage therapy in BU, as a novel therapeutic approach against this disease, particularly in advanced stages where bacteria are found primarily in an extracellular location in the subcutaneous tissue, and thus immediately accessible by lytic phages.This work was supported by a grant from the Health Services of Fundacao Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/64032/2009, SFRH/BD/41598/2007, and SFRH/BPD/68547/2010 to GT, TGM, and AGF, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript