Ivone M. Martins

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction.

β(1,3)glucan is critical for contractile ring positioning and for coupling septum synthesis to constriction of the contractile ring and plasma membrane extension during cytokinesis.

Differential activities of three families of specific β(1,3)glucan synthase inhibitors in wild-type and resistant strains of fission yeast

Three specific β(1,3)glucan synthase (GS) inhibitor families, papulacandins, acidic terpenoids, and echinocandins, have been analyzed in Schizosaccharomyces pombe wild-type and papulacandin-resistant cells and GS activities. Papulacandin and enfumafungin produced similar in vivo effects, different from that of echinocandins. Also, papulacandin was the strongest in vitro GS inhibitor (IC50 103–104-fold lower than with enfumafungin or pneumocandin), but caspofungin was by far the most efficient antifungal because of the following. 1) It was the only drug that affected resistant cells (minimal inhibitory concentration close to that of the wild type). 2) It was a strong inhibitor of wild-type GS (IC50 close to that of papulacandin). 3) It was the best inhibitor of mutant GS. Moreover, caspofungin showed a special effect for two GS inhibition activities, of high and low affinity, separated by 2 log orders, with no increase in inhibition. pbr1-8 and pbr1-6 resistances are due to single substitutions in the essential Bgs4 GS, located close to the resistance hot spot 1 region described in Saccharomyces and Candida Fks mutants. Bgs4pbr1-8 contains the E700V change, four residues N-terminal from hot spot 1 defining a larger resistance hot spot 1-1 of 13 amino acids. Bgs4pbr1-6 contains the W760S substitution, defining a new resistance hot spot 1-2. We observed spontaneous revertants of the spherical pbr1-6 phenotype and found that an additional A914V change is involved in the recovery of the wild-type cell shape, but it maintains the resistance phenotype. A better understanding of the mechanism of action of the antifungals available should help to improve their activity and to identify new antifungal targets.

Transglutaminases : recent achievements and new sources

Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, tissues, and body fluids, that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein or peptide-bound glutamine. Besides forming these bonds, that exhibit high resistance to proteolytic degradation, transglutaminases also form extensively cross-linked, generally insoluble, protein biopolymers that are indispensable for the organism to create barriers and stable structures. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have also been explored, but in a much lesser extent. Our group has identified a transglutaminase in the oomycete Phytophthora cinnamomi, which is able to induct defense responses and disease-like symptoms. In this mini-review, we report the achievements in this area in order to illustrate the importance and the versatility of transglutaminases.

Scientifically advanced solutions for chestnut ink disease

On the north regions of Portugal and Spain, the Castanea sativa Mill. culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, the causal agent being the oomycete Phytophthora cinnamomi. This oomycete is also responsible for the decline of many other plant species in Europe and worldwide. P. cinnamomi and Phytophthora cambivora are considered, by the generality of the authors, as the C. sativa ink disease causal agents. Most Phytophthora species secrete large amounts of elicitins, a group of unique highly conserved proteins that are able to induce hypersensitive response (HR) and enhances plant defense responses in a systemic acquired resistance (SAR) manner against infection by different pathogens. Some other proteins involved in mechanisms of infection by P. cinnamomi were identified by our group: endo-1,3-beta-glucanase (complete cds); exo-glucanase (partial cds) responsible by adhesion, penetration, and colonization of host tissues; glucanase inhibitor protein (GIP) (complete cds) responsible by the suppression of host defense responses; necrosis-inducing Phytophthora protein 1 (NPP1) (partial cds); and transglutaminase (partial cds) which inducts defense responses and disease-like symptoms. In this mini-review, we present some scientifically advanced solutions that can contribute to the resolution of ink disease.

Bioactive properties and functional constituents of Hypericum androsaemum L.: A focus on the phenolic profile

Hypericum androsaemum L. ethanol:water extract acted as a lipid peroxidation inhibitor and free radical scavenger. A marked inhibition of the growth of breast, lung, cervical and hepatocellular human carcinoma cell lines was also observed, whereas no toxicity was shown against non-tumor porcine liver cells (>400μg/mL). The extract was also effective in inhibiting nitric oxide production, as an indicator of the anti-inflammatory potential. The anti-Candida effects varied among different strains of the same species, C. glabrata and C. tropicalis being the most sensible species with an effect directly related with the extract concentrations tested. A significant anti-biofilm formation potential was also observed, namely for C. glabrata and C. tropicalis (biofilm reduction >90%). 5-O-Caffeoylquinic and 3-O-caffeoylquinic acids were the most abundant phenolic compounds identified in the extract, and might be related with the observed bioactive effects. Nevertheless, future studies should be carried out to obtain dose-response curves of the isolated active compounds, in order to perform further pre-clinically testing to quantify the presence of the most active compounds in the extract.

Isolation and Sequencing of Actin1, Actin2 and Tubulin1 Genes Involved in Cytoskeleton Formation in Phytophthora cinnamomi

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. On the Nordeste Transmontano region (northeast Portugal), the Castanea sativa chestnut culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, caused by Phytophthora cinnamomi which is one of the most widely distributed Phytophthora species, with nearly 1000 host species. The knowledge about molecular mechanisms responsible for pathogenicity is an important tool in order to combat associate diseases of this pathogen. Complete open reading frames (ORFs) of act1, act2 and tub1 genes who participate in cytoskeleton formation in P. cinnamomi were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) polymerase chain reaction (PCR). act1 gene comprises a 1128 bp ORF, encoding a deduced protein of 375 amino acids (aa) and 41,972 kDa. act2 ORF comprises 1083 bp and encodes a deduced protein of 360 aa and 40,237 kDa. tub1 has a total length of 2263 bp and encodes a 453 aa protein with a molecular weight of 49.911 kDa. Bioinformatics analyses shows that actin1 is ortholog to the act1 genes of Phytophthora infestans, Phytophthora megasperma and Phytophthora melonis; actin2 is ortholog to the act2 genes of P. infestans, Phytophthora brassicae, P. melonis and Pythium splendens and tubulin1 shows the highest orthology to P. infestans and P. capsici α-tubulin genes. Analysed 3D structure of the three putative proteins revealed a spatial conformation highly similar to those described for orthologous proteins obtained by X-ray diffraction.

Selection of Novel Peptides Homing the 4T1 CELL Line: Exploring Alternative Targets for Triple Negative Breast Cancer.

The use of bacteriophages to select novel ligands has been widely explored for cancer therapy. Their application is most warranted in cancer subtypes lacking knowledge on how to target the cancer cells in question, such as the triple negative breast cancer, eventually leading to the development of alternative nanomedicines for cancer therapeutics. Therefore, the following study aimed to select and characterize novel peptides for a triple negative breast cancer murine mammary carcinoma cell line– 4T1. Using phage display, 7 and 12 amino acid random peptide libraries were screened against the 4T1 cell line. A total of four rounds, plus a counter-selection round using the 3T3 murine fibroblast cell line, was performed. The enriched selective peptides were characterized and their binding capacity towards 4T1 tissue samples was confirmed by immunofluorescence and flow cytometry analysis. The selected peptides (4T1pep1 –CPTASNTSC and 4T1pep2—EVQSSKFPAHVS) were enriched over few rounds of selection and exhibited specific binding to the 4T1 cell line. Interestingly, affinity to the human MDA-MB-231 cell line was also observed for both peptides, promoting the translational application of these novel ligands between species. Additionally, bioinformatics analysis suggested that both peptides target human Mucin-16. This protein has been implicated in different types of cancer, as it is involved in many important cellular functions. This study strongly supports the need of finding alternative targeting systems for TNBC and the peptides herein selected exhibit promising future application as novel homing peptides for breast cancer therapy.

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

BackgroundClaudin-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 relevant for an early diagnosis. Additionally, claudin-low breast carcinoma peptide ligands can be used to design powerful drug delivery systems that specifically target this type of breast cancer.MethodsIn 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, such as phage forming units and ELISA, were performed to select the most interesting peptides (i.e., specific to the target cells) and bioinformatics approaches were applied to putatively identify the biomarkers to which these peptides bind.ResultsTwo 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.ConclusionsThe peptides herein identified may contribute to an earlier detection of claudin-low breast carcinomas and possibly to develop more individualized therapies.

Artificial virus particles

This chapter intends to provide an overview of the use of viruses for medical applications. The first part describes the basic characteristics of viral particles regarding their chemical composition and size, as well as their structure and assembly/disassembly abilities. Next, a discussion about the newest and most important strategies for virus particles modification including genetic, chemical, and self-assembly/encapsulation engineering towards the development of new virus particles for biomedical applications, including targeted delivery and therapy, molecular imaging for disease detection, vaccine development, and bacterial infection control.

Genes found in partial sequencing od Phytophthora cinnamomi

The Project COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Found, supported this work.