To identify genes specifically expressed during early stages of actinorhizal nodule development, a cDNA library made from poly(A) RNA from root nodules of Alnus glutinosa was screened differentially with nodule and root cDNA, respectively. Seven nodule-enhanced and four nodule-specific cDNA clones were isolated. By using in situ hybridization, two of the nodule-specific cDNAs were shown to be expressed at the highest levels in infected cells before the onset of nitrogen fixation; one of them, ag12 (A. glutinosa), was examined in detail. Sequencing showed that ag12 codes for a serine protease of the subtilisin (EC 3.4.21.14) family. Subtilisins previously appeared to be limited to microorganisms. However, subtilisin-like serine proteases have recently been found in archaebacteria, fungi, and yeasts as well as in mammals; a plant subtilisin has also been sequenced. In yeast and mammals, subtilases are responsible for processing peptide hormones. A homolog of ag12, ara12, was identified in Arabidopsis; it was expressed in all organs, and its expression levels were highest during silique development. Hence, our study shows that subtilases are also involved in both symbiotic and nonsymbiotic processes in plant development.

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SUMMARY The beverage cash crop coffee (Coffea arabica L.) is subject to severe losses caused by the rust fungus Hemileia vastatrix. In naturally resistant coffee plants, a specific hypersensitive reaction (HR) may be elicited early to stop fungal infection. To isolate host genes involved in HR, we undertook an expressed sequence tags (ESTs) analysis. Two cDNA libraries were constructed using suppression subtractive hybridization (SSH) and 527 non-redundant ESTs were generated from 784 randomly picked clones. Classification of the ESTs into several functional categories showed that more than one-quarter of the predicted proteins might encode disease resistance (R) proteins, stress- and defence-proteins, and components of signal transduction pathways. Twenty-eight differentially screened sequences (DSSs) were selected after differential hybridization of 1000 cDNA clones from each library. Investigation of the expression patterns of a subset of 13 DSSs showed higher levels of gene expression in inoculated plants compared with control plants. HR-up-regulation of transcript accumulation occurred for 9 out of the 13 genes 24 and 48 h after H. vastatrix challenge. Two genes encoded homologues of the Arabidopsis DND1 and NDR1 proteins, suggesting conservation of resistance signalling pathways in perennial plants. Other HR-regulated sequences matched receptor kinases, AP2 domain- and WRKY transcription factors, cytochromes P450, heat shock 70 proteins, glucosyltransferases and proteins of unknown function. The ESTs reported here provide a useful resource for studying coffee resistance responses and for improving C. arabica for durable disease resistance.

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 μg/mL) to induce the same effects as the AMPs (500–7.8 μg/mL) or EOs (365.2–19.7 μg/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5–7.8 μg/mL and 39.3–19.7 μg/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

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Nanotechnology has expanded into a broad range of clinical applications. In particular, metal nanoparticles (MNPs) display unique antimicrobial properties, a fundamental function of novel medical devices. Combining MNPs with commercial antimicrobial drugs (e. g., antibiotics, antifungals and antivirals) may offer several opportunities to overcome some disadvantages of their individual use and enhance effectiveness. MNPs-conjugates display multiple advantages. As drug delivery systems, the conjugates can extend the circulation of the drugs in the body, facilitate intercellular targeting, improve drug stabilization, and possess superior delivery. Concomitantly, they reduce the required drug dose, minimize toxicity and broaden the antimicrobial spectrum. In this work, the common strategies to combine MNPs with clinically used antimicrobial agents are underscored. Furthermore, a comprehensive survey about synergistic antimicrobial effects, mechanism of action and cytotoxicity is depicted. SilverAntimicrobial, drug deliver, anticancer, anti-angiogenic and biosensors 46,47 Gold Drug delivery, catalyst for medical therapy, antimicrobial conjugations, anticancer, gene therapy and diagnostic 48,49 Copper and copper oxide Antimicrobial and catalysis 50, 51 Iron and iron oxideAnticancer therapy, magnetic resonance imaging, targeted drug delivery and cell separation catalysis [52][53][54] Zinc oxide Personal care products, coatings, drug delivery, anticancer and antimicrobial 55, 56Aluminum oxide Drug delivery, biosensing, cancer therapy, antimicrobial, biomolecular preservation, immunotherapy 57 Titanium oxideDrug delivery, photodynamic therapy, cell imaging, biosensors, and genetic engineering, antimicrobial 58,59 Platinum Biomedical devices, anticancer therapies, cardiovascular diseases, bioimaging, nanozymes, biosensors, antimicrobial 60, 61 PalladiumPhotothermal agents, photoacoustic agents, antimicrobial, anticancer, gene/drug carriers, prodrug activators and biosensors 62,63 Scheme 1. MNPs used in biomedical applications. Silver Nanoparticles (AgNPs)AgNPs are the most prevalent inorganic nanoparticles applied as antimicrobial agent. AgNPs have demonstrated high antimicrobial activity comparable to Ag ionic form. However, several concerns have emerged regarding their cytotoxicity. The toxicity mechanisms, long-term accumulation effects and dose-response relationship are still grievously unknown. 64 Gold Nanoparticles (AuNPs)AuNPs are extremely valuable in developing antibacterial agents due to their low toxicity, high propensity for functionalization, eclectic effects, easy detection and photothermal activity. AuNPs per se possess very low antimicrobial activity, but numerous studies on the antimicrobial activity of AuNPs conjugated with small molecules, such as drugs, vaccines and antibodies, have been reported.

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.