Plant Virus Vectors 3.0: Transitioning into Synthetic Genomics.

Plant viruses were first implemented as heterologous gene expression vectors more than three decades ago. Since then, the methodology for their use has varied, but we propose it was the merging of technologies with virology tools, which occurred in three defined steps discussed here, that has driven viral vector applications to date. The first was the advent of molecular biology and reverse genetics, which enabled the cloning and manipulation of viral genomes to express genes of interest (vectors 1.

The major facilitator superfamily transporter ZIFL2 modulates cesium and potassium homeostasis in Arabidopsis.

Potassium (K(+)) is an essential mineral nutrient for plant growth and development, with numerous membrane transporters and channels having been implicated in the maintenance and regulation of its homeostasis. The cation cesium (Cs(+)) is toxic for plants but shares similar chemical properties to the K(+) ion and hence competes with its transport. Here, we report that K(+) and Cs(+) homeostasis in Arabidopsis thaliana also requires the action of ZIFL2 (Zinc-Induced Facilitator-Like 2), a member of the Major Facilitator Superfamily (MFS) of membrane transporters.

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: scale-up challenges.

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale.

On the dual effect of glucose during production of pBAD/AraC-based minicircles.

Minicircles are promising vectors for DNA vaccination, gene or cell therapies due to their increased transfection efficacy and transgene expression. The in vivo production of these novel vectors involves the arabinose inducible excision of a parental molecule into a minicircircle and a miniplasmid bacterial backbone. Tight control of recombination is crucial to maximize minicircle yields and purity.

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin.

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells. We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels.

The genome sequence of the highly acetic acid-tolerant Zygosaccharomyces bailii-derived interspecies hybrid strain ISA1307, isolated from a sparkling wine plant.

In this work, it is described the sequencing and annotation of the genome of the yeast strain ISA1307, isolated from a sparkling wine continuous production plant. This strain, formerly considered of the Zygosaccharomyces bailii species, has been used to study Z. bailii physiology, in particular, its extreme tolerance to acetic acid stress at low pH.

Intertidal macrofauna and environmental stress at a riverine-marine boundary.

A field experiment was carried out to test the effect of pore water salinity on the macrobenthic assemblages in an estuarine region of the Tagus estuary (Portugal) subjected to wide fluctuations in salinity. The conditions at the experimental site ranged from freshwater (minimum salinity 0.2) to mesohaline (maximum salinity 15.

The bacterial protein azurin impairs invasion and FAK/Src signaling in P-cadherin-overexpressing breast cancer cell models.

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells.

Multivariate analysis applied to agglomerated macrobenthic data from an unpolluted estuary.

We agglomerated species into higher taxonomic aggregations and functional groups to analyse environmental gradients in an unpolluted estuary. We then applied non-metric Multidimensional Scaling and Redundancy Analysis (RDA) for ordination of the agglomerated data matrices. The correlation between the ordinations produced by both methods was generally high.

Engineering of bacterial strains and their products for cancer therapy.

The use of live bacteria in cancer therapies offers exciting possibilities. Nowadays, an increasing number of genetically engineered bacteria are emerging in the field, with applications both in therapy and diagnosis. In parallel, purified bacterial products are also gaining relevance as new classes of bioactive products to treat and prevent cancer growth and metastasis.

Characterization of an estuarine environment by means of an index based on intertidal macrofauna.

Macrobenthic intertidal assemblages from five Atlantic Iberian estuaries were analyzed to develop an estuarine index. An R-mode analysis revealed a close association between the isopod Cyathura carinata, the polychaete Hediste diversicolor and the bivalve Scrobicularia plana. Although these species are abundant in all the estuaries considered, they tend to be absent from sites at the marine and freshwater ends of the environmental gradient.

Genetically engineered stem cell-based strategies for articular cartilage regeneration.

Cartilage is frequently injured, often as a result of inflammatory rheumatic diseases or sports-related trauma. Given its nonvascular nature, articular cartilage has a limited capability for self-repair and currently the few therapeutic options still have uncertain long-term outcomes. Cell-based surgical therapies using autologous chondrocytes to repair cartilage injury have been used in the clinic for over a decade, but this approach has shown mixed results mainly due to the low number of harvested chondrocytes and the loss of cartilage-related phenotype and functionality after several passages of in vitro culture.

A BCAM0223 mutant of Burkholderia cenocepacia is deficient in hemagglutination, serum resistance, adhesion to epithelial cells and virulence.

Burkholderia cepacia complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs).

The Pht1;9 and Pht1;8 transporters mediate inorganic phosphate acquisition by the Arabidopsis thaliana root during phosphorus starvation.

• The activation of high-affinity root transport systems is the best-conserved strategy employed by plants to cope with low inorganic phosphate (Pi) availability, a role traditionally assigned to Pi transporters of the Pht1 family, whose respective contributions to Pi acquisition remain unclear. • To characterize the Arabidopsis thaliana Pht1;9 transporter, we combined heterologous functional expression in yeast with expression/subcellular localization studies and reverse genetics approaches in planta. Double Pht1;9/Pht1;8 silencing lines were also generated to gain insight into the role of the closest Pht1;9 homolog.

Genomic expression analysis reveals strategies of Burkholderia cenocepacia to adapt to cystic fibrosis patients' airways and antimicrobial therapy.

Pulmonary colonization of cystic fibrosis (CF) patients with Burkholderia cenocepacia or other bacteria of the Burkholderia cepacia complex (Bcc) is associated with worse prognosis and increased risk of death. During colonization, the bacteria may evolve under the stressing selection pressures exerted in the CF lung, in particular, those resulting from challenges of the host immune defenses, antimicrobial therapy, nutrient availability and oxygen limitation. Understanding the adaptive mechanisms that promote successful colonization and long-term survival of B.

The antibacterial properties of docosahexaenoic omega-3 fatty acid against the cystic fibrosis multiresistant pathogen Burkholderia cenocepacia.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens that cause multiresistant pulmonary infections in patients with cystic fibrosis (CF). In this study, we evaluated the in vitro antimicrobial efficacy of eight unsaturated fatty acids against Burkholderia cenocepacia K56-2, a CF epidemic strain. Docosahexaenoic acid (DHA) was the most active compound.

Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress.

The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5'-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3'.

Law-medicine interfacing: patenting of human genes and mutations.

Mutations, Single Nucleotide Polymorphisms (SNPs), deletions and genetic rearrangements in specific genes in the human genome account for not only our physical characteristics and behavior, but can lead to many in-born and acquired diseases. Such changes in the genome can also predispose people to cancers, as well as significantly affect the metabolism and efficacy of many drugs, resulting in some cases in acute toxicity to the drug. The testing of the presence of such genetic mutations and rearrangements is of great practical and commercial value, leading many of these genes and their mutations/deletions and genetic rearrangements to be patented.

Mutation detection in plasmid-based biopharmaceuticals.

As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of high-quality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.

Microbial-based therapy of cancer: current progress and future prospects.

The use of bacteria in the regression of certain forms of cancer has been recognized for more than a century. Much effort, therefore, has been spent over the years in developing wild-type or modified bacterial strains to treat cancer. However, their use at the dose required for therapeutic efficacy has always been associated with toxicity problems and other deleterious effects.

Trimeric autotransporter adhesins in members of the Burkholderia cepacia complex: a multifunctional family of proteins implicated in virulence.

Trimeric autotransporter adhesins (TAAs) are multimeric surface proteins exclusively found in bacteria. They are involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance, and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains.

Picomolar detection limit on a magnetoresistive biochip after optimization of a thiol-gold based surface chemistry.

The surface biochemistry plays a crucial role in the development of stable and reproducible bioanalytical devices. Very often, it represents the bottleneck of a successful integration of magnetoelectronic transducers with the biological receptors on its interface. Here is discussed how a thiolgold surface chemistry can be tailored and optimized in order to allow the biofunctionalization of a magnetoresistive biochip, preventing loss of viability by corrosion while improving its sensitivity.

Photo-response behavior of electrospun nanofibers based on spiropyran-cyclodextrin modified polymer.

Tunable and durable photochromic materials are a rapidly expanding area of interest, with applications ranging from biomedical devices to industrial-fields. Here we examine electrospun poly (methacrylic acid) PMAA nanofibers covalently modified with the highly photochromic molecule, spiropyran (SP) or a derivate SP which is firstly coupled to a cyclodextrin molecule (βCD). The photochromic properties of the starting materials and of the nanofibers were investigated.

A microwell platform for the scale-up of a multistep bioconversion to bench-scale reactors: sitosterol side-chain cleavage.

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to complex whole cell multistep bioconversions, requiring prolonged incubation periods. The present study aims to contribute to filling this gap. The side-chain cleavage of sitosterol to androstenedione (AD) with Mycobacterium sp.

Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of the ET-12 lineage.

Members of the Burkholderia cepacia complex (Bcc) are respiratory pathogens in patients with cystic fibrosis (CF). Close repetitive DNA sequences often associate with surface antigens to promote genetic variability in pathogenic bacteria. The genome of Burkholderia cenocepacia J2315, a CF isolate belonging to the epidemic lineage Edinburgh-Toronto (ET-12), was analysed for the presence of close repetitive DNA sequences.