Microencapsulated Chitosan-Based Nanocapsules: A New Platform for Pulmonary Gene Delivery.

In this work, we propose chitosan (CS)-based nanocapsules (NCs) for pulmonary gene delivery. Hyaluronic acid (HA) was incorporated in the NCs composition (HA/CS NCs) aiming to promote gene transfection in the lung epithelium. NCs were loaded with a model plasmid (pCMV-ßGal) to easily evaluate their transfection capacity. The plasmid encapsulation efficiencies were of approx. 90%. To facilitate their administration to the lungs, the plasmid-loaded NCs were microencapsulated in mannitol (Ma) microspheres (MS) using a simple spray-drying technique, obtaining dry powders of adequate properties. In vivo, the MS reached the deep lung, where the plasmid-loaded CS-based NCs were released and transfected the alveolar cells more homogeneously than the control formulation of plasmid directly microencapsulated in Ma MS. The HA-containing formulation achieved the highest transfection efficiency, in a more extended area and more homogeneously distributed than the rest of tested formulations. The new micro-nanostructured platform proposed in this work represents an efficient strategy for the delivery of genetic material to the lung, with great potential for the treatment of genetic lung diseases.

Bacillus safensis subsp. osmophilus subsp. nov., isolated from condensed milk, and description of Bacillus safensis subsp. safensis subsp. nov.

A bacterial strain, designated BC09T, was isolated from a contaminated sample of condensed milk. Phylogenetic analyses based on 16S rRNA gene sequences placed strain BC09T into the genus Bacillus with its closest relatives being Bacillus safensis and Bacillus australimaris with 100 and 99.9 % similarity, respectively. Analysis of the gyrB gene confirmed the closeness of strain BC09T with respect to the species B. safensis since it presented 97.8 and 95.2 % similarity values, respectively, to the type strains of B. safensis and B. australimaris. DNA-DNA hybridization confirmed these results showing averages of 67 and 56 %, respectively, between strain BC09T and the type strains of B. safensis and B. australimaris. Average nucleotide identity blast values obtained for BC09T compared to the closest relative type strains were 95.7 and 67.6 %, respectively, and predicted DNA-DNA hybridization values were 93.1 and 51.9 %, respectively. However, strain BC09T differs from the type strains of its closest relatives in several phenotypic characteristics. MK-7 was the only menaquinone detected and iso-C15:0 and anteiso-C15:0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, two unidentifed glycolipids, three unidentified lipids and one unidentifed phosphoglycolipid. Meso-diaminopimelic acid was detected in the peptidoglycan. The G+C content was 40.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BC09T represents a new subspecies of B. safensis, for which the name Bacillus safensis subsp. osmophilus subsp. nov. is proposed. The type strain is BC09T (=LMG 30124T, =CECT 9344T).

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris.

Galium verum, also known as Lady's Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and ß-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

Considerations on bacterial nucleoids.

The classic genome organization of the bacterial chromosome is normally envisaged with all its genetic markers linked, thus forming a closed genetic circle of duplex stranded DNA (dsDNA) and several proteins in what it is called as "the bacterial nucleoid." This structure may be more or less corrugated depending on the physiological state of the bacterium (i.e., resting state or active growth) and is not surrounded by a double membrane as in eukayotic cells. The universality of the closed circle model in bacteria is however slowly changing, as new data emerge in different bacterial groups such as in Planctomycetes and related microorganisms, species of Borrelia, Streptomyces, Agrobacterium, or Phytoplasma. In these and possibly other microorganisms, the existence of complex formations of intracellular membranes or linear chromosomes is typical; all of these situations contributing to weakening the current cellular organization paradigm, i.e., prokaryotic vs eukaryotic cells.

Recent patents on microbial proteases for the dairy industry.

This paper reviews the general characteristics of exo and endopeptidases of microbial origin currently used in the milk industry. It also includes recent patents developed either to potentiate the enzymatic activity or to improve the resulting milk derivatives. The main application of these proteases is in the cheese-making industry. Although this industry preferentially uses animal rennets, and in particular genetically engineered chymosins, it also utilizes milk coagulants of microbial origin. Enzymes derived from Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica are currently used to replace the conventional milk-clotting enzymes. In addition, the dairy industry uses microbial endo and exoproteases for relatively new applications, such as debittering and flavor generation in cheese, accelerated cheese ripening, manufacture of protein hydrolysates with improved functional properties, and production of enzyme-modified cheeses. Lactic acid bacteria play an essential role in these processes, hence these bacteria and the proteases they produce are currently being investigated by the dairy industry and are the subject of many of their patent applications.

cDNA cloning of a novel gene codifying for the enzyme lycopene ß-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (ß-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic ß-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, ß-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-ß from Ficus carica (Lyc-ß Fc), which codes for the enzyme lycopene ß-cyclase (ß-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. ß-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to ß-carotene as a result of the enzyme's action. The ß-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the ß-carotene. The lycopene to ß-carotene conversion rate was 90%. The experiments carried out in this work showed that ß-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to ß-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing ß-LCY in E. coli, we have obtained a new gene for ß-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.

Genetic stabilization of Saccharomyces cerevisiae oenological strains by using benomyl.

Wild-type oenological strains of Saccharomyces cerevisiae are usually aneuploid and heterozygotes; thus, when they are used as starters in must fermentation the resulting wine characteristics may vary from year to year. Treatment of a wild-type S. cerevisiae oenological strain with benomyl (methyl-l-butylcarbamoyl-2-benzimidazole carbamate), an antifungal agent shown to cause chromosome loss in yeasts, resulted in a stable starter strain in which the parental oenological traits were unchanged. The oenological S. cerevisiae strain was treated with benomyl in two different ways (A and B), and sporulation ability and spore viability were subsequently assayed. Treatment A resulted in both the highest numbers of tetrads and a reduction in DNA cell content, while treatment B increased spore viability. Fermentation assays were carried out with spore clones obtained from treatment A, and the concentrations of glycerol, lactic acid, acetic acid, and ethanol resulting from the treated strains were found to be similar to those of the parental strain. Benomyl treatment thus achieved stable, highly sporulating oenological S. cerevisiae strains of low ploidy, but preserved the desirable oenological properties of the parental strain.

Genetic stabilization of Saccharomyces cerevisiae oenological strains by using benomyl

Wild-type oenological strains of Saccharomyces cerevisiae are usually aneuploid and heterozygotes; thus, when they are used as starters in must fermentation the resulting wine characteristics may vary from year to year. Treatment of a wild-type S. cerevisiae oenological strain with benomyl (methyl-l-butylcarbamoyl-2-benzimidazole carbamate), an antifungal agent shown to cause chromosome loss in yeasts, resulted in a stable starter strain in which the parental oenological traits were unchanged. The oenological S. cerevisiae strain was treated with benomyl in two different ways (A and B), and sporulation ability and spore viability were subsequently assayed. Treatment A resulted in both the highest numbers of tetrads and a reduction in DNA cell content, while treatment B increased spore viability. Fermentation assays were carried out with spore clones obtained from treatment A, and the concentrations of glycerol, lactic acid, acetic acid, and ethanol resulting from the treated strains were found to be similar to those of the parental strain. Benomyl treatment thus achieved stable, highly sporulating oenological S. cerevisiae strains of low ploidy, but preserved the desirable oenological properties of the parental strain (AU)

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