Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus.

Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis.

BACKGROUND: Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. RESULTS: Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. CONCLUSIONS: Inserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis.

Probiotic Characteristics of Lactiplantibacillus plantarum CECT 9435 and Its Survival and Competitive Properties Under Simulated Conditions of the Child Gut Microbiota.

Probiotics are valuable microorganisms effective in reducing malnutrition-related infections in children. In this work, a collection of lactobacilli strains representative of traditional Andean fermented beverages was in vitro screened for their capability to survive the gastrointestinal transit, to adhere to the intestinal epithelium and to compete under simulated conditions of the child gut microbiota. The results allowed the selection of the riboflavin overproducing strain Lactiplantibacillus plantarum CECT 9435 based on its good rate of survival under in vitro gastrointestinal conditions when included in a food matrix representing the fortified food supplement Incaparina. The strain also showed good adhesion to HT29 cells producing mucus and outstanding performance in E. coli competition for the adhesion to this epithelial cell line. L. plantarum CECT 9435 gut performance was also evaluated in the child intestinal microbiota simulated in a dynamic gut model (BFBL simulator). The viability of the probiotic candidate in the gut conditions was high during the 7-day intervention period, reaching over 1 × 107 counts in each of the reactors simulating the three colonic regions. The transient viability of L. plantarum CECT 9435 within the child gut microbiota and its adhesion capacity to intestinal cells could facilitate the strain potential benefits as probiotic added to fortified supplementary foods destined to malnourished children.

Stability of plasma electrolytic oxidation coating on titanium in artificial saliva.

Bioactive PEO coating on titanium with high Ca/P ratio was fabricated and characterized with respect to its morphology, composition and microstructure. Long-term electrochemical stability of the coating and Ti(4+) ion release was evaluated in artificial saliva. Influence of the lactic acid and fluoride ions on corrosion protection mechanism of the coated titanium was assessed using AC and DC electrochemical tests. The PEO-treated titanium maintained high passivity in the broad range of potentials up to 2.5 V (Ag/AgCl) for up to 8 weeks of immersion in unmodified saliva and exhibited Ti(4+) ion release <0.002 µg cm(-2) days(-1). The high corrosion resistance of the coating is determined by diffusion of reacting species through the coating and resistance of the inner dense part of the coating adjacent to the substrate. Acidification of saliva in the absence of fluoride ions does not affect the surface passivity, but the presence of 0.1 % of fluoride ions at pH ≤4.0 causes loss of adhesion of the coating due to inwards migration of fluoride ions and their adsorption at the substrate/coating interface in the presence of polarisation.

Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli.

Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.

Bioactive multi-elemental PEO-coatings on titanium for dental implant applications.

Bioactive PEO (Plasma Electrolytic Oxidation) coatings were generated on Grade I commercially pure titanium for dentistry applications using a Ca/P-based electrolyte with added Si, Mg, Zn or F species. Surface characteristics, chemical composition and ion liberation of the coatings were characterized using SEM/EDS, X-ray diffraction, optical profilometry, contact angle and ICP-OES. Corrosion resistance (OCP and DC polarization) was evaluated in SBF. Osteoblastogenesis and osteoclastogenesis processes on PEO-coated Ti and non-coated Ti controls were assessed after 7 days and 5 days of cell culture, respectively. Monolayer formation and metabolic activity were evaluated for the MC3T3 preosteoblastic cell line. All PEO coatings favoured differentiation processes over proliferation and presented three times greater quantity of secreted collagen than non-coated Ti control. All coating enabled osteoclast differentiation, with differences in number and size of the osteoclasts between the materials.

PEO coatings design for Mg-Ca alloy for cardiovascular stent and bone regeneration applications.

Four bioactive PEO (plasma electrolytic oxidation) coatings were generated on Mg0.8Ca alloy using a Ca/P-based electrolyte and adding Si or Fas necessary. Surface characteristics, chemical composition and ion liberation of the coatings were characterized using SEM/EDS (Scanning Electron Microscopy/Energy Dispersive X-ray spectroscopy), X-ray diffraction, optical profilometry and ICP-OES (inductively coupled plasma optical emission spectrometry). Direct biocompatibility studies were performed by seeding premyoblastic, endothelial and preosteoblastic cell lines over the coatings. Biocompatibility of the coatings was also evaluated with respect to murine endothelial, preosteoblastic, preosteoclastic and premyoblastic cell cultures using extracts obtained by the immersion degradation of the PEO-coated specimens. The coatings reduced the degradation of magnesium alloy and released Mg Ca, P, Si and F. Of all the studied compositions, the Si-containing PEO coating exhibited the optimal characteristics for use in all potential applications, including bone regeneration and cardiovascular applications. Coatings with high F content negatively influenced the endothelial cells. RAW 264.7, MC3T3 and co-culture differentiation studies using extracts of PEO coated Mg0.8Ca demonstrated improved osteoclastogenesis and osteoblastogenesis processes compared to bare alloy.

[Follow-up of serum monoclonal gammopathy at Guadalajara Health Area]. / Seguimiento de una cohorte de pacientes con gammapatía monoclonal sérica en el Area Sanitaria de Guadalajara.

OBJECTIVE: To study the clinical course of patients with a serum monoclonal protein at Guadalajara Health Area. MATERIAL AND METHODS: Prospective study of 186 patients with a newly diagnosed monoclonal component. They have been collected during the years 1999 and 2000. RESULTS: The cumulative transformation probability at 43 months was 4.99% for those patients whose monoclonal gammopathy was overlooked, and 2% at 23 months for patients with monoclonal gammopathy of undetermined significance. The cumulative probability of survival for patients with multiple myeloma was 66.7% at 21 months. The conditional mortality rate (patients/months) at 4 years due to haematological disease was 4.48 x 10(-4) for overlooked patients, 0 for diagnosed of monoclonal gammopathy of undetermined significance and 1.388 x 10(-2) for multiple myeloma diagnosed. DISCUSSION: A non malignant M component must be followed up due to it could increase patients survival rate in relation with transformation in malignant disease.

Draft Genome Sequence of Pediococcus parvulus 2.6, a Probiotic ß-Glucan Producer Strain.

We report here the draft genome sequence of the probiotic Pediococcus parvulus 2.6, a lactic acid bacterial strain isolated from ropy cider. The bacterium produces a prebiotic and immunomodulatory exopolysaccharide, and this is the first strain of the P. parvulus species whose genome has been characterized.

Enhancement of 2-methylbutanal formation in cheese by using a fluorescently tagged Lacticin 3147 producing Lactococcus lactis strain.

The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains L. lactis T1 and L. lactis IFPL730, showing branched chain amino acid aminotransferase and alpha-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry.

Metal release from ceramic coatings for dental implants.

OBJECTIVES: Two types of ceramic coatings on commercially pure titanium for dental implant applications with different Ca/P ratios in the range from 1.5 to 4.0, and two different thicknesses (∼5 and ∼15µm) were examined with the aim of underpinning the effect of coating composition, thickness and microstructure on the corrosion behavior and hydroxyapatite forming ability in SBF. METHODS: Bioactive coatings were formed on Ti by plasma electrolytic oxidation (PEO). The composition, structure, and morphology of the materials were characterized before and after the immersion in simulated body fluid solution (SBF) at 37°C for up to 4 weeks. All the materials were screened with respect to metal ion release into SBF. RESULTS: Only thick PEO coating with overstoichiometric Ca/P ratio of 4.0 exhibited capacity to induce the precipitation of hydroxyapatite over the short period of 1 week. Long term Ti(4+) ion release from all PEO-coated materials was 2-3 times lower than from the uncoated Ti. Metal ion release is attributed mostly to chemical dissolution of the coating at initial stages of immersion. SIGNIFICANCE: The long term stability was greater for thin PEO coating with overstoichiometric Ca/P ratio of 2.0, which exhibited ∼95ngcm(-2) of Ti(4+) ions release over 4 weeks. Thin PEO coatings present economically more viable option.

Bioactive plasma electrolytic oxidation coatings--the role of the composition, microstructure, and electrochemical stability.

A Plasma electrolytic oxidation (PEO) process was used to produce bioactive coatings on Ti. PEO coatings with Ca/P atomic ratio of 1.7 and 4.0 were fabricated and characterized with respect to their morphology, composition, and microstructure. AC and DC electrochemical tests were used to evaluate the effect of (i) organic additives (amino acids, proteins, vitamins, and antibiotics) in alpha-minimum essential medium (α-MEM) on electrochemical stability of noncoated and PEO-coated Ti and (ii) coating composition, microstructure, and corrosion behavior on the cell response in α-MEM. PEO-coated Ti showed higher corrosion resistance than the noncoated Ti in MEM with and without organic additives by an order of magnitude. The corrosion resistance in α-MEM decreased with time for nonmodified Ti and increased for PEO-coated Ti; the latter was because of the adsorption of the proteins in the coating pores which increased the diffusion resistance. The presence of Ca and P in titanium oxide coating at the Ca/P ratio exceeding that of any stoichiometric Ca-P-O and Ca-P-O-H compounds facilitates faster osteoblast cell adhesion.

Evidence that the essential response regulator YycF in Streptococcus pneumoniae modulates expression of fatty acid biosynthesis genes and alters membrane composition.

The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism.

[Monoclonal gammopathy: a frequently overlooked diagnosis]. / Gammapatía monoclonal: un diagnóstico frecuentemente soslayado.

OBJECTIVE: To know the rate of occurrence of monoclonal bands in a clinical laboratory as an estimate of monoclonal gammopathy incidence, and to detect the proportion of these bands in which an explicit clinical diagnosis or follow-up request is not established. Other objectives are to describe its distribution and the characteristics of the patients. PATIENTS AND METHODS: 200 patients were studied in which a monoclonal band had been detected de novo. RESULTS: The incidence was 6.59 x 10(-4) year(-1). In 59.5% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was monoclonal gammopathy with uncertain meaning. Average age of patients was 74.4 years and the difference between percentages by sex was statistically significant. DISCUSSION: The data suggest a lack of clinical effort when the result is the appearance of a monoclonal band. There is no discrepancy in the distribution of the bands and in the characteristics of the patients with regard to what is described in other studies.