Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms.

Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of ß-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics.

Painful shoulder in a patient receiving anticoagulation therapy with rivaroxaban. / Hombro doloroso en paciente anticoagulado con rivaroxabán.

We report the case of an 83-year-old man on rivaroxaban treatment, with pain in right shoulder, back of the upper arm, forearm and wrist, and inability to extend the wrist and fingers, as the result of an injury. The radiographs were normal, but thoracic CT showed an axillary artery pseudoaneurysm and a secondary hematoma that compressed the brachial plexus. This should be borne in mind in patients with painful shoulder, treated with anticoagulant therapy and without associated radiographic changes.

The yin-yang of kinase activation and unfolding explains the peculiarity of Val600 in the activation segment of BRAF.

Many driver mutations in cancer are specific in that they occur at significantly higher rates than - presumably - functionally alternative mutations. For example, V600E in the BRAF hydrophobic activation segment (AS) pocket accounts for >95% of all kinase mutations. While many hypotheses tried to explain such significant mutation patterns, conclusive explanations are lacking. Here, we use experimental and in silico structure-energy statistical analyses, to elucidate why the V600E mutation, but no other mutation at this, or any other positions in BRAF's hydrophobic pocket, is predominant. We find that BRAF mutation frequencies depend on the equilibrium between the destabilization of the hydrophobic pocket, the overall folding energy, the activation of the kinase and the number of bases required to change the corresponding amino acid. Using a random forest classifier, we quantitatively dissected the parameters contributing to BRAF AS cancer frequencies. These findings can be applied to genome-wide association studies and prediction models.

Large-scale metabolome analysis and quantitative integration with genomics and proteomics data in Mycoplasma pneumoniae.

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Rice straw pulp obtained by using various methods.

Rice straw was used as an alternative raw material to obtain cellulosics pulps. Pulping was done by using classics reagents as soda (with anthraquinone and parabenzoquinone as aditives), potassium hydroxide and Kraft process. The holocellulose, alpha-cellulose and lignin contents of rice straw (viz. 60.7, 41.2 and 21.9 wt%, respectively) are similar to those of some woody raw materials such as pine and eucalyptus, and various non-wood materials including olive tree prunings, wheat straw and sunflower stalks. Pulping tests were conducted by using soda, soda and anthraquinone at 1 wt%, soda and parabenzoquinone at 1 wt%, potassium hydroxide and sodium sulphate (Kraft process) under two different sets of operating conditions, namely: (a) a 10 wt% reagent concentration, 170 degrees C and 60 min; and (b) 15 wt% reagent, 180 degrees C and 90 min. The solid/liquid ratio was 6 in both cases. Paper sheets made from pulp extracted by cooking with soda (15 wt%) and AQ (1 wt%) at 180 degrees C and 90 min pulp exhibit the best drainage index, breaking length, stretch and burst index (viz. 23 degrees SR, 3494 m, 3.34% and 2.51 kN/g, respectively).

Use of high-boiling point organic solvents for pulping oil palm empty fruit bunches.

Oil palm empty fruit bunches were used as an alternative raw material to obtain cellulosic pulp. Pulping was done by using high-boiling point organic solvents of decreased polluting power relative to classical (Kraft, sulphite) solvents but affording operation at similar pressure levels. The holocellulose, alpha-cellulose and lignin contents of oil palm empty fruit bunches (viz. 66.97%, 47.91% and 24.45%, respectively) are similar to those of some woody raw materials such as pine and eucalyptus, and various non-wood materials including olive tree prunings, wheat straw and sunflower stalks. Pulping tests were conducted by using ethyleneglycol, diethyleneglycol, ethanolamine and diethanolamine under two different sets of operating conditions, namely: (a) a 70% solvent concentration, 170 degrees C and 90 min; and (b) 80% solvent, 180 degrees C and 150 min. The solid/liquid ratio was six in both cases. The amine solvents were found to provide pulp with better properties than did the glycol solvents. Ethanolamine pulp exhibited the best viscosity and drainage index (viz. 636 mL/g and 17 degrees SR, respectively), and paper made from it the best breaking length (1709 m), stretch (1.95%), burst index (0.98 kN/g) and tear index (0.33 mNm(2)/g). Operating costs can be reduced by using milder conditions, which provide similar results. In any case, the amines are to be preferred to the glycols as solvents for this purpose.

Habilidad de los médicos para palpar nódulos en modelos mamarios de silicona / The hability of the physicians to detect nodules in silicone breast models

El examen físico de la mama es la técnica más empleada para la detección de nódulos mamarios, sin embargo, poco se sabe acerca de la capacidad de los profesionales de nuestro país para realizarlo. En este estudio se pretendió evaluar la capacidad de los médicos para realizar el examen de las mamas, midiendo su habilidad en descubrir nódulos en modelos mamarios de silicona, para lo cual se entrevistó a 46 médicos de diversas especialidades y a 18 matronas a los cuales se les pidió examinar un modelo mamario de silicona que contiene nódulos de diferente tamaño, consistencia y ubicación. Se midió el tiempo tomado en el examen, el número y tipo de nódulos encontrados y se registró diversos parámetros de la técnica utilizada. El 75,4 por ciento de los nódulos fue descubierto. Los ginecólogos detectaron en promedio más nódulos durante el examen, seguidos por los internistas y los cirujanos. Los determinantes de un mayor índice de detección fueron: tiempo tomado en el examen, uso de mayor número de dedos, examen a diferentes profundidades, ejecución de movimientos rotatorios de los dedos durante el examen y realización de un examen ordenado