Study of stability and biophysical characterization of ranibizumab and aflibercept.

The anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab (Lucentis®) and aflibercept (EyLea®) are currently used as monthly or bimonthly intravitreal injections to treat potentially retinal diseases such as wet age-related macular degeneration (AMD) or diabetic macular edema (DME). Because of the complications associated with repeated intra-vitreal injections, there is considerable interest in developing a sustained delivery system. The purpose of this study was to examine the stability of both therapeutic proteins under physiological conditions as well as when incorporated into drug delivery systems (DDS). First, thermotropic properties in physiological conditions and at different pH values were evaluated by differential scanning calorimetry (DSC) to determine the protein denaturation temperature. Second, the effects of pH and incubation time on conformational changes and aggregation were evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, and size-exclusion chromatography (SEC). Also, the ability of both proteins to bind to VEGF was tested in the aforementioned experimental conditions for up to 30 days. Finally, we investigated the stability of both proteins after a rapid screening method that simulates the first homogenizing step during the protein microencapsulation process. This method allowed the development of stable ranibizumab and aflibercept formulations that may be useful to entrap these proteins into microparticles selecting the most convenient organic solvent and protein stabilizers.

Nueva estrategia docente: la enseñanza de la microbiología a través de su historia / A new teaching strategy to teach microbiology through its history

Ante el nuevo reto de la implantación del Espacio Europeo de Educación Superior los métodos de estudio deben adaptarse y servirse de herramientas innovadoras. Una buena estrategia para comprender una ciencia, su metodología y objetivos, es conocer su historia. Esta estrategia se hace especialmente útil en el caso de la Microbiología por ser una disciplina joven, que se ha cimentado precisamente en la metodología que sus creadores han ido elaborando.El objetivo de este trabajo ha sido crear una herramienta de utilidad en el aprendizaje de la Microbiología. Para ello hemos tomado como punto de apoyo su historia, es decir, explicar la Microbiología mediante la descripción de los descubrimientos y hechos que contribuyeron al desarrollo de esta ciencia. Para ello se ha creado una página web con dos aplicaciones independientes que operan sobre una base de datos común.1. La primera de carácter público para los alumnos aunque con posibilidad de acceso restringido; se pueden consultar los contenidos sin posibilidad de alterarlos.2. La otra aplicación es privada, los profesores pueden administrar los contenidos, y hacer uso de diferentes herramientas que facilitan la gestión de los mismos(AU)

In the light of the challenge presented by the European Higher Education Area, study strategies will have to adapt themselves and take advantage of innovative tools provided by modern information technology. One good way of understanding a science, its methodology and objectives is by taking an interest in its history. This approach is especially useful with regard to Microbiology, which is a fairly young discipline founded upon and made cohesive by the methods devised and constantly elaborated on by its designers.The aim of this work has been to offer guidance towards learning Microbiology. To this end we have chosen as our starting point, and indeed one of the keystones of our approach, the history of the subject itself; that is to say, an explanation of Microbiology via a description of the discoveries and milestones that have contributed to the development of this science. Thus we have set up a web page with two separate applications operating on one common database.1. The first is openly available to the students, although allowing the possibility of restricted access. Its contents may be consulted but not altered.2. The second is accessible only by lecturers, who may administer its contents and resort to different tools to facilitate the management of the information available on the site(AU)

The role and application of enterococci in food and health.

The genus Enterococcus is the most controversial group of lactic acid bacteria. Studies on the microbiota of many traditional cheeses in the Mediterranean countries have indicated that enterococci play an important role in the ripening of these cheeses, probably through proteolysis, lipolysis, and citrate breakdown, hence contributing to their typical taste and flavour. Enterococci are also present in other fermented foods, such as sausages and olives. However, their role in these products has not been fully elucidated. Furthermore, the production of bacteriocins by enterococci is well documented. Moreover, enterococci are nowadays used as probiotics. At the same time, however, enterococci have been associated with a number of human infections. Several virulence factors have been described and the number of vancomycin-resistant enterococci is increasing. The controversial nature of enterococci has prompted an enormous increase in scientific papers and reviews in recent years, where researchers have been divided into two groups, namely pro and contra enterococci. To the authors' impression, the negative traits have been focused on very extensively. The aim of the present review is to give a balanced overview of both beneficial and virulence features of this divisive group of microorganisms, because it is only acquaintance with both sides that may allow their safe exploitation as starter cultures or co-cultures.

Enterococcus faecium RZS C5, an interesting bacteriocin producer to be used as a co-culture in food fermentation.

Enterocins, bacteriocins produced by enterococci, are gaining interest because of their industrial potential. Due to its bacteriocin production, Enterococcus faecium RZS C5, a natural cheese isolate, has a strong activity towards Listeria monocytogenes. For this reason, the strain may be applicable as a bacteriocin-producing co-culture in food fermentation in order to reduce the risk on Listeria outgrowth. The strain displays remarkable bacteriocin production kinetics. Whereas most lactic acid bacteria produce bacteriocin in a growth-associated way until the beginning of the stationary phase, bacteriocin production by E. faecium RZS C5 in MRS broth at controlled pH values below 7.5 is characterised by a boost of bacteriocin activity levels in the very early growth phase. In addition, bacteriocin production kinetics are closely linked to the environmental and cultural conditions. However, no straightforward statement about the effect of environmental stress on bacteriocin production can be made since the effect is dependent on the type of stress applied. Kinetic experiments in milk and on pilot scale, applying Cheddar cheese-making conditions, have indicated that the strain may be effective as a bacteriocin-producing co-culture. Further research is needed to evaluate the use of E. faecium RZS C5 as a co-culture for the production of fermented sausage.

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins.

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources.

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.

Applicability of a bacteriocin-producing Enterococcus faecium as a co-culture in Cheddar cheese manufacture.

Two strains, Enterococcus faecium RZS C5 and E. faecium DPC 1146, produce listericidal bacteriocins, so-called enterocins. E. faecium RZS C5 was studied during batch fermentation in both a complex medium (MRS) and in milk to understand the influence of environmental factors, characteristic for milk and cheese, on both growth and bacteriocin production. Fermentation conditions were chosen in view of the applicability of in situ enterocin production during Cheddar cheese production. Enterocin production by E. faecium RZS C5 in MRS started in the early logarithmic growth phase, and enterocin activity decreased during the stationary phase. The effect of pH on enterocin production and decrease of activity was as intense as the effect on bacterial growth. Higher enterocin production took place at pH 5.5 compared with pH 6.5. The use of lactose instead of glucose increased the production of enterocin, and at higher lactose concentration, production increased more and loss of activity decreased. The production in skimmed milk compared to MRS was lower and was detected mainly in the stationary phase. When casein hydrolysate was added to the milk, enterocin production was higher and started earlier, indicating the importance of an additional nitrogen source for growth of E. faecium in milk. For co-cultures of E. faecium RZS C5 with the starters used during Cheddar cheese manufacture, no enterocin activity was detected during the milk fermentation. Furthermore, the applicability of E. faecium RZS C5 and E. faecium DPC 1146 strains was tested in Cheddar cheese manufacture on pilot scale. Enterocin production took place from the beginning of the cheese manufacturing and was stable during the whole ripening phase of the cheese. This indicates that both an early and late contamination of the milk or cheese can be combated with a stable, in situ enterocin production. The use of such a co-culture is an additional safety provision beyond good manufacturing practices.

Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium.

AIMS: Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s). METHODS AND RESULTS: LAB were present in high numbers in final products as well as during processing. Isolates, Enterococcus faecium B1 and E. faecium B2 (E. faecium LMG 19827 and E. faecium LMG 19828, respectively) inhibited Gram-positive indicators, including Listeria monocytogenes. Partially purified bacteriocins showed a proteinaceous nature. Activity was stable after heat-treatment except at alkaline pH values. Both strains displayed a bacteriostatic mode of action. Bacteriocin production was associated with late exponential/early stationary growth. Molecular mass, calculated by SDS-PAGE, was 3.4 kDa for B1 bacteriocin, and 3.4 kDa and 5.8 kDa for B2 bacteriocins. PCR screening of enterocin-coding genes revealed three amplified fragments in total genomic DNA that may correspond with PCR signals for enterocin P, enterocin L50A and enterocin L50B. Both B1 and B2 contained a 42-kb plasmid. No differences in bacteriocinogenic capacity were found between wild type strains and plasmid-cured strains. CONCLUSIONS: It was possible to isolate bacteriocinogenic E. faecium active against various Gram-positive bacteria from final products of tempeh. SIGNIFICANCE AND IMPACT OF THE STUDY: A first step in applying biopreservation to fermented South-east Asian foods is to obtain bacteriocinogenic LAB from this source. Such isolates may also be used for biopreservation of mould-fermented foods in general, including various types of mould-ripened cheese.

[Evaluation of health status using Survey SF-36: preliminary results in Mexico]. / Evaluación del estado de salud con la Encuesta SF-36: resultados preliminares en México.

OBJECTIVE: To establish a multidimensional profile of the health status in a population of southeastern Mexico and analyze the psychometric properties of a translation of the SF-36 survey authorized by the International Quality of Life Assessment Project. MATERIAL AND METHODS: The SF-36 was administered to 257 volunteers of a clinic and a control group in a governmental institution. The methodology proposed by the author of the survey to build the health scales was used without modifications. The validity and reliability of the SF-36 adaptation to Mexico was psychometrically analyzed. RESULTS: Eight scales or health concepts related to physical function, physical role, body pain, general health, vitality, social function, emotional role and mental health were constructed. In the health service volunteers, the scale with the lowest mean was general health (63), and that with the highest (89) was physical role. In the control population, the lowest mean was general health (73) and the highest (94.6) was physical function. A comparison of the health scales of both groups showed significant differences in physical function, physical role, body pain, general health and vitality. CONCLUSIONS: Based on the psychometric evaluation, the SF-36 shows satisfactory consistency with its supposed validity and reliability, although the translation of certain items will be further analyzed for subsequent modifications.

Cross-sectional deformation of the aorta as measured with magnetic resonance imaging.

Vessel geometry is commonly accepted as one of the primary factors influencing blood flow patterns. The vessels near the heart present a particular challenge because myocardial contraction creates dynamic changes in vessel geometry due to the movement created by the contraction of the myocardial muscle. The importance of vessel movement and deformation on blood flow patterns in the coronary arteries has been previously demonstrated. For larger vessels such as the aorta, the effects are less well understood, partially because no estimates of the dynamic variations in vessel cross section shape geometry have been reported. This study was undertaken to provide an estimate of the amount of dynamic variation in cross-sectional shape present in the aorta. Two young healthy male subjects were used, with measurements taken in the ascending aorta, aortic arch, and descending thoracic aorta using Magnetic Resonance Imaging (MRI). The magnitude of elliptical deformation was measured throughout the cardiac cycle by taking a discrete Fourier transform of the radius versus angle plot. Deformations of more than 7 percent of the mean vessel radius were noted. This level of deformation may be enough to influence flow patterns in the aorta significantly, and thus should be included in future flow studies.

Role of the renin-angiotensin system in the development of thyroxine-induced hypertension.

OBJECTIVE: We evaluated the influence of chronic blockade of the renin-angiotensin system on hypertension induced by long-term thyroxin (T4) administration. To this end, we determined the effects of chronic treatment with captopril on blood pressure, cardiac hypertrophy and other renal and metabolic variables of hypertensive hyperthyroid rats. METHODS: T4 was administered s.c. at 0.38 mumol/kg per day and captopril was given in the drinking water (1.38 mmol/l). Both treatments were maintained for 6 weeks. Control rats received tap water. After the treatment period, the rats were placed in metabolic cages. Later, blood pressure was measured in conscious rats by intra-arterial determination. RESULTS: T4-treated rats showed an increased mean arterial pressure (MAP) whereas, in rats treated with T4 plus captopril, MAP was similar to that of the control group. Captopril did not affect the increased heart rate or ventricular weight/body weight ratio of hyperthyroid rats, but it improved the reduced creatinine clearance of these animals. CONCLUSIONS: The elevation in blood pressure produced by long-term T4 administration was prevented by chronic blockade of the renin-angiotensin system. Captopril improved the renal function of hyperthyroid rats, but did not affect the relative cardiac hypertrophy of these animals.

[Prophylaxis of bacterial endocarditis in children with congenital heart disease: current status of parents' knowledge]. / Profilaxis de endocarditis bacteriana en niños con cardiopatía congénita: Estado actual de conocimientos por parte de los padres.

OBJECTIVE: The purpose of this study was to assess the knowledge that parents of children with congenital cardiac disease have about bacterial endocarditis (EB) and its prophylaxis (PEB). PATIENTS AND METHODS: To this end, an elusive questionnaire was prepared and offered to one hundred parents of children who needed PEB. The group was classified according to the age of the children, the cultural level of the parents an the event of a dental or if another septic focus was known in the past. Likewise, the parents' knowledge of their child's disease was analyzed. RESULTS: Of the results obtained, it stood-out that the parents appeared well informed about their child's illness, but their information was insufficient in relationship to EB and PEB. Of those tested, 85% defined the child's heart disease, while only 14% did so for EB. Likewise, only 67.7% of the children who underwent an intervention for a septic focus performed PEB correctly. The knowledge of this theme are in relationship to the family's cultural formation. CONCLUSIONS: We emphasize the need to reinforce the information about PEB that the cardiologists, pediatricians and general physicians give to the families in order to prevent the morbidity and mortality caused by EB.

Mechanism studies of coomassie blue and silver staining of proteins.

A relatively high complexation affinity has been found for coomassie blue G-250 and the following amino acids: arginine; tyrosine; lysine; and histidine. A linear relationship was observed between log molar absorptivity and log molecular weight of 52 of 69 proteins, polypeptides, and di- and tripeptides that were allowed to react with coomassie blue G-250 in solution. The solution complexation results were used in a study of the detection of the following model proteins: bovine serum albumin, lysozyme, recombinant DNA derived human insulin, and calmodulin. Interactions between coomassie blue stained gels and silver detection reagents were determined and used as the basis for studies of enhanced sensitivity of detection of electrophoretically developed proteins. Sensitivity enhancements of up to eight-fold were observed when various sulfonic acid dye complexed proteins were detected with silver reagents versus the use of silver reagents alone. A site-directed nucleation of silver caused by the protein complexed sulfonic acid dyes is proposed as a mechanism for the observed enhancements.

Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure.

A combined Coomassie blue-silver stain method has been developed in sodium dodecyl sulfate-polyacrylamide gels for the detection of proteins using the model compounds bovine serum albumin, lysozyme, and recombinant DNA-derived human insulin. Sensitivity was enhanced 2.2 to 8.6 times by the new method relative to that of silver staining alone. The new method may also be useful in enhancing detection sensitivities of other proteins.