Improvement of the Pour Plate Method by Separate Sterilization of Agar and Other Medium Components and Reduction of the Agar Concentration.

Although the pour plate method is widely employed in microbiological quality control, it has certain drawbacks, including having to melt the culture medium before seeding. In this study, the preparation of the culture medium was modified by using a lower concentration of agar (10 g/L), which was separated from the nutrients during sterilization. The new protocol was assessed in media frequently used in microbiological quality control of food, cosmetics, and pharmaceutical products, with tryptic soy agar (TSA), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). In comparison with the conventionally produced media, the modifications significantly improved the growth of Saccharomyces cerevisiae in SDA, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium, and Candida albicans in TSA and Escherichia coli ATCC 8739 and ATCC 25922 and S. Typhimurium in VRBG. The modified VRBG was also more selective for Pseudomonas aeruginosa. Regarding physicochemical properties, a significantly lower pH was observed in TSA and VRBG and lower strength values in TSA. Sterilizing agar separately from the other components of the medium and reducing the agar concentration to 10 g/L can improve microorganism growth and enhance the selectivity of differential media in the pour plate method. These modifications could facilitate the automation of this culture technique. IMPORTANCE In the era of rapid microbiological methods, there is a need to improve long-established culture techniques. Drawbacks of the pour plate method include having to melt each medium separately before seeding. For this technique, we demonstrate that separating the agar from the other components of commonly used media during sterilization and reducing the agar concentration to 10 g/L can enhance microbial growth. The new protocol could have advantages in routine laboratory practice because less agar is required and the same molten agar suspension can be used to prepare different media. Moreover, these modifications could facilitate the automation of the pour plate method.

Consequences of excessive plasticity in the hippocampus induced by perinatal asphyxia.

Perinatal asphyxia (PA) is one of the most frequent risk factors for several neurodevelopmental disorders (NDDs) of presumed multifactorial etiology. Dysfunction of neuronal connectivity is thought to play a central role in the pathophysiology of NDDs. Because underlying causes of some NDDs begin before/during birth, we asked whether this clinical condition might affect accurate establishment of neural circuits in the hippocampus as a consequence of disturbed brain plasticity. We used a murine model that mimics the pathophysiological processes of perinatal asphyxia. Histological analyses of neurons (NeuN), dendrites (MAP-2), neurofilaments (NF-M/Hp) and correlative electron microscopy studies of dendritic spines were performed in Stratum radiatum of the hippocampal CA1 area after postnatal ontogenesis. Protein and mRNA analyses were achieved by Western blot and RT-qPCR. Behavioral tests were also carried out. NeuN abnormal staining and spine density were increased. RT-qPCR assays revealed a ß-actin mRNA over-expression, while Western blot analysis showed higher ß-actin protein levels in synaptosomal fractions in experimental group. M6a expression, protein involved in filopodium formation and synaptogenesis, was also increased. Furthermore, we found that PI3K/Akt/GSK3 pathway signaling, which is involved in synaptogenesis, was activated. Moreover, asphyctic animals showed habituation memory changes in the open field test. Our results suggest that abnormal synaptogenesis induced by PA as a consequence of excessive brain plasticity during brain development may contribute to the etiology of the NDDs. Consequences of this altered synaptic maturation can underlie some of the later behavioral deficits observed in NDDs.

Hippocampal dendritic spines modifications induced by perinatal asphyxia.

Perinatal asphyxia (PA) affects the synaptic function and morphological organization. In previous works, we have shown neuronal and synaptic changes in rat neostriatum subjected to hypoxia leading to long-term ubi-protein accumulation. Since F-actin is highly concentrated in dendritic spines, modifications in its organization could be related with alterations induced by hypoxia in the central nervous system (CNS). In the present study, we investigate the effects of PA on the actin cytoskeleton of hippocampal postsynaptic densities (PSD) in 4-month-old rats. PSD showed an increment in their thickness and in the level of ubiquitination. Correlative fluorescence-electron microscopy photooxidation showed a decrease in the number of F-actin-stained spines in hippocampal excitatory synapses subjected to PA. Although western blot analysis also showed a slight decrease in ß-actin in PSD in PA animals, the difference was not significant. Taken together, this data suggests that long-term actin cytoskeleton might have role in PSD alterations which would be a spread phenomenon induced by PA.

Mejora de la calidad asistencial a los pacientes de la unidad de dolor torácico de un hospital comarcal / Improvement in health care quality for patients from the thoracic/chest pain unit in a regional hospital

Objetivos. Evaluar un ciclo de mejora en pacientes con dolor torácico en urgencias hospitalarias, especialmente los que se benefician de la realización de test de isquemia precoz en nuestro entorno. Material y métodos. Se diseñó un protocolo de atención por grupo multidisciplinario que identifica oportunidades de mejora y prioriza abordar que «la realización de test de isquemia de forma precoz era menor que lo recomendado». Se analizan las causas (diagrama de Ishikawa) y se definen seis criterios de calidad. Se evaluaron estos en una muestra aleatoria de 30 pacientes del total a los que se realizó ergometría en el hospital en el primer semestre de 2007 (n=180) y con encuesta a facultativos. Se introdujeron medidas correctivas: difusión, accesibilidad en intranet, información explícita a nuevos facultativos. La segunda evaluación se realizó durante el primer semestre de 2008 en otra muestra similar de 30 pacientes (n=120). Resultados. En la primera evaluación la clasificación de riesgo según protocolo fue muy baja (incumplimiento del 100%) y se derivaba a consultas de cardiología a pacientes subsidiarios de ingreso en la unidad de dolor torácico y test de isquemia precoz (incumplimiento del criterio del 74%). Tras medidas correctivas, se obtiene una mejora general, pero muy significativa en los anteriores, reduciendo incumplimientos al 17% en clasificación y el 23% en derivaciones. Conclusiones. El ciclo estructurado ha facilitado la solución del problema priorizado en un plazo corto. Las medidas adoptadas han sido fundamentalmente organizativas, dependientes de los profesionales y con coste muy bajo. Enfoques sencillos pero con metodología ordenada deben valorarse antes de la incorporación de tecnologías de mayor coste(AU)

Objectives. The evaluation of an improvement cycle in patients suffering thoracic/chest pain in hospital emergencies, especially in those who could benefit from the early Bruce Treadmill Test. Material and methods. A multidisciplinary group care protocol was designed, which identified improvement opportunities and gave priority to the fact that «an early Bruce Treadmill Test was carried out on fewer occasions than recommended». Causes were analysed (Ishikawa diagram) and six quality criteria were defined. These criteria were evaluated in a random sample of 30 patients out of the total of 180 who used the ergometer at the Hospital in the first six months of 2007, as well as questionnaire for the doctors. Corrective measures were introduced: circulation, accessibility through intranet and explicit information for new employees (doctors). The second evaluation was carried out during the first six-months of 2008 using another random sample of 30 patients from a total of 120. Results. In the first evaluation, the classification of the risk according to the protocol was very low (100% non-compliance) and patients whose admission to the Chest Pain Unit was recommended and an early Bruce Treadmill Test (74% criteria failure) were referred to cardiology clinics. After implementation of the corrective measures, we obtain a general improvement in all the criteria, but very significant from the previous ones, with non-compliances being reduced to 17% in classification and to the 23% in referrals. Conclusions. The structured cycle has helped resolve the priority problem in the short-term. The adopted measures have mainly been organisational, dependent on the professionals involved, and at a very low cost. Simple but organised methodological approaches should be taken into account before the incorporation of higher cost technologies(AU)

[Improvement in health care quality for patients from the thoracic/chest pain unit in a regional hospital]. / Mejora de la calidad asistencial a los pacientes de la unidad de dolor torácico de un hospital comarcal.

OBJECTIVES: The evaluation of an improvement cycle in patients suffering thoracic/chest pain in hospital emergencies, especially in those who could benefit from the early Bruce Treadmill Test. MATERIAL AND METHODS: A multidisciplinary group care protocol was designed, which identified improvement opportunities and gave priority to the fact that «an early Bruce Treadmill Test was carried out on fewer occasions than recommended¼. Causes were analysed (Ishikawa diagram) and six quality criteria were defined. These criteria were evaluated in a random sample of 30 patients out of the total of 180 who used the ergometer at the Hospital in the first six months of 2007, as well as questionnaire for the doctors. Corrective measures were introduced: circulation, accessibility through intranet and explicit information for new employees (doctors). The second evaluation was carried out during the first six-months of 2008 using another random sample of 30 patients from a total of 120. RESULTS: In the first evaluation, the classification of the risk according to the protocol was very low (100% non-compliance) and patients whose admission to the Chest Pain Unit was recommended and an early Bruce Treadmill Test (74% criteria failure) were referred to cardiology clinics. After implementation of the corrective measures, we obtain a general improvement in all the criteria, but very significant from the previous ones, with non-compliances being reduced to 17% in classification and to the 23% in referrals. CONCLUSIONS: The structured cycle has helped resolve the priority problem in the short-term. The adopted measures have mainly been organisational, dependent on the professionals involved, and at a very low cost. Simple but organised methodological approaches should be taken into account before the incorporation of higher cost technologies.

Enzymes involved in arachidonic acid release in adrenal and Leydig cells.

Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.

Arachidonic acid regulation of steroid synthesis: new partners in the signaling pathway of steroidogenic hormones.

Although the role of arachidonic acid (AA) in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl-CoA thioesterase (MTE-I). We have shown that recombinant MTE-I hydrolyses arachidonoyl-CoA to release free AA. An acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2-mediated pathway. Inhibition of ACS4 and MTE-I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH-stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl-CoA synthetase and the acyl-CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2-mediated pathway that involves a hormone-induced acyl-CoA synthetase and a hormone-regulated acyl-CoA thioesterase.

N-acetyl-D-glucosamine induces germination in Candida albicans through a mechanism sensitive to inhibitors of cAMP-dependent protein kinase.

The present study examines the involvement of cAMP-dependent protein kinase (PKA) in the dimorphic transition of Candida albicans by assessing the in vivo effect of two permeable PKA inhibitors on N-acetyl-D-glucosamine (GlcNAc)- and serum-induced differentiation. The permeable myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI), which inhibited C. albicans PKA in vitro, caused a concentration-dependent inhibition of germ-tube formation in cultures induced to germinate by GlcNAc; germination halted irrespective of the time of addition of the inhibitor. MyrPKI also blocked dibutyryl-cAMP (dbcAMP)- and glucagon-stimulated germination but did not affect serum-induced germination. H-89, another highly specific PKA inhibitor, displayed the same effect on germination. Neither MyrPKI nor H-89 had any effect on budding of yeast cells. In conclusion, our results indicate that cAMP-mediated activation of PKA plays a pivotal role in the biochemical mechanism underlying morphogenesis.

Reassessment of the effect of glucagon and nucleotides on Candida albicans germ tube formation.

The role of cyclic AMP in the process of germ tube formation in Candida albicans was investigated. The exogenous supply of the nucleotide or of agents that raise its intracellular levels stimulated germination induced by N-acetyl-D-glucosamine; glucagon showed this same stimulatory effect on yeast cell transition to the hyphal form. Compounds, included glucagon, that stimulated hyphal formation, also notably enhanced the development of hyphae. The stimulatory effect of glucagon on germination was blocked by the specific antagonist des His1 [Glu9] glucagon amide, probably indicating an interaction of the hormone with a glucagon-like receptor on the membrane of the cells. Indirect immunofluorescence experiments showed that glucagon binds to the yeast cell surface. When N-acetyl-D-glucosamine was replaced by serum as inducing agent of germination, the stimulatory effect of glucagon was substantially augmented, the resulting of germination being more than 2.5-fold greater than that attained in the presence of N-acetyl-D-glucosamine; moreover, the glucagon concentration needed for half maximal stimulatory activity with serum as inducing agent was at least 50-fold lower than with N-acetyl-D-glucosamine. Monoclonal and polyclonal anti-glucagon antibodies blocked the effect of the hormone. An interesting result observed during these experiments was the fact that a definite period of incubation of C. albicans yeast cells with N-acetyl-D-glucosamine as inducer commits them to hyphal development. When serum was used as inducer, only yeast cells evaginated during the initial incubation period evolved to the hyphal form upon further incubation in the absence of serum.

Malaria rates and fate: a socioeconomic study of malaria in Brazil.

This paper examines behavioural risk factors for malaria in the Machadinho resettlement area in the Amazonian forests of Brazil. Analysis suggests that economic status and knowledge of the importance and behaviour of the mosquito in transmitting malaria are significant factors in determining prevalence risk, irrespective of whether preventive precautions (DDT spraying of houses, and clearing vector breeding sites) are undertaken in the endemic area. However, a higher economic status combined with better knowledge of the vector and DDT spraying decreases the risks of infection considerably. The results suggest that economic status--which is not easily subject to intervention--plays a more important role in transmission than is normally suspected, although preventive actions diminish the disease burden significantly. One might conclude that the landless and impoverished migrants who seek income, and independence in the jungle are destined to have malaria as one of their many burdens. A more positive implication is that control programmes must work harder and more intensively on behalf of poorer migrants in order to diminish the disease burden for these groups.

Alteraciones bioquímicas y citoquímicas de hidrolasas ácidas lisosomales en leucemias / Biochemical and citochemical alterations of lisosomal acid hydrolases in leukemias

La actividad enzimática total de ß-galactosidasa (ß-gal), hexosaminidasa (hex) y fosfatasa ácida (Fac) fue determinada bioquímicamente, tanto en suero como en sobrenadantes de homogenatos celulares de sujetos normales y pacientes portadores de leucemias, empleándose sustratos paranitrofenilados específicos. La actividad de ß-gal en leucemias mieloides, tanto agudas (LMA-M1) como crónicas (LMC), sólo mostró un incremento significativo en sobrenadantes de homogenato celulares, respecto a los valores de neutrófilos normales. En leucemias linfoidales agudas (LLA), como crónicas (LLC), su comportamiento no ofreció variaciones. Tanto los sueros como los sobrenadantes de homogenatos celulares de LMA-M1 y LMC mostraron un franco incremento en la actividad de hex, mientras en LLA y LLC esta actividad no mostró variaciones. La actividad sérica de fosfatasa ácida estuvo incrementada en el 86% de las LMC. En los sobrenadantes de homogenatos celulares de LLA y LLC, esta actividad enzimática se mostró significativamente disminuida respecto de los valores de linfocitos normales. En los tres casos de LMA-M4 analizados, fue observado en el contenido celular niveles elevados de ß-gal, hex y Fac (lo que estaría correlacionado con la presencia de monocitos y/o monoblastos normales, con alto contenido de hidrolasas ácidas). Citoquímicamente fue demostrado en médula ósea y sangre periférica de pacientes con LMA-M1 una ligera o nula actividad de hex, en tanto que en LLA la reacción fue localizada asimétricamente en un polo celular o en gránulos citoplasmáticos. Los resultados encontrados demuestran una gran heterogeneidad en el contenido lisosomal de los diferentes tipos de leucemias

Alteraciones bioquímicas y citoquímicas de hidrolasas ácidas lisosomales en leucemias / Biochemical and citochemical alterations of lisosomal acid hydrolases in leukemias

La actividad enzimática total de ß-galactosidasa (ß-gal), hexosaminidasa (hex) y fosfatasa ácida (Fac) fue determinada bioquímicamente, tanto en suero como en sobrenadantes de homogenatos celulares de sujetos normales y pacientes portadores de leucemias, empleándose sustratos paranitrofenilados específicos. La actividad de ß-gal en leucemias mieloides, tanto agudas (LMA-M1) como crónicas (LMC), sólo mostró un incremento significativo en sobrenadantes de homogenato celulares, respecto a los valores de neutrófilos normales. En leucemias linfoidales agudas (LLA), como crónicas (LLC), su comportamiento no ofreció variaciones. Tanto los sueros como los sobrenadantes de homogenatos celulares de LMA-M1 y LMC mostraron un franco incremento en la actividad de hex, mientras en LLA y LLC esta actividad no mostró variaciones. La actividad sérica de fosfatasa ácida estuvo incrementada en el 86% de las LMC. En los sobrenadantes de homogenatos celulares de LLA y LLC, esta actividad enzimática se mostró significativamente disminuida respecto de los valores de linfocitos normales. En los tres casos de LMA-M4 analizados, fue observado en el contenido celular niveles elevados de ß-gal, hex y Fac (lo que estaría correlacionado con la presencia de monocitos y/o monoblastos normales, con alto contenido de hidrolasas ácidas). Citoquímicamente fue demostrado en médula ósea y sangre periférica de pacientes con LMA-M1 una ligera o nula actividad de hex, en tanto que en LLA la reacción fue localizada asimétricamente en un polo celular o en gránulos citoplasmáticos. Los resultados encontrados demuestran una gran heterogeneidad en el contenido lisosomal de los diferentes tipos de leucemias

Giant T waves simulating apical hypertrophic myocardiopathy that disappear with sodium nitroprusside administration. Case report of pheochromocytoma.

Giant inverted T waves (amplitude greater than 1 mV) in precordial leads have been described in various disease processes. However, the pathophysiology of these anomalies has not been studied in depth. This paper analyzes repolarization abnormalities occurring in a case of pheochromocytoma in which giant T waves were unaffected by alpha-adrenergic blockade but were affected by administration of sodium nitroprusside. It is postulated that the probable cause of this aberration is myocardial ischemia of noncoronary origin resulting from an imbalance of supply and demand of oxygen that is relieved by the hemodynamic action of nitroprusside when left ventricular wall stress is reduced.

[Postoperative course of prosthetic valve replacement]. / Evolución postoperatoria en el recambio valvular protésico.

In order to know the post-operative outcome of patients with valvular replacement due to prosthetic dysfunction, we reviewed the clinical charts of 94 patients operated at the Instituto Nacional de Cardiología "Ignacio Chávez" between January 1986 and December 1988. Eighty four cases were replaced by the first time the remaining 10 by a second time. Diagnosis of prosthetic dysfunction was made by clinical, radiological, echocardiographic and haemodynamic parameters. The most frequent causes of dysfunction were the rupture of prosthetic leaflets, stenosis with calcific deposition and paravalvular leaks. The global mortality rate was 19.15%, higher than the native valve replacement group. The most important predictors of surgical mortality were: 1) poor ventricular function (functional classes III and IV), 2) aortic clamping period, 3) the need of a second prosthetic replacement and 4) the time of prosthetic dysfunction. Thus, we conclude that it is of great importance the early recognition of prosthetic valve dysfunction. The need of special surgical procedures in these cases should be evaluated in order to reduce morbidity and mortality.