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Erosion and tillage changes negatively the soil physical structure, which directly impacts agricultural systems and consequently food security. To mitigate these adverse modifications, different polymeric materials from synthetic and natural sources, have been used as soil conditioners to improve the hydro-mechanical behavior of affected soils. One of the most interesting and used natural polymers is the alginate hydrogel. Although commercially available alginate hydrogels are primarily sourced from algal, they can also be sourced from bacteria. The gelation capacity of these hydrogels is determined by their molecular properties, which, in turn, are influenced by the production conditions. Bacterial alginate hydrogel production offers the advantage of precise control over environmental conditions during cultivation and extraction, thereby maintaining and enhancing their molecular properties. This, in turn, results in higher molecular weight and improved gelation capacity. In this study, we compared the effects of bacterial alginate (BH) and algal alginate (AH) hydrogels over the mechanical, hydraulic, and structural behavior of coarse quartz sand as a model soil. Mechanically, it was observed that the treatment with the lowest concentration of bacteria alginate hydrogel (BH1) reached higher values of yield strength, Young's modulus (E), shear modulus (G) and strain energy (U) than those treatments with algal alginate hydrogel (AH). Furthermore, the increase in the aggregate stability could be associated with the improvement of mechanical parameters. On the other hand, a greater water retention capacity was observed in the BH treatments, as well as a greater decrease in hydraulic conductivity with respect to the AH and control treatments. All these changes could be explained by the formation of bridge-like structures between the sand particles and the hydrogel, and this alteration may result in a shift in the mechanical and wettability characteristics of the treated soils. Finally, our findings emphasize the superior impact of bacterial alginate hydrogel on enhancing the mechanical and hydraulic properties of coarse quartz sand compared to traditional algal alginate. Besides, the use of bacterial alginate hydrogel could be useful to counteract erosion and water scarcity scenarios in agricultural systems.

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Acidiphilium cryptum is an acidophilic, heterotrophic, and metallotolerant bacteria able to use dissolved oxygen or Fe(III) as an electron sink. The ability of this extremophile to accumulate poly(3-hydroxybutyrate) (PHB) and secrete extracellular polymeric substances (EPS) has also been reported. Hence, the aim of this work is to characterize the production of PHB and EPS by the wild strain DSM2389 using glycerol in shaken flasks and bioreactor. Results showed that maximum PHB accumulation (37-42% w/w) was obtained using glycerol concentrations of 9 and 15 g L-1, where maximum dry cell weight titers reached 3.6 and 3.9 g L-1, respectively. The culture in the bioreactor showed that PHB accumulation takes place under oxygen limitation, while the redox potential of the culture medium could be used for online monitoring of the PHB production. Recovered EPS was analyzed by Fourier-transform infrared spectroscopy and subjected to gas chromatography-mass spectrometry after cleavage and derivatization steps. These analyses showed the presence of sugars which were identified as mannose, rhamnose and glucose, in a proportion near to 3.2:2.3:1, respectively. Since glycerol had not been used in previous works, these findings suggest the potential of A. cryptum to produce biopolymers from this compound at a large scale with a low risk of microbial contamination due to the low pH of the fermentation process.

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Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a polymer produced by Azotobacter vinelandii OP. In the bioreactor, PHBV production and its molar composition are affected by aeration rate. PHBV production by A. vinelandii OP was evaluated using extended batch cultures at different aeration rates, which determined different oxygen transfer rates (OTR) in the cultures. Under the conditions evaluated, PHBV with different 3-hydroxyvalerate (3HV) fractions were obtained. In the cultures with a low OTR (6.7 mmol L-1 h-1, at 0.3 vvm), a PHBV content of 38% w w-1 with 9.1 mol % 3HV was achieved. The maximum PHBV production (72% w w-1) was obtained at a high OTR (18.2 mmol L-1 h-1, at 1.0 vvm), both at 48 h. Thus, PHBV production increased in the bioreactor with an increased aeration rate, but not the 3HV fraction in the polymer chain. An OTR of 24.9 mmol L-1 h-1 (at 2.1 vvm) was the most suitable for improving the PHBV content (61% w w-1) and a high 3HV fraction of 20.8 mol % (at 48 h); and volumetric productivity (0.15 g L-1 h-1). The findings indicate that the extended batch culture at 2.1 vvm is the most adequate mode of cultivation to produce higher biomass and PHBV with a high 3HV fraction. Overall, the results have shown that the PHBV production and 3HV fraction could be affected by the aeration rate and the proposed approach could be applied to implement cultivation strategies to optimize PHBV production for different biotechnological applications.

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Agricultural systems are facing the negative impacts of erosion and water scarcity, directly impacting the hydro-mechanical behavior of soil aggregation. Several technologies have been proposed to reduce hydro-mechanical soil-related problems in agriculture. Biopolymer-based hydrogels have been reported to be a great tool to tackle these problems in soils. In this study, we investigated the hydro-mechanical behavior of different soils media treated with Ca-bacterial alginate hydrogel. We used an unconfined uniaxial compression test, aggregate stability test and hydraulic conductivity measurements to investigate the mechanical and hydraulic behavior of treated soils media. Our results from unconfined uniaxial compression test showed that yield stress (i.e., strength) increased in treated soils with higher kaolinite and water content (i.e., HCM3), compared with untreated coarse quartz sand (i.e., CM1). Furthermore, we found that temperature is an important factor in the gelation capacity of our hydrogel. At room temperature, HCM3 displayed the higher aggregate stability, almost 5.5-fold compared with treated coarse quartz sand (HCM1), while this differential response was not sustained at warm temperature. In general, the addition of different quantities of kaolinite decreased the saturated hydraulic conductivity for all treatments. Finally, bright field microscopy imaging represents the soil media matrix between sand and clay particles with Ca-bacterial alginate hydrogel that modify the hydro-mechanical behavior of different soils media. The results of this study could be helpful for the soil-related problems in agriculture facing the negative effects of climate change.

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Bionanocomposites based on Polylactide (PLA) and Polyhydroxybutyrate (PHB) blends were successfully obtained through a combined extrusion and impregnation process using supercritical CO2 (scCO2). Graphene oxide (GO) and cinnamaldehyde (Ci) were incorporated into the blends as nano-reinforcement and an active compound, respectively, separately, and simultaneously. From the results, cinnamaldehyde quantification values varied between 5.7% and 6.1% (w/w). When GO and Ci were incorporated, elongation percentage increased up to 16%, and, therefore, the mechanical properties were improved, with respect to neat PLA. The results indicated that the Ci diffusion through the blends and bionanocomposites was influenced by the nano-reinforcing incorporation. The disintegration capacity of the developed materials decreased with the incorporation of GO and PHB, up to 14 and 23 days of testing, respectively, without compromising the biodegradability characteristics of the final material.

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BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

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Alginates can be used to elaborate hydrogels, and their properties depend on the molecular weight (MW) and the guluronic (G) and mannuronic (M) composition. In this study, the MW and G/M ratio were evaluated in cultures of Azotobacter vinelandii to 3 and 30 L scales at different oxygen transfer rates (OTRs) under diazotrophic conditions. An increase in the maximum OTR (OTRmax) improved the alginate production, reaching 3.3 ± 0.2 g L-1. In the cultures conducted to an OTR of 10.4 mmol L-1 h-1 (500 rpm), the G/M increased during the cell growth phase and decreased during the stationary phase; whereas, in the cultures at 19.2 mmol L-1 h-1 was constant throughout the cultivation. A higher alginate MW (520 ± 43 kDa) and G/M ratio (0.86 ± 0.01) were obtained in the cultures conducted at 10.4 mmol L-1 h-1. The OTR as a criterion to scale up alginate production allowed to replicate the concentration and the alginate production rate; however, it was not possible reproduce the MW and G/M ratio. Under a similar specific oxygen uptake rate (qO2) (approximately 65 mmol g-1 h-1) the alginate MW was similar (approximately 365 kDa) in both scales. The evidences revealed that the qO2 can be a parameter adequate to produce alginate MW similar in two bioreactor scales. Overall, the results have shown that the alginate composition could be affected by cellular respiration, and from a technological perspective the evidences contribute to the design process based on oxygen consumption to produce alginates defined.

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Poly-3-hydroxybutyrate (P3HB) is a biopolymer, which presents characteristics similar to those of plastics derived from the petrochemical industry. The thermomechanical properties and biodegradability of P3HB are influenced by its molecular weight (MW). The aim of the present study was to evaluate the changes of the molecular weight of P3HB as a function of oxygen transfer rate (OTR) in the cultures using two strains of Azotobacter vinelandii, a wild-type strain OP, and PhbZ1 mutant with a P3HB depolymerase inactivated. Both strains were grown in a bioreactor under different OTR conditions. An inverse relationship was found between the average molecular weight of P3HB and the OTRmax, obtaining a polymer with a maximal MW (8000-10,000 kDa) from the cultures developed at OTRmax of 5 mmol L-1 h-1 using both strains, with respect to the cultures conducted at 8 and 11 mmol L-1 h-1, which produced a P3HB between 4000 and 5000 kDa. The increase in MW of P3HB was related to the activity of enzymes involved in the synthesis and depolymerization. Overall, our results show that it is possible to modulate the average molecular weight of P3HB by manipulating oxygen transfer conditions with both strains (OP and PhbZ1 mutant) of A. vinelandii.

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Resumen Introducción: La hipercolesterolemia familiar homocigótica (HFHo) se caracteriza por niveles muy elevados de cLDL y por enfermedad aterosclerótica temprana. Aunque la frecuencia es baja (1/300.000), las complicaciones son muy severas y pueden ser evitadas. Encontrar y tratar esta población de manera temprana podría reducir la mortalidad. Se describen 36 casos en Colombia, en donde se calcula que haya entre 160 y 200 casos. Resultados: Un total de 36 pacientes con fenotipo sugestivo de HFHo fueron identificados y tratados en un período de observación de cuatro años. La media de edad fue 27 años (24 mujeres). 34 pacientes tuvieron un puntaje según la Red de Clínicas de Lípidos de Holanda (RCLH) mayor de 8 (diagnóstico definitivo) y los restantes 2 tenían puntaje equivalente a diagnóstico probable. Un cuarto de los casos procedían de la costa norte colombiana. En las pruebas genéticas, 14 fueron homocigóticos verdaderos para mutación del gen que codifica para el receptor de LDL (LDLR), 12 heterocigóticos compuestos, 2 heterocigóticos dobles y uno autosómico recesivo (LDLRAP1); 5 pacientes fueron heterocigóticos simples (LDLR) y 2 pacientes no autorizaron la prueba. En los homocigóticos verdaderos, la variante más frecuente encontrada fue la c.11G>A. 14 pacientes cursaron con enfermedad coronaria, 9 con estenosis carotídea, 8 con estenosis aórtica y 2 tuvieron ataques cerebrovasculares (ACV). 34 pacientes recibían estatinas (24 rosuvastatina), 30 recibían ezetimibe, 2 recibían evolocumab y 20 recibían lomitapide (dosis promedio 12,7mg). Ninguno recibió aféresis de cLDL. Los medicamentos, en general, fueron bien tolerados y la reducción promedio de cLDL con la terapia fue de 533,7mg/dl a 245,1mg/dl (54%). Conclusiones: Todos los pacientes recibieron tratamiento hipolipemiante y se encontraron alteraciones genéticas diagnósticas en todos aquellos que autorizaron el examen. Los niveles elevados de cLDL conllevan tanto riesgo que el tratamiento debe establecerse aún sin conocer el diagnóstico genético.

Abstract Background: Homozygous familial hypercholesterolemia (HoFH) is characterized for very high levels of cLDL and early cardiovascular disease. Although incidence is low (1/300 000), complications are very severe and can be avoided. Finding and treating this population promptly could reduce mortality. We describe 36 cases in Colombia, where 160 to 200 cases are expected. Results: 36 patients with phenotype of HoHF were identified and treated in a follow-up of 4 years. The mean age was 27 years (24 women). 34 of them had at least 8 points in the FH Dutch Lipid Clinic Criteria (definitive diagnosis) and two had probable diagnosis. A quarter of the cases came from the Colombian North Coast. In molecular tests, 14 were true homozygous for LDLR, 12 were compound heterozygous for LDLR, 2 double heterozygous and one was autosomal recessive; 5 were heterozygous and 2 patients did not authorized genetic test. In true homozygous subjects, the most frequent variant was c.11G>A. 14 patients had coronary disease, 9 carotid stenosis, 8 aortic stenosis and 2 had stroke. 34 patients were on statins (25 rosuvastatin), 30 were receiving ezetimibe, 2 were receiving a PSCK9 inhibitor (evolocumab) and 20 were on lomitapide with mean doses of 12.7mg. None received lipoprotein apheresis. Medications were very well tolerated. Changes in cLDL after therapy was from 533.7 mg/dL to 245 mg/dL, (54%). Conclusions: Treatment was started in all patients. We found genetic mutations in all patients with genetic tests. The high levels of cLDL mean such a high risk that treatment must be started promptly, even without a genetic test.

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Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

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Alcoholic fermentation is fundamentally an adaptation process, in which the yeast Saccharomyces cerevisiae outperforms its competitors and takes over the fermentation process itself. Although wine yeast strains appear to be adapted to the stressful conditions of alcoholic fermentation, nitrogen limitations in grape must cause stuck or slow fermentations, generating significant economic losses for the wine industry. One way to discover the genetic bases that promote yeast adaptation to nitrogen-deficient environments are selection experiments, where a yeast population undergoes selection under conditions of nitrogen restriction for a number of generations, to then identify by sequencing the molecular characteristics that promote this adaptation. In this work, we carried out selection experiments in bioreactors imitating wine fermentation under nitrogen-limited fermentation conditions (SM60), using the heterogeneous SGRP-4X yeast population, to then sequence the transcriptome and the genome of the population at different time points of the selection process. The transcriptomic results showed an overexpression of genes from the NA strain (North American/YPS128), a wild, non-domesticated isolate. In addition, genome sequencing and allele frequency results allowed several QTLs to be mapped for adaptation to nitrogen-limited fermentation. Finally, we validated the ECM38 allele of NA strain as responsible for higher growth efficiency under nitrogen-limited conditions. Taken together, our results revealed a complex pattern of molecular signatures favouring adaptation of the yeast population to nitrogen-limited fermentations, including differential gene expression, allele frequency changes and loss of the mitochondrial genome. Finally, the results suggest that wild alleles from a non-domesticated isolate (NA) may have a relevant role in the adaptation to the assayed fermentation conditions, with the consequent potential of these alleles for the genetic improvement of wine yeast strains.

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In the present study, the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Azotobacter vinelandii was evaluated in shake flasks and bioreactors, utilizing different precursors and oxygen transfer rates (OTRs). In shake flask cultures, the highest PHBV yield from sucrose (0.16 g g-1) and 3-hydroxyvalerate (3HV) fraction in the PHA chain (27.4 mol%) were obtained with valerate (1.0 g L-1). In the bioreactor, the cultures were grown under oxygen-limited conditions, and the maximum OTR (OTRmax) was varied by adjusting the agitation rate. In the cultures grown at low OTRmax (4.3 mmol L-1 h-1), the intracellular content of PHBV (73% w w-1) was improved, whereas a maximum 3HV fraction (35 mol %) was obtained when a higher OTRmax (17.2 mmol L-1 h-1, to 600 rpm) was employed. The findings obtained suggest that the PHBV production and the content of 3HV incorporated into the polymer were affected by the OTR. Based on the evidence, it is possible to produce PHBV with a different composition by varying the OTR of the culture; thus, the approach in this study could be used to scale up PHBV production.

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Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain of a specific length depending on the organism. We constructed an engineered Escherichia coli strain (menF) unable to produce demethylmenaquinone and menaquinone, compounds that compete for both chorismate, precursor of the benzoquinone ring, and the isoprenoid chain involved in CoQ biosynthesis. In addition, a mutant strain (entC) unable to produce enterobactin, high-affinity siderophore, synthesized from chorismate, and a double mutant (entC-menF) were constructed. The use of glucose or glycerol as carbon sources was also evaluated for the production of CoQ8 in these strains. The double mutant (entC-menF) showed 18% increase in CoQ8-specific content compared to the control strain; however, the single-mutant strains did not show statistically significant differences in CoQ8-specific content respect to the control, in glucose medium in bioreactor experiments. Glycerol was significantly superior compared to glucose for the production of CoQ8 in E. coli, where the CoQ8-specific content increased 126% and 53% in the control and double-mutant strain, respectively. The expression of genes related to CoQ8 biosynthesis is reported, where the entC-menF double-mutant strain showed a significant increase in the expression of CoQ8 biosynthesis-related genes when glycerol was used as sole carbon source. The control strain did not show gene expression difference between both carbon sources, indicating a possible regulation at a different level.

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Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6 mmol L-1 h-1) and specific oxygen uptake rate (11.6 mmol g-1 h-1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6 w w-1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.

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Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1-4)-ß-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.

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Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g-1 h-1 by changes in the dilution rate (D) from 0.06 to 0.10 h-1, whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

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The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (µ) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same µ at either 22 or 30 °C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10-10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.

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Reseña: El propósito de este estudio es evaluar la aplicación clínica e indicación de los criterios apropiados para la ecocardiografía de estrés de acuerdo a la Fundación del Colegio Americano de Cardiología/Sociedad Americana de Ecocardiografía, en la práctica diaria de un centro cardiovascular de referencia. Materiales y métodos: Fueron incluidos los estudios consecutivos realizados entre enero de 2012 y julio de 2013. Los datos fueron revisados, analizados y clasificados por cuatro cardiólogos expertos teniendo en cuenta: la demografía, la especialidad que refiere, la indicación primaria y su clasificación final como apropiados, inapropiados o inciertos. Resultados: Un total de 1.556 estudios fueron incluidos inicialmente, 450 fueron excluidos por estar relacionados con programas de trasplante de órgano sólido y en 170 no se pudo determinar una indicación clara para el estudio. Los 936 estudios restantes (85%) fueron analizados para la determinación de criterios apropiados. De estos 679 (73%) tenían una indicación apropiada, 70 (7%) eran inciertos y 187 (20%) fueron inapropiados. Cuatrocientos veintiocho (46%) fueron referidos por medicina interna, 375 (40%) por cardiología, 51 (5%) por anestesiología y 82 (10%) por medicina general y otras especialidades. Un total de 159 (17%) estudios arrojaron un resultado anormal, todos ellos estaban apropiadamente indicados, ninguno fue anormal en los inapropiadamente indicados. Conclusiones: La revisión de la aplicación de los criterios apropiados para la ecocardiografía de estrés no solo permite una implementación clínica efectiva en un centro cardiovascular de referencia, sino que también estratifica razonablemente la posibilidad de tener un resultado anormal en aquellos con una indicación apropiada. Sin embargo, esto debería validarse extensamente en una cohorte multicéntrica.

Background: The purpose of this study is to evaluate the clinical application and assessment of appropriate criteria for the indication of stress echocardiograms according to the American College of Cardiology Foundation/American Society of Echocardiography in daily clinical practice at a cardiovascular reference center. Materials and methods: All consecutive studies carried out between January 2012 and July 2013 were included. The data was reviewed, analyzed and classified by four expert cardiologists regarding issues such as demography, referred specialty, primary indication and its final classification as appropriate, inappropriate or uncertain. Results: A total of 1,556 studies were initially included, 450 which were excluded as being related to program of solid organ transplantation and 170 as no clear indication of the study could be determined. Therefore, the remaining 936 were analyzed for the evaluation of appropriateness criteria. Of these 679 (73%) had appropriate indication, 70 (7%) were uncertain and 187 (20%) inappropriate. 428 (46%) were referred by internal medicine, 375 (40%) by cardiology, 51 (5%) by anesthesiology and 82 (10%) by general medicine and other specialties. A total of 159 (17%) studies rendered an abnormal result, all of them which were properly indicated, and no one was abnormal in the inappropriately indicated. Conclusions: Reviewing appropriateness criteria for SE not only allows an effective characterization and clinical performance in a single center, but also reasonably stratifies the possibility of having an abnormal result in those with an appropriate indication. However, this should be validated extensively in a multicenter cohort.

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BACKGROUND: Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. METHODS: In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). RESULTS AND CONCLUSION: Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.

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Alginates are polysaccharides used as food additives and encapsulation agents in biotechnology, and their functional properties depend on its molecular weight. In this study, different steady-states in continuous cultures of A. vinelandii were established to determine the effect of the dilution rate (D) and the agitation rate on alginate production and expression of genes involved in alginate polymerization and depolymerization. Both, the agitation and dilution rates, determined the partitioning of the carbon utilization from sucrose into alginate and CO2 under oxygen-limiting conditions. A low D (0.07 h(-1)) and 500 rpm resulted in the highest carbon utilization into alginate (25%). Quantitative real-time polymerase chain reaction was used to determine the transcription level of six genes involved in alginate polymerization and depolymerization. In chemostat cultures at 0.07 h(-1), the gene expression was affected by changes in the agitation rate. By increasing the agitation rate from 400 to 600 rpm, the algE7 gene expression decreased tenfold, whereas alyA1, algL and alyA2 gene expression increased between 1.5 and 2.8 times under similar conditions evaluated. Chemostat at 0.07 h(-1) showed a highest alginate molecular weight (580 kDa) at 500 rpm whereas similar molecular weights (480 kDa) were obtained at 400 and 600 rpm. The highest molecular weight was not explained by changes in the expression of alg8 and alg44 (genes involved in alginate polymerization). Nonetheless, a different expression pattern observed for lyases could explain the highest alginate molecular weight obtained. Overall, the results suggest that the control of alginate molecular weight in A. vinelandii cells growing in continuous mode is determined by a balance between the gene expression of intracellular and extracellular lyases in response to oxygen availability. These findings better our understanding of the biosynthesis of bacterial alginate and help us progress toward obtain tailor-made alginates.

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