Study of the Degradation of a TPS/PCL/Fique Biocomposite Material in Soil, Compost, and Water.

The degradability of the biocomposite produced from a binary mixture of thermoplastic banana starch (TPS) and polycaprolactone (PCL) reinforced with fique fibers (Fs) was evaluated in three different environments (soil, compost, water). An experimental design with two factors (soil and compost) and three levels (5, 10, and 20 cm) was used, with additional tests for a third aqueous environment (water from the lake of the Universidad del Valle) at a depth of 20 cm. The biocomposite was prepared from the implementation of a twin-screw extrusion process of the binary mixture TPS/PCL and fique fibers (54, 36, and 10% composition, respectively), followed by hot compression molding, and after that, generating ASTM D638 type V specimens using a stainless-steel die. The specimens were dried and buried according to the experimental design, for a total experimental time of 90 days, and removing samples every 30 days. After 90 days, all samples showed signs of degradation, where the best results were obtained in the compost at a depth of 20 cm (34 ± 4% mass loss and a decrease in tensile strength of 77.3%, which indicates that the material lost mechanical properties). TPS was the fastest disappearing component and promoted the degradation of the composite material as it disappeared. Finally, the aqueous media presented the lowest degradation results, losing only 20% of its initial mass after 90 days of the experiment, being the least effective environment in which the biocomposite can end up.

Bibliometric analysis of global research output on teaching and learning of human anatomy / Análisis bibliométrico de la producción mundial de investigación sobre la enseñanza y el aprendizaje de la anatomía humana

SUMMARY: The study and teaching of human anatomy is one of the cornerstones of education of basis science in health professionals. The aim of this study was to present a bibliometric analysis of the global outputs of research on the teaching and learning of human anatomy in the last two decades. The Scopus database was used to search and retrieve studies related to this topic between 2001 and February 10, 2021. A total of 10,481 documents were found through a systematic search strategy. A growing trend in publishing research results was evidenced, starting in 2001 with a considerable increase between 2012 and 2015. Four clusters were identified in studies related to teaching-learning methodologies of human anatomy. These clusters correspond to traditional methods and emerging methodologies such as the use of information and communication technologies, 3D impressions and diagnostic images. In addition, the results of this study indicate that the United States, the United Kingdom and Germany were the countries with the highest production in the number of publications on this topic. Although new methodologies have been included in teaching and learning human anatomy, such as the use of information and communication technologies, the trend in these processes continues to be mediated by the traditional method of cadaveric dissection. However, there is an increase in the immersion of virtual resources as part of these methodologies that should be integrated.

RESUMEN: La enseñanza y el aprendizaje de la anatomía humana es uno de los pilares de la educación científica básica para los profesionales de la salud. El objetivo de este estudio fue presentar un análisis bibliométrico de los resultados globales de la investigación sobre la enseñanza y el aprendizaje de la anatomía humana en las últimas dos décadas. Se utilizó la base de datos Scopus para buscar y recuperar estudios relacionados con este tema entre 2001 y el 10 de febrero de 2021. Se encontraron un total de 10.481 documentos mediante una estrategia de búsqueda sistemática. Hubo una tendencia creciente en la publicación de resultados de investigación, comenzando en 2001 con un aumento considerable entre 2012 y 2015. Se identificaron cuatro grupos en estudios relacionados con metodologías de enseñanza-aprendizaje de anatomía humana. Estos clusters corresponden a métodos tradicionales, metodologías emergentes como el uso de tecnologías de información y comunicación, impresión 3D e imágenes de diagnósticas. Además, los resultados de este estudio indican que Estados Unidos, Reino Unido y Alemania fueron los países con mayor producción en el número de publicaciones sobre este tema. Si bien se han incluido nuevas metodologías en la enseñanza y el aprendizaje de la anatomía humana, como el uso de tecnologías de la información y la comunicación, la tendencia en estos procesos continúa siendo a través del método tradicional y la disección cadavérica. Sin embargo, hay un incremento en la inclusión de recursos virtuales que se integran al aula.

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

Robustifying Experimental Tracer Design for13C-Metabolic Flux Analysis.

13C metabolic flux analysis (MFA) has become an indispensable tool to measure metabolic reaction rates (fluxes) in living organisms, having an increasingly diverse range of applications. Here, the choice of the13C labeled tracer composition makes the difference between an information-rich experiment and an experiment with only limited insights. To improve the chances for an informative labeling experiment, optimal experimental design approaches have been devised for13C-MFA, all relying on some a priori knowledge about the actual fluxes. If such prior knowledge is unavailable, e.g., for research organisms and producer strains, existing methods are left with a chicken-and-egg problem. In this work, we present a general computational method, termed robustified experimental design (R-ED), to guide the decision making about suitable tracer choices when prior knowledge about the fluxes is lacking. Instead of focusing on one mixture, optimal for specific flux values, we pursue a sampling based approach and introduce a new design criterion, which characterizes the extent to which mixtures are informative in view of all possible flux values. The R-ED workflow enables the exploration of suitable tracer mixtures and provides full flexibility to trade off information and cost metrics. The potential of the R-ED workflow is showcased by applying the approach to the industrially relevant antibiotic producer Streptomyces clavuligerus, where we suggest informative, yet economic labeling strategies.

Chemistry and Anticancer Activity of Hybrid Molecules and Derivatives based on 5-Fluorouracil.

Considering that cancer continues to be an important cause of death worldwide, several conventional anticancer treatments are widely used. However, most of them display low selectivity against malignant cells and induce many adverse side effects. Among these, the use of therapies based on 5-Fluorouracil (5-FU) has been one of the most efficient, with a broad-spectrum. Due to these circumstances, various modifications of 5-FU have been developed to improve drug delivery and reduce side effects. Among the optimization strategies, modifications of 5-FU at N1 or N3 position are made, usually including the incorporation of pharmacologically active compounds with anticancer activity (called hybrid molecule) and functionalization with other groups of compounds (called conjugates). Several studies have been conducted in the search for new alternative therapies against cancer. Many of them have evidenced that hybrid compounds exhibit good anticancer activity, which has emerged as a promising strategy in this field of drug discovery and development. Furthermore, the binding of 5-FU to amino acids, peptides, phospholipids, polymers, among others, improves metabolic stability and absorption. This review highlights the potential of hybrids and derivatives based on 5-FU as a scaffold for the development of antitumor agents. Besides, it also presents a detailed description of the different strategies employed to design and synthesized these compounds, together with their biological activities and structure-activity relationship (SAR) analysis.

Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing.

Clavulanic acid (CA) is an irreversible ß-lactamase enzyme inhibitor with a weak antibacterial activity produced by Streptomyces clavuligerus (S. clavuligerus). CA is typically co-formulated with broad-spectrum ß­lactam antibiotics such as amoxicillin, conferring them high potential to treat diseases caused by bacteria that possess ß­lactam resistance. The clinical importance of CA and the complexity of the production process motivate improvements from an interdisciplinary standpoint by integrating metabolic engineering strategies and knowledge on metabolic and regulatory events through systems biology and multi-omics approaches. In the large-scale bioprocessing, optimization of culture conditions, bioreactor design, agitation regime, as well as advances in CA separation and purification are required to improve the cost structure associated to CA production. This review presents the recent insights in CA production by S. clavuligerus, emphasizing on systems biology approaches, strain engineering, and downstream processing.

Double-Ring Epimerization in the Biosynthesis of Clavulanic Acid.

All reaction steps during the biosynthesis of suicidal clavulanic acid (coformulated with ß-lactam antibiotics and used to fight bacterial infections) are known, except for the crucial 3S,5S → 3R,5R double epimerization needed to produce a biologically active stereoisomer, for which mechanistic hypothesis is subject to debate. In this work, we provide evidence for a reaction channel for the double inversion of configuration that involves a total of six reaction steps. When mediated by an enzyme with a terminal S-H bond, this highly complex reaction is spontaneous in the absence of solvents. Polarizable continuum models introduce reaction barriers in aqueous environments because of the strong destabilization of the first transition state. Molecular geometries and electronic structures in both cases indicate that solvent-free spontaneity and aqueous medium barriers are both firmly rooted in a substantial reorganization of the electron density right at the onset of the reaction, mostly involving a cyclic evolution/involution of large regions of π delocalization used to stabilize the excess charge left after the initial proton abstraction.

A Genome-Scale Insight into the Effect of Shear Stress During the Fed-Batch Production of Clavulanic Acid by Streptomyces Clavuligerus.

Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the ß-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the Streptomyces genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of Streptomyces clavuligerus was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress. A flux balance analysis based on experimental evidence revealed that clavulanic acid biosynthetic reaction fluxes are favored in conditions of phosphate limitation, and this is correlated with enhanced activity of central and amino acid metabolism, as well as with enhanced oxygen uptake. In silico and experimental results show a possible slowing down of tricarboxylic acid (TCA) due to reduced oxygen availability in low shear stress conditions. In contrast, high shear stress conditions are connected with high intracellular oxygen availability favoring TCA activity, precursors availability and clavulanic acid (CA) production.

Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production.

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of ß-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.

Data of clavulanic acid and clavulanate-imidazole stability at low temperatures.

Clavulanic acid (CA) is a ß-lactam antibiotic with a strong inhibitory effect on ß-lactamase enzymes. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus (S. clavuligerus). CA is an unstable molecule in aqueous solution and its stability depends strongly on temperature and concentration. In this contribution, the experimental data of CA stability, produced in chemically defined media and exposed to temperatures between -80 and 25 °C, are presented. The chromophore clavulanate-imidazole (CAI) is commonly used for analysis and quantification of CA samples by High Performance Liquid Chromatography (HPLC); nevertheless, this molecule is also susceptible to suffer degradation in aqueous solution, potentially affecting the quantification of CA. Data of CAI concentration for samples conserved at 4 °C and 25 °C are also presented. A reversible-irreversible kinetic model was applied to estimate the degradation rate of CA. Data from numerical simulations of CA degradation using the proposed kinetic model are also graphically presented. The data show the clavulanic acid instability in fermentation broths, in a range of temperatures of interest for bioprocess operation, downstream processing, samples quantification, conservation and storage.

Data set of in silico simulation for the production of clavulanic acid and cephamycin C by Streptomyces clavuligerus using a genome scale metabolic model.

Streptomyces clavuligerus (S. clavuligerus) is a Gram-positive bacterium which produced clavulanic acid (CA) and cephamycin C (CephC). In this data article, a curated genome scale metabolic model of S. clavuligerus is presented. A total of eighteen objective functions were evaluated for a better representation of CA and CephC production by S. clavuligerus. The different objective functions were evaluated varying the weighting factors of CA and CephC between 0, 1 y 2, whereas for the case of biomass the weight factor was varied between 1 and 2. A robustness analysis, by mean of flux balance analysis, showed five different metabolic phenotypes of S. clavuligerus as a function of oxygen uptake: (I) and (II) biomass production, (III) biomass and CephC production, (IV) simultaneous production of biomass, CA and CephC and (V) production of biomass and CA. Data of shadow prices and reduced cost are also presented.

Degradation Kinetics of Clavulanic Acid in Fermentation Broths at Low Temperatures.

Clavulanic acid (CA) is a ß-lactam antibiotic inhibitor of ß-lactamase enzymes, which confers resistance to bacteria against several antibiotics. CA is produced in submerged cultures by the filamentous Gram-positive bacterium Streptomyces clavuligerus; yield and downstream process are compromised by a degradation phenomenon, which is not yet completely elucidated. In this contribution, a study of degradation kinetics of CA at low temperatures (-80, -20, 4, and 25 °C) and pH 6.8 in chemically-defined fermentation broths is presented. Samples of CA in the fermentation broths showed a fast decline of concentration during the first 5 h followed by a slower, but stable, reaction rate in the subsequent hours. A reversible-irreversible kinetic model was applied to explain the degradation rate of CA, its dependence on temperature and concentration. Kinetic parameters for the equilibrium and irreversible reactions were calculated and the proposed kinetic model was validated with experimental data of CA degradation ranging 16.3 mg/L to 127.0 mg/L. Degradation of the chromophore CA-imidazole, which is commonly used for quantifications by High Performance Liquid Chromatography, was also studied at 4 °C and 25 °C, showing a rapid rate of degradation according to irreversible first-order kinetics. A hydrolysis reaction mechanism is proposed as the cause of CA-imidazole loss in aqueous solutions.

Bibliometric Analysis of Global Research on Clavulanic Acid.

Clavulanic acid (CA), a potent inhibitor of the ß-lactam, ase enzyme, is frequently co-formulated with a broad spectrum of antibiotics to treat infections caused by ß-lactamase-producing pathogens. In order to evaluate the impact and the progress of CA studies in the last four decades, a bibliometric analysis of the global scientific production of CA was carried out. A total of 39,758 records in the field of CA were indexed in the Scopus database for a 43-year period of study (1975⁻2017). The results indicated that CA studies have grown, showing three phases (1975⁻1999, 2000⁻2003 and 2004⁻2017) based on records of publications; the results showed a sigmoidal profile. Medicine was the main subject area for CA studies, whereas biochemistry, genetics and molecular biology were areas of research for CA production by Streptomyces clavuligerus (S. clavuligerus). Nevertheless, chemical engineering (as a subject area) had the highest increase in the percentage of publications related to CA production by S. clavuligerus. The United States, France, the United Kingdom, Spain and Brazil were the leading countries in the scientific production of studies on both CA and CA related to S. clavuligerus. This analysis allowed the identification of the area of knowledge with the highest impact on CA studies, the top researchers and their geographic distribution, and also helped to highlight the existence of antibiotic-resistant bacteria as an emergent area in CA research.

Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis.

Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection.

BACKGROUND: Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb's vulnerability and metabolic adaptation have been carried out. RESULTS: A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. CONCLUSIONS: This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Inversion of the stereochemical configuration (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid: A computationally-assisted approach based on experimental evidence.

Clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes, is produced by Streptomyces clavuligerus (Sc) cultivation processes, for which low yields are commonly obtained. Improved knowledge of the clavam biosynthetic pathway, especially the steps involved in the inversion of 3S-5S into 3R-5R stereochemical configuration, would help to eventually identify bottlenecks in the pathway. In this work, we studied the role of acetate in CA biosynthesis by a combined continuous culture and computational simulation approach. From this we derived a new model for the synthesis of N-acetyl-glycyl-clavaminic acid (NAG-clavam) by Sc. Acetylated compounds, such as NAG-clavam and N-acetyl-clavaminic acid, have been reported in the clavam pathway. Although the acetyl group is present in the ß-lactam intermediate NAG-clavam, it is unknown how this group is incorporated. Hence, under the consideration of the experimentally proven accumulation of acetate during CA biosynthesis, and the fact that an acetyl group is present in the NAG-clavam structure, a computational evaluation of the tentative formation of NAG-clavam was performed for the purpose of providing further understanding. The proposed reaction mechanism consists of two steps: first, acetate reacts with ATP to produce a reactive acylphosphate intermediate; second, a direct nucleophilic attack of the terminal amino group of N-glycyl-clavaminic on the carbonyl carbon of the acylphosphate intermediate leads to a tetrahydral intermediate, which collapses and produces ADP and N-acetyl-glycyl-clavaminic acid. The calculations suggest that for the proposed reaction mechanism, the reaction proceeds until completion of the first step, without the direct action of an enzyme, where acetate and ATP are involved. For this step, the computed activation energy was ≅2.82kcal/mol while the reaction energy was ≅2.38kcal/mol. As this is an endothermic chemical process with a relatively small activation energy, the reaction rate should be considerably high. The calculations offered in this work should not be considered as a definite characterization of the potential energy surface for the reaction between acetate and ATP, but rather as a first approximation that provides valuable insight about the reaction mechanism. Finally, a complete route for the inversion of the stereochemical configuration from (3S, 5S)-clavaminic acid into (3R, 5R)-clavulanic acid is proposed, including a novel alternative for the double epimerization using proline racemase and NAG-clavam formation.

An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus.

Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial ß-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.