Preclinical Study of DCD and Normothermic Perfusion for Visceral Transplantation.

Considering recent clinical and experimental evidence, expectations for using DCD-derived intestines have increased considerably. However, more knowledge about DCD procedure and long-term results after intestinal transplantation (ITx) is needed. We aimed to describe in detail a DCD procedure for ITx using normothermic regional perfusion (NRP) in a preclinical model. Small bowel was obtained from pigs donors after 1 h of NRP and transplanted to the recipients. Graft Intestinal samples were obtained during the procedure and after transplantation. Ischemia-reperfusion injury (Park-Chiu score), graft rejection and transplanted intestines absorptive function were evaluated. Seven of 8 DCD procedures with NRP and ITx were successful (87.5%), with a good graft reperfusion and an excellent recovery of the recipient. The architecture of grafts was well conserved during NRP. After an initial damage of Park-chiu score of 4, all grafts recovered from ischemia-reperfusion, with no or very subtle alterations 2 days after ITx. Most recipients (71.5%) did not show signs of rejection. Only two cases demonstrated histologic signs of mild rejection 7 days after ITx. Interestingly intestinal grafts showed good absorptive capacity. The study's results support the viability of intestinal grafts from DCD using NRP, contributing more evidence for the use of DCD for ITx.

SIMUNEO: Control and Monitoring System for Lung Ultrasound Examination and Treatment of Neonatal Pneumothorax and Thoracic Effusion.

Training with real patients is a critical aspect of the learning and growth of doctors in training. However, this essential step in the educational process for clinicians can potentially compromise patient safety, as they may not be adequately prepared to handle real-life situations independently. Clinical simulators help to solve this problem by providing real-world scenarios in which the physicians can train and gain confidence by safely and repeatedly practicing different techniques. In addition, obtaining objective feedback allows subsequent debriefing by analysing the situation experienced and learning from other people's mistakes. This article presents SIMUNEO, a neonatal simulator in which professionals are able to learn by practicing the management of lung ultrasound and the resolution of pneumothorax and thoracic effusions. The article also discusses in detail the hardware and software, the main components that compose the system, and the communication and implementation of these. The system was validated through both usability questionnaires filled out by neonatology residents as well as through follow-up sessions, improvement, and control of the system with specialists of the department. Results suggest that the environment is easy to use and could be used in clinical practice to improve the learning and training of students as well as the safety of patients.

Global transcriptomic response of Escherichia coli to p-coumaric acid.

The aromatic compound p-coumaric acid (p-CA) is a secondary metabolite produced by plants. This aromatic acid and derived compounds have positive effects on human health, so there is interest in producing them in biotechnological processes with recombinant Escherichia coli strains. To determine the physiologic response of E. coli W3110 to p-CA, dynamic expression analysis of selected genes fused to a fluorescent protein reporter as well as RNA-seq and RT-qPCR were performed. The observed transcriptional profile revealed the induction of genes involved in functions related to p-CA active export, synthesis of cell wall and membrane components, synthesis of amino acids, detoxification of formaldehyde, phosphate limitation, acid stress, protein folding and degradation. Downregulation of genes encoding proteins involved in energy production, carbohydrate import and metabolism, as well as several outer and plasma membrane proteins was detected. This response is indicative of cell envelope damage causing the leakage of intracellular components including amino acids and phosphate-containing compounds. The cellular functions responding to p-CA that were identified in this study will help in defining targets for production strains improvement.

The aminoshikimic acid pathway in bacteria as source of precursors for the synthesis of antibacterial and antiviral compounds.

The aminoshikimic acid (ASA) pathway comprises a series of reactions resulting in the synthesis of 3-amino-5-hydroxybenzoic acid (AHBA), present in bacteria such as Amycolatopsis mediterranei and Streptomyces. AHBA is the precursor for synthesizing the mC7N units, the characteristic structural component of ansamycins and mitomycins antibiotics, compounds with important antimicrobial and anticancer activities. Furthermore, aminoshikimic acid, another relevant intermediate of the ASA pathway, is an attractive candidate for a precursor for oseltamivir phosphate synthesis, the most potent anti-influenza neuraminidase inhibitor treatment of both seasonal and pandemic influenza. This review discusses the relevance of the key intermediate AHBA as a scaffold molecule to synthesize diverse ansamycins and mitomycins. We describe the structure and control of the expression of the model biosynthetic cluster rif in A. mediterranei to synthesize ansamycins and review several current pharmaceutical applications of these molecules. Additionally, we discuss some relevant strategies developed for overproducing these chemicals, focusing on the relevance of the ASA pathway intermediates kanosamine, AHAB, and ASA.

Design and Development of an Interactive Web-Based Simulator for Trauma Training: A Pilot Study.

Trauma is the leading cause of death in people under 45 years old and one of the leading causes of death in the world. Therefore, specific trauma training during medical school as well as after it is crucial. Web-based learning is an important tool in education, offering the possibility to create realistic trauma scenarios. A web-based simulator has been developed and a pilot study has been accomplished to trial the simulator. A pelvic trauma scenario was created and 41 simulations were performed, 28 by medical students and 13 by doctors. The data analyzed are the actions taken to treat the trauma patient, the evolution of the vital signs of the patient, the timing spent on deciding which action to take, when each action was performed and the consequence that it had on the patient. Moreover, a post-simulation questionnaire was completed related to the usability of the simulator. The clinical treatment performance of doctors is better than the performance of medical students performing more actions correctly and in the right sequence as per ATLS recommendations. Moreover, significant differences are obtained in the time response provided to the patients which is key in trauma. With respect to the usability of the tool, responses provide a positive usability rating. In conclusion, this pilot study has demonstrated that the web-based training developed can be used to train and evaluate trauma management. Moreover, this research has highlighted a different approach to trauma treatment between medical students and doctors.

New insights into transport capability of sugars and its impact on growth from novel mutants of Escherichia coli.

The fast-growing capability of Escherichia coli strains used to produce industrially relevant metabolites relies on their capability to transport efficiently glucose or potential industrial feedstocks such as sucrose or xylose as carbon sources. E. coli imports extracellular glucose into the periplasmic space across the outer membrane porins: OmpC, OmpF, and LamB. As the internal membrane is an impermeable barrier for sugars, the cell employs several primary and secondary active transport systems, and the phosphoenolpyruvate (PEP)-sugar phosphotransferase (PTS) system for glucose transport. PTS:glucose is the preferred system by E. coli to transport and phosphorylate the periplasmic glucose; nevertheless, PTS imposes a strict metabolic control mechanism on the preferential consumption of glucose over other carbon sources in sugar mixtures such as glucose and xylose resulting from the hydrolysis of lignocellulosic biomass, by the carbon catabolite repression. In this contribution, we summarize the major sugar transport systems for glucose and disaccharide transport, the exhibited substrate plasticity, and their impact on the growth of E. coli, highlighting the relevance of PTS in the control of the expression of genes for the transport and catabolism of other sugars as xylose. We discuss the strategies developed by evolved mutants of E. coli during adaptive laboratory evolution experiments to overcome the nutritional stress condition imposed by inactivation of PTS as a strategy for the selection of fast-growing derivatives in glucose, xylose, or mixtures of glucose:xylose. This approach results in the recruitment of other primary and secondary active transporters, demonstrating relevant sugar plasticity in derivative-evolved mutants. Elucidation of the molecular and biochemical basis of sugar-transport substrate plasticity represents a consistent approach for sugar-transport system engineering for the design of efficient E. coli derivative strains with improved substrate assimilation for biotechnological purposes.

Metabolic reconstruction of Pseudomonas chlororaphis ATCC 9446 to understand its metabolic potential as a phenazine-1-carboxamide-producing strain.

Pseudomonas chlororaphis is a plant-associated bacterium with reported antagonistic activity against different organisms and plant growth-promoting properties. P. chlororaphis possesses exciting biotechnological features shared with another Pseudomonas with a nonpathogenic phenotype. Part of the antagonistic role of P. chlororaphis is due to its production of a wide variety of phenazines. To expand the knowledge of the metabolic traits of this organism, we constructed the first experimentally validated genome-scale model of P. chlororaphis ATCC 9446, containing 1267 genes and 2289 reactions, and analyzed strategies to maximize its potential for the production of phenazine-1-carboxamide (PCN). The resulting model also describes the capability of P. chlororaphis to carry out the denitrification process and its ability to consume sucrose (Scr), trehalose, mannose, and galactose as carbon sources. Additionally, metabolic network analysis suggested fatty acids as the best carbon source for PCN production. Moreover, the optimization of PCN production was performed with glucose and glycerol. The optimal PCN production phenotype requires an increased carbon flux in TCA and glutamine synthesis. Our simulations highlight the intrinsic H2O2 flux associated with PCN production, which may generate cellular stress in an overproducing strain. These results suggest that an improved antioxidative strategy could lead to optimal performance of phenazine-producing strains of P. chlororaphis. KEY POINTS : • This is the first publication of a metabolic model for a strain of P. chlororaphis. • Genome-scale model is worthy tool to increase the knowledge of a non model organism. • Fluxes simulations indicate a possible effect of H2O2 on phenazines production. • P. chlororaphis can be a suitable model for a wide variety of compounds.

Evolution of an Escherichia coli PTS- strain: a study of reproducibility and dynamics of an adaptive evolutive process.

Adaptive laboratory evolution (ALE) has been used to study and solve pressing questions about evolution, especially for the study of the development of mutations that confer increased fitness during evolutionary processes. In this contribution, we investigated how the evolutionary process conducted with the PTS- mutant of Escherichia coli PB11 in three parallel batch cultures allowed the restoration of rapid growth with glucose as the carbon source. The significant findings showed that genomic sequence analysis of a set of newly evolved mutants isolated from ALE experiments 2-3 developed some essential mutations, which efficiently improved the fast-growing phenotypes throughout different fitness landscapes. Regulator galR was the target of several mutations such as SNPs, partial and total deletions, and insertion of an IS1 element and thus indicated the relevance of a null mutation of this gene in the adaptation of the evolving population of PB11 during the parallel ALE experiments. These mutations resulted in the selection of MglB and GalP as the primary glucose transporters by the evolving population, but further selection of at least a second adaptive mutation was also necessary. We found that mutations in the yfeO, rppH, and rng genes improved the fitness advantage of evolving PTS- mutants and resulted in amplification of leaky activity in Glk for glucose phosphorylation and upregulation of glycolytic and other growth-related genes. Notably, we determined that these mutations appeared and were fixed in the evolving populations between 48 and 72 h of cultivation, which resulted in the selection of fast-growing mutants during one ALE experiments in batch cultures of 80 h duration.Key points• ALE experiments selected evolved mutants through different fitness landscapes in which galR was the target of different mutations: SNPs, deletions, and insertion of IS.• Key mutations in evolving mutants appeared and fixed at 48-72 h of cultivation.• ALE experiments led to increased understanding of the genetics of cellular adaptation to carbon source limitation.

Xylose-glucose co-fermentation to ethanol by Escherichia coli strain MS04 using single- and two-stage continuous cultures under micro-aerated conditions.

BACKGROUND: Simultaneous co-fermentation of mixed sugars is an important feature to consider in the production of ethanol from lignocellulosic biomass hydrolysates because it enhances the overall ethanol yield and volumetric productivity during fermentation. Continuous cultures can be used during ethanol production from lignocellulosic hydrolysates to prevent catabolite repression by glucose on other sugars, such as xylose, and thus promote the simultaneous and total consumption of sugars and reduce fermentation time. The use of single- and two-stage continuous cultures under micro-aerated conditions for simultaneous consumption of xylose and glucose, and fermentation to ethanol by ethanologenic Escherichia coli strain MS04 was studied. Mineral medium supplemented with glucose, xylose and sodium acetate, was used to compare continuous cultures performance to batch cultures. RESULTS: Single-stage continuous cultures under micro-aerated conditions allowed the total co-consumption of a mixture of glucose and xylose (7.5 and 42.5 g/L, respectively) in mineral medium, with steady state ethanol production of 18 g/L, and a volumetric ethanol productivity of 0.9 g/L h, when low dilution rates (0.05 h-1) were used. However, the volumetric productivity was lower than the batch process under similar conditions (1.3 g/L h). Conversely, micro-aerated two-stage continuous culture enhanced the volumetric productivity up to 1.6 g/L h at a dilution rate of 0.15 h-1, with a total consumption of sugars and a slight reduction of the overall ethanol yield. CONCLUSIONS: The total and simultaneous consumption of glucose and xylose by the ethanologenic E. coli strain MS04 was accomplished by using two-stage continuous culture under micro-aerated conditions with an increase in the volumetric ethanol productivity of 23% and 78% when compared to batch and single-stage continuous cultures, respectively. Multi-stage continuous cultivation can be used to promote the simultaneous consumption of all sugars contained in biomass hydrolysates, and thus increase the volumetric ethanol productivity of the fermentation process.

Controlled clinical trial comparing the effectiveness of a mindfulness and self-compassion 4-session programme versus an 8-session programme to reduce work stress and burnout in family and community medicine physicians and nurses: MINDUUDD study protocol.

BACKGROUND: Health personnel are susceptible to high levels of work stress and burnout due to the psychological and emotional demands of their work, as well as to other aspects related to the organisation of that work. This paper describes the rationale and design of the MINDUUDD study, the aim of which is to evaluate the effectiveness of a mindfulness and self-compassion 4-session programme versus the standard 8-session programme to reduce work stress and burnout in Family and Community Medicine and Nursing tutors and residents. METHODS: The MINDUDD study is a multicentre cluster randomised controlled trial with three parallel arms. Six Teaching Units will be randomised to one of the three study groups: 1) Experimental Group-8 (EG8); 2) Experimental Group-4 (EG4) Control group (CG). At least 132 subjects will participate (66 tutors/66 residents), 44 in the EG8, 44 in the EG4, and 44 in the CG. Interventions will be based on the Mindfulness-Based Stress Reduction (MBSR) program, including some self-compassion practices of the Mindful Self-Compassion (MSC) programme. The EG8 intervention will be implemented during 8 weekly face-to-face sessions of 2.5 h each, while the EG4 intervention will consist of 4 sessions of 2.5 h each. The participants will have to practice at home for 30 min/day in the EG8 and 15 min/day in the EG4. The Five Facet Mindfulness Questionnaire (FFMQ), Self-Compassion Scale (SCS), Perceived Stress Questionnaire (PSQ), Maslach Burnout Inventory (MBI), Jefferson Scale of Physician Empathy (JSPE), and Goldberg Anxiety-Depression Scale (GADS) will be administered. Measurements will be taken at baseline, at the end of the programs, and at three months after completion. The effect of the interventions will be evaluated by bivariate and multivariate analyses (Multiple Linear Regression). DISCUSSION: If the abbreviated mindfulness programme is at least as effective as the standard program, its incorporation into the curriculum and training plans will be easier and more appropriate. It will also be more easily applied and accepted by primary care professionals because of the reduced resources and means required for its implementation, and it may also extend beyond care settings to academic and teaching environments as well. TRIAL REGISTRATION: The study has been registered at ClinicalTrials.gov ( NCT03629457 ; date of registration: 13.08.2018).

[Accomplishment of preventive activities proposed by resident doctors of Family Medicine in Primary Care and its relationship with communication skills]. / Oferta de actividades preventivas propuestas por médicos residentes de Medicina Familiar en Atención Primaria y su relación con las habilidades comunicacionales.

OBJECTIVE: To determine the offer of preventive activities by resident physicians of family medicine in the Primary Care consultations and the relation with their communication habilities. DESIGN: A descriptive multicentre study assessing medical consultations video recording. LOCATION: Eight Primary Healthcare centres in Jaen (Andalucia). PARTICIPANTS: Seventy-three resident physicians (4th year) filmed and observed with patients. PRINCIPAL MEASUREMENTS: Offer of preventive activities (according to the Spanish Program of Preventive Activities and Health Promotion -PAPPS-). Doctor, patient and consultation characteristics. Peer-review of the communication between physicians and patients, using a CICAA scale. A descriptive, bivariate, logistic regression analysis was performed. RESULTS: Two hundred and sixty interviews were evaluated (duration 8.5±4.0min) of 73 residents (50.7% women, mean age 32.9±7.7 years, 79% urban environment). The patient is more frequently a woman (60%) who comes alone (72%) due to acute processes (80%) and with 2.1±1.0 demands. Preventive activities are offered in 47% (duration less than one minute) of primary (70%) and secondary (59%) prevention, offered through advice (72%) or screening (52%) and focused on the cardiovascular area (52%) and lifestyles (53%). Eighty percent related to the patient's reason for consultation. Communication skills 41% improvable, 26% adequate, 23% excellent. The offer of preventive activities is related to the duration of the consultation (OR=1.1, 95% CI 1.01; 1.16) and communication skills (OR=1.03, 95% CI 1.01; 1.10). CONCLUSIONS: Preventive activities are carried out in almost half of the consultations, although focused on advice and screening and linked to the patient's demand. Consultation time and communication skills favor a greater preventive offer.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

Communication with patients and the duration of family medicine consultations.

OBJECTIVE: To determine the distribution of consultation times, the factors that determine their length, and their relationship with a more participative, patient-centred consulting style. DESIGN: Cross-sectional multicentre study. LOCATION: Primary Healthcare Centres in Andalusia, Spain. PARTICIPANTS: A total of 119 tutors and family medicine physician residents. PRINCIPAL MEASUREMENTS: Consultation length and communication with the patient were analysed using the CICCAA scale (Connect, Identify, Understand, Consent, Help) during 436 interviews in Primary Care. RESULTS: The mean duration of consultations was 8.8min (sd: 3.6). The consultation tended to be longer when the physician had a patient-centred approach (10.37±4.19min vs 7.54±2.98min; p=0.001), and when there was joint decision-making (9.79±3.96min vs 7.73±3.42min: p=0.001). In the multivariable model, longer consultations were associated with obtaining higher scores on the CICAA scale, a wider range of reasons for consultation, whether they came accompanied, in urban centres, and a smaller number of daily visits (r2=0.32). There was no correlation between physician or patient gender, or problem type. CONCLUSION: A more patient centred medical profile, increased shared decision-making, a wider range of reasons for consultation, whether they came accompanied, in urban centres, and less professional pressure all seem to be associated with a longer consultation.

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc.

Physiological effects of pH gradients on Escherichia coli during plasmid DNA production.

A two-compartment scale-down system was used to mimic pH heterogeneities that can occur in large-scale bioreactors. The system consisted of two interconnected stirred tank reactors (STRs) where one of them represented the conditions of the bulk of the fluid and the second one the zone of alkali addition for pH control. The working volumes ratio of the STRs was set to 20:1 in order to simulate the relative sizes of the bulk and alkali addition zones, respectively, in large-scale bioreactors. Residence times (tR ) in the alkali addition STR of 60, 120, 180, and 240 s were simulated during batch cultures of an engineered Escherichia coli strain that produced plasmid DNA (pDNA). pH gradients of up to 0.9 units, between the two compartments, were attained. The kinetic, stoichiometric, and pDNA topological changes due to the pH gradients were studied and compared to cultures at constant pH of 7.2 and 8.0. As the tR increased, the pDNA and biomass yields, as well as pDNA final titer decreased, whereas the accumulation of organic acids increased. Furthermore, the transcriptional response of 10 selected genes to alkaline stress (pH 8.0) and pH gradients was monitored at different stages of the cultures. The selected genes coded for ion transporters, amino acids catabolism enzymes, and transcriptional regulators. The transcriptional response of genes coding for amino acids catabolism, in terms of relative transcription level and stage of maximal expression, was different when the alkaline stress was constant or transient. This suggests the activation of different mechanisms by E. coli to cope with pH fluctuations compared to constant alkaline pH. Moreover, the transcriptional response of genes related to negative control of DNA synthesis did not correlate with the lower pDNA yields. This is the first study that reports the effects of pH gradients on pDNA production by E. coli cultures. The information presented can be useful for the design of better bioreactor scale-up strategies.

Biosynthesis of catechol melanin from glycerol employing metabolically engineered Escherichia coli.

BACKGROUND: Melanins comprise a chemically-diverse group of polymeric pigments whose function is related to protection against physical and chemical stress factors. These polymers have current and potential applications in the chemical, medical, electronics and materials industries. The biotechnological production of melanins offers the possibility of obtaining these pigments in pure form and relatively low cost. In this study, Escherichia coli strains were engineered to evaluate the production of melanin from supplemented catechol or from glycerol-derived catechol produced by an Escherichia coli strain generated by metabolic engineering. RESULTS: It was determined that an improved mutant version of the tyrosinase from Rhizobium etli (MutmelA), could employ catechol as a substrate to generate melanin. Strain E. coli W3110 expressing MutmelA was grown in bioreactor batch cultures with catechol supplemented in the medium. Under these conditions, 0.29 g/L of catechol melanin were produced. A strain with the capacity to synthesize catechol melanin from a simple carbon source was generated by integrating the gene MutmelA into the chromosome of E. coli W3110 trpD9923, that has been modified to produce catechol by the expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, transketolase and anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. In batch cultures with this strain employing complex medium with 40 g/L glycerol as a carbon source, 1.21 g/L of catechol melanin were produced. The melanin was analysed by employing Fourier transform infrared spectroscopy, revealing the expected characteristics for a catechol-derived polymer. CONCLUSIONS: This constitutes the first report of an engineered E. coli strain and a fermentation process for producing a catechol melanin from a simple carbon source (glycerol) at gram level, opening the possibility of generating a large quantity of this polymer for its detailed characterization and the development of novel applications.

Engineering of a microbial coculture of Escherichia coli strains for the biosynthesis of resveratrol.

BACKGROUND: Resveratrol is a plant natural product with many health-protecting effects which makes it an attractive chemical both for academic studies and industrial purposes. However, the low quantities naturally produced by plants as well as the unsustainable procedures of extraction, purification and concentration have prompted many biotechnological approaches to produce this chemical in large quantities from renewable sources. None of these approaches have considered a microbial coculture strategy to produce this compound. The aim of this study was to prove the functionality of a microbial coculture for the biosynthesis of resveratrol. RESULTS: In this work, we have successfully applied a coculture system strategy comprised of two populations of Escherichia coli strains, each with a partial and complementary section of the pathway leading to the biosynthesis of the stilbene resveratrol. The first strain is a pheA knockout mutant previously engineered to excrete p-coumaric acid into the medium through the overexpression of genes encoding a tyrosine ammonia lyase from Rhodothorula glutinis, a feedback resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and a transketolase. The second strain in the coculture was engineered to express the second part of the resveratrol biosynthetic pathway through the introduction of synthetic genes encoding the 4-coumaroyl-CoA ligase from Streptomyces coelicolor A2 and the stilbene synthase either from the peanut Arachis hypogaea or the grapevine Vitis vinifera, the latter synthesized employing a gene harmonization strategy and showing better resveratrol production performance. Batch cultures were performed in mineral medium with glycerol as the sole carbon source, where a final titer of 22.6 mg/L of resveratrol was produced in 30 h. CONCLUSIONS: To our knowledge, this is the first time that a coculture of bacterial strains is used for the biosynthesis of resveratrol from glycerol, having the potential for a greater improvement in the product yield and avoiding the use of precursors such as p-coumaric acid, yeast extract or an expensive inhibitor such as cerulenin.

Communication skills of tutors and family medicine physician residents in Primary Care clinics.

AIM: To determine the communicative profiles of family physicians and the characteristics associated with an improved level of communication with the patient. DESIGN: A descriptive multicentre study. LOCATION: Primary Healthcare Centres in Almeria, Granada, Jaen and Huelva. PARTICIPANTS: 119 family physicians (tutors and 4th year resident physicians) filmed and observed with patients. PRINCIPAL MEASUREMENTS: Demographic and professional characteristics. Analysis of the communication between physicians and patients, using a CICAA (Connect, Identify, Understand, Agree and Assist, in English) scale. A descriptive, bivariate, multiple linear regression analysis was performed. RESULTS: There were 436 valid interviews. Almost 100% of physicians were polite and friendly, facilitating a dialogue with the patient and allowing them to express their doubts. However, few physicians attempted to explore the state of mind of the patient, or enquire about their family situation or any important stressful events, nor did they ask open questions. Furthermore, few physicians summarised the information gathered. The mean score was 21.43±5.91 points (maximum 58). There were no differences in the total score between gender, city, or type of centre. The linear regression verified that the highest scores were obtained from tutors (B: 2.98), from the duration of the consultations (B: 0.63), and from the age of the professionals (B: -0.1). CONCLUSION: Physicians excel in terms of creating a friendly environment, possessing good listening skills, and providing the patient with information. However the ability to empathise, exploring the psychosocial sphere, carrying out shared decision-making, and asking open questions must be improved. Being a tutor, devoting more time to consultations, and being younger, results in a significant improvement in communication with the patient.

Production of cinnamic and p-hydroxycinnamic acid from sugar mixtures with engineered Escherichia coli.

BACKGROUND: The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates. RESULTS: In this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate. CONCLUSIONS: In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

Deletion of the 2-acyl-glycerophosphoethanolamine cycle improve glucose metabolism in Escherichia coli strains employed for overproduction of aromatic compounds.

BACKGROUND: As a metabolic engineering tool, an adaptive laboratory evolution (ALE) experiment was performed to increase the specific growth rate (µ) in an Escherichia coli strain lacking PTS, originally engineered to increase the availability of intracellular phosphoenolpyruvate and redirect to the aromatic biosynthesis pathway. As result, several evolved strains increased their growth fitness on glucose as the only carbon source. Two of these clones isolated at 120 and 200 h during the experiment, increased their µ by 338 and 373 %, respectively, compared to the predecessor PB11 strain. The genome sequence and analysis of the genetic changes of these two strains (PB12 and PB13) allowed for the identification of a novel strategy to enhance carbon utilization to overcome the absence of the major glucose transport system. RESULTS: Genome sequencing data of evolved strains revealed the deletion of chromosomal region of 10,328 pb and two punctual non-synonymous mutations in the dhaM and glpT genes, which occurred prior to their divergence during the early stages of the evolutionary process. Deleted genes related to increased fitness in the evolved strains are rppH, aas, lplT and galR. Furthermore, the loss of mutH, which was also lost during the deletion event, caused a 200-fold increase in the mutation rate. CONCLUSIONS: During the ALE experiment, both PB12 and PB13 strains lost the galR and rppH genes, allowing the utilization of an alternative glucose transport system and allowed enhanced mRNA half-life of many genes involved in the glycolytic pathway resulting in an increment in the µ of these derivatives. Finally, we demonstrated the deletion of the aas-lplT operon, which codes for the main components of the phosphatidylethanolamine turnover metabolism increased the further fitness and glucose uptake in these evolved strains by stimulating the phospholipid degradation pathway. This is an alternative mechanism to its regeneration from 2-acyl-glycerophosphoethanolamine, whose utilization improved carbon metabolism likely by the elimination of a futile cycle under certain metabolic conditions. The origin and widespread occurrence of a mutated population during the ALE indicates a strong stress condition present in strains lacking PTS and the plasticity of this bacterium that allows it to overcome hostile conditions.