Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio.

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

The complete genome of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina platforms.

We report the complete genome and the plasmid (F' episome) sequences of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina data. The resulting genome is a single contig of 4,524,963 bp, and the plasmid consists of a single contig of 197,186 bp.

The complete genome of two Lactiplantibacillus plantarum isolates from the traditional Mexican fermented pulque beverage assembled with a combination of PacBio and Illumina platforms.

We report the sequence of the complete genome and associated plasmids of two Lactiplantibacillus plantarum isolates from the traditional Mexican pulque beverage assembled with a combination of PacBio and Illumina data. The resulting complete genome for strain LB1_P46 is 3,287,706 bp; for strain LB2_P47, the complete genome is 3,289,072 bp.

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.

Glucose Transport in Escherichia coli: From Basics to Transport Engineering.

Escherichia coli is the best-known model for the biotechnological production of many biotechnological products, including housekeeping and heterologous primary and secondary metabolites and recombinant proteins, and is an efficient biofactory model to produce biofuels to nanomaterials. Glucose is the primary substrate used as the carbon source for laboratory and industrial cultivation of E. coli for production purposes. Efficient growth and associated production and yield of desired products depend on the efficient sugar transport capabilities, sugar catabolism through the central carbon catabolism, and the efficient carbon flux through specific biosynthetic pathways. The genome of E. coli MG1655 is 4,641,642 bp, corresponding to 4702 genes encoding 4328 proteins. The EcoCyc database describes 532 transport reactions, 480 transporters, and 97 proteins involved in sugar transport. Nevertheless, due to the high number of sugar transporters, E. coli uses preferentially few systems to grow in glucose as the sole carbon source. E. coli nonspecifically transports glucose from the extracellular medium into the periplasmic space through the outer membrane porins. Once in periplasmic space, glucose is transported into the cytoplasm by several systems, including the phosphoenolpyruvate-dependent phosphotransferase system (PTS), the ATP-dependent cassette (ABC) transporters, and the major facilitator (MFS) superfamily proton symporters. In this contribution, we review the structures and mechanisms of the E. coli central glucose transport systems, including the regulatory circuits recruiting the specific use of these transport systems under specific growing conditions. Finally, we describe several successful examples of transport engineering, including introducing heterologous and non-sugar transport systems for producing several valuable metabolites.

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.

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.

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.

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.

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.

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.

[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.

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).

Metabolic modeling and response surface analysis of an Escherichia coli strain engineered for shikimic acid production.

BACKGROUND: Classic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media. RESULTS: The obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process. CONCLUSIONS: The combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.

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.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

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.

Draft Genome Sequence of Pseudomonas chlororaphis ATCC 9446, a Nonpathogenic Bacterium with Bioremediation and Industrial Potential.

Pseudomonas chlororaphis strain ATCC 9446 is a biocontrol-related organism. We report here its draft genome sequence assembled into 35 contigs consisting of 6,783,030 bp. Genome annotation predicted a total of 6,200 genes, 6,128 coding sequences, 81 pseudogenes, 58 tRNAs, 4 noncoding RNAs (ncRNAs), and 41 frameshifted genes.