Dashing Growth Curves: a web application for rapid and interactive analysis of microbial growth curves.

BACKGROUND: Recording and analyzing microbial growth is a routine task in the life sciences. Microplate readers that record dozens to hundreds of growth curves simultaneously are increasingly used for this task raising the demand for their rapid and reliable analysis. RESULTS: Here, we present Dashing Growth Curves, an interactive web application ( http://dashing-growth-curves.ethz.ch/ ) that enables researchers to quickly visualize and analyze growth curves without the requirement for coding knowledge and independent of operating system. Growth curves can be fitted with parametric and non-parametric models or manually. The application extracts maximum growth rates as well as other features such as lag time, length of exponential growth phase and maximum population size among others. Furthermore, Dashing Growth Curves automatically groups replicate samples and generates downloadable summary plots for of all growth parameters. CONCLUSIONS: Dashing Growth Curves is an open-source web application that reduces the time required to analyze microbial growth curves from hours to minutes.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells.

Endosymbioses are cellular mergers in which one cell lives within another cell and have led to major evolutionary transitions, most prominently to eukaryogenesis. Generation of synthetic endosymbioses aims to provide a defined starting point for studying fundamental processes in emerging endosymbiotic systems and enable the engineering of cells with novel properties. Here, we tested the potential of different bacteria for artificial endosymbiosis in mammalian cells. To this end, we adopted the fluidic force microscopy technology to inject diverse bacteria directly into the cytosol of HeLa cells and examined the endosymbiont-host interactions by real-time fluorescence microscopy. Among them, Escherichia coli grew exponentially within the cytoplasm, however, at a faster pace than its host cell. To slow down the intracellular growth of E. coli, we introduced auxotrophies in E. coli and demonstrated that the intracellular growth rate can be reduced by limiting the uptake of aromatic amino acids. In consequence, the survival of the endosymbiont-host pair was prolonged. The presented experimental framework enables studying endosymbiotic candidate systems at high temporal resolution and at the single cell level. Our work represents a starting point for engineering a stable, vertically inherited endosymbiosis.

Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle.

Methanol is a liquid with high energy storage capacity that holds promise as an alternative substrate to replace sugars in the biotechnology industry. It can be produced from CO2 or methane and its use does not compete with food and animal feed production. However, there are currently only limited biotechnological options for the valorization of methanol, which hinders its widespread adoption. Here, we report the conversion of the industrial platform organism Escherichia coli into a synthetic methylotroph that assimilates methanol via the energy efficient ribulose monophosphate cycle. Methylotrophy is achieved after evolution of a methanol-dependent E. coli strain over 250 generations in continuous chemostat culture. We demonstrate growth on methanol and biomass formation exclusively from the one-carbon source by 13C isotopic tracer analysis. In line with computational modeling, the methylotrophic E. coli strain optimizes methanol oxidation by upregulation of an improved methanol dehydrogenase, increasing ribulose monophosphate cycle activity, channeling carbon flux through the Entner-Doudoroff pathway and downregulating tricarboxylic acid cycle enzymes. En route towards sustainable bioproduction processes, our work lays the foundation for the efficient utilization of methanol as the dominant carbon and energy resource.

Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle.

Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.

Author Correction: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Utilization of surgical safety checklists by urological surgeons in Germany: a nationwide prospective survey.

OBJECTIVES: We aimed to investigate the contemporary usage rate and habits of the WHO Surgical Safety Checklist (SSC) in German urological departments. METHODS: We designed a 26-item questionnaire that was sent to all urological departments in Germany. The primary aim of this study was to evaluate the usage rate of the SSC. Secondary aims were to compare perioperative characteristics of users vs. non-users of the SSC and to assess circumstances of the SSC application. RESULTS: A total of 213 of 234 (91 %) urological departments were users of the SSC, and 21 (9 %) were non-users. SSC users had more often a standard protocol, took less time and had fewer people involved for checking perioperative patient data compared to non-users. Financial budgeting for the SSC existed in 55 (24 %) departments and for patient safety in 73 (32 %) departments. CONCLUSIONS: The usage rate of the SSC in urological departments in Germany is high despite restricted financial budgeting. Users of the SSC profit by saving time and manpower for checking perioperative patient data.

Derivation of a GIS-based watershed-scale conceptual model for the St. Jones River Delaware from habitat-scale conceptual models.

Conceptual modeling is a useful tool for identifying pathways between drivers, stressors, Valued Ecosystem Components (VECs), and services that are central to understanding how an ecosystem operates. The St. Jones River watershed, DE is a complex ecosystem, and because management decisions must include ecological, social, political, and economic considerations, a conceptual model is a good tool for accommodating the full range of inputs. In 2002, a Four-Component, Level 1 conceptual model was formed for the key habitats of the St. Jones River watershed, but since the habitat level of resolution is too fine for some important watershed-scale issues we developed a functional watershed-scale model using the existing narrowed habitat-scale models. The narrowed habitat-scale conceptual models and associated matrices developed by Reiter et al. (2006) were combined with data from the 2002 land use/land cover (LULC) GIS-based maps of Kent County in Delaware to assemble a diagrammatic and numerical watershed-scale conceptual model incorporating the calculated weight of each habitat within the watershed. The numerical component of the assembled watershed model was subsequently subjected to the same Monte Carlo narrowing methodology used for the habitat versions to refine the diagrammatic component of the watershed-scale model. The narrowed numerical representation of the model was used to generate forecasts for changes in the parameters "Agriculture" and "Forest", showing that land use changes in these habitats propagated through the results of the model by the weighting factor. Also, the narrowed watershed-scale conceptual model identified some key parameters upon which to focus research attention and management decisions at the watershed scale. The forecast and simulation results seemed to indicate that the watershed-scale conceptual model does lead to different conclusions than the habitat-scale conceptual models for some issues at the larger watershed scale.

Is a circular polypropylene mesh appropriate for application at the esophageal hiatus? Results from an experimental study in a porcine model.

BACKGROUND: Mesh reinforcement in hiatal hernia surgery is debated. Randomized controlled trials have shown that recurrences may be reduced, but there is also the fear of mesh-related complications. Experimental studies on the characteristics of specific mesh types with regard to the risk of such complications are rare. The current study aimed to investigate the properties of a circular heavy-weight polypropylene mesh in terms of stenosis, migration, erosions, and adhesions in a porcine model. METHODS: A 55 x 55-mm heavy-weight polypropylene mesh with a 16.5-mm eccentric hole for the esophagus corresponding to a calculated mesh area of 2811 mm(2) and a hole area of 214 mm(2) were implanted in nine German Landrace pigs. Six weeks later, the meshes were explanted and investigated for size, shrinkage, migration and adhesions. RESULTS: The total mesh area shrank to a mean of 2,040 +/- 178 mm(2) (p < 0.001), and the hole for the esophagus showed a trend toward an increase to 239 +/- 38 mm(2) (p = 0.108). In not a single location did the mesh overhang the hiatal margin. The mean distance of retraction from the hiatal margin was 4.3 +/- 2.8 mm. Therefore, no stenoses, migrations, or erosions occurred. CONCLUSIONS: A circular heavy-weight polypropylene mesh seems to be appropriate for the application at the esophageal hiatus in terms of safety and stability. This means that it is characterized by a position-stable centered fixation around the esophagus without a tendency toward stenosis, migration, or erosion.

Robotic suturing: technique and benefit in advanced laparoscopic surgery.

Suturing is one of the main tasks in advanced laparoscopic surgery, but limited degrees of freedom, 2D vision, fulcrum and pivoting effect make it difficult to perform. Robotic systems provide corresponding solutions as three-dimensional (3D) view, intuitive motion and additional degrees of freedom. This review evaluates these benefits for their impact on suturing in experimental and clinical studies. The Medline database was searched for "robot*, telemanipulat* and laparoscop*". A total of 1150 references were found and further limited to "suturing" for experimental evaluation, finding 89 references. All references were considered for information on robotic suturing in advanced laparoscopy. Further references were obtained through cross-referencing the bibliography cited in each work. In experimental studies current robotic systems have proven their superior suturing capabilities compared to conventional laparoscopic techniques, mainly attributed to 3D visualization and full seven degrees of freedom. In clinical studies these benefits have not yet been sufficiently reproduced. Robotic systems have to prove the benefits shown in experimental studies for suturing tasks in clinical applications. Robotic devices shorten the learning curve of laparoscopic procedures. Further clinical trials focusing on anastomosis time are needed to assess this question.