Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-d-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

     

Use of a Mutated Protoporphyrinogen Oxidase Gene as an Effective In Vitro Selectable Marker System that Also Conveys <Emphasis Type="Italic">in planta</Emphasis> Herbicide Resistance in Sugarcane

Genetic engineering can be used to introduce economically important traits in sugarcane cultivars. Part of any transformation process involves the selection of genetically transformed cells. In this study, an efficient sugarcane in vitro selection system was developed using mutated protophorhyrinogen oxidase (PPO) genes as selectable markers. Two PPO genes, that encode proteins targeted either to the mitochondria or plastid, were isolated from tobacco and maize. Site-directed mutagenesis was used to alter the nucleotide sequence of these genes so that the resulting proteins are less sensitive to diphenylether type herbicides. Sugarcane callus was genetically transformed through particle bombardment with constructs allowing expression of either transgene, and putative transgenic calli were selected on fomesafen. It took approximately 4 weeks to select herbicide resistant calli clones on 10 mg/l fomesafen in the presence of light, which increased the selection pressure, and a further 8 weeks to regenerate resistant plantlets. PCR analysis confirmed that all regenerated putative transgenic sugarcane plants contained the transgene. All transgenic plants showed levels of herbicide resistance when planted in soil.     

Learning without Borders: A Review of the Implementation of Medical Error Reporting in Médecins Sans Frontières

Objective

To analyse the results from the first 3 years of implementation of a medical error reporting system in Médecins Sans Frontières-Operational Centre Amsterdam (MSF) programs.

Methodology

A medical error reporting policy was developed with input from frontline workers and introduced to the organisation in June 2010. The definition of medical error used was “the failure of a planned action to be completed as intended or the use of a wrong plan to achieve an aim.” All confirmed error reports were entered into a database without the use of personal identifiers.

Results

179 errors were reported from 38 projects in 18 countries over the period of June 2010 to May 2013. The rate of reporting was 31, 42, and 106 incidents/year for reporting year 1, 2 and 3 respectively. The majority of errors were categorized as dispensing errors (62 cases or 34.6%), errors or delays in diagnosis (24 cases or 13.4%) and inappropriate treatment (19 cases or 10.6%). The impact of the error was categorized as no harm (58, 32.4%), harm (70, 39.1%), death (42, 23.5%) and unknown in 9 (5.0%) reports. Disclosure to the patient took place in 34 cases (19.0%), did not take place in 46 (25.7%), was not applicable for 5 (2.8%) cases and not reported for 94 (52.5%). Remedial actions introduced at headquarters level included guideline revisions and changes to medical supply procedures. At field level improvements included increased training and supervision, adjustments in staffing levels, and adaptations to the organization of the pharmacy.

Conclusion

It was feasible to implement a voluntary reporting system for medical errors despite the complex contexts in which MSF intervenes. The reporting policy led to system changes that improved patient safety and accountability to patients. Challenges remain in achieving widespread acceptance of the policy as evidenced by the low reporting and disclosure rates.     

Demonstration of gonadotropin releasing-hormone receptors on gonadotrophs and somatotrophs of the goldfish: an electron microscope study.

Dispersed pituitary cells of the goldfish were incubated with biotinylated [D-Lys6, Pro9-N-ethylamide] salmon gonadotropin-releasing hormone (sGnRH-A) then avidingold (10 nm), and were fixed, embedded and sectioned. Cells were identified as gonadotrophs, somatotrophs, or prolactin cells using specific hormone antisera and protein-A gold (20 nm) as a marker. Attachment of the biotinylated sGnRH-A to the pituitary cell sections was determined by scanning cell surfaces for the smaller gold particles using the transmission electron microscope. Attachment was observed on gonadotrophs and somatotrophs, but was negligible on prolactin cells. Preincubation with unlabelled salmon gonadotropin-releasing hormone or chicken II gonadotropin-releasing hormone, or omission of the salmon gonadotropin-releasing hormone analog, prevented the reaction. The direct visualization of specific gonadotropin-releasing hormone receptors on gonadotrophs and somatotrophs supports the existence of direct stimulatory actions of gonadotropin-releasing hormone on gonadotropin and somatotropin release in gold-fish.