13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.     

Comparison and transfer testing of multiplex ligation detection methods for GM plants

Background  

With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.     

Effects of assessing the productivity of faculty in academic medical centres: a systematic review

Background:

Many academic medical centres have introduced strategies to assess the productivity of faculty as part of compensation schemes. We conducted a systematic review of the effects of such strategies on faculty productivity.

Methods:

We searched the MEDLINE, Healthstar, Embase and PsycInfo databases from their date of inception up to October 2011. We included studies that assessed academic productivity in clinical, research, teaching and administrative activities, as well as compensation, promotion processes and satisfaction.

Results:

Of 531 full-text articles assessed for eligibility, we included 9 articles reporting on eight studies. The introduction of strategies for assessing academic productivity as part of compensation schemes resulted in increases in clinical productivity (in six of six studies) in terms of clinical revenue, the work component of relative-value units (these units are nonmonetary standard units of measure used to indicate the value of services provided), patient satisfaction and other departmentally used standards. Increases in research productivity were noted (in five of six studies) in terms of funding and publications. There was no change in teaching productivity (in two of five studies) in terms of educational output. Such strategies also resulted in increases in compensation at both individual and group levels (in three studies), with two studies reporting a change in distribution of compensation in favour of junior faculty. None of the studies assessed effects on administrative productivity or promotion processes. The overall quality of evidence was low.

Interpretation:

Strategies introduced to assess productivity as part of a compensation scheme appeared to improve productivity in research activities and possibly improved clinical productivity, but they had no effect in the area of teaching. Compensation increased at both group and individual levels, particularly among junior faculty. Higher quality evidence about the benefits and harms of such assessment strategies is needed.Academic productivity can be defined as a measurable output of a faculty member related to clinical, research, education or administrative activities. Achieving the best possible academic productivity is essential for academic medical centres to maintain or nurture recognition and good reputation.¹ Furthermore, clinical productivity is essential for the survival of academic departments given the economic realities in medicine.²Strategies for productivity assessment help in identifying highly productive faculty, determining areas for faculty and departmental improvement,³ and implementing processes for promotion and tenure.⁴ When coupled with reward schemes, these strategies may improve productivity and compensation at both individual and departmental levels. In the long-term, they may enhance the ability to recruit and retain high-quality faculty and achieve the academic mission of the department. However, these strategies may have some unintended effects such as using time dedicated to education to do more clinical work.³ In addition, they may be challenging to implement.³We conducted a systematic review of the effects of strategies introduced in academic medical centres to assess faculty productivity, compensation, promotion processes and satisfaction.     

Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing

The phenotype and fate of fetal microchimeric cells transfered into the maternal circulation during pregnancy are not well described. Since progenitors from distal sites mobilize during wound healing, we analyzed the recruitment and plasticity of fetal progenitors into maternal wounds. Wounds were generated on normal and bleomycin-induced fibrotic skin of parous or pregnant wild-type females with fluorescent GFP(+) fetuses. Analyses were performed on skin and blood specimens through PCR, immunohistochemistry, and flow cytometry. Controls consisted of parous and pregnant females without wounds and virgin females with wounds. Fetal cells were detected in all skin specimens of parous mice as long as healing was not achieved. During early stages of wound healing, fetal cells expressed mainly leukocyte markers, while in later phases endothelial markers prevailed. Fetally derived vessels connected to maternal circulation were also found, demonstrating the transfer of fetal endothelial progenitor cells. Wounding mobilized fetal CD34(+)ckit(-) cells into the blood during pregnancy. Most of this population was CD11b(-)VEGFR2(-). Another part was CD11b(+) with a fraction expressing VEGFR2. VEGFa-spiked Matrigel plugs partially mimicked this fetal progenitor recruitment and mobilization into the blood. In summary, fetal cells that mobilize in response to wounding are mainly progenitor cells and participate in angiogenesis and inflammation.     

Measuring dynamic stability requirements during sitting pivot transfers using stabilizing and destabilizing forces in individuals with complete motor paraplegia

Dynamic stability requirements have never been quantified when long-term manual wheelchair users transfer themselves in a seated position from an initial surface to a target surface, a functional task commonly referred to as sitting pivot transfers (SPTs). Ten individuals with spinal cord injury (SCI), who rely on a manual wheelchair for mobility, underwent a comprehensive biomechanical SPT assessment. SPTs performed toward a target seat of same height (even) and a seat 10cm higher than the initial seat (uneven), repeated three times for each task, were assessed. A dynamic equilibrium model, continuously measuring the theoretical forces required to move the center of pressure to the limit of the base of support (destabilizing force) and to neutralize the kinetic energy and stop the displacement of the center of mass at the limit of the base of support (stabilizing force) at each instance during the performance of SPTs, was used to identify the phases of greatest instability during the SPT tasks. The greatest levels of instability were reached around the time the buttocks lost contact with the initial seat and around the time the buttocks landed on the target seat (pre- and post-lift transition phases). These transition periods, characterized by the lowest destabilizing force (424.7-487.1N) and the greatest stabilizing force (24.2-33.2N), confirmed the greatest level of instability. The height of the target seat had no significant effect (p=0.278-0.739) on dynamic postural stability requirements during the SPTs. During SPTs towards even and uneven target seats, the greatest postural instability occurs during the transition phases in individuals with complete motor thoracic SCI.     

Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides

Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application. This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class. To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln. mesenteroides. pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e. the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion. A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene. Ln. mesenteroides DSM20484(pDMJF01) was unable to produce MesY105. The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF. DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin. MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF. This indicates that the MesY105 DTS permits the transport of MesB105. MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated. Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.     

A functional OGG1 homologue from Arabidopsis thaliana

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH₄, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.