Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei.

<div><p>Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that <i>Trypanosoma brucei</i> hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form <i>T</i>. <i>brucei</i>. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.</p></div

A method for the reconstruction of unknown non-monotonic growth functions in the chemostat

We propose an adaptive control law that allows one to identify unstable steady states of the open-loop system in the single-species chemostat model without the knowledge of the growth function. We then show how one can use this control law to trace out (reconstruct) the whole graph of the growth function. The process of tracing out the graph can be performed either continuously or step-wise. We present and compare both approaches. Even in the case of two species in competition, which is not directly accessible with our approach due to lack of controllability, feedback control improves identifiability of the non-dominant growth rate.Comment: expansion of ideas from proceedings paper (17 pages, 8 figures), proceedings paper is version v

Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10–16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer–template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI

Mice Lacking Three Loci Encoding 14 Glutathione Transferase Genes: A Novel Tool for Assigning Function to the GSTP, GSTM, and GSTT Families

Glutathione S-transferases (GSTs) form a superfamily defined by their ability to catalyze the conjugation of glutathione with electrophilic substrates. These enzymes are proposed to play a critical role in protection of cellular components from damage mediated by reactive metabolites. Twenty-two cytosolic GSTs, grouped into seven families, are recognized in mice. This complexity hinders the assignment of function to a subset or family of these genes. We report generation of a mouse line in which the locus encoding three GST gene families is deleted. This includes the four Gstt genes spanning 65 kb on chromosome 10 and the seven Gstm genes found on a 150 kb segment of DNA chromosome 3. In addition, we delete two Gstp genes on chromosome 19 as well as a third related gene located 15 kb telomeric to Gstp1 and Gstp2, which we identify as a potential new member of this gene family. We show that, despite the loss of up to 75% of total GST activity in some tissues from these animals, the mice are healthy and fertile, with normal life expectancy. The normal development and health of these animals make them an appropriate model for defining the role of these families in redox homeostasis and metabolism of drugs and environmental pollutants

Targeting N-myristoylation for therapy of B-cell lymphomas

N-myristoyltransferases (NMTs) target many signaling proteins to membranes. Here the authors show an NMT inhibitor named PCLX-001 selectively kills lymphoma cells by shutting down their main survival signaling pathway and offers an additional treatment strategy for lymphoma patients

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not infective to mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both

Organizational configuration of hospitals succeeding in attracting and retaining nurses

Organizational configuration of hospitals succeeding in attracting and retaining nurses. This paper contrasts structural and managerial characteristics of low- and high-turnover hospitals, and describes the organizational configuration of attractive hospitals. In countries facing nurse shortages and turnover, some hospitals succeed in recruiting and retaining nurses. In Magnet Hospitals, managerial practices and environmental characteristics increase nurses\u2019 job satisfaction and their commitment to the organization, which in turn decreases nurse turnover. Such an approach suggests that organizations are best understood as clusters of interconnected structures and practices, i.e. organizational configurations rather than entities whose components can be understood in isolation. From a sample of 12 hospitals whose nurse turnover was studied for 1 year, structural and organizational features of hospitals in the first and fourth quartiles, i.e. attractive (turnover11\uc68%) were contrasted. A questionnaire, including perceptions of health-related factors, job demands, stressors, work schedules, organizational climate, and work adjustments antecedent to turnover, was received from 401 nurses working in attractive hospitals (response rate - 53\uc68%) and 774 nurses in conventional hospitals (response rate \ubc 54\uc65%). Structural characteristics did not differentiate attractive and conventional hospitals, but employee perceptions towards the organization differed strikingly. Differences were observed for risk exposure, emotional demands, role ambiguity and conflicts, work-family conflicts, effort-reward imbalance and the meaning of work, all in favour of attractive hospitals (P < 0.01). Relationships with nursing management, work ability and satisfaction with working time, handover shifts and schedules were also better in attractive hospitals (P < 0.001). Job satisfaction and commitment were higher in attractive hospitals, whereas burnout and intention to leave were lower (P < 0.001). Organizational characteristics are key factors in nurse attraction and retention. Nurses face difficulties in their work situations, but some hospitals are perceived as healthy organizations. The concept of attractive institutions could serve as a catalyst for improvement in nurses\u2019 work environments in Europe

Heterogeneous antiretroviral drug distribution and HIV/SHIV detection in the gut of three species

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3+ T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-μm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3+ T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography-mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3+ T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence

Transposon based tagging: IRAP, REMAP, and iPBS

Retrotransposons are a major component of virtually all eukaryotic genomes, which makes them useful as molecular markers. Various molecular marker systems have been developed that exploit the ubiquitous nature of these genetic elements and their property of stable integration into dispersed chromosomal loci that are polymorphic within species. To detect polymorphisms for retrotransposon insertions, marker systems generally rely on PCR amplification between the retrotransposon termini and some component of flanking genomic DNA. The main methods of IRAP, REMAP, RBIP, and SSAP all detect the polymorphic sites at which the retrotransposon DNA is integrated into the genome. Marker systems exploiting these methods can be easily developed and are inexpensively deployed in the absence of extensive genome sequence data. Here, we describe protocols for the IRAP, REMAP and iPBS techniques, including methods for PCR amplification with a single primer or with two primers, agarose gel electrophoresis of the product using optimal electrophoresis buffers, we also describe iPBS techniques for the rapid isolation of retrotransposon termini and full-length elements.Peer reviewe