Characterization of resistance genes to Cladosporium fulvum on the short arm of chromosome 1 of tomato

Plant breeders generally use qualitative resistance that is associated with a hypersensitive reaction (HR) to obtain cultivars that are resistant to pathogens and pests. The genetics of this resistance is based on the gene-for-gene relationship, which involves the product of a plant resistance gene and the product of an avirulence gene of the pathogen occurs. The interaction between leaf mold ( Cladosporium fulvum ) and its solely host, tomato ( Lycopersicon esculentum ), complies with this model. In the last few years, the isolation of several avirulence ( Avr ) and resistance ( R ) genes have contributed to an increase in our knowledge on this interaction. Several resistance genes to C. fulvum ( Cf genes) have been isolated from tomato. These Cf genes are located on two different clusters on the tomato genome, which contain not only functional Cf genes, but also several homologs with yet unknown function.. The short arm of Chromosome 1 harbors one of these clusters, designated "Milky Way", comprising functional Cf genes ( Cf-4 , Cf-4A , Cf-9 ). Moreover, two other clusters are located on the short arm of Chromosome 1, designated "Northern Lights" and "Southern Cross", which only harbor homologs ( Hcr9 s), but no functional Cf genes. Also, there are several reports about the presence of other Cf genes on the short arm of Chromosome 1.To increase our knowledge on the genetic and molecular organization of Cf genes on the short arm of Chromosome 1, an experimental approach was chosen to identify Cf genes with novel specificities that map on the short arm of Chromosome 1. To saturate the tomato genome with molecular markers, an integrated high-density AFLP-RFLP map was constructed using two different L. esculentum x L. pennellii F 2 mapping populations. Although 1175 AFLP markers were mapped on the tomato genome, covering 1482 cM, only nine AFLP markers were detected between the RFLP markers CT233 and TG51, which mark a 23.6 cM interval, comprising several Hcr9 clusters, on the short arm of Chromosome 1. This relatively low number of markers is due to the clustering of most Eco RI/ Mse I AFLP markers around the centromeres (Chapter 2).Testcross populations of 66 C. fulvum resistant Lycopersicon accessions were obtained by crossing these accessions with the near isogenic line Moneymaker-Cf4 and subsequent crossing of the F 1 with the susceptible tomato cultivar Moneymaker (Chapter 3). Using disease resistance tests with C. fulvum race 0 on only 24 plants of these testcross populations, susceptible plants were identified. An under-representation of susceptible plants identified Cf resistance linked to Cf-4 , and hence location of the unknown resistance on the short arm of Chromosome 1. Out of the 21 resistant accessions that have been tested in this way, ten showed a Cf-4 linked Cf gene. Among these ten accessions, one accession specifically recognized the extracellular protein ECP5 of C. fulvum and the corresponding gene was designated Cf-ECP5 . This gene was more accurately mapped using a testcross population of 338 plants and an F 2 population of a cross between Moneymaker and CfECP5, consisting of 233 individuals. Cf-ECP5 mapped 4 cM proximal to the Hcr9 locus Milky Way and the corresponding Cf locus was designated Aurora. An amplification product that cosegregated completely with the Cf-ECP5 gene, was cloned and nine clones were sequenced (Chapter 6). These nine clones could be classified into four groups, indicating that the Aurora locus comprises several Hcr9 s.Of the 66 resistant Lycopersicon accessions mentioned above, 64 have been screened for the presence of Cf-4 and/or Cf-9 , using PVX:: Avr4 and PVX:: Avr9 , respectively. A relatively large proportion of these accessions all harbored the functional genes Cf-4 and Cf-4A (Chapter 4). Sequence analysis of the 3' end of Cf-4 and part of the 3' untranslated region of Cf-4 showed no differences from the previously published Cf-4 sequences, hence these lines contain an introgression fragment with identical Cf-4 and Cf-4A genes. Since several of these lines were previously designated with different Cf digits, a change in nomenclature is proposed.Five out of the 66 accessions studied, showed an HR upon specific recognition of ECP2 and therefore harbor the corresponding resistance gene Cf-ECP2 (Chapter 5). Using two different testcross populations and one F 2 population from a cross between Moneymaker and CfECP2, representing in total 282 individuals, Cf-ECP2 was accurately mapped. Cf-ECP2 cosegregates with the molecular marker CT116, which is located proximal to the Milky Way and Aurora clusters, but distal to the Southern Cross locus. Southern hybridization, using Cf-9 as a probe, showed a hybridizing band of7.5 kb cosegregating with Cf-ECP2 , indicating that Cf-ECP2 is a member of a previously unidentified Hcr9 locus, that has been designated Orion.Studies in Chapters 3, 5 and 6 show that functional Cf genes can be located on several different Hcr9 loci on the short arm of Chromosome 1 and that these Hcr9 loci are highly polymorphic.</p

Quantitative analysis of amino acid metabolism in liver cancer links glutamate excretion to nucleotide synthesis

Many cancer cells consume glutamine at high rates; counterintuitively, they simultaneously excrete glutamate, the first intermediate in glutamine metabolism. Glutamine consumption has been linked to replenishment of tricarboxylic acid cycle (TCA) intermediates and synthesis of adenosine triphosphate (ATP), but the reason for glutamate excretion is unclear. Here, we dynamically profile the uptake and excretion fluxes of a liver cancer cell line (HepG2) and use genome-scale metabolic modeling for in-depth analysis. We find that up to 30% of the glutamine is metabolized in the cytosol, primarily for nucleotide synthesis, producing cytosolic glutamate. We hypothesize that excreting glutamate helps the cell to increase the nucleotide synthesis rate to sustain growth. Indeed, we show experimentally that partial inhibition of glutamate excretion reduces cell growth. Our integrative approach thus links glutamine addiction to glutamate excretion in cancer and points toward potential drug targets

Does total hip replacement affect sexual quality of life?

Background: Total Hip Replacement (THR) is an effective treatment for end-stage hip osteoarthritis. Since the introduction of total joint replacement, the effect on the Sexual Quality of Life (SQoL) following THR has been addressed in scant studies. The aim of our study was to systematically review the literature, to summarise effects of THR on patients’ SQoL. Methods: We searched PubMed, EMBASE and PsycINFO between January 1970 and February 9th, 2015 with search terms including Total Hip, Osteoarthritis, SQoL, and THR. Eligible studies were identified and two independent authors extracted data including details of SQoL, study quality and risk of bias. Results: There were 12 eligible studies, which included a total of 2099 patients with an age range of 20–85 years. The methodological quality of ten studies was rated as low, and of two as moderate. Amongst the majority of patients, SQoL improved after surgery, both in terms of physical-functional and psychosocial well-being. However, changes between pre-operative and postoperative SQoL ranged extensively: for example, Sexual Dysfunction Δ 8–51 % and Sexual Activity (SA) Δ 0–77 %. Three studies reported that some patients never resumed SA again after surgery. Conclusion: In over 40 years of THR treatment, scant studies have examined the effect of THR on patients’ SQoL. This review suggests that SQol improves after THR, although the magnitude of effects varies highly. However, the quality of the supporting evidence was rated as low to moderate. This suggests a need for more high quality evidence about the effects of THR on SQoL

PREhabilitation of CAndidates for REnal Transplantation (PreCareTx) study:protocol for a hybrid type I, mixed method, randomised controlled trial

INTRODUCTION: Kidney transplant candidates (KTCs) need to be in optimal physical and psychological condition prior to surgery. However, KTCs often experience compromised functional capacity which can be characterised as frailty. Prehabilitation, the enhancement of a person's functional capacity, may be an effective intervention to improve the health status of KTCs. The PREhabilitation of CAndidates for REnal Transplantation (PreCareTx) study aims to examine the effectiveness of a multimodal prehabilitation programme on the health status of KTCs, and to explore the potential of implementation of prehabilitation in daily clinical practice.METHODS AND ANALYSIS: This study uses a single centre, effectiveness-implementation hybrid type I study design, comprised of a randomised controlled trial and a mixed-methods study. Adult patients who are currently on the transplant waiting list or are waitlisted during the study period, at a university medical centre in The Netherlands, will be randomly assigned to either prehabilitation (n=64) or care as usual (n=64) groups. The prehabilitation group will undergo a 12-week home-based, tailored prehabilitation programme consisting of physical and/or nutritional and/or psychosocial interventions depending on the participant's deficits. This programme will be followed by a 12-week maintenance programme in order to enhance the incorporation of the interventions into daily life. The primary endpoint of this study is a change in frailty status as a proxy for health status. Secondary endpoints include changes in physical fitness, nutritional status, psychological well-being, quality of life and clinical outcomes. Tertiary endpoints include the safety, feasibility and acceptability of the prehabilitation programme, and the barriers and facilitators for further implementation.ETHICS AND DISSEMINATION: Medical ethical approval was granted by the Medical Ethics Committee Groningen, Netherlands (M22.421). Written informed consent will be obtained from all participants. The results will be disseminated at international conferences and in peer-reviewed journals.TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT05489432.</p

Integrative analysis of the Trypanosoma brucei gene expression cascade predicts differential regulation of mRNA processing and unusual control of ribosomal protein expression

Background: Trypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model. Results: We refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated. Conclusions: Levels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism

Metabolomics guides rational development of a simplified cell culture medium for drug screening against <i>Trypanosoma brucei</i>

n vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form &lt;i&gt;Trypanosoma brucei&lt;/i&gt;. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (&#60;3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening

Pulmonary MTBVAC vaccination induces immune signatures previously correlated with prevention of tuberculosis infection

To fight tuberculosis, better vaccination strategies are needed. Live attenuated Mycobacterium tuberculosis-derived vaccine, MTBVAC, is a promising candidate in the pipeline, proven to be safe and immunogenic in humans so far. Independent studies have shown that pulmonary mucosal delivery of Bacillus Calmette-Guérin (BCG), the only tuberculosis (TB) vaccine available today, confers superior protection over standard intradermal immunization. Here we demonstrate that mucosal MTBVAC is well tolerated, eliciting polyfunctional T helper type 17 cells, interleukin-10, and immunoglobulins in the airway and yielding a broader antigenic profile than BCG in rhesus macaques. Beyond our previous work, we show that local immunoglobulins, induced by MTBVAC and BCG, bind to M. tuberculosis and enhance pathogen uptake. Furthermore, after pulmonary vaccination, but not M. tuberculosis infection, local T cells expressed high levels of mucosal homing and tissue residency markers. Our data show that pulmonary MTBVAC administration has the potential to enhance its efficacy and justifies further exploration of mucosal vaccination strategies in preclinical efficacy studies

CD28 between tolerance and autoimmunity: The side effects of animal models [version 1; referees: 2 approved]

Regulation of immune responses is critical for ensuring pathogen clearance and for preventing reaction against self-antigens. Failure or breakdown of immunological tolerance results in autoimmunity. CD28 is an important co-stimulatory receptor expressed on T cells that, upon specific ligand binding, delivers signals essential for full T-cell activation and for the development and homeostasis of suppressive regulatory T cells. Many in vivo mouse models have been used for understanding the role of CD28 in the maintenance of immune homeostasis, thus leading to the development of CD28 signaling modulators that have been approved for the treatment of some autoimmune diseases. Despite all of this progress, a deeper understanding of the differences between the mouse and human receptor is required to allow a safe translation of pre-clinical studies in efficient therapies. In this review, we discuss the role of CD28 in tolerance and autoimmunity and the clinical efficacy of drugs that block or enhance CD28 signaling, by highlighting the success and failure of pre-clinical studies, when translated to humans