Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis

Presence of depression and anxiety before and after coronary artery bypass graft surgery and their relationship to age

<p>Abstract</p> <p>Background</p> <p>Scientific literature on depression and anxiety in patients with coronary heart disease (CHD) consistently reports data of elevated anxiety and depression scores indicating clinically relevant quantities of these psychopathological conditions. Depression is considered to be a risk factor for the development of CHD and deteriorates the outcome after cardiac rehabilitation efforts. The aim of our study was to evaluate the presence of clinically relevant anxiety and depression in patients before and after coronary artery bypass grafting (CABG). Additionally we evaluated their relationship to age because of the increasing number of elderly patients undergoing CABG surgery.</p> <p>Methods</p> <p>One hundred and forty-two consecutive patients who underwent CABG in our hospital were asked to fill in the "Hospital Anxiety and Depression Scale – German Version (HADS)" to measure depression and anxiety scores two days before and ten days after CABG surgery. Differences between these pre- and post-surgical scores were then calculated as means for changes, and the amount of elevated scores were appraised. In order to investigate the relationship between age and anxiety and depression, respectively, Spearman correlations between age and the difference scores were calculated. In addition, ANOVA procedures with the factor "age group" and McNemar tests were calculated. Therefore the sample was divided into four equally sized age groups.</p> <p>Results</p> <p>25.8% of the patients were clinically depressed before and 17.5% after surgery; 34.0% of the patients were clinically anxious before and 24.7% after surgery. This overall change is not significant. We found a significant negative correlation between age and the difference between the two time points for anxiety (Spearman rho = -.218; p = 0.03), but not for depression (Spearman rho = -.128; p = 0.21). ANOVA and McNemar-Tests revealed that anxiety scores and the number of patients high in anxiety declined statistically meaningful only in the youngest patient group. Such a relationship could not be found for depression.</p> <p>Conclusion</p> <p>Our data show a relationship between age and anxiety. Younger patients are more anxious before CABG surgery than older ones and show a decline in symptoms while elderly patients show hardly any change.</p

Biology of Francisella tularensis Subspecies holarctica Live Vaccine Strain in the Tick Vector Dermacentor variabilis

Background: The c-proteobacterium Francisella tularensis is the etiologic agent of seasonal tick-transmitted tularemia epizootics in rodents and rabbits and of incidental infections in humans. The biology of F. tularensis in its tick vectors has not been fully described, particularly with respect to its quanta and duration of colonization, tissue dissemination, and transovarial transmission. A systematic study of the colonization of Dermacentor variabilis by the F. tularensis subsp. holarctica live vaccine strain (LVS) was undertaken to better understand whether D. variabilis may serve as an inter-epizootic reservoir for F. tularensis. Methodology/Principal Findings: Colony-reared larva, nymph, and adult D. variabilis were artificially fed LVS via glass capillary tubes fitted over the tick mouthparts, and the level of colonization determined by microbial culture. Larvae and nymphs were initially colonized with 8.860.8610 1 and 1.160.03610 3 CFU/tick, respectively. Post-molting, a significant increase in colonization of both molted nymphs and adults occurred, and LVS persisted in 42 % of molted adult ticks at 126 days post-capillary tube feeding. In adult ticks, LVS initially colonized the gut, disseminated to hemolymph and salivary glands by 21 days, and persisted up to 165 days. LVS was detected in the salivary secretions of adult ticks after four days post intra-hemocoelic inoculation, and LVS recovered from salivary gland was infectious to mice with an infectious dose 50 % of 3 CFU. LVS in gravid female ticks colonized via the intra-hemocoelic route disseminated to the ovaries and then t

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depo-lymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites

A combined prediction strategy increases identification of peptides bound with high affinity and stability to porcine MHC class I molecules SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01

Affinity and stability of peptides bound by major histocompatibility complex (MHC) class I molecules are important factors in presentation of peptides to cytotoxic T lymphocytes (CTLs). In silico prediction methods of peptide-MHC binding followed by experimental analysis of peptide-MHC interactions constitute an attractive protocol to select target peptides from the vast pool of viral proteome peptides. We have earlier reported the peptide binding motif of the porcine MHC-I molecules SLA-1*04:01 and SLA-2*04:01, identified by an ELISA affinity-based positional scanning combinatorial peptide library (PSCPL) approach. Here, we report the peptide binding motif of SLA-3*04:01 and combine two prediction methods and analysis of both peptide binding affinity and stability of peptide-MHC complexes to improve rational peptide selection. Using a peptide prediction strategy combining PSCPL binding matrices and in silico prediction algorithms (NetMHCpan), peptide ligands from a repository of 8900 peptides were predicted for binding to SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01 and validated by affinity and stability assays. From the pool of predicted peptides for SLA-1*04:01, SLA-2*04:01, and SLA-3*04:01, a total of 71, 28, and 38 % were binders with affinities below 500 nM, respectively. Comparison of peptide-SLA binding affinity and complex stability showed that peptides of high affinity generally, but not always, produce complexes of high stability. In conclusion, we demonstrate how state-of-the-art prediction and in vitro immunology tools in combination can be used for accurate selection of peptides for MHC class I binding, hence providing an expansion of the field of peptide-MHC analysis also to include pigs as a livestock experimental model.Fil: Pedersen, Lasse Eggers. Technical University of Denmark; DinamarcaFil: Rasmussen, Michael. Universidad de Copenhagen; DinamarcaFil: Harndahl, Mikkel. Universidad de Copenhagen; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús) | Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas (subsede Chascomús); ArgentinaFil: Buus, Søren. Universidad de Copenhagen; DinamarcaFil: Jungersen, Gregers. Technical University of Denmark; Dinamarc

Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

The activation of nuclear factor of activated T cells 5(NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.open3

The role of entry screening in case finding of tuberculosis among asylum seekers in Norway

<p>Abstract</p> <p>Background</p> <p>Most new cases of active tuberculosis in Norway are presently caused by imported strains and not transmission within the country. Screening for tuberculosis with a Mantoux test of everybody and a chest X-ray of those above 15 years of age is compulsory on arrival for asylum seekers.</p> <p>We aimed to assess the effectiveness of entry screening of a cohort of asylum seekers. Cases detected by screening were compared with cases detected later. Further we have characterized cases with active tuberculosis.</p> <p>Methods</p> <p>All asylum seekers who arrived at the National Reception Centre between January 2005 - June 2006 with an abnormal chest X-ray or a Mantoux test ≥ 6 mm were included in the study and followed through the health care system. They were matched with the National Tuberculosis Register by the end of May 2008.</p> <p>Cases reported within two months after arrival were defined as being detected by screening.</p> <p>Results</p> <p>Of 4643 eligible asylum seekers, 2237 were included in the study. Altogether 2077 persons had a Mantoux ≥ 6 mm and 314 had an abnormal chest X-ray. Of 28 cases with tuberculosis, 15 were detected by screening, and 13 at 4-27 months after arrival. Abnormal X-rays on arrival were more prevalent among those detected by screening. Female gender and Somalian origin increased the risk for active TB.</p> <p>Conclusion</p> <p>In spite of an imperfect follow-up of screening results, a reasonable number of TB cases was identified by the programme, with a predominance of pulmonary TB.</p