Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI

<p>Abstract</p> <p>Background</p> <p>The anaerobe <it>Clostridium difficile </it>produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI).</p> <p>Results</p> <p>Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results.</p> <p>Conclusion</p> <p>Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation.</p

The use of urinary proteomics in the assessment of suitability of mouse models for ageing

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8–96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing

Особенности школьной организационной культуры

Рассматривается основное содержание понятия «школьная организационная культура», функции и потенциал использования организационной культуры в общеобразовательных учреждениях. Выявляются особенности школьной организационной культуры. Обосновывается взаимосвязь организационной культуры и социально-психологического климата общеобразовательного учреждени

Практический опыт развития комплексной системы экологического просвещения в образовательной организации

В статье представлены разработанные и апробированные в образовательной организации новые подходы, механизмы и инструменты по одному из приоритетных направлений развития страны в части формирования экологического культуры вузовской молодежи с целью повышения познавательной активности, уровня экологических знаний в области гармоничного развития человека и природы, устойчивого интереса к экологическим проблемам современности, воспитания и привития бережного отношения к окружающей природе.The article presents new approaches, mechanisms and tools developed and tested in the educational organization in one of the priority directions of the country's development in terms of forming the ecological culture of university youth in order to increase cognitive activity, the level of ecological knowledge in the field of harmonious development of man and nature, and a steady interest in environmental problems of modernity, upbringing and inculcation of respect for nature

Alpha-1 antitrypsin deficiency impairs lung antibacterial immunity in mice

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency

Intrathecal immunoglobulin A and G antibodies to synapsin in a patient with limbic encephalitis

To report on the identification of intrathecally synthesized immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies to synapsin, a synaptic vesicle-associated protein, in a patient with limbic encephalitis

Vector and Axial-Vector Spectral Functions and QCD

We present new results of the tau hadronic spectral function analysis using data accumulated by the ALEPH detector at LEP during the years 1991-94. In addition to the vector spectral functions, the axial-vector spectral functions and, separately, the tau --> 3pi nu as well as the tau --> pi 2pi0 nu spectral functions are determined from their respective unfolded, i.e., physical invariant mass spectra. The spectral functions are applied to QCD chiral sum rules in order to extract information about saturation at the tau mass scale. Using the the semi-leptonic tau decay rate for vector and axial-vector currents in addition to spectral moments, we obtain precise measurements of the strong coupling constant alpha_s(M_tau) and the contributing non-perturbative power terms. The evolution to the Z mass yields alpha_s(M_Z) = 0.1219 +/- 0.0019.Comment: 15 pages, 12 figures, LaTex, Talk given at the Fourth International Workshop on Tau Lepton Physics (TAU96), Colorado, September 199

C-reactive protein (CRP) recognizes uric acid crystals and recruits proteases C1 and MASP1

Gout is caused by crystallization of uric acid in the form of monosodium urate (MSU) crystals, which induce a sterile inflammatory response that is hardly distinguishable from microbe-induced inflammatory responses. It is unclear, if MSU crystals (like microbes) are recognized by specific pattern recognition receptors. To identify possible soluble pattern recognition molecules for MSU crystals, we purified MSU-binding proteins from human body fluids. We identified C-reactive protein (CRP) as a major MSU-binding protein. Binding of CRP was strong enough to specifically deplete CRP from human serum. We found that CRP was required for fixation of complement components C1q, C1r, C1s and MASP1. Thus, we have identified a pattern recognition molecule for MSU crystals that links to the activation of complement. Notably, CRP does not show an even binding to the complete surface of the crystals. It rather binds to edges or distinct faces of the crystals

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin

In-medium chiral perturbation theory beyond the mean-field approximation

An explicit expression of the generating functional of two-flavor low-energy QCD with external sources in the presence of non-vanishing nucleon densities has been derived recently [1]. Within this approach we derive power counting rules for the calculation of in-medium pion properties. We develop the so-called standard rules for residual nucleon energies of the order of the pion mass and a modified scheme (non-standard counting) for vanishing residual nucleon energies. We also establish the different scales for the range of applicability of this perturbative expansion, which are \sqrt{6}\pi f_\pi\simeq 0.7 GeV for the standard and 6\pi^2 f_\pi^2/2m_N\simeq 0.27 GeV for non-standard counting, respectively. We have performed a systematic analysis of n-point in-medium Green functions up to and including next-to-leading order when the standard rules apply. These include the in-medium contributions to quark condensates, pion propagators, pion masses and couplings of the axial-vector, vector and pseudoscalar currents to pions. In particular, we find a mass shift for negatively charged pions in heavy nuclei that agrees with recent determinations from deeply bound pionic Pb-207. We have also established the absence of in-medium renormalization in the \pi^0 \to \gamma\gamma decay amplitude up to the same order. The study of \pi\pi scattering requires the use of the non-standard counting and the calculation is done at leading order. Even at that order we establish new contributions not considered so far. We also point towards further possible improvements of this scheme and touch upon its relation to more conventional many-body approaches.Comment: 40 pages, 12 figures, version to appear in Ann. Phy