Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe‐associated molecular patterns (MAMPs) and/or damage‐associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP‐triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co‐receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well‐known class of DAMPs, are oligomers of α‐1,4‐linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG‐triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin‐related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG‐activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.     

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes

Background

Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic.

Methods

RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes.

Results

Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication.

Conclusions

Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

     

Differential susceptibility of Popillia japonica 3rd instars to Heterorhabditis bacteriophora (Italian strain) at three different seasons

The scarab beetle Popillia japonica, a pest native to northern Japan, has been recently found in Italy. Entomopathogenic nematodes are useful for biological control of this invasive insect. Previous work showed that 1st and 2nd larval stages are more susceptible to nematodes than 3rd instars. We tested the effectiveness of Heterorhabditis bacteriophora in the laboratory against P. japonica 3rd instars. Experiments were conducted in Italy with larvae field collected in the fall, winter and spring, showing a significant decrease in effectiveness from the fall to spring.     

Sulfoximines as ATR inhibitors: Analogs of VE-821

The ATM- and Rad3-related (ATR) kinases play a key role in DNA repair processes and thus ATR is an attractive target for cancer therapy. Here we designed and synthesized sulfilimidoyl- and sulfoximidoyl-substituted analogs of the sulfone VE-821, a reported ATR inhibitor. The properties of these analogs have been investigated by calculating physicochemical parameters and studying their potential to specifically inhibit ATR in cells. Prolonged inhibition of ATR by the analogs in a Burkitt lymphoma cell line resulted in enhanced DNA damage and a substantial amount of apoptosis. Together our findings suggest that the sulfilimidoyl- and sulfoximidoyl-substituted analogs are efficient ATR inhibitors.     

Development and validation of a HPLC-ESI-MS/MS method for the determination of 5-aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma

A new HPLC method for the determination of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) in human plasma was developed and validated. Plasma samples were analyzed after protein precipitation with methanol and the two analytes were separated using a C18 column with a mobile phase composed of 17.5mmol/L acetic acid (pH 3.3):acetonitrile=85:15 (v/v) at 0.2mL/min flow rate. 4-ASA and N-Ac-4-ASA were used as internal standards. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in negative ionization mode and in multiple reaction monitoring acquisition (m/z 152-->108 for 5-ASA; m/z 194-->150 and 194-->107 for N-Ac-5-ASA). The limit of quantification (LOQ) was 50ng/mL for both analytes (0.2ng injected) and matrix-matched standard curves showed linearity up to 4000ng/mL. In the entire analytical range the within- and between-batch precision (R.S.D.%) values were respectively 90% for 5-ASA and >95% for N-Ac-5-ASA (R.S.D.%相似文献   

Exploring the binding features of rimonabant analogues and acyclic CB<Subscript>1</Subscript> antagonists: docking studies and QSAR analysis

In order to elucidate the structural requirements for human CB₁ receptor antagonism, 78 antagonists belonging to five different chemical classes were selected from the literature and docked into the receptor binding site, built by homology modeling techniques. To further explore the structure-activity relationships within the considered chemical classes, a pharmacophore model and a QSAR analysis were developed. In a first step five alignments, one for each group of compounds were generated. All of them were then submitted to a MOE pharmacophore search in order to obtain a final pharmacophore model representative of the whole dataset which was used to elaborate the following 3D-QSAR analysis, by means of the CoMFA methodology. The results of these investigations are expected to be useful in the process of design and development of new potent CB₁ antagonists. Figure Compounds 1-78 are aligned into the putative CB1 receptor binding site. The three key features shared by all of them are reported in coloured spheres. The hydrophobic/aromatic ones are depicted in purple while the acceptor functions are coloured in blue.     

The effect of serum withdrawal on the protein profile of quiescent human dermal fibroblasts in primary cell culture

The effect of serum deprivation on proliferating cells is well known, in contrast its role on primary cell cultures, at confluence, has not been deeply investigated. Therefore, in order to explore the response of quiescent cells to serum deprivation, ubiquitous mesenchymal cells, as normal human dermal fibroblasts, were grown, for 48 h after confluence, in the presence or absence of 10% FBS. Fibroblast behaviour (i.e. cell morphology, cell viability, ROS production and elastin synthesis) was evaluated morphologically and biochemically. Moreover, the protein profile was investigated by 2-DE and differentially expressed proteins were identified by MS. Serum withdrawal caused cell shrinkage but did not significantly modify the total cell number. ROS production, as evaluated by the dihydroethidium (DH2) probe, was increased after serum deprivation, whereas elastin synthesis, measured by a colorimetric method, was markedly reduced in the absence of serum. By proteome analysis, 41 proteins appeared to significantly change their expression, the great majority of protein changes were related to the cytoskeleton, the stress response and the glycolytic pathway. Data indicate that human dermal fibroblasts in primary cell culture can adapt themselves to environmental changes, without significantly altering cell viability, at least after a few days of treatment, even though serum withdrawal represents a stress condition capable to increase ROS production, to influence cell metabolism and to interfere with cell behaviour, favouring the expression of several age-related features.     

Fully immunocompetent CD8+ T lymphocytes are present in autologous haematopoietic stem cell transplantation recipients despite an ineffectual T-helper response

Background

Reduced CD4 T lymphocytes counts can be observed in HIV infection and in patients undergoing autologous haematopoietic stem cell transplantation (ASCT). Nevertheless, whereas opportunistic infections (OI) are frequent in HIV-infected individuals with low cell counts, OI are uncommon in ASCT patients.

Methodology/Principal Findings

To verify whether this observation could be secondary to intrinsic HIV-correlated T cell defects, we performed in-depth immunologic analyses in 10 patients with comparable CD4 counts in whom lymphopenia was secondary either to HIV-infection or ASCT-associated immunosuppressive therapy and compared them to age-matched healthy subjects. Results showed the presence of profound alterations in CD4+ T lymphocytes in both groups of patients with respect to healthy controls. Thus, a low percentage of CCR7+ CD4+ T cells and a compensative expansion of CD45RA−CCR7− CD4+ T cells, a reduced IL-2/IFN-γ cytokine production and impaired recall antigens-specific proliferative responses were detected both in ASCT and HIV patients. In stark contrast, profound differences were detected in CD8+ T-cells between the two groups of patients. Thus, mature CD8+ T cell prevailed in ASCT patients in whom significantly lower CD45RA−CCR7− cells, higher CD45RA+CCR7− CD8+ cells, and an expansion of CCR7+CD8+ cells was detected; this resulted in higher IFN-γ +/TNFα production and granzyme CD8+ expression. The presence of strong CD8 T cells mediated immune responses justifies the more favorable clinical outcome of ASCT compared to HIV patients.

Conclusion/Significance

These results indicate that CD8 T cells maturation and functions can be observed even in the face of a profound impairment of CD4+ T lymphocytes in ASCT but not in HIV patients. Primary HIV-associated CD8 defects or an imprinting by an intact CD4 T cell system in ASCT could justify these results.