Comparative Analysis of Proteins Regulated during Cadmium Sulfide Quantum Dots Response in Arabidopsis thaliana Wild Type and Tolerant Mutants

In previous work, two independent Arabidopsis thaliana Ac/Ds transposon insertional mutant lines, atnp01 and atnp02, were identified that showed a higher level of tolerance than the wild type (wt) line to cadmium sulfide quantum dots (CdS QDs). The tolerance response was characterized at physiological, genetic and transcriptomic levels. In this work, a comparative analysis was performed on protein extracts from plantlets of the two mutants and of wt, each treated with 80 mg L-1 CdS QDs. A comparative protein analysis was performed by 2D-PAGE, and proteins were characterized by MALDI-TOF/TOF mass spectrometry. Of 250 proteins identified from all three lines, 98 showed significant changes in relative abundance between control and CdS QD-treated plantlets. The wt, atnp01, and atnp02 control-treated pairs respectively showed 61, 31, and 31 proteins with differential expression. The two mutants had a different response to treatment in terms of type and quantity of up- and downregulated proteins. This difference became more striking when compared to wt. A network analysis of the proteins differentially expressed in atnp01 and atnp02 included several of those encoded by putative genes accommodating the transposons, which were responsible for regulation of some proteins identified in this study. These included nifu-like protein 3 (Nfu3), involved in chloroplast assembly, elongator complex 3 (Elo3), involved in transcriptional elongation, magnesium-chelate subunit-2 (Chli2), involved in chlorophyll biosynthesis, and protein phosphatase 2C (PP2C) which mediates abiotic stress response

Proteomic Analysis Identifies Markers of Exposure to cadmium Sulphide Quantum Dots (CdS QDs)

The use of cadmium sulphide quantum dot (CdS QD)-enabled products has become increasingly widespread. The prospect of their release in the environment is raising concerns. Here we have used the yeast model Saccharomyces cerevisiae to determine the potential impact of CdS QD nanoparticles on living organisms. Proteomic analyses and cell viability assays performed after 9 h exposure revealed expression of proteins involved in oxidative stress and reduced lethality, respectively, whereas oxidative stress declined, and lethality increased after 24 h incubation in the presence of CdS QDs. Quantitative proteomics using the iTRAQ approach (isobaric tags for relative and absolute quantitation) revealed that key proteins involved in essential biological pathways were differentially regulated over the time course of the experiment. At 9 h, most of the glycolytic functions increased, and the abundance of the number of heat shock proteins increased. This contrasts with the situation at 24 h where glycolytic functions, some heat shock proteins as well as oxidative phosphorylation and ATP synthesis were down-regulated. It can be concluded from our data that cell exposure to CdS QDs provokes a metabolic shift from respiration to fermentation, comparable to the situation reported in some cancer cell

Infants hospitalized for Bordetella pertussis infection commonly have respiratory viral coinfections

Background: Whether viral coinfections cause more severe disease than Bordetella pertussis (B. pertussis) alone remains unclear. We compared clinical disease severity and sought clinical and demographic differences between infants with B. pertussis infection alone and those with respiratory viral coinfections. We also analyzed how respiratory infections were distributed during the 2 years study. Methods: We enrolled 53 infants with pertussis younger than 180 days (median age 58 days, range 17–109 days, 64. 1% boys), hospitalized in the Pediatric Departments at “Sapienza” University Rome and Bambino Gesù Children’s Hospital from August 2012 to November 2014. We tested in naso-pharyngeal washings B. pertussis and 14 respiratory viruses with real-time reverse-transcriptase-polymerase chain reaction. Clinical data were obtained from hospital records and demographic characteristics collected using a structured questionnaire. Results: 28/53 infants had B. pertussis alone and 25 viral coinfection: 10 human rhinovirus (9 alone and 1 in coinfection with parainfluenza virus), 3 human coronavirus, 2 respiratory syncytial virus. No differences were observed in clinical disease severity between infants with B. pertussis infection alone and those with coinfections. Infants with B. pertussis alone were younger than infants with coinfections, and less often breastfeed at admission. Conclusions: In this descriptive study, no associations between clinical severity and pertussis with or without co-infections were found

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

<p>Abstract</p> <p>Background</p> <p>In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in <it>Nicotiana benthamiana</it>, followed by a two-step affinity purification protocol of plant-derived Nef.</p> <p>Results</p> <p>The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.</p> <p>Conclusion</p> <p>We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.</p

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy

Altered sphingolipid metabolism in N-(4-hydroxyphenyl) retinamide resistant A2780 human ovarian carcinoma cells

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells

Fasting-Mimicking Diet Promotes Ngn3-Driven β-Cell Regeneration to Reverse Diabetes

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing β cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models