Interactions between p300 and multiple NF-Y trimers govern cyclin B2 promoter function

The CCAAT box is one of the most common elements in eukaryotic promoters and is activated by NF-Y, a conserved trimeric transcription factor with histone-like subunits. Usually one CCAAT element is present in promoters at positions between -60 and -100, but an emerging class of promoters harbor multiple NF-Y sites. In the triple CCAAT-containing cyclin B2 cell-cycle promoter, all CCAAT boxes, independently from their NF-Y affinities, are important for function. We investigated the relationships between NF-Y and p300. Chromatin immunoprecipitation analysis found that NF-Y and p300 are bound to the cyclin B2 promoter in vivo and that their binding is regulated during the cell cycle, positively correlating with promoter function. Cotransfection experiments determined that the coactivator acts on all CCAAT boxes and requires a precise spacing between the three elements. We established the order of in vitro binding of the three NF-Y complexes and find decreasing affinities from the most distal Y1 to the proximal Y3 site. Binding of two or three NF-Y trimers with or without p300 is not cooperative, but association with the Y1 and Y2 sites is extremely stable. p300 favors the binding of NF-Y to the weak Y3 proximal site, provided that a correct distance between the three CCAAT is respected. Our data indicate that the precise spacing of multiple CCAAT boxes is crucial for coactivator function. Transient association to a weak site might be a point of regulation during the cell cycle and a general theme of multiple CCAAT box promoters

Heat exchanger/reactors (HEX reactors): Concepts, technologies: State-of-the-art

Process intensification is a chemical engineering field which has truly emerged in the past few years and is currently rapidly growing. It consists in looking for safer operating conditions, lower waste in terms of costs and energy and higher productivity; and away to reach such objectives is to develop multifunctional devices such as heat exchanger/reactors for instance. This review is focused on the latter and makes a point on heat exchanger/reactors. After a brief presentation of requirements due to transposition from batch to continuous apparatuses, heat exchangers/reactors at industrial or pilot scales and their applications are described

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Chronic hyperglycemia induces trans-differentiation of human pancreatic stellate cells and enhances the malignant molecular communication with human pancreatic cancer cells

BACKGROUND: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment. METHODOLOGY/PRINCIPAL FINDINGS: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-β1 treatment (3.09-fold with a 2.73-fold without TGF-β1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour. CONCLUSIONS: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration

Long-term and realistic global change manipulations had low impact on diversity of soil biota in temperate heathland

In a dry heathland ecosystem we manipulated temperature (warming), precipitation (drought) and atmospheric concentration of CO(2) in a full-factorial experiment in order to investigate changes in below-ground biodiversity as a result of future climate change. We investigated the responses in community diversity of nematodes, enchytraeids, collembolans and oribatid mites at two and eight years of manipulations. We used a structural equation modelling (SEM) approach analyzing the three manipulations, soil moisture and temperature, and seven soil biological and chemical variables. The analysis revealed a persistent and positive effect of elevated CO(2) on litter C:N ratio. After two years of treatment, the fungi to bacteria ratio was increased by warming, and the diversities within oribatid mites, collembolans and nematode groups were all affected by elevated CO(2) mediated through increased litter C:N ratio. After eight years of treatment, however, the CO(2)-increased litter C:N ratio did not influence the diversity in any of the four fauna groups. The number of significant correlations between treatments, food source quality, and soil biota diversities was reduced from six to three after two and eight years, respectively. These results suggest a remarkable resilience within the soil biota against global climate change treatments in the long term

Microbial Patterns Signaling via Toll-Like Receptors 2 and 5 Contribute to Epithelial Repair, Growth and Survival

Epithelial cells (ECs) continuously interact with microorganisms and detect their presence via different pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs). Ligation of epithelial TLRs by pathogens is usually associated with the induction of pro-inflammatory mediators and antimicrobial factors. In this study, using human airway ECs as a model, we found that detection of microbial patterns via epithelial TLRs directly regulates tissue homeostasis. Staphylococcus aureus (S. aureus) and microbial patterns signaling via TLR2 and TLR5 induce a set of non-immune epithelial responses including cell migration, wound repair, proliferation, and survival of primary and cancerous ECs. Using small interfering RNA (siRNA) gene targeting, receptor-tyrosine kinase microarray and inhibition studies, we determined that TLR and the epidermal growth factor receptor (EGFR) mediate the stimulating effect of microbial patterns on epithelial repair. Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival. This effect is independent of hematopoietic and other cells as well as inflammatory cytokines suggesting that epithelia are able to regulate their integrity in an autonomous non-inflammatory manner by sensing microbes directly via TLRs

Pro-autophagic signal induction by bacterial pore-forming toxins

Pore-forming toxins (PFT) comprise a large, structurally heterogeneous group of bacterial protein toxins. Nucleated target cells mount complex responses which allow them to survive moderate membrane damage by PFT. Autophagy has recently been implicated in responses to various PFT, but how this process is triggered is not known, and the significance of the phenomenon is not understood. Here, we show that S. aureus α-toxin, Vibrio cholerae cytolysin, streptolysin O and E. coli haemolysin activate two pathways leading to autophagy. The first pathway is triggered via AMP-activated protein kinase (AMPK). AMPK is a major energy sensor which induces autophagy by inhibiting the target of rapamycin complex 1 (TORC1) in response to a drop of the cellular ATP/AMP-ratio, as is also observed in response to membrane perforation. The second pathway is activated by the conserved eIF2α-kinase GCN2, which causes global translational arrest and promotes autophagy in response to starvation. The latter could be accounted for by impaired amino acid transport into target cells. Notably, PKR, an eIF2α-kinase which has been implicated in autophagy induction during viral infection, was also activated upon membrane perforation, and evidence was obtained that phosphorylation of eIF2α is required for the accumulation of autophagosomes in α-toxin-treated cells. Treatment with 3-methyl-adenine inhibited autophagy and disrupted the ability of cells to recover from sublethal attack by S. aureus α-toxin. We propose that PFT induce pro-autophagic signals through membrane perforation–dependent nutrient and energy depletion, and that an important function of autophagy in this context is to maintain metabolic homoeostasis

Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs

Characterization of Profilin Polymorphism in Pollen with a Focus on Multifunctionality

Profilin, a multigene family involved in actin dynamics, is a multiple partners-interacting protein, as regard of the presence of at least of three binding domains encompassing actin, phosphoinositide lipids, and poly-L-proline interacting patches. In addition, pollen profilins are important allergens in several species like Olea europaea L. (Ole e 2), Betula pendula (Bet v 2), Phleum pratense (Phl p 12), Zea mays (Zea m 12) and Corylus avellana (Cor a 2). In spite of the biological and clinical importance of these molecules, variability in pollen profilin sequences has been poorly pointed out up until now. In this work, a relatively high number of pollen profilin sequences have been cloned, with the aim of carrying out an extensive characterization of their polymorphism among 24 olive cultivars and the above mentioned plant species. Our results indicate a high level of variability in the sequences analyzed. Quantitative intra-specific/varietal polymorphism was higher in comparison to inter-specific/cultivars comparisons. Multi-optional posttranslational modifications, e.g. phosphorylation sites, physicochemical properties, and partners-interacting functional residues have been shown to be affected by profilin polymorphism. As a result of this variability, profilins yielded a clear taxonomic separation between the five plant species. Profilin family multifunctionality might be inferred by natural variation through profilin isovariants generated among olive germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, affecting the mechanisms underlying the transmission of signals throughout signaling pathways in response to different stress environments. Moreover, elucidating the effect of profilin polymorphism in adaptive responses like actin dynamics, and cellular behavior, represents an exciting research goal for the future