New type of vulnerability curve gives insight in the hydraulic capacitance and conductivity of the xylem

Drought vulnerability of trees and other woody plants is much debated in the context of climate change, which creates a high interest in understanding plant water relations. The role and functioning of internal water storage is crucial, but still insufficiently understood. Drought vulnerability is typically assessed by considering loss in conductivity in function of decreasing xylem water potential, in a so-called ‘vulnerability curve’. The xylem water potential at which a certain percentage of conductivity is lost (usually 50%) gives an indication of the vulnerability to cavitation. In a ‘desorption curve’, we can examine the release of water from internal storage tissues with decreasing water potential. Both curves are very valuable, but rely on a sequence of manual measurements (xylem water potential, hydraulic conductivity and water content) and are time-consuming. Therefore, we propose a new type of vulnerability curve that is based on continuous measurements of diameter shrinkage and ultrasonic acoustic emissions (UAE). We monitored weight loss, xylem diameter shrinkage and UAE and measured xylem water potential during the dehydration of excised branches of Vitis vinifera L. ‘Johanniter’. The vulnerability curves could be interpreted in terms of water loss in elastic and inelastic tissues. The proposed method can be a tool to assess hydraulic capacitance and conductivity of the xylem

Modulation of the unfolded protein response by tauroursodeoxycholic acid counteracts apoptotic cell death and fibrosis in a mouse model for secondary biliary liver fibrosis

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death

Dissection of the Human Multipotent Adult Progenitor Cell (MAPC) Secretome by Proteomic Analysis

Digitalitzat per Artypla

Effect of prolyl hydroxylase domain 2 haplodeficiency on liver progenitor cell characteristics in early mouse hepatocarcinogenesis

Activation of the hypoxia-inducible factor (HIF)-pathway in hepatocellular carcinoma (HCC) induces therapy resistant tumours, characterized by increased liver progenitor cell (LPCs) characteristics and poor prognosis. We previously reported corresponding results in mice with HCC in which hypoxia was mimicked by prolyl hydroxylase domain (PHD) inhibition. Here, we aimed at investigating whether induction of LPC characteristics occurs during the onset of hepatocarcinogenesis and if this is associated with activation of Notch signalling. Dietheylnitrosamine (DEN) was used to induce hepatic tumours in PHD2 haplodeficient (PHD2(+/-)) mice which were euthanized at 5, 10, 15 and 17 weeks following DEN during neoplastic transformation, before tumour formation. Neoplasia and mRNA expression of LPC and Notch markers were evaluated by histology and qPCR on isolated livers. PHD2 haplodeficiency resulted in enhanced expression of HIF target genes after 17 weeks of DEN compared to wild type (WT) littermates but had no effect on the onset of neoplastic transformation. The mRNA expression of Afp and Epcam was increased at all time points following DEN whereas CK19, Prom1 and Notch3 were increased after 17 weeks of DEN, without difference between PHD2(+/-) and WT mice. MDR1 mRNA expression was increased in all DEN treated mice compared to saline control with increased expression in PHD2(+/-) compared to WT from 15 weeks. These results indicate that the effects of PHD2 haplodeficiency on the expression of LPC and Notch markers manifest during tumour nodule formation and not early on during neoplastic transformatio

Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators

Placental growth factor inhibition modulates the interplay between hypoxia and unfolded protein response in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses. Methods: PlGF knockout mice and validated monoclonal anti-PlGF antibodies were used in a diethylnitrosamine-induced mouse model for HCC. We examined the interactions among hypoxia, UPR activation and PlGF induction in HCC cells. Results: Both the genetic and pharmacological inhibitions of PlGF reduced the chaperone levels and the activation of the PKR-like endoplasmic reticulum kinase (PERK) pathway of the UPR in diethylnitrosamine-induced HCC. Furthermore, we identified that tumour hypoxia was attenuated, as shown by reduced pimonidazole binding. Interestingly, hypoxic exposure markedly activated the PERK pathway in HCC cells in vitro, suggesting that PlGF inhibition may diminish PERK activation by improving oxygen delivery. We also found that PlGF expression is upregulated by different chemical UPR inducers via activation of the inositol-requiring enzyme 1 pathway in HCC cells. Conclusions: PlGF inhibition attenuates PERK activation, likely by tempering hypoxia in HCC via vessel normalisation. The UPR, in turn, is able to regulate PlGF expression, suggesting the existence of a feedback mechanism for hypoxia-mediated UPR that promotes the expression of the angiogenic factor PlGF. These findings have important implications for our understanding of the effect of therapies normalising tumour vasculature

Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure

Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2a (eIf2a) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-kappa B inhibitor I kappa Ba. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

A proteomic analysis of the immune response of Caenorhabditis elegans.

De toenemende resistentie van talrijke pathogene micro-organismen tegen een breed spectrum aan antibiotica heeft de zoektocht naar alternatieve antimicrobiële middelen geopend. Gedurende het voorbije decennium is de interesse in het gebruik van modelorganismen in immuniteitsonderzoek opm erkelijk gestegen. Omwille van zijn gesequeneerd genoom, de toegankelijk heid voor genetische analyses en de eenvoudige kweek is de vrijlevende bodemnematode C. elegans dan ook uitg egroeid tot één van de meest succesvolle modellen voor experimentee l onderzoek. In zijn natuurlijke habitat wordt deze kleine worm continu omgeven door micro-organismen, die tegelijkertijd een noodzakelijk voedi ngsbron maar onvermijdelijk ook een bron van pathogenen vormen. Aangezie n de worm geen adaptief immuunsysteem bezit, is hij volledig afhankelijk van zijn aangeboren immuniteit om deze uitdagende levenswijze heelhuids te doorstaan. Daarom vertegenwoordigt C. elegans het model bij uitstek o m meer inzicht te verwerven in het mechanisme van aangeboren immuniteit, dat sterk geconserveerd is zowel bij vertebraten als invertebraten. Dit mechanisme bestaat uit receptoren die de potentiële pathogenen detecter en, een complex netwerk van immuun-signaalwegen en effectormoleculen die de microben uiteindelijk vernietigen. Bij de aanvang van mijn doctoraat was de kennis over het immuunsysteem v an C. elegans voornamelijk gebaseerd op genetische studies. Toch waren e r nog heel wat onduidelijkheden over de mechanismen die het immuunsystee m reguleren. Er was nagenoeg niets geweten over de veranderingen die opt reden ter hoogte van het proteïne-niveau na infectie. Een proteomisch on derzoek kan ons waardevolle informatie opleveren en proteïnen naar voor schuiven die een potentieel belangrijke functie vervullen in de verdedig ingsstrategieën van de worm. Deze aanpak vormt dan ook de basis van mijn proefschrift, meer bepaald het bestuderen van gastheer-pathogeen intera cties met behulp van twee dimensionele differentiële gel elektroforese ( 2D-DIGE). Eerst heb ik de juiste condities voor het infecteren van worme n vastgesteld (waarbij de overleving van wormen op verschillende media, bezaaid met verschillende pathogenen getest werd) en een proteomics prot ocol voor wormstalen geoptimaliseerd. Daarna heb ik drie onafhankelijke DIGE experimenten uitgevoerd van stalen genomen op 1, 3 en 5 dagen na in fectie met de Gram-negatieve bacterie Aeromonas hydrophila. Vervolgens h eb ik 4 onafhankelijke DIGE experimenten uitgevoerd van stalen genomen o p 1, 4, 8 en 24 uur na infectie met de Gram-positieve bacterie Staphyloc occus aureus. Deze 7 DIGE experimenten resulteerden in een uitgebreide l ijst van meer dan 150 proteïnen die een gewijzigd expressiepatroon verto nen na bacteriële infectie. De studie bevestigt de betrokkenheid van gal ectines, C-type lectines en lipiden-bindende proteïnen in het immuunsysteem van C. elegans. Daarnaast werden ook een aantal ongekende proteïnen geïdentificeerd die voordien nog niet met immuniteit geassocieerd waren. Verder hebben we de voorkeur van C. elegans voor de verschillende experi mentele bacteriën (A. hydrophila, S. aureus en E. coli OP50) onderz ocht. Hieruit konden we besluiten dat de worm niet louter een niet-patho gene bacterie verkiest boven een pathogeen maar dat wellicht ook de voed ingswaarde van de bacteriën een rol speelt. Onze hypothese is dat een vo ortdurende kosten-baten analyse noodzakelijk is opdat de worm de juiste keuze kan maken tussen enerzijds een niet-pathogene bacterie met een lag e voedingswaarde en anderzijds een licht-pathogene bacterie met een hoge voedingswaarde. Met dit proefschrift hebben we voor de eerste maal getracht de complexe veranderingen die optreden in de worm na infectie op proteïne-niveau te bestuderen. De studie heeft geleid tot een lijst van potentieel interess ante kandidaat immuun proteïnen die nu verder onderzocht kunnen worden om hun functioneel belang in C. elegans immuniteit ontcijferen.nrpages: 138status: publishe

Antimicrobial peptides in Caenorhabditis elegans

The nematode Caenorhabditis elegans is one of the most successful model species for experimental research because of its sequenced genome, the versatile genetic toolkit and the straightforward breeding among others. In natural conditions however, this tiny worm is constantly surrounded by micro-organisms, simultaneously a source of indispensable nutrition and inevitable pathogens. Lacking an adaptive immune system, the worm solely relies on its innate immune defence to cope with its challenging life style. Hence C. elegans is an excellent model to gain more insight in innate immunity, which is remarkably preserved between invertebrate and vertebrate animals. The innate defence consists of receptors to detect potential pathogens, a complex network of signalling pathways and last but not least, effector molecules to abolish harmful microbes. In this review, we focus on the antimicrobial peptides, a vital subgroup of effector molecules. We summarise the current knowledge of the different families of C. elegans antimicrobial peptides, comprising NLPs, caenacins, ABFs, caenopores, and a recently discovered group with antifungal activity among which thaumatin-like proteins.status: publishe