Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes

BACKGROUND: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. RESULTS: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. CONCLUSIONS: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function

A Ce: LiCAF UV laser pumped by an intracavity frequency-doubled radiation at 532 nm

We examine here the lasing conditions of a Ce: LiCAF laser crystal placed intracavity with a BBO nonlinear crystal and pumped longitudinally throughout an input dichroic mirror by the 532 nm radiation of a frequency-doubled diode-pumped Nd: YAG laser. The comparison with the results obtained with an off-axis configuration shows lower laser slope efficiencies but similar laser performance in terms of threshold absorbed pump fluences (around 200 mJ/cm 2). A model based on revisited spectroscopic parameters is developed to account for these laser performance

Sphingolipids: towards an integrated view of metabolism during the plant stress response

Plants exist in an environment of changing abiotic and biotic stresses. They have developed a complex set of strategies to respond to these stresses and over recent years it has become clear that sphingolipids are a key player in these responses. Sphingolipids are not universally present in all three domains of life. Many bacteria and archaea do not produce sphingolipids but they are ubiquitous in eukaryotes and have been intensively studied in yeast and mammals. During the last decade there has been a steadily increasing interest in plant sphingolipids. Plant sphingolipids exhibit structural differences when compared to their mammalian counterparts and it is now clear that they perform some unique functions. Sphingolipids are recognized as critical components of the plant plasma membrane and endomembrane system. Besides being important structural elements of plant membranes, their particular structure contributes to the fluidity and biophysical order. Sphingolipids are also involved in multiple cellular and regulatory processes including vesicle trafficking, plant development and defense. This review will focus on our current knowledge as to the function of sphingolipids during plant stress responses, not only as structural components of biological membranes, but also as signaling mediators

Pancreatic cancer intrinsic PI3Kα activity accelerates metastasis and rewires macrophage component.

Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP3 , with a selective decrease of C36:2 PI-3,4,5-P3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

The virus family Arenaviridae includes several hemorrhagic fever causing agents such as Lassa, Guanarito, Junin, Machupo, and Sabia virus that pose a major public health concern to the human population in West African and South American countries. Current treatment options to control fatal outcome of disease are limited to the ribonucleoside analogue ribavirin, although its use has some significant limitations. The lack of effective treatment alternatives emphasizes the need for novel antiviral therapeutics to counteract these life-threatening infections. Maturation cleavage of the viral envelope glycoprotein by the host cell proprotein convertase site 1 protease (S1P) is critical for infectious virion production of several pathogenic arenaviruses. This finding makes this protease an attractive target for the development of novel anti-arenaviral therapeutics. We demonstrate here that highly selective S1P-adapted α1-antitrypsins have the potential to efficiently inhibit glycoprotein processing, which resulted in reduced Lassa virus replication. Our findings suggest that S1P should be considered as an antiviral target and that further optimization of modified α1-antitrypsins could lead to potent and specific S1P inhibitors with the potential for treatment of certain viral hemorrhagic fevers

Somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium

Angiogenesis is characterised by activation, migration and proliferation of endothelial cells and is central to the pathology of cancer, cardiovascular disease and chronic inflammation. Somatostatin is an inhibitory polypeptide that acts through five receptors (sst 1, 2, 3, 4, 5). Sst has previously been reported in endothelium, but their role remains obscure. Here, we report the expression of sst in human umbilical vein endothelial cells (HUVECs) in vitro, during proliferation and quiescence. A protocol for culturing proliferating and quiescent HUVECs was established, and verified by analysing cell cycle distribution in propidium-iodide-stained samples using flow cytometry. Sst mRNA was then quantified in nine proliferating and quiescent HUVEC lines using quantitative reverse transcriptase–polymerase chain reaction. Sst 2 and 5 were preferentially expressed in proliferating HUVECs. All samples were negative for sst 4. Sst 1 and 3 expression and cell cycle progression were unrelated. Immunostaining for sst 2 and 5 showed positivity in proliferating but not quiescent cells, confirming sst 2 and 5 protein expression. Inhibition of proliferating cells with somatostatin analogues Octreotide and SOM230, which have sst 5 activity, was found (Octreotide 10−10–10−6 M: 48.5–70.2% inhibition; SOM230 10−9–10−6 M: 44.9–65.4% inhibition) in a dose-dependent manner, suggesting that sst 5 may have functional activity in proliferation. Dynamic changes in sst 2 and 5 expression during the cell cycle and the inhibition of proliferation with specific analogues suggest that these receptors may have a role in angiogenesis

Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells

Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets

Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing

α1Proteinase Inhibitor Regulates CD4+ Lymphocyte Levels and Is Rate Limiting in HIV-1 Disease

Background: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE CS), it acts not as a proteinase, but as a receptor for a 1proteinase inhibitor (a 1PI, a 1antitrypsin, SerpinA1). Binding of a1PI to HLECS forms a motogenic complex. We previously demonstrated that a1PI deficiency attends HIV-1 disease and that a1PI augmentation produces increased numbers of immunocompetent circulating CD4 + lymphocytes. Herein we investigated the mechanism underlying the a 1PI deficiency that attends HIV-1 infection. Methods and Findings: Active a 1PI in HIV-1 subjects (median 17 mM, n = 35) was significantly below normal (median 36 mM, p,0.001, n = 30). In HIV-1 uninfected subjects, CD4 + lymphocytes were correlated with the combined factors a1PI, HLECS + lymphocytes, and CXCR4 + lymphocytes (r 2 = 0.91, p,0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with.220 CD4 cells/ml, CD4 + lymphocytes were correlated solely with active a 1PI (r 2 =0.93,p,0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human a 1PI. Chimpanzee a 1PI differs from human a1PI by a single amino acid within the 3F5-binding epitope. Unlike human a1PI, chimpanzee a1PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4 + lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-a 1PI immune complexes correlated with decreased CD4 + lymphocytes in HIV-1 subjects

CRISPR/Cas9: transcending the reality of genome editing

With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics