Proteomic Identification of VEGF-dependent Protein Enrichment to Membrane Caveolar-raft Microdomains in Endothelial Progenitor Cells

Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.On recruitment from bone marrow, endothelial progenitor cells (EPCs)¹ are involved in adult neovascularization, a process referred to as “postnatal vasculogenesis” (1, 2). In a way similar to mature endothelial cells (ECs), EPCs form endothelial colonies in vitro, migrate and differentiate into tubular-like structures, showing at the same time a high proliferation potential, which is uncommon for mature ECs. For this reason, EPCs have been intensively investigated as a source of cells potentially able to produce a more efficient revascularization with respect to mature ECs.We have previously shown (3) that vascular endothelial growth factor (VEGF)-dependent angiogenesis by endothelial colony forming cells (ECFCs), a particular subset which recapitulates all the characteristics of EPCs coupled with a particularly high proliferation potential (4, 5), requires localization of the full-length form of the urokinase plasminogen activator receptor (uPAR) in caveolar-rafts. uPAR is critical in angiogenesis because it is involved both in urokinase plasminogen activator (uPA)-mediated ECM degradation and uPAR-dependent cell adhesion (6). VEGF stimulates a caveolar-raft redistribution of uPAR coupled with inhibition of EPC production of matrix-metalloproteinase-12 (MMP12) (3), the main enzyme responsible for cleavage of uPAR between its domain 1 and 2. Such a cleavage deprives uPAR of its angiogenesic properties by elimination of its domain-1-uPA-binding ability and by loss of uPAR integrity, which is required in order for uPAR to retain its domain-2/3 affinity for vitronectin (VN) and EC membrane integrins (7). In ECFCs uPAR, a typical glycosyl-phosphatidyl-inositol (GPI)-anchored protein, preferentially partitions on caveolar-rafts. Lipid-rafts are dynamic microdomains of the cell membrane, rich in cholesterol, sphingolipids and glycolipids, trans-membrane protein receptors, integrins and a large number of signaling molecules (8).Caveolar-raft microdomains (also referred to as caveolae) are functionally and morphologically distinct forms of lipid-raft microdomains characterized by the presence of the protein caveolin-1. They are particularly abundant in ECs, playing a fundamental role in their function (9). VEGF localizes in caveolar-raft microdomains on interaction with its type-2 receptor (VEGFR2), thus providing a “functional platform,” together with other signaling molecules, involved in angiogenesis (10, 11). Thus, the overall emerging picture clearly indicates that the angiogenesis activity of VEGF involves a coordinated sequence of events taking place on caveolar-raft microdomains.To identify candidate proteins whose occurrence in ECFC caveolar-raft microdomains was modified by VEGF, we performed a proteomic analysis coupled with mass spectrophotometry of caveolar-rafts, before and after VEGF stimulation. We identified 28 protein spots, including several proteins endowed with anti-apoptotic functions and involved in stress response. Our observations offer new insights into how changes in protein caveolar-raft microdomain organization may affect prosurvival pathways, giving further support to the concept of caveolar-rafts as “floating islands of death or survival” (12).     

Parsimony analysis of endemicity and analysis of endemicity: A fair comparison

Parsimony Analysis of Endemicity (PAE) has been compared with other methods regarding its performance to identify areas of endemism. It is frequently compared with the Analysis of Endemicity (AE), which seems to perform better than PAE to identify these areas. Here I compare PAE and AE considering the sympatric taxa diagnosed as endemic, being as strictly close as possible to sympatry, and using previously published data of Sciobius (Coleoptera: Curculionidae). AE identified more candidate areas of endemism than PAE, but the number of highly restricted endemic taxa to these areas was insufficient to support them as areas of endemism. Considering strictly sympatry (homopatry), PAE performed better than AE; however, both methods may identify areas with some grade of sympatry, but the recognition of which areas constitute real areas of endemism in the strict sense depends on the interpretation of the researcher.     

Neoadjuvant oral vs. infusional chemoradiotherapy on locally advanced rectal cancer: Prognostic factors

Aim

To evaluate the prognostic factors and impact on survival of neoadjuvant oral and infusional chemoradiotherapy in patients with locally advanced rectal cancer.

Background

There is still no definitive consensus about the prognostic factors and the impact of neoadjuvant chemoradiotherapy on survival. Some studies have pointed to an improvement in overall survival (OS) and progression-free survival (PFS) in patients with tumor downstaging (TD) and nodal downstaging (ND).

Materials and methods

A set of 159 patients with LARC were treated preoperatively. Group A – 112 patients underwent concomitant oral chemoradiotherapy: capecitabine or UFT + folinic acid. Group B – 47 patients submitted to concomitant chemoradiation with 5-FU in continuous infusion. 63.6% of patients were submitted to adjuvant chemotherapy.

Results

Group A: pathologic complete response (pCR) – 18.7%; TD – 55.1%; ND – 76%; loco-regional response – 74.8%. Group B: pCR – 11.4%; TD – 50%; ND – 55.8%; LRR – 54.5%. The loco-regional control was 95.6%. There was no difference in survival between both groups. Those with loco-regional response had better PFS.

Conclusions

Tumor and nodal downstaging, loco-regional response and a normal CEA level turned out to be important prognostic factors in locally advanced rectal cancer. Nodal downstaging and loco-regional response were higher in Group A. Those with tumor downstaging and loco-regional response from Group A had better OS. Adjuvant chemotherapy had no impact on survival except in those patients with loco-regional response who achieved a higher PFS.     

Harnessing autophagy for cell fate control gene therapy

We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.     

11α‐Ethoxy‐β‐boswellic Acid and Nizwanone,a New Boswellic Acid Derivative and a New Triterpene,Respectively, from Boswellia sacra

A new boswellic acid derivative, 11α‐ethoxy‐β‐boswellic acid (EBA; 1 ) and a new ursane‐type triterpene, named nizwanone ( 2 ), were isolated from Omani frankincense Boswellia sacra Flueck . together with two known compounds papyriogenin B and rigidenol. The structures of 1 and 2 were elucidated by detailed spectroscopic analysis using ¹H‐ and ¹³C‐NMR, ¹H,¹H‐COSY, HMQC, HMBC, and HR‐EI‐MS techniques. The relative configurations of 1 and 2 were assigned by comparative analysis of the NMR spectral data with those of known analogs together with NOESY experiments. Structures of known compounds were identified by comparison with the reported data.     

Cytogenetic and Molecular Analyses Reveal a Divergence between Acromyrmex striatus (Roger, 1863) and Other Congeneric Species: Taxonomic Implications

The leafcutter ants, which consist of Acromyrmex and Atta genera, are restricted to the New World and they are considered the main herbivores in the neotropics. Cytogenetic studies of leafcutter ants are available for five species of Atta and 14 species of Acromyrmex, both including subspecies. These two ant genera have a constant karyotype with a diploid number of 22 and 38 chromosomes, respectively. The most distinct Acromyrmex species from Brazil is A. striatus, which is restricted to the southern states of Santa Catarina and Rio Grande do Sul. Several cytogenetic and phylogenetic studies have been conducted with ants, but the karyotypic characterization and phylogenetic position of this species relative to leafcutter ants remains unknown. In this study, we report a diploid number of 22 chromosomes for A. striatus. The phylogenetic relationship between A. striatus and other leafcutter ants was estimated based on the four nuclear genes. A. striatus shared the same chromosome number as Atta species and the majority of metacentric chromosomes. Nuclear data generated a phylogenetic tree with a well-supported cluster, where A. striatus formed a different clade from other Acromyrmex spp. This combination of cytogenetic and molecular approaches provided interesting insights into the phylogenetic position of A. striatus among the leafcutter ants and the tribe Attini.     

Snapshot of Viral Infections in Wild Carnivores Reveals Ubiquity of Parvovirus and Susceptibility of Egyptian Mongoose to Feline Panleukopenia Virus

The exposure of wild carnivores to viral pathogens, with emphasis on parvovirus (CPV/FPLV), was assessed based on the molecular screening of tissue samples from 128 hunted or accidentally road-killed animals collected in Portugal from 2008 to 2011, including Egyptian mongoose (Herpestes ichneumon, n = 99), red fox (Vulpes vulpes, n = 19), stone marten (Martes foina, n = 3), common genet (Genetta genetta, n = 3) and Eurasian badger (Meles meles, n = 4). A high prevalence of parvovirus DNA (63%) was detected among all surveyed species, particularly in mongooses (58%) and red foxes (79%), along with the presence of CPV/FPLV circulating antibodies that were identified in 90% of a subset of parvovirus-DNA positive samples. Most specimens were extensively autolysed, restricting macro and microscopic investigations for lesion evaluation. Whenever possible to examine, signs of active disease were not present, supporting the hypothesis that the parvovirus vp2 gene fragments detected by real-time PCR possibly correspond to viral DNA reminiscent from previous infections. The molecular characterization of viruses, based on the analysis of the complete or partial sequence of the vp2 gene, allowed typifying three viral strains of mongoose and four red fox’s as feline panleukopenia virus (FPLV) and one stone marten’s as newCPV-2b type. The genetic similarity found between the FPLV viruses from free-ranging and captive wild species originated in Portugal and publicly available comparable sequences, suggests a closer genetic relatedness among FPLV circulating in Portugal.Although the clinical and epidemiological significance of infection could not be established, this study evidences that exposure of sympatric wild carnivores to parvovirus is common and geographically widespread, potentially carrying a risk to susceptible populations at the wildlife-domestic interface and to threatened species, such as the wildcat (Felis silvestris) and the critically endangered Iberian lynx (Lynx pardinus).     

Invasion Is a Community Affair: Clandestine Followers in the Bacterial Community Associated to Green Algae,Caulerpa racemosa,Track the Invasion Source

Biological invasions rank amongst the most deleterious components of global change inducing alterations from genes to ecosystems. The genetic characteristics of introduced pools of individuals greatly influence the capacity of introduced species to establish and expand. The recently demonstrated heritability of microbial communities associated to individual genotypes of primary producers makes them a potentially essential element of the evolution and adaptability of their hosts. Here, we characterized the bacterial communities associated to native and non-native populations of the marine green macroalga Caulerpa racemosa through pyrosequencing, and explored their potential role on the strikingly invasive trajectory of their host in the Mediterranean. The similarity of endophytic bacterial communities from the native Australian range and several Mediterranean locations confirmed the origin of invasion and revealed distinct communities associated to a second Mediterranean variety of C . racemosa long reported in the Mediterranean. Comparative analysis of these two groups demonstrated the stability of the composition of bacterial communities through the successive steps of introduction and invasion and suggested the vertical transmission of some major bacterial OTUs. Indirect inferences on the taxonomic identity and associated metabolism of bacterial lineages showed a striking consistency with sediment upheaval conditions associated to the expansion of their invasive host and to the decline of native species. These results demonstrate that bacterial communities can be an effective tracer of the origin of invasion and support their potential role in their eukaryotic host’s adaptation to new environments. They put forward the critical need to consider the ''meta-organism'' encompassing both the host and associated micro-organisms, to unravel the origins, causes and mechanisms underlying biological invasions.