Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Susceptibility of different populations of ticks to entomopathogenic fungi

This study aimed to evaluate the in vitro effect of the entomopathogenic fungi Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana sensu lato (s.l.) on two distinct populations of Rhipicephalus microplus, from two different experimental farms. Bioassays were performed with engorged females, eggs and larvae. Fungal infection was evaluated based on biological parameters of treated engorged females, percentage of hatch from treated eggs, and percentage of mortality and mean lethal time (LT90) of treated larvae. When the treatments were compared between the two populations, there were significant differences in the following parameters: pre-oviposition period, hatching period and egg production index. Moreover, the results showed that B. bassiana s.l., isolate Bb 986, was more virulent than M. anisopliae s.l., isolate 959, for engorged females, showing a control percentage of 49%. In the bioassay with eggs, the hatching percentage ranged from 3.1% to 49.5% in one population and from 3.4% to 42.7% in the other, with no significant difference between the two populations. In the bioassay with unfed larvae, the mortality percentage ranged from 91.8% to 98.7% in one population and from 71.0% to 94.0% in the other. The LT(90) varied from 19.52 to 27.51 days in one of the populations and 22.89 to 37.31 days in the other. These results suggest that populations of R. microplus show distinct variation in their susceptibility to B. bassiana s.l. and M. anisopliae s.l.     

Red bat fatality: Geographic extents through deuterium and niche models

Bat fatality at wind energy facilities is a conservation issue, but its effect on bat populations is difficult to estimate. We have little understanding of wind turbine effects on bat population persistence, in part because we have poor knowledge of bat migration pathways and hence the source populations for individual fatalities. We used deuterium ratio analysis combined with genetic algorithm for rule-set prediction and the web-based isoscapes modeling, analysis, and prediction in a geographic information system environment as a novel approach. Our objectives were to explore the utility of these methods together and map the geographic extents of eastern red bat (Lasiurus borealis) specimens salvaged in 2008–2010 from a single, 92-km² wind energy facility in Illinois, USA. Results indicate that combining these methods can be successful and support their use with species where ranges may be less well defined. Because of the migratory nature of this species and the range of deuterium values of pixels in our isotope model, we predicted that 18% and 82% of the specimens would have isotope results inside and outside of the wind facility's isocline respectively. We concluded that 71.4% of the specimens had isotope signatures placing them outside the wind facility's isocline. It could be argued that the wide distribution of bat fatalities dilutes the overall effect of those fatalities on the bat species; however, if other facilities show a similar pattern, each facility could have cumulative and far reaching population-level effects. © 2019 The Wildlife Society.     

Trypanosoma cruzi: cell type dependent distribution of a tubulin domain in mammalian stages

The distribution of tubulin domains in the mammalian stages of Trypanosoma cruzi was investigated by using monoclonal antibodies elicited against bovine brain tubulin. Western blotting performed on T. brucei trypomastigotes and T. cruzi epimastigotes showed that the monoclonal antibodies 16D3 and 24E3 reacted only with tubulin in these cell types. Indirect immunofluorescence revealed that, whereas 16D3 stained all microtubules, including subpellicular microtubules, the epitope defined by 24E3 was found in only a part of the tubulin pool of amastigotes and intermediate stages infecting murine fibroblasts and of broad trypomastigotes; the staining was limited to the basal bodies and the distal region of the flagellar adhesion zone in these developmental forms. By contrast, slender trypomastigotes did not exhibit any reaction with 24E3. These results are consistent with a transformation of broad trypomastigotes into slender trypomastigotes during which the tubulin domain recognized by 24E3 would undergo modifications leading to its complete masking in slender forms. The morphogenesis of the mammalian stages of T. cruzi would involve modifications of the tubulin molecule.     

Sequence determinants in the cathelicidin LL-37 that promote inflammation via presentation of RNA to scavenger receptors

Cathelicidins such as the human 37-amino acid peptide (LL-37) are peptides that not only potently kill microbes but also trigger inflammation by enabling immune recognition of endogenous nucleic acids. Here, a detailed structure–function analysis of LL-37 was performed to understand the details of this process. Alanine scanning of 34-amino acid peptide (LL-34) showed that some variants displayed increased antimicrobial activity against Staphylococcus aureus and group A Streptococcus. In contrast, different substitutions clustered on the hydrophobic face of the LL-34 alpha helix inhibited the ability of those variants to promote type 1 interferon expression in response to U1 RNA or to present U1 to the scavenger receptor (SR) B1 on the keratinocyte cell surface. Small-angle X-ray scattering experiments of the LL-34 variants LL-34, F5A, I24A, and L31A demonstrated that these peptides form cognate supramolecular structures with U1 characterized by inter-dsRNA spacings of approximately 3.5 nm, a range that has been previously shown to activate toll-like receptor 3 by the parent peptide LL-37. Therefore, while alanine substitutions on the hydrophobic face of LL-34 led to loss of binding to SRs and the complete loss of autoinflammatory responses in epithelial and endothelial cells, they did not inhibit the ability to organize with U1 RNA in solution to associate with toll-like receptor 3. These observations advance our understanding of how cathelicidin mediates the process of innate immune self-recognition to enable inert nucleic acids to trigger inflammation. We introduce the term “innate immune vetting” to describe the capacity of peptides such as LL-37 to enable certain nucleic acids to become an inflammatory stimulus through SR binding prior to cell internalization.     

The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.     

Helicobacter pylori virulence genes and host IL-1RN and IL-1beta genes interplay in favouring the development of peptic ulcer and intestinal metaplasia

Helicobacter pylori infection outcome might depend on genotypic polymorphisms of both the bacterium and the host. We ascertained: (1) the functionality of H. pylori oipA gene; (2) the polymorphism of the hostinterleukin (IL-1beta) gene (-31 C/T) and of the IL-1RN gene (intron 2 VNTR); (3) the association between the above genes and the histological and pathological outcome of H. pylori infection. One hundred and sixty-five H. pylori positive and 137 H. pylori negative subjects (23 gastric adenocarcinoma, 58 peptic ulcer, 221 gastritis) were studied. oipA was sequenced, IL-1beta was RFLP analysed. Antral and body mucosal biopsies were histologically evaluated. Functional oipA genes were correlated with cagA gene; both genes were significantly associated with gastritis activity, peptic ulcer and gastric adenocarcinoma. In these patients heterozygousIL-1RN 1/2 and IL-1beta C/T genotypes were more frequent than in gastritis patients. Intestinal metaplasia was associated with cagA, functional oipA and IL-1RN 2 allele. In conclusion, peptic ulcer and the preneoplastic intestinal metaplasia are associated with H. pylori virulence genes and with IL-1RN 2 host allele. An interplay between bacterial virulence factors and cytokines genotypes, is probably the main route causing H. pylori infection to lead to benign mild disease, benign severe disease or preneoplastic lesions.     

Volatile organic compounds: a potential direct long-distance mechanism for antagonistic action of Fusarium oxysporum strain MSA 35

Fusarium oxysporum MSA 35 [wild-type (WT) strain] is an antagonistic Fusarium that lives in association with a consortium of bacteria belonging to the genera Serratia, Achromobacter , Bacillus and Stenotrophomonas in an Italian soil suppressive to Fusarium wilt. Typing experiments and virulence tests provided evidence that the F. oxysporum isolate when cured of the bacterial symbionts [the cured (CU) form], is pathogenic, causing wilt symptoms identical to those caused by F. oxysporum f. sp. lactucae . Here, we demonstrate that small volatile organic compounds (VOCs) emitted from the WT strain negatively influence the mycelial growth of different formae speciales of F. oxysporum. Furthermore, these VOCs repress gene expression of two putative virulence genes in F. oxysporum lactucae strain Fuslat10, a fungus against which the WT strain MSA 35 has antagonistic activity. The VOC profile of the WT and CU fungus shows different compositions. Sesquiterpenes, mainly caryophyllene, were present in the headspace only of WT MSA 35. No sesquiterpenes were found in the volatiles of ectosymbiotic Serratia sp. strain DM1 and Achromobacte r sp. strain MM1. Bacterial volatiles had no effects on the growth of the different ff. spp. of F. oxysporum examined. Hyphae grown with VOC from WT F. oxysporum f. sp. lactucae strain MSA 35 were hydrophobic whereas those grown without VOCs were not, suggesting a correlation between the presence of volatiles in the atmosphere and the phenotype of the mycelium. This is the first report of VOC production by antagonistic F. oxysporum MSA 35 and their effects on pathogenic F. oxysporum. The results obtained in this work led us to propose a new potential direct long-distance mechanism for antagonism by F. oxysporum MSA 35 mediated by VOCs . Antagonism could be the consequence of both reduction of pathogen mycelial growth and inhibition of pathogen virulence gene expression.