A sea urchin in vivo model to evaluate Epithelial‐Mesenchymal Transition

Epithelial‐mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti‐EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti‐EMT candidates.     

Differential interleukin 1 elaboration by unfractionated and density fractionated human alveolar macrophages and blood monocytes: relationship to cell maturity

The elaboration of interleukin 1 (IL 1) by mononuclear phagocytes is important in the regulation of human inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we studied the elaboration of IL 1 by unfractionated and density-fractionated human alveolar macrophages and blood monocytes. Stimulated blood monocytes elaborated more IL 1 than stimulated alveolar macrophages. In addition, denser alveolar macrophages and blood monocytes elaborated more IL 1 than less dense alveolar macrophages and monocytes. Lastly, as monocytes matured in vitro, they lost their ability to elaborate IL 1 and became less dense. Thus, there is variability between and within mononuclear phagocyte cell populations in their ability to elaborate IL 1. These differences may result in part from differences in cell maturation.     

Direct probing of RNA structures and RNA-protein interactions in the HIV-1 packaging signal by chemical modification and electrospray ionization fourier transform mass spectrometry

RNA hairpins of the HIV-1 packaging signal and their complexes with the nucleocapsid protein p7 (NC) were probed by solvent-accessibility reagents and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The combination of dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMCT) offers the full range of information on base-pairing and solvent exposure concerning the four more abundant ribonucleotides. ESI-FTMS provides a universal method to achieve a direct and unambiguous characterization of alkylated structures, with no need for the different probe-specific procedures required by established methodologies based on gel electrophoresis. It enables us to streamline the optimization of the conditions for probe administration to minimize the incidence of probe-induced distortion of the structures under investigation. Nucleotides located in the single-stranded loops of hairpins SL2, SL3 and SL4 manifested different levels of protection, which were correlated directly to their conformation and structural surroundings. A common feature noted for all the hairpins was the limited susceptibility observed for the guanine base located at the 5'-end of each tetraloop, which assumes a stacked position upon the last base-pair of the double-stranded stems. The remaining loop bases were found to be clearly accessible by modifying reagents in free RNA, but were effectively protected in the NC-hairpin complexes. While this finding is consistent with the proven participation of SL2 and SL3 loops in interactions with NC, it contrasts with prior suggestions that tetraloop bases in SL4 might not be involved directly in NC binding. Alkylation was detected for stem nucleotides, which are not involved in the normal base-pairing and stacking typical of double-stranded structures, such as adenine 15 of the SL2 triple-base platform. Modification of the blunt ends of the double-stranded stems was found to be absent or extremely limited, due to the annealing stabilization introduced by the presence of G-C pairs at the end of the stems structures. Previously undetected alkylation of guanine 3 and guanine 13 in SL4 provides direct evidence of the destabilizing effects induced by the tandem G.U wobbles on the double-stranded structure of this stem, which is thought to be important for the hairpin's biological function.     

Type-1 (CB1) Cannabinoid Receptor Promotes Neuronal Differentiation and Maturation of Neural Stem Cells

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB₁ and CB₂ in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB₁ is present at higher levels than CB₂ in NSCs. The eCB anandamide (AEA) or the CB₁-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB₂-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB₁, but not CB₂, antagonist, corroborating the specificity of the response. CB₁ activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB₁ stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB₁ activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB₁ activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB₁ in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.     

Bioreactor production of 2,3-butanediol by Pantoea agglomerans using soybean hull acid hydrolysate as substrate

Production of 2,3-butanediol (2,3-BD) by Pantoea agglomerans strain BL1 was investigated using soybean hull hydrolysate as substrate in batch reactors. The cultivation media consisted of a mixture of xylose, arabinose, and glucose, obtained from the hemicellulosic fraction of the soybean hull biomass. We evaluated the influence of oxygen supply, pH control, and media supplementation on the growth kinetics of the microorganism and on 2,3-BD production. P. agglomerans BL1 was able to simultaneously metabolize all three monosaccharides present in the broth, with average conversions of 75% after 48 h of cultivation. The influence of aeration conditions employed demonstrated the mixed acid pathway of 2,3-BD formation by enterobacteria. Under fully aerated conditions (2 vvm of air), up to 14.02 g L⁻¹ of 2.3-BD in 12 h of cultivation were produced, corresponding to yields of 0.53 g g⁻¹ and a productivity of 1.17 g L⁻¹ h⁻¹, the best results achieved. These results suggest the production potential of 2,3-BD by P. agglomerans BL1, which has been recently isolated from an environmental consortium. The present work proposes a solution for the usage of the hemicellulosic fraction of agroindustry biomasses, carbohydrates whose utilization are not commonly addressed in bioprocess.

     

Candidatus Renichlamydia lutjani, a Gram-negative bacterium in internal organs of blue-striped snapper Lutjanus kasmira from Hawaii

The blue-striped snapper Lutjanus kasmira (Perciformes, Lutjanidae) are cosmopolitan in the Indo-Pacific but were introduced into Oahu, Hawaii, USA, in the 1950s and have since colonized most of the archipelago. Studies of microparasites in blue-striped snappers from Hawaii revealed chlamydia-like organisms (CLO) infecting the spleen and kidney, characterized by intracellular basophilic granular inclusions containing Gram-negative and Gimenez-positive bacteria similar in appearance to epitheliocysts when seen under light microscopy. We provide molecular evidence that CLO are a new member of Chlamydiae, i.e. Candidatus Renichlamydia lutjani, that represents the first reported case of chlamydial infection in organs other than the gill in fishes.     

Diffusione pollinica e caratteristiche immunologiche e broncoreattive di soggetti atopici in Pietra Ligure (Savona)

Riassunto Sono state rilevate durante un monitoraggio aerobiologico effettuato a Pietra Ligure (Savona) nel corso del 1987 le concentrazioni polliniche di 50 taxa ed è stata valutata l'influenza relativa dei fattori meteorologici. Le osservazioni palinologiche sono state rapportate alle concentrazioni sieriche delle IgE specifiche, alla reattività bronchiale specifica ed aspecifica valutate in 101 pazienti allergici (rinitici ed asmatici), sensibilizzati a Graminaceae ed Urticaceae (Parietaria) al fine di riconoscere correlazioni tra le caratteristiche aerobiologiche di questi allergeni edi meccanismi patogenetici che sostengono la reattività bronchiale.      

Assessment of the genotoxic potential of benzo(a)pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha)

The single-cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test were carried out with haemocytes of Zebra mussel (Dreissena polymorpha) specimens to evaluate the potential genotoxicity of benzo(a)pyrene (BaP) and pp'-dichlorodiphenyldichloroethylene (pp'-DDE, a metabolite of pp'-DDT). Mussels were exposed to three different concentrations (0.1 microg/L, 2 microg/L, 10 microg/L) of each chemical in water during 168 h (SCGE assay) and 96 h (MN test) of exposure under laboratory conditions. These levels correspond to nominal molar concentrations of 0.4 nM, 7.9 nM and 40 nM for BaP and 0.3 nM, 6.2 nM and 31 nM for pp'-DDE, respectively. Concurrently, the levels of toxicants were measured in soft tissues of the mussels by gas-chromatographic analyses, to evaluate their temporal trends and the dose/response relationships. Significant increases of the ratio between the comet length and the diameter of the comet head (LDR) and of micronucleus frequencies in comparison with baseline levels were observed not only for all concentrations of BaP, but also for pp'-DDE (except 0.3 nM). The concentration above which DNA damage starts to be significantly increased was 0.8 nmol/g lipids for BaP and 1.6 nmol/g lipids for pp'-DDE, respectively. The results of these experiments show a clear genotoxic effect on this non-target organism not only for the well-known genotoxicant BaP, but also for the final metabolite of pp'-DDT at soft-tissue concentrations that have been found in several aquatic ecosystems worldwide.