Importance of subaerial biofilms and airborne microflora in the deterioration of stonework: a molecular study

The study characterized the sessile microbial communities on mortar and stone in Milan University's Richini's Courtyard and investigated the relationship between airborne and surface-associated microbial communities. Active colonization was found in three locations: green and black patinas were present on mortar and black spots on stone. Confocal laser scanning microscopy, scanning electron microscopy and culture-independent molecular methods revealed that the biofilm causing deterioration was dominated by green algae and black fungi. The mortar used for restoration contained acrylic and siloxane resins that could be used by microorganisms as carbon and energy sources thereby causing proliferation of the biofilm. Epifluorescence microscopy and culture-based methods highlighted a variety of airborne microflora. Bacterial and fungal counts were quantitatively similar to those reported in other investigations of urban areas, the exception being fungi during summer (1–2 orders of magnitude higher). For the first time in the cultural heritage field, culture-independent molecular methods were used to resolve the structure of airborne communities near discoloured surfaces, and to investigate the relationship between such communities and surface-associated biofilms.     

Shift Work or Food Intake during the Rest Phase Promotes Metabolic Disruption and Desynchrony of Liver Genes in Male Rats

In the liver, clock genes are proposed to drive metabolic rhythms. These gene rhythms are driven by the suprachiasmatic nucleus (SCN) mainly by food intake and via autonomic and hormonal pathways. Forced activity during the normal rest phase, induces also food intake, thus neglecting the signals of the SCN, leading to conflicting time signals to target tissues of the SCN. The present study explored in a rodent model of night-work the influence of food during the normal sleep period on the synchrony of gene expression between clock genes and metabolic genes in the liver. Male Wistar rats were exposed to forced activity for 8 h either during the rest phase (day) or during the active phase (night) by using a slow rotating wheel. In this shift work model food intake shifts spontaneously to the forced activity period, therefore the influence of food alone without induced activity was tested in other groups of animals that were fed ad libitum, or fed during their rest or active phase. Rats forced to be active and/or eating during their rest phase, inverted their daily peak of Per1, Bmal1 and Clock and lost the rhythm of Per2 in the liver, moreover NAMPT and metabolic genes such as Pparα lost their rhythm and thus their synchrony with clock genes. We conclude that shift work or food intake in the rest phase leads to desynchronization within the liver, characterized by misaligned temporal patterns of clock genes and metabolic genes. This may be the cause of the development of the metabolic syndrome and obesity in individuals engaged in shift work.     

The ESR2 AluI 1730G>A (rs4986938) gene polymorphism is associated with fibrinogen plasma levels in postmenopausal women

The incidence of cardiovascular disease (CVD) and resultant morbidity and mortality are highly increased in postmenopausal women. Recent observations indicate the involvement of estrogen receptor beta in the pathogenesis of CVD, and the potential role of ESR2 gene polymorphisms as independent risk factors for CVD. We aimed to investigate the possible association between the ESR2 AluI 1730G>A gene polymorphism (rs4986938) with different CVD risk markers, such as body mass index (BMI), blood fibrinogen, glucose and insulin, homeostasis model assessment of insulin resistance and urinary F2-isoprostanes, in 89 postmenopausal women. Genotyping for ESR2 1730G>A polymorphism showed the higher prevalence of heterozygous GA1730 genotype than either wild-type GG1730 or homozygously mutated AA1730 genotype (50.6 vs 34.8 or 14.6%, respectively). Statistical analysis of between-group variability revealed that mean levels of the examined CVD risk markers, except BMI and fibrinogen, were within the normal range in all subjects grouped to different ESR2 1730G>A genotypes. Interestingly, only fibrinogen levels were statistically different in AA1730 carriers compared with other genotypes. The analysis of genotype relative risk showed a significant elevation of plasma fibrinogen in AA1730 carriers compared with GG + GA ones. The present data strongly indicate that genotyping for the ESR2 AluI 1730G>A gene variant should be included in a screening panel for assessment of cardiovascular risk in menopausal women.     

p38α Protein Negatively Regulates T Helper Type 2 Responses by Orchestrating Multiple T Cell Receptor-associated Signals

Mitogen-activated protein kinase p38α is a critical regulator of certain inflammatory diseases. However, its role in T helper type 2 (Th2) responses and allergic inflammation remains unknown. Here we show an increase in the production of interleukin-4 (IL-4) in p38α^−/− CD4⁺ T cells in response to antigen stimulation. p38α-deficient naïve CD4⁺ T cells preferentially differentiate into Th2 cells through increased endogenous production of IL-4. Consistent with those results, we also observed decreased expression of p38α during T helper cell differentiation. Furthermore, deficiency of p38α alters the balance in the expression of NFATc1 and NFATc2 under steady-state conditions and enhances the expression and nuclear translocation of NFATc1 in CD4⁺ T cells upon antigen stimulation. Knockdown of NFATc1 significantly inhibits Th2 differentiation in p38α^−/− T cells but not in p38α^+/− T cells. p38α deficiency also inhibits the activation of Akt but enhances the activation of ERK in response to T cell receptor engagement without impacting IL-2/Stat5 signaling. In a model of ovalbumin-induced acute allergic airway inflammation, mice with induced deletion of p38α show elevated serum ovalbumin-specific IgE level, increased infiltration of eosinophils, and higher concentrations of Th2 cytokines including IL-4 and IL-5 in the bronchoalveolar lavage fluid relative to control mice. Taken together, p38α regulates multiple T cell receptor-associated signals and negatively influences Th2 differentiation and allergic inflammation.     

Structure-guided design,synthesis and biological evaluation of novel DNA ligase inhibitors with in vitro and in vivo anti-staphylococcal activity

A series of 2-amino-[1,8]-naphthyridine-3-carboxamides (ANCs) with potent inhibition of bacterial NAD⁺-dependent DNA ligases (LigAs) evolved from a 2,4-diaminopteridine derivative discovered by HTS. The design was guided by several highly resolved X-ray structures of our inhibitors in complex with either Streptococcus pneumoniae or Escherichia coli LigA. The structure–activity-relationship based on the ANC scaffold is discussed. The in-depth characterization of 2-amino-6-bromo-7-(trifluoromethyl)-[1,8]-naphthyridine-3-carboxamide, which displayed promising in vitro (MIC Staphylococcus aureus 1 mg/L) and in vivo anti-staphylococcal activity, is presented.     

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.     

Ras GTPase-activating protein binds to Akt and is required for its activation

RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras. To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen. This resulted in discovering its interaction with Akt. Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase. Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway. To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression. Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation. Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473. The data suggest that this effect is independent of Ras activity.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

CXCR7 contributes to the aggressive phenotype of cholangiocarcinoma cells

Development of cholangiocarcinoma (CCA) is dependent on a cross-talk with stromal cells, which release different chemokines including CXCL12, that interacts with two different receptors, CXCR4 and CXCR7. The aim of the present study was to investigate the role of CXCR7 in CCA cells. CXCR7 is overexpressed by different CCA cell lines and in human CCA specimens. Knock-down of CXCR7 in HuCCT-1 cells reduced migration, invasion, and CXCL12-induced adhesion to collagen I. Survival of CCA was also reduced in CXCR7-silenced cells. The ability of CXCL12 to induce cell migration and survival was also blocked by CCX733, a CXCR7 antagonist. Similar effects of CXCR7 activation were observed in CCLP-1 cells and in primary iCCA cells. Enrichment of tumor stem-like cells by a 3D culture system resulted in increased CXCR7 expression compared to cells grown in monolayers, and genetic knockdown of CXCR7 robustly reduced sphere formation both in HuCCT-1 and in CCLP-1 cells. In HuCCT-1 cells CXCR7 was found to interact with β-arrestin 2, which was necessary to mediate CXCL12-induced migration, but not survival. In conclusion, CXCR7 is widely expressed in CCA, and contributes to the aggressive phenotype of CCA cells, inducing cell migration, invasion, adhesion, survival, growth and stem cell-like features. Cell migration induced by CXCR7 requires interaction with β-arrestin 2.