Reduction of excavation face collapse risk in tunnelling.

Two large road tunnels, recently developed near Florence, showed instabilities of the excavation face which subsequently caused sixteen collapses. Due to the risk for workers' safety, the public authority for occupational health and safety (ASL) has monitored the failure rate and other background variables in order to assess the possible correlations between risk reductions, its own actions, and those of the various safety actors involved. OBJECTIVES: To evaluate if the interventions carried out by the design team were able to reduce the risks of collapse and which of the ASL actions and/or which other factors were more effective in changing the attitudes of the parties involved, leading to a more expensive but safer project variant. RESULTS: After adoption of the second of two project variants, no more collapses were observed. No correlation was found between trend of ASL inspections and observed variation of collapse rate. Conversely, the adoption of strongly coercive measures and investigation reporting by local media coincided with periods of risk reduction, even if the low number of events does not allow for statistical evaluation. CONCLUSIONS: These findings appear to be coherent with the ratio of the cost of penalties related to health and safety infringements (thousands of euros) to the overall cost of the safer project variant (a hundred times greater).  The safer variant required 7% more labour but avoided forced interruptions caused by the collapses, allowing a 13% faster excavation rate.

A novel DAG-dependent mechanism links PKCɑ and Cyclin B1 regulating cell cycle progression.

Through the years, different studies showed the involvement of Protein Kinase C (PKC) in cell cycle control, in particular during G1/S transition. Little is known about their role at G2/M checkpoint. In this study, using K562 human erythroleukemia cell line, we found a novel and specific mechanism through which the conventional isoform PKCα positively affects Cyclin B1 modulating G2/M progression of cell cycle. Since the kinase activity of this PKC isoform was not necessary in this process, we demonstrated that PKCα, physically interacting with Cyclin B1, avoided its degradation and stimulated its nuclear import at mitosis. Moreover, the process resulted to be strictly connected with the increase in nuclear diacylglycerol levels (DAG) at G2/M checkpoint, due to the activity of nuclear Phospholipase C ß1 (PLCß1), the only PLC isoform mainly localized in the nucleus of K562 cells. Taken together, our findings indicated a novel DAG dependent mechanism able to regulate the G2/M progression of the cell cycle.

PLC-beta 1 regulates the expression of miR-210 during mithramycin-mediated erythroid differentiation in K562 cells.

PLC-beta 1 (PLCß1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCß1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCß1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCß1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCß1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCß1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effects were the same encountered in cells overexpressing PLCß1. Therefore we suggest a novel role for PLCß1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCß1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression.

K562 cell proliferation is modulated by PLCß1 through a PKCα-mediated pathway.

Phospholipase C ß1 (PLCß1) is known to play an important role in cell proliferation. Previous studies reported an involvement of PLCß1 in G 0-G 1/S transition and G 2/M progression in Friend murine erythroleukemia cells (FELC). However, little has been found about its role in human models. Here, we used K562 cell line as human homologous of FELC in order to investigate the possible key regulatory role of PLCß1 during cell proliferation of this human cell line. Our studies on the effects of the overexpression of both these isoforms showed a specific and positive connection between cyclin D3 and PLCß1 in K562 cells, which led to a prolonged S phase of the cell cycle and a delay in cell proliferation. In order to shed light on this mechanism, we decided to study the possible involvement of protein kinases C (PKC), known to be direct targets of PLC signaling and important regulators of cell proliferation. Our data showed a peculiar decrease of PKCα levels in cells overexpressing PLCß1. Moreover, when we silenced PKCα, by RNAi technique, in order to mimic the effects of PLCß1, we caused the same upregulation of cyclin D3 levels and the same decrease of cell proliferation found in PLCß1-overexpressing cells. The key features emerging from our studies in K562 cells is that PLCß1 targets cyclin D3, likely through a PKCα-mediated-pathway, and that, as a downstream effect of its activity, K562 cells undergo an accumulation in the S phase of the cell cycle.

The physiology and pathology of inositide signaling in the nucleus.

Nuclear inositide signaling is nowadays a well-established issue and a growing field of investigation, even though the very first evidence came out at the end of the 1980's. The understanding of its biological role is supported by the recent acquisitions dealing with pathology and namely hematological malignancies. Here, we review this issue highlighting the main achievements in the last years.

Hypoxia-induced down-modulation of PKCepsilon promotes trail-mediated apoptosis of tumor cells.

Tumor oxygen status is considered as a prognostic marker that impacts on malignant progression and outcome of tumor therapy. TNF-related apoptosis inducing ligand (TRAIL) plays a key role in cancer immunity, with potential applications in cancer therapy. Protein kinase C (PKC)epsilon, a transforming oncogene, has a role in the protection of cardiomyocytes and neurons from hypoxia-induced damage while, it can also modulate the susceptibility of tumor cells to TRAIL-induced cell death. Here we demonstrate that hypoxia induces a tumor cell phenotype highly sensitive to the cytotoxic effects of TRAIL. Based on the observation that: i) PKCepsilon expression levels are impaired during hypoxia, ii) the overexpression of PKCepsilon, but not of a kinase-inactive PKCepsilon mutant, is able to revert the hypoxia-induced sensitivity to TRAIL, iii) the down-modulation of PKCepsilon levels by RNA interference, on the contrary, induces the highly TRAIL-sensitive phenotype, iv) the inhibition of hypoxia-inducible transcription factor-1alpha (HIF-1alpha) by specific siRNA blocks both the hypoxia-induced down-modulation of PKCepsilon and the induction of the highly TRAIL-sensitive phenotype; we conclude that the HIF-1alpha upregulation during hypoxia is associated to PKCepsilon down-modulation that likely represents the key molecular event promoting the apoptogenic effects of TRAIL in hypoxic tumor cells.

A role for PKCepsilon during C2C12 myogenic differentiation.

In a previous report we have demonstrated that PLCgamma1 is involved in the differentiation process of C2C12 myoblasts, induced by insulin administration. In order to identify the downstream targets of PLCgamma1-dependent signalling, we have analyzed the expression of DAG-dependent PKC isoforms during muscle differentiation. We show that during myotube formation, there is a marked increase of PKCepsilon and eta expression, and that PKCepsilon is able to form a complex with PLCgamma1. The increase in PKCepsilon amount during myogenic differentiation is associated to an increase in PKCepsilon activity as well. Immunofluorescence analysis indicated that in growing C2C12 cells both PLCgamma1 and PKCepsilon localize in the cytoplasm with a distinct perinuclear accumulation. In insulin-treated cells, the expression of PLCgamma1 and PKCepsilon increases and the two proteins are still distributed mainly in the perinuclear region of the myotubes. We show that PLCgamma1-PKCepsilon complex co-localizes with protein 58K, a specific Golgi marker. Moreover, our results indicate that the Golgi-associated PKCepsilon form, i.e. PKCepsilon phosphorylated at Ser 729, is increased in differentiated myoblasts. Since it has been previously demonstrated that in C2C12 cells after insulin administration cyclin D3 levels could be modulated by PLCgamma1, we analyzed the effect on cyclin D3 expression of either PKCepsilon overexpression or silencing, in order to investigate whether PKCepsilon could also affect cyclin D3 expression. The results showed that either a modification of PKCepsilon expression or a change in its catalytic activity determines a variation of cyclin D3 levels and muscle differentiation in terms of myogenin expression. These data support a role for PKCepsilon in regulating insulin inositide-dependent PLCgamma1 signalling in skeletal muscle differentiation.

Efficient platelet delta-granule release induced by [Ca2+]i elevation is modulated by GPIIbIIIa.

Intracellular Ca2+ elevation generates a cascade of events that leads to platelet activation and degranulation. The GPIIbIIIa-ligand molecular complex plays a central role in several aspects of platelet activation. Taking advantage of the flow cytometric simultaneous analysis of surface GPIIbIIIa expression and intracellular serotonin content, we demonstrate here that the functional inhibition of GPIIbIIIa generates an impairment of delta-granule release even upon maximal intracellular Ca2+ elevation. In healthy subjects, the GPIIbIIIa inhibitor tirofiban impairs platelet delta-granule release. Analogously, Glanzmann thrombasthenia patients show an impairment of delta-granule release that is proportional to their residual expression of platelet GPIIbIIIa. These data show that platelet surface expression of functional GPIIbIIIa is required for a fully efficient secretion of delta-granules and serotonin release. The implications of our findings are discussed in the light of the complex interplay between vesicle release and ligand-receptor triggering during platelet activation.

Nuclear phospholipase C beta1 (PLCbeta1) affects CD24 expression in murine erythroleukemia cells.

Inositide-specific phospholipase C (PLC) beta1 is a key enzyme in nuclear lipid signal transduction affecting cell cycle progression and may be directly involved in regulation of gene expression and hematopoiesis. By microarrays, we compared the effect of nuclear PLCbeta1 overexpression with that of PLC M2b cytoplasmatic mutant, which is exclusively located in the cytoplasm, in murine erythroleukemia cells. Out of 9000 genes analyzed, the CD24 gene, coding for an antigen involved in differentiation and hematopoiesis as well, was up-regulated in cells overexpressing nuclear PLCbeta1 as compared with both cells overexpressing the M2b cytoplasmatic mutant and the wild type cells. Here we show that nuclear PLCbeta1 up-regulated the expression of CD24. The correlation was strengthened by the observation that when PLCbeta1 expression was silenced by means of small interfering RNA, CD24 expression was down-regulated. We also demonstrated that PLCbeta1-dependent up-modulation of CD24 was mediated, at least in part, at the transcriptional level, in that PLCbeta1 affected the CD24 promoter activity. Moreover, the up-regulation of CD24 was higher during erythroid differentiation of murine erythroleukemia cells. Altogether our findings, obtained by combining microarrays, phenotypic analysis, and small interfering RNA technology, identify CD24 as an molecular effector of nuclear PLCbeta1 signaling pathway in murine erythroleukemia cells and strengthen the contention that nuclear PLCbeta1 constitutes a key step in erythroid differentiation in vitro.

Expression of signal transduction proteins during the differentiation of primary human erythroblasts.

The high number (>10(8-10)) of primary human pro-erythroblasts (CD36high/CD235alow) obtainable in HEMA culture (Migliaccio et al., 2002) is exploited here to analyse the expression of proteins implicated in erythropoietin (EPO)-signalling (STATs, PI-3K, and PLCs) during the process of erythroid maturation. Human pro-erythroblasts progressed in 4 days of culture with EPO into basophilic- (CD36high/CD235amedium, 24 h), polychromatic-(CD36high/CD235ahigh, 48 h), and, finally, orthochromatic-(CD36low/CD235ahigh, 72-96 h) erythroblasts. During this maturation, STAT-1 was expressed up to the orthochromatic stage, expression of STAT-5, as well as of its target proteins BclxL and IRF1, remained constant up to 48 h (polychromatic-erythroblasts) but decreased by 96 h (orthochromatic-erythroblasts), while that of STAT-3 decreased constantly from 24 h on and became undetectable by 96 h. Expression of PI-3K rapidly decreased with differentiation since only 50% of original protein levels were detected by 48 h. On the other hand, among the members of PLC families investigated, PLC beta4 was not expressed, PLC beta2, delta1, and gamma2 were expressed at constant levels throughout the maturation process, while expression of PLC beta3 and of PLC gamma1 decreased, as PI-3K, by 24 h and that of PLC beta1 was induced by 6 h and became undetectable by 24 h. In conclusion, these data depict the dynamic signalling scenario associated with the maturation of erythroid cells and provide the first indication that members of PLC families (PLC beta1, beta3, and gamma1) might be involved in the control of erythroid differentiation in humans.

Phospholipase-C beta1 is predominantely expressed in the granular layer of rat cerebellar cortex.

The beta1 isoform of phospholipase-C is exclusively present in the nucleus of several hematopoietic and non-hematopoietic cell lines and primary cells of different species. When present, it represents the key enzyme for initiating the nuclear phospholipid breakdown that is involved in the cellular response to proliferating and differentiating stimuli. We have studied the expression of this enzyme isoform in the rat cerebellar cortex. We demonstrate that phospholipase-C beta1 (PLCbeta1) is predominantly expressed in the neurons of the granular layer, while it is virtually absent in the molecular and Purkinje cell layers of rat cerebellar cortex. This pattern of expression is partially different from that of the mouse cerebellar cortex, where not only granular cells, but also Purkinje cells express PLCbeta1. The high level of synaptic inputs that converge on granular cells may imply a constantly active nuclear phospholipid metabolism that may not be strictly required for the appropriate cellular responses of the other cell types of rat cerebellar cortex.

Membrane technologies applied to textile wastewater treatment.

This paper describes the experimental results of a pilot-scale application of membrane technologies to textile wastewater advanced treatment, downstream of a biological activated sludge process, aimed at water reuse in textile technology processes. The chosen approach consisted of sand filtration as a pretreatment, a microfiltration (MF) or ultrafiltration (UF) membrane process, and a final separation treatment performed by means of a nanofiltration (NF) or a reverse osmosis (RO) membrane. An experimental study to compare spiral wound membranes, operating under pressure, to flat membranes, operating under vacuum was conducted. The technical results and a preliminary economic analysis indicate the possibility of technological transfer of the membrane technologies to an industrial scale for textile wastewater reclamation.

Nuclear PLCbeta(1) acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells.

It is well established that phospholipase C (PLC) beta(1) plays a role in the nuclear compartment and is involved in the signalling pathway that controls the switching of the erythroleukemia cells programming from an undifferentiated to a differentiated state. Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend cells differentiation. For further characterization, we investigated the localization of PLCbeta(1)a and PLCbeta(1)b in Friend cells by fusing their cDNA to enhanced green fluorescent protein (GFP). To investigate the potential target of nuclear PLCbeta(1) in Friend differentiation, we studied the expression of p45/NF-E2 transcription factor, which is an enhancer binding protein for expression of the beta-globin gene and the expression of GATA proteins that are important for the survival and differentiation of erythroid cells. Our data suggest that the overexpression of PLCbeta(1) (both 1a and 1b) only in the nuclear compartment significantly reduces the expression of p45/NF-E2. The effect observed is attributable to the specific action of nuclear PLCbeta(1) signalling given that GATA-1 and GATA-3 are not affected at all. Here we show the existence of a unique target, i.e. the transcription factor p45/NF-E2, whose expression is specifically inhibited by the nuclear signalling evoked by PLCbeta(1) forms.