Lung Cancer: Are we up to the Challenge?

Lung cancer is the leading cause of cancer deaths worldwide among both men and women, with more than 1 million deaths annually. Non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers.Although recent advances have been made in diagnosis and treatment strategies, the prognosis of NSCLC patients is poor and it is basically due to a lack of early diagnostic tools.However, in the last years genetic and biochemical studies have provided more information about the protein and gene's mutations involved in lung tumors. Additionally, recent proteomic and microRNA's approaches have been introduced to help biomarker discovery.Here we would like to discuss the most recent discoveries in lung cancer pathways, focusing on the genetic and epigenetic factors that play a crucial role in malignant cell proliferation, and how they could be helpful in diagnosis and targeted therapy.     

Carbon exchange and water use efficiency of a growing, irrigated olive orchard

We measured eddy covariance fluxes of CO₂ and H₂O over a flat irrigated olive orchard during growth, in different periods from Leaf Area Index (LAI) of 0.3–1.9; measurements of soil respiration were also collected. The daily net ecosystem exchange flux (F_NEE) was practically zero at LAI around 0.4 or when the orchard intercepted 11% of the incoming daily radiation; at the end of the experiment, with LAI of 1.9 (and the fraction of intercepted daily radiation close to 0.5), F_NEE was around 10 g CO₂ m⁻² day⁻¹. The night-time ecosystem respiration (R_eco), calculated from eddy fluxes in well-mixed night conditions, show a clear but non-linear dependence with LAI; it ranged from 0.05 to 0.15 mg CO₂ m⁻² s⁻¹ (in average), being the lower limit ideally close to the heterotrophic soil respiration at the site. The gross primary production flux (F_GPP) was linearly related to LAI within the LAI range of this experiment (with 11 g CO₂ m⁻² day⁻¹ increments per unit of LAI) and to the fraction of intercepted radiation. The maximum rates of F_GPP (0.75 mg CO₂ m⁻² s⁻¹) were obtained in the summer mornings of 2002, at LAI close to 1.9. F_GPP was strongly modulated by vapour pressure deficit (VPD) through the canopy conductance, even in absence of water stress. Hence, especially in the summer, the maximum rates of carbon assimilation are reached always before noon. The daily course of F_GPP shows a two-phase pattern, first related to irradiance and then to canopy conductance. The water use efficiency (WUE) was, in average, 3.8, 6.3 and 7 g CO₂ L⁻¹ in 1999, 2001 and 2002, respectively, with maxima always in the early morning. Hourly WUE was strongly related to VPD (WUE = −10.25 + 22.52 × VPD^−0.34). Our results suggest that drip irrigated orchards in general, and olive in particular, deserve specific carbon exchange and carbon budget studies and cannot be easily included in other biomes.     

Firing rates of motor units in human vastus lateralis muscle during fatiguing isometric contractions.

We investigated the firing rate of motor units in the vastus lateralis muscle in five healthy young men (mean = 21.4 yr, SD = 0.9) during a sequence of isometric constant-torque contractions repeated to exhaustion. The contractions were sustained at 20% of the maximal voluntary level, measured at the beginning of the test sequence. Electromyographic (EMG) signals were recorded via quadrifilar fine-wire electrodes and subsequently decomposed into their constituent motor unit action potentials to obtain the motor unit firing times. In addition, we measured the whole muscle mechanical properties during the fatigue task using electrical stimulation. The firing rate of motor units first decreased within the first 10-20% of the endurance time of the contractions and then increased. The firing rate increase was accompanied by recruitment of additional motor units as the force output remained constant. The elicited twitch and tetanic torque responses first increased and then decreased. The two processes modulated in a complementary fashion at the same time. Our data suggest that, when the vastus lateralis muscle is activated to maintain a constant torque output, its motoneuron pool receives a net excitatory drive that first decreases to compensate for the short-lived potentiation of the muscle force twitch and then increases to compensate for the diminution of the force twitch. The underlying inverse relationship between the firing rate and the recruitment threshold that has been reported for nonfatigued contractions is maintained. We, therefore, conclude that the central nervous system control of vastus lateralis motor units remains invariant during fatigue in submaximal isometric isotonic contractions.     

Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion

In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 microg lipase ml(-1) of culture was achieved in sorbitol-based medium. However, a large amount of recombinant protein was retained intracellularly throughout the fermentation, pointing to the transport step as a major bottleneck. Therefore a translational fusion with the green fluorescent protein (GFP) was constructed that was expressed and transported similarly to the native lipase and retained catalytic activity. This analytical tool enables a rapid monitoring of product localization and amount, based on GFP-associated fluorescence.     

Arterially perfused neurosphere-derived cells distribute outside the ischemic core in a model of transient focal ischemia and reperfusion in vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.     

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.     

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.