Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar)

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15 g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 < pH < 9.0, P⁺Q_B⁻ recombines with a pH independent average rate constant <k> more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which <k> increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH > 11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH > 9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q_AQ_B⁻ state is stabilized by about 40 meV at 6.5 < pH < 9.0, while it is destabilized at pH > 11. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and ³¹P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

Two Different Serum MiRNA Signatures Correlate with the Clinical Outcome and Histological Subtype in Pleural Malignant Mesothelioma Patients

Pleural malignant mesothelioma (MPM) is a detrimental neoplasm affecting pleural sheets and determining a high rate of mortality. In this study, we have enrolled 14 consecutive patients (13 males and 1 female) with MPM (mean age: 70.3 ± 4.6 years). We have collected serum for the determination of a miRNA profiling using a low-density microarray real time PCR system in the serum of patients and comparing it with that one of 10 control counterparts affected by not-cancer-related pleural effusions. In the patients 5 miRNAs were up-regulated (miR101, miR25, miR26b, miR335 and miR433), 2 miRNA were downregulated (miR191, miR223) and two miRNAs were expressed exclusively in patients (miR29a and miR516). Based upon the changes in the expression of the above mentioned miRNAs we detected two distinctive miRNA signatures predicting histotype and survival in these patients: I) patients with more than 3/9 upregulated miRNAs or 3/9 upregulated miRNAs and miR516 not recordable or unchanged (signature A); II) patients with at least 3/9 downregulated or unchanged miRNAs and/or miR29a downregulated (signature B). Based upon these criteria, 5 patients were stratified in signature A and the remaining 9 in signature B. Patients with signature A had a significant shorter median survival than those with signature B (7 months vs. 17 months, 95% CI: 0.098–1.72, p = 0.0021), had a sarcomatoid or mixed histological MPM subtype and were diagnosed in stage II (3/5) and stage III (2/5). In conclusion, we suggest that miRNA signature A is predictive of sarcomatoid histotype and of worse prognosis in MPM.     

Zona Occludens-2 Inhibits Cyclin D1 Expression and Cell Proliferation and Exhibits Changes in Localization along the Cell Cycle

Here, we have studied the effect of the tight junction protein zona occludens (ZO)-2 on cyclin D1 (CD1) protein expression. CD1 is essential for cell progression through the G1 phase of the cell cycle. We have found that in cultures of synchronized Madin-Darby canine kidney cells, ZO-2 inhibits cell proliferation at G0/G1 and decreases CD1 protein level. These effects occur in response to a diminished CD1 translation and an augmented CD1 degradation at the proteosome triggered by ZO-2. ZO-2 overexpression decreases the amount of Glycogen synthase kinase-3β phosphorylated at Ser9 and represses β-catenin target gene expression. We have also explored the expression of ZO-2 through the cell cycle and demonstrate that ZO-2 enters the nucleus at the late G1 phase and leaves the nucleus when the cell is in mitosis. These results thus explain why in confluent quiescent epithelia ZO-2 is absent from the nucleus and localizes at the cellular borders, whereas in sparse proliferating cultures ZO-2 is conspicuously present at the nucleus.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

Theoretical analysis of alendronate and risedronate effects on canine vertebral remodeling and microdamage

Bisphosphonates suppress bone remodeling activity, increase bone volume, and significantly reduce fracture risk in individuals with osteoporosis and other metabolic bone diseases. The objectives of the current study were to develop a mathematical model that simulates control and 1 year experimental results following bisphosphonate treatment (alendronate or risedronate) in the canine fourth lumbar vertebral body, validate the model by comparing simulation predictions to 3 year experimental results, and then use the model to predict potential long term effects of bisphosphonates on remodeling and microdamage accumulation. To investigate the effects of bisphosphonates on bone volume and microdamage, a mechanistic biological model was modified from previous versions to simulate remodeling in a representative volume of vertebral trabecular bone in dogs treated with various doses of alendronate or risedronate, including doses equivalent to those used for treatment of post-menopausal osteoporosis in humans. Bisphosphonates were assumed to affect remodeling by suppressing basic multicellular unit activation and reducing resorption area. Model simulation results for trabecular bone volume fraction, microdamage, and activation frequency following 1 year of bisphosphonate treatment are consistent with experimental measurements. The model predicts that trabecular bone volume initially increases rapidly with 1 year of bisphosphonate treatment, and continues to slowly rise between 1 and 3 years of treatment. The model also predicts that microdamage initially increases rapidly, 0.5–1.5-fold for alendronate or risedronate during the first year of treatment, and reaches its maximum value by 2.5 years before trending downward for all dosages. The model developed in this study suggests that increasing bone volume fraction with long term bisphosphonate treatment may sufficiently reduce strain and damage formation rate so that microdamage does not accumulate above that which is initiated in the first two years of treatment.     

Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A

Background

Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.

Methods

Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.

Results

After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.

Conclusion

We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).     

Long-Term Evolution of Email Networks: Statistical Regularities,Predictability and Stability of Social Behaviors

In social networks, individuals constantly drop ties and replace them by new ones in a highly unpredictable fashion. This highly dynamical nature of social ties has important implications for processes such as the spread of information or of epidemics. Several studies have demonstrated the influence of a number of factors on the intricate microscopic process of tie replacement, but the macroscopic long-term effects of such changes remain largely unexplored. Here we investigate whether, despite the inherent randomness at the microscopic level, there are macroscopic statistical regularities in the long-term evolution of social networks. In particular, we analyze the email network of a large organization with over 1,000 individuals throughout four consecutive years. We find that, although the evolution of individual ties is highly unpredictable, the macro-evolution of social communication networks follows well-defined statistical patterns, characterized by exponentially decaying log-variations of the weight of social ties and of individuals’ social strength. At the same time, we find that individuals have social signatures and communication strategies that are remarkably stable over the scale of several years.