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Epidermal growth factor (EGF) and insulin receptor tyrosine kinases (RTKs) exemplify how receptor location is coupled to signal transduction. Extracellular binding of ligands to these RTKs triggers their concentration into vesicles that bud off from the cell surface to generate intracellular signaling endosomes. On the exposed cytosolic surface of these endosomes, RTK autophosphorylation selects the downstream signaling proteins and lipids to effect growth factor and polypeptide hormone action. This selection is followed by the recruitment of protein tyrosine phosphatases that inactivate the RTKs and deliver them by membrane fusion and fission to late endosomes. Coincidentally, proteinases inside the endosome cleave the EGF and insulin ligands. Subsequent inward budding of the endosomal membrane generates multivesicular endosomes. Fusion with lysosomes then results in RTK degradation and downregulation. Through the spatial positioning of RTKs in target cells for EGF and insulin action, the temporal extent of signaling, attenuation, and downregulation is regulated.

Objectives: The aim of this study was to establish a conversion factor for assessing the scattered radiation produced during intraoral radiography with a rectangular collimator in areas where the remnant beam is not present. The dose level in the remnant beam will be addressed in another study. Methods: A radiological phantom (Rando) in the upright position was exposed to radiation delivered by a Planmeca Intra X-ray unit over 9 exposure geometries (right side of a full mouth series). For each geometry, the scattered radiation dose was measured at 5 locations in the horizontal plane and 4 locations in the vertical plane in front of the patient. The measurements were corrected for background and leakage radiation. An inventory of the distributions of the different geometries with respect to their share of the total workload was created in order to assign the correct weightings of the total ambient scattered radiation for the different geometries. The weightings were used to construct a scattered radiation pattern for a representative mix of exposures. The maximum value found in this pattern was used to arrive at a safe conversion factor. Results: The scattered ambient radiation dose was evenly distributed over the horizontal plane. The scattered ambient radiation dose ranged from 20 to 65% lower in the vertical plane. Conclusions: The conversion factor established for scattered ambient radiation was 0.05 µSv per mAs at 1 meter. This factor is 3-fold lower than the conversion factors reported previously.

Bats are excellent models for studying the molecular basis of sensory adaptation. In Chiroptera, a sensory trade-off has been proposed between the visual and auditory systems, though the extent of this association has yet to be fully examined. To investigate whether variation in visual performance is associated with echolocation, we experimentally assayed the dim-light visual pigment rhodopsin from bat species with differing echolocation abilities. While spectral tuning properties were similar among bats, we found that the rate of decay of their light-activated state was significantly slower in a nonecholocating bat relative to species that use distinct echolocation strategies, consistent with a sensory trade-off hypothesis. We also found that these rates of decay were remarkably slower compared with those of other mammals, likely indicating an adaptation to dim light. To examine whether functional changes in rhodopsin are associated with shifts in selection intensity upon bat Rh1 sequences, we implemented selection analyses using codon-based likelihood clade models. While no shifts in selection were identified in response to diverse echolocation abilities of bats, we detected a significant increase in the intensity of evolutionary constraint accompanying the diversification of Chiroptera. Taken together, this suggests that substitutions that modulate the stability of the light-activated rhodopsin state were likely maintained through intensified constraint after bats diversified, being finely tuned in response to novel sensory specializations. Our study demonstrates the power of combining experimental and computational approaches for investigating functional mechanisms underlying the evolution of complex sensory adaptations.

Deep-sea mining is likely to result in biodiversity loss, and the significance of this to ecosystem function is not known. “Out of kind” biodiversity offsets substituting one ecosystem type (e.g., coral reefs) for another (e.g., abyssal nodule fields) have been proposed to compensate for such loss. Here we consider a goal of no net loss (NNL) of biodiversity and explore the challenges of applying this aim to deep seabed mining, based on the associated mitigation hierarchy (avoid, minimize, remediate). We conclude that the industry cannot at present deliver an outcome of NNL. This results from the vulnerable nature of deep-sea environments to mining impacts, currently limited technological capacity to minimize harm, significant gaps in ecological knowledge, and uncertainties of recovery potential of deep-sea ecosystems. Avoidance and minimization of impacts are therefore the only presently viable means of reducing biodiversity losses from seabed mining. Because of these constraints, when and if deep-sea mining proceeds, it must be approached in a precautionary and step-wise manner to integrate new and developing knowledge. Each step should be subject to explicit environmental management goals, monitoring protocols, and binding standards to avoid serious environmental harm and minimize loss of biodiversity. “Out of kind” measures, an option for compensation currently proposed, cannot replicate biodiversity and ecosystem services lost through mining of the deep seabed and thus cannot be considered true offsets. The ecosystem functions provided by deep-sea biodiversity contribute to a wide range of provisioning services (e.g., the exploitation of fish, energy, pharmaceuticals, and cosmetics), play an essential role in regulatory services (e.g., carbon sequestration) and are important culturally. The level of “acceptable” biodiversity loss in the deep sea requires public, transparent, and well informed consideration, as well as wide agreement. If accepted, further agreement on how to assess residual losses remaining after the robust implementation of the mitigation hierarchy is also imperative. To ameliorate some of the inter-generationalinequity caused by mining-associated biodiversity losses, and only after all NNL measures have been used to the fullest extent, potential compensatory actions would need to be focused on measures to improve the knowledge and protection of the deep sea and to demonstrate benefits that will endure for future generations.&nbsp

Aims Patients with acute coronary syndromes (ACSs) who are managed without coronary revascularization represent a mixed and understudied population that seems to receive suboptimal pharmacological treatment. Methods and results We assessed patterns of antithrombotic therapies employed during the hospitalization and in-hospital clinical events of medically managed patients with ACS enrolled in the prospective, multicentre, nationwide EYESHOT (EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in iTalian cardiac care units) registry. Among the 2585 consecutive ACS patients enrolled in EYESHOT, 783 (30.3%) did not receive any revascularization during hospital admission. Of these, 478 (61.0%) underwent coronary angiography (CA), whereas 305 (39.0%) did not. The median GRACE and CRUSADE risk scores were significantly higher among patients who did not undergo CA compared with those who did (180 vs. 145, P < 0.0001 and 50 vs. 33, P < 0.0001, respectively). Antithrombotic therapies employed during hospitalization significantly differ between patients who received CA and those who did not with unfractioned heparin and novel P2Y12 inhibitors more frequently used in the first group, and low-molecular-weight heparins and clopidogrel in the latter group. During the index hospitalization, patients who did not receive CA presented a higher incidence of ischaemic cerebrovascular events and of mortality compared with those who underwent CA (1.6 vs. 0.2%, P = 0.04 and 7.9 vs. 2.7%, P = 0.0009, respectively). Conclusion Almost one-third of ACS patients are managed without revascularization during the index hospitalization. In this population, a lower use of recommended antiplatelet therapy and worse clinical outcome were observed in those who did not undergo CA when compared with those who did. Clinical Trial Registration Unique identifier: [NCT02015624][1], . [10.1093/ehjcvp/pvv017][2] [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02015624&atom=%2Fehjcardpharm%2F1%2F3%2F168.atom [2]: /lookup/doi/10.1093/ehjcvp/pvv017

Background: Late preterm infants show a major fat mass accretion from birth to term. The contribution of preterm birth to the development of the metabolic syndrome is still under investigation. Objectives: To evaluate body composition changes in late preterm infants during the first 3 months and to investigate their insulin sensitivity and resistance. Methods: We conducted an observational, longitudinal study. A total of 216 late preterm infants underwent body composition assessment using an air displacement plethysmograph at term and at 3 months of corrected age. In a subgroup of infants (n = 48) the blood glucose and insulin concentration were determined at term and insulin resistance (homeostasis model assessment for insulin resistance; HOMA-IR) and sensitivity (quantitative insulin sensitivity check index; QUICKI) were then calculated. The reference group comprised 71 healthy term infants. Results: The mean birth weight and gestational age were 2,390 ± 391 g and 35.2 ± 0.8 weeks, respectively. At term the fat mass index (kg/m2) of late preterm infants, born adequate for their gestational age and small for their gestational age, was higher than that of term infants (2.08 ± 0.82 vs. 1.62 ± 0.64 vs. 1.03 ± 0.36, p < 0.005, respectively), whereas at 3 months of corrected age no difference was found among the groups. The mean values of glucose, insulin, HOMA-IR, and QUICKI were within the 5th and 95th percentiles. Conclusions: On the basis of these preliminary findings, fat mass accretion of late preterm infants appears not to be associated with perturbation of the glucose homeostasis.

Cichlids encompass one of the most diverse groups of fishes in South and Central America, and show extensive variation in life history, morphology, and colouration. While studies of visual system evolution in cichlids have focussed largely on the African rift lake species flocks, Neotropical cichlids offer a unique opportunity to investigate visual system evolution at broader temporal and geographic scales. South American cichlid colonization of Central America has likely promoted accelerated rates of morphological evolution in Central American lineages as they encountered reduced competition, renewed ecological opportunity, and novel aquatic habitats. To investigate whether such transitions have influenced molecular evolution of vision in Central American cichlids, we sequenced the dim-light rhodopsin gene in 101 Neotropical cichlid species, spanning the diversity of the clade. We find strong evidence for increased rates of evolution in Central American cichlid rhodopsin relative to South American lineages, and identify several sites under positive selection in rhodopsin that likely contribute to adaptation to different photic environments. We expressed a Neotropical cichlid rhodopsin protein invitro for the first time, and found that while its spectral tuning properties were characteristic of typical vertebrate rhodopsin pigments, the rate of decay of its active signalling form was much slower, consistent with dim light adaptation in other vertebrate rhodopsins. Using site-directed mutagenesis combined with spectroscopic assays, we found that a key amino acid substitution present in some Central American cichlids accelerates the rate of decay of active rhodopsin, which may mediate adaptation to clear water habitats.

Convergent evolution in response to similar selective pressures is a well-known phenomenon in evolutionary biology. Less well understood is how selection drives convergence in protein function, and the underlying mechanisms by which this can be achieved. Here, we investigate functional convergence in the visual system of two distantly related lineages of high-altitude adapted Andean and Himalayan catfishes. Statistical analyses revealed in the two high-altitude lineages, a parallel acceleration of evolutionary rates in rhodopsin, the dim-light visual pigment. However, the elevated rates were found to be accompanied by substitutions at different sites in the protein. Experiments substituting Andean- or Himalayan-specific residues significantly accelerated the kinetic rates of rhodopsin, destabilizing the ligand-bound forms. As found in cold-adapted enzymes, this phenotype likely compensates for a cold-induced decrease in kinetic rates, properties of rhodopsin mediating rod sensitivity and visual performance. Our study suggests that molecular convergence in protein function can be driven by parallel shifts in evolutionary rates but via nonparallel molecular mechanisms. Signatures of natural selection may therefore be a powerful guide for identifying complex instances of functional convergence across a wider range of protein systems.

Aims In the last decades, several new therapies have emerged for the treatment of acute coronary syndromes (ACS). We sought to describe real-world patterns of use of antithrombotic treatments in the catheterization laboratory for ACS patients undergoing percutaneous coronary interventions (PCI). Methods EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in iTalian cardiac care units was a nationwide, prospective registry aimed to evaluate antithrombotic strategies employed in ACS patients in Italy. Results Over a 3-week period, a total of 2585 consecutive ACS patients have been enrolled in 203 cardiac care units across Italy. Among these patients, 1755 underwent PCI (923 with ST-elevation myocardial infarction and 832 with non-ST-elevation ACS). In the catheterization laboratory, unfractioned heparin was the most used antithrombotic drug in both ST-elevation myocardial infarction (64.7%) and non-ST-elevation ACS (77.5%) undergoing PCI and, as aspirin, bivalirudin and glycoprotein IIb/IIIa inhibitors (GPIs) more frequently employed before or during PCI compared with the postprocedural period. Any crossover of heparin therapy occurred in 36.0% of cases, whereas switching from one P2Y12 inhibitor to another occurred in 3.7% of patients. Multivariable analysis yielded several independent predictors of GPIs and of bivalirudin use in the catheterization laboratory, mainly related to clinical presentation, PCI complexity and presence of complications during the procedure. Conclusion In our contemporary, nationwide, all-comers cohort of ACS patients undergoing PCI, antithrombotic therapies were commonly initiated before the catheterization laboratory. In the periprocedural period, the most frequently employed drugs were unfractioned heparin, leading to a high rate of crossover, followed by GPIs and bivalirudin, mainly used during complex PCI. Clinical trial registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02015624.

Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.

IntroductionPreterm and small for gestational age (SGA) infants are at increased risk of poor growth, disability and delayed development. While growing up they are also at increased risk of obesity, diabetes and later heart disease. The risk of such adverse outcomes may be altered by how preterm and SGA infants are fed after birth. Faltering postnatal growth is common due to failure to achieve recommended high energy and protein intakes, and thus preterm and SGA infants are often provided with supplemental nutrition soon after birth. Enhanced nutrition has been associated with improved early growth and better cognitive development. However, limited evidence suggests that faster growth may increase the risk for later adiposity, metabolic and cardiovascular disease, and that such risks may differ between girls and boys.Methods and analysisWe will search Ovid MEDLINE, Embase, Cochrane CENTRAL, Cochrane Database of Systematic Reviews, controlled-trials.com, ClinicalTrials.gov and anzctr.org.au for randomised trials that studied the effects of macronutrient supplements for preterm and SGA infants on (i) developmental and metabolic and (ii) growth outcomes after hospital discharge. The outcomes will be (i) cognitive impairment and metabolic risk (co-primary) and (ii) body mass index. Individual participant data (IPD) from all available trials will be included using an intention-to-treat approach. A one-stage procedure for IPD meta-analysis (MA) will be used, accounting for clustering of participants within studies. Exploratory subgroup analyses will further investigate sources of heterogeneity, including sex and size of infants, different timing, duration and type of supplements.Ethics and disseminationThis IPD-MA is approved by the University of Auckland Human Participants Ethics Committee (reference number: 019874). Individual studies have approval from relevant local ethics committees. Results will be disseminated in a peer-reviewed journal and presented at international conferences.PROSPERO registration numberCRD42017072683

The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the shikimate dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the 'core' structure of the enzyme.

Epithelial-to-mesenchymal transition (EMT) is controlled by cellular signaling pathways that trigger the loss of cell-cell adhesion and lead to the restructuring of the cell cytoskeleton. Transforming growth factor β (TGF-β) has been shown to regulate cell plasticity through the phosphorylation of Par6 on a conserved serine residue (S345) by the type II TGF-β receptor. We show here that atypical protein kinase C (aPKC) is an essential component to this signaling pathway in non-small-cell lung cancer (NSCLC) cells. We show that the aPKC, PKCι, interacts with TGF-β receptors through Par6 and that these proteins localize to the leading edge of migrating cells. Furthermore, Par6 phosphorylation on serine 345 by TGF-β receptors is enhanced in the presence of aPKC. aPKC kinase activity, as well as an association with Par6, were found to be important for Par6 phosphorylation. In effect, small interfering RNA-targeting aPKC reduces TGF-β-induced RhoA and E-cadherin loss, cell morphology changes, stress fiber production, and the migration of NSCLC cells. Interestingly, reintroduction of a phosphomimetic Par6 (Par6-S345E) into aPKC-silenced cells rescues both RhoA and E-cadherin loss with TGF-β stimulation. In conclusion, our results suggest that aPKCs cooperate with TGF-β receptors to regulate phospho-Par6-dependent EMT and cell migration.

BACKGROUND The number of elderly patients undergoing major surgical interventions and then needing admission to intensive care unit (ICU) grows steadily. We investigated this issue in a cohort of 232,278 patients admitted in five years (2011-2015) to 163 Italian general ICUs. METHODS Surgical patients older than 75 registered in the GiViTI MargheritaPROSAFE project were analyzed. The impact on hospital mortality of important chronic conditions (severe COPD, NYHA class IV, dementia, end-stage renal disease, cirrhosis with portal hypertension) was investigated with two prognostic models developed yearly on patients staying in the ICU less or more than 24 hours. RESULTS 44,551 elderly patients (19.2%) underwent emergency (47.3%) or elective surgery (52.7%). At least one severe comorbidity was present in 14.6% of them, yielding a higher hospital mortality (32.4%, vs. 21.1% without severe comorbidity). In the models for patients staying in the ICU 24 hours or more, cirrhosis, NYHA class IV, and severe COPD were constant independent predictors of death (adjusted odds ratios [ORs] range 1.67-1.97, 1.54-1.91, and 1.34-1.50, respectively), while dementia was statistically significant in four out of five models (adjusted ORs 1.23-1.28). End-stage renal disease, instead, never resulted to be an independent prognostic factor. For patients staying in the ICU less than 24 hours, chronic comorbidities were only occasionally independent predictors of death. CONCLUSIONS Our study confirms that elderly surgical patients represent a relevant part of all ICUs admissions. About one of seven bear at least one severe chronic comorbidity, that, excluding end-stage renal disease, are all strong independent predictors of hospital death.

Synthetic triterpenoids are a class of anti-cancer compounds that target many cellular functions, including apoptosis and cell growth in both cell culture and animal models. We have shown that triterpenoids inhibit cell migration in part by interfering with Arp2/3-dependent branched actin polymerization in lamellipodia (To et al., 2010). Our current studies reveal that Glycogen Synthase Kinase 3 beta (GSK3β), a kinase that regulates many cellular processes, including cell adhesion dynamics, is a triterpenoid-binding protein. Furthermore, triterpenoids were observed to inhibit GSK3β activity and increase cellular focal adhesion size. To further examine whether these effects on focal adhesions in triterpenoid-treated cells were GSK3β-dependent, GSK3β inhibitors (lithium chloride and SB216763) were used to examine cell adhesion and morphology as well as cell migration. Our results showed that GSK3β inhibitors also altered cell adhesion sizes. Moreover, these inhibitors blocked cell migration and displaced proteins at the leading edge of migrating cells, consistent with what was observed in triterpenoid-treated cells. Therefore, the triterpenoids may affect cell migration via a mechanism that targets and alters the activity and localization of GSK3β.

Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (dN/dS) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity–independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Transforming growth factor (TGF)-β1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein α2-HS-glycoprotein (AHSG) blocks TGF-β1 binding to cell surface receptors, suppresses TGF-β signal transduction, and inhibits TGF-β-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg−/− mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-β signaling in vivo. Furthermore, TGF-β-mediated suppression of immune cell function was exaggerated in Ahsg−/− animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate–induced cutaneous inflammation. Reconstitution of Ahsg−/− mice with bovine Ahsg suppressed endogenous TGF-β-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-β.

In this study, we evaluated the prevalence and association with thrombosis and/or thrombocytopenia of IgG and IgM antibodies to cardiolipin (aCL), phosphatidic acid (aPA), phosphatidylinositol (aPI), phosphatidylserine, and beta(2)-glycoprotein I (abeta(2)-GPI) in systemic lupus erythematosus (SLE). Sera were obtained from 87 patients affected by SLE (77 of the 87 patients were females), 41 of them with a history of arterial and/or venous thrombosis. Antiphospholipid antibodies and abeta(2)-GPI were evaluated by enzyme-linked immunosorbent assay. IgG-aCL, IgG-aPA, IgG-aPI, IgG-aPS, and IgG-abeta(2)-GPI were found in 53%, 37%, 32%, 38%, and 24% of patients, respectively. IgM-aCL, IgM-aPA, IgM-aPI, IgM-aPS, and IgM-abeta(2)-GPI were detected in 15%, 17%, 18%, 14%, and 16%, respectively. With respect to antibody titer, IgG-aCL strongly correlated with all other antiphospholipid antibodies and abeta(2)-GPI of IgG isotype. Thrombosis was significantly associated with IgG-aPA (p = 0.044), IgG-aPI (p = 0.038), IgG-aPS (p = 0.026), IgG-abeta(2)-GPI, IgM-aPA (p = 0.044), IgM-aPI (p = 0.024), and IgM-aPS (p = 0.01), irrespective of antibody titer, whereas IgG-aCL were associated with thrombosis and thrombocytopenia when taken at medium-high titer (p = 0.009 and p = 0.046, respectively). Our results confirm that, besides aCL and abeta(2)-GPI, other antibodies to negatively-charged phospholipids are present in a large percentage of patients with SLE. However, it remains doubtful whether these other antiphospolipid antibodies actually represent an important parameter predictive of thrombosis and thrombocytopenia in SLE.

Transforming growth factor b (TGFb) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g., Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFb receptor trafficking and signaling. Here, we report that downstream TGFb-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFb pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb dependent apoptosis.

Transforming growth factor b (TGFb) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g., Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGFb receptor trafficking and signaling. Here, we report that downstream TGFb-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGFb pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGFb dependent apoptosis.

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non–small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non–small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Cancer Prev Res; 7(7); 727–37. ©2014 AACR.

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (γ-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941–54]