Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

A simple method of generating axenic derivatives of Dictyostelium strains

We describe a generally applicable method of adapting Dictyostelium from growth on a bacterial food source to axenic growth. Cells are initially selected by growth on a plastic substratum but subsequently acquire the ability to grow in suspension culture. These strains can be transformed efficiently by DNA-mediated gene transfer.     

The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.     

ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N₃-ATP) and 8-azidoadenosine 5′-monophosphate (8-N₃-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N₃-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N₃-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P⁵-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.     

Tracking reveals behavioural coordination driven by environmental constraints in the Black-vented Shearwater Puffinus opisthomelas

During the breeding season, seabird foraging trips are constrained by nest attendance schedule and are necessarily colony centred. Oceanographic cues play a major role in the choice of foraging areas to minimize the time spent away from the nest. Here, we analysed the foraging tracks of Black-vented Shearwaters Puffinus opisthomelas during the incubation and chick-rearing periods of 2016 and 2017 at Isla Natividad (Mexico). We applied expectation-maximization binary clustering to track data to clusterize different behaviour patterns during foraging flights. We then applied binary generalized linear mixed models to characterize of foraging areas based on of environmental variables. We finally used kernel estimation techniques to describe main foraging areas. In 2016, breeding shearwaters used two core areas for foraging and resting on the water; the core area delineated by males was located northward from the colony in the Vizcaino Bay and the core area for females was located southward from the colony at the entrance of San Ignacio Lagoon. In 2017, males and females used the same areas with no evident segregation. Our study provided the first information on Black-vented Shearwater foraging areas during the breeding season and indicated that sexual segregation within coastal waters off the central Baja California Peninsula might be a foraging strategy during years of warmer ocean, likely less productive regimes. Factors including ocean-climate-mediated sexual segregation at sea, leading to interannual variation in foraging areas, should be considered when evaluating management actions intended to protect critical foraging habitats for Black-vented Shearwaters.     

Predictors of Chikungunya rheumatism: a prognostic survey ancillary to the TELECHIK cohort study

Introduction

Long-lasting relapsing or lingering rheumatic musculoskeletal pain (RMSP) is the hallmark of Chikungunya virus (CHIKV) rheumatism (CHIK-R). Little is known on their prognostic factors. The aim of this prognostic study was to search the determinants of lingering or relapsing RMSP indicative of CHIK-R.

Methods

Three hundred and forty-six infected adults (age ≥ 15 years) having declared RMSP at disease onset were extracted from the TELECHIK cohort study, Reunion island, and analyzed using a multinomial logistic regression model. We also searched for the predictors of CHIKV-specific IgG titres, assessed at the time of a serosurvey, using multiple linear regression analysis.

Results

Of these, 111 (32.1%) reported relapsing RMSP, 150 (43.3%) lingering RMSP, and 85 (24.6%) had fully recovered (reference group) on average two years after acute infection. In the final model controlling for gender, the determinants of relapsing RMSP were the age 45-59 years (adjusted OR: 2.9, 95% CI: 1.0, 8.6) or greater or equal than 60 years (adjusted OR: 10.4, 95% CI: 3.5, 31.1), severe rheumatic involvement (fever, at least six joints plus four other symptoms) at presentation (adjusted OR: 3.6, 95% CI: 1.5, 8.2), and CHIKV-specific IgG titres (adjusted OR: 3.2, 95% CI: 1.8, 5.5, per one unit increase). Prognostic factors for lingering RMSP were age 45-59 years (adjusted OR: 6.4, 95% CI: 1.8, 22.1) or greater or equal than 60 years (adjusted OR: 22.3, 95% CI: 6.3, 78.1), severe initial rheumatic involvement (adjusted OR: 5.5, 95% CI: 2.2, 13.8) and CHIKV-specific IgG titres (adjusted OR: 6.2, 95% CI: 2.8, 13.2, per one unit increase). CHIKV specific IgG titres were positively correlated with age, female gender and the severity of initial rheumatic symptoms.

Conclusions

Our data support the roles of age, severity at presentation and CHIKV specific IgG titres for predicting CHIK-R. By identifying the prognostic value of the humoral immune response of the host, this work also suggest a significant contribution of the adaptive immune response to the physiopathology of CHIK-R and should help to reconsider the paradigm of this chronic infection primarily shifted towards the involvement of the innate immune response.     

Frondoside A Suppressive Effects on Lung Cancer Survival,Tumor Growth,Angiogenesis, Invasion,and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.     

Here Today,Gone Tomorrow – Adaptation to Change in Memory-Guided Visual Search

Visual search for a target object can be facilitated by the repeated presentation of an invariant configuration of nontargets (‘contextual cueing’). Here, we tested adaptation of learned contextual associations after a sudden, but permanent, relocation of the target. After an initial learning phase targets were relocated within their invariant contexts and repeatedly presented at new locations, before they returned to the initial locations. Contextual cueing for relocated targets was neither observed after numerous presentations nor after insertion of an overnight break. Further experiments investigated whether learning of additional, previously unseen context-target configurations is comparable to adaptation of existing contextual associations to change. In contrast to the lack of adaptation to changed target locations, contextual cueing developed for additional invariant configurations under identical training conditions. Moreover, across all experiments, presenting relocated targets or additional contexts did not interfere with contextual cueing of initially learned invariant configurations. Overall, the adaptation of contextual memory to changed target locations was severely constrained and unsuccessful in comparison to learning of an additional set of contexts, which suggests that contextual cueing facilitates search for only one repeated target location.