The CD85J/leukocyte inhibitory receptor-1 distinguishes between conformed and beta 2-microglobulin-free HLA-G molecules

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with beta(2)m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.     

Self-Reported Knee Symptoms Assessed by KOOS Questionnaire in Downhill Runners (Skyrunners)

Background

The knee is the weight-bearing joint most commonly associated with sports injuries, and therefore is most at risk of developing degenerative changes, including osteoarthritis. Skyrunners can be considered to be at risk of developing symptoms of post-traumatic osteoarthritis due to downhill running.

Aim

The aim of this study was to analyze the health of the knee joints of a large group of these athletes via a specific self-report questionnaire.

Methods

This study was carried out by asking the participants of seven official Skyraces (22.4±3.1 km length; 1596±393 m elevation) to fill out a questionnaire. Information regarding age, sex, downhill elevation (m) during training and competitions over the last month, and history of previous knee injury was also collected before the participants filled out the Knee injury and Osteoarthritis Outcome Score (KOOS), which is a reliable and validated instrument designed to assess patients’ opinions about their knees and associated problems that can result in post-traumatic osteoarthritis. Athletes were divided into six age groups (from 17 to 70 years) and 12 groups based on the downhill gradient they had covered over the last month (from 1,000 to 40,000 m).

Results

Six hundred twenty-one questionnaires were collected from 45% of the participants in the seven races. Multivariate analysis revealed that self-reported KOOS scores were unrelated to age, sex and monthly downhill gradient. Only 74 (12%) of the participants reported previous knee injuries. Significant differences in the five subscales of the KOOS were found between skyrunners with and without previous knee injuries (P<0.01).

Conclusions

In the studied population, regular training for downhill running and participation in Skyraces could not be considered risk factors for subjective knee symptoms. Skyrunners with self-reported histories of knee injuries scored worse on all five subscales of the KOOS.     

On the Estimation of Time Dependent Lift of a European Starling (Sturnus vulgaris) during Flapping Flight

We study the role of unsteady lift in the context of flapping wing bird flight. Both aerodynamicists and biologists have attempted to address this subject, yet it seems that the contribution of unsteady lift still holds many open questions. The current study deals with the estimation of unsteady aerodynamic forces on a freely flying bird through analysis of wingbeat kinematics and near wake flow measurements using time resolved particle image velocimetry. The aerodynamic forces are obtained through two approaches, the unsteady thin airfoil theory and using the momentum equation for viscous flows. The unsteady lift is comprised of circulatory and non-circulatory components. Both approaches are presented over the duration of wingbeat cycles. Using long-time sampling data, several wingbeat cycles have been analyzed in order to cover both the downstroke and upstroke phases. It appears that the unsteady lift varies over the wingbeat cycle emphasizing its contribution to the total lift and its role in power estimations. It is suggested that the circulatory lift component cannot assumed to be negligible and should be considered when estimating lift or power of birds in flapping motion.     

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion,Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.Despite the efforts for colorectal cancer (CRC)¹ prevention using different strategies (1–6), 30–40% of patients have regionally advanced disease or suffer from metastasis when diagnosed (7). Moreover, half of the CRC patients will develop recurrence and liver metastasis within 5 years (8). Although genetic changes leading to the development of sporadic colorectal cancer primary tumors in intestinal cells have been relatively well characterized (9), further efforts are necessary to better understand the biology of CRC metastasis and to identify associated markers that can be used as diagnostic/prognostic biomarkers or potential drug targets. Metastasis is a complex process involving different steps from extravasation to liver colonization and requires the concerted action of a large number of proteins to modulate different effects on adhesion, migration, invasion, and survival at the target organ (10).Cancer cells secrete proteins or protein fragments to body fluids, such as blood, that can be used as biomarkers (11, 12) and/or potential therapeutic targets (13). In the case of CRC, there are only three proteins currently used as biomarkers: the carcinoembryonic antigen (CEA) for recurrence and metastasis (1), deleted in colorectal carcinoma (DCC), and vascular endothelial growth factor (VEGF). The secretome constitutes a rich source of information not only for the identification of biomarkers but for the characterization of altered molecules like growth factors, cytokines, proteases, etc., which are vital for cancer progression and metastasis.We are using the well known human KM12 cell system (14) to study the biology of CRC metastasis. KM12SM cells, which possess high metastatic capacity to liver, were isolated from liver metastases in nude mice after five cycles of intrasplenic injection of the poorly metastatic cell line KM12C (14, 15). Multiple studies support a good correlation between the findings observed in the KM12 cell model and patient samples, indicating that KM12 isogenic cell lines recapitulate quite effectively some of the critical issues in CRC metastasis (16–21). In a previous study, we carried out a characterization of plasma membrane proteins of metastatic KM12 cells using a SILAC assay but with a low accuracy and resolution linear ion trap (17). About 60 proteins that showed ≥1.5-fold-change between both types of cells were identified. Recent studies applied iTRAQ or label-free quantification to other pairs of isogenic, nonmetastatic-metastatic colorectal cancer cell lines, SW480 and SW620, for the characterization of protein differences in the whole cell proteome (22) and secretome (23), respectively. The SW620 cell line was isolated from a metastatic lymph node of the same patient as SW480 (24). In contrast, KM12SM cells were chosen based on their capacity for liver metastasis, which makes them most appropriate for the study of liver homing and late stages of metastasis.We are analyzing different fractions of KM12 cells, including the secretome, for a deeper analysis of functionally relevant proteins in metastasis. In a previous report, we analyzed the cytokine/chemokine profiles released in the conditioned media by colorectal metastatic cancer KM12SM cells compared with KM12C using antibody microarrays (20). We found an important role for T_H2 cytokine IL-13 and its receptor IL13Rα2 in cell adhesion, migration, invasion, and liver colonization. Here, we continued this in-depth characterization of the secretome compartment using SILAC analysis with a high accuracy and resolution mass spectrometer, the linear ion trap Orbitrap Velos. The proteomic characterization resulted in the identification and quantification of 1337 and 1098 proteins, respectively, in the conditioned medium. In silico studies demonstrated a predominant association of deregulated proteins in metastatic cells to adhesion, migration, and invasion processes. Three candidates (GDF15, S100A8/A9, and SERPINI1) showed promise as CRC diagnostic markers in serum samples from CRC patients using ELISA. Functional studies using siRNA silencing and antibody blocking experiments demonstrated important tumorigenic and invasive properties in some previously uncharacterized proteins in CRC. In addition, three proteins, EFNA3, CD137L/TNFSF9, and SOSTDC1, demonstrated a critical role in liver homing for metastasis. Finally, meta-analysis of mRNA alterations data indicated that CD137L/TNFSF9, CTSS, SOSTDC1, ZG16B, EFNA3, and MDK were associated with poor prognosis in colorectal cancer.     

Feedback regulation of EGFR signalling: decision making by early and delayed loops

Human-made information relay systems invariably incorporate central regulatory components, which are mirrored in biological systems by dense feedback and feedforward loops. This type of system control is exemplified by positive and negative feedback loops (for example, receptor endocytosis and dephosphorylation) that enable growth factors and receptor Tyr kinases of the epidermal growth factor receptor (EGFR)/ERBB family to regulate cellular function. Recent studies show that the collection of feedback regulatory loops can perform computational tasks - such as decoding ligand specificity, transforming graded input signals into a digital output and regulating response kinetics. Aberrant signal processing and feedback regulation can lead to defects associated with pathologies such as cancer.     

Adenosine and TNF-alpha exert similar inotropic effect on heart cultures, suggesting a cardioprotective mechanism against hypoxia

When cardiomyocytes were subjected to hypoxia, tumor necrosis factor-α (TNF-α; 3-50 ng/ml) or adenosine (1-100 μM), decreased hypoxic damage as was detected by lactate dehydrogenase (LDH) release, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) absorbance, ROS (reactive oxygen species) measurement or desmin immunostaining. This cardioprotection was not prevented in TNF-α-treated cultures by 5-hydroxydecanoic acid (5-HD). Our aim was to elucidate whether adenosine and TNF-α mediate a similar protective mechanism against hypoxia in primary heart cultures and in H9c2 cardiomyocytes. Adenosine and TNF-α are known for their negative inotropic effects on the heart. We have suggested that deoxyglucose uptake reflects heart contractility in cell cultures; therefore, we assayed its accumulation under various conditions. Treatment for 20 min with adenosine, R-PIA [(−)-N(6)-phenylisopropyladenosine] (10 μM), or TNF-α reduced ³H-deoxyglucose uptake in primary heart cultures and also in H9c2 cardiomyocytes by 30-50%. Isoproterenol accelerated ³H-deoxyglucose uptake by 50%. Adenosine, R-PIA, or TNF-α attenuated the stimulatory effect of isoproterenol on ³H-deoxyglucose uptake to control levels. Hypoxia reduced ³H-deoxyglucose uptake by 50%, as in the treatment of the hypoxic cultures with TNF-α or adenosine. Glibenclamide (2 μM), 5-HD (300 μM), or diazoxide (50 μM) increased ³H-deoxyglucose uptake by 50-80%. Adenosine (100 μM) and TNF-α (50 ng/ml) stimulated ⁸⁶Rb efflux. Glibenclamide attenuated this effect. We demonstrate that TNF-α, like adenosine, accelerated Ca²⁺ uptake into the sarcoplasmic reticulum (SR) by 50-100% and therefore prevented cardiomyocyte Ca²⁺ overload. Our findings further suggest that TNF-α, as well as adenosine, may mediate an adaptive effect in the heart by preventing Ca²⁺ overload via activation of SR Ca-ATPase (SERCA₂a).     

Notation-dependent and -independent representations of numbers in the parietal lobes

It is a commonly held view that numbers are represented in an abstract way in both parietal lobes. This view is based on failures to find differences between various notational representations. Here we show that by using relatively smaller voxels together with an adaptation paradigm and analyzing subjects on an individual basis it is possible to detect specialized numerical representations. The current results reveal a left/right asymmetry in parietal lobe function. In contrast to an abstract representation in the left parietal lobe, the numerical representation in the right parietal lobe is notation dependent and thus includes nonabstract representations. Our results challenge the commonly held belief that numbers are represented solely in an abstract way in the human brain.     

Begging in the absence of parents: a "quick on the trigger" strategy to minimize costly misses

Nestling begging in the absence of parents may reflect "falsealarms" due to cognitive constraints or signaling activity towardnest mates (sibling negotiation). According to signal detectiontheory, cognitive constraints should result in both false alarms(begging in the absence of parents or to inappropriate stimuli)and misses (failure to beg during parental visits). In our studyof house sparrows, nestling begging in the absence of parentscomprised up to 50% of the begging events at the nest and wasmore frequent at an early age and among hungrier (lower ranked)nestlings. In contrast, the probability of begging during parentalvisits was constantly high (80% or more), suggesting that therate of misses must have been low even at an early age. Theseresults have 2 main implications. First, the observation thatbegging in the absence of parents decreases with nestling agefavors the cognitive constraints hypothesis over functionalexplanations such as the sibling negotiation hypothesis. Second,the low proportion of "misses" among young nestlings suggeststhat nestling respond to their cognitive constraints by usinglow decision criteria (a "quick on the trigger" strategy) thatincreases the frequency of false alarms but minimizes costlymisses.