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We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it.  相似文献   
3.
In signal transduction research natural or synthetic molecules are commonly used to target a great variety of signaling proteins. For instance, forskolin, a diterpene activator of adenylate cyclase, has been widely used in cellular preparations to increase the intracellular cAMP level. However, it has been shown that forskolin directly inhibits some cloned K+ channels, which in excitable cells set up the resting membrane potential, the shape of action potential and regulate repetitive firing. Despite the growing evidence indicating that K+ channels are blocked by forskolin, there are no studies yet assessing the impact of this mechanism of action on neuron excitability and firing patterns. In sympathetic neurons, we find that forskolin and its derivative 1,9-Dideoxyforskolin, reversibly suppress the delayed rectifier K+ current (IKV). Besides, forskolin reduced the spike afterhyperpolarization and enhanced the spike frequency-dependent adaptation. Given that IKV is mostly generated by Kv2.1 channels, HEK-293 cells were transfected with cDNA encoding for the Kv2.1 α subunit, to characterize the mechanism of forskolin action. Both drugs reversible suppressed the Kv2.1-mediated K+ currents. Forskolin inhibited Kv2.1 currents and IKV with an IC50 of ~32 μM and ~24 µM, respectively. Besides, the drug induced an apparent current inactivation and slowed-down current deactivation. We suggest that forskolin reduces the excitability of sympathetic neurons by enhancing the spike frequency-dependent adaptation, partially through a direct block of their native Kv2.1 channels.  相似文献   
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Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the ‘alternative clamp loader’ known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved ‘Pol ϵ binding module’ in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.  相似文献   
6.
Phosphatases are recognized to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (βGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85%; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of βGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.  相似文献   
7.

Background

Meteorin (METRN) is a recently described neutrophic factor with angiogenic properties. This is a nested case-control study in a longitudinal cohort study that describes the serum profile of METRN during different periods of gestation in healthy and preeclamptic pregnant women. Moreover, we explore the possible application of METRN as a biomarker.

Methods and Findings

Serum METRN was measured by ELISA in a longitudinal prospective cohort study in 37 healthy pregnant women, 16 mild preeclamptic women, and 20 healthy non-pregnant women during the menstrual cycle with the aim of assessing serum METRN levels and its correlations with other metabolic parameters. Immunostaining for METRN protein was performed in placenta. A multivariate logistic regression model was proposed and a classifier model was formulated for predicting preeclampsia in early and middle pregnancy. The performance in classification was evaluated using measures such as sensitivity, specificity, and the receiver operating characteristic (ROC) curve. In healthy pregnant women, serum METRN levels were significantly elevated in early pregnancy compared to middle and late pregnancy. METRN levels are significantly lower only in early pregnancy in preeclamptic women when compared to healthy pregnant women. Decision trees that did not include METRN levels in the first trimester had a reduced sensitivity of 56% in the detection of preeclamptic women, compared to a sensitivity of 69% when METRN was included.

Conclusions

The joint measurements of circulating METRN levels in the first trimester and systolic blood pressure and weight in the second trimester significantly increase the probabilities of predicting preeclampsia.  相似文献   
8.
Some epiphytic Hymenophyllaceae are restricted to lower parts of the host (<60 cm; 10–100 μmol photons m-2 s-1) in a secondary forest of Southern Chile; other species occupy the whole host height (≥10 m; max PPFD >1000 μmol photons m-2 s-1). Our aim was to study the photosynthetic light responses of two Hymenophyllaceae species in relation to their contrasting distribution. We determined light tolerance of Hymenoglossum cruentum and Hymenophyllum dentatum by measuring gas exchange, PSI and PSII light energy partitioning, NPQ components, and pigment contents. H. dentatum showed lower maximum photosynthesis rates (Amax) than H. cruentum, but the former species kept its net rates (An) near Amax across a wide light range. In contrast, in the latter one, An declined at PPFDs >60 μmol photons m-2 s-1. H. cruentum, the shadiest plant, showed higher chlorophyll contents than H. dentatum. Differences in energy partitioning at PSI and PSII were consistent with gas exchange results. H. dentatum exhibited a higher light compensation point of the partitioning of absorbed energy between photochemical Y(PSII) and non-photochemical Y(NPQ) processes. Hence, both species allocated energy mainly toward photochemistry instead of heat dissipation at their light saturation points. Above saturation, H. cruentum had higher heat dissipation than H. dentatum. PSI yield (YPSI) remained higher in H. dentatum than H. cruentum in a wider light range. In both species, the main cause of heat dissipation at PSI was a donor side limitation. An early dynamic photo-inhibition of PSII may have caused an over reduction of the Qa+ pool decreasing the efficiency of electron donation to PSI. In H. dentatum, a slight increase in heat dissipation due to acceptor side limitation of PSI was observed above 300 μmol photons m-2s-1. Differences in photosynthetic responses to light suggest that light tolerance and species plasticity could explain their contrasting vertical distribution.  相似文献   
9.
Physiological and behavioural constraints hinder albino individuals. Albino animals are rare in the wild; this trait is associated with easy detection by predators, non-native or damaged environments, and exclusively aphotic environments in total darkness. The social aspect of albinism is reported only for human beings, and the effect is distinguishable in time and space when social benefits, are used to a limited the extent. Thus far, the social consequences of albinism for animals remain unknown. We used socially established groups of the pigmented catfish, (Silurus glanis), to observe space and temporal distance detachment of albino specimens in laboratory conditions. The albino fish were separated at larger distances from the group than pigmented individuals with the same social status determined by familiarity, and this asymmetry also varied in time. Albinism-related ostracism results in a solitary existence, usually followed by enhanced predation risk. The motivation for an individual’s exclusion from a group appears to be the avoidance of the predation risk that increases not only for an odd individual but also for conspecifics within a group. Our findings indicate a role for albinism in behavioural processes related to sociality in a group of conspecifics.  相似文献   
10.
Insulin resistance is a key feature of Type 2 diabetes and an important therapeutic target to address glycemic control to prevent diabetic complications. Lifestyle advice is the first step in the ADA/EASD consensus guidelines followed by metformin therapy. Aerobic exercise (AE) can increase insulin sensitivity by several molecular pathways including upregulation of insulin transporters in the cellular membrane of insulin-dependent cells. In addition, AE improves insulin sensitivity by amelioration of the pathophysiologic pathways involved in insulin resistance such as the reduction of adipokines, inflammatory and oxidative stress responses, and improvement of insulin signal transduction via different molecular pathways. This review details the molecular pathways by which AE induces beneficial effects on insulin resistance  相似文献   
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