Temporal Variability of Urinary Phthalate Metabolite Levels in Men of Reproductive Age

Phthalates are a family of multifunctional chemicals widely used in personal care and other consumer products. The ubiquitous use of phthalates results in human exposure through multiple sources and routes, including dietary ingestion, dermal absorption, inhalation, and parenteral exposure from medical devices containing phthalates. We explored the temporal variability over 3 months in urinary phthalate metabolite levels among 11 men who collected up to nine urine samples each during this time period. Eight phthalate metabolites were measured by solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry. Statistical analyses were performed to determine the between- and within-subject variance apportionment, and the sensitivity and specificity of a single urine sample to classify a subject’s 3-month average exposure. Five of the eight phthalates were frequently detected. Monoethyl phthalate (MEP) was detected in 100% of samples; monobutyl phthalate, monobenzyl phthalate, mono-2-ethylhexyl phthalate (MEHP), and monomethyl phthalate were detected in > 90% of samples. Although we found both substantial day-to-day and month-to-month variability in each individual’s urinary phthalate metabolite levels, a single urine sample was moderately predictive of each subject’s exposure over 3 months. The sensitivities ranged from 0.56 to 0.74. Both the degree of between- and within-subject variance and the predictive ability of a single urine sample differed among phthalate metabolites. In particular, a single urine sample was most predictive for MEP and least predictive for MEHP. These results suggest that the most efficient exposure assessment strategy for a particular study may depend on the phthalates of interest

Attenuated reovirus displays oncolysis with reduced host toxicity

Background: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. Methods: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. Results: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated ?1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. Conclusion: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy

Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells

Chondrogenesis is the complex process that leads to the establishment of cartilage and bone formation. Due to their ability to differentiate in vitro and mimic development, embryonic stem cells (ESCs) show great potential for investigating developmental processes. In this study, we used chondrogenic differentiation of ESCs as a model to analyze morphogenetic events during chondrogenesis.ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.We demonstrate that ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages. The study of chondrogenesis via ESC differentiation may be informative to our further understanding of skeletal growth and regeneration

Mesodermal fate decisions of a stem cell: the Wnt switch

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis

Determinants of Functional Coupling between Astrocytes and Respiratory Neurons in the Pre-Bötzinger Complex

Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive

Cortical Layer 1 and Layer 2/3 Astrocytes Exhibit Distinct Calcium Dynamics In Vivo

Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca2+) concentration is important for astrocytes as Ca2+ surges co-occur with gliotransmission and neurotransmitter reception. Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca2+ activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca2+ activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca2+ dynamics by investigating two distinct EEG states (“synchronized” vs. “de-synchronized” states). We found that astrocytes in L1 had nearly twice higher Ca2+ activity than L2/3. Furthermore, Ca2+ fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca2+ activity. These results suggest that spontaneous astrocytic Ca2+ surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity

Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populations

Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 mu V for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm(2)) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 mu V in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.Portuguese Foundation for Science and Technology (FCT), through the project "Implantable organic devices for advanced therapies" (INNOVATE) [PTDC/EEI-AUT/5442/2014]; Instituto de Telecomunicacoes [UID/Multi/04326/2013]; Associated Laboratory - Institute of Nanoscience and Nanotechnology [POCI-01-0145-FEDER-016623]; [PTDC/CTM-NAN/3146/2014

Principles of cartilage tissue engineering in TMJ reconstruction

Diseases and defects of the temporomandibular joint (TMJ), compromising the cartilaginous layer of the condyle, impose a significant treatment challenge. Different regeneration approaches, especially surgical interventions at the TMJ's cartilage surface, are established treatment methods in maxillofacial surgery but fail to induce a regeneration ad integrum. Cartilage tissue engineering, in contrast, is a newly introduced treatment option in cartilage reconstruction strategies aimed to heal cartilaginous defects. Because cartilage has a limited capacity for intrinsic repair, and even minor lesions or injuries may lead to progressive damage, biological oriented approaches have gained special interest in cartilage therapy. Cell based cartilage regeneration is suggested to improve cartilage repair or reconstruction therapies. Autologous cell implantation, for example, is the first step as a clinically used cell based regeneration option. More advanced or complex therapeutical options (extracorporeal cartilage engineering, genetic engineering, both under evaluation in pre-clinical investigations) have not reached the level of clinical trials but may be approached in the near future. In order to understand cartilage tissue engineering as a new treatment option, an overview of the biological, engineering, and clinical challenges as well as the inherent constraints of the different treatment modalities are given in this paper

Membrane Potential Controls Adipogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Background: Control of stem cell behavior is a crucial aspect of developmental biology and regenerative medicine. While the functional role of electrophysiology in stem cell biology is poorly understood, it has become clear that endogenous ion flows represent a powerful set of signals by means of which cell proliferation, differentiation, and migration can be controlled in regeneration and embryonic morphogenesis. Methodology/Principal Findings: We examined the membrane potential (Vmem) changes exhibited by human mesenchymal stem cells (hMSCs) undergoing adipogenic (AD) and osteogenic (OS) differentiation, and uncovered a characteristic hyperpolarization of differentiated cells versus undifferentiated cells. Reversal of the progressive polarization via pharmacological modulation of transmembrane potential revealed that depolarization of hMSCs prevents differentiation. In contrast, treatment with hyperpolarizing reagents upregulated osteogenic markers. Conclusions/Significance: Taken together, these data suggest that the endogenous hyperpolarization is a functiona

Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes

Recent years have witnessed an increasing interest in neuron-glia communication. This interest stems from the realization that glia participates in cognitive functions and information processing and is involved in many brain disorders and neurodegenerative diseases. An important process in neuron-glia communications is astrocyte encoding of synaptic information transfer: the modulation of intracellular calcium dynamics in astrocytes in response to synaptic activity. Here, we derive and investigate a concise mathematical model for glutamate-induced astrocytic intracellular Ca2+ dynamics that captures the essential biochemical features of the regulatory pathway of inositol 1,4,5-trisphosphate (IP3). Starting from the well-known two-state Li-Rinzel model for calcium-induced-calcium release, we incorporate the regulation of the IP3 production and phosphorylation. Doing so we extended it to a three-state model (referred as the G-ChI model), that could account for Ca2+ oscillations triggered by endogenous IP3 metabolism as well as by IP3 production by external glutamate signals. Compared to previous similar models, our three-state models include a more realistic description of the IP3 production and degradation pathways, lumping together their essential nonlinearities within a concise formulation. Using bifurcation analysis and time simulations, we demonstrate the existence of new putative dynamical features. The cross-couplings between IP3 and Ca2+ pathways endows the system with self-consistent oscillator properties and favor mixed frequency-amplitude encoding modes over pure amplitude modulation ones. These and additional results of our model are in general agreement with available experimental data and may have important implications on the role of astrocytes in the synaptic transfer of information.Comment: 42 pages, 16 figures, 1 table. Figure filenames mirror figure order in the paper. Ending "S" in figure filenames stands for "Supplementary Figure". This article was selected by the Faculty of 1000 Biology: "Genevieve Dupont: Faculty of 1000 Biology, 4 Sep 2009" at http://www.f1000biology.com/article/id/1163674/evaluatio