High-throughput organ-on-chip platform with integrated programmable fluid flow and real-time sensing for complex tissue models in drug development workflows.

Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technology provides advanced culture capabilities to generate physiologically appropriate, human-based tissue in vitro, therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-standard form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry standard plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiologically relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addition, we integrate electrical sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiologically-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.

[Can the addition of clonidine improve the analgesic efficacy of low dose intrathecal morphine? A randomised double-blind trial]. / Verbessert der Zusatz von Clonidin zur Spinalanästhesie die analgetische Wirkung niedrig dosierten intrathekalen Morphins? Eine randomisierte doppelblinde Studie.

OBJECTIVE: To evaluate the influence of intrathecal clonidine on spinal morphine analgesia and adverse effects after major orthopaedic surgery. METHODS: The study was approved by the local Ethics Committee.After written informed consent, 45 ASA I-III patients scheduled for hip or knee replacement were included. Patients were randomly allocated to receive either placebo, 0.1 mg morphine or 0.1 mg morphine+50 microg clonidine in addition to 15 mg bupivacaine intrathecally. The primary outcome parameter was the time to first opioid request. Statistical differences were calculated with U-test or Fisher's exact test. RESULTS: Clonidine did not result in a significant improvement of postoperative analgesia. The mean time until first opioid request was for placebo 10.3+/-7.9 h, for 0.1 mg morphine 23.0+/-3.9 h and for 0.1 mg morphine+ 50 microg clonidine 21+/-6.9 h, respectively. Clonidine significantly increased the rate of adverse effects. CONCLUSION: Our trial did not confirm an improved analgesia with the combination of intrathecal morphine and clonidine. Due to increased adverse effects the combination of intrathecal clonidine and morphine does not seem to be a reasonable alternative in the management of postoperative pain after orthopaedic surgery.

Validation of a new algorithm for the BPM-100 electronic oscillometric office blood pressure monitor.

BACKGROUND: To test the accuracy of a new algorithm for the BPM-100, an automated oscillometric blood pressure (BP) monitor, using stored data from an independently conducted validation trial comparing the BPM-100(Beta) with a mercury sphygmomanometer. DESIGN: Raw pulse wave and cuff pressure data were stored electronically using embedded software in the BPM-100(Beta), during the validation trial. The 391 sets of measurements were separated objectively into two subsets. A subset of 136 measurements was used to develop a new algorithm to enhance the accuracy of the device when reading higher systolic pressures. The larger subset of 255 measurements (three readings for 85 subjects) was used as test data to validate the accuracy of the new algorithm. METHODS: Differences between the new algorithm BPM-100 and the reference (mean of two observers) were determined and expressed as the mean difference +/- SD, plus the percentage of measurements within 5, 10, and 15 mmHg. RESULTS: The mean difference between the BPM-100 and reference systolic BP was -0.16 +/- 5.13 mmHg, with 73.7% < or = 5 mmHg, 94.9% < or = 10 mmHg and 98.8% < or = 15 mmHg. The mean difference between the BPM-100 and reference diastolic BP was -1.41 +/- 4.67 mmHg, with 78.4% < or = 5 mmHg, 92.5% < or = 10 mmHg, and 99.2% < or = 15 mmHg. These data improve upon that of the BPM-100(Beta) and pass the AAMI standard, and 'A' grade BHS protocol. CONCLUSION: This study illustrates a new method for developing and testing a change in an algorithm for an oscillometric BP monitor utilizing collected and stored electronic data and demonstrates that the new algorithm meets the AAMI standard and BHS protocol.

Wnt signalling: antagonistic Dickkopfs.

Dickkopf proteins are secreted antagonists of the Wnt cell signalling molecules, which have a novel mode of action. Dickkopf1 binds to the LRP5/6 Wnt co-receptor and prevents the formation of active Wnt--Frizzled--LRP5/6 receptor complexes, thus blocking the canonical Wnt--beta-catenin pathway.

Swift is a novel BRCT domain coactivator of Smad2 in transforming growth factor beta signaling.

Transforming growth factor beta (TGFbeta) signaling is transduced via Smad2-Smad4-DNA-binding protein complexes which bind to responsive elements in the promoters of target genes. However, the mechanism of how the complexes activate the target genes is unclear. Here we identify Xenopus Swift, a novel nuclear BRCT (BRCA1 C-terminal) domain protein that physically interacts with Smad2 via its BRCT domains. We examine the activity of Swift in relation to gene activation in Xenopus embryos. Swift mRNA has an expression pattern similar to that of Smad2. Swift has intrinsic transactivation activity and activates target gene transcription in a TGFbeta-Smad2-dependent manner. Inhibition of Swift activity results in the suppression of TGFbeta-induced gene transcription and defective mesendoderm development. Blocking Swift function affects neither bone morphogenic protein nor fibroblast growth factor signaling during early development. We conclude that Swift is a novel coactivator of Smad2 and that Swift has a critical role in embryonic TGFbeta-induced gene transcription. Our results suggest that Swift may be a general component of TGFbeta signaling.

Gene expression in the embryonic Xenopus liver.

In recent years, significant progress has been made in uncovering the molecular basis of endoderm specification in Xenopus. Much less is understood, however, about endodermal patterning and how endoderm-derived organs such as the liver are formed. Progress has been hampered by the lack of good molecular markers of presumptive liver tissue. Here, we have examined the embryonic expression of a number of marker genes during liver organogenesis, including the transcription factors hex, sox17alpha, and hnf3beta, as well as a number of proteins specific to the adult liver. Interestingly, sox17alpha appears to specifically mark the gall bladder precursors. At 7 days of development expression of the liver differentiation markers albumin, alpha1-microglobulin/bikunin precursor, fibrinogen, transferrin and transthyretin is restricted to the differentiating liver bud. Surprisingly, however, at 3 days of development most of these genes have a more widespread endodermal expression pattern. In addition to expression in the undifferentiated liver bud they were expressed extensively throughout the presumptive intestinal tissue, which may reflect some general feature of how the hepatic gene program is developmentally regulated.

Nonradioactive in situ hybridization to xenopus tissue sections.

We describe a protocol for the localization of specific messenger RNAs in Xenopus laevis embryo tissue sections using a nonradioactive detection method. After fixation, embryos are embedded in paraffin wax, sectioned, mounted on slides, and subjected to a series of prehybridization treatments which improve the accessibility of the probe to the target mRNA and reduce nonspecific binding. These treatments are followed by hybridization in situ with single-stranded antisense RNA probe generated by in vitro transcription and labeled with digoxigenin. The hybridization products are detected with preabsorbed alkaline phosphatase-coupled digoxigenin antibody and subsequently localized using a chromogenic substrate that generates a colored precipitate at the hybridization site. The nonradioactive in situ hybridization method we describe is reproducible and has a detection sensitivity akin to those methods that use antisense RNA probes labeled with radioisotopes; however, it is faster, safer, and easier to perform. Sectioning of prestained whole-mount X. laevis embryos does not always show the complete expression pattern of many genes, particularly those in deep endodermal structures, due to inadequate probe penetration. Therefore thorough analysis of gene expression patterns often requires in situ hybridization on presectioned material whereby probe has equal accessibility to all tissue.

eFGF and its mode of action in the community effect during Xenopus myogenesis.

The community effect is an interaction among a group of many nearby precursor cells, necessary for them to maintain tissue-specific gene expression and differentiate co-ordinately. During Xenopus myogenesis, the muscle precursor cells must be in group contact throughout gastrulation in order to develop into terminally differentiated muscle. The molecular basis of this community interaction has not to date been elucidated. We have developed an assay for testing potential community factors, in which isolated muscle precursor cells are treated with a candidate protein and cultured in dispersion. We have tested a number of candidate factors and we find that only eFGF protein is able to mediate a community effect, stimulating stable muscle-specific gene expression in demonstrably single muscle precursor cells. In contrast, Xwnt8, bFGF, BMP4 and TGF(&bgr;)2 do not show this capacity. We show that eFGF is expressed in the muscle precursor cells at the right time to mediate the community effect. Moreover, the time when the muscle precursor cells are sensitive to eFGF corresponds to the period of the endogenous community effect. Finally, we demonstrate that FGF signalling is essential for endogenous community interactions. We conclude that eFGF is likely to mediate the community effect in Xenopus myogenesis.

Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis.

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.

Searching for a general anaesthesia protocol for rapid detoxification from opioids.

The technique for ultra rapid opioid detoxification is designed to shorten the detoxification period by precipitating withdrawal by the administration of opioid antagonists such as naloxone or naltrexone. This procedure is performed under deep sedation or general anaesthesia to ensure that the patient does not consciously experience the acute withdrawal phase. This strategy has aroused controversy regarding the risk of sedation or anaesthesia in this situation. In the present study, ultra rapid opioid detoxification was carried out in 12 opiate-addicted patients by infusion of naloxone 4 mg for a period of 5 h using controlled ventilation during general anaesthesia, induced and maintained with midazolam, propofol and atracurium. Invasive cardiovascular and respiratory monitoring was performed, and withdrawal signs were evaluated using a graduated scale. Anaesthesia was maintained for another hour after the completion of the naloxone infusion. The validity of this anaesthesia protocol was confirmed by the relative lack of change in the patients' haemodynamic values associated with mild signs of withdrawal.

Single cells can sense their position in a morphogen gradient.

Xenopus blastula cells show a morphogen-like response to activin by expressing different genes according to the concentration of activin to which they are exposed. To understand how cells recognize their position in a concentration gradient, it is essential to know whether each cell responds individually to activin concentration. An alternative idea, proposed by previous work, is that cells need to interact with their neighbours to generate a concentration-related response. To distinguish between these ideas, we have cultured blastula cells under conditions which provide different degrees of contact with other cells, allowing nil to maximum communication with their neighbours. The cultures include cells attached to fibronectin and cells resting unattached on an agarose base. The cultures also include cells that have no contact with any cell except their clonal progeny, cells that have lateral contact to neighbouring cells, and cells that are completely enveloped by other cells in a reaggregate. We have used RNase protection and in situ hybridization to assay the expression of the activin-responsive Xenopus genes Xbra, Xgsc, Xeomes, Xapod, Xchordin, Mix1, Xlim1 and Cerberus. We find no difference in gene expression between cells attached to fibronectin and those unattached on agarose. Most importantly, we find that cells respond to activin in a concentration-related way irrespective of their degree of contact with other cells. Therefore interaction among cells is not required for the interpretation of morphogen concentration, at least in the case of the early genes studied here. We conclude that isolated blastula cells can sense and respond individually to activin by expressing genes in a concentration-dependent way.

Regulation of Wnt signaling by Sox proteins: XSox17 alpha/beta and XSox3 physically interact with beta-catenin.

Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17 beta. Ectopic expression of XSox17 beta ventralizes embryos by inhibiting the Wnt pathway downstream of beta-catenin but upstream of the Wnt-responsive gene Siamois. XSox17 beta also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and beta-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses beta-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17 beta maps to a region C-terminal to the HMG box; this region of XSox17 beta physically interacts with the Armadillo repeats of beta-catenin. Two additional Sox proteins, XSox17 alpha and XSox3, likewise bind to beta-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.

Differential expression of VegT and Antipodean protein isoforms in Xenopus.

The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. We conclude that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors.

Anterior endomesoderm specification in Xenopus by Wnt/beta-catenin and TGF-beta signalling pathways.

In Xenopus, XHex and cerberus are early marker genes of the anterior endomesoderm (AE), a subset of endoderm cells fated to form the liver and foregut and implicated in head induction. Using XHex and cerberus as markers we have examined the signals underlying AE induction. We show that the AE is specified by the early blastula in the absence of mesodermal signals but that cell-cell contact between presumptive AE cells is required. In overexpression experiments maternal Wnt/beta-catenin and TGF-beta signals (Vg1, Xnr1-2) can induce ectopic XHex and cerberus. Inhibiting these pathways with dominant interfering signalling components blocks endogenous XHex and cerberus expression. We assess the role of signals from the organiser and show that the BMP antagonists noggin and chordin are important for maintaining XHex and cerberus expression. Finally, ventral injection of XHex mRNA can induce ectopic cerberus. Our results indicate that endodermal and mesodermal patterning are closely coordinated and that the AE is likely to be specified by the combined action of dorsal Wnt/beta-catenin signals and endoderm-specific factors mediated by TGF-beta signalling. These results provide a starting point for understanding the molecular events underlying the progressive determination of endodermally derived organs, such as the liver and foregut.

Activin has direct long-range signalling activity and can form a concentration gradient by diffusion.

BACKGROUND: Activin has strong mesoderm-inducing properties in the early Xenopus embryo, and has a long-range signalling activity that activates genes in cells distant from a source in a concentration-dependent way. It has not yet been established what mechanism of signal transmission accounts for this and other examples of long-range signalling in vertebrates. Nor is it known whether activin itself acts on distant cells or whether other kinds of molecules are used for long-range signalling. Here we have used a well characterised model system, involving animal caps of Xenopus blastulae treated with activin or transforming growth factor beta, to analyze some fundamental properties of long-range signalling and of the formation of a morphogen gradient. RESULTS: We find that cells distant from the source of activin require functional activin receptors to activate Xbrachyury, a result suggesting that activin itself acts directly on distant cells and that other secondary signalling molecules are not required. We also find that the signals can be transmitted across a tissue that cannot respond to it; this argues against a relay process. We provide direct evidence that labelled activin forms a concentration gradient emanating from its source and extending to the distant cells that express Xbrachyury. Lastly, we show that there is no inherent polarity in the responding tissue that influences either the direction or rate of signalling. CONCLUSIONS: The long-range signalling mechanism by which activin initiates the transcription of genes in a concentration-dependent manner depends on a process of rapid diffusion and the establishment of an activin gradient across the tissue. It cannot be explained by a relay or wave propagation mechanism. Activin itself is the signalling molecule to which distant cells respond.

The KH domain protein encoded by quaking functions as a dimer and is essential for notochord development in Xenopus embryos.

Mutations in the mouse indicate that quaking gene function is essential for both embryogenesis and for development of the nervous system. Recent isolation of the mouse quaking gene identified a putative RNA-binding protein containing a single KH domain. We have previously isolated the Xenopus homolog of quaking, Xqua, and shown that the sequence is highly conserved through evolution. Here, we report experimental data on the biochemical function of the quaking protein and its role during development. We demonstrate that the quaking protein expressed during early embryogenesis, pXqua357, can bind RNA in vitro, and we have mapped the regions of the protein that are essential for RNA binding. We present evidence that pXqua can form homodimers and that dimerization may be required for RNA binding. Oocyte injection experiments show that pXqua357 is located in both the nucleus and cytoplasm. In the Xenopus embryo, Xqua is first expressed during gastrulation in the organizer region and its derivative, the notochord. In later stage embryos, Xqua is expressed in a number of mesodermal and neural tissues. We demonstrate that disruption of normal Xqua function, by overexpression of a dominant inhibitory form of the protein, blocks notochord differentiation. Xqua function appears to be required for the accumulation of important mRNAs such as Xnot, Xbra, and gsc. These results indicate an essential role for the quaking RNA-binding protein during early vertebrate embryogenesis.

Cell-cell signalling: frog frizbees.

The recent discovery that Frizzled proteins are receptor for Wnts has been quickly followed by the identification of a secreted protein, Frzb, that is related to Frizzled, expressed by the Spemann organizer in frog embryos and can bind to and antagonize Wnt developmental signalling molecules.

Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein.

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3' UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm.

Although axons are generally considered to lack the ability to synthesize proteins, the Mauthner axon (M-axon) of the goldfish has been reported to contain some of the basic components of the translational machinery, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and ribosomes. To determine if the M-axon also contains mRNA, we isolated samples of M-axoplasm free of glial contamination as demonstrated by the absence of glial-specific mRNA and protein. Reverse transcription-polymerase chain reaction (RT-PCR) of M-axoplasmic cDNA in the presence of primers for the goldfish medium-weight neurofilament (NF-M) gene produced a single product of the expected length for RT-PCR amplification of goldfish NF-M mRNA. This mRNA might direct protein synthesis of NF-M within the M-axoplasm.