High-speed imaging of supersaturated cavitation clouds and the vibration modes of the radiation surface of high-power transducers.

The vibration mode of the radiation surface of transducer (or structure of supersaturated cavitation cloud in thin liquid) is investigated experimentally by high-speed photography. The classification of saturated, supersaturated and undersaturated cavitation clouds was proposed, and a comparison was made between saturated and supersaturated cavitation cloud structures in liquid thin layers. The characteristics and formation mechanism of supersaturated cavitation cloud structure were investigated. Based on the close correspondence and rapid response between the distribution of supersaturated cavitation clouds and vibration modes of radiation surface, a new approach is proposed to measure the vibration mode of transducer operating at high power and large amplitude in real time.

Super enhancer regulation of cytokine-induced chemokine production in alcoholic hepatitis.

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.

Circular motion of submillimeter-sized acoustic bubbles attached to a boundary by high-speed image analysis.

The circular motion of submillimeter-sized bubbles attached to a boundary in an 18.5 kHz ultrasonic field are investigated experimentally by high-speed photography and image analysis. It is found that the vibration of gas bubbles with diameters of 0.2-0.4 mm is between spherical radial vibration and regular surface fluctuation. Different from the circular motion of suspended bubbles in water, the circular motion of gas bubbles attached to a boundary presents some new characteristics. These bubbles attached to a boundary (wandering bubbles) will rotate around a fixed bubble array (holding bubbles). Both the wondering bubbles and holding bubbles are "degas" bubbles. The primary Bjerknes force acting on wandering bubbles in the acoustic wave field and the secondary Bjerknes force between the wandering bubbles and the holding bubbles strongly affects the circular motion. The circling and residence behavior of gas bubbles is described and analyzed in detail, which is helpful to understand and improve industrial applications such as ultrasonic cleaning, sonochemical treatment, aeration and cavitation reduction.

Integrin ß1-enriched extracellular vesicles mediate monocyte adhesion and promote liver inflammation in murine NASH.

BACKGROUND & AIMS: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin ß1 (ITGß1), which promotes monocyte adhesion and liver inflammation in murine NASH. METHODS: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGß1 neutralizing antibody (ITGß1Ab) or control IgG isotype. RESULTS: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGß1. Furthermore, we showed that LPC treatment in hepatocytes activates ITGß1 and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGß1Ab. FFC-fed, ITGß1Ab-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGß1Ab reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGß1Ab treatment significantly ameliorated liver injury and fibrosis. CONCLUSIONS: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGß1-dependent mechanism. ITGß1Ab ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGß1 is a potential anti-inflammatory therapeutic strategy in human NASH. LAY SUMMARY: Herein, we report that a cell adhesion molecule termed integrin ß1 (ITGß1) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGß1 is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGß1 reduces liver inflammation, injury and fibrosis. Hence, ITGß1 inhibition may serve as a new therapeutic strategy for NASH.

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension.

BACKGROUND & AIMS: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. METHODS: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice. RESULTS: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel. CONCLUSIONS: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.

Fabrication of composite microfluidic devices for local control of oxygen tension in cell cultures.

Oxygen tension is a central component of the cellular microenvironment and can serve as a trigger for changes in cell phenotype and function. There is a strong need to precisely control and modulate oxygen tension in cell culture systems in order to more accurately model the physiology and pathophysiology observed in vivo. The objective of this paper was to develop a simple, yet effective strategy for local control of oxygen tension in microfluidic cell cultures. Our strategy relied on fabrication of microfluidic devices using oxygen-permeable and impermeable materials. This composite device was designed so as to incorporate regions of gas permeability into the roof of the cell culture chamber and was outfitted with a reservoir for the oxygen-consuming chemical pyrogallol. When assembled and filled with pyrogallol, this device allowed oxygen depletion to occur within a specific region of the microfluidic culture chamber. The geometry and dimensions of the hypoxic region inside a microfluidic chamber were controlled by features fabricated into the oxygen-impermeable layer. Oxygen tension as low as 0.5% could be achieved using this strategy. To prove the utility of this device, we demonstrated that hypoxia induced anaerobic metabolism in a group of liver cancer cells, and that neighboring cancer cells residing under normoxic conditions upregulated the expression of transporters for taking up lactate - a product of anaerobic respiration. The microfluidic devices described here may be broadly applicable for mimicking multiple physiological scenarios where oxygen tension varies on the length scale of tens of micrometers including the cancer microenvironment, liver zonation, and luminal microenvironment of the gut.

Riluzole induces LTD of spinal nociceptive signaling via postsynaptic GluR2 receptors.

PURPOSE: Riluzole - a major therapeutic medicine for patients with amyotrophic lateral sclerosis - reportedly has anti-nociceptive and anti-allodynic efficacies in neuropathic pain models. However, little is known about its effect on neurotransmission in the spinal superficial dorsal horn (SDH). The present study aims to investigate the effects of riluzole on the synaptic transmission of SDH nociceptive pathways in both physiological and pathological conditions. MATERIALS AND METHODS: Spinal nerve ligation was used to produce a neuropathic pain model. Mechanical allodynia behavior was assessed with Von Frey filaments. Riluzole's effects on nociceptive synaptic transmission under both physiological and pathological conditions were examined by patch-clamp recordings in rat SDH neurons. RESULTS: The principal findings of the present study are three-fold. First, we affirm that riluzole has a remarkable long-lasting analgesic effect on both in vitro and in vivo pathological pain models. Second, the prolonged inhibitory effects of riluzole on spinal nociceptive signaling are mediated by both presynaptic and postsynaptic mechanisms. Finally, endocytosis of post-synaptic GluR2 contributes to the riluzole-induced long-term depression (LTD) of the spinal nociceptive pathway. CONCLUSION: The present study finds that riluzole induces LTD of nociceptive signaling in the SDH and produces long-lasting anti-allodynia effects in nerve injury-induced neuropathic pain conditions via postsynaptic AMPA receptors associated with the endocytosis of GluR2.

Important Role of Intermolecular Interaction in Cobalt(II) Single-Ion Magnet from Single Slow Relaxation to Double Slow Relaxation.

Two cobalt complexes with similar structures were synthesized using quinoline-2-carboxylic acid (HL) as the ligand. Both complexes are six-coordinated in antitriangular prism coordination geometries. There are one and four molecule units per cell for 1 and 2, respectively, with nearest Co-Co distances of 7.129 and 5.855 Å, respectively, which lead to their intermolecular interactions zj'. Both complexes are field-induced single-ion magnets. Complex 1 shows single slow relaxation under Hdc = 1.5 kOe attributed to the moment reversal, while complex 2 shows double slow relaxation resulting from intermolecular dipolar interaction and moment reversal, respectively.

Adaptive Unscented Kalman Filter Phase Unwrapping Method and Its Application on Gaofen-3 Interferometric SAR Data.

Phase unwrapping (PU) is a key step in the reconstruction of digital elevation models (DEMs) and the monitoring of surface deformation from interferometric synthetic aperture radar (SAR, InSAR) data. In this paper, an improved PU method that combines an amended matrix pencil model, an adaptive unscented kalman filter (AUKF), an efficient quality-guided strategy based on heapsort, and a circular median filter is proposed. PU theory and the existing UKFPU method are covered. Then, the improved method is presented with emphasis on the AUKF and the circular median filter. AUKF has been well used in other fields, but it is for the first time applied to interferometric images PU, to the best of our knowledge. First, the amended matrix pencil model is used to estimate the phase gradient. Then, an AUKF model is used to unwrap the interferometric phase based on an efficient quality-guided strategy based on heapsort. Finally, the key results are obtained by filtering the results using a circular median. The proposed method is compared with the minimum cost network flow (MCF), statistical cost network flow (SNAPHU), regularized phase tracking technique (RPTPU), and UKFPU methods using two sets of simulated data and two sets of experimental GF-3 SAR data. The improved method is shown to yield the greatest accuracy in the interferometric phase maps compared to the methods considered in this paper. Furthermore, the improved method is shown to be the most robust to noise and is thus most suitable for PU of GF-3 SAR data in high-noise and low-coherence regions.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

Hepatocyte-Derived Lipotoxic Extracellular Vesicle Sphingosine 1-Phosphate Induces Macrophage Chemotaxis.

Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms.

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-ß. Chromatin IP assay of the PDGFR-ß promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion.

Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator-activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward ß-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.

Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury.

Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-ß1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-ß inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury.

Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels.

The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-ß1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.

Embryonic Stem Cells Cultured in Microfluidic Chambers Take Control of Their Fate by Producing Endogenous Signals Including LIF.

It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced ∼140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. Stem Cells 2016;34:1501-1512.

Functional imaging of neuron-astrocyte interactions in a compartmentalized microfluidic device.

Traditional approaches in cultivating neural cells in a dish without orienting their interactions have had only limited success in revealing neural network properties. To enhance the experimental capabilities of studying neural circuitry in vitro, we designed an experimental system combining concepts of micropatterned surfaces, microfluidic devices and genetically encoded biosensors. Micropatterning was used to position neurons and astrocytes in defined locations and guide interactions between the two cell types. Microfluidic chambers were placed atop micropatterned surfaces to allow delivery of different pharmacological agents or viral vectors to the desired cell types. In this device, astrocytes and neurons communicated through grooves molded into the floor of the microfluidic device. By combining microfluidics with genetically encoded calcium indicators as functional readouts, we further demonstrated the utility of this device for analyzing neuron-neuron and neuron-astrocyte interactions in vitro under both healthy and pathophysiological conditions. We found that both spontaneous and evoked calcium dynamics in astrocytes can be modulated by interactions with neurons. In the future, we foresee employing the microdevices described here for studying mechanisms of neurological disorders.

Erratum: Functional imaging of neuron-astrocyte interactions in a compartmentalized microfluidic device.

[This corrects the article DOI: 10.1038/micronano.2015.45.].

Upregulation of EMMPRIN (OX47) in Rat Dorsal Root Ganglion Contributes to the Development of Mechanical Allodynia after Nerve Injury.

Matrix metalloproteinases (MMPs) are widely implicated in inflammation and tissue remodeling associated with various neurodegenerative diseases and play an important role in nociception and allodynia. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) plays a key regulatory role for MMP activities. However, the role of EMMPRIN in the development of neuropathic pain is not clear. Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunofluorescence were performed to determine the changes of messenger RNA and protein of EMMPRIN/OX47 and their cellular localization in the rat dorsal root ganglion (DRG) after nerve injury. Paw withdrawal threshold test was examined to evaluate the pain behavior in spinal nerve ligation (SNL) model. The lentivirus containing OX47 shRNA was injected into the DRG one day before SNL. The expression level of both mRNA and protein of OX47 was markedly upregulated in ipsilateral DRG after SNL. OX47 was mainly expressed in the extracellular matrix of DRG. Administration of shRNA targeted against OX47 in vivo remarkably attenuated mechanical allodynia induced by SNL. In conclusion, peripheral nerve injury induced upregulation of OX47 in the extracellular matrix of DRG. RNA interference against OX47 significantly suppressed the expression of OX47 mRNA and the development of mechanical allodynia. The altered expression of OX47 may contribute to the development of neuropathic pain after nerve injury.

Microfluidic co-cultures with hydrogel-based ligand trap to study paracrine signals giving rise to cancer drug resistance.

Targeted cancer therapies are designed to deactivate signaling pathways used by cancer cells for survival. However, cancer cells are often able to adapt by activating alternative survival pathways, thereby acquiring drug resistance. An emerging theory is that autocrine or paracrine growth factor signaling in the cancer microenvironment represent an important mechanism of drug resistance. In the present study we wanted to examine whether paracrine interactions between groups of melanoma cells result in resistance to vemurafenib - an FDA approved drug targeting the BRAF mutation in metastatic melanoma. We used a vemurafenib-resistant melanoma model which secretes fibroblast growth factor (FGF)-2 to test our hypothesis that this is a key paracrine mediator of resistance to vemurafenib. Sensitive cells treated with media conditioned by resistant cells did not protect from the effects of vemurafenib. To query paracrine interactions further we fabricated a microfluidic co-culture device with two parallel compartments, separated by a 100 µm wide hydrogel barrier. The gel barrier prevented resorting/contact of cells while permitting paracrine cross-talk. In this microfluidic system, sensitive cells did become refractive to the effects of vemurafenib when cultured adjacent to resistant cells. Importantly, incorporation of FGF-2 capture probes into the gel barrier separating the two cell types prevented onset of resistance to vemurafenib. Microfluidic tools described here allow for more sensitive analysis of paracrine signals, may help better understand signaling in the cancer microenvironment and may enable development of more effective cancer therapies.