Integrated Control of Microfluidics – Application in Fluid Routing, Sensor Synchronization, and Real-Time Feedback Control

Microfluidic applications range from combinatorial chemical synthesis to high-throughput screening, with platforms integrating analog perfusion components, digitally controlled microvalves, and a range of sensors that demand a variety of communication protocols. A comprehensive solution for microfluidic control has to support an arbitrary combination of microfluidic components and to meet the demand for easy-to-operate system as it arises from the growing community of unspecialized microfluidics users. It should also be an easy to modify and extendable platform, which offer an adequate computational resources, preferably without a need for a local computer terminal for increased mobility. Here we will describe several implementation of microfluidics control technologies and propose a microprocessor-based unit that unifies them. Integrated control can streamline the generation process of complex perfusion sequences required for sensor-integrated microfluidic platforms that demand iterative operation procedures such as calibration, sensing, data acquisition, and decision making. It also enables the implementation of intricate optimization protocols, which often require significant computational resources. System integration is an imperative developmental milestone for the field of microfluidics, both in terms of the scalability of increasingly complex platforms that still lack standardization, and the incorporation and adoption of emerging technologies in biomedical research. Here we describe a modular integration and synchronization of a complex multicomponent microfluidic platform

Transcriptional Regulation of Human and Rat Hepatic Lipid Metabolism by the Grapefruit Flavonoid Naringenin: Role of PPARα, PPARγ and LXRα

Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARα, PPARγ, and LXRα. We show it activates the ligand-binding domain of both PPARα and PPARγ, while inhibiting LXRα in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRα, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARα co-activator, PGC1α. The flavonoid activates PPAR response element (PPRE) while suppressing LXRα response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRα-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRα inhibitor

Multiple effects of silymarin on the hepatitis C virus lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell

Effect of SARS-CoV-2 proteins on vascular permeability.

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein–protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While vali-dating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-1

Enhancement of Naringenin Bioavailability by Complexation with Hydroxypropoyl-β-Cyclodextrin

The abundant flavonoid aglycone, naringenin, which is responsible for the bitter taste in grapefruits, has been shown to possess hypolipidemic and anti-inflammatory effects both in vitro and in vivo. Recently, our group demonstrated that naringenin inhibits hepatitis C virus (HCV) production, while others demonstrated its potential in the treatment of hyperlipidemia and diabetes. However, naringenin suffers from low oral bioavailability critically limiting its clinical potential. In this study, we demonstrate that the solubility of naringenin is enhanced by complexation with β-cyclodextrin, an FDA approved excipient. Hydroxypropoyl-β-cyclodextrin (HPβCD), specifically, increased the solubility of naringenin by over 400-fold, and its transport across a Caco-2 model of the gut epithelium by 11-fold. Complexation of naringenin with HPβCD increased its plasma concentrations when fed to rats, with AUC values increasing by 7.4-fold and Cmax increasing 14.6-fold. Moreover, when the complex was administered just prior to a meal it decreased VLDL levels by 42% and increased the rate of glucose clearance by 64% compared to naringenin alone. These effects correlated with increased expression of the PPAR co-activator, PGC1α in both liver and skeletal muscle. Histology and blood chemistry analysis indicated this route of administration was not associated with damage to the intestine, kidney, or liver. These results suggest that the complexation of naringenin with HPβCD is a viable option for the oral delivery of naringenin as a therapeutic entity with applications in the treatment of dyslipidemia, diabetes, and HCV infection.National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (K01DK080241)Harvard Clinical Nutrition Research Center (P30-DK040561)European Research Council (Starting Grant (TMIHCV 242699))Massachusetts General Hospital (BioMEMS Resource Center (P41 EB-002503))Alexander Silberman Institute of Life Science

HCV Causes Chronic Endoplasmic Reticulum Stress Leading to Adaptation and Interference with the Unfolded Protein Response

BACKGROUND: The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance. METHODS AND FINDINGS: The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2-5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells. CONCLUSIONS: Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways

Propofol induces ERK-dependant expression of c-Fos and Egr-1 in neuronal cells

This study explored the effects of propofol on c-Fos and Egr-1 in neuroblastoma (N2A) cells. We demonstrate that propofol induced the expression of c-Fos and Egr-1 within 30 and 60 min of exposure time. At 16.8 μM concentration, propofol induced a 6 and 2.5-fold expression of c-Fos and Egr-1, respectively. However, at concentrations above 100 μM, propofol failed to induce expression of c-Fos or Egr-1. Propofol-induced c-Fos and Egr-1 transcription was unaffected by bicuculline, a γ-aminobutyric acid-A receptor antagonist, but was abolished by PD98059, a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor. Our study shows that clinically relevant concentrations of propofol induce c-Fos and Egr-1 expression through an extracellular signalregulated kinase mediated and γ-aminobutyric acid-A independent pathway

Laser-guided direct writing for three-dimensional tissue engineering

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis.status: publishe