Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor PPAR-γ. Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase (iNOS) 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) towards β-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.

The fabrication of hydrogel microstructures based upon poly(ethylene glycol) diacrylates, dimethacrylates, and tetraacrylates patterned photolithographically on silicon or glass substrates is described. A silicon/silicon dioxide surface was treated with 3-(trichlorosilyl)propyl methacrylate to form a self-assembled monolayer (SAM) with pendant acrylate groups. The SAM presence on the surface was verified using ellipsometry and time-of-flight secondary ion mass spectrometry. A solution containing an acrylated or methacrylated poly(ethylene glycol) derivative and a photoinitiator (2,2-dimethoxy-2-phenylacetophenone) was spin-coated onto the treated substrate, exposed to 365 nm ultraviolet light through a photomask, and developed with either toluene, water, or supercritical CO2. As a result of this process, three-dimensional, cross-linked PEG hydrogel microstructures were immobilized on the surface. Diameters of cylindrical array members were varied from 600 to 7 micrometers by the use of different photomasks, while height varied from 3 to 12 micrometers, depending on the molecular weight of the PEG macromer. In the case of 7 micrometers diameter elements, as many as 400 elements were reproducibly generated in a 1 mm2 square pattern. The resultant hydrogel patterns were hydrated for as long as 3 weeks without delamination from the substrate. In addition, micropatterning of different molecular weights of PEG was demonstrated. Arrays of hydrogel disks containing an immobilized protein conjugated to a pH sensitive fluorophore were also prepared. The pH sensitivity of the gel-immobilized dye was similar to that in an aqueous buffer, and no leaching of the dye-labeled protein from the hydrogel microstructure was observed over a 1 week period. Changes in fluorescence were also observed for immobilized fluorophore labeled acetylcholine esterase upon the addition of acetyl acholine.

This manuscript presents a microfabrication-derived approach for controlling mammalian cell-surface interactions. Poly(ethylene glycol)-diacrylate (PEG-DA) was patterned, in a process analogous to photolithography, to manufacture high-density arrays of micrometer-scale PEG hydrogel wells on glass. Individual wells consisted of PEG walls and glass attachment pads; thus, as a result of the biological inertness of PEG, microwell patterning created a highly ordered biointerface with modulating-cell or protein-repellent properties. Fabricated hydrogel microstrucures proved very effective in guiding and confining adhesion of transformed 3T3 fibroblasts and primary rat hepatocytes to defined regions on the glass substrate. PEG-patterned glass surfaces were selectively modified with collagen (type I) to induce hepatocyte attachment. Localization of the fluorescein-conjugated collagen within the glass attachment pads of PEG hydrogel microwells was visualized by fluorescence microscopy. Further surface analysis was performed by tapping mode atomic force microscopy conducted within individual PEG wells. Proteinmodified regions inside the wells had a root-mean-square roughness of 1.13 ( 0.03 nm compared to 0.7 ( 0.04 nm for alkylsilane-treated regions lacking protein. The cell occupancy of 96.7 ( 1.9% within the arrays of 30 × 30 µm individual wells was demonstrated for 3T3 fibroblasts. At the same time, cells remained effectively isolated in the individual PEG microwells. Primary hepatocytes attached and became fully confluent within the collagen-coated PEG after 24 h of incubation. Each 30 × 30 µm well contained one to three hepatocytes. Cells patterned on the surface remained viable after 24 h of incubation.

In this paper, we describe the development of an electrochemical DNA aptamer-based biosensor for detection of IFN-γ. A DNA hairpin containing IFN-γ-binding aptamer was thiolated, conjugated with Methylene Blue (MB) redox tag and immobilized on a gold electrode by self-assembly. Binding of IFN-γ caused the aptamer hairpin to unfold, pushing MB redox molecules away from the electrode and decreasing electron-transfer efficiency. The change in redox current was quantified using Square Wave Voltammetry (SWV) and was found to be highly sensitive to IFN-γ concentration. The limit of detection for optimized biosensor was 0.06 nM with linear response extending to 10 nM. This aptasensor was specific to IFN-γ in the presence of overabundant serum proteins. Importantly, the same aptasensor could be regenerated by disrupting aptamer-IFN-γ complex in urea buffer and reused multiple times. Unlike standard sandwich immunoassays, the aptasensor described here allowed to detect IFN-γ binding directly without the need for multiple washing steps and reagents. An electrochemical biosensor for simple and sensitive detection of IFN-γ demonstrated in this paper will have future applications in immunology, cancer research and infectious disease monitoring.

Biosensors are of great significance because of their capability to resolve a potentially large number of analytical problems and challenges in very diverse areas such as defense, homeland security, agriculture and food safety, environmental monitoring, medicine, pharmacology, industry, etc. The expanding role of biosensing in society and a real-world environment has led to an exponential growth of the R&D efforts around the world. The world market for biosensor devices, according to Global Industry Analysts, Inc., is expected to reach $12 billion by 2015. Such expedient growth is driven by several factors including medical and health problems, such as a growing population with a high risk of diabetes and obesity, and the rising incidence of chronic diseases such as heart disease, stroke, cancer, chronic respiratory diseases, tuberculosis, etc.; significant problems with environmental monitoring; and of course serious challenges in security and military applications and agriculture/food safety. A review paper in the biosensor technology area may be structured based on (i) the principles of detection, such as the type of transducer platform, bioanalytical principles (affinity or kinetic), and biorecognition elements origin/properties (i.e. antibodies, enzymes, cells, aptamers, etc.), and (ii) the application area. This review follows the latter strategy and focuses on the applications. This allows discussion on how different sensing strategies are brought to bear on the same problem and highlights advantages/disadvantages of these sensing strategies. Given the broad range of biosensor related applications, several particularly relevant areas of application were selected for review: biological threat agents, chemical threat agents, and medicine.

Primary hepatocytes are commonly used as liver surrogates in toxicology and tissue engineering fields, therefore, maintenance of functional hepatocytes in vitro is an important topic of investigation. This paper sought to characterize heparin-based hydrogel as a three-dimensional scaffold for hepatocyte culture. The primary rat hepatocytes were mixed with a prepolymer solution comprised of thiolated heparin and acrylated poly(ethylene glycol) (PEG). Raising the temperature from 25° to 37°C initiated Michael addition reaction between the thiol and acrylated moieties and resulted in formation of hydrogel with entrapped cells. Analysis of liver-specific products, albumin and urea, revealed that the heparin hydrogel was non-cytotoxic to cells and, in fact, promoted hepatic function. Hepatocytes entrapped in the heparin-based hydrogel maintained high levels of albumin and urea synthesis after three weeks in culture. Because heparin is known to bind growth factors, we incorporated hepatocyte growth factor (HGF) -an important liver signaling molecule -into the hydrogel. HGF release from heparin hydrogel matrix was analyzed using enzyme linked immunoassay (ELISA) and was shown to occur in a controlled manner with only 40% of GF molecules released after 30 days in culture. Importantly, hepatocytes cultured within HGF-containing hydrogels exhibited significantly higher levels of albumin and urea synthesis compared to cells cultured in the hydrogel alone. Overall, heparin-based hydrogel showed to be a promising matrix for encapsulation and maintenance of difficult-to-culture primary hepatocytes. In the future, we envision employing heparin-based hyrogels as matrices for in vitro differentiation of hepatocytes or stem cells and as vehicles for transplantation of these cells.

Exosomes are small (50-100 nm in diameter) vesicles secreted from various mammalian cells. Exosomes have been correlated with tumor antigens and anti-tumor immune responses and may represent cancer biomarkers. Herein, we report on the development of an aptamer-based electrochemical biosensor for quantitative detection of exosomes. Aptamers specific to exosome transmembrane protein CD63 were immobilized onto gold electrode surfaces and incorporated into a microfluidic system. Probing strands pre-labeled with redox moieties were hybridized onto aptamer molecules anchored on the electrode surface. In the presence of exosomes these beacons released probing strands with redox reporters causing electrochemical signal to decrease. These biosensors could be used to detect as few as 1×10(6) particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays relying on anti-CD63 antibodies. Given the importance of exosome-mediated signal transmission among cells, our study may represent an important step towards development of a simple biosensor that detects exosomes without washing or labeling steps in complex media.

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.