Microfabricated regular sieving structures hold great promise as an alternative to gels to improve biomolecule separation speed and resolution. In contrast to disordered gel porous networks, these regular structures also provide well-defined environments ideal for study of molecular dynamics in confining spaces. However, previous regular sieving structures have been limited for separation of long DNA molecules, and separation of smaller, physiologically-relevant macromolecules, such as proteins, still remains as a challenge. Here we report a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array (Anisotropic Nanofilter Array: ANA). The designed structural anisotropy in the ANA causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow sizebased separation of DNA and proteins as well as electrostatic separation of proteins were achieved, thus demonstrating the potential of the ANA as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system. Efficient methods of separating and purifying biomolecules from a complex mixture are of utmost importance in biology and biomedical engineering. Currently, nucleic acids and proteins are routinely separated based on size by gel filtration chromatography or by gel electrophoresis 1,2 . Both techniques use gelatinous materials that consist of a cross-linked, three-dimensional pore network, where the sieving interaction with the migrating macromolecules determines the separation efficiency 3,4 . Both gel-based techniques represent the current standard for size-based macromolecule separation. However poor separation resolution in gel filtration chromatography and difficult sample recovery with gel electrophoresis make neither method optimal in separating complex mixtures for downstream analysis 1 . Liquid and solid gelatinous materials have also been integrated in microchip-based *Correspondence should be addressed to Jongyoon Han [J. Han (email address: jyhan@mit.edu, Tel: 617-253-2290, Fax: 617-258-5846) systems for rapid separation of biomolecules (e.g., DNA, proteins and carbohydrates) with high resolution 5-7 . However, the foreign sieving matrices pose intrinsic difficulties for the integration of automated multi-step bioanalysis microsystems. Furthermore, these microchipbased systems are limited to analytical separation of biomolecules, due to the difficulty of harvesting purified biomolecules for downstream analysis.Recently, there has been great interest in switching from disordered porous gel media to patterned regular sieving structures, either by colloidal templating of self-assembled bead arrays 8,9 or by microfabrication techniques 10-15 . While significantly more efficient than gels in terms of separation speed and resolution, these regular sieving structures still largely resemble gels in the sense that separation is achieved by repeated sieving through multiple, identical "pores"....

The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1␤, TNF-␣, or mechanical compression injury in vitro and to interpret this release in the context of anabolic-catabolic shifts known to occur in cartilage in response to these insults in vitro and their implications in vivo. Bovine calf cartilage explants from 6 -12 animals were subjected to injurious compression, TNF-␣ (100 ng/ml), IL-1␤ (10 ng/ml), or no treatment and cultured for 5 days in equal volumes of medium. The pooled medium from each of these four conditions was labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time-of-flight instrument. Data were analysed by ProQuant for peptide identification and quantitation. k-means clustering and biological pathways analysis were used to identify proteins that may correlate with known cartilage phenotypic responses to such treatments. IL-1␤ and TNF-␣ treatment caused a decrease in the synthesis of collagen subunits (p < 0.05) as well as increased release of aggrecan G2 and G3 domains to the medium (p < 0.05). MMP-1, MMP-3, MMP-9, and MMP-13 were significantly increased by all treatments compared with untreated samples (p < 0.10). Increased release of proteins involved in innate immunity and immune cell recruitment were noted following IL-1␤ and TNF-␣ treatment, whereas increased release of intracellular proteins was seen most dramatically with mechanical compression injury. Proteins involved in insulin-like growth factor and TGF-␤ superfamily pathway modulation showed changes in pro-anabolic pathways that may represent early repair signals. At the systems level, two principal components were sufficient to describe 97% of the covariance in the data. A strong correlation was noted between the proteins released in response to IL-1␤ and TNF-␣; in contrast, mechanical injury resulted in both similarities and unique differ-

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5′-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5′-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Objective. To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣), by characterizing proteins lost to the medium from cartilage explant culture.Methods. Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL-1␤ or 100 ng/ml of TNF␣ or were subjected to uniaxial, radiallyunconfined injurious compression (50% strain; 100%/ second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel-based separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis.Results. More than 250 proteins were detected, including extracellular matrix (ECM) structural proteins, pericellular matrix proteins important in cell-cell interactions, and novel cartilage proteins CD109, platelet-derived growth factor receptor-like, angiopoietin-like 7, and adipocyte enhancer binding protein 1. IL-1␤ and TNF␣ caused increased release of chitinase 3-like protein 1 (CHI3L1), CHI3L2, complement factor B, matrix metalloproteinase 3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins, including Grp58, Grp78, ␣4-actinin, pyruvate kinase, and vimentin. Injurious compression also caused increased release and evidence of proteolysis of type VI collagen subunits, cartilage oligomeric matrix protein, and fibronectin.Conclusion. Overload compression injury caused a loss of cartilage integrity, including matrix damage and cell membrane disruption, which likely occurred through strain-induced mechanical disruption of cells and matrix. IL-1␤ and TNF␣ caused the release of proteins associated with an innate immune and stress response by the chondrocytes, which may play a role in host defense against pathogens or may protect cells against stress-induced damage.Osteoarthritis (OA) is characterized by cartilage degeneration, which results from an imbalance between matrix synthesis and matrix degradation. Development of OA secondary to traumatic joint injury occurs in ϳ15-75% of patients over followup periods of 14-22 years, equivalent to an average relative risk or odds ratio of between 3 and 20 of developing OA postinjury (for review, see ref. 1). Moreover, corrective surgery has little or no impact on the risk of developing OA following traumatic joint injury (1). In vitro models of joint injury have helped to understand the contribution of

Objective. To evaluate the effects of injurious compression on the biosynthesis of lubricin at different depths within articular cartilage and to examine alterations in structure and function of the articular surface following mechanical injury.Methods. Bovine cartilage explants were subdivided into level 1, with intact articular surface, and level 2, containing middle and deep zone cartilage. Following mechanical injury, lubricin messenger RNA (mRNA) levels were monitored by quantitative reverse transcriptase-polymerase chain reaction, and soluble or cartilage-associated lubricin protein was analyzed by Western blotting and immunohistochemistry. Cartilage morphology was assessed by histologic staining, and tissue functionality was assessed by friction testing.Results. Two days after injury, lubricin mRNA expression was up-regulated ϳ3-fold for level 1 explants and was down-regulated for level 2 explants. Lubricin expression in level 1 cartilage returned to control levels after 6 days in culture. Similarly, lubricin protein synthesis and secretion increased in response to injury for level 1 explants and decreased for level 2 cartilage. Histologic staining revealed changes in the articular surface of level 1 explants following injury, with respect to glycosaminoglycan and collagen content. Injured level 1 explants displayed an increased coefficient of friction relative to controls. Conclusion. Our findings indicate that increased lubricin biosynthesis appears to be an early transient response of surface-layer cartilage to injurious compression. However, distinct morphologic changes occur with injury that appear to compromise the frictional properties of the tissue.Osteoarthritis (OA) is characterized by the degeneration of articular cartilage, leading to matrix fibrillation, fissuring, and the development of lesions. In the final stages of the disease, erosion of cartilage leads to painful bone-on-bone contact. The etiology of OA is complex and involves multiple biochemical, biomechanical, and genetic factors in addition to aging (1-3). Cartilage injury in young individuals is a prominent predisposing factor leading to increased risk of the subsequent development of OA (4,5) and, as such, represents a discrete pathologic event. Damage to the meniscus or ligaments sustained during traumatic joint injury causes instability, subjecting articular cartilage to abnormal biomechanical forces and resulting in the release of mediators of inflammation (6). Several animal models of OA are thus based on the observation that joint instability, i.e., via anterior cruciate ligament transaction or perturbation of the meniscus (7), results in the rapid onset of articular cartilage degeneration with an OA-like phenotype. The initial events following joint injury are thought to be crucial, since surgical interventions to restore joint stability do not seem to reduce the risk of developing posttraumatic OA (8).

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [(3)H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha treatment while having no effect on IL-1beta treated cartilage explants.

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