ROOT AERENCHYMA DEVELOPMENT IN SPARTINA PATENS IN RESPONSE TO FLOODING

Root aerenchyma, developed in response to flooding, was measured as specific gravity in previously existing (old) and newly developed (new) roots of Spartina patens in a 25-day greenhouse experiment. Root specific gravity was related to porosity (fractional volume of aerenchyma): porosity = 1.026 – 0.969 × specific gravity, over a wide range of specific gravities (0.5 to 0.9). The specific gravity of flooded roots (new and old) decreased from 0.78 to 0.58 between day 5 and 25, while the specific gravity of old roots that remained drained did not change. After 5 days growth, newly produced roots were found to have less aerenchyma than their older counterparts, but after 25 days the specific gravity of new roots equaled that of old roots. In addition, flooding increased mortality of preexisting roots and inhibited growth of new roots.     

Mov10 and APOBEC3G Localization to Processing Bodies Is Not Required for Virion Incorporation and Antiviral Activity

Mov10 and APOBEC3G (A3G) localize to cytoplasmic granules called processing bodies (P bodies), incorporate into human immunodeficiency virus type 1 (HIV-1) virions, and inhibit viral replication. The functional relevance of Mov10/A3G P-body localization to virion incorporation and antiviral activity has not been fully explored. We found that a helicase V mutant of Mov10 exhibits significantly reduced localization to P bodies but still efficiently inhibits viral infectivity via virion incorporation. Disruption of the P bodies by DDX6 knockdown also confirmed Mov10 antiviral activity without P-body localization. In addition, overexpression of SRP19, which binds to 7SL RNA, depleted A3G from P bodies but did not affect its virion incorporation. Sucrose gradient sedimentation assays revealed that the majority of Mov10, A3G, HIV-1 RNA, and Gag formed high-molecular-mass (HMM) complexes that are converted to low-molecular-mass (LMM) complexes after RNase A treatment. In contrast, the P-body markers DCP2, LSM1, eIF4e, DDX6, and AGO1 were in LMM complexes, whereas AGO2, an effector protein of the RNA-induced silencing complex that localizes to P bodies, was present in both LMM and HMM complexes. Depletion of AGO2 indicated that RNA-induced silencing function is required for Mov10''s ability to reduce Gag expression upon overexpression, but not its virion incorporation or effect on virus infectivity. We conclude that the majority of Mov10 and A3G are in HMM complexes, whereas most of the P-body markers are in LMM complexes, and that virion incorporation and the antiviral activities of Mov10 and A3G do not require their localization to P bodies.     

IL-12 attenuates bleomycin-induced pulmonary fibrosis

Interleukin (IL)-12 is a potent inducer of interferon (IFN)-gamma. We postulated that IL-12 would attenuate bleomycin-induced pulmonary fibrosis. To test this hypothesis, we administered IL-12 or murine serum albumin to bleomycin-treated mice by daily intraperitoneal injection until day 12. Mice treated with IL-12 demonstrated decreased hydroxyproline levels compared with control treated mice. Furthermore, administration of IL-12 led to a time-dependent increase in both lung and bronchoalveolar lavage fluid IFN-gamma. The antifibrotic effect of IL-12 could be attenuated with simultaneous administration of neutralizing anti-IFN-gamma antibodies. These findings support the notion that IL-12 attenuates bleomycin-induced pulmonary fibrosis via modulation of IFN-gamma production.     

Obesity and Weight Control Practices in 2000 Among Veterans Using VA Facilities

Objective: To examine obesity prevalence and weight control practices among veterans who use Department of Veterans Affairs (VA) medical facilities (VA users). Research Methods and Procedures: Data from the 2000 Behavioral Risk Factor Surveillance System, a telephone survey of 184, 450 adults, were analyzed. Outcome measures included BMI, weight control practices (the intent to manage weight, and diet and physical activity patterns), and receipt of professional weight control advice. Results: Of VA users, 44% were overweight and 25% were obese. After controlling for demographic factors, VA users were somewhat less likely to be overweight (odds ratio, 0.86; 95% confidence interval, 0.74 to 1.00) but equally likely to be obese (odds ratio, 1.08; 95% confidence interval, 0.92 to 1.27), compared with non‐VA users. Among obese VA users, 75% reported trying to lose weight, and another 17% reported trying to maintain weight. Of these, only 40% decreased both calorie and fat intake. Only 27% of obese VA users who reported increasing exercise to lose weight followed recommendations for regular and sustained physical activity. Of obese VA users, 59% were inactive or irregularly active. Only 51% of obese VA users received professional advice to lose weight. Obese VA users were more likely than obese non‐VA users to report trying to lose weight, modifying diet to lose weight by decreasing both calories and fat intake, and receiving professional weight control advice. Discussion: Interventions for weight management programs in VA facilities need to take into account the high prevalence of overweight/obesity among VA users and should emphasize effective weight control practices.     

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).     

CXCR3/CXCR3 ligand biological axis impairs RENCA tumor growth by a mechanism of immunoangiostasis

Metastatic renal cell carcinoma (RCC) responds poorly to chemo- or radiation therapy but appears to respond to systemic immunotherapy (i.e., IL-2 and/or IFN-alpha), albeit with only 5-10% durable response. The CXCR3/CXCR3 ligand biological axis plays an important role in mediating type 1 cytokine-dependent cell-mediated immunity, which could be beneficial for attenuating RCC if optimized. We found that systemic IL-2 induced the expression of CXCR3 on circulating mononuclear cells but impaired the CXCR3 ligand chemotactic gradient from plasma to tumor by increasing circulating CXCR3 ligand levels in a murine model of RCC. Moreover, the antitumor effect of systemic IL-2 was CXCR3-dependent, as IL-2 failed to inhibit tumor growth and angiogenesis in CXCR3-/- mice. We hypothesized that the immunotherapeutic effect of the CXCR3/CXCR3 ligand biological axis could be optimized by first priming with systemic IL-2 to induce CXCR3 expression on circulating mononuclear cells followed by enhancing the intratumor CXCR3 ligand levels to establish optimal CXCR3-dependent chemotactic gradient. We found that combined systemic IL-2 with an intratumor CXCR3 ligand (CXCL9) lead to significantly greater reduction in tumor growth and angiogenesis, increased tumor necrosis, and increased intratumor infiltration of CXCR3+ mononuclear cells, as compared with either IL-2 or CXCL9 alone. The enhanced antitumor effect of the combined strategy was associated with a more optimized CXCR3-dependent chemotactic gradient and increased tumor-specific immune response. These data suggest that the combined strategy of systemic IL-2 with intratumor CXCR3 ligand is more efficacious than either strategy alone for reducing tumor-associated angiogenesis and augmenting tumor-associated immunity, the concept of immunoangiostasis.     

Fluid shear regulates the kinetics and molecular mechanisms of activation-dependent platelet binding to colon carcinoma cells

This study was undertaken to investigate the kinetics and molecular requirements of platelet binding to tumor cells in bulk suspensions subjected to a uniform linear shear field, using a human colon adenocarcinoma cell line (LS174T) as a model. The effects of shear rate (20-1000 s(-1)), shear exposure time (30-300 s), shear stress (at constant shear rate by adjusting the viscosity of the medium from 1.3-2.6 cP), cell concentration, and platelet activation on platelet-LS174T heteroaggregation were assessed. The results indicate that hydrodynamic shear-induced collisions augment platelet-LS174T binding, which is further potentiated by thrombin/GPRP-NH(2). Peak adhesion efficiency occurs at low shear and decreases with increasing shear. Intercellular contact duration is the predominant factor limiting heteroaggregation at shear rates up to 200 s(-1), whereas these interactions become shear stress-sensitive at > or = 400 s(-1). Heteroaggregation increases with platelet concentration due to an elevation of the intercellular collision frequency, whereas adhesion efficiency remains nearly constant. Moreover, hydrodynamic shear affects the receptor specificity of activation-dependent platelet binding to LS174T cells, as evidenced by the transition from a P-selectin-independent/Arg-Gly-Asp (RGD)-dependent process at 100 s(-1) to a P-selectin/alpha(IIb)beta(3)-dependent interaction at 800 s(-1). This study demonstrates that platelet activation and a fluid-mechanical environment representative of the vasculature affect platelet-tumor cell adhesive interactions pertinent to the process of blood-borne metastasis.     

The role of peroxisome proliferator-activated receptor-beta/delta in epithelial cell growth and differentiation

The physiological and pharmacological roles of peroxisome proliferator-activated receptor-beta (PPARbeta-also referred to as PPARdelta) are just beginning to emerge. It has recently become clear that PPARbeta has a function in epithelial tissues, but controversy exists due to inconsistencies in the literature. There is strong evidence that ligand activation of PPARbeta can induce terminal differentiation of keratinocytes, with a concomitant inhibition of cell proliferation. However, the role of PPARbeta in keratinocyte-specific apoptosis is less clear. Additionally, the role of PPARbeta in colonic epithelium remains unclear due to conflicting evidence suggesting that ligand activation of PPARbeta can potentiate, as well as attenuate, intestinal cancer. Recent studies revealed that ligand activation of PPARbeta can induce fatty acid catabolism in skeletal muscle and is associated with improved insulin sensitivity, attenuated weight gain and elevated HDL levels thus demonstrating promising potential for targeting PPARbeta for treating obesity, dyslipidemias and type 2 diabetes. Therefore, it becomes critical to determine the safety of PPARbeta ligands. This review focuses on recent literature describing the role of PPARbeta in epithelial tissues and highlights critical discrepancies that need to be resolved for a more comprehensive understanding of how this receptor modulates epithelial homeostasis.     

Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks

Hyaluronic acid is a natural polysaccharide found abundantly throughout the body with many desirable properties for application as a biomaterial, including scaffolding for tissue engineering. In this work, hyaluronic acid with molecular weights ranging from 50 to 1100 kDa was modified with methacrylic anhydride and photopolymerized into networks with a wide range of physical properties. With macromer concentrations from 2 to 20 wt %, networks exhibited volumetric swelling ratios ranging from approximately 42 to 8, compressive moduli ranging from approximately 2 to over 100 kPa, and degradation times ranging from less than 1 day up to almost 38 days in the presence of 100 U/mL of hyaluronidase. When 3T3-fibroblasts were photoencapsulated in the hydrogels, cells remained viable with low macromer concentrations but decreased sequentially as the macromer concentration increased. Finally, auricular swine chondrocytes produced neocartilage when photoencapsulated in the hyaluronic acid networks. This work presents a next step toward the development of advanced in vivo curable biomaterials.     

A Regional Assessment of Salt Marsh Restoration and Monitoring in the Gulf of Maine

We compiled salt marsh monitoring datasets from 36 complete or imminent restoration projects in the Gulf of Maine to assess regional monitoring and restoration practices. Data were organized by functional indicators and restoration project types (culvert replacement, excavation works, or ditch plugging) then pooled to generate mean values for indicators before restoration, after restoration, and at reference sites. Monitoring data were checked against the regional standards of a voluntary protocol for the Gulf of Maine. Data inventories showed that vegetation and salinity indicators were most frequently collected (89 and 78% of sites, respectively), whereas nekton, bird, and hydrologic measures were collected at only about half of the sites. Reference conditions were monitored at 72% of sites. Indicators were analyzed to see if project sites were degraded relative to reference areas and to detect ecological responses to restoration activities. Results showed that compared to reference areas, prerestoration sites had smaller tidal ranges, reduced salinity levels, greater cover of brackish plants species, and lower cover of halophyte plants. Following restoration, physical factors rebounded rapidly with increased flood and salinity levels after about one year, especially for culvert projects. Biological responses were less definitive and occurred over longer time frames. Plant communities trended toward recovered halophytes and reduced brackish species at 3+ years following restoration. Nekton and avian indicators were indistinguishable among reference, impacted, and restored areas. The protocol was successful in demonstrating restoration response for the region, but results were limited by regional inconsistencies in field practices and relatively few multiyear datasets. To improve future assessment capabilities, we encourage greater adherence to the standard protocol throughout the Gulf of Maine salt marsh restoration community.