Fractal Characterization of Chromatin Decompaction in Live Cells

Chromatin organization has a fundamental impact on the whole spectrum of genomic functions. Quantitative characterization of the chromatin structure, particularly at submicron length scales where chromatin fractal globules are formed, is critical to understanding this structure-function relationship. Such analysis is currently challenging due to the diffraction-limited resolution of conventional light microscopy. We herein present an optical approach termed inverse spectroscopic optical coherence tomography to characterize the mass density fractality of chromatin, and we apply the technique to observe chromatin decompaction in live cells. The technique makes it possible for the first time, to our knowledge, to sense intracellular morphology with length-scale sensitivity from ∼30 to 450 nm, thus primarily probing the higher-order chromatin structure, without resolving the actual structures. We used chromatin decompaction due to inhibition of histone deacytelases and measured the subsequent changes in the fractal dimension of the intracellular structure. The results were confirmed by transmission electron microscopy and confocal fluorescence microscopy.     

15-Hydroxyprostaglandin Dehydrogenase Generation of Electrophilic Lipid Signaling Mediators from Hydroxy Ω-3 Fatty Acids

15-Hydroxyprostaglandin dehydrogenase (15PGDH) is the primary enzyme catalyzing the conversion of hydroxylated arachidonic acid species to their corresponding oxidized metabolites. The oxidation of hydroxylated fatty acids, such as the conversion of prostaglandin (PG) E₂ to 15-ketoPGE₂, by 15PGDH is viewed to inactivate signaling responses. In contrast, the typically electrophilic products can also induce anti-inflammatory and anti-proliferative responses. This study determined that hydroxylated docosahexaenoic acid metabolites (HDoHEs) are substrates for 15PGDH. Examination of 15PGDH substrate specificity was conducted in cell culture (A549 and primary human airway epithelia and alveolar macrophages) using chemical inhibition and shRNA knockdown of 15PGDH. Substrate specificity is broad and relies on the carbon position of the acyl chain hydroxyl group. 14-HDoHE was determined to be the optimal DHA substrate for 15PGDH, resulting in the formation of its electrophilic metabolite, 14-oxoDHA. Consistent with this, 14-HDoHE was detected in bronchoalveolar lavage cells of mild to moderate asthmatics, and the exogenous addition of 14-oxoDHA to primary alveolar macrophages inhibited LPS-induced proinflammatory cytokine mRNA expression. These data reveal that 15PGDH-derived DHA metabolites are biologically active and can contribute to the salutary signaling actions of Ω-3 fatty acids.     

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE^−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.     

Design and performance of combined infrared canopy and belowground warming in the B4WarmED (Boreal Forest Warming at an Ecotone in Danger) experiment

Conducting manipulative climate change experiments in complex vegetation is challenging, given considerable temporal and spatial heterogeneity. One specific challenge involves warming of both plants and soils to depth. We describe the design and performance of an open‐air warming experiment called Boreal Forest Warming at an Ecotone in Danger (B4WarmED) that addresses the potential for projected climate warming to alter tree function, species composition, and ecosystem processes at the boreal‐temperate ecotone. The experiment includes two forested sites in northern Minnesota, USA, with plots in both open (recently clear‐cut) and closed canopy habitats, where seedlings of 11 tree species were planted into native ground vegetation. Treatments include three target levels of plant canopy and soil warming (ambient, +1.7 °C, +3.4 °C). Warming was achieved by independent feedback control of voltage input to aboveground infrared heaters and belowground buried resistance heating cables in each of 72‐7.0 m² plots. The treatments emulated patterns of observed diurnal, seasonal, and annual temperatures but with superimposed warming. For the 2009 to 2011 field seasons, we achieved temperature elevations near our targets with growing season overall mean differences (?T_below) of +1.84 °C and +3.66 °C at 10 cm soil depth and (?T^above) of +1.82 °C and +3.45 °C for the plant canopies. We also achieved measured soil warming to at least 1 m depth. Aboveground treatment stability and control were better during nighttime than daytime and in closed vs. open canopy sites in part due to calmer conditions. Heating efficacy in open canopy areas was reduced with increasing canopy complexity and size. Results of this study suggest the warming approach is scalable: it should work well in small‐statured vegetation such as grasslands, desert, agricultural crops, and tree saplings (<5 m tall).