Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides

In a previous report (M.S. Wadhwa et al . (1997) Bioconjugate Chem. 8, 81–88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK 18 ) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK 18 . Surprisingly, AlkCWK 18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000–4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK 18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK 18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK 18 and K 20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK 18 or K 20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK 18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK 18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74767/1/j.1399-3011.1999.00104.x.pd

Balancing Uncertainty and Complexity to Incorporate Fire Spread in an Eco-Hydrological Model

Wildfire affects the ecosystem services of watersheds, and climate change will modify fire regimes and watershed dynamics. In many eco-hydrological simulations, fire is included as an exogenous force. Rarely are the bidirectional feedbacks between watersheds and fire regimes integrated in a simulation system because the eco-hydrological model predicts variables that are incompatible with the requirements of fire models. WMFire is a fire-spread model of intermediate complexity designed to be integrated with the Regional Hydro-ecological Simulation System (RHESSys). Spread in WMFire is based on four variables that (i) represent known influences on fire spread: litter load, relative moisture deficit, wind direction and topographic slope, and (ii) are derived directly from RHESSys outputs. The probability that a fire spreads from pixel to pixel depends on these variables as predicted by RHESSys. We tested a partial integration between WMFire and RHESSys on the Santa Fe (New Mexico) and the HJ Andrews (Oregon State) watersheds. Model assessment showed correspondence between expected spatial patterns of spread and seasonality in both watersheds. These results demonstrate the efficacy of an approach to link eco-hydrologic model outputs with a fire spread model. Future work will develop a fire effects module in RHESSys for a fully coupled, bidirectional model

COMPILING, SYNTHESIZING AND ANALYZING EXISTING BOREAL FOREST FIRE HISTORY DATA IN ALASKA

Wildland fires play a critical role in maintaining the ecological integrity of boreal forests in Alaska. Identifying and maintaining natural fire regimes is an important component of fire management. There are numerous research projects that directly or indirectly address historical fire regimes in the Alaskan boreal forest, but many are unpublished, have many unprocessed dendrochronological (tree age and fire scar) samples, or their data were used for other purposes. Furthermore, no assessment of these data exists to understand how fire has historically affected the boreal forest ecosystems of Alaska. The goal of this project was to compile and synthesize existing Alaska boreal-forest fire-history literature and datasets (http://frames.nbii.gov/alaska/borealfirehistory). We include a literature review and synthesis of publications related to fire regimes in boreal forests in Alaska (the pending general technical report “Fire Regimes of the Alaskan Boreal Forest”), and incorporate the reference information into the Alaska Fire Effects Reference Database (http://frames.nbii.gov/alaska; funded by JFSP as part of project 05-4-2-03: Expanding FIREHouse to Alaska). Fourteen published and unpublished fire-history or stand-age datasets were compiled and processed into the Alaska Fire History Database (http://frames.nbii.gov/documents/alaska/fire_history/ak_fire_history_db.zip), and data summarized by plot are available through a dynamic map interface (within the Alaska Fire and Fuels Research Map; http://afsmaps.blm.gov/imf/imf.jsp?site=firehouse). Data compiled in the Alaska Fire History Database have also been submitted to the International Multiproxy Paleofire Database (IMPD). Finally, some of the project funds were used to clean up and improve data within the Alaska Large Fire Database, a database started in the early 1990s that includes reported fire locations since 1939 and fire perimeters since 1942 (http://afsmaps.blm.gov/imf/imf.jsp?site=firehistory)

Formulation of highly soluble poly(ethylene glycol)-peptide DNA condensates

Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys 18 (CWK 18 ) resulting in the formation of nonreducible (PEG-VS-CWK 18 ) and reducible (PEG-SS-CWK 18 ) PEG- peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by 1H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK 18 , resulting in DNA condensates with a mean diameter of 80–90 nm and ζ (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0.05–2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK 18 DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK 18 DNA conden- sates was over two orders of magnitude lower than PEG-SS-CWK 18 DNA condensates and three orders of magnitude lower than AlkCWK 18 DNA condensates. PEG-VS-CWK 18 efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34495/1/10_ftp.pd

Nonlinear Diffusive Shock Acceleration with Magnetic Field Amplification

We introduce a Monte Carlo model of nonlinear diffusive shock acceleration allowing for the generation of large-amplitude magnetic turbulence. The model is the first to include strong wave generation, efficient particle acceleration to relativistic energies in nonrelativistic shocks, and thermal particle injection in an internally self-consistent manner. We find that the upstream magnetic field can be amplified by large factors and show that this amplification depends strongly on the ambient Alfven Mach number. We also show that in the nonlinear model large increases in the magnetic field do not necessarily translate into a large increase in the maximum particle momentum a particular shock can produce, a consequence of high momentum particles diffusing in the shock precursor where the large amplified field converges to the low ambient value. To deal with the field growth rate in the regime of strong fluctuations, we extend to strong turbulence a parameterization that is consistent with the resonant quasi-linear growth rate in the weak turbulence limit. We believe our parameterization spans the maximum and minimum range of the fluctuation growth and, within these limits, we show that the nonlinear shock structure, acceleration efficiency, and thermal particle injection rates depend strongly on the yet to be determined details of wave growth in strongly turbulent fields. The most direct application of our results will be to estimate magnetic fields amplified by strong cosmic-ray modified shocks in supernova remnants.Comment: Accepted in ApJ July 2006, typos corrected in this versio

In vivo gene transfer using sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates

Recent interest in sulfhydryl cross-linked nonviral gene delivery systems, designed to trigger the intracellular release of DNA, has inspired studies to establish their utility in vitro . To determine if this concept can be extrapolated to in vivo gene delivery, sulfhydryl cross-linking peptides (dp 20), derivatized with either an N-glycan or polyethylene glycol (PEG), were used to generate sulfhydryl cross-linked gene formulations. The biodistribution, metabolism, cell-type targeting, and gene expression of sulfhydryl cross-linked PEG-peptide/glycopeptide DNA co-condensates were examined following i.v. dosing in mice. Optimal targeting to hepatocytes was achieved by condensing 125 I-DNA with an add-mixture of 10 mol % triantennary glycopeptide, 5 mol % PEG-peptide, and 85 mol % backbone peptide. Four backbone peptides were substituted into the formulation to examine the influence of peptide metabolism and disulfide bond strength on the rate of DNA metabolism and the level of gene expression in vivo . The half-life of DNA in liver was extended from 1 to 3 h using a backbone peptide composed of d -amino acids, whereas substituting penicillamine for cysteine failed to further increase the metabolic stability of DNA. Optimized gene delivery formulations transiently expressed secreted alkaline phosphatase in mouse serum for 12 days. The results suggest that disulfide bond reduction in liver hepatocytes proceeds rapidly, followed by peptide metabolism, ultimately limiting the metabolic half-life of sulfhydryl cross-linked DNA condensates in vivo . © 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1174–1185, 2003Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34509/1/10384_ftp.pd

Large-area 2D selective area growth for photonic crystal surface emitting lasers

We report an investigation into large-area selective area growth of InGaAs/GaAs quantum wells by metalorganic vapour phase epitaxy. The emission wavelength tuning range, growth enhancement, and uniformity of material deposited within square masked regions with central square growth windows with widths in the range of 100–300 μm are studied. Micro-photoluminescence measurements at the centre point of each of the growth windows reveals a total wavelength tuning range of 86 nm across all samples, with a typical tuning range of 30 nm for a given window width, dependent upon dielectric mask width. The thickness enhancement in each of features, as determined by white-light interferometric profiling, indicates that centre point growth rate enhancements of between 1.19 and 2.23× are achieved with respect to the nominal epitaxial structure. By comparing the observed emission wavelengths with those simulated using the enhanced quantum well thicknesses, a range of indium concentrations between 12 and 17 % is calculated for the material at the centre of each feature. Two-dimensional analysis of selected features reveals that areas with uniform emission wavelength up to 100 × 100 μm2 in size can be achieved for the mask patterns used, indicating suitability for future applications in the fabrication of monolithically integrated multi-wavelength photonic crystal surface emitting laser arrays

An elaborated feeding cycle model for reductions in vectorial capacity of night-biting mosquitoes by insecticide-treated nets

BACKGROUND: Insecticide Treated Nets (ITNs) are an important tool for malaria control. ITNs are effective because they work on several parts of the mosquito feeding cycle, including both adult killing and repelling effects. METHODS: Using an elaborated description of the classic feeding cycle model, simple formulas have been derived to describe how ITNs change mosquito behaviour and the intensity of malaria transmission, as summarized by vectorial capacity and EIR. The predicted changes are illustrated as a function of the frequency of ITN use for four different vector populations using parameter estimates from the literature. RESULTS: The model demonstrates that ITNs simultaneously reduce mosquitoes' lifespans, lengthen the feeding cycle, and by discouraging human biting divert more bites onto non-human hosts. ITNs can substantially reduce vectorial capacity through small changes to all of these quantities. The total reductions in vectorial capacity differ, moreover, depending on baseline behavior in the absence of ITNs. Reductions in lifespan and vectorial capacity are strongest for vector species with high baseline survival. Anthropophilic and zoophilic species are affected differently by ITNs; the feeding cycle is lengthened more for anthrophilic species, and the proportion of bites that are diverted onto non-human hosts is higher for zoophilic species. CONCLUSION: This model suggests that the efficacy of ITNs should be measured as a total reduction in transmission intensity, and that the quantitative effects will differ by species and by transmission intensity. At very high rates of ITN use, ITNs can generate large reductions in transmission intensity that could provide very large reductions in transmission intensity, and effective malaria control in some areas, especially when used in combination with other control measures. At high EIR, ITNs will probably not substantially reduce the parasite rate, but when transmission intensity is low, reductions in vectorial capacity combine with reductions in the parasite rate to generate very large reductions in EIR

Collection of Aerosolized Human Cytokines Using Teflon® Filters

Background: Collection of exhaled breath samples for the analysis of inflammatory biomarkers is an important area of research aimed at improving our ability to diagnose, treat and understand the mechanisms of chronic pulmonary disease. Current collection methods based on condensation of water vapor from exhaled breath yield biomarker levels at or near the detection limits of immunoassays contributing to problems with reproducibility and validity of biomarker measurements. In this study, we compare the collection efficiency of two aerosol-to-liquid sampling devices to a filter-based collection method for recovery of dilute laboratory generated aerosols of human cytokines so as to identify potential alternatives to exhaled breath condensate collection. Methodology/Principal Findings: Two aerosol-to-liquid sampling devices, the SKC® Biosampler and Omni 3000™, as well as Teflon® filters were used to collect aerosols of human cytokines generated using a HEART nebulizer and single-pass aerosol chamber setup in order to compare the collection efficiencies of these sampling methods. Additionally, methods for the use of Teflon® filters to collect and measure cytokines recovered from aerosols were developed and evaluated through use of a high-sensitivity multiplex immunoassay. Our results show successful collection of cytokines from pg/m3 aerosol concentrations using Teflon® filters and measurement of cytokine levels in the sub-picogram/mL concentration range using a multiplex immunoassay with sampling times less than 30 minutes. Significant degradation of cytokines was observed due to storage of cytokines in concentrated filter extract solutions as compared to storage of dry filters. Conclusions: Use of filter collection methods resulted in significantly higher efficiency of collection than the two aerosol-to-liquid samplers evaluated in our study. The results of this study provide the foundation for a potential new technique to evaluate biomarkers of inflammation in exhaled breath samples

On Imprimitive Representations of Finite Reductive Groups in Non-defining Characteristic

In this paper, we begin with the classification of Harish-Chandra imprimitive representations in non-defining characteristic. We recall the connection of this problem to certain generalizations of Iwahori-Hecke algebras and show that Harish-Chandra induction is compatible with the Morita equivalence by Bonnaf\'{e} and Rouquier, thus reducing the classification problem to quasi-isolated blocks. Afterwards, we consider imprimitivity of unipotent representations of certain classical groups. In the case of general linear and unitary groups, our reduction methods then lead to results for arbitrary Lusztig series