Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells

Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.     

Effects of temperature on energy cost and timing of embryonic and larval development of the terrestrially breeding moss frog, Bryobatrachus nimbus

The Australian moss frog, Bryobatrachus nimbus, oviposits four to 16 large eggs in terrestrial nests constructed in moss or lichen in subalpine regions of southern Tasmania. Nidicolous larvae overwinter beneath snow, reaching metamorphosis without feeding after 395 d, the longest development time known for an endotrophic anuran. However, a few clutches develop more quickly and metamorphose before winter. This study examines the effect of temperature on development time and energy expenditure by measuring temperatures and developmental stages in field nests as well as rates of oxygen consumption (Vo2), developmental stage, body mass, and energy content in the laboratory at three relevant temperatures (5 degrees, 10 degrees, 15 degrees C). Eggs and larvae reared at 5 degrees C differentiated very slowly, and their development time far exceeded those in natural nests, but development times at 10 degrees and 15 degrees C averaged 277 and 149 d, respectively, and were shorter than field incubation times. Generally, respiration rates of aquatic hatchlings were low in comparison with other species but increased with larval age and jumped about 25% higher near metamorphosis when larvae were able to air breathe. The mean energy density was 26.0 J mg(-1) for the dry ova and 20.6 J mg(-1) for a dry gut-free froglet, and total production efficiency was 61.5%. We developed a model based on the relationships between incubation temperature and V&d2;o2 to estimate the respiratory cost of development to metamorphosis, the first such study for an amphibian. The cost was 177 J at 15 degrees C, 199 J at 10 degrees C, and at least 249 J at 5 degrees C, and we predicted that continual development at 5 degrees C would lead to premature yolk depletion because it equalled the 249 J contained in fresh ova. Continuously logged field-nest temperatures and interpolation of laboratory data provided estimates of development rates, Vo2, and respiratory energy costs in field nests. Development to metamorphosis required between 185 and 234 J when larvae overwintered, but completion of metamorphosis before winter saved 123 J. However, the advantage of emergence in warmer months, when conditions are suitable for feeding and growth, may offset the greater energy cost of overwintering.     

The principle of laplace and scaling of ventricular wall stress and blood pressure in mammals and birds

Maximum left ventricular wall stress is calculated at end-diastolic volume and systemic arterial diastolic blood pressure, according to a thick-walled model for the principle of Laplace. Stress is independent of body mass and averages 13.9 kPa (+/-2.3; 95% confidence interval) in 24 species of mammals weighing 0.025-4,000 kg and 15.5 kPa (+/-4.7) in 12 birds weighing 0.014-110 kg. Birds have higher arterial blood pressures and larger hearts than mammals. Systolic and diastolic arterial blood pressures increase with body mass according to M(0.05) in mammals, and heart mass increases according to M(1.06) in the same species, further supporting the principle. However, blood pressure in birds is independent of body mass, and heart mass scales isometrically. End-diastolic stress values, calculated according to Laplace, are about one-third of peak stresses recorded in isolated mammalian myocardial preparations.     

Hearts, neck posture and metabolic intensity of sauropod dinosaurs

Hypothesized upright neck postures in sauropod dinosaurs require systemic arterial blood pressures reaching 700 mmHg at the heart. Recent data on ventricular wall stress indicate that their left ventricles would have weighed 15 times those of similarly sized whales. Such dimensionally, energetically and mechanically disadvantageous ventricles were highly unlikely in an endothermic sauropod. Accessory hearts or a siphon mechanism, with sub-atmospheric blood pressures in the head, were also not feasible. If the blood flow requirements of sauropods were typical of ectotherms, the left-ventricular blood volume and mass would have been smaller; nevertheless, the heart would have suffered the serious mechanical disadvantage of thick walls. It is doubtful that any large sauropod could have raised its neck vertically and endured high arterial blood pressure, and it certainly could not if it had high metabolic rates characteristic of endotherms.     

Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

Comparing Injury and Illness Risk Assessments for Occupational Hazards

One common framework for describing the evaluation and assessment of hazards in the workplace includes the four steps of hazard identification, exposure assessment, exposure-response modeling, and risk characterization (NAS, 1983). We discuss hazards for occupational injury and illness in light of this framework, and we contrast the evaluation of injury hazards with the evaluation of illness hazards. In particular, the nature of the hazards, typical exposure patterns, quantification of exposure, and the attribution of outcome to exposure are discussed. Finally, we discuss the management of occupational illness and injury hazards and issues encountered when evaluating efforts designed to mitigate the effects of occupational hazards.