Optical scattering measurements of laser induced damage in the intraocular lens

This study optically determines whether the amount of light scatter due to laser-induced damage to the intraocular lens (IOL) is significant in relation to normal straylight values in the human eye. Two IOLs with laser-induced damage were extracted from two donor eyes. Each IOL had 15 pits and/or cracks. The surface area of each pit was measured using a microscope. For 6 pits per intraocular lens the point spread function (PSF) in terms of straylight was measured and the total straylight for all 15 pits was estimated. The damage in the IOLs was scored as mild/moderate. The total damaged surface areas, for a 3.5 mm pupil, in the two IOLs were 0.13% (0.0127 mm(2)) and 0.66% (0.064 mm(2)), respectively. The angular dependence of the straylight caused by the damage was similar to that of the normal PSF. The total average contribution to straylight was log(s) = -0.82 and -0.42, much less than the straylight value of the normal eye.The straylight due to normal levels of laser induced damage of the IOL is much lower than normal straylight values found clinically for the normal eye and may therefore be considered not significant.     

Epigenetic variation during the adult lifespan: cross-sectional and longitudinal data on monozygotic twin pairs

The accumulation of epigenetic changes was proposed to contribute to the age-related increase in the risk of most common diseases. In this study on 230 monozygotic twin pairs (MZ pairs), aged 18-89 years, we investigated the occurrence of epigenetic changes over the adult lifespan. Using mass spectrometry, we investigated variation in global (LINE1) DNA methylation and in DNA methylation at INS, KCNQ1OT1, IGF2, GNASAS, ABCA1, LEP, and CRH, candidate loci for common diseases. Except for KCNQ1OT1, interindividual variation in locus-specific DNA methylation was larger in old individuals than in young individuals, ranging from 1.2-fold larger at ABCA1 (P = 0.010) to 1.6-fold larger at INS (P = 3.7 × 10(-07) ). Similarly, there was more within-MZ-pair discordance in old as compared with young MZ pairs, except for GNASAS, ranging from an 8% increase in discordance each decade at CRH (P = 8.9 × 10(-06) ) to a 16% increase each decade at LEP (P = 2.0 × 10(-08) ). Still, old MZ pairs with strikingly similar DNA methylation were also observed at these loci. After 10-year follow-up in elderly twins, the variation in DNA methylation showed a similar pattern of change as observed cross-sectionally. The age-related increase in methylation variation was generally attributable to unique environmental factors, except for CRH, for which familial factors may play a more important role. In conclusion, sustained epigenetic differences arise from early adulthood to old age and contribute to an increasing discordance of MZ twins during aging.     

High-throughput fluorescence-based isolation of live C. elegans larvae

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.     

Strain-time cell-death threshold for skeletal muscle in a tissue-engineered model system for deep tissue injury

Deep tissue injury (DTI) is a severe pressure ulcer that results from sustained deformation of muscle tissue overlying bony prominences. In order to understand the etiology of DTI, it is essential to determine the tolerance of muscle cells to large mechanical strains. In this study, a new experimental method of determining the time-dependent critical compressive strains for necrotic cell death (E(zz)(c)(t)) in a planar tissue-engineered construct under static loading was developed. A half-spherical indentor is used to induce a non-uniform, concentric distribution of strains in the construct, and E(zz)(c)(t) is calculated from the radius of the damage region in the construct versus time. The method was employed to obtain E(zz)(c)(t) for bio-artificial muscles (BAMs) cultured from C2C12 murine cells, as a model system for DTI. Specifically, propidium iodine was used to fluorescently stain the development of necrosis in BAMs subjected to strains up to 80%. Two groups of BAMs were tested at an extracellular pH of 7.4 (n=10) and pH 6.5 (n=5). The lowest strain levels causing cell death in the BAMs were determined every 15min, during 285-min-long trials, from confocal microscopy fluorescent images of the size of the damage regions. The experimental E(zz)(c)(t) data fitted a decreasing single-step sigmoid of the Boltzmann type. Analysis of the parameters of this sigmoid function indicated a 95% likelihood that cells could tolerate engineering strains below 65% for 1h, whereas the cells could endure strains below 40% over a 285min trial period. The decrease in endurance of the cells to compressive strains occurred between 1-3h post-loading. The method developed in this paper is generic and suitable for studying E(zz)(c)(t) in virtually any planar tissue-engineered construct. The specific E(zz)(c)(t) curve obtained herein is necessary for extrapolating biological damage from muscle-strain data in biomechanical studies of pressure ulcers and DTI.     

Biogeography of the Purple Nonsulfur Bacterium Rhodopseudomonas palustris

The biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as R. palustris based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (C_MH), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (C_MH, ≥0.95) but there was almost no similarity between locations that were ≥9 m apart (C_MH, ≤0.25). These calculations showed there was a gradual decrease in similarity among the five locations as a function of distance and that clones of R. palustris were lognormally distributed along the linear 10-m transect. These data indicate that natural populations of R. palustris are assemblages of genetically distinct ecotypes and that the distribution of each ecotype is patchy.     

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis.

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model. We were unable to show a correlation between the local PpIX fluorescence increase and the distance from a blood vessel. The spatial distribution of PpIX fluorescence within normal tissue or tumour is not changed in response to the illumination. These observations suggest that there is no diffusion of PpIX into the treated tissue. Cooling the tissue to 12 degrees C, a temperature at which PpIX synthesis is inhibited, inhibited the PpIX fluorescence increase normally observed after illumination. We also found a strong correlation between local PpIX photobleaching during illumination and the fluorescence intensity 1 h after illumination similar to what we have observed in patients treated with ALA-PDT. Therefore we conclude that the increase in PpIX fluorescence after illumination is due to local cellular re-synthesis.     

Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae

The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal, Saccharomyces cerevisiae could potentially shape the immune response in a significant way. We observed that S. cerevisiae cells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity both in vitro and in vivo. These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.     

Small animal imaging with magnetic resonance microscopy

Small animal magnetic resonance microscopy (MRM) has evolved significantly from testing the boundaries of imaging physics to its expanding use today as a tool in noninvasive biomedical investigations. MRM now increasingly provides functional information about living animals, with images of the beating heart, breathing lung, and functioning brain. Unlike clinical MRI, where the focus is on diagnosis, MRM is used to reveal fundamental biology or to noninvasively measure subtle changes in the structure or function of organs during disease progression or in response to experimental therapies. High-resolution anatomical imaging reveals increasingly exquisite detail in healthy animals and subtle architectural aberrations that occur in genetically altered models. Resolution of 100 mum in all dimensions is now routinely attained in living animals, and (10 mum)(3) is feasible in fixed specimens. Such images almost rival conventional histology while allowing the object to be viewed interactively in any plane. In this review we describe the state of the art in MRM for scientists who may be unfamiliar with this modality but who want to apply its capabilities to their research. We include a brief review of MR concepts and methods of animal handling and support, before covering a range of MRM applications-including the heart, lung, and brain-and the emerging field of MR histology. The ability of MRM to provide a detailed functional and anatomical picture in rats and mice, and to track this picture over time, makes it a promising platform with broad applications in biomedical research.     

Epigenetic signature of birth weight discordance in adult twins

Background

A low birth weight has been extensively related to poor adult health outcomes. Birth weight can be seen as a proxy for environmental conditions during prenatal development. Identical twin pairs discordant for birth weight provide an extraordinary model for investigating the association between birth weight and adult life health while controlling for not only genetics but also postnatal rearing environment. We performed an epigenome-wide profiling on blood samples from 150 pairs of adult monozygotic twins discordant for birth weight to look for molecular evidence of epigenetic signatures in association with birth weight discordance.

Results

Our association analysis revealed no CpG site with genome-wide statistical significance (FDR < 0.05) for either qualitative (larger or smaller) or quantitative discordance in birth weight. Even with selected samples of extremely birth weight discordant twin pairs, no significant site was found except for 3 CpGs that displayed age-dependent intra-pair differential methylation with FDRs 0.014 (cg26856578, p = 3.42e-08), 0.0256 (cg15122603, p = 1.25e-07) and 0.0258 (cg16636641, p = 2.05e-07). Among the three sites, intra-pair differential methylation increased with age for cg26856578 but decreased with age for cg15122603 and cg16636641. There was no genome-wide statistical significance for sex-dependent effects on intra-pair differential methylation in either the whole samples or the extremely discordant twins.

Conclusions

Genome-wide DNA methylation profiling did not reveal epigenetic signatures of birth weight discordance although some sites displayed age-dependent intra-pair differential methylation in the extremely discordant twin pairs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1062) contains supplementary material, which is available to authorized users.