Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.     

Hands-Free Image Capture,Data Tagging and Transfer Using Google Glass: A Pilot Study for Improved Wound Care Management

Chronic wounds, including pressure ulcers, compromise the health of 6.5 million Americans and pose an annual estimated burden of $25 billion to the U.S. health care system. When treating chronic wounds, clinicians must use meticulous documentation to determine wound severity and to monitor healing progress over time. Yet, current wound documentation practices using digital photography are often cumbersome and labor intensive. The process of transferring photos into Electronic Medical Records (EMRs) requires many steps and can take several days. Newer smartphone and tablet-based solutions, such as Epic Haiku, have reduced EMR upload time. However, issues still exist involving patient positioning, image-capture technique, and patient identification. In this paper, we present the development and assessment of the SnapCap System for chronic wound photography. Through leveraging the sensor capabilities of Google Glass, SnapCap enables hands-free digital image capture, and the tagging and transfer of images to a patient’s EMR. In a pilot study with wound care nurses at Stanford Hospital (n=16), we (i) examined feature preferences for hands-free digital image capture and documentation, and (ii) compared SnapCap to the state of the art in digital wound care photography, the Epic Haiku application. We used the Wilcoxon Signed-ranks test to evaluate differences in mean ranks between preference options. Preferred hands-free navigation features include barcode scanning for patient identification, Z(15) = -3.873, p < 0.001, r = 0.71, and double-blinking to take photographs, Z(13) = -3.606, p < 0.001, r = 0.71. In the comparison between SnapCap and Epic Haiku, the SnapCap System was preferred for sterile image-capture technique, Z(16) = -3.873, p < 0.001, r = 0.68. Responses were divided with respect to image quality and overall ease of use. The study’s results have contributed to the future implementation of new features aimed at enhancing mobile hands-free digital photography for chronic wound care.     

The Unique GGA Clathrin Adaptor of Drosophila melanogaster Is Not Essential

The Golgi-localized, γ-ear-containing, ARF binding proteins (GGAs) are a highly conserved family of monomeric clathrin adaptor proteins implicated in clathrin-mediated protein sorting between the trans-Golgi network and endosomes. GGA RNAi knockdowns in Drosophila have resulted in conflicting data concerning whether the Drosophila GGA (dGGA) is essential. The goal of this study was to define the null phenotype for the unique Drosophila GGA. We describe two independently derived dGGA mutations. Neither allele expresses detectable dGGA protein. Homozygous and hemizygous flies with each allele are viable and fertile. In contrast to a previous report using RNAi knockdown, GGA mutant flies show no evidence of age-dependent retinal degeneration or cathepsin missorting. Our results demonstrate that several of the previous RNAi knockdown phenotypes were the result of off-target effects. However, GGA null flies are hypersensitive to dietary chloroquine and to starvation, implicating GGA in lysosomal function and autophagy.     

Ammonia emissions during vermicomposting of sheep manure

The effect of C:N ratio, temperature and water content on ammonia volatilization during two-phase composting of sheep manure was evaluated. The aerobic phase was conducted under field conditions. This was followed by Phase II, vermicomposting, conducted in the laboratory under controlled conditions of water content (70% and 80%) and temperature (15 and 22 °C). The addition of extra straw lead to a 10% reduction in NH3 volatilization compared to sheep manure composted without extra straw. Temperature and water content significantly effected ammonia volatilization at 0 day in Phase II, with a water content of 70% and temperature of 22 °C leading to greater losses of ammonia. Nitrogen loss by ammonia volatilization during vermicomposting ranged from 8% to 15% of the initial N content. The addition of extra straw did not result in significant differences in total carbon content following vermicomposting.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Patch area, substrate depth, and richness affect giving-up densities: a test with mourning doves and cottontail rabbits

We compared the foraging behavior of mourning doves Zenaida macroura and cottontail rabbits Sylvilagus floridanus in patches that varied in initial food abundance, surface area and substrate depth. We measured giving‐up densities (GUD), food harvest and proportion of food harvested to investigate their ability to respond to characteristics of resource patches. GUDs have been analyzed in three ways: grams of per patch, grams per unit surface area (GUD_AREA), and grams per unit volume of sand (GUD_VOL). Mourning doves and cottontails exhibited similar responses to resource density and sand depth. Both foragers detected and responded to variation in initial food abundance. The proportion of food harvested from a patch increased from 40.7, 43.8 to 48.3% (for the doves) and 34.9, 35.8 to 38.4% (for the rabbits) in patches of low, medium and high initial food abundance, respectively. Deeper substrates reduced the foragers’ encounter probability with food, decreased patch quality and resulted in higher GUDs (60% higher in the deepest relative to shallowest substrate) and lower harvests. A significant interaction between initial food abundance and substrate depth showed that both species were willing to dig deeper in patches with higher resource density. Patch size (surface area) had no effect on food harvest or the proportion of food harvested. Consequently, GUD_AREA and GUD_VOL increased in patches with a smaller surface area. Smaller patches appeared to hamper the dove's and cottontail's movement across the surface. Our results revealed that mourning doves and cottontails forage under imperfect information. Both species were able to respond to patch properties by biasing their feeding efforts toward rich and easy opportunities, however, mourning doves were more efficient at food harvesting. The interaction of patch area, volume and food abundance directly influenced food harvest. Such resource characters occur under natural situations where food varies in abundance, area of distribution, and accessibility.     

Targeting molecular interactions essential for Plasmodium sexual reproduction

Malaria remains one of the most devastating infectious diseases, killing up to a million people every year. Whereas much progress has been made in understanding the life cycle of the parasite in the human host and in the mosquito vector, significant gaps of knowledge remain. Fertilization of malaria parasites, a process that takes place in the lumen of the mosquito midgut, is poorly understood and the molecular interactions (receptor–ligand) required for Plasmodium fertilization remain elusive. By use of a phage display library, we identified FG1 (Female Gamete peptide 1), a peptide that binds specifically to the surface of female Plasmodium berghei gametes. Importantly, FG1 but not a scrambled version of the peptide, strongly reduces P. berghei oocyst formation by interfering with fertilization. In addition, FG1 also inhibits P. falciparum oocyst formation suggesting that the peptide binds to a molecule on the surface of the female gamete whose structure is conserved. Identification of the molecular interactions disrupted by the FG1 peptide may lead to the development of novel malaria transmission‐blocking strategies.     

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.     

Humidity – The overlooked variable in the thermal biology of mosquito-borne disease

Vector-borne diseases cause significant financial and human loss, with billions of dollars spent on control. Arthropod vectors experience a complex suite of environmental factors that affect fitness, population growth and species interactions across multiple spatial and temporal scales. Temperature and water availability are two of the most important abiotic variables influencing their distributions and abundances. While extensive research on temperature exists, the influence of humidity on vector and pathogen parameters affecting disease dynamics are less understood. Humidity is often underemphasized, and when considered, is often treated as independent of temperature even though desiccation likely contributes to declines in trait performance at warmer temperatures. This Perspectives explores how humidity shapes the thermal performance of mosquito-borne pathogen transmission. We summarize what is known about its effects and propose a conceptual model for how temperature and humidity interact to shape the range of temperatures across which mosquitoes persist and achieve high transmission potential. We discuss how failing to account for these interactions hinders efforts to forecast transmission dynamics and respond to epidemics of mosquito-borne infections. We outline future research areas that will ground the effects of humidity on the thermal biology of pathogen transmission in a theoretical and empirical framework to improve spatial and temporal prediction of vector-borne pathogen transmission.     

Gene promoter methylation is associated with lung function in the elderly: The normative aging study

Lung function is a strong predictor of mortality. While inflammatory markers have been associated with lung function decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline mechanisms. We studied the cross-sectional association between DNA methylation in nine inflammatory genes and lung function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999–2006. We used separate multivariate mixed effects regression models to study the association between each lung function measurement and DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was significantly associated with lower lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV₁), respectively by 2.94% (p < 10⁻⁴) and 2.47% (p < 10⁻³) for F3 and by 2.10% (p < 10⁻²) and 2.42% (p < 10⁻³) for TLR2. Decreased IFNγ and IL6 methylation was significantly associated with better lung function. One IQR decrease in DNA methylation was associated with higher FEV₁ by 1.75% (p = 0.02) and 1.67% (p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of negative feedback.Key words: DNA methylation, genes, spirometry, FEV₁, lungs, TLR2, F3, INOS, GCR, OGG1