Characterization of a Distinct Population of Circulating Human Non-Adherent Endothelial Forming Cells and Their Recruitment via Intercellular Adhesion Molecule-3

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133⁺ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.     

Transcontinental Phylogeography of the Daphnia pulex Species Complex

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex – a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.     

Type I Antifreeze Proteins Enhance Ice Nucleation above Certain Concentrations

In this study, we examined the effects that antifreeze proteins have on the supercooling and ice-nucleating abilities of aqueous solutions. Very little information on such nucleation currently exists. Using an automated lag time apparatus and a new analysis, we show several dilution series of Type I antifreeze proteins. Our results indicate that, above a concentration of ∼8 mg/ml, ice nucleation is enhanced rather than hindered. We discuss this unexpected result and present a new hypothesis outlining three components of polar fish blood that we believe affect its solution properties in certain situations.     

Decline in American marten occupancy rates at Sagehen Experimental Forest,California

We compared the distribution and frequency of American marten (Martes americana) detections during historic surveys and a recent survey on the Sagehen Experimental Forest (SEF) in the Sierra Nevada Mountains, California. This area has been the location of 9 previous marten surveys during 1980–1993, each involving a systematic detection/non-detection survey on the same grid. These data are a time series of information on the occupancy of martens that can be related to habitat change in the study area. Our objectives were to 1) resurvey martens in SEF using methodology similar to previous studies to assess current marten occupancy; 2) evaluate changes in marten occupancy during the period 1980–2008; and 3) examine associations between marten occurence and changes in habitat and landscape metrics. Current marten occupancy was estimated using surveys conducted in summer 2007, winter 2007–2008, and summer 2008. From 1978 to 2007 there was a decrease in predicted habitat patch size, core area, and total amount of marten habitat in the study area, as well as an increase in distance between patches. Marten detections in 2007–2008 were approximately 60% lower than in surveys in the 1980s. We detected no martens in the summers of 2007 and 2008, and 10 detections in winter 2007–2008 were limited to higher elevations in the southwestern portion of SEF. No martens were detected in the lower elevations where most of the recent forest management activity occurred. We suggest that the marten population at SEF has been negatively affected by the loss and fragmentation of habitat. We recommend that future management of forests in the Sagehen basin focus on restoring and connecting residual marten habitat to improve habitat quality for martens. © 2011 The Wildlife Society.     

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

Multiple sclerosis is an immune-mediated, demyelinating and neurodegenerative disease that currently lacks any neuroprotective treatments. Innovative neuroprotective trial designs are required to hasten the translational process of drug development. An ideal target to monitor the efficacy of strategies aimed at treating multiple sclerosis is the visual system, which is the most accessible part of the human central nervous system. A novel C57BL/6 mouse line was generated that expressed transgenes for a myelin oligodendrocyte glycoprotein-specific T cell receptor and a retinal ganglion cell restricted-Thy1 promoter-controlled cyan fluorescent protein. This model develops spontaneous or induced optic neuritis, in the absence of paralytic disease normally associated with most rodent autoimmune models of multiple sclerosis. Demyelination and neurodegeneration could be monitored longitudinally in the living animal using electrophysiology, visual sensitivity, confocal scanning laser ophthalmoscopy and optical coherence tomography all of which are relevant to human trials. This model offers many advantages, from a 3Rs, economic and scientific perspective, over classical experimental autoimmune encephalomyelitis models that are associated with substantial suffering of animals. Optic neuritis in this model led to inflammatory damage of axons in the optic nerve and subsequent loss of retinal ganglion cells in the retina. This was inhibited by the systemic administration of a sodium channel blocker (oxcarbazepine) or intraocular treatment with siRNA targeting caspase-2. These novel approaches have relevance to the future treatment of neurodegeneration of MS, which has so far evaded treatment.     

Salivary Oxytocin Concentrations in Males following Intranasal Administration of Oxytocin: A Double-Blind,Cross-Over Study

The use of intranasal oxytocin (OT) in research has become increasingly important over the past decade. Although researchers have acknowledged a need for further investigation of the physiological effects of intranasal administration, few studies have actually done so. In the present double-blind cross-over study we investigated the longevity of a single 24 IU dose of intranasal OT measured in saliva in 40 healthy adult males. Salivary OT concentrations were significantly higher in the OT condition, compared to placebo. This significant difference lasted until the end of testing, approximately 108 minutes after administration, and peaked at 30 minutes. Results showed significant individual differences in response to intranasal OT administration. To our knowledge this is the largest and first all-male within-subjects design study to demonstrate the impact of intranasal OT on salivary OT concentrations. The results are consistent with previous research in suggesting that salivary OT is a valid matrix for OT measurement. The results also suggest that the post-administration ‘wait-time’ prior to starting experimental tasks could be reduced to 30 minutes, from the 45 minutes typically used, thereby enabling testing during peak OT concentrations. Further research is needed to ascertain whether OT concentrations after intranasal administration follow similar patterns in females, and different age groups.