Developing human laboratory models of smoking lapse behavior for medication screening

Use of human laboratory analogues of smoking behavior can provide an efficient, cost-effective mechanistic evaluation of a medication signal on smoking behavior, with the result of facilitating translational work in medications development. Although a number of human laboratory models exist to investigate various aspects of smoking behavior and nicotine dependence phenomena, none have yet modeled smoking lapse behavior. The first instance of smoking during a quit attempt (i.e. smoking lapse) is highly predictive of relapse and represents an important target for medications development. Focusing on an abstinence outcome is critical for medication screening as the US Food and Drug Administration approval for cessation medications is contingent on demonstrating effects on smoking abstinence. This paper outlines a three-stage process for the development of a smoking lapse model for the purpose of medication screening. The smoking lapse paradigm models two critical features of lapse behavior: the ability to resist the first cigarette and subsequent ad libitum smoking. Within the context of the model, smokers are first exposed to known precipitants of smoking relapse (e.g. nicotine deprivation, alcohol, stress), and then presented their preferred brand of cigarettes. Their ability to resist smoking is then modeled and once smokers 'give in' and decide to smoke, they participate in a tobacco self-administration session. Ongoing and completed work developing and validating these models for the purpose of medication screening is discussed.     

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

AIM: To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging.METHODS: Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers.RESULTS: Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively.CONCLUSION: This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.     

Soil warming alters seed-bank responses across the geographic range of freshwater Taxodium distichum (Cupressaceae) swamps

? Premise of the Study: Climate warming is predicted to have far-reaching effects on the distribution of species, but those effects may depend on the flexibility of regenerating species in responding to climate gradients. We conducted a study to determine whether the variation in the response of seed banks to temperature varied across the latitudinal range of Taxodium distichum swamps in North America. ? Methods: The soil was collected in a long-term research network and heated experimentally to three current-day spring normal soil temperatures (average maximum daily spring normal soil temperatures during May in Illinois, Arkansas, and Louisiana, USA, respectively: 22°, 25°, and 29°C). A "normal" is the mean temperature calculated over a 30-yr interval (1971-2000). Seed-bank density and biomass responses were examined in relation to latitude and difference in the soil temperature of the experiment and the spring normal. ? Key Results: Using first- and second-order regression analysis, we determined that the variation in total germination density was lowest mid-range and in experimental soil temperatures similar to the spring normal. For some dominant species, the variance in germination density was higher in the northern (Cephalanthus occidentalis) or the southern part of the network (Saururus cernuus and Polygonum pensylvanicum). Overall, the variance of total biomass (root, shoot, whole plant) was higher if the experimental soil temperature was warmer than the spring normal. ? Conclusions: Our results suggest that the regeneration of some populations of swamp species may have more flexibility to respond to climate warming than others.     

New species of Cercopithecoides from Haasgat, North West Province, South Africa

Analyses of new cercopithecid fossil specimens from the South African site of Haasgat point to craniofacial affinities with the genus Cercopithecoides. Detailed metric and non-metric comparisons with South African Cercopithecoides williamsi, and other East African Cercopithecoides species, Cercopithecoides kimeui, Cercopithecoides meaveae, Cercopithecoides kerioensis, and Cercopithecoides alemyehui demonstrate that the Haasgat fossils have distinct craniofacial morphology and dental metrics. Specifically, material from Haasgat probably represents one of the smaller Cercopithecoides, differing from the others in its particular suite of features that vary within the genus. It is unique in its more vertical ramus, associated with a relatively lengthened mandibular body. Haasgat Cercopithecoides has a particularly narrow interorbital region between relatively larger ovoid orbits, with articulation of the maxillary bones at a suture above the triangular nasal bones. Furthermore, the maxillary arcade is more rounded than other Cercopithecoides, converging at the M² and M³. The conclusion drawn from this analysis is that the Pleistocene Haasgat fossils are colobines representing a distinct taxon of Cercopithecoides, Cercopithecoides haasgati, thus adding a second species of the genus to southern Africa.     

A cell-autonomous requirement for neutral sphingomyelinase 2 in bone mineralization

A deletion mutation called fro (fragilitas ossium) in the murine Smpd3 (sphingomyelin phosphodiesterase 3) gene leads to a severe skeletal dysplasia. Smpd3 encodes a neutral sphingomyelinase (nSMase2), which cleaves sphingomyelin to generate bioactive lipid metabolites. We examined endochondral ossification in embryonic day 15.5 fro/fro mouse embryos and observed impaired apoptosis of hypertrophic chondrocytes and severely undermineralized cortical bones in the developing skeleton. In a recent study, it was suggested that nSMase2 activity in the brain regulates skeletal development through endocrine factors. However, we detected Smpd3 expression in both embryonic and postnatal skeletal tissues in wild-type mice. To investigate whether nSMase2 plays a cell-autonomous role in these tissues, we examined the in vitro mineralization properties of fro/fro osteoblast cultures. fro/fro cultures mineralized less than the control osteoblast cultures. We next generated fro/fro;Col1a1-Smpd3 mice, in which osteoblast-specific expression of Smpd3 corrected the bone abnormalities observed in fro/fro embryos without affecting the cartilage phenotype. Our data suggest tissue-specific roles for nSMase2 in skeletal tissues.     

A Novel Method to Evaluate the Community Built Environment Using Photographs – Environmental Profile of a Community Health (EPOCH) Photo Neighbourhood Evaluation Tool

Background

Previous research has shown that environments with features that encourage walking are associated with increased physical activity. Existing methods to assess the built environment using geographical information systems (GIS) data, direct audit or large surveys of the residents face constraints, such as data availability and comparability, when used to study communities in countries in diverse parts of the world. The aim of this study was to develop a method to evaluate features of the built environment of communities using a standard set of photos. In this report we describe the method of photo collection, photo analysis instrument development and inter-rater reliability of the instrument.

Methods/Principal Findings

A minimum of 5 photos were taken per community in 86 communities in 5 countries according to a standard set of instructions from a designated central point of each community by researchers at each site. A standard pro forma derived from reviewing existing instruments to assess the built environment was developed and used to score the characteristics of each community. Photo sets from each community were assessed independently by three observers in the central research office according to the pro forma and the inter-rater reliability was compared by intra-class correlation (ICC). Overall 87% (53 of 60) items had an ICC of ≥0.70, 7% (4 of 60) had an ICC between 0.60 and 0.70 and 5% (3 of 60) items had an ICC ≤0.50.

Conclusions/Significance

Analysis of photos using a standardized protocol as described in this study offers a means to obtain reliable and reproducible information on the built environment in communities in very diverse locations around the world. The collection of the photographic data required minimal training and the analysis demonstrated high reliability for the majority of items of interest.     

Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

The influence of climate change scenarios on populations of the mayfly Cloeon dipterum

Populations of the mayfly Cloeon dipterum from 48 ponds (3000 l fibre-glass tanks of 1 m depth) were monitored over the course of 1 year. To simulate possible patterns of climatic change, the ponds were subject to three temperature treatments: continuous heating to 3 °C above ambient; heating to 3 °C above ambient during the summer only; and no heating. Further experimental complexity included enhanced nutrient input into the ponds and the presence or absence of fish, giving a factorial combination of 3 temperature regimes × 2 nutrient levels × presence/absence of fish predation.Few nymphs were found in the presence of fish. Where fish were absent, the temperature treatments did not significantly affect nymph abundances, and only marginally influenced mean nymph body-lengths. In contrast, the nutrient treatment had significant effects on both nymph abundance and size, with greater numbers of generally larger nymphs occurring in those fish-free ponds receiving additional nutrients. Adult emergence began earlier in the year from the heated ponds, particularly those ponds receiving additional nutrients. Adult body-length differed between temperature treatments, but consistent patterns were difficult to ascertain because of interactions with nutrient treatment and seasonal effects.Our results show that during the short term at least, elevated temperature as a simulation of climate change does not have an overwhelming influence on either mayfly abundance or size. The influence of temperature is subtle and subject to complex interaction with other habitat variables. We therefore suggest that the direct consequences of small changes in temperature will likely be of little significance to C. dipterum, relative to indirect effects operating through interactions with predation and nutrient input.