Spotted substrates for focal presentation of proteins to cells

Neurites and other cell processes encounter localized deposits of signaling factors as they grow. The difficulty in generating patterned artificial substrates has hindered the analysis of these instructive factors in vitro. Here we report a simple method for presenting cultured cells with small spots of protein on an otherwise uniform substrate. We use a biolistic device called a gene gun to deposit 0.1-5 microm fluorescent dots of pure protein on or beneath a growth-promoting substrate. Using this technique, we demonstrate local presynaptic differentiation of motoneurons in response to dots of a polycation. We also show that biotin-avidin and antibody-antigen interactions can be used to prepare spots from more dilute, more labile, or less abundant proteins. This method should prove useful for analyzing extracellular signaling molecules that act focally on neurons or other cell types.     

An Overlooked Mandibular-Rubbing Behavior Used during Recruitment by the African Weaver Ant,Oecophylla longinoda

In Oecophylla, an ant genus comprising two territorially dominant arboreal species, workers are known to (1) use anal spots to mark their territories, (2) drag their gaster along the substrate to deposit short-range recruitment trails, and (3) drag the extruded rectal gland along the substrate to deposit the trails used in long-range recruitment. Here we study an overlooked but important marking behavior in which O. longinoda workers first rub the underside of their mandibles onto the substrate, and then—in a surprising posture—tilt their head and also rub the upper side of their mandibles. We demonstrate that this behavior is used to recruit nestmates. Its frequency varies with the rate at which a new territory, a sugary food source, a prey item, or an alien ant are discovered. Microscopy analyses showed that both the upper side and the underside of the mandibles possess pores linked to secretory glands. So, by rubbing their mandibles onto the substrate, the workers probably spread a secretion from these glands that is involved in nestmate recruitment.     

The effects of released pheasants on invertebrate populations in and around woodland release sites

The release of gamebirds for recreational shooting exerts a series of effects on the ecosystems into which they are placed. Pheasants (Phasianus colchicus) are omnivorous and eat invertebrates, especially when young or, if females, when breeding. Consequently, the release of large numbers of pheasants into woodland release pens may affect local invertebrate populations. Previous studies have reported mixed evidence. We conducted pitfall trapping at 13 sites (49 pens) in central England over 2 years (totaling 65 pen measures), comprising three surveys annually, immediately prior to releases in mid‐summer, 4 weeks later when most birds were still in the pens, and another 5 weeks later when most birds had dispersed. We compared traps inside and 25 m outside pens in the same wood. We considered release densities and whether the birds had prior experience of eating invertebrates. While accounting for overall seasonal declines in invertebrates trapped, we detected changes for total invertebrate biomass and total counts indicative of released pheasants causing local decreases inside pens, either directly by predation or indirectly by modifying vegetation. There were also relative decreases outside pens when the pheasants start to disperse, suggesting that the released pheasants may be affecting invertebrates in those nearby areas or that their earlier effects inside the pen, modifying vegetation or increasing invertebrate activity, increased the relative susceptibility of invertebrates there to trapping. However, these effects were not seen for specific invertebrate groups believed to be especially susceptible to pheasants. For slugs and detritivores, we detected small population increases inside pens. Across the study period, decreases for most measures were greater outside pens than inside them. We conclude that any effects pheasants have on invertebrate communities at release sites in woodlands are complex and that long‐term and taxon‐specific studies should be considered to understand the local net ecological effects of gamebird releases on invertebrates.     

Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging

Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (<0.1 microg/ml) and contain high concentrations of contaminating protein (>10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays.     

Blow fly artifacts from blood and putrefaction fluid on various surfaces: a source for forensic STR typing

Adult blow flies can deposit small spots (‘artifacts’) containing biological fluids like blood on various surfaces at a crime scene, either by regurgitation or defecation, and thereby create new or modify existing stains. These minute traces may represent evidence for the past presence of a certain person or victim in crime scene scenarios where nobody is present or all other crime‐related biological traces have been cleaned up. We analyzed artifacts on crime scene relevant surfaces (glass, wood, concrete, wallpaper, carpet, plastic wrap), produced by the forensically important blow fly Calliphora vicina Robineau‐Desvoidy (Diptera: Calliphoridae) after ingesting fresh and degraded porcine blood as well as putrefaction fluid from a pig's trotter and evaluated the potential of these spots for short tandem repeat (STR) analysis at various time intervals after deposition. Genotyping was possible on single fly artifacts on all surfaces up to 300 days after deposition by the flies after both blood or putrefaction fluid intake. The results indicate that the suitability for STR typing of those spots mainly depends on the time since deposition. The activity of flies at a crime scene can lead to artifacts which are – even 300 days after deposition – a valuable source of case‐related DNA. Sometimes those traces may even be the only source of information for DNA profiling.     

Changes in activity and object manipulation before tail damage in finisher pigs as an early detector of tail biting

Tail damage within the production of finisher pigs is an animal welfare problem. Recent research suggests that removal of known risk factors may not be enough to eliminate tail biting, especially in undocked pigs, thus a different strategy is worth investigating. This could be early detection of tail biting, using behavioural changes observed before tail damage. If these early stages of tail biting can be detected before tail damage occurs, then tail damage could be prevented by early interventions. The first step in developing such a strategy is to identify the types of behaviour changes that emerge during early stages of tail biting. Thus, the aim of the current study was to investigate whether pen level activity and object manipulation evolved differently during the last 7 days before the scoring of tail damage (day 0) for pens scored with tail damage (tail damage pens) and pens not scored with tail damage (matched control pens). The study included video recordings for twenty-four tail damage pens and thirty-two matched control pens. Activity level and object manipulation were observed the last 7 days before day 0 during the morning (0600 to 0800 h), afternoon (1600 to 1800 h) and evening (2200 to 2400 h, only activity level). Both activity level and object manipulation were analysed using generalised linear mixed effects models with a binomial distribution for activity level and a negative binomial distribution for object manipulation. The probability of being active was higher in tail damage pens compared to control pens during the afternoon the last 5 days before day 0 (P<0.001). This was seen due to a decrease in activity level in the control pens, which makes it difficult to identify future tail damage pens from this difference. Object manipulation was lower in tail damage pens compared to the control pens on all 7 days before day 0, but only in pens with undocked pigs (P<0.01). Thus, it is still unknown when this difference in object manipulation initiated. It was concluded that both activity level and object manipulation seemed related to ongoing tail biting and should be investigated through more detailed observations and for a longer time to establish the normal behaviour pattern for a particular pen. Thus, it is suggested that future research focusses on developing automatic monitoring methods for pen level activity and object manipulation and applies algorithms that establish and detect deviations from the normal behaviour pattern of the pen before tail damage.     

An Update on Insulin Injection Devices

Background: Over the past 15 years, it has become clear that better glycemic control can lead to a substantial reduction in diabetic complications and that such control often requires the use of insulin therapy. However, a number of barriers exist to starting such therapy in patients with diabetes mellitus (DM). Many of these barriers to treatment are related to the use of a syringe to inject the medication. In the past ~20 years, various "pen" devices have become available that help to reduce the stigma associated with insulin injection, allowing more patients to achieve the glycemic control that they require.Objectives: This article provides an overview of the various pen devices available in the United States for subcutaneous insulin delivery and discusses the benefits these devices can provide to patients; their disadvantages are also discussed. Third-party reimbursement for these devices is highlighted.Methods: A MEDLINE search was performed (1980-2007) to identify relevant articles (English-language only) using appropriate key terms, including insulin pen and insulin delivery device. Technical specifications and availability information for the various pen devices were obtained directly from their manufacturers. Insurance coverage data were provided by major national insurance carriers.Results: There are a number of excellent pen devices available for administering each of the currently offered basal and bolus insulin analogues as well as neutral protamine Hagedorn and regular insulins. These devices range from disposable pens—which are supplied to the patient prefilled from the pharmacy, used until empty, and then discarded—to refillable digital pens, some of which have the ability to “remember” prior insulin doses. Data from various studies indicate that both patients and their physicians generally prefer insulin pens over the traditional vial and syringe delivery method. These devices are simple to use, allow patients to be more discreet in social situations, can easily be carried in a shirt pocket or purse, do not need to be refrigerated while in use, are associated with a lesser degree of injection anxiety, and may even be more accurate at lower doses. They may also be the preferred delivery system for the visually impaired, given their larger displays and the audible/tactile “click” produced each time the dose is increased by 1 U. Insulin pens lead to increased patient confidence and satisfaction and improved attitude toward insulin therapy. Most major insurance plans provide some coverage for the disposable pens as well as the cartridges for refillable pens, but this is often under tier 2 and some insurers may require prior authorization.Conclusions: Insulin pens provide DM patients with a number of advantages over a vial and syringe and can often help them overcome major barriers to the initiation of insulin therapy. The use of insulin pens leads to increased patient compliance and potential improvements in glycosylated hemoglobin, but data on glycemic control are not available. The pens should be offered to virtually all patients who require insulin therapy, except in instances in which these pens are financially prohibited.     

One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry

We describe a simple and direct zymographic method for the detection of proteases using quenched fluorescent substrates. The proteases were separated using one- and two-dimensional electrophoresis, and the gel subsequently was incubated with the quenched fluorescent substrate. After a short incubation, the released fluorescence allowed the localization of the proteases directly using UV light. The protease spots could then be cut directly from the gel and processed for identification by mass spectrometry. This method could easily be used to develop or test whether a substrate is specific or not and also to detect the proteases that are able to cleave this substrate in a complex biological fluid. This also allowed direct identification of proteases without complex purification.     

Drepanocladus trifarius– an example of unsuspected niche widths among mosses

The habitat of the pleurocarpous moss Drepanocladus trifarius is commonly described as mineral‐rich wet fens. We sampled individual D. trifarius shoots at 214 pre‐defined randomly distributed spots in an area of ca 15 km² in a sloping fen in central–western Sweden. We assessed the habitat variation of the sampling spots in this area by means of a multi‐variate analysis (DCA) based on the species identity of the ten shoots adjacent to D. trifarius, and by indicator values for light availability and substrate moisture, acidity, and nutrient availability of the associated species. Both approaches indicated that D. trifarius occurred in well lit and constantly moist, wet or submerged places. Most spots were weakly acid to sub‐neutral, but some spots had distinctly acid conditions. Generally the spots were nutrient‐poor, but a few were nutrient‐ rich. DCA revealed gradients from relatively wet, sub‐neutral to basic towards drier and more acid habitats, and from nutrient‐poor to ‐richer environments. The species obviously exhibits a broader than expected niche width with respect to substrate acidity and nutrients. We suggest that succession, disturbance, or climatic factors may explain the species’ occurrences in different habitats.     

Insulin Pen Injection Devices for Management of Patients with Type 2 Diabetes: Considerations Based on an Endocrinologist’s Practical Experience in the United States

ObjectiveTo review the features of several insulin pens currently available in the United States, discuss the validity of concerns about certain pen devices, and provide specific training information for clinicians to increase the accuracy of insulin administration and patient satisfaction with the use of insulin pens.MethodsThe published literature on insulin pens and Internet-available, product-specific information are reviewed. In addition, special practical considerations regarding insulin pen selection based on personal experience in a high-volume endocrinology practice are highlighted by presentation of case vignettes.ResultsFor some patients with diabetes, the need for performance of self-injection can be a barrier to acceptance of insulin therapy. Insulin pen devices provide a delivery option that may be more acceptable and more convenient to use in comparison with traditional vials and syringes and thus may promote patient compliance, which can enhance the ability to achieve and maintain glycemic control. When choosing a specific insulin pen for an individual patient, the clinician should consider the patient’s insulin regimen, lifestyle, and factors that may affect the ability to use a particular device, such as motor dexterity and visual acuity.ConclusionInsulin pens offer convenience and can potentially increase patient satisfaction and compliance with therapy. Because certain characteristics of a given insulin pen may make it preferable for specific patients, it is important for clinicians to be aware of individual needs. Provision of thorough training for patients in the correct use of insulin pens is important because user error can affect pen performance and accuracy of the dose administered. Manufacturers should be notified of any recurring problems. (Endocr Pract. 2007;13:672-678)     

Scent‐Mark Identification and Scent‐Marking Behaviour in African Wild Dogs (Lycaon pictus)

Scent‐marking is common in mammals, but where signals are carried by urine and faeces, distinguishing between scent‐marking and mere elimination is problematic. To do so, we documented behaviours and context variables associated with urination and defecation in free‐ranging endangered African wild dogs (Lycaon pictus) and tested whether these were related to the responses of other dogs to deposits. We found that distinct postures were almost uniquely associated with deposits by dominant wild dogs, were more common during urination than defecation, and increased the likelihood that these deposits would be investigated by other wild dogs. The likelihood of investigation depended on the sex and dominance status of the depositor, the type of deposit and the substrate. Urine from dominant females was more likely to be investigated by other wild dogs than any other deposits, and deposits placed on vegetation were more likely to be investigated than those on bare ground. The likelihood that a deposit would be overmarked was affected by the deposit type and the sex and dominance status of the last depositor. Collectively, these results suggest that dominant wild dog urine is of greatest interest to other dogs. Our results show that some deposits by African wild dogs are not scent‐marks and that detailed observations of behaviours and context variables during elimination events can be used to distinguish deposits that are likely to be of communication value.     

Effect of pens with an elevated platform on space utilization,skin lesions and growth performance in nursery pigs

Environmental enrichment, adequate space and the ability to separate a living area into suitable functional areas are key elements for pig welfare. In this study, a two-level pen for nursery pigs was explored with the aim of analysing the use of space and the impact on pig behaviour, health and performance. Therefore, per batch, three experimental groups in pens with elevated platforms (two-level pens) and one control group in a standard pen were formed after weaning at the age of 4 weeks. Thereafter, groups were studied for a period of 6 weeks. In a total of eight batches (n = 882 pigs), the occurrence of skin lesions was determined per individual using a lesion score, and in seven batches (n = 761 pigs) individual daily weight gain was analysed. In five batches (n = 450 pigs), the individual use of space and the behaviour of pigs were investigated by direct observation (4 h per day on 2 days in weeks 1, 3 and 6 of the housing period, respectively). Results revealed that 98.9% of pigs (n = 445) were observed on the elevated platform at least once. The probability that an individual pig used the platform was significantly higher than 0.95 (P < 0.0001, confidence interval = 0.977, 1). The use of the platform was not affected by sex (odds ratio (OR) = 1.013, P = 0.937). However, the probability of observing an individual on the elevated platform increased with increasing BW during the experimental period (OR = 1.043, P < 0.0001). The presence of a platform decreased the probability of seeing a pig fighting (P = 0.014) and increased the probability of observing locomotor behaviour as opposed to lying (P < 0.0001). In two-level pens, pigs sustained fewer skin injuries than in standard pens (day 41: OR = 0.731, P < 0.0001). Over 41 days of experiment, pigs in two-level pens had higher daily weight gains than animals in standard pens (416 versus 393 g/day, P = 0.006). We conclude that usable space for nursery pigs can be effectively extended by introducing elevated platforms into the pigs’ pen. Furthermore, pigs may benefit from two-level systems by establishing spaces for activity as well as for retreat from other pen mates, thereby reducing aggressive behaviour and social stress. The installation of two-level pens is therefore encouraged, if they are intended to provide more space than legally stipulated.     

Preslaughter Holding Environment in Pork Plants Is Highly Contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied (~150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period (~2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.     

Preslaughter holding environment in pork plants is highly contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied ( approximately 150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period ( approximately 2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.     

Sexually dimorphic melanin‐based colour polymorphism,feather melanin content,and wing feather structure in the barn owl (Tyto alba)

Feathers confer protection against biophysical agents and determine flying ability. The geometry and arrangement of the barbs, together with the keratin and pigments deposited in the feathers, determine the mechanical stability of the vane, and its stiffness and resistance to abrasive agents. In colour‐polymorphic species, individuals display alternative colour morphs, which can be associated with different foraging strategies. Each morph may therefore require specific flying abilities, and their feathers may be exposed to different abrasive agents. Feathers of differently coloured individuals may thus have a specific structure, and colour pigments may help resist abrasive agents and improve stiffness. We examined these predictions in the barn owl (Tyto alba), a species for which the ventral body side varies from white to dark reddish pheomelanic, and in the number and size of black spots located at the tip of the feathers. White and reddish birds show different foraging strategies, and the size of black feather spots is associated with several phenotypic attributes. We found that birds displaying a darker reddish coloration on the ventral body side deposit more melanin pigments in their remiges, which also have fewer barbs. This suggests that wear resistance increases with darkness, whereas feathers of lighter coloured birds may bend less easily. Accordingly, individuals displaying a lighter reddish coloration on the ventral body side, and those displaying larger black spots, displayed more black transverse bars on their remiges: as larger‐spotted individuals are heavier and longer‐winged birds also have more transverse bars, these bars may reduce feather bending when flying. We conclude that differently coloured individuals produce wing feathers of different strengths to adopt alternative behavioural and life history strategies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 562–573.     

Root Distribution and Nutrient Status of Mycorrhizal and Non-mycorrhizal Pinus sylvestris L. Seedlings Growing in a Sandy Substrate with Lignite Fragments

Reclaimed mine soils of the Lusatian mining district are characterised by small-scale heterogeneous distribution of lignite fragments of varying size embedded in a matrix of Tertiary and Quaternary sandy material. Despite amelioration with basic fly ashes, ongoing pyrite oxidation and the subsequent acidification generate a high physical and chemical heterogeneity within the substrate, which could negatively affect root proliferation. We hypothesised that this limitation for the root system may be compensated for by intensive exploration of the porous lignite fragments by roots and/or mycorrhizal hyphae to access water and nutrients stored in these fragments. To test this hypothesis, we compared growth, shoot nutrient content, and root distribution of mycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings in lignite-containing and lignite-free sandy substrate. Rhizotrons used for this experiment were filled with a sandy matrix with 6–9 evenly distributed spots of lignite fragments. Treatments included different levels of water and nutrient availability. After 8 months of growth, root tip vitality as well as growth and shoot nutrient concentration of the plants was higher for treatments with lignite spots in the sandy substrate than for sandy substrate without such amendments. Compared to the non-mycorrhizal plants, the seedlings inoculated with Paxillus involutus (Batsch) Fr. had a higher root dry mass, an increased number of root tips and a higher root length. These results confirm our hypothesis that the lignite fragments are an important nutrient and water reservoir for plants in these mine soils and they indicate that mycorrhizal colonisation may allow an intensive exploration of porous lignite fragments by mycorrhizal hyphae.     

Effects of the observation method (direct v. from video) and of the presence of an observer on behavioural results in veal calves

This study aimed at assessing the effect of the observation method (direct or from video) and the effect of the presence of an observer on the behavioural results in veal calves kept on a commercial farm. To evaluate the effect of the observation method, 20 pens (four to five calves per pen) were observed by an observer for 60 min (two observation sessions of 30 min) and video-recorded at the same time. To evaluate the effect of the presence of the observer in front of the pen, 24 pens were video-recorded on 4 consecutive days and an observer was present in front of each pen for 60 min (two observation sessions of 30 min) on the third day. Behaviour was recorded using instantaneous scan sampling. For the study of the observer's effect, the analysis was limited to the posture, abnormal oral behaviour and manipulation of substrates. The two observation methods gave similar results for the time spent standing, but different results for all other behaviours. The presence of an observer did not affect the behaviour of calves at day level; however, their behaviour was affected when the observer was actually present in front of the pens. A higher percentage of calves were standing and were manipulating substrate in the presence of the observer, but there was no effect on abnormal oral behaviour. In conclusion, direct observations are a more suitable observation method than observations from video recordings for detailed behaviours in veal calves. The presence of an observer has a short-term effect on certain behaviours of calves that will have to be taken into consideration when monitoring these behaviours.     

Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.     

Higher preweaning mortality in free farrowing pens compared with farrowing crates in three commercial pig farms

If loose-housed farrowing systems are to be an alternative to traditional farrowing crates, it is important that they can deliver the same production results as can be achieved in farrowing crates under commercial conditions. The aim of this study was to compare preweaning mortality in farrowing crates and free farrowing pens (FF-pens) within herds that had both systems. The study was conducted over 2 years in three commercial Danish herds that had FF-pens as well as traditional farrowing crates in their farrowing unit. Piglet mortality was analysed in two periods: before litter equalisation and after litter equalisation. Linear models were used to analyse effects of housing (crate or pen), herd (Herd A, B or C), parity (parities 1, 2, 3 to 4 or 5 to 8) as well as the effect of number of total born piglets on mortality before litter equalisation, and the effect of equalised litter size on piglet mortality after litter equalisation. All corresponding interactions were included in the models. Before litter equalisation piglet mortality was higher (P<0.001) in pens (13.7%) than in crates (11.8%). Similarly, piglet mortality after litter equalisation was higher in pens than in crates in all three herds, but the difference between pens and crates were dissimilar (P<0.05) in the different herds. In addition, piglet mortality, both before (P<0.001) and after litter equalisation (P<0.001), grew with increasing parity of the sows. Mortality before litter equalisation moreover increased with increasing number of total born piglets per litter (P<0.001), and mortality after equalisation increased when equalised litter size increased (P<0.001). No significant interactions were detected between housing and parity or housing and litter size for any of the analysed variables. In conclusion, there is knowledge how to design pens for free farrowing; but this study showed a higher preweaning mortality in the FF-pen. Nonetheless a noteworthy proportion of the sows in the FF-pens delivered results comparable to those farrowing in crates. This indicates that FF-pens are not yet a robust type of housing for farrowing sows.     

The development and validation of a fast and robust dried blood spot based lipid profiling method to study infant metabolism

Early life exposures and metabolic programming are associated with later disease risk. In particular lipid metabolism is thought to play a key role in the development of the metabolic syndrome and insulin resistance in later life. Investigative studies of metabolic programming are limited by the ethics and practicalities of sample collection in small infants. Dried blood spots on filter paper, derived from heel pricks are considered as the most suitable option for this age group. We validated a novel lipid profiling method, based on high resolution mass spectrometry to successfully determine the lipid composition of infants using dried blood spots. The spotting and air drying of blood on paper has noticeable effects on many of the lipids, leading to lipid oxidation and hydrolysis, which demand careful interpretation of the obtained data. We compared the lipid profiles from plasma or whole blood samples and the results from dried blood spots to determine if these revealed the same inter-subject differences. The results from dried blood spots were no less reproducible than other lipid profiling methods which required comparatively larger sample volumes. Therefore, lipid profiles obtained from dried blood spots can be successfully used to monitor infancy lipid metabolism and we show significant differences in the lipid metabolism of infants at age 3 versus 12 months.