Development of an ELISA for Evaluation of Swab Recovery Efficiencies of Bovine Serum Albumin

After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32–80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0–60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.     

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus‐infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV‐1‐infected cells. By combining an unbiased large‐scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV‐1‐infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor‐mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL‐mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL‐mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti‐HIV‐1 activity of NK cells but also possesses a multifunctional role beyond receptor‐mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.     

Transgenic or tumor-induced expression of heparanase upregulates sulfation of heparan sulfate

Heparan sulfate proteoglycans (HSPGs) interact with numerous proteins of importance in animal development and homeostasis. Heparanase, which is expressed in normal tissues and upregulated in angiogenesis, cancer and inflammation, selectively cleaves beta-glucuronidic linkages in HS chains. In a previous study, we transgenically overexpressed heparanase in mice to assess the overall effects of heparanase on HS metabolism. Metabolic labeling confirmed extensive fragmentation of HS in vivo. In the current study we found that in liver showing excessive heparanase overexpression, HSPG turnover is accelerated along with upregulation of HS N- and O-sulfation, thus yielding heparin-like chains without the domain structure typical of HS. Heparanase overexpression in other mouse organs and in human tumors correlated with increased 6-O-sulfation of HS, whereas the domain structure was conserved. The heavily sulfated HS fragments strongly promoted formation of ternary complexes with fibroblast growth factor 1 (FGF1) or FGF2 and FGF receptor 1. Heparanase thus contributes to regulation of HS biosynthesis in a way that may promote growth factor action in tumor angiogenesis and metastasis.     

Three-dimensional epidermis-like growth of human mesenchymal stem cells on dermal equivalents: contribution to tissue organization by adaptation of myofibroblastic phenotype and function

Abstract Human mesenchymal stem cells (hMSC) are able to differentiate into mature cells of various mesenchymal tissues. Recent studies have reported that hMSC may even give rise to cells of ectodermal origin. This indication of plasticity makes hMSC a promising donor source for cell-based therapies. This study explores the differentiation potential of hMSC in a tissue-specific microenvironment simulated in vitro . HMSC were cultured air-exposed on dermal equivalents (DEs) consisting of collagen types I and III with dermal fibroblasts and subjected to conditions similar to those used for tissue engineering of skin with keratinocytes. Culture conditions were additionally modified by pre-treating the cells with 5-azacytidine or supplementing the medium with all trans retinoic acid (RA). HMSC were capable of adaptation to epidermis-specific conditions without losing their mesenchymal multipotency. However, despite the viability and evident three-dimensional epidermis-like growth pattern, hMSC showed a persistent expression of mesenchymal but not of epithelial markers, thus indicating a lack of epidermal (trans) differentiation. Further, electron microscopy and immunohistochemical analyses demonstrated that hMSC cultured under epidermis-specific conditions adopted a myofibroblastic phenotype and function, promoted in particular by air exposure. In conclusion, multipotent hMSC failed to differentiate into E-cadherin- or cytokeratin-expressing cells under optimized organotypic culture conditions for keratinocytes but differentiated into myofibroblast-like cells contracting the extracellular matrix, a phenomenon that was enhanced by RA and 5-azacytidine. These results indicate that hMSC might contribute to wound-healing processes by extracellular matrix reorganization and wound contraction but not by differentiation into keratinocytes.     

Identifying Drivers of China's Provincial Wastewater Reuse Outcomes Using Qualitative Comparative Analysis

In water‐scarce regions of China, wastewater reuse is increasingly considered as a potential component of China's future water resource management strategy. Currently, the percentage of wastewater reuse varies substantially across Chinese provinces, but conditions leading to a high rate of wastewater reuse have not been elucidated clearly. In this work, we use fuzzy‐set qualitative comparative analysis (fsQCA) to identify the drivers of high and low percentages of wastewater reuse in water‐stressed Chinese provinces in 2013. We find that among the five conditions studied (per capita water availability, urban population, access to sea, access to urban space, and access to agricultural land), a high percentage of wastewater reuse is primarily driven by water stress and access to urban green space. Consequently, policies should consider targeting provinces with these attributes where wastewater reuse is more likely to be successful. Further, our results show that there is asymmetry in the conditions that lead to high and low percentages of wastewater reuse, and that the drivers for and against reuse identified in this study are not completely analogous to those identified in previous studies. As such, the drivers for and against wastewater reuse should not be generalized without due consideration of the local context.     

Comparison of two prepill cortisol concentrations in dogs with hypercortisolism treated with trilostane

Background

The ideal method for monitoring trilostane therapy in dogs with hypercortisolism is still open to debate. Recently, determination of the pre-trilostane (prepill) cortisol concentration has been proposed to be more repeatable than either post-trilostane or post-ACTH cortisol. The aim of this study was to compare two prepill cortisol concentrations in dogs with hypercortisolism during trilostane therapy. Sixteen client-owned dogs with naturally occurring hypercortisolism were prospectively included and cortisol concentrations were measured twice, 1?h apart, before the morning trilostane dose (prepill 1 and 2 cortisol).

Results

A total of 47 prepill cortisol measurement pairs were included. Compared to prepill 1, prepill 2 cortisol was higher in 15, equal in 8 and lower in 24 pairs. Group agreement between prepill 1 and 2 cortisol was 70% (moderate agreement - weighted kappa 0.55). In 30% of the pairs, group assignment was discrepant, implying a different therapeutic decision. In some dogs certain circumstances (e.g. excessive barking, difficulties during blood collection, excitement at arrival) were identified as potential factors explaining the discrepancy between prepill 1 and 2 cortisol measurements.

Conclusions

In a substantial number of dogs treated with trilostane, the two prepill cortisol concentrations differed. Part of this difference might be ascribable to stressful events during test performance. When using prepill cortisol measurements to monitor trilostane therapy, recording of any incident during handling that might affect cortisol release might be helpful to make a reliable decision about a trilostane dose adaptation.

     

Effects of a sea barrier on large-scale migration patterns studied by a network of weather radars

Capsule: Nocturnal passerine migration patterns were studied by a network of weather radars within the East-Atlantic flyway providing large-scale information on the effect of a geographical barrier.

Aims: The aim of this study was to obtain a large-scale spatial overview of the effects of a sea barrier on migratory flyways in northern Spain/western France.

Methods: Weather radar data were used from five sites at the Bay of Biscay during nights in spring 2015 to calculate flight directions and migration traffic rates (MTRs).

Results: The highest MTRs were registered by the radars at the southeastern edge of the bay, with a gradual decrease northwards. Spring migration direction was generally NNE/NE. Continuous nocturnal migration pattern indicated migration over land in the south. The radar half way up the French coast exhibited bimodal migration intensities at night, indicating sea crossing, and the northernmost corner of the bay showed little migration.

Conclusions: Radar patterns indicated migration over land and sea during spring migration. Sea crossing occurred with flight distances of up to about 500?km. Most migration activity was observed in the radars along the southeastern section of the bay, indicating that the general migration flyway from Spain funnels through the eastern side of the north Iberian Peninsula.     

Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation

Summary Direct evidence is presented that the mechanism which discriminates against low efficiency markers in transformation of Diplococcus pneumoniae of genotype hex ⁺ acts on them after the formation of donor-recipient heteroduplexes. This conclusion is based on assays of the transforming activity of donor markers in lysates made after various times of incubation of recipient cells following exposure to DNA. The activity of a low efficiency marker rises substantially, indicating formation of native-like heteroduplex structures, and then falls. At 37° C the process is essentially completed 10 minutes after entry, and the apparent half life of a susceptible heteroduplex is 1.5 to 2 minutes. Data from these and other experiments imply that about as many of the surviving low efficiency markers have simply escaped attack as have been inserted into both strands by the excision-repair process suggested by Ephrussi-Taylor.