Basic strategies for valid cytometry using image analysis

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals. This revised version was published online in November 2006 with corrections to the Cover Date.     

Salinity thresholds for understory plants in coastal wetlands

The effects of sea level rise and coastal saltwater intrusion on wetland plants can extend well above the high-tide line due to drought, hurricanes, and groundwater intrusion. Research has examined how coastal salt marsh plant communities respond to increased flooding and salinity, but more inland coastal systems have received less attention. The aim of this study was to identify whether ground layer plants exhibit threshold responses to salinity exposure. We used two vegetation surveys throughout the Albemarle-Pamlico Peninsula (APP) of North Carolina, USA to assess vegetation in a low elevation landscape (≤?3.8 m) experiencing high rates of sea level rise (3–4 mm/year). We examined the primary drivers of community composition change using Non-metric Multidimensional Scaling (NMDS) and used Threshold Indicator Taxa Analysis (TITAN) to detect thresholds of compositional change based on indicator taxa, in response to potential indicators of exposure to saltwater (Na, and the Σ Ca?+?Mg) and elevation. Salinity and elevation explained 64% of the variation in community composition, and we found two salinity thresholds for both soil Na⁺ (265 and 3843 g Na⁺/g) and Ca⁺ +?Mg⁺ (42 and 126 µeq/g) where major changes in community composition occur on the APP. Similar sets of species showed sensitivity to these different metrics of salt exposure. Overall, our results showed that ground layer plants can be used as reliable indicators of salinity thresholds in coastal wetlands. These results can be used for monitoring salt exposure of ecosystems and for identifying areas at risk for undergoing future community shifts.

     

Evaluation of the prevalence and economic burden of adverse drug reactions presenting to the medical emergency department of a tertiary referral centre: a prospective study

Background  

Adverse drug reactions (ADRs) are now recognized as an important cause of hospital admissions, with a proportion ranging from 0.9–7.9%. They also constitute a significant economic burden. We thus aimed at determining the prevalence and the economic burden of ADRs presenting to Medical Emergency Department (ED) of a tertiary referral center in India     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

Epigenetic landscape correlates with genetic subtype but does not predict outcome in childhood acute lymphoblastic leukemia

Although children with acute lymphoblastic leukemia (ALL) generally have a good outcome, some patients do relapse and survival following relapse is poor. Altered DNA methylation is highly prevalent in ALL and raises the possibility that DNA methylation-based biomarkers could predict patient outcome. In this study, genome-wide methylation analysis, using the Illumina Infinium HumanMethylation450 BeadChip platform, was carried out on 52 diagnostic patient samples from 4 genetic subtypes [ETV6-RUNX1, high hyperdiploidy (HeH), TCF3-PBX1 and dic(9;20)(p11–13;q11)] in a 1:1 case-control design with patients who went on to relapse (as cases) and patients achieving long-term remission (as controls). Pyrosequencing assays for selected loci were used to confirm the array-generated data. Non-negative matrix factorization consensus clustering readily clustered samples according to genetic subgroups and gene enrichment pathway analysis suggested that this is in part driven by epigenetic disruption of subtype specific signaling pathways. Multiple bioinformatics approaches (including bump hunting and individual locus analysis) were used to identify CpG sites or regions associated with outcome. However, no associations with relapse were identified. Our data revealed that ETV6-RUNX1 and dic(9;20) subtypes were mostly associated with hypermethylation; conversely, TCF3-PBX1 and HeH were associated with hypomethylation. We observed significant enrichment of the neuroactive ligand-receptor interaction pathway in TCF3-PBX1 as well as an enrichment of genes involved in immunity and infection pathways in ETV6-RUNX1 subtype. Taken together, our results suggest that altered DNA methylation may have differential impacts in distinct ALL genetic subtypes.     

Effects of crop field characteristics on nocturnal winter use by American woodcock

Since the late 1960s, American woodcock (Scolopax minor) have undergone population declines because of habitat loss. Previous research suggested ridge and furrow topography in conventionally tilled soybean fields provided critical nocturnal cover as birds foraged on earthworms. However, the use of no-till technology has increased and many fields now lack ridge and furrow topography. We assessed woodcock winter nocturnal foraging habitat use given recent changes in agricultural technology, and investigated how field treatment, earthworm abundance, and environmental variables affect the selection of nocturnal foraging sites. We counted woodcock along transects in 5 field treatments twice in each of 67 fields during December–March 2008–2009 and 72 fields during December–March 2009–2010. During both seasons, we collected earthworm and soil samples from a subset of fields of each field treatment. Woodcock densities were at least twice as high in no-till soybean fields planted after corn and in undisked corn fields with mowed stalks than in other field treatments. No-till soybean planted after corn and undisked corn fields contained ridge and furrow topography, whereas other crops did not, and earthworms were at least 1.5 times more abundant in no-till soybean fields than other field treatments. Ridges and furrows in no-till soybean fields planted after corn and undisked corn fields may provide wintering woodcock with thermal protection and concealment from predators. No-till soybean fields planted after corn offered the additional benefit of relatively high food availability. The presence of ridge and furrow topography can be used to predict woodcock field use on the wintering grounds in agricultural areas. Farmers can provide nocturnal winter foraging sites for woodcock by delaying field disking and leaving ridge and furrow topography in crop fields. © 2011 The Wildlife Society.     

Comprehensive gene-expression survey identifies wif1 as a modulator of cardiomyocyte differentiation

During chicken cardiac development the proepicardium (PE) forms the epicardium (Epi), which contributes to several non-myocardial lineages within the heart. In contrast to Epi-explant cultures, PE explants can differentiate into a cardiomyocyte phenotype. By temporal microarray expression profiles of PE-explant cultures and maturing Epi cells, we identified genes specifically associated with differentiation towards either of these lineages and genes that are associated with the Epi-lineage restriction. We found a central role for Wnt signaling in the determination of the different cell lineages. Immunofluorescent staining after recombinant-protein incubation in PE-explant cultures indicated that the early upregulated Wnt inhibitory factor-1 (Wif1), stimulates cardiomyocyte differentiation in a similar manner as Wnt stimulation. Concordingly, in the mouse pluripotent embryogenic carcinoma cell line p19cl6, early and late Wif1 exposure enhances and attenuates differentiation, respectively. In ovo exposure of the HH12 chicken embryonic heart to Wif1 increases the Tbx18-positive cardiac progenitor pool. These data indicate that Wif1 enhances cardiomyogenesis.