Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.     

Fertilization effects with P on accumulated leaf area duration,biomass and yield of three cultivars of maize in Toluca,Mexico

The effect of six phosphorus levels (0, 40, 80, 120, 160 and 200 kg/ha) on the duration of cumulative leaf area, biomass and agronomic yield was determined in the maize cultivars: Amarillo Almoloya, Cacahuacintle and Condor in 2010 and 2011. Such cultivars were sown in the Cerrillo Piedras Blancas Mexico. A completely randomized complete block design with factorial arrangement was utilized. High phosphorus levels (120, 160 and 200 kg/ha) positively affected the duration of cumulative leaf area; greatest values were obtained in Cacahuacintle. A greater duration of accumulated leaf area contributes to determine high values of biomass accumulation and grain yield in this cultivar. Leaf area duration appeared to be a useful tool for evaluating different genotypes in a given environment.     

Tissue factor as a proinflammatory agent

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4^-Mac1⁺ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1α and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.     

Stem cell competition: how speeding mutants beat the rest

How mutations lead to tumor formation is a central question in cancer research. Although cellular changes that follow the occurrence of common mutations are well characterized, much less is known about their effects on the population level. Now, two recent studies reveal in what way oncogenic aberrations alter stem cell dynamics to provide cells with an evolutionary advantage over their neighbors (Amoyel et al, 2014 ; Baker et al, 2014 ).     

Tissue factor as a proinflammatory agent

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4-Mac1+ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1alpha and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.     

Genetic analysis of life-history constraint and evolution in a wild ungulate population

Trade-offs among life-history traits are central to evolutionary theory. In quantitative genetic terms, trade-offs may be manifested as negative genetic covariances relative to the direction of selection on phenotypic traits. Although the expression and selection of ecologically important phenotypic variation are fundamentally multivariate phenomena, the in situ quantification of genetic covariances is challenging. Even for life-history traits, where well-developed theory exists with which to relate phenotypic variation to fitness variation, little evidence exists from in situ studies that negative genetic covariances are an important aspect of the genetic architecture of life-history traits. In fact, the majority of reported estimates of genetic covariances among life-history traits are positive. Here we apply theory of the genetics and selection of life histories in organisms with complex life cycles to provide a framework for quantifying the contribution of multivariate genetically based relationships among traits to evolutionary constraint. We use a Bayesian framework to link pedigree-based inference of the genetic basis of variation in life-history traits to evolutionary demography theory regarding how life histories are selected. Our results suggest that genetic covariances may be acting to constrain the evolution of female life-history traits in a wild population of red deer Cervus elaphus: genetic covariances are estimated to reduce the rate of adaptation by about 40%, relative to predicted evolutionary change in the absence of genetic covariances. Furthermore, multivariate phenotypic (rather than genetic) relationships among female life-history traits do not reveal this constraint.     

Factor VIIa/tissue factor generates a form of factor V with unchanged specific activity, resistance to activation by thrombin, and increased sensitivity to activated protein C

Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.     

The predictive value of transcranial duplex sonography for the clinical diagnosis in undiagnosed parkinsonian syndromes: comparison with SPECT scans

Background  

Transcranial duplex sonography (TCD) of the substantia nigra has emerged as a promising, non-invasive tool to diagnose idiopathic Parkinson's disease (IPD). However, its diagnostic accuracy in patients with undefined parkinsonism remains to be determined.     

Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.