Detection of residual aneuploid leukemic cells by "continuous gating".

BACKGROUND: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list-mode-datasets with a new software called "Continuous Gating". The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. METHODS: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode-data were analyzed with the new "Windows"-based "Continuous Gating" software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1-peak of possible residual tumor-cells should be found. Ten thousand overlapping gates of size 200 x 200 channels (out of 1,023 x 1,023 channels) were set automatically by the program into the side-scatter (SSC)/CD34 dot-plot, calculating the percentage of aneuploid G0/G1-phase cells for every specific gate. RESULTS: The results are plotted in a contour-plot. In dot-plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour-plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. CONCLUSIONS: The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.     

Electron availability in CO2, CO and H2 mixtures constrains flux distribution,energy management and product formation in Clostridium ljungdahlii

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO₂ footprint by converting industrial emissions containing CO₂, CO and H₂ into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO₂ + H₂, CO and CO + CO₂ + H₂ (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD⁺ provider for CO₂ + H_2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd_red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd_red formation when CO is used as substrate, whereas Fd_red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO₂ + H₂. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.     

Ecological importance of large-diameter trees in a temperate mixed-conifer forest

Large-diameter trees dominate the structure, dynamics and function of many temperate and tropical forests. Although both scaling theory and competition theory make predictions about the relative composition and spatial patterns of large-diameter trees compared to smaller diameter trees, these predictions are rarely tested. We established a 25.6 ha permanent plot within which we tagged and mapped all trees ≥1 cm dbh, all snags ≥10 cm dbh, and all shrub patches ≥2 m(2). We sampled downed woody debris, litter, and duff with line intercept transects. Aboveground live biomass of the 23 woody species was 507.9 Mg/ha, of which 503.8 Mg/ha was trees (SD?=?114.3 Mg/ha) and 4.1 Mg/ha was shrubs. Aboveground live and dead biomass was 652.0 Mg/ha. Large-diameter trees comprised 1.4% of individuals but 49.4% of biomass, with biomass dominated by Abies concolor and Pinus lambertiana (93.0% of tree biomass). The large-diameter component dominated the biomass of snags (59.5%) and contributed significantly to that of woody debris (36.6%). Traditional scaling theory was not a good model for either the relationship between tree radii and tree abundance or tree biomass. Spatial patterning of large-diameter trees of the three most abundant species differed from that of small-diameter conspecifics. For A. concolor and P. lambertiana, as well as all trees pooled, large-diameter and small-diameter trees were spatially segregated through inter-tree distances <10 m. Competition alone was insufficient to explain the spatial patterns of large-diameter trees and spatial relationships between large-diameter and small-diameter trees. Long-term observations may reveal regulation of forest biomass and spatial structure by fire, wind, pathogens, and insects in Sierra Nevada mixed-conifer forests. Sustaining ecosystem functions such as carbon storage or provision of specialist species habitat will likely require different management strategies when the functions are performed primarily by a few large trees as opposed to many smaller trees.     

The ligand-binding loops in the tunicate C-type lectin TC14 are rigid

C-type lectin-like domains are very common components of extracellular proteins in animals. They bind to a variety of ligands, including carbohydrates, proteins, ice, and CaCO3 crystals. Their structure is characterized by long surface loops in the area of the protein usually involved in ligand binding. The C-type lectin TC14 from Polyandrocarpa misakiensis specifically binds to D-galactose by coordination of the sugar to a bound calcium atom. We have studied the dynamic properties of TC14 by measuring 15N longitudinal and transverse relaxation rates as well as [1H-15N] heteronuclear NOEs. Relaxation rates and heteronuclear NOE data for holo-TC14 show minimal variations, indicating that there is no substantial difference in rigidity between the elements of regular secondary structure and the extended surface loops. Anisotropic tumbling of the elongated TC14 dimer can account for the main fluctuations in relaxation rates. Loss of the bound calcium does not significantly alter the internal dynamics, suggesting that the stability of the loop region is intrinsic and not dependent on the coordination of the calcium ion. Chemical shift differences between the holo and apo form show that main structural changes occur in the calcium-binding site, but smaller structural changes are propagated throughout the molecule without affecting the overall fold. The disappearance of two resonances for residues following the conserved cis-proline 87 (which is located in the calcium-binding site) in the apo form indicates conformational change on an NMR time scale between the cis and trans configurations of this peptide bond in the absence of calcium. Possible implications of these findings for the ligand binding in C-type lectin-like domains are discussed.     

IL-1beta-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes

Chronic inflammation appears to play a critical role in type 2 diabetes and its complications. Here we tested the hypothesis that this inflammatory dysregulation affects the IL-1beta system and has functional consequences in the brain. Diabetic, db/db, and nondiabetic, db/+, mice were administered i.p. LPS, a potent cytokine inducer, at a dose of 100 microg/kg/mouse. db/db mouse innate immune-associated sickness behavior was 14.8, 33, 44.7, and 34% greater than that of db/+ mice at 2, 4, 8, and 12 h, respectively. When a fixed dose of LPS was used (5 microg/mouse), db/db mouse sickness was again enhanced 18.4, 22.2, and 14.5% at 4, 8, and 12 h as compared with db/+ mice. In diabetic mice, peritoneal macrophages produced more IL-1beta in response to LPS, and peritoneal levels of IL-1beta induced by LPS were increased. Importantly, IL-1R antagonist and type 2 IL-1 receptor (IL-1R2) failed to up-regulate in response to LPS in db/db mice. Finally, both peripheral and central administration of IL-1beta, itself, induced sickness in db/db mice that mimicked the effects of peripheral LPS and was significantly greater than that seen in db/+ mice. Taken together, these results indicate that IL-1beta-mediated innate immunity is augmented in db/db mice both at the periphery and in the brain, and the mechanism is due to diabetes-associated loss of IL-1beta counterregulation.     

A diversity of U1 small nuclear RNAs in the silk moth Bombyx mori

Variants of U1 small nuclear RNAs (snRNAs) have been previously detected in a permanent cell line (BmN) of the silk moth Bombyx mori. In this study, the existence of U1 snRNA isoforms in the silk gland (SG) of the organism is investigated. The polyploidy (approximately 200,000X the 2N somatic value) state of the B. mori silk gland cells represents a unique system to explore the potential presence and differential expression of multiple U1 variants in a normal tissue. B. mori U1-specific RT-PCR libraries from the silk gland were generated and five U1 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were examined in these variants and compared to the previously reported isoforms from the transformed BmN cell line. In all these SG U1 variants, variant sites and inter-species differences are located in moderately conserved regions. Substitutional or compensatory changes were found in the double stranded areas and clustered in moderately conserved regions. Some of the changes generate stronger base pairing. Calculated free energy (DeltaG) values for the entire U1 snRNA secondary structures and for the individual stem/loops (I, II, III and IV) domains of the isoforms were generated and compared to determine their structural stability. Using phylogenetic analysis, an evolutionary parallelism is observed between the polymorphic sites in B. mori and variant locations found among animal and plant species.     

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine‐5′‐triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule. Separation by LC coupled with analysis by MS/MS (LC‐MS/MS) confidently identified 206 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N‐?‐acetyl‐lysine, seven of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron. The MS data have been deposited in the ProteomeXchange with the identifier PXD000121.