A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation

Пленум Наукової ради«Українська мова» Українська лексикографія та лексикологія: проблеми, завдання

10–11 листопада 2011року у Ніжинському державному університеты імені Миколи Гоголя відбувся Пленум Наукової ради “Українська мова” Інституту української мови НАН України на тему “Українська лексикографія та лексикологія: проблеми, завдання”

MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation

The exosome is a complex of 3′→5′ exoribonucleases which is involved in many RNA metabolic processes. To regulate these functions distinct proteins are believed to recruit the exosome to specific substrate RNAs. Here, we demonstrate that M-phase phosphoprotein 6 (MPP6), a protein reported previously to co-purify with the TAP-tagged human exosome, accumulates in the nucleoli of HEp-2 cells and associates with a subset of nuclear exosomes as evidenced by co-immunoprecipitation and biochemical fractionation experiments. In agreement with its nucleolar accumulation, siRNA-mediated knock-down experiments revealed that MPP6 is involved in the generation of the 3′ end of the 5.8S rRNA. The accumulation of the same processing intermediates after reducing the levels of either MPP6 or exosome components strongly suggests that MPP6 is required for the recruitment of the exosome to the pre-rRNA. Interestingly, MPP6 appeared to display RNA-binding activity in vitro with a preference for pyrimidine-rich sequences, and to bind to the ITS2 element of pre-rRNAs. Our data indicate that MPP6 is a nucleolus-specific exosome co-factor required for its role in the maturation of 5.8S rRNA

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur. CONCLUSIONS: We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients

Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space

Performance model for “Just-in-Time” problems in real-time multimedia applications

Over the last few years, the use of large-scale multimedia data applications has been growing tremendously, and this growth is not likely to slow down in the near future. Many multimedia applications operate in a real-time environment (e.g., surveillance cameras, iris scans), which must meet strict time constraints, i.e. to analyze video frames at the same rate as a camera produces them. To meet this requirement, Grid computing is rapidly becoming indispensable. However, the variabilities of the software and the hardware in grid environment cause the strong burstiness in the transmission delay of video frames. Because the burstiness is unknown beforehand, it is difficult to determine the right sending moments of video frames. If the time interval between sending two sequential frames is too large, then the service utilization may be low. If use large buffer to guarantee the service utilization, then video frames may be outof- date because of the long waiting time at buffer in the server side. This problem is referred to as “Just-in-time” problem. To solve this problem, it is essential to determine the right sending moments of video frames, properly dealing with the trade-off between the service utilization and the “up-to-date” of video frames. Motivated by this, in this paper we develop an adaptive control method that react to the continuously changing circumstances in grid system so as to obtain the highest service utilization on the one hand and to keep the video frame up-to-date on the other hand. Extensive experimental validation in our DAS-3 testbed and the trace-driven simulation show that our method is indeed highly effective

Особенности немецкого языка переселенцев из бывшего СССР в Германии

Previous studies have suggested that murine peritoneal cavity-derived B-1a cells possess similarities with described regulatory B cell subsets. The aim of the current study was to examine the potential immunoregulatory function of peritoneal cavity-derived B(-1a) cells. In vitro activation of peritoneal cavity-derived B- and B-1a cells shows that activation of these B cells with anti-CD40 and LPS induces these cells to secrete more IL-10, IL-6 and IgM as compared to splenic B cells. In a suppression assay, CD40/TLR4-activated peritoneal cavity B cells possess regulatory B cell functions as they inhibit the capacity of CD4(+) T cells to produce both tumor necrosis factor-α and interferon-γ. Splenic B cells did not show this, whereas non-activated peritoneal cavity B cells augmented the capacity of CD4(+) T cells to produce tumor necrosis factor-α, while the ability to produce interferon-γ was not altered. The current paper compares splenic B cells to peritoneal cavity B(-1a) cells in an in vitro activation- and an suppression-assay and concludes that peritoneal cavity B(-1a) cells possess properties that appear similar to splenic autoimmune-suppressive regulatory B cell subsets described in the literature

Effective and Efficient Stand Magnifier Use in Visually Impaired Children

Contains fulltext : 167758.pdf (publisher's version ) (Open Access)PURPOSE: The main objective of this study was to analyze the effectiveness and efficiency of magnifier use in children with visual impairment who did not use a low vision aid earlier, in an ecologically valid goal-directed perceptuomotor task. METHODS: Participants were twenty-nine 4- to 8-year-old children with visual impairment and 47 age-matched children with normal vision. After seeing a first symbol (an Lea Hyvarinen [LH] symbol), children were instructed to (1) move the stand magnifier as quickly as possible toward a small target symbol (another LH symbol that could only be seen by using the magnifier), (2) compare the two symbols, and (3) move the magnifier to one of two response areas to indicate whether the two symbols were identical. Performance was measured in terms of accuracy, response time, identification time, and movement time. Viewing distance, as well as hand and eye dominance while using the magnifier was assessed. RESULTS: There were no significant differences between the two groups in accuracy, reaction time, and movement time. Contrary to the prediction, children with visual impairment required less time to identify small symbols than children with normal vision. Both within-subject and between-subject variability in viewing distance were smaller in the visually impaired group than in the normally sighted group. In the visually impaired group, a larger viewing distance was associated with shorter identification time, which in turn was associated with higher accuracy. In the normally sighted group, a faster movement with the magnifier and a faster identification were associated with increasing age. CONCLUSION: The findings indicate that children with visual impairment can use the stand magnifier adequately and efficiently. The normally sighted children show an age-related development in movement time and identification time and show more variability in viewing distance, which is not found in visually impaired children. Visually impaired children seem to choose a standard but less adaptive strategy in which they primarily used their preferred hand to manipulate the magnifier and their preferred eye to identify the symbol. TRIAL REGISTRATION: Registered at http://www.trialregister.nl; NTR2380.11 p

The contributions of diverse sense organs in the control of leg movement by a walking insect

Cruse H, Dean J, Suilmann M. The contributions of diverse sense organs in the control of leg movement by a walking insect. Journal of Comparative Physiology, A. 1984;154(5):695-705