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81.
82.
We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity. 相似文献
83.
Complex network dynamics have been analyzed with models of systems of coupled switches or systems of coupled oscillators. However, many complex systems are composed of components with diverse dynamics whose interactions drive the system's evolution. We, therefore, introduce a new modeling framework that describes the dynamics of networks composed of both oscillators and switches. Both oscillator synchronization and switch stability are preserved in these heterogeneous, coupled networks. Furthermore, this model recapitulates the qualitative dynamics for the yeast cell cycle consistent with the hypothesized dynamics resulting from decomposition of the regulatory network into dynamic motifs. Introducing feedback into the cell-cycle network induces qualitative dynamics analogous to limitless replicative potential that is a hallmark of cancer. As a result, the proposed model of switch and oscillator coupling provides the ability to incorporate mechanisms that underlie the synchronized stimulus response ubiquitous in biochemical systems. 相似文献
84.
Chen R Mias GI Li-Pook-Than J Jiang L Lam HY Chen R Miriami E Karczewski KJ Hariharan M Dewey FE Cheng Y Clark MJ Im H Habegger L Balasubramanian S O'Huallachain M Dudley JT Hillenmeyer S Haraksingh R Sharon D Euskirchen G Lacroute P Bettinger K Boyle AP Kasowski M Grubert F Seki S Garcia M Whirl-Carrillo M Gallardo M Blasco MA Greenberg PL Snyder P Klein TE Altman RB Butte AJ Ashley EA Gerstein M Nadeau KC Tang H Snyder M 《Cell》2012,148(6):1293-1307
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86.
A model for the growth of a size-structured cell population reproducing by fission into two identical daughters is formulated and analysed. The model takes the form of a linear first order partial differential equation (balance law) in which one term has a transformed argument. Using semigroup theory and compactness arguments we establish the existence of a stable size distribution under a certain condition on the growth rate of the individuals. An example shows that one cannot dispense with this condition. 相似文献
87.
Cornelia Richter Sebastian Thieme Joanna Bando?a Magdalena Laugsch Konstantinos Anastassiadis Sebastian Brenner 《PloS one》2013,8(4)
Dendritic cells are the professional antigen presenting cells of innate immunity and key players in maintaining the balance of immune responses. Studies with dendritic cells are mainly limited by their low numbers in vivo and their difficult maintenance in vitro. We differentiated bone marrow cells from transgenic mice expressing an inducible SV40 large T-antigen into dendritic cells. When immortalized by dexamethasone and doxycycline, these cells were stable in long-term culture. In the absence of dexamethasone and doxycycline (de-induction), dendritic cells displayed properties of primary cells, characterized by expression of classical dendritic cell surface markers CD11c, CD11b, MHCII, CD40 and CD86. Furthermore, de-induced lipopolysaccharide activated dendritic cells secreted IL-1β, IL-6, TNFα and IL-12. De-induced, Ovalbumin-loaded dendritic cells polarize CD4+ T cells into Th1, Th17 and Th2 cells, indicating their correct antigen presenting property. Consistent with intratracheal application of Ovalbumin-loaded primary dendritic cells into mice, the application of de-induced dendritic cells resulted in recruitment of lymphocytes to the lungs. In summary, we successfully expanded dendritic cells using conditional immortalization. The generated dendritic cells demonstrate the characteristic immunophenotype of primary dendritic cells and will facilitate further studies on immunomodulatory properties of dendritic cells. 相似文献
88.
Salivary alpha amylase-cortisol asymmetry in maltreated youth 总被引:1,自引:0,他引:1
BACKGROUND: Maltreatment represents a major stressor in the lives of many youth. Given the known effects of stress exposure on subsequent functioning of biological stress response systems, researchers have been interested in the effects of maltreatment on the functioning of these systems. Experimental studies reveal that previous exposure to stress affects the symmetry between components of the physiological stress response to subsequent stress. The present study examined asymmetry between salivary alpha amylase (sAA), a sympathetic indicator, and cortisol reactivity to a social stressor among maltreated and comparison youth age 9 to 14 years. Consistent with earlier studies suggesting that stress leads to asymmetry between hypothalamic-pituitary-adrenal axis and sympathetic nervous system activity, we expected that maltreated youth would exhibit greater sAA-cortisol asymmetry than would comparison youth. METHODS: Forty-seven maltreated and 37 comparison youth visited the laboratory and engaged in a social stress protocol. We collected 2 saliva samples before the stressor and 4 after, at 0 min post-stress and every 10 min for 30 min. RESULTS: Maltreatment status moderated the relation between sAA and cortisol activity in response to the stressor. Comparison youth showed significant links between the sAA and cortisol responses; maltreated youth had no significant associations between responses in the two biomarkers. CONCLUSION: The data were consistent with sAA-cortisol asymmetry among maltreated youth. Further research should seek to replicate this finding and investigate its implication for developmental trajectories. 相似文献
89.
A possible control strategy against the spread of an infectious disease is the treatment with antimicrobials that are given prophylactically to those who had a contact with an infective person. The treatment continues until recovery or until it becomes obvious that there was no infection in the first place. The model considers susceptible, treated uninfected exposed, treated infected, (untreated) infectious, and recovered individuals. Since treatment lengths have an arbitrary distribution, the model system consists of ordinary differential and integral equations. A sharp threshold condition is established in terms of a basic replacement ratio (disease reproduction number) that separates disease extinction from uniform disease persistence. We use results from dynamical systems persistence theory proving the existence of a global compact attractor along the way. Existence and multiplicity of endemic equilibria are also studied. 相似文献
90.