A rapid and scalable method for selecting recombinant mouse monoclonal antibodies

<p>Abstract</p> <p>Background</p> <p>Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.</p> <p>Results</p> <p>Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.</p> <p>Conclusions</p> <p>This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.</p

Assessing the Effects of a Paired TBL Session and Patient Simulation on Pharmacy Student HIV Treatment Knowledge

Description of the Problem: Pharmacists can play a crucial role in monitoring, counseling, and providing adherence checks across practice pharmacy settings; but they may not gain experience in this area until after graduating from pharmacy school. Statement of Innovation: Students participated in an intentionally aligned team-based learning session followed by completion of an HIV patient treatment worksheet and an HIV patient care simulation. This sequence was assessed using the HIV Treatment Knowledge Scale. Description of the Innovation: Second-year pharmacy students (N=48, 98% response rate) participated in a baseline knowledge assessment before a four-hour HIV team-based learning (TBL) session, which included the use of an online HIV Patient Management Simulator. Students were administered the scale again post-session. Three days before the simulation, students had access to an HIV patient treatment worksheet that was required to be completed before the simulation. Ten days after the initial assessment, students participated in an HIV patient simulation where they proposed a new antiretroviral plan while also addressing monitoring, barriers, and maximizing adherence for the patient. Post-simulation, students were again administered the scale. Data were analyzed using descriptive statistics, Wilcoxon and paired t-tests, as appropriate. Critical Analysis: A total of 48 second-year pharmacy students participated. HIV knowledge increased significantly post-TBL (p \u3c 0.001). Post-simulation, scores improved, but not significantly (p = 0.291). Knowledge on 15 of the 21 items on the HIV Treatment Knowledge Scale significantly improved from pre-TBL to post-simulation (p ≤ 0.025). Next Steps: Future investigation should focus on the impact that HIV simulation training has on skills, abilities, confidence, and empathy

A comprehensive modelling framework for demand side flexibility in smart grids

The increasing share of renewable energy generation in the electricity system comes with significant challenges, such as the volatility of renewable energy sources. To tackle those challenges, demand side management is a frequently mentioned remedy. However, measures of demand side management need a high level of exibility to be successful. Although extensive research exists that describes, models and optimises various processes with exible electrical demands, there is no unified notation. Additionally, most descriptions are very process-specific and cannot be generalised. In this paper, we develop a comprehensive modelling framework to mathematically describe demand side exibility in smart grids while integrating a majority of constraints from different existing models. We provide a universally applicable modelling framework for demand side exibility and evaluate its practicality by looking at how well Mixed-Integer Linear Program (MIP) solvers are able to optimise the resulting models, if applied to artificially generated instances. From the evaluation, we derive that our model improves the performance of previous models while integrating additional exibility characteristics

Molecular and cytological features of the mouse B-cell lymphoma line iMyc(Eμ)-1

BACKGROUND: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMyc(Eμ )mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMyc(Eμ )mice that carry a His(6)-tagged mouse Myc cDNA, Myc(His), just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell tumors. The predominant tumor (~50%) that arose in the iMyc(Eμ )mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMyc(Eμ)-1, for the in-depth evaluation of LBL in vitro. METHODS: The morphological features and the surface marker expression profile of the iMyc(Eμ)-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMyc(Eμ)-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted Myc(His )gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMyc(Eμ)-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip(© )and Superarray(© )cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. RESULTS: Consistent with their derivation from LBL, the iMyc(Eμ)-1 cells were found to be neoplastic IgM(high)IgD(low )lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMyc(Eμ)-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed Myc(His )at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip(© )microarrays that were consistent with Myc(His)-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray(© )cDNA macroarrays, the iMyc(Eμ)-1 cells showed the highest number of changes on the NFκB array. CONCLUSION: The iMyc(Eμ)-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro

The Prethalamus Is Established during Gastrulation and Influences Diencephalic Regionalization

The vertebrate neural plate contains distinct domains of gene expression, prefiguring the future brain areas. In this study, we draw an extended expression map of the rostral neural plate that reveals discrete domains inside the presumptive posterior forebrain. We show, by fate mapping, that these well-defined cell populations will develop into specific diencephalic regions. To address whether these early subterritories are already committed to restricted identities, we began to analyse the consequences of ablation and transplantation of these specific cell populations. We found that precursors of the prethalamus are already specified and irreplaceable at late gastrula stage, because ablation of these cells results in loss of prethalamic markers. Moreover, when transplanted into the ectopic environment of the presumptive hindbrain, these cells still pursue their prethalamic differentiation program. Finally, transplantation of these precursors, in the rostral-most neural epithelium, induces changes in cell identity in the surrounding host forebrain. This cell–non-autonomous property led us to propose that these committed prethalamic precursors may play an instructive role in the regionalization of the developing diencephalon