Operational Changes in a Shared Resource Laboratory with the Use of a Product Lifecycle Management Approach: A Case Study.

Shared Resource Laboratories (SRLs) provide investigators access to necessary scientific and resource expertise to leverage complex technologies fully for advancing high-quality biomedical research in a cost-effective manner. At the University of Nebraska Medical Center, the Flow Cytometry Research Facility (FCRF) offered access to exceptional technology, but the methods of operation were outdated and unsustainable. Whereas technology has advanced and the institute has expanded, the operations at the facility remained unchanged for 35 yr. To rectify this, at the end of 2013, we took a product lifecycle management approach to affect large operational changes and align the services offered with the SRL goal of education, as well as to provide service to researchers. These disruptive operational changes took over 10 mo to complete and allowed for independent end-user acquisition of flow cytometry data. The results have been monitored for the past 12 mo. The operational changes have had a positive impact on the quality of research, increased investigator-facility interaction, reduced stress of facility staff, and increased overall use of the resources. This product lifecycle management approach to facility operations allowed us to conceive of, design, implement, and monitor effectively the changes at the FCRF. This approach should be considered by SRL management when faced with the need for operationally disruptive measures.

Loss of tumor suppressor RPL5/RPL11 does not induce cell cycle arrest but impedes proliferation due to reduced ribosome content and translation capacity.

Humans have evolved elaborate mechanisms to activate p53 in response to insults that lead to cancer, including the binding and inhibition of Hdm2 by the 60S ribosomal proteins (RPs) RPL5 and RPL11. This same mechanism appears to be activated upon impaired ribosome biogenesis, a risk factor for cancer initiation. As loss of RPL5/RPL11 abrogates ribosome biogenesis and protein synthesis to the same extent as loss of other essential 60S RPs, we reasoned the loss of RPL5 and RPL11 would induce a p53-independent cell cycle checkpoint. Unexpectedly, we found that their depletion in primary human lung fibroblasts failed to induce cell cycle arrest but strongly suppressed cell cycle progression. We show that the effects on cell cycle progression stemmed from reduced ribosome content and translational capacity, which suppressed the accumulation of cyclins at the translational level. Thus, unlike other tumor suppressors, RPL5/RPL11 play an essential role in normal cell proliferation, a function cells have evolved to rely on in lieu of a cell cycle checkpoint.

The abundance of Rad51 protein in mouse embryonic stem cells is regulated at multiple levels.

DNA double-strand breaks (DSBs) in embryonic stem (ES) cells are repaired primarily by homologous recombination (HR). The mechanism by which HR is regulated in these cells, however, remains enigmatic. To gain insight into such regulatory mechanisms, we have asked how protein levels of Rad51, a key component of HR, are controlled in mouse ES cells and mouse embryo fibroblasts (MEFs). The Rad51 protein level is about 15-fold higher in ES cells than in MEFs. The level of Rad51 mRNA, however, is only ~2-fold higher, indicating that the differences in mRNA levels due to rates of transcription or mRNA stability are not sufficient to account for the large difference in the abundance of Rad51 protein. Comparison of Rad51 half-lives between ES cells and MEFs also did not explain the elevated level of Rad51 protein in the ES cells. A comparative assessment of the Rad51 translation level demonstrated that it is translated with much greater efficacy in ES cells than in MEFs. To determine whether this high level of translation in ES cells is a general phenomenon in these cells or whether it is a characteristic of specific proteins, such as those involved with recombination and cell cycle progression, we compared mechanisms that regulate the level of Pcna in ES cells with those that regulate Rad51. The half-life of Pcna and its rate of synthesis were considerably different from those of Rad51 in ES cells, demonstrating that regulation of Rad51 abundance cannot be generalized to other ES cell proteins and not to proteins involved in DNA replication and cell cycle control. Finally, we show that only a small proportion of the abundant Rad51 protein population is activated under basal conditions in ES cells and recruited to DNA DSBs and/or stalled replication forks.

Optimization and application of a flow cytometric PU.1 assay for murine immune cells.

PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. There was a dose dependent increase in the amount of PU.1 detected with increasing amounts of PU.1 antibody that was not seen when blocking peptide was used. Methanol or ethanol gave equivalent results as permeabilization agents, but the latter allowed easier detection of surface antigens when surface staining was performed prior to permeabilization. T cells had little if any PU.1, while B cells had intermediate levels of PU.1, and myeloid cells had high levels of PU.1. Monocytes had higher levels of PU.1 than did neutrophils or spleen macrophages. Plasmacytoid dendritic cells had lower levels of PU.1 than did conventional dendritic cells. Immature myeloid cells had higher levels of PU.1 than did mature myeloid cells. In addition, PU.1 levels were higher in proliferating cells than the corresponding non proliferating cells. Myeloid cells derived from burn mice tended to have higher levels of PU.1 than did unburned, but proliferating cells from burn or sham mice showed no difference in their levels of PU.1. This assay should be a useful addition to the tools used to study the function of PU.1 in health and disease.