Ablation of Proliferating Marrow with 5‐Fluorouracil Allows Partial Purification of Mesenchymal Stem Cells

The ability to identify and maintain mesenchymal stem cells in vitro is a prerequisite for the ex vivo expansion of cells capable of effecting mesenchymal tissue regeneration. The aim of this investigation was to develop an assay to enrich and ultimately purify mesenchymal stem cells. To enrich the population of mesenchymal stem cell‐like cells, rats or mice were administered 5‐fluorouracil (5‐FU) in vivo. Limiting dilution analysis demonstrated that 5‐FU‐treated bone marrow had the potential to form colony‐forming units‐fibroblastic (CFU‐F) at a 10‐fold or sixfold enrichment compared to normal bone marrow in rats or mice, respectively. In vivo and in vitro differentiation assays supported the enrichment and purification effects. In vitro, bone marrow cultures from 5‐FU‐treated bone marrow demonstrated lineage‐specific gene expression in lineage‐specific medium conditions in contrast to the multilineage gene expression of control bone marrow cultures. In vivo implantation of 5‐FU‐treated cells that were not expanded in culture generated ossicles containing an intact bone cortex and mature hematopoietic components, whereas non‐5‐FU‐treated bone marrow only formed fibrous tissues. Our results demonstrate that enrichment of a quiescent cell population in the bone marrow by in vivo treatment of 5‐FU spares those undifferentiated mesenchymal stem cells and influences the differentiation of bone marrow stromal cells in vitro and in vivo. This prospective identification of a population of mesenchymal cells from the marrow that maintain their multilineage potential should lead to more focused studies on the characterization of a true mesenchymal stem cell.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90309/1/241573_ftp.pd

Characterization of the plasma membrane proteins and receptor-like kinases associated with secondary vascular differentiation in poplar

The constituents of plasma membrane proteins, particularly the integral membrane proteins, are closely associated with the differentiation of plant cells. Secondary vascular differentiation, which gives rise to the increase in plant stem diameter, is the key process by which the volume of the plant body grows. However, little is known about the plasma membrane proteins that specifically function in the vascular differentiation process. Proteomic analysis of the membrane proteins in poplar differentiating secondary vascular tissues led to the identification 226 integral proteins in differentiating xylem and phloem tissues. A majority of the integral proteins identified were receptors (55 proteins), transporters (34 proteins), cell wall formation related (27 proteins) or intracellular trafficking (17 proteins) proteins. Gene expression analysis in developing vascular cells further demonstrated that cambium differentiation involves the expression of a group of receptor kinases which mediate an array of signaling pathways during secondary vascular differentiation. This paper provides an outline of the protein composition of the plasma membrane in differentiating secondary vascular tissues and sheds light on the role of receptor kinases during secondary vascular development

Leptin Functions Peripherally to Regulate Differentiation of Mesenchymal Progenitor Cells

Leptin functions through a well-documented central neuroendocrine pathway to regulate bone mass. However, the ability of leptin to modulate bone mass through a peripheral mechanism has been debated due to conflicting in vitro results and lack of sufficient in vivo models. We utilized mice with LoxP sites introduced into the long-form leptin receptor (ObRb) gene to determine how leptin regulates mesenchymal progenitor cell (MPC) differentiation and osteoblast function in vitro and in vivo . Rapid phosphorylation of Stat3 after leptin treatment of bone marrow stromal cells (BMSCs) from mice with conditional deletion of ObRb in macrophages (LysM Cre+F/F ) confirmed expression of functional leptin receptors by BMSCs. Adenovirus-Cre mediated disruption of ObRb in primary stromal cells decreased mineralization and increased adipogenesis. In contrast, BMSCs harvested from leptin-signaling deficient Ob/Ob or Db/Db mice showed increased mineralization. To determine the physiologic relevance of these differences, mice with cell-specific deletion of ObRb in mesenchymal precursors (3.6 Cre+F/F ) or osteoblasts (2.3 Cre+F/F ) were generated. Although the 2.3 Cre+F/F mice were grossly normal, the 3.6 Cre+F/F mice displayed mild obesity that was not attributed to food intake. Femurs of 3.6 Cre+F/F animals showed a 58%–61.9% increase in trabecular bone volume and a 65.5%–74% increase in bone mineral density. Cortical volume and mineral content were also increased 18%–22%. Primary 3.6 Cre+F/F BMSCs recapitulated the high mineralization phenotype of Ob/Ob and Db/Db BMSCs. We conclude that leptin may have multiple peripheral roles depending on the differentiation state of MPC. Leptin (a) helps maintain MPCs in an undifferentiated state and (b) promotes mineralization of more differentiated osteoblasts. S TEM C ells 2010;28:1071–1080Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77453/1/432_ftp.pd

Handheld optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging with high spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We developed a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes, the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary bed in a mouse ear and both the capillary bed and a mole on a human volunteer

High-speed widefield photoacoustic microscopy of small-animal hemodynamics

Optical-resolution photoacoustic microscopy (OR-PAM) has become a popular tool in small-animal hemodynamic studies. However, previous OR-PAM techniques variously lacked a high imaging speed and/or a large field of view, impeding the study of highly dynamic physiologic and pathophysiologic processes over a large region of interest. Here we report a high-speed OR-PAM system with an ultra-wide field of view, enabled by an innovative water-immersible hexagon-mirror scanner. By driving the hexagon-mirror scanner with a high-precision DC motor, the new OR-PAM has achieved a cross-sectional frame rate of 900 Hz over a 12-mm scanning range, which is 3900 times faster than our previous motor-scanner-based system and 10 times faster than the MEMS-scanner-based system. Using this hexagon-scanner-based OR-PAM system, we have imaged epinephrine-induced vasoconstriction in the whole mouse ear and vascular reperfusion after ischemic stroke in the mouse cortex in vivo, with a high spatial resolution and high volumetric imaging speed. We expect that the hexagon-scanner-based OR-PAM system will become a powerful tool for small animal imaging where the hemodynamic responses over a large field of view are of interest

Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus

<p>Abstract</p> <p>Background</p> <p>The transcription regulator PhoP has been shown to be important for <it>Y. pestis </it>survival in macrophages and under various <it>in vitro </it>stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in <it>Y. pestis</it>. A series of biochemical experiments were done herein to study members of the PhoP regulon of <it>Y. pestis </it>biovar <it>Microtus</it>.</p> <p>Results</p> <p>By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in <it>Y. pestis</it>. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation.</p> <p>Conclusion</p> <p>The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which <it>Y. pestis </it>survives the antibacterial strategies employed by host macrophages.</p

Non-TAL Effectors From Xanthomonas oryzae pv. oryzae Suppress Peptidoglycan-Triggered MAPK Activation in Rice

Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice, depends on its type III secretion system and associated effector proteins to grow and colonize the vascular tissues of rice plants. The type III effectors include a family of closely related transcription activator-like (TAL) effectors and the rest of diverse effectors, so-called non-TAL effectors. Our understanding of non-TAL effectors for pathogenesis in rice blight is still limited. Here we report a feasible method to rapidly detect the activation of mitogen-activated protein kinase pathway in rice mesophyll protoplasts by the X. oryzae pv. oryzae derived peptidoglycan and screen for virulent effectors that can suppress the pathogen-associated molecular pattern triggered immunity (PTI) response. Amongst 17 non-TAL effectors transiently expressed in rice cells, we found that three effectors (XopZ, XopN, and XopV) were able to suppress the peptidoglycan-triggered MAPK activation. The triple mutant of the X. oryzae pv. oryzae strain PXO99A lacking XopZ, XopN, and XopV showed additively reduced virulence. Adding back either of genes restored the virulence of the triple mutant. Our results demonstrate the collective and redundant ability of defense suppression by non-TAL effectors in causing bacterial blight of rice

Handheld optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) offers label-free in vivo imaging with high spatial resolution by acoustically detecting optical absorption contrasts via the photoacoustic effect. We developed a compact handheld OR-PAM probe for fast photoacoustic imaging. Different from benchtop microscopes, the handheld probe provides flexibility in imaging various anatomical sites. Resembling a cup in size, the probe uses a two-axis water-immersible microelectromechanical system mirror to scan both the illuminating optical beam and resultant acoustic beam. The system performance was tested in vivo by imaging the capillary bed in a mouse ear and both the capillary bed and a mole on a human volunteer