The dynamic interplay between a PTK (Kit) and a PTP (Shp2) in hematopoietic stem and progenitor cells

Comment on: Zhu HH, et al. Blood 2011; 117:5350-61 and Chan G, et al. Blood 2011; 117:4253-61.     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.     

Simulation of stent deployment in a realistic human coronary artery

Background  

The process of restenosis after a stenting procedure is related to local biomechanical environment. Arterial wall stresses caused by the interaction of the stent with the vascular wall and possibly stress induced stent strut fracture are two important parameters. The knowledge of these parameters after stent deployment in a patient derived 3D reconstruction of a diseased coronary artery might give insights in the understanding of the process of restenosis.     

Salt-avoidance tropism in Arabidopsis thaliana

The orientation of plant root growth is modulated by developmental as well as environmental cues. Among the environmental factors, gravity has been extensively studied because of its overpowering effects in modulating root growth direction. However, our knowledge of the effects of other abiotic signals that influence root growth direction is largely unknown. Recently, we have shown that high salinity can modify root growth direction by inducing rapid amyloplast degradation in root columella cells of Arabidopsis thaliana. By exploiting salt overly sensitive (sos) mutants and PIN2 expression analyses, we have shown that the altered root growth direction in response to salt is mediated by ion disequilibrium and is correlated with PIN2 mRNA abundance and expression and localization of the protein. Our study demonstrates that the SOS pathway may mediate this process. Here we discuss our data from broader perspectives. We propose that salt-induced modification of root growth direction is a salt-avoidance behavior, which is an active adaptive mechanism for plants grown under saline conditions. Furthermore, high salinity also stimulates alteration of gravitropic growth of shoots. These findings illustrate that plants have a fine and sophisticated sensory and communication system that enable plants to dynamically and efficiently cope with rapidly changing environment.Key words: abidopsis, adaptation, avoidance, root, salt stress, tropic growthOwing to their sessile nature, plant roots are constantly bombarded with various environmental stimuli from the soil, such as gravity, physical obstacles and imbalanced distribution of water and/or nutrients and high salinity. Where to grow is an important developmental decision in the life cycle of a plant that is crucial for its adaptation and the subsequent reproductive success. The proper orientation of root growth is shaped by both the developmental inputs and external signals.^1,2 The overwhelming environmental factor that modulates root growth direction is gravity, and plant primary roots grow downward toward the gravity vector. This directed growth of root in response to gravity is named as tropic growth to gravity or gravitropism. Studies of gravity perception and signaling pathway of the root cap at the primary root of Arabidopsis strongly support the starch statolith hypothesis.³ In this hypothesis, the columella cells in the root cap, which contain sedimentable amyloplasts, are the gravity-perceptive site in roots. The inner columella cells of the second tier have been proposed as making the greatest contribution to root gravitropism.⁴ Upon gravity stimulation, cytosolic ions such as Ca²⁺ and rapid cytoplasmic alkalization may be involved in gravity signal transduction.^5–7 Asymmetric distribution of auxin in roots caused by basipetal transport mainly through the auxin efflux carrier PIN-FORMED2 (PIN2), which is distributed asymmetrically within the cells, results in gravitropic root response of the root elongation zone.^8,9In contrast to our understanding of gravitropism of root, our knowledge of tropistic responses of root to other major environmental stimuli, such as water availability, imbalanced nutrient distribution and high salinity, and the interplay between these stimuli in determining the directional growth of root remains enigmatic. Recent studies have confirmed the existence of hydrotropism and the molecular genetic basis of the tropistic growth of root to water in determining the final direction of root growth starts to be deciphered.^10–12 Hydrotropic growth of roots is an important trait for plants to actively find water and to optimize their fitness under drought condition. Salinity is another major constraint to root system development, and limits the productivity of agricultural crops and the distribution of plant species.^13–15 It is known that salt stress-induced disturbed balance of ions is the primary cause for inhibition of plant growth and subsequent yield reduction. How does root minimize entrance of harmful ions and subsequently avoid salt injury? Does plant have capacity to sense salt signal, and prevent potentially harmful ions reaching root and shoot?Previous studies have shown that plant use different strategies to avoid salt injury at various levels. After Na⁺ enters the root cells, the Casparian strip can restrict the movements of the harmful ion into the xylem.¹⁶ Root cells also avoid salt injury by extruding Na⁺ actively back to the outside solution. This energy-dependent ion efflux from cytosol across the plasma membrane is mediated by SOS1 gene, a Na⁺-H⁺ antiporter, which is regulated by at least other genes, SOS3 (calcium binding protein) and SOS2 (serine/threonine kinase). This is the well characterized SOS (Salt Overly Sensitive) signaling pathway.^17,18 Another way for plant root cells to avoid ion injury is to accumulate Na⁺ into vacuole. Vacuolar compartmentation of Na⁺ is also in part regulated by Na⁺-H⁺ antiporters, such as AtNHX1.¹⁹ These findings reveal mechanisms of how plants avoid Na⁺ injury after passive entrance of sodium ions into root cells. We questioned whether a plant is capable of actively preventing the harmful ions from reaching root cells or escape from high salinity in the environment, and how plant roots respond to changing salt conditions, because salt distribution is unbalanced under natural saline conditions, especially after rain and irrigation. With a new assay that allows us to specifically address how plant roots respond to changing salt levels, we discovered an alternative adaptive mechanism for plant root to avoid salt injury.²⁰We set up a two-layer medium assay in which a sodium ion gradient would be generated. A normal nutrient agar medium is at the top of the growth bottle and an agar with salt-stressed medium is in the bottom of the bottle. This simple assay allows us to monitor root growth and orientation. The roots of the wild type seedlings penetrated the interface of the layers and grew straight downwards exhibiting gravitropism, when both layers were MS media. In contrast, when the bottom medium contained NaCl, roots of seedlings grew downward first, and then curved and grew upward toward the lower levels of salt. Roots started to bend upward at an early stage even before contacting high-salt medium (250 mM NaCl) on the bottom. The results indicate that roots can sense ion gradients in the growing environment and transduce the signal, combine with internal signals to make decisions that enable roots to stay away from high salt.^21,22 Here, we would like to propose this salt-induced tropic growth as a salt-avoidance tropism, which is an important adaptive behavior for plant roots to avoid salt injury and direct them toward their goal of optimal fitness.²³ Because salt stress inhibits root elongation, we tested impact of salt-induced negative gravitropism on the root growth. The results showed that inhibitory effect of salt on root growth was largely alleviated with this tropic curve (Fig. 1), further verifying our hypothesis that the salt-induced developmental plasticity is a salt-avoidance behavior (Fig. 2).Open in a separate windowFigure 1Effects of salt on root elongation of Arabidopsis thaliana seedlings from different salt treatments. The inhibitory effect of salt stress on root growth was greatly alleviated in the wild type (Col-0) when root growth of the seedlings was analyzed using a two-layer medium assay (black bars). The MS nutrient medium is on the top, and NaCl concentrations in the media on the bottom are 0, 150 and 250 mM. More severe inhibition of root growth of the seedlings by various levels of NaCl in a root bending assay (white bars) was observed. Data represents means of measurements from >40 individuals from three independent experiments. Bars represent standard error.Open in a separate windowFigure 2An illustrative model of the sensing and response by the plant root when grown under different saline conditions. This model proposes two major mechanisms of salt responses by plants, where salt tolerance is the ability to function while stressed; Salt avoidance is the capacity to stay away from salt stress when growing in changing saline conditions.Another important point that we would like to bring out based on our observation in this work is that salinity also stimulated shoot positive gravitropism or negative phototropism. The observation implicates long-distance communication from root to shoot during plant salt response in the stressed plants. The exact biological function of shoot tropic growth, the signals in this long-distance communication, and underlying molecular mechanism still remains unknown.In conclusion, our study has revealed a novel complex adaptive mechanism that provides plants a capacity for avoiding injury from salt. The hypothesis we have proposed here should provide novel insights into plant stress avoidance. Further analysis using a combinatorial approach, mutant analysis and genomics, is required to decipher the molecular network underlying this salt-avoidance behavior.     

Transgenic nonhuman primates for neurodegenerative diseases

Animal models that represent human diseases constitute an important tool in understanding the pathogenesis of the diseases, and in developing effective therapies. Neurodegenerative diseases are complex disorders involving neuropathologic and psychiatric alterations. Although transgenic and knock-in mouse models of Alzheimer's disease, (AD), Parkinson's disease (PD) and Huntington's disease (HD) have been created, limited representation in clinical aspects has been recognized and the rodent models lack true neurodegeneration. Chemical induction of HD and PD in nonhuman primates (NHP) has been reported, however, the role of intrinsic genetic factors in the development of the diseases is indeterminable. Nonhuman primates closely parallel humans with regard to genetic, neuroanatomic, and cognitive/behavioral characteristics. Accordingly, the development of NHP models for neurodegenerative diseases holds greater promise for success in the discovery of diagnoses, treatments, and cures than approaches using other animal species. Therefore, a transgenic NHP carrying a mutant gene similar to that of patients will help to clarify our understanding of disease onset and progression. Additionally, monitoring disease onset and development in the transgenic NHP by high resolution brain imaging technology such as MRI, and behavioral and cognitive testing can all be carried out simultaneously in the NHP but not in other animal models. Moreover, because of the similarity in motor repertoire between NHPs and humans, it will also be possible to compare the neurologic syndrome observed in the NHP model to that in patients. Understanding the correlation between genetic defects and physiologic changes (e.g. oxidative damage) will lead to a better understanding of disease progression and the development of patient treatments, medications and preventive approaches for high risk individuals. The impact of the transgenic NHP model in understanding the role which genetic disorders play in the development of efficacious interventions and medications is foreseeable.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.     

Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.