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The mission of the United States Department of Energy’s Office of Environmental Management is to complete the safe cleanup of the environmental legacy brought about from five decades of nuclear weapons development and government-sponsored nuclear energy research. Some of the wastes that that must be managed have been identified as good candidates for disposal in a deep borehole in crystalline rock (SNL 2014a). In particular, wastes that can be disposed of in a small package are good candidates for this disposal concept. A canister-based system that can be used for handling these wastes during the disposition process (i.e., storage, transfers, transportation, and disposal) could facilitate the eventual disposal of these wastes. This report provides information for a program plan for developing specifications regarding a canister-based system that facilitates small waste form packaging and disposal and that is integrated with the overall efforts of the DOE’s Office of Nuclear Energy Used Fuel Disposition Campaign's Deep Borehole Field Test. Groundwork for Universal Canister System Development September 2015 ii Wastes to be considered as candidates for the universal canister system include capsules containing cesium and strontium currently stored in pools at the Hanford Site, cesium to be processed using elutable or nonelutable resins at the Hanford Site, and calcine waste from Idaho National Laboratory. The initial emphasis will be on disposal of the cesium and strontium capsules in a deep borehole that has been drilled into crystalline rock. Specifications for a universal canister system are derived from operational, performance, and regulatory requirements for storage, transfers, transportation, and disposal of radioactive waste. Agreements between the Department of Energy and the States of Washington and Idaho, as well as the Deep Borehole Field Test plan provide schedule requirements for development of the universal canister system. Future work includes collaboration with the Hanford Site to move the cesium and strontium capsules into dry storage, collaboration with the Deep Borehole Field Test to develop surface handling and emplacement techniques and to develop the waste package design requirements, developing universal canister system design options and concepts of operations, and developing system analysis tools. Areas in which further research and development are needed include material properties and structural integrity, in-package sorbents and fillers, waste form tolerance to heat and postweld stress relief, waste package impact limiters, sensors, cesium mobility under downhole conditions, and the impact of high pressure and high temperature environment on seals design. September 2015 Groundwork for Universal Canister System Development

The process of repair of wounded skin involves intricate orchestration not only between the epidermal and dermal compartments but also between the resident and immigrant cells and the local microenvironment. Only now are we beginning to appreciate the complex roles played by the matrix in directing the outcome of the repair processes, and how this impacts the signals from the various cells. Recent findings speak of dynamic and reciprocal interactions that occurs among the matrix, growth factors, and cells that underlies this integrated process. Further confounding this integration are the physiologic and pathologic situations that directly alter the matrix to impart at least part of the dysrepair that occurs. These topics will be discussed with a call for innovative model systems of direct relevance to the human situation.

Epidermal growth factor [EGF] mediated stimulation of its receptor in endothelial cell [EC] is accompanied by phosphorylation of the EGF-receptor [EGFR] and activation of phospholipase C-γ, resulting in the breakdown of phosphatidylinositol(4,5)-bisphosphate and generating inositol (1,4,5)-trisphosphate [IP3] and diacylglycerol. IP3 thus formed can be further converted to inositol (1,3,4,5)-tetrakisphosphate [IP4] by an enzyme called IP3-kinase [IP3K]. In this study we have investigated the effect of modulation of intracellular IP3K activity by the use of an inhibitor, 2-trifluoromethyl [6-(4-nitrobenzyl)-purine] [IP3KI] and siRNA against IP3KB on EGF-induced ERK-phosphorylation and cell motility. EGF stimulated ERK-phosphorylation that has been implicated in EGF-stimulated cell migration was inhibited by both IP3KI and siRNA against IP3KB. Inhibition of ERK-phosphorylation was accompanied by decreased cell migration in the presence of IP3KI.

Selection of appropriate chemotherapy, including identification of platinum resistance, is critical to effective management of advanced epithelial ovarian cancer (EOC). ChemoFx®, a multiple treatment marker (chemoresponse assay), has been developed to address this challenge and to improve outcomes in patients with advanced EOC. While much work has been done that has demonstrated the analytical validity of this assay, more recent studies have highlighted the unique clinical benefits offered by the assay. A prospective, multicenter trial has shown an increase in overall survival (OS) of 14 months and an increase in progression-free survival (PFS) by 3 months in patients with recurrent EOS treated by a "sensitive" therapy based on ChemoFx results. Along with other studies showing similar gains in OS and PFS, ChemoFx has been shown to be both a prognostic and predictive marker in patients with recurrent EOC where current treatment options are sorely lacking. In addition to these clinical benefits, economic analyses have shown that ChemoFx is a cost-effective intervention. Current guidelines and technology assessments relating to ChemoFx are largely outdated and refer primarily to metrics of analytical validity. Thus, in addition to analytical validity, the clinical validity, clinical utility and economic impact of ChemoFx are reviewed herein, including published literature, technology assessments by independent parties, and regulatory approvals of this marker.

Deeper understanding of the anatomical intermediaries for disease and other complex genetic traits is essential to understanding mechanisms and developing new interventions. Existing ontology tools provide functional annotations for many genes in the genome and they are widely used to develop mechanistic hypotheses based on genetic and transcriptomic data. Yet, information about where a set of genes is expressed may be equally useful in interpreting results and forming novel mechanistic hypotheses for a trait. Therefore, we developed a framework for statistically testing the relationship between gene expression across the body and sets of candidate genes from across the genome. We validated this tool and tested its utility on three applications. First, using thousands of loci identified by GWA studies, our framework identifies the number of disease-associated genes that have enriched expression in the disease-affected tissue. Second, we experimentally confirmed an underappreciated prediction highlighted by our tool: variation in skin expressed genes are a major quantitative genetic modulator of white blood cell count - a trait considered to be a feature of the immune system. Finally, using gene lists derived from sequencing data, we show that human genes under constrained selective pressure are disproportionately expressed in nervous system tissues.

Pericytes are mural cell that have been found to play important roles in promoting blood vessel development and regulating blood flow. The signals that attract pericytes to maturing vessels during the resolution phase of wound healing are unknown. In this study, we examine the role of the chemokine receptor CXC receptor 3 (CXCR3) ligands, as they are produced by maturing endothelial cells. Pericytes isolated from muscle and retina were found to by and large only express the B-isoform of CXCR3 (CXCR3B), with expression being independent of the mitotic state of the cells. Pericyte stimulation with the CXCR3 ligands Mig (CXCL9), IP-9/I-TAC (CXCL11), or IP-10 (CXCL10) resulted in the activation of ERK but not AKT. Treatment with Mig or IP-9, but not IP-10, enhanced p38(MAPK) phosphorylation. Interestingly, while cyclic adenosine monophosphate is generated downstream of CXCR3B in other cells, protein kinase A activation was not observed in these pericytes when treated with these three CXCR3 ligands. The increase in ERK activity resulted in a slight increase in cell transmigration, with the inhibition of ERK leading to a decrease in CXCR3B mediated migration and inhibition of p38(MAPK) reducing transmigration through small pores. These ligands did not affect proliferation. These data are the first to characterize CXCR3B as the predominant isoform expressed on pericytes, and was found on these diverse cells isolated from both muscle and eye. We also show that CXCR3B signaling stimulates transmigration of barrier pores in pericytes as opposed to its inhibitory affects on endothelial cells and fibroblasts. These findings characterize a novel role for the CXCR3B in regulating cellular function. Taken together these data show a role for CXCR3B in regulating pericyte function.

CXCR3 is a G-protein coupled receptor which binds to ELR-negative CXC chemokines that have been found to impact immune responses, vascular develop, and wound repair. More recently, CXCR3 has been examined in the context of cancer and increased expression in many human tumors has been correlated with poor prognosis in breast, melanoma, colon and renal cancer patients. Three variants of CXCR3 are identified so far (CXCR3-A, CXCR3-B and CXCR3-alt) with the two primary ones, CXCR3-A and CXCR3-B, considered to induce opposite physiological functions. Generally, CXCR3-A, the predominant form in hematopoietic cells, appears to mediate tumor “go” signaling via promoting cell proliferation, survival, chemotaxis, invasion and metastasis; while CXCR3-B, the main form on formed elements including epithelial cells, appears to mediate tumor “stop” signaling via promoting growth suppression, apoptosis and vascular involution. Thus, aberrant expression of the isoforms CXCR3-A and CXCR3-B could affect tumor progression. In this review, we have discussed the profiles of CXCR3 variants and related signaling, as well as the role of CXCR3 variants in cancer.

To investigate hepatocyte growth factor (HGF) primed fibroblasts and decorin application on skin and vocal fold fibroblasts in vitro and in vivo in rabbit vocal fold scar model.Vocal fold and skin fibroblasts underwent five in vitro treatment conditions: control, epidermal growth factor, HGF, both decorin and HGF, and decorin alone. Hyaluronic acid and collagen enzyme-linked immunosorbent assays were performed. In vivo, 12 rabbits underwent unilateral vocal fold stripping. Injured vocal folds were then injected with skin fibroblasts, HGF, HGF-primed fibroblasts and decorin, or decorin. Outcomes included histologic and lamina propria height analyses.In vitro, HGF increased hyaluronic acid synthesis in vocal fold fibroblasts (P0.001). HGF and decorin treatment diminished collagen secretion (P0.01). In vivo, histologic findings indicated minimal difference in collagen amount between treatment groups.HGF and decorin together may decrease collagen production by skin and vocal fold fibroblasts. Fibroblast transplantation into scarred vocal folds has equivocal benefit.

Phospholipase C-gamma (PLCgamma) has been implicated in tumor cell motility required for invasiveness and metastasis. Diminished tumor dissemination has been demonstrated in xenograft models, but studies in naturally-occurring tumors are lacking, having been limited by the timing of the interventions. Therefore, we generated mice that express a doxycycline (DOX)-inducible dominant-negative fragment of PLCgamma, PLCz; this approach avoids the in utero lethality caused by the absence of PLCgamma. As we targeted two de novo-occurring carcinomas of the mammary (MMTV-driven polyoma middle T antigen model, PyVmT) and prostate (TRAMP model) glands, we limited expression to these epithelial cells by driving DOX transactivator from the prostatein C3 promoter. This avoids the confounding variable of potentially abrogating motility in stromal and endothelial cells. These mice developed normally in the presence of DOX, except for limited mammary development if treated before 6 weeks and immaturity of the prostate gland if treated before 2 weeks of age. DOX-mediated induction of PLCz from age 8 to 16 weeks in PyVmT mice decreased the number of lung metastases by >10-fold (P

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and β1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.

Calpain activity is required for de-adhesion of the cell body and rear to enable productive locomotion of adherent cells during wound repair and tumor invasion. Growth factors activate m-calpain (calpain 2, CAPN2) via ERK/mitogen-activated protein kinases, but only when these kinases are localized to the plasma membrane. We thus hypothesized that m-calpain is activated by epidermal growth factor (EGF) only when it is juxtaposed to the plasma membrane secondary to specific docking. Osmotic disruption of NR6 fibroblasts expressing the EGF receptor demonstrated m-calpain being complexed with the substratum-adherent membrane with this increasing in an EGF-dependent manner. m-Calpain colocalized with phosphoinositide biphosphate (PIP(2)) with exogenous phospholipase C removal of phosphoinositides, specifically, PI(4,5)P(2) but not PI(4)P(1) or PIP(3), releasing the bound m-calpain. Downregulation of phosphoinositide production by 1-butanol resulted in diminished PIP(2) in the plasma membrane and eliminated EGF-induced calpain activation. This PIP(2)-binding capacity resided in domain III of calpain, which presents a putative C2-like domain. This active conformation of this domain appears to be partially masked in the holoenzyme as both activation of m-calpain by phosphorylation at serine 50 and expression of constitutively active phosphorylation mimic glutamic acid-increased m-calpain binding to the membrane, consistent with blockade of this cascade diminishing membrane association. Importantly, we found that m-calpain was enriched toward the rear of locomoting cells, which was more pronounced in the plasma membrane footprints; EGF further enhanced this enrichment, in line with earlier reports of loss of PIP(2) in lamellipodia of motile cells. These data support a model of m-calpain binding to PIP(2) concurrent with and likely to enable ERK activation and provides a mechanism by which cell de-adhesion is directed to the cell body and tail as phospholipase C-gamma hydrolyzes PIP(2) in the protruding lamellipodia.

Fibroblasts stimulated by EGF within collagen matrices generate contraction forces that are likely of importance to cell migration and matrix compaction during wound healing. We have employed an in vitro fibroblast-embedded collagen matrix compaction assay to ascertain signaling pathway components downstream of EGFR activation leading to generation and transmission of contractile force. EGF compacts this floating collagen matrix to a similar extent as PDGF. We demonstrate that compaction requires EGFR kinase activity, yet is maximal in magnitude at intermediate EGF concentrations. This suggests that transmission of EGFR-induced contractile force to the matrix can be mitigated by consequent anti-adhesive effects of EGFR signaling in a dose-dependent manner. Treatment with pharmacological inhibitors demonstrated involvement of the signaling components extracellular signal-regulated kinase (ERK), Rho kinase, and myosin light chain kinase (MLCK) in the force generation and/or transmission process. Moreover, treatment with the pan-calpain inhibitor ALLN and isoform-specific downregulation of m-calpain (CAPN2) using RNA interference determined m-calpain to be a key component of the EGF-induced force response. ALLN treatment modulated the compaction response in a biphasic manner, enhancing matrix deformation to the greatest extent at intermediate concentrations. Our findings have thus identified key signals downstream of EGFR, which integrate in a complex manner to generate and transmit contractile forces to yield matrix deformation.

Ten years have passed since the Graylyn Conference Report on Laboratory Medicine/Clinical Pathology training was issued. During that period, the Accreditation Council for Graduate Medical Education (ACGME) substantially revised the requirements for training programs, the American Board of Pathology (ABP) amended the requirements and the time needed for certification, and the discipline itself along with the broader discipline of pathology, evolved significantly. Recently, a curriculum proposal in anatomic pathology was published as a potential template to be used by training programs to help meet these new and evolving needs. Toward the same end, the Academy of Clinical Laboratory Physicians and Scientists has developed a template for a curriculum in clinical pathology (laboratory medicine), taking into account newly designated and revised areas of residency core competency, the alterations in training requirements promulgated by the ACGME and ABP, and the rapidly developing nature of the discipline itself The proposed clinical pathology curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by pathology residency training programs.

Prostate cancer is frequent among men over 45 years of age, but it generally only becomes lethal with metastasis. In this study, we identified a gene called cellular stress response 1 (CSR1) that was frequently down-regulated and methylated in prostate cancer samples. Survival analysis indicated that methylation of the CSR1 promoter, and to a lesser extent down-regulation of CSR1 protein expression, was associated with a high rate of prostate cancer metastasis. Forced expression of CSR1 in prostate cancer cell lines DU145 and PC3 resulted in a two- to threefold decrease in colony formation and a 10-fold reduction in anchorage-independent growth. PC3 cells stably expressing CSR1 had an average threefold decrease in their ability to invade in vitro. Expression of CSR1 in PC3 cell xenografts produced a dramatic reduction (>8-fold) in tumor size, rate of invasion (0 versus 31%), and mortality (13 versus 100%). The present findings suggest that CSR1 is a potent tumor sup-pressor gene.

The study objective was to determine the effectiveness of a phenotypic chemoresponse assay in predicting response to chemotherapy measured by progression-free interval (PFI) in a retrospective series of ovarian cancer patients whose tumor specimens had been tested with the ChemoFx assay. A statistically significant correlation between assay prediction of response and PFI was observed in 256 cases with an exact or partial match between drug(s) assayed and received. In 135 cases with an exact match, the hazard ratio for progression of the resistant group was 2.9 (confidence interval [CI]: 1.4-6.3; P0.01) compared to the sensitive group and 1.7 (CI: 1.2-2.5) for the intermediate compared to the sensitive group. The median PFI for patients treated with drugs assayed as resistant was 9 months, 14 months for those with drugs assayed as intermediately sensitive, and PFI had not been achieved for those with drugs assayed as sensitive. These data indicate that the ChemoFx assay is predictive of PFI in ovarian cancer. As the majority of ovarian cancers display different degrees of response to different chemotherapy agents ex vivo, the incorporation of assay information into treatment selection has the potential to improve clinical outcomes in ovarian cancer patients.

The renal roles of physiologically relevant doses of angiotensin II (Ang II) and C-type natriuretic peptide (CNP) were investigated in the dogfish, Scyliorhinus canicula , using an in situ perfused trunk preparation. Perfusion with 10 −9 M Ang II resulted in a glomerular antidiuresis and decreases in perfusate flow rate, transport maxima for glucose and the proportion of filtering glomeruli. In addition, the renal clearances and excretion of urea, sodium, and chloride were significantly reduced, whereas the relative clearances of these parameters remained unchanged. In contrast, perfusion of 10 −9 M CNP caused a glomerular diuresis, an increase in transport maxima for glucose, but no significant change in the proportion of filtering glomeruli. In addition, the renal clearances of urea, sodium, and chloride were significantly increased but there was no effect on the relative clearances of urea, sodium, or chloride. Perfusion with 10 −10 M Ang II or CNP had no significant renal effects. Our results suggest that these hormones act at the level of the glomeruli rather than at a tubular level.

Contains fulltext : 35572.pdf (Publisher’s version ) (Closed access) For wild, sea-run brown trout (Salmo trutta) smolts, the physiological consequences of abrupt transfer to seawater and simultaneous challenge with copepodid larvae of the sea louse, Lepeophtheirus salmonis (Kr�yer, 1837), were investigated in the laboratory. Analysis of osmoregulatory, metabolic, and stress markers allowed the derivation of a sublethal threshold burden of L. salmonis, above which the host suffers major physiological stress. Noticeable lice effects, consistent across all measured markers, were not observed until L. salmonis developed to the mobile preadult and adult stages. Preadult L. salmonis caused significant increases in plasma chloride, osmolality, glucose, lactate, and cortisol and a significant reduction in haematocrit. Piecewise linear statistical approaches allowed the determination of abrupt changes in these physiological markers, attributable to the intensity of L. salmonis infestation on individual fish, and identification of overall threshold lice burdens. Thirteen mobile lice�fish�1 (weight range 19�70 g) was a consistent breakpoint across several physiological measures. This information will provide a valuable, objectively derived tool to aid in the formulation of effective wild fisheries management policy concerning S. trutta conservation.

The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process.

As academic pathology departments have become increasingly based in large, regional medical systems spread across hundreds of square miles, new methods are needed to tie these increasingly distributed departments together as integrated units. An important part of that integration is the ability to share academic and teaching conferences across long distances. In this article we present an effective, low-cost webcasting system that has evolved at the University of Pittsburgh Medical Center Department of Pathology over the past several years based on inexpensive, widely available software. To date, the system has broadcast and archived more than 400 conferences and currently serves approximately 80 to 100 requests each week. Important factors in the success of the program include the creation of a faculty steering committee to control resources and manage growth, the availability of informatics faculty and support for technical staff, and the decision to operate the service as part of the core information technology infrastructure of the department. Webcasting will likely become an even more important academic and operational tool in the future as more of the department's conferences, seminars, and even working meetings are communicated through the webcasting infrastructure.

Recently, extracellular matrix components have been shown to contain domains that can interact with and activate receptors with intrinsic tyrosine kinase activity. These receptor tyrosine kinases are strong mediators of the cell responses of proliferation, migration, differentiation, and dedifferentiation. However, an interesting question is raised as to why cells would present growth factor receptor ligands in such a manner, as the majority of growth factors are small, soluble, or only transiently tethered ligands. With the exception of the discoidin domain receptors that bind collagen, the other described domains interact with a receptor that binds ubiquitous soluble peptide growth factors, the epidermal growth factor receptor. Unlike traditional growth factors, these individual "matrikine" domains within tenascin-C, laminin, collagen, and decorin possess relatively low binding affinity (high nanomolar or micromolar) and are often presented in multiple valency. The presentation of ligands within the extracellular matrix in this fashion might allow for unique biochemical and physiological outcomes. This new class of "matrikine" ligand may be critical for wound healing, as the majority of known extracellular matrix components possessing matrikines play a strong role, or are presented uniquely, during skin repair. Tenascin-C expression, for instance, is uniquely regulated spatially and has been proposed to present pro-migratory tracks during skin repair through its epidermal growth factor-like repeats. The epidermal growth factor-like repeats of laminin-5 act as cryptic ligands revealed upon matrix metalloproteinase-2 degradation of the surrounding extracellular matrix. The deletion of the discoidin domain receptors 1 and 2 for collagen have negative consequences on the role of fibroblasts and epithelial cells for matrix metalloproteinase production, migration, proliferation, and extracellular matrix turnover. Finally, decorin can bind to, inhibit, and down-regulate epidermal growth factor receptor levels and signaling, suggesting a tonic role of the epidermal growth factor binding domain of decorin in the resolution of wound healing. We provide a model framework for further studies into this emerging class of signals.

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Keloids, which overgrow the boundaries of the original injury, represent aberrations in the fundamental process of wound healing that include over-abundant cell in-migration, cell proliferation, and inflammation, as well as increased extracellular matrix synthesis and defective remodeling. To understand the key events that result in the formation of these abnormal scars would open new avenues for better understanding of excessive repair, and might provide new therapeutic options. We examined epidermal growth factor receptor (EGFR)-induced cell motility in keloid fibroblasts, as this receptor initiates cell migration during normal wound repair. We show that keloid fibroblasts respond to EGF-induced cell migration but the response is somewhat diminished compared to normal adult fibroblasts (approximately 30% reduced); the mitogenic response was similarly blunted (approximately 5% reduced). Keloid fibroblasts express near normal levels of EGFR (82%), but show a much more attenuated activation of EGFR itself and the motility-associated phospholipase C-gamma. This was reflected in part by rapid loss of EGFR upon exposure to EGF. Interestingly, while extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) activation was relatively robust in keloid fibroblasts, the downstream triggering of the motility-associated calpain activity was blunted. This was reflected by high cell-substratum adhesiveness in the keloid fibroblasts. Thus, the blunted migratory response to EGF noted in keloid fibroblasts appears due to limited activation of two important biochemical switches for cell motility.

The two best known calpains, μ- and m-calpain, are Ca 2+ -dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca 2+ -linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (μ-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine–Dalgarno ribosome binding site. The results provide a simple method to obtain ∼10 mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.

Since the landmark contributions of Homer Smith and co-workers in the 1930s there has been a considerable advance in our knowledge regarding the osmoregulatory strategy of elasmobranch fish. Smith recognised that urea was retained in the body fluids as part of the 'osmoregulatory ballast' of elasmobranch fish so that body fluid osmolality is raised to a level that is iso- or slightly hyper-osmotic to that of the surrounding medium. From studies at that time he also postulated that many marine dwelling elasmobranchs were not capable of adaptation to dilute environments. However, more recent investigations have demonstrated that, at least in some species, this may not be the case. Gradual acclimation of marine dwelling elasmobranchs to varying environmental salinities under laboratory conditions has demonstrated that these fish do have the capacity to acclimate to changes in salinity through independent regulation of Na(+), Cl(-) and urea levels. This suggests that many of the presumed stenohaline marine elasmobranchs could in fact be described as partially euryhaline. The contributions of Thomas Thorson in the 1970s demonstrated the osmoregulatory strategy of a fully euryhaline elasmobranch, the bull shark, Carcharhinus leucas, and more recent investigations have examined the mechanisms behind this strategy in the euryhaline elasmobranch, Dasyatis sabina. Both partially euryhaline and fully euryhaline species utilise the same physiological processes to control urea, Na(+) and Cl(-) levels within the body fluids. The role of the gills, kidney, liver, rectal gland and drinking process is discussed in relation to the endocrine control of urea, Na(+) and Cl(-) levels as elasmobranchs acclimate to different environmental salinities.

Cell motility is actuated by a host of intracellular signaling cascades that result in movement of the cell in one direction, even without an external gradient. Phospholipase C-gamma (PLCgamma) has been shown to be important for growth factor-induced lamellipodial protrusion at the front of the cell while Cdc42 has been implicated in both filopodium formation at the leading edge and control of polarity of migrating cells. We asked whether these asymmetries in effector molecules may be linked. When we overexpressed either constitutively active, dominant negative, or GFP-tagged Cdc42, wild-type NR6 fibroblasts lost directionality, as expected. On epidermal growth factor (EGF) exposure these cells produced multiple, transient protrusions in every direction; these extensions failed to result in productive motility. GFP-tagged Cdc42 appeared transiently at edges of newly formed protrusions in EGF-stimulated cells while they moved haphazardly. While PLCgamma is distributed throughout the cell, the ratio of active, tyrosyl-phosphorylated PLCgamma was increased at the leading edge, where phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis is concentrated. This co-localization of activities may be due to Cdc42 directing PLCgamma to the cell front, as PLCgamma associated with Cdc42 in an EGF-dependent manner. We conclude that Cdc42 controls cell polarity, likely in part, through its binding to active PLCgamma.

The vast majority of cancer morbidity and mortality arises from tumor progression beyond the primary tumor site. Unfortunately, most therapies are not effective for advanced stage disease with regional extension or distant metastases. Thus, new treatments are needed to target rate limiting steps in tumor progression. The ability of cancers to invade and metastasize requires the acquisition of specific cell behaviors that enable the cell to escape from the localized site, breach the defined boundaries, reach a hospitable ectopic site and grow in this new locale. Recently, dysregulation of cell motility as stimulated by various extracellular factors has gained credence as a rate-limiting alteration in tumor progression in carcinomas and some other solid tumors. This has focused attention on initiators of signaling cascades that regulate tumor migration. In this effort, one molecule, phospholipase C-γ1 (PLCγ), has been shown to function as a key molecular switch.

Do cell-surface growth-factor receptors and their ligands accumulate in the nucleoplasm under physiological conditions? And, if so, how do they get there and what function do they serve in this location? Recent advances have provided tantalizing hints to the answers to these questions, and hold the key to identifying a new mode of signal transduction.

Calpains are a large family of intracellular proteases whose precise and limited cleavage of specific proteins might be an integral regulatory aspect of signaling pathways. This intriguing mechanism for transducing biochemical and biophysical information from the external milieu seems to operate during cell motility. The two first described and ubiquitous isoforms, mu-calpain and M-calpain, have been implicated in enabling cell spreading by modifying adhesion sites and in promoting locomotion of adherent cells by facilitating rear-end detachment. Recent elucidation of the molecular structure of calpain opens the door for understanding how these pluripotential signal proteins are regulated to help govern migration. Armed with this knowledge, the precise roles of calpains in inflammation, wound repair and tumor progression can be ascertained and offer novel therapeutic targets.

Tumor progression to the invasive and metastatic states dramatically enhances the morbidity and mortality of cancer. Rational therapeutic interventions will only be possible when we understand the molecular mechanisms governing the cell behavior underlying this transformation. For invasion, a subpopulation of tumor cells must recognize the extracellular matrix barrier, modify the barrier, migrate through the barrier, and then proliferate in the adjacent but ectopic locale. Prevention of any one of these steps would prevent invasion, but determining the most sensitively dysregulated step should provide the most promising therapeutic index. In many invasive tumors, upregulation of active motility is stimulated by growth factor receptor signaling, the EGF receptor being the most frequently implicated. Two key downstream molecular switches, PLCgamma and m-calpain, are required for growth factor-induced motility but not basal, matrix-stimulated motility. Inhibition of either of these enzymes blocks in vitro and in vivo invasion of prostate, breast, and bladder carcinomas and glioblastomas. These represent novel and potentially selective targets for drug development. Future advances in the imaging of tumors in animals and ex vivo organ culture systems should provide additional new targets.

The cholesterol reducing drugs, statins, exhibit anti-tumor effects against cancer stem cells and various cancer cell lines, exert potent additivity or synergy with existing chemotherapeutics in animal models of cancer and may reduce cancer incidence and cancer related mortality in humans. However, not all tumor cell lines are sensitive to statins, and clinical trials have demonstrated mixed outcomes regarding statins as anticancer agents. Here, we show that statin-induced reduction in intracellular cholesterol levels correlate with the growth inhibition of cancer cell lines upon statin treatment. Moreover, statin sensitivity segregates with abundant cytosolic vimentin expression and absent cell surface E-cadherin expression, a pattern characteristic of mesenchymal-like cells. Exogenous expression of cell surface E-cadherin converts statin- sensitive cells to a partially resistant state implying that statin resistance is in part dependent on the tumor cells attaining an epithelial phenotype. As metastasizing tumor cells undergo epithelial to mesenchymal transition during the initiation of the metastatic cascade, statin therapy may represent an effective approach to targeting the cells most likely to disseminate.

Tenascin-C (TNC), a multifunctional matricellular glyco-protein, is highly expressed in the majority of melanoma cell lines and has been implicated in the progression of melanoma. A growing body of evidence has implicated the role of TNC in the process of invasion and metastasis for melanoma. However, the mechanism and individual signaling pathways by which TNC drives melanoma progression have not been illuminated. Herein we provide perspectives from the investigation of TNC in other settings that may hint at the mechanistic role of TNC in this disease.

Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human a-actinin-4 (ACTN4), Tyr[superscript 4] and Tyr[superscript 31] are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr[superscript 4] site. We found that introducing a phosphomimetic mutation at only Tyr[superscript 31] was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr[superscript 31] is mostly buried and that phosphorylation of Tyr[superscript 4] would increase the solvent exposure and thus kinase accessibility of Tyr[superscript 31]. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr[superscript 4], whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr[superscript 4] and Tyr[superscript 31]. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr[superscript 31] can be dictated by the kinase affinity for Tyr[superscript 4]. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site., National Institutes of Health (U.S.) (Grant R01 GM69668)

Introduction Bone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation. Methods MSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers. Results We found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin. Conclusions Our findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis., National Institute of General Medical Sciences (U.S.) (Grant GM069668), National Institute of Dental and Craniofacial Research (U.S.) (Grant DE019523), National Institute on Aging (Grant AG026389)

Prostate cancer disproportionately afflicts African-American men. As such, we feel that it is critically important to recruit researchers from this population if we are to conquer this disease. Numerous programs have attempted to recruit minorities to biomedical research and prostate cancer in particular. Often this involves a short period of research immersion during a summer semester. However, it has been shown that many of these trainees do not persevere in the selected area due to the singular nature of the experience. Our goal is to formalize a program to broaden the scope of and enlarge Tuskegee University s prostate cancer research , which will be accomplished through tatargeting interested undergraduate students early during their science studies at Tuskegee University and enabling them to participate in summer research and education training periods at the University of Pittsburgh and the University of Pittsburgh Cancer Institute as part of their overall prostate cancer education.

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability., National Institutes of Health (U.S.) (Grant 1UH2TR000496-01), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal–regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-“tethered” ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of “insoluble” growth factor ligands and a novel mode of activation for growth factor receptors.

Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates.

The most ominous development in tumor progression is the transition to an invasive and metastatic phenotype. Little is known, however, about the molecular alterations that cause a tumor to become invasive. In view of this, we have used microarray expression analysis to evaluate the expression profiles of a unique panel of human DU145 prostate cancer sublines that vary in their invasive potential. The three DU145 sublines expressed epidermal growth factor (EGF) receptors that differed in their ability to activate phospholipase C-gamma (PLC gamma). Three-way analyses yielded 11 genes out of 4608 genes screened that associated directly or inversely with invasive potential. The gene whose expression correlated most strongly with lack of invasion was identified as a potential invasion suppressor and called prostin-1. Pharmacological inhibition of PLC gamma (U73122) confirmed that PLC gamma signaling suppressed prostin-1 in that U73122 treatment caused induction of prostin-1 in PLC gamma competent cells. The prostin-1 gene, conserved through phylogeny, is induced by androgen in LNCaP cells and encodes a 92 amino acid protein. The protein shares no extensive homologies with other known genes, yet was recently identified as a small stabilizer subunit of the dolichol-phosphate-mannose (DPM) synthase complex. That DPM3/prostin-1 might suppress tumor progression was supported by the finding that exogenous expression in COS cells leads to apoptosis. These findings support the use of model cell lines to identify putative tumor suppressors and promoters.

(2001). Nationality, Race, and gender on the American pop charts: What happened in the ‘90s? Popular Music and Society: Vol. 25, Nineteenth‐Century American Popular Music, pp. 221-231.

In mammals, a principal bioactive component of the renin-angiotensin system (RAS), angiotensin II (ANG II), is known to be vasopressor, dipsogenic, a stimulant of adrenocortical secretion and to control glomerular and renal tubular function. Historically, a RAS analogous to that found in mammals was thought to have first evolved in the bony fishes. Recent research has identified the unusually structured elasmobranch [Asp(1)-Pro(3)-Ile(5)] ANG II. Physiological studies have demonstrated that ANG II in elasmobranchs is vasopressor, and stimulates interrenal gland production of the elasmobranch corticosteroid 1alpha-hydroxycorticosterone. The specific binding of ANG II in elasmobranchs has been reported in gills, heart, interrenal gland, gut and rectal gland. The precise osmoregulatory role ANG II plays in cartilaginous fishes is not yet known; however, putative evidence is emerging for a role in the control of drinking rate, rectal gland secretion, and kidney function.

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A “kinase-dead” EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.