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Tau hyperphosphorylation and aggregation is a common feature of many dementia-causing neurodegenerative diseases. Tau can be phosphorylated at up to 85 different sites, and there is increasing interest in whether tau phosphorylation at specific epitopes, by specific kinases, plays an important role in disease progression. The AMP-activated protein kinase (AMPK)-related enzyme NUAK1 has been identified as a potential mediator of tau pathology, whereby NUAK1-mediated phosphorylation of tau at Ser356 prevents the degradation of tau by the proteasome, further exacerbating tau hyperphosphorylation and accumulation. This study provides a detailed characterisation of the association of p-tau Ser356 with progression of Alzheimer's disease pathology, identifying a Braak stage-dependent increase in p-tau Ser356 protein levels and an almost ubiquitous presence in neurofibrillary tangles. We also demonstrate, using sub-diffraction-limit resolution array tomography imaging, that p-tau Ser356 co-localises with synapses in AD postmortem brain tissue, increasing evidence that this form of tau may play important roles in AD progression. To assess the potential impacts of pharmacological NUAK inhibition in an ex vivo system that retains multiple cell types and brain-relevant neuronal architecture, we treated postnatal mouse organotypic brain slice cultures from wildtype or APP/PS1 littermates with the commercially available NUAK1/2 inhibitor WZ4003. Whilst there were no genotype-specific effects, we found that WZ4003 results in a culture-phase-dependent loss of total tau and p-tau Ser356, which corresponds with a reduction in neuronal and synaptic proteins. By contrast, application of WZ4003 to live human brain slice cultures results in a specific lowering of p-tau Ser356, alongside increased neuronal tubulin protein. This work identifies differential responses of postnatal mouse organotypic brain slice cultures and adult human brain slice cultures to NUAK1 inhibition that will be important to consider in future work developing tau-targeting therapeutics for human disease., (© 2024. The Author(s).)

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are therapeutic targets for diseases such as cancer, neurodegeneration and immunological disorders as they are key components in regulating cell signalling pathways. In an effort to make probe molecules available for further exploring these targets, we have previously reported PI5P4Kα-selective and PI5P4Kγ-selective ligands. Herein we report the rational design of PI5P4Kα/γ dual inhibitors, using knowledge gained during the development of selective inhibitors for these proteins. ARUK2007145 (39) is disclosed as a potent, cell-active probe molecule with ADMET properties amenable to conducting experiments in cells., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) play a central role in regulating cell signalling pathways and, as such, have become therapeutic targets for diseases such as cancer, neurodegeneration and immunological disorders. Many of the PI5P4Kα inhibitors that have been reported to date have suffered from poor selectivity and/or potency and the availability of better tool molecules would facilitate biological exploration. Herein we report a novel PI5P4Kα inhibitor chemotype that was identified through virtual screening. The series was optimised to deliver ARUK2002821 (36), a potent PI5P4Kα inhibitor (pIC 50 = 8.0) which is selective vs. other PI5P4K isoforms and has broad selectivity against lipid and protein kinases. ADMET and target engagement data are provided for this tool molecule and others in the series, as well as an X-ray structure of 36 solved in complex with its PI5P4Kα target., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

Owing to their central role in regulating cell signaling pathways, the phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive therapeutic targets in diseases such as cancer, neurodegeneration, and immunological disorders. Until now, tool molecules for these kinases have been either limited in potency or isoform selectivity, which has hampered further investigation of biology and drug development. Herein we describe the virtual screening workflow which identified a series of thienylpyrimidines as PI5P4Kγ-selective inhibitors, as well as the medicinal chemistry optimization of this chemotype, to provide potent and selective tool molecules for further use. In vivo pharmacokinetics data are presented for exemplar tool molecules, along with an X-ray structure for ARUK2001607 ( 15 ) in complex with PI5P4Kγ, along with its selectivity data against >150 kinases and a Cerep safety panel.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 ( 1 ). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kβ. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

Bifunctional molecules known as PROTACs simultaneously bind an E3 ligase and a protein of interest to direct ubiquitination and clearance of that protein, and they have emerged in the past decade as an exciting new paradigm in drug discovery. In order to investigate the permeability and properties of these large molecules, we synthesized two panels of PROTAC molecules, constructed from a range of protein-target ligands, linkers, and E3 ligase ligands. The androgen receptor, which is a well-studied protein in the PROTAC field was used as a model system. The physicochemical properties and permeability of PROTACs are discussed., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Remediation and restoration of the Nation's nuclear legacy of radiological and chemical contaminated areas is an ongoing and costly challenge for the U.S. Department of Energy (DOE). For large sites, such as the Hanford and Savannah River Sites, successful remediation involves complex decisions related to remedies, end-states, timing, and sequencing of cleanup of separate and related contaminated units within a site. Hanford Site cannot clean up every unit simultaneously due to limits in funding, personnel, and technology. This paper addresses one of the major considerations - the consequences of delaying remediation of a unit on different receptors (e.g. people, ecological, and eco-cultural resources), using the DOE Hanford Site as a case study. We develop a list of attributes that managers should consider for successful remediation, examine how delaying remediation could affect workers, the public and ecological resources (including water resources), and use some examples to illustrate potential effects of delays. The factors to consider when deciding whether and how long to delay remediation of a unit include personnel, information and data, funding, equipment, structural integrity, contaminant source, and resource vulnerability. Each of these factors affects receptors differently. Any remediation task may be dependent on other remediation projects, on the availability of transport, containers, interim storage and ultimate disposition decisions, or the availability of trained personnel. Delaying remediation may have consequences for people (e.g. workers, site neighbors), plants, animals, ecosystems, and eco-cultural resources (i.e. those cultural values that depend upon ecological resources). The risks, benefits, and uncertainties for evaluating the consequences of delaying remediation are described and discussed. Assessing the advantages and disadvantages of delaying remediation is important for health professionals, ecologists, resource trustees, regulators, Tribal members, recreationists, fishermen, hunters, conservationists, and a wide range of other stakeholders., (Copyright © 2018. Published by Elsevier B.V.)

Background: Hospitals face growing pressure to meet the dual but often competing goals of providing a safe environment while controlling operating costs. Evidence-based data are needed to provide insight for facility management practices to support these goals., Methods: The quality of the air in 3 operating rooms was measured at different ventilation rates. The energy cost to provide the heating, ventilation, and air conditioning to the rooms was estimated to provide a cost-benefit comparison of the effectiveness of different ventilation rates currently used in the health care industry., Results: Simply increasing air change rates in the operating rooms tested did not necessarily provide an overall cleaner environment, but did substantially increase energy consumption and costs. Additionally, and unexpectedly, significant differences in microbial load and air velocity were detected between the sterile fields and back instrument tables., Conclusions: Increasing the ventilation rates in operating rooms in an effort to improve clinical outcomes and potentially reduce surgical site infections does not necessarily provide cleaner air, but does typically increase operating costs. Efficient distribution or management of the air can improve quality indicators and potentially reduce the number of air changes required. Measurable environmental quality indicators could be used in lieu of or in addition to air change rate requirements to optimize cost and quality for an operating room and other critical environments., (Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Sufficient quantities of quality air and controlled, unidirectional flow are important elements in providing a safe building environment for operating rooms., Methods: To make dynamic assessments of an operating room environment, a validated method of testing the multiple factors influencing the air quality in health care settings needed to be constructed. These include the following: temperature, humidity, particle load, number of microbial contaminants, pressurization, air velocity, and air distribution. The team developed the name environmental quality indicators (EQIs) to describe the overall air quality based on the actual measurements of these properties taken during the mock surgical procedures. These indicators were measured at 3 different hospitals during mock surgical procedures to simulate actual operating room conditions. EQIs included microbial assessments at the operating table and the back instrument table and real-time analysis of particle counts at 9 different defined locations in the operating suites. Air velocities were measured at the face of the supply diffusers, at the sterile field, at the back table, and at a return grille., Results: The testing protocol provided consistent and comparable measurements of air quality indicators between institutions. At 20 air changes per hour (ACH), and an average temperature of 66.3°F, the median of the microbial contaminants for the 3 operating room sites ranged from 3-22 colony forming units (CFU)/m 3 at the sterile field and 5-27 CFU/m 3 at the back table. At 20 ACH, the median levels of the 0.5-µm particles at the 3 sites were 85,079, 85,325, and 912,232 in particles per cubic meter, with a predictable increase in particle load in the non-high-efficiency particulate air-filtered operating room site. Using a comparison with cleanroom standards, the microbial and particle counts in all 3 operating rooms were equivalent to International Organization for Standardization classifications 7 and 8 during the mock surgical procedures., Conclusions: The EQI protocol was measurable and repeatable and therefore can be safely used to evaluate air quality within the health care environment to provide guidance for operational practices and regulatory requirements., (Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are enigmatic lipid kinases with physiological functions that are incompletely understood, not the least because genetic deletion and cell transfection have led to contradictory data. Here, we used the genetic tractability of DT40 cells to create cell lines in which endogenous PI5P4Kα was removed, either stably by genetic deletion or transiently (within 1 h) by tagging the endogenous protein genomically with the auxin degron. In both cases, removal impacted Akt phosphorylation, and by leaving one PI5P4Kα allele present but mutating it to be kinase-dead or have PI4P 5-kinase activity, we show that all of the effects on Akt phosphorylation were dependent on the ability of PI5P4Kα to synthesize phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] rather than to remove PI5P. Although stable removal of PI5P4Kα resulted in a pronounced decrease in Akt phosphorylation at Thr308 and Ser473, in part because of reduced plasma membrane PIP3, its acute removal led to an increase in Akt phosphorylation only at Ser473. This process invokes activation primarily of mammalian target of rapamycin complex 2 (mTORC2), which was confirmed by increased phosphorylation of other mTORC2 substrates. These findings establish PI5P4Kα as a kinase that synthesizes a physiologically relevant pool of PI(4,5)P2 and as a regulator of mTORC2, and show a phenomenon similar to the "butterfly effect" described for phosphatidylinositol 3-kinase Iα [Hart JR, et al. (2015) Proc Natl Acad Sci USA 112(4):1131-1136], whereby through apparently the same underlying mechanism, the removal of a protein's activity from a cell can have widely divergent effects depending on the time course of that removal., Competing Interests: The authors declare no conflict of interest.

The chicken B-cell line DT40 has two isoforms of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K), α and β, which are likely to exist as a mixture of obligate homo- and hetero-dimers. Previous work has led us to speculate that an important role of the β isoform may be to target the more active PI5P4Kα isoform to the nucleus. In the present study we expand upon that work by genomically tagging the PI5P4Ks with fluorochromes in the presence or absence of stable or acute depletions of PI5P4Kβ. Consistent with our original hypothesis we find that PI5P4Kα is predominantly (possible entirely) cytoplasmic when PI5P4Kβ is stably deleted from cells. In contrast, when PI5P4Kβ is inducibly removed within 1 h PI5P4Kα retains its wild-type distribution of approximately 50:50 between cytoplasm and nucleus even through a number of cell divisions. This leads us to speculate that PI5P4Kα is chromatin-associated. We also find that when cells are in the exponential phase of growth PI5P4Kβ is primarily cytoplasmic but translocates to the nucleus upon growth into the stationary phase or upon serum starvation. Once again this is not accompanied by a change in PI5P4Kα localization and we show, using an in vitro model, that this is possible because the dimerization between the two isoforms is dynamic. Given this shift in PI5P4Kβ upon nutrient deprivation we explore the phenotype of PI5P4K B-null cells exposed to this stress and find that they can sustain a greater degree of nutrient deprivation than their wild-type counterparts possibly as a result of up-regulation of autophagy., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

NIH-12848 (NCGC00012848-02), a putative phosphatidylinositol 5-phosphate 4-kinase γ (PI5P4Kγ) inhibitor, was explored as a tool for investigating this enigmatic, low activity, lipid kinase. PI5P4K assays in vitro showed that NIH-12848 inhibited PI5P4Kγ with an IC50 of approximately 1 μM but did not inhibit the α and β PI5P4K isoforms at concentrations up to 100 μM. A lack of inhibition of PI5P4Kγ ATPase activity suggested that NIH-12848 does not interact with the enzyme's ATP-binding site and direct exploration of binding using hydrogen-deuterium exchange (HDX)-MS (HDX-MS) revealed the putative PI5P-binding site of PI5P4Kγ to be the likely region of interaction. This was confirmed by a series of mutation experiments which led to the identification of a single PI5P4Kγ amino acid residue that can be mutated to its PI5P4Ks α and β homologue to render PI5P4Kγ resistant NIH-12848 inhibition. NIH-12848 (10 μM) was applied to cultured mouse principal kidney cortical collecting duct (mpkCCD) cells which, we show, express PI5P4Kγ that increases when the cells grow to confluence and polarize. NIH-12848 inhibited the translocation of Na⁺/K⁺-ATPase to the plasma membrane that occurs when mpkCCD cells grow to confluence and also prevented reversibly their forming of 'domes' on the culture dish. Both these NIH-12848-induced effects were mimicked by specific RNAi knockdown of PI5P4Kγ, but not that of PI5P4Ks α or β. Overall, the data reveal a probable contribution of PI5P4Kγ to the development and maintenance of epithelial cell functional polarity and show that NIH-12848 is a potentially powerful tool for exploring the cell physiology of PI5P4Ks.

Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The family of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) is emerging from a comparative backwater in inositide signalling into the mainstream, as is their substrate, phosphatidylinositol 5-phosphate (PI5P). Here we review some of the key questions about the PI5P4Ks, their localisation, interaction, and regulation and also we summarise our current understanding of how PI5P is synthesised and what its cellular functions might be. Finally, some of the evidence for the involvement of PI5P4Ks in pathology is discussed., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammals have genes coding for three PI5P4Ks (PtdIns5P 4-kinases), and these have different cellular localizations, tissue distributions and lipid kinase activities. We describe in the present paper a detailed molecular exploration of human PI5P4Ks α, β and γ, as well as their fly and worm homologues, to understand how and why these differences came to be. The intrinsic ATPase activities of the three isoforms are very similar, and we show that differences in their G-loop regions can account for much of their wide differences in lipid kinase activity. We have also undertaken an extensive in silico evolutionary study of the PI5P4K family, and show experimentally that the single PI5P4K homologues from Caenorhabditis elegans and Drosophila melanogaster are as widely different in activity as the most divergent mammalian isoforms. Finally we show that the close association of PI5P4Ks α and γ is a true heterodimerization, and not a higher oligomer association of homodimers. We reveal that structural modelling is consistent with this and with the apparently random heterodimerization that we had earlier observed between PI5P4Kα and PI5P4Kβ [Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke (2010), Biochem. J. 430, 215-221]. Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.

The effects of aligning fields on models of polymer liquid crystals were simulated using the dissipative particle dynamics method. Exposing a liquid crystal of rod-like particles to a directional field causes a stabilization of the phases with orientational order, shifts the isotropic-nematic and nematic-smectic-A phase transitions to higher temperatures, makes the transitions continuous beyond a critical field strength, and induces weak para-nematic alignment in the zero-field isotropic phase. The interplay of liquid-crystalline ordering, microphase separation, and an alignment field endows the diblock and triblock copolymers studied here with rich phase behavior. The simulations suggest that field-induced orientational ordering can give rise to positional ordering. Reversely, positional ordering resulting from rod-coil demixing may be accompanied by orientational ordering, which is enhanced by external fields. For highly asymmetric rod-coil copolymers, the microphase separation pattern formed by the rigid segments can be altered by an aligning field.

During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.

PtdIns5P 4-kinases IIalpha and IIbeta are cytosolic and nuclear respectively when transfected into cells, including DT40 cells [Richardson, Wang, Clarke, Patel and Irvine (2007) Cell. Signalling 19, 1309-1314]. In the present study we have genomically tagged both type II PtdIns5P 4-kinase isoforms in DT40 cells. Immunoprecipitation of either isoform from tagged cells, followed by MS, revealed that they are associated directly with each other, probably by heterodimerization. We quantified the cellular levels of the type II PtdIns5P 4-kinase mRNAs by real-time quantitative PCR and the absolute amount of each isoform in immunoprecipitates by MS using selective reaction monitoring with 14N,13C-labelled internal standard peptides. The results suggest that the dimerization is complete and random, governed solely by the relative concentrations of the two isoforms. Whereas PtdIns5P 4-kinase IIbeta is >95% nuclear, as expected, the distribution of PtdIns4P 4-kinase IIalpha is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIalpha was 2000-fold more active as a PtdIns5P 4-kinase than the IIbeta isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIbeta may be to target the more active IIalpha isoform into the nucleus.

The role of cellular phosphatidylinositol 5-phosphate (PtdIns5P), as a signalling molecule or as a substrate for the production of small, compartmentalized pools of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], may be dependent on cell type and subcellular localization. PtdIns5P levels are primarily regulated by the PtdIns5P 4-kinases (type II PIP kinases or PIP4Ks), and we have investigated the expression and localization in the brain of the least-studied PIP4K isoform, PIP4Kgamma. In situ hybridization and immunohistochemistry, using antisense oligonucleotide probes and a PIP4Kgamma-specific antibody, revealed that this isoform has a restricted CNS expression profile. The use of antibodies to different cell markers showed that this expression is limited to neurons, particularly the cerebellar Purkinje cells, pyramidal cells of the hippocampus, large neuronal cell types in the cerebral cortex including pyramidal cells, and mitral cells in the olfactory bulb and is not expressed in cerebellar, hippocampal formation, or olfactory bulb granule cells. In neurons expressing this enzyme, PIP4Kgamma has a vesicular distribution and shows partial colocalization with markers of cellular compartments of the endomembrane trafficking pathway. The PIP4Kgamma isoform expression is established after day 7 of postnatal development. Overall, our data suggest that PIP4Kgamma may have a role in neuron function, specifically in the regulation of vesicular transport, in specific regions of the developed brain.

The groundwater/surface water interface (GWSWI) represents an important transition zone between groundwater and surface water environments that potentially changes the nature and flux of contaminants exchanged between the two systems. Identifying dominant and rate-limiting contaminant transformation processes is critically important for estimating contaminant fluxes and compositional changes across the GWSWI. A new, user-friendly, spreadsheet- and Visual Basic-based analytical screening tool that assists in evaluating the dominance of controlling kinetic processes across the GWSWI is presented. Based on contaminant properties, first-order processes that may play a significant role in solute transport/transformation are evaluated in terms of a ratio of process importance (P(i)) that relates the process rate to the rate of fluid transfer. Besides possessing several useful compilations of contaminant and process data, the screening tool also includes 1-D analytical models that assist users in evaluating contaminant transport across the GWSWI. The tool currently applies to 29 organics and 10 inorganics of interest within the context of the GWSWI. Application of the new screening tool is demonstrated through an evaluation of natural attenuation at a site with trichloroethylene and 1,1,2,2-tetrachloroethane contaminated groundwater discharging into wetlands.

Covenant language resides at the heart of all great spiritual journeys. The covenant is rigid and flexible, secure and dangerous, constant and ever changing. On the surface a covenant can be understood as an agreement between the client and the therapist. Beneath the surface lies the dynamic nature of the covenant, a nature based on the emotional quality a client brings, couple with the therapist capacity to support emotional integrity in the process of covenant development. In its simplicity the covenant becomes the guiding force that leads the client and therapist into the complex world of the unconscious. This article seeks to access the theological and psychological ramifications of integrating covenant theory into the psychotherapeutic process. Herein we encounter a dynamic that animates change in the client while at the same time making significant demands upon the therapist.

Allogeneic blood transfusion has an immunomodulatory capacity on its recipients through accumulation of immunologically active substances with blood storage, and prestorage leukoreduction reduces many of these mediators. We investigated lipopolysaccharide (LPS)-induced cytokine response of peripheral blood mononuclear cells (PBMCs) exposed to packed red blood cell (PRBC) supernatants from leukoreduced (LR) or non-leukoreduced (NLR) units with variable duration of storage. PRBC units were collected with or without leukoreduction on Day 0 before routine storage. The plasma fraction (supernatant) was isolated from LR and NLR units after 1 day (D1) or 42 days (D42) of storage and exposed to PBMCs versus control media for 24 h, then with LPS for an additional 24 h. Cell supernatants were analyzed for IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha by cytokine bead array. IL-1beta, TNF-alpha, and IL-6 were significantly elevated in PRBC groups versus control. D42 NLR PRBC supernatant significantly increased secretion of IL-1beta and IL-6 compared to D1 NLR PRBC supernatant. LR significantly attenuated the cytokine response of IL-1beta. Thus, PRBC supernatant potentiates proinflammatory LPS-induced cytokine secretion from PBMCs. This response is accentuated with storage duration and partially attenuated with leukoreduction. These findings may partially explain the immune activation seen clinically after blood transfusion.

Microphase separation and liquid-crystalline ordering in diblock and triblock rod-coil copolymers (with rod-to-coil fraction f=0.5) were investigated using the dissipative particle dynamics method. When the isotropic disordered phases of these systems were cooled down below their order-disorder transition temperatures T(ODT), lamellar structures were observed. For rod-coil diblock copolymers, the lamellar layers were obtained below T=2.0. This temperature was found to be higher than the T(ODT) for normal coil-coil diblock copolymers. Significant ordering of the rods was observed only below T=0.9 which is the isotropic-nematic transition temperature for rodlike fluids. For the triblock rod-coil copolymers, both microphase separation and rod ordering occurred at T=0.9. Normal coil-coil triblock copolymers were found to undergo microphase separation at T=0.8, which is about half the T(ODT) of the normal diblock copolymers. Investigations of the mean square displacement and the parallel and the perpendicular components of the spatial distribution function revealed that at low temperatures, the rod-coil diblock copolymers exhibit smectic-A and crystalline phases, while the triblock copolymers show smectic-C and crystalline phases. No nematic phases were observed at the density and interaction parameters used in this study.

PIP4Ks (type II phosphatidylinositol 4-phosphate kinases) are phosphatidylinositol 5-phosphate (PtdIns5P) 4-kinases, believed primarily to regulate cellular PtdIns5P levels. In this study, we investigated the expression, localization, and associated biological activity of the least-studied PIP4K isoform, PIP4Kgamma. Quantitative RT-PCR and in situ hybridization revealed that compared with PIP4Kalpha and PIP4Kbeta, PIP4Kgamma is expressed at exceptionally high levels in the kidney, especially the cortex and outer medulla. A specific antibody was raised to PIP4Kgamma, and immunohistochemistry with this and with antibodies to specific kidney cell markers showed a restricted expression, primarily distributed in epithelial cells in the thick ascending limb and in the intercalated cells of the collecting duct. In these cells, PIP4Kgamma had a vesicular appearance, and transfection of kidney cell lines revealed a partial Golgi localization (primarily the matrix of the cis-Golgi) with an additional presence in an unidentified vesicular compartment. In contrast to PIP4Kalpha, bacterially expressed recombinant PIP4Kgamma was completely inactive but did have the ability to associate with active PIP4Kalpha in vitro. Overall our data suggest that PIP4Kgamma may have a function in the regulation of vesicular transport in specialized kidney epithelial cells.

This article is an exploration of the psychological and theological themes of freedom and meaning within the world of those who experience emotional pain. The article focuses on how freedom and meaning can contribute to the development of a pastorally rigorous diagnostic protocol. It is in the examination of emotional freedom and the search for meaning that practitioners of the pastoral arts come to an understanding of the nature of suffering. The article offers an overview of the state of pastoral diagnosis highlighting and drawing from those resources that emphasize the need for pastoral psychology to construct a diagnostic protocol that is both theological and resonate with the lived experience of clients.

For many crops, a narrowing genetic base is becoming an increasingly significant problem for improvements made through breeding. Commonly used breeding procedures systematically reduce genetic diversity within elite gene pools. Here we describe a new technique for activation of genes in lines carrying Spm or dSpm transposon insertions. Activation of genes in Arabidopsis harbouring Spm or dSpm insertions in their promoters can be induced by over-expression of the TnpA-VP16 fusion protein, which binds Spm ends and activates local transcription. As a result, a variety of phenotypes are recovered from multiple-copy Spm lines in Arabidopsis. Application of this technique to a number of Spm insertion collections in Arabidopsis provides a valuable approach for new insights into plant gene functions. It also provides a proof-of-principle demonstration that the method could be used to generate new variation in elite lines of maize.

Step-down inhibitory avoidance (IA) memory formation involves association of stepping-down from a platform present in a training box (conditioned stimulus) with a footshock (unconditioned stimulus). A single short training session is enough to induce a lasting and strong memory trace expressed as an increase in step-down latency. Repeated nonreinforced retrieval, however, induces extinction of the IA response, a process involving a new learning that overrules the original one to indicate that the conditioned stimulus no longer predicts the unconditioned stimulus. Although the molecular requirements of IA memory consolidation are well understood, comparatively less is known about the signaling pathways involved in its extinction. Here we report that, when given into dorsal CA1 immediately but not 180 min after daily nonreinforced retrieval sessions, SP60015, a specific inhibitor of the mitogen-activated protein kinase, c-Jun N-terminal kinase, impaired IA memory extinction in a dose-dependent manner without producing any motor or perceptual impairment or damaging the hippocampal formation. Our results suggest that, as happens during consolidation, extinction of IA long-term memory also requires c-Jun N-terminal kinase activity in the CA1 region of the dorsal hippocampus.

Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type IIbeta PtdIns5P 4-kinase (PIPkin IIbeta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-591 2000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin IIbeta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIbeta is nuclear, we have used the high homologous recombination frequency of DT40 cells to knock an epitope tag (Mosedale et al., Nat Struct Biol. 12, 763-771 2005) into one of the alleles of the DT40 PIPkin IIbeta gene. We show that PIPkin IIbeta is expressed as a tagged protein, is active as revealed by immunoprecipitation and enzyme assay, and that cellular fractionation reveals that it is indeed nuclear. Genomic tagging of endogenous proteins in DT40 cells is a technique that offers unique advantages in studying endogenous signalling proteins.

The regulation of the synthesis of PtdIns(4,5)P2 is emerging as being as complex as we might expect from the multi-functional nature of this lipid. In the present chapter we focus on one aspect of inositide metabolism, which is the functions of the Type II PIPkins (Type II PtdInsP kinases). These are primarily PtdIns5P 4-kinases, although in vitro they will also phosphorylate PtdIns3P to PtdIns(3,4)P2. Thus they have three, not necessarily exclusive, functions: to make PtdIns(4,5)P2 by a quantitatively minor route, to remove PtdIns5P and to make PtdIns(3,4)P2 by a route that does not involve a Class I PtdIns 3-kinase. None of these three possible functions has yet been unambiguously proven or ruled out. Of the three isoforms, alpha and beta are widely expressed, the IIalpha being predominantly cytosolic and the IIbeta primarily nuclear. PIPkin IIgamma has a much more restricted tissue expression pattern, and appears to be localized primarily to intracellular vesicles. Here we introduce in turn each of the three Type II PIPkins, and discuss what we know about their localization, their regulation and their function.

Mesh material affects complications following hernia repair. Medical device reports on the use of surgical mesh for hernia repair were reviewed from the Food and Drug Administration's (FDA) Manufacturer User Facility Device Experience Database from January 1996 to September 2004. We analyzed 252 adverse event reports related to the use of surgical mesh for hernia repair. Adverse events included infection (42%, 107 reports), mechanical failure (18%, 46), pain (9%, 23), reaction (8%, 20), intestinal complications (7%, 18), adhesions (6%, 14), seroma (4%, 9), erosion (2%, 6), and other (4%, 9). Compared to all other mesh types, Sepra/polypropylene mesh had more mechanical failures (80 vs 14%, p < 0.05), biomaterial mesh had more reactions (57 vs 7%, p < 0.05), polytetrafluoroethylene (PTFE)/polypropylene mesh had more intestinal complications (14 vs 7%, p < 0.05), and PTFE mesh tended to have more infections (75 vs 41% all other, p = 0.07). Death occurred in 2% (5). We conclude that specific mesh materials are related to specific complications.

Background: Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells. Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. We hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling., Study Design: Melanoma conditioned media (MCM) was generated from B16 melanoma cells. Peritoneal macrophages from TLR-4 competent or TLR-4 incompetent mice were exposed to control or MCM for 24 hours; then stimulated with lipopolysaccharide. TNF-alpha secretion, TNF-alpha mRNA production, nuclear factor-kappaB (NF-kappaB) activation, and TLR-4 surface expression were measured., Results: Peritoneal macrophages exposed to MCM produced considerably less TNF-alpha in response to stimulus than controls (691 pg/mL versus 2,066 pg/mL, p < 0.001). TNF-alpha production by TLR-4 incompetent macrophages was not affected by MCM (454 pg/mL versus 480 pg/mL). Stimulated TNF-alpha mRNA and activated NF-kappaB were decreased in MCM treated C57BL/6 macrophages (by 38% and 33%, respectively). TLR-4 surface expression, however, was not decreased by exposure to MCM., Conclusions: Melanoma inhibits macrophage activation by suppressing TLR-4 signaling downstream of the TLR-4 receptor.

Phosphatidylinositol 5-phosphate (PtdIns5P) is a relatively recently discovered inositol lipid whose metabolism and functions are not yet clearly understood. We have transfected cells with a number of enzymes that are potentially implicated in the synthesis or metabolism of PtdIns5P, or subjected cells to a variety of stimuli, and then measured cellular PtdIns5P levels by a specific mass assay. Stable or transient overexpression of Type IIalpha PtdInsP kinase, or transient overexpression of Type Ialpha or IIbeta PtdInsP kinases caused no significant change in cellular PtdIns5P levels. Similarly, subjecting cells to oxidative stress or EGF stimulation had no significant effect on PtdIns5P, but stimulation of HeLa cells with a phosphoinositide-specific PLC-coupled agonist, histamine, caused a 40% decrease within 1 min. Our data question the degree to which inositide kinases regulate PtdIns5P levels in cells, and we discuss the possibility that a significant part of both the synthesis and removal of this lipid may be regulated by phosphatases and possibly phospholipases.

We have carried out first principles plane wave density-functional theory calculations to study the adsorption of CO molecule on a clean and unreconstructed Cu (110) surface at 1/12 monolayer coverage and have investigated the subsequent oxidation by preadsorbed oxygen atoms. As found experimentally, the CO adsorbs perpendicular to the surface plane through the carbon atom; the top site was found to be the most favorable position for CO adsorption although the short-bridge site is only slightly less stable. Surprisingly, for a sparely oxidized surface with O atoms adsorbed in hollow sites the coadsorption energy is slightly negative for only the above two CO sites which have therefore been used as starting points to explore the energy surface of the oxidation reaction. We have confirmed the existence of bent CO(2) surface intermediate as previously suggested from experimental studies. Using the nudged elastic band method, we have characterized a two step reaction which involves the formation of this intermediate. The results suggest that the rate determining step of the oxidation reaction is the formation of the intermediate and the energy barrier (200 meV) is close to although smaller than experimentally estimated values., ((c) 2004 American Institute of Physics)

Using mesoscale dissipative particle dynamics (DPD) simulations, which ignore all atomistic details, we show the formation of lamella mesophases by cooling a fully disordered system composed of symmetric (A7B7) rod-coil diblock copolymers. Equilibration is achieved very rapidly using DPD, and isotropic, smectic A and crystalline phases of the rod-like blocks can be observed either by heating or cooling. An interesting pseudo-smectic phase can be characterized when the order-disorder transition temperature is above the clearing temperature. This phase gradually fades into a normal microphase-separated structure as the system is heated through the clearing temperature. Simulations of pure rods, however, show the formation of isotropic, nematic, smectic A and crystalline phases., (Copyright 2004 The Royal Society)

The development of engineered containment and control systems for contaminated sites must consider the environmental setting of each site. The behaviors of both contaminated materials and engineered systems are affected by environmental conditions that will continue to evolve over time as a result of such natural processes as climate change, ecological succession, pedogenesis, and landform changes. Understanding these processes is crucial to designing, implementing, and maintaining effective systems for sustained health and environmental protection. Traditional engineered systems such as landfill liners and caps are designed to resist natural processes rather than working with them. These systems cannot be expected to provide long-term isolation without continued maintenance. In some cases, full-scale replacement and remediation may be required within 50 years, at an effort and cost much higher than for the original cleanup. Approaches are being developed to define smarter containment and control systems for stewardship sites, considering lessons learned from implementing prescriptive waste disposal regulations enacted since the 1970s. These approaches more effectively involve integrating natural and engineered systems; enhancing sensors and predictive tools for evaluating performance; and incorporating information on failure events, including precursors and consequences, into system design and maintenance. An important feature is using natural analogs to predict environmental conditions and system responses over the long term, to accommodate environmental change in the design process, and, as possible, to engineer containment systems that mimic favorable natural systems. The key emphasis is harmony with the environment, so systems will work with and rely on natural processes rather than resisting them. Implementing these new integrated systems will reduce current requirements for active management, which are resource-intensive and expensive.

Many physiological targets have been suggested for polyphosphoinositol lipids, but two out of the three monophosphorylated PIPs appeared to be no more than metabolic precursors. Recent work has shown that they also have distinct binding proteins and functions.

The DEFORMED ROOTS AND LEAVES1 (DRL1) gene is single copy in the Arabidopsis genome, and based on overall amino acid similarity and conservation of functional domains, the DRL1 protein is homologous with yeast TOT4/KTI12. TOT4/KTI12 associates with Elongator, a multisubunit complex that binds the RNA polymerase II transcription elongation complex. Recessive mutations at the DRL1 locus caused defective organ formation indicative of disorganized shoot, inflorescence, flower, and root meristems. DRL1 is a putative ATP/GTP binding protein; in addition, calmodulin binding activity was demonstrated in vitro for the C terminus of the DRL1 protein. Phenotypic and genetic data position DRL1 relative to regulatory loci for leaf development, in which it acts early. We identified Arabidopsis homologs for the six Elongator components and hypothesize that DRL1 regulates transcription elongation through a putative plant Elongator. Upregulation of the ANGUSTIFOLIA transcript in the strong drl1-2 allele supports this model.

Expressions are obtained for the pressure tensor in the canonical and the microcanonical ensemble for both isolated and periodic systems, using the same geometric approach to thermodynamic derivatives as has been used previously to define the configurational temperature. The inherent freedom of the method leads to a straightforward proof of the equivalence of atomic and molecular pressures, for short molecules and for molecules exceeding the dimensions of a periodic simulation box. The effect of holonomic constraints on the pressure is discussed. Expressions for the elastic constants are derived in the same manner.

Previous data suggest the existence of discrete pools of inositol lipids, which are components of a nuclear phosphoinositide (PI) cycle. However, it is not known whether the contents of these pools are regulated during cell proliferation. In the present study we demonstrate that the mass levels of three important constituents of the nuclear PI cycle are regulated during the cell cycle. Radioactive label incorporation into PtdIns(4,5)P(2) was seen to increase dramatically as synchronized cells entered S-phase. This did not coincide with any significant changes in the nuclear mass levels of this lipid, suggesting that the rate of turnover of this molecule was increased. Levels of PtdIns4P, the major substrate for PtdIns(4,5)P(2) production by Type I PtdInsP kinases (PIPkins), were regulated during the cell cycle and indicated a complex relationship between these two lipids. An alternative substrate for PtdIns(4,5)P(2), PtdIns5P, phosphorylated by Type II PIPkins, was present in nuclei at much smaller amounts than the PtdIns4P, and thus is unlikely to contribute significantly to PtdIns(4,5)P(2) turnover. However, a large increase in nuclear PtdIns5P mass was observed when murine erythroleukaemia cells are in G(1), and this could represent a potential pool of nuclear inositol lipid that has a specific signalling role. Analysis of extracted lipid fractions indicated the absence of any PtdIns3P in these nuclei.

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA. gave the most effective kappa number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

It is now clear that phosphoinositides, which play a major role in the regulation of a variety of cellular processes in the cytoplasm, are found within the nucleus. Their role in this subcellular compartment is still contentious: however, data has suggested that nuclear inositides generate substrates, such as PtdIns(4,5)P2, utilised by a number of nuclear signalling pathways: for example, nuclear phospholipase C and the PtdIns 3-kinase cascade. There is also evidence that PtdIns(4,5)P2 may play a role in the localisation and regulation of a number of nuclear proteins such as the BAF complex, which is involved in the regulation of chromatin structure. Although the presence of nuclear inositides has been demonstrated in a number of different cell types, suggesting that it is ubiquitous, there are many inconsistencies within the literature concerning the locations and isotypes of enzymes that are involved in their regulation and in the potential second messengers which are generated by them. This review aims to highlight some of these inconsistencies in order to focus on areas that need further characterisation.