Electron holography on HfO2/HfO2-x bilayer structures with multilevel resistive switching properties.

Unveiling the physical nature of the oxygen-deficient conductive filaments (CFs) that are responsible for the resistive switching of the HfO2-based resistive random access memory (RRAM) devices represents a challenging task due to the oxygen vacancy related defect nature and nanometer size of the CFs. As a first important step to this goal, we demonstrate in this work direct visualization and a study of physico-chemical properties of oxygen-deficient amorphous HfO2-x by carrying out transmission electron microscopy electron holography as well as energy dispersive x-ray spectroscopy on HfO2/HfO2-x bilayer heterostructures, which are realized by reactive molecular beam epitaxy. Furthermore, compared to single layer devices, Pt/HfO2/HfO2-x /TiN bilayer devices show enhanced resistive switching characteristics with multilevel behavior, indicating their potential as electronic synapses in future neuromorphic computing applications.

Justice and impoverished women: the ethical implications of work-based welfare.

Current U.S. welfare policy, Temporary Assistance for Needy Families, requires impoverished people to work in order to receive welfare, and it limits cash support to 5 years. Most of the people who have used this program are single-parent women, and a disturbing number have been terminated at 5 years, not having made a successful transition to work. The purpose of this longitudinal study was to explore the barriers to success and the social justice of the program from the perspective of single-parent women who were terminated. In all, 41 women were recruited through community-based purposive sampling, and the primary research methods were a qualitative, narrative interview approach and narrative analysis. Data from the semistructured interview guide are reported here. Findings describe health and socioeconomic burdens, and barriers that lie within the social policy. The study has ethical implications for nursing advocacy, and it informs nursing interventions for impoverished women and their families.

Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1 by high content imaging.

Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC50: ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.

Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence.

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.

Substitution of Yor1p NBD1 residues improves the thermal stability of Human Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508. To investigate substitutions that can stabilize this domain, we constructed chimeras between human CFTR and its closest yeast homolog Yor1p. The α-subdomain of Yor1p was replaced with that of CFTR in Saccharomyces cerevisiae. Cellular localization of green fluorescence protein-tagged Yor1p-CFTR chimeras was analyzed by fluorescence microscopy and quantitative multispectral imaging flow cytometry, steady-state protein levels were compared by SDS-PAGE and protein function probed by a phenotypic oligomycin resistance assay. The chimeras exhibited ER retention in yeast characteristic of defective protein folding/processing. Substitution of seven CFTR α-subdomain residues that are highly conserved in Yor1p and other transporters but differ in CFTR (S495P/R516K/F533L/A534P/K536G/I539T/R553K) improved Yor1p-CFTR chimera localization to the yeast plasma membrane. When introduced into human CFTR expressed in mammalian cells, the same substitutions improve the purified protein thermal stability. This stabilized human CFTR protein will be directly useful for structural and biophysical studies that have been limited by the thermal sensitivity of wild type CFTR. The insights into critical structural residues within CFTR could facilitate development of effective therapeutics for CF-causing mutations.

FIB based fabrication of an operative Pt/HfO2/TiN device for resistive switching inside a transmission electron microscope.

Recent advances in microelectromechanical systems (MEMS) based chips for in situ transmission electron microscopy are opening exciting new avenues in nanoscale research. The capability to perform current-voltage measurements while simultaneously analyzing the corresponding structural, chemical or even electronic structure changes during device operation would be a major breakthrough in the field of nanoelectronics. In this work we demonstrate for the first time how to electrically contact and operate a lamella cut from a resistive random access memory (RRAM) device based on a Pt/HfO2/TiN metal-insulator-metal (MIM) structure. The device was fabricated using a focused ion beam (FIB) instrument and an in situ lift-out system. The electrical switching characteristics of the electron-transparent lamella were comparable to a conventional reference device. The lamella structure was initially found to be in a low resistance state and could be reset progressively to higher resistance states by increasing the positive bias applied to the Pt anode. This could be followed up with unipolar set/reset operations where the current compliance during set was limited to 400 µA. FIB structures allowing to operate and at the same time characterize electronic devices will be an important tool to improve RRAM device performance based on a microstructural understanding of the switching mechanism.

Mitotic motors in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.

Measurement of branched-chain alpha-keto acid dehydrogenase flux rates in perfused heart and liver.

The methods described here represent a flexible set of procedures for investigating the metabolism of the branched-chain alpha-keto acids and other substances in perfused organs, notably the rat heart and liver. These procedures have been used to investigate many aspects of the metabolism of the branched-chain alpha-keto acids not discussed here, such as the effects on branched-chain alpha-keto acid metabolism by exposure to alpha-adrenergic agents, by inhibition of the monocarboxylate translocator, and by the coinfusion of other metabolites.

A model for community involvement in health (CIH) program development.

Community involvement in health (CIH), a central concept in health development, is a participatory approach to health care that is organized from the perspective of the recipient. Putting CIH into practice represents a learning experience for the community, the health professionals involved and those responsible for the national climate in which this change takes place. The CIH process was operationalized over a two-year period in a black township in South Africa. A community survey identified the health needs and capacities related to the elderly, their families and their support system. Community groups and individuals, in partnership with the researcher, prioritized the needs that had been identified and then implemented four programs related to those needs. A process model was developed that provided the structure for initiating and maintaining these programs. The model helped people who were new to the community organizing to focus on general principles. It was flexible so that programs could be interpreted and implemented in the context of local culture and resources. The model was functional in guiding community nurses, lay community members and employees in health-related programs through the process of starting new programs. This approach empowered participants to move beyond only hoping for change or being puzzled by its elusiveness.

The effects of salicylate on enzymes of vitamin K metabolism.

The mechanism of salicylate-induced hypoprothrombinaemia has been investigated in the rat. Salicylate administration produced an increase in the percentage of the total liver vitamin that was present as vitamin K 2,3-epoxide, but the addition of salicylate did not influence vitamin K epoxide reductase activity in-vitro. Neither did it influence vitamin K-dependent carboxylase or vitamin K epoxidase activity. Both cytosolic and microsomal DT-diaphorase activities were, however, inhibited about 50% by 75 microM sodium salicylate. Salicylate inhibition was also observed when vitamin K quinone and NADH or dithiothreitol were used to support carboxylation. To achieve 50% inhibition required 0.5 mM salicylate with NADH as a reductant and 4 mM salicylate when dithiothreitol was the reductant. These results suggest that the main effect of salicylate on vitamin K metabolism is to inhibit quinone reductases and may be useful in explaining the inhibition of the biosynthesis of vitamin K-dependent clotting factors that occurs in salicylate-induced hypothrombinaemia. These data also demonstrate that the percentage of total liver vitamin present as vitamin K epoxide can be increased by agents that do not have a direct effect on the vitamin K epoxide reductase in-vitro.

Indirect inhibition of vitamin K epoxide reduction by salicylate.

Salicylate antagonizes the vitamin K-dependent biosynthesis of clotting factors in the rat and produces an elevation of the ratio of vitamin K epoxide to vitamin K in the liver. Vitamin K epoxide is reduced to vitamin K by a vitamin K epoxide reductase, and 1 mM salicylate was required to cause a 50% inhibition of the dithiothreitol-dependent in-vitro reduction of vitamin K epoxide by this enzyme. This enzyme was, however, inhibited 50% by as little as 70-80 microM salicylate when reducing equivalents for the reaction were furnished by endogenous cytosolic reductants. This effect on the cytosolic reductant supply was shown to be unrelated to a previously demonstrated inhibition of DT-diaphorase by salicylate. The concentrations of salicylate at which significant inhibitory effects are exerted in-vitro (50-100 microM) are below the 200 microM levels observed in the livers of rats given an anticoagulating dose of salicylate.

The training and use of research assistants for a survey in a Third World setting.

This article describes an approach to gathering data in a black township. It emphasizes the importance of using indigenous interviewers and offers suggestions for their training. Innovative techniques were used to help the field workers to understand and apply the concept of randomization to the streets and houses of their Township. It emphasizes the need to supervise and reinforce the research standards throughout the data collection process.

Prevention of atherosclerosis and end-organ damage: a basis for antihypertensive interventional strategies.

AIM: To review the nature of the complex relationships between essential hypertension and cardiovascular end-organ damage, with a particular focus on the pathogenesis and prevention of coronary heart disease, the major complication of untreated hypertension. RISK FACTORS FOR CORONARY HEART DISEASE: Both atherosclerosis and hypertension have their origins in childhood; in the second and third decades of life development of the more advanced fibrous plaques accelerates, emphasizing the need for early diagnosis and intervention. Perplexing and complex relationships have been found among the principal risk factors for coronary heart disease, hyperinsulinemia, insulin resistance, dyslipidemia and hypertension. In the pathogenesis of atherosclerosis at the cellular and molecular level, the important features are the effects of monocyte-macrophages, oxidant stress, lipoprotein modification, inflammatory mediators and the focal hemodynamic environment. Even brief periods of experimental hypertension can accentuate atherogenesis, the effects of which are greatest but not limited to the cervical and cerebral arteries. Further, acute hypertension lasting for even a few minutes causes a 'leakage' of plasma proteins and particulate probes into the artery wall, which has far-reaching implications for antihypertensive therapy. Recent work has shown that 24-h blood pressure variability is correlated with target-organ damage in hypertensive patients. THERAPY: Antihypertensive therapy should not only lower blood pressure but also prevent significant short-term blood pressure fluctuations. The trough: peak ratio has been used to assess the effect of antihypertensive treatment on blood pressure variability. CONCLUSION: More intensive research is required to clarify the nature of the interface between hypertension and atherogenesis.

Preceptors: a perspective of what works.

This article provides a perspective of clinical preceptor contributions to advanced practice nurse education. It supports quality clinical education in the face of the proliferation of academic programs, and the economics of the resulting competition. The strategies of 2 universities are used as examples of how nurse practitioner programs respond to issues such as who precepts students, how preceptors are identified, what draws preceptors to the role, and the effects of students on clinic productivity.

Building community participation in health care: a model and example from South Africa.

A conceptual model was developed for a community-based intervention study in a Black township in South Africa. The model shows a useful way to structure the complex role public health nurses play as they meet community health needs using a community's priorities and building toward community involvement in health and self-care. The model was applied over a 2-year period in an under-developed community of 100,00 people where the unemployment rate was over 50%, fewer than 10% of the homes had electricity, and only one-third had access to the sewage removal system. Over half of older adults interviewed were illiterate. The residents, in collaboration with the nurse researcher, gathered data, prioritized needs, and chose projects to produce solutions. The model guided activities for community empowerment through a deliberate transfer of information and expertise from the nurse to members of the community. Conceptual models or paradigms are useful to focus nursing strategies, to guide professional nursing practice, and to support interdisciplinary goals for cooperative efforts. The principles are also applicable in the United States and other developed countries as more effective ways to achieve health goals are sought.

Focus groups and vulnerable populations. Insight into client strengths and needs in complex community health care environments.

Focus groups are a useful qualitative research technique to assist in interpreting quantitative community assessment data. Data obtained from focus groups can provide sociological and psychological insights into the perceptions of population subgroups and suggest answers to the "why" questions raised by descriptive data about such issues as teen pregnancy, poverty, immunization levels, or lifestyle-related morbidity and mortality. Application of these insights can lead to the better use of community strengths and the creation of community-specific responses to barriers to health care. Focus groups work well for involving hard-to-reach members of a community in program development, planning, and evaluation. They may be more effective than face-to-face interviews and questionnaires because people often have not thought about how they feel and tend not to form opinions in isolation (1). The information sought through the use of focus groups is not randomly distributed in the population. Thus, groups are not randomly selected, and data are not gathered with the intent to generalize to all populations.

Survey data collection: operationalizing the research design.

This article describes how indigenous interviewers were used to collect data about the health needs and resources in a black South African township. The survey was done during the dismantling of the apartheid political system of South Africa. The political unrest, distrust, and tension were barriers to carrying out a survey and threatened the quality of the data collection. A vulnerability of survey research is the difficulty in controlling the variables of the community and the interviewer during the process of data collection. How this survey was carried out, in this unstable setting, influenced the quality of the data and the validity and reliability of the research. The data-collection requirements were carried out in a way that was functional in the real-world setting while maintaining research standards. The criteria used for hiring interviewers and the content and delivery of training were effective in this tense, educationally disadvantaged community setting. Methods that were used to motivate and supervise interviewers were successful and are recommended for use in similar survey research.

Phosphoamino acid analysis of protein immobilized on polyvinylidene difluoride membrane.

The direct analysis of phosphorylated proteins bound to polyvinylidene difluoride membrane (PVDFm) has been examined. Use of 14C-methylated marker proteins demonstrated that proteins electroblotted on PVDFm were quantitatively retained through a series of test conditions, which included 1 M hydroxylamine (25 degrees C, 30 min), 0.1 M NaOH (37 degrees C, 30 min), 0.1 M HCl (55 degrees C, 2 h), and 6 U/ml alkaline phosphatase (pH 9.5, 37 degrees C, 24 h). Approximately half the protein remained bound following 2-h treatment in 1 M KOH (55 degrees C). The same series of test conditions were employed to assess the stability of phosphorylated residues in 32P-labeled protein immobilized on PVDFm, in order to assign them as carboxyl-,N-, or O-linked groups. The properties of phosphorylated proteins as determined by this method were comparable to the properties that have been reported for soluble proteins. Use of the PVDFm immobilization step affords simplification of the experimental procedures and permits rapid, quantitative sample recovery using submicrogram quantities of protein. Further, the PVDFm-bound phosphoproteins could be subjected to partial acid hydrolysis directly on the membrane and required no further purification for subsequent identification of the labeled phosphohydroxyamino acids. Definitive identification of labeled phosphoserine residues in histone, phosphoserine and phosphothreonine residues in myelin basic protein and insulin receptor, and phosphotyrosine residues in autophosphorylated insulin receptor was accomplished with as little as 0.2 nCi in about 50 ng of phosphorylated protein.