Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes.

We present evidence that the microtubule-associated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein. Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

Electrostatic force microscope for probing surface charges in aqueous solutions.

A scanning force microscope was converted to an electrostatic force microscope by charging the usually neutral cantilever with phospholipids. The electrostatic force microscope was used to study surface electrostatic charges of samples in aqueous solutions. Lysozymes, DEAE-Sephadex beads, 3-propyltriethoxysilane-treated glass and mica were imaged in water or phosphate buffer with electrostatic force microscopy. The adhesion force measured when a charged probe and oppositely charged specimen interacted was up to 500 times greater than when a bare probe was used. This dramatic increase in measured adhesion force can be attributed to the energy required to break the salt bridges formed between the charged probe and the specimen. The use of phospholipids to functionalize the cantilever tip allows the incorporation of other biomolecules and ligands that can be used as biologically specific tips (e.g., receptors, drugs) for the study of intermolecular interactions.

Identification of auxin-responsive elements of parB and their expression in apices of shoot and root.

Detailed analysis of transgenic tobaccos containing a series of chimeric parB promoter/beta-glucuronidase (GUS) gene constructs allowed us to define two auxin-responsive elements (AREs) of 48 bp and 95 bp (positions -210 to -163 and -374 to -280) in the parB promoter. The two AREs responded independently to physiological concentrations of auxin. Gel retardation assays revealed binding of nuclear protein(s) to the sequence conserved between ARE I and ARE II. The auxin responsiveness of the parB promoter did not mediate the pathway through the as-1 element and transcription factor ASF-1. AREs I and II were responsive to auxin at physiological concentrations, whereas as-1 responded only to higher concentrations of auxin which may be interpreted as stress, though as-1 had been reported to be a minimal ARE [Liu, X. & Lam, E. (1994) J. Biol. Chem. 269, 668-675]. Histochemical staining of transgenic tobacco that contained a parB promoter/GUS construct demonstrated the expression of GUS activity in the shoot apex as well as in the root tips, suggesting the involvement of parB expression in meristematic activity or differentiation. The drastic change in auxin responsiveness in the transgenic plants between the 6th and 10th day after imbibition of seeds implies the development or the activation of auxin signal transduction systems during plant development.

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.

In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.

A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.

Ensaio clínico randomizado do uso do curativo gel de clorexidina para a prevenção da colonização do cateter venoso central em pacientes adultos críticos

Os acessos venosos são indispensáveis para assistência do paciente em situação crítica. O cateter venoso central (CVC) é um acesso que viabiliza a terapêutica dessa clientela, mas o seu uso pode levar à infecções. Estas infecções ocasionam maior permanência hospitalar, elevam os custos totais das instituições e aumentam a morbidade e a mortalidade do paciente. O uso de curativos como cobertura do sítio de saída do CVC é eficaz na prevenção das infecções relacionadas a estes cateteres, em particular, o uso de curativos impregnados com antissépticos como o curativo gel de clorexidina. Este estudo teve como objetivo comparar a efetividade do curativo gel de clorexidina com a do filme transparente de poliuretano na prevenção da colonização do cateter venoso central em pacientes adultos críticos. Trata-se de estudo experimental, do tipo ensaio clínico randomizado, com tratamentos em paralelo, prospectivo e monocêntrico, realizado de acordo com as recomendações do Consolidated Standards of Reporting Trials (CONSORT). O estudo foi realizado na Unidade de Terapia Intensiva e na Unidade Coronariana de um hospital de ensino do interior do Estado de São Paulo. Participaram do estudo 102 indivíduos hospitalizados nestes locais, divididos aleatoriamente em dois grupos: grupo intervenção, no qual o tipo de cobertura utilizada foi o curativo de gel de clorexidina e grupo controle, que utilizou como cobertura o filme transparente de poliuretano. O desfecho primário mensurado foi a colonização do cateter e os desfechos secundários foram a infecção clínica do sítio de saída, a infecção microbiológica do sítio de saída e a infecção da corrente sanguínea relacionada ao cateter. Para a coleta de dados foi elaborado um instrumento, e este validado quanto ao seu conteúdo e forma por 13 enfermeiros pertencentes aos locais do estudo. Estes profissionais foram treinados para a realização dos curativos e coleta das pontas dos cateteres centrais, swabs dos sítios de saída e hemoculturas. Análises descritivas foram usadas para todas as variáveis do estudo. O teste Exato de Fisher foi utilizado para comparar as proporções de cada desfecho nos grupos de intervenção e controle, e a regressão logística para explorar se a colonização no CVC poderia ser associada com o tempo de uso do cateter e com o Acute Physiology and Chronic Health Evaluation II (APACHE II) dos pacientes do estudo. De acordo com os resultados não houve diferença estatisticamente significante entre a colonização nos dois grupos (p valor = 1.00), para a infecção microbiológica do sítio de saída (p valor = 0.08), para a infecção clínica do sítio de saída (p valor = 0.77) e para as infecções da corrente sanguínea relacionadas ao cateter (p valor = 1,00). Conclui-se que o presente estudo pode contribuir para que as unidades de saúde tenham subsídios para realizar a escolha do tipo de curativo baseado em suas necessidades institucionais e no desenvolvimento de protocolos relacionados à medidas de inserção e manutenção do cateter, bem como medidas educativas permanentes

Influência do LPS e do zinco na interação mãe e filhote

O comportamento materno consiste em um conjunto de mudanças comportamentais e fisiológicas, exercidas pelos indivíduos adultos em torno dos indivíduos reprodutivamente imaturos, garantindo sua sobrevivência e a propagação de sua espécie. A interação mãe e filhote é tida tipicamente como simbiótica. Os filhotes quando separados da mãe sinalizam para serem recolhidos através de dicas olfativas, visuais e da vocalização que representa uma forma de comunicação filhote e mãe. O modelo de febre clássico e amplamente empregado envolve a utilização do lipopolissacarídeo (LPS), principal componente da parede celular de bactérias Gram-Negativas. Além da febre, as infecções apresentam uma cadeia de respostas não especificas do hospedeiro que se sabe estarem envolvidos em muitas das funções vitais, incluindo a resposta imune estas incluem a hipozinquemia. Sendo assim, fêmeas virgens, gestantes e lactantes receberam LPS (100 µg/kg, i.p.) e foram tratadas com zinco (2 mg/kg, s.c.) O peso corporal, consumo de água, ração, e a temperatura corporal foram medidas por noventa e seis horas, duas horas após a administração do LPS. No quinto dia de lactação foram observados o comportamento maternal, a atividade geral em campo aberto e a vocalização ultrassônica nos filhotes. No dia do desmame os filhotes dessas fêmeas receberam um desafio com LPS (50 µg/kg, i.p.) e duas horas após a administração, foram observados a atividade geral em campo aberto, e o burst e fagocitose de neutrófilos. Observamos que: 1) Em ratas virgens, gestantes e lactantes, a exposição ao LPS e o tratamento com zinco modificou de forma específica a temperatura e peso corporal, consumo de água e ração e a atividade geral observadas em campo aberto; 2) No período de lactação, houve redução da latência para busca do primeiro filhote. Na prole das fêmeas lactantes verificou-se que: 3) Houve alteração no padrão de vocalização dos filhotes; 4) houve alteração na atividade geral observada em campo aberto e no burst e fagocitose de neutrófilos no vigésimo primeiro dia pós natal, após um desafio com a endotoxina, Assim, os resultados indicam que a administração de LPS e o tratamento com zinco têm seus efeitos modulados conforme o estágio fisiológico em que a fêmea se encontra, e interfere com a interação mãe/filhote, resultando em efeitos de curto e longo prazo sobre o comportamento dos filhotes. A partir deste trabalho, a possibilidade da exposição de mães à endotoxina bacteriana e da modulação de seus efeitos pelo zinco programar as respostas inflamatórias dos filhos torna-se factível

How I Learned to Stop Worrying and Love the Bits: Archival Motivation in the Digital Age

Why do people become archivists? Historically (and anecdotally) it was a deep love of musty, old records that drew people to the profession. While there have been many other motivating forces that inspired would-be archivists, it is most often that one hears of people seeking jobs in archives for love of “the stuff,” as evidenced in Kate Thiemer’s blog post, Honest tips for wannabe archivists (2012). As a result of the continually advancing presence of digitized and born digital archival collections, the physical nature of archival “stuff” is changing. While there remains the physical imprint of digital information on floppy disks, CDs, DVDs, hard drives, and old computers; the aspects of these physical artifacts might not evoke the same visceral pull to the profession as musty, raspy, paper-based documents. In light of this shift in physical presentation of information, we are faced with the question: how does love of archival “stuff” translate to work in digital archives? What is and/or will be the pull to become a digital archivist? To answer these questions, we will perform a survey-based study where we will invite archivists who work with both traditional and digital archival material to answer questions related to the aspects of their work that inspired or motivated them to join the profession. What motivates people to become archivists? What aspects of digital archives do or can potentially motivate people to seek out a career as an archivist? What, if any, motivational factors for becoming a traditional archivist are the same as those for becoming a digital archivist? What, if any, motivational factors for becoming a traditional archivist are different from those for becoming a digital archivist? By answering these questions, we hope to expand the archival discussion on what it means to be an archivist in the digital age. What compelling intrinsic, evidential, or informational values are present in digital archival content that will draw professionals to the field? Are there other values inherent in digital content that are currently unexplored? In our poster, we will present our discussion of the topic, our survey design, and results we have at the time of the Institute. Thiemer, K. (2012). Honest tips for wannabe archivists. Archivesnext blog. Retrieved from http://www.archivesnext.com/?p=2849

How Does the Denver Public Library System Respond to its Customer's Requests for Global Information?

The interaction between globally available information and public library users is a changing one. Global information is readily available yet provider and user struggle to find efficiencies of time and resources. As a primary resource of global information the Denver Public Library (DPL) is approaching this challenge by providing changing technology to a changing user and by providing a customized approach to immigrant populations. DPL provides global information to library users through collections, programs and Internet. Internet and collections global information usage cannot be directly measured due to privacy restrictions. Only 12.5% of general user programs focus on global information. Four percent of budget serves the immigrant users. This is greater than national averages.

Time course modifications in organotypic culture of human neuroretina

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell® dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Mechanical annealing of metallic electrodes at the atomic scale

The process of creating an atomically defined and robust metallic tip is described and quantified using measurements of contact conductance between gold electrodes and numerical simulations. Our experiments show how the same conductance behavior can be obtained for hundreds of cycles of formation and rupture of the nanocontact by limiting the indentation depth between the two electrodes up to a conductance value of approximately 5G0 in the case of gold. This phenomenon is rationalized using molecular dynamics simulations together with density functional theory transport calculations which show how, after repeated indentations (mechanical annealing), the two metallic electrodes are shaped into tips of reproducible structure. These results provide a crucial insight into fundamental aspects relevant to nanotribology or scanning probe microscopies.

Spin dynamics of current-driven single magnetic adatoms and molecules

A scanning tunneling microscope can probe the inelastic spin excitations of a single magnetic atom in a surface via spin-flip assisted tunneling in which transport electrons exchange spin and energy with the atomic spin. If the inelastic transport time, defined as the average time elapsed between two inelastic spin flip events, is shorter than the atom spin-relaxation time, the scanning tunnel microscope (STM) current can drive the spin out of equilibrium. Here we model this process using rate equations and a model Hamiltonian that describes successfully spin-flip-assisted tunneling experiments, including a single Mn atom, a Mn dimer, and Fe Phthalocyanine molecules. When the STM current is not spin polarized, the nonequilibrium spin dynamics of the magnetic atom results in nonmonotonic dI/dV curves. In the case of spin-polarized STM current, the spin orientation of the magnetic atom can be controlled parallel or antiparallel to the magnetic moment of the tip. Thus, spin-polarized STM tips can be used both to probe and to control the magnetic moment of a single atom.

Understanding the structure of the first atomic contact in gold

We have studied experimentally jump-to-contact (JC) and jump-out-of-contact (JOC) phenomena in gold electrodes. JC can be observed at first contact when two metals approach each other, while JOC occurs in the last contact before breaking. When the indentation depth between the electrodes is limited to a certain value of conductance, a highly reproducible behaviour in the evolution of the conductance can be obtained for hundreds of cycles of formation and rupture. Molecular dynamics simulations of this process show how the two metallic electrodes are shaped into tips of a well-defined crystallographic structure formed through a mechanical annealing mechanism. We report a detailed analysis of the atomic configurations obtained before contact and rupture of these stable structures and obtained their conductance using first-principles quantum transport calculations. These results help us understand the values of conductance obtained experimentally in the JC and JOC phenomena and improve our understanding of atomic-sized contacts and the evolution of their structural characteristics.

Empirical study on the maintainability of Web applications: Model-driven Engineering vs Code-centric

Model-driven Engineering (MDE) approaches are often acknowledged to improve the maintainability of the resulting applications. However, there is a scarcity of empirical evidence that backs their claimed benefits and limitations with respect to code-centric approaches. The purpose of this paper is to compare the performance and satisfaction of junior software maintainers while executing maintainability tasks on Web applications with two different development approaches, one being OOH4RIA, a model-driven approach, and the other being a code-centric approach based on Visual Studio .NET and the Agile Unified Process. We have conducted a quasi-experiment with 27 graduated students from the University of Alicante. They were randomly divided into two groups, and each group was assigned to a different Web application on which they performed a set of maintainability tasks. The results show that maintaining Web applications with OOH4RIA clearly improves the performance of subjects. It also tips the satisfaction balance in favor of OOH4RIA, although not significantly. Model-driven development methods seem to improve both the developers’ objective performance and subjective opinions on ease of use of the method. This notwithstanding, further experimentation is needed to be able to generalize the results to different populations, methods, languages and tools, different domains and different application sizes.

Difference schemes for numerical solutions of lagging models of heat conduction

Non-Fourier models of heat conduction are increasingly being considered in the modeling of microscale heat transfer in engineering and biomedical heat transfer problems. The dual-phase-lagging model, incorporating time lags in the heat flux and the temperature gradient, and some of its particular cases and approximations, result in heat conduction modeling equations in the form of delayed or hyperbolic partial differential equations. In this work, the application of difference schemes for the numerical solution of lagging models of heat conduction is considered. Numerical schemes for some DPL approximations are developed, characterizing their properties of convergence and stability. Examples of numerical computations are included.