Selection of Aptamers for Amyloid β-Protein,the Causative Agent of Alzheimer's Disease

Alzheimer''s disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult¹. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies^2,3.Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD^4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood⁶. Specific molecular recognition tools may unravel the relationships amongst Aβ assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies^7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)^9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).Despite emergence of aptamers as tools in modern biotechnology and medicine¹¹, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)^12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-β¹⁷, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils¹⁸. Aptamers generated using monomeric and several forms of fibrillar β₂-microglobulin (β₂m) were found to bind fibrils of certain other amyloidogenic proteins besides β₂m fibrils¹⁹. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ40²⁰. Unexpectedly, these aptamers bound fibrillar Aβ40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Aβ40²¹ generated using photo-induced cross-linking of unmodified proteins (PICUP)^22,23. Similar to previous findings^17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope²¹. Here, we present the SELEX methodology used in production of these aptamers²¹.Download video file.^{(175M, mp4)}     

Conflict of use for multi-purpose tree species in the state of Pará, eastern Amazonia, Brazil

Although diversified forest management is promoted as a strategy aimed at slowing tropical deforestation, little is known about the viability of integrating timber and non-timber forest products in the same forest management plans. In this study we offer an initial characterization of multi-purpose tree species in the State of Pará, the principal Amazonian logging region. We identify the species used for both timber and non-timber extraction, and classify these according to their commercial value. We relate multi-purpose species to their ecological traits, the type of non-timber forest use and the fraction of the tree harvested. Although a high number of species present a potential conflict of use, this conflict is only relevant in four of them: D. odorata, T. serratifolia, T. impetiginosa and H. courbaril. Nevertheless, the nature and relevance of this conflict will ultimately depend on the importance that the non-timber use has for the livelihoods of forest-dependant people, the commercial value and the ecological resilience of these species.     

Interaction of photons with molecules – cross-sections for photoabsorption, photoionization, and photodissociation

A survey is given of recent progress in measurements of photoabsorption, photoionization, and photodissociation cross-sections of molecules in the wavelength range of photons in the vacuum ultraviolet (VUV), where the optical oscillator-strengths of most molecules are predominantly distributed. Some remarks are presented on molecules in the condensed phase. Particular emphasis is placed on the current understanding of spectroscopy and dissociation dynamics of molecules in the superexcited states which are produced through the interaction of photons in this wavelength range. In the VUV range, most of the observed superexcited states are assigned to high-Rydberg states which are vibrationally (and/or rotationally), doubly, or inner-core excited, and converge to each of the ion states. Received: 2 September 1998 / Accepted in revised form: 10 September 1999     

A highly selective, fluorescent chemosensor for bioimaging of Fe³⁺

A fluorescent sensor for Fe(3+) has been synthesized based on rhodamine-lactam, which shows excitation (531 nm) and emission (557 nm) wavelength, displays an excellent selectivity for Fe(3+) and can be used for imaging Fe(3+) in living cells. The pK(a) of the sensor is as low as of 3.2. It can be used in the range of pH 5-9.     

Cereal phytochromes: targets of selection, targets for manipulation?

Plants respond to shading through an adaptive syndrome termed shade avoidance. In high-density crop plantings, shade avoidance generally increases extension growth at the expense of yield and can be at odds with the agronomic performance of the crop as a whole. Studies in Arabidopsis are beginning to reveal the essential role phytochromes play in regulating this process and to identify genes underlying the response. In this article, we focus on how phytochrome signaling networks have been targeted in cereal breeding programs in the past and discuss the potential to alter these pathways through breeding and transgenic manipulation to develop crops that perform better under typical high density conditions.     

Characteristics of Tφ3, a Bacteriophage for Bacillus stearothermophilus

A bacteriophage (Tphi3) which infects the thermophilic bacterium Bacillus stearothermophilus ATCC 8005 was isolated and characterized. Infection of the bacterium by the bacteriophage was carried out at 60 C, the optimal growth temperature of the host. At 60 C, the phage had a latent period of 18 min and a burst size of about 200. The phage was comparatively thermostable in broth. The halflife of Tphi3 was 400 min at 60 C, 120 min at 65 C, 40 min at 70 C, and 12 min at 75 C. The activation energy for the heat inactivation of Tphi3 was 56,000 cal. The buoyant density of Tphi3 in a cesium chloride density gradient was 1.526 g/ml. Electron micrographs of Tphi3 indicate that the phage has a head that is 57 mmu long. The dimensions and shape of the head are compatible with those of a regular icosahedron. Each edge of the head is 29 mmu long. The tail of Tphi3 is 125 mmu long and 10 mmu wide. There are about 30 cross-striations that are spaced at 3.9-mmu intervals along the tail. Under the conditions investigated, Tphi3 adsorbed slowly to the host. Only 2.8% of the phage adsorbed in 10 min at 60 C, the normal incubation temperature that was used. Tphi3 was not infective to four other thermophilic strains or to two mesophilic strains of bacteria.     

Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for Bacillus stearothermophilus

It is shown that the individual strands of bacteriophage Tphi3 DNA are intact and that heat-denatured Tphi3 DNA forms a bimodal distribution in a neutral CsCl density gradient.     

NEX-TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α- and β-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences.     

Transfer of IP₃ through gap junctions is critical, but not sufficient, for the spread of apoptosis

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP₃) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP₃ in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP₃ signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP₃ receptors or stimulating IP₃ degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP₃ permeability. However, IP₃ by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP₃ is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.     

Weather,microclimate, and energy costs of thermoregulation for breeding Adélie Penguins

Summary We measured meteorological conditions and estimated the energy costs of thermoregulation for young and adult Adélie Penguins (Pygoscelis adeliae) at a breeding colony near the Antarctic Peninsula. Air temperatures averaged < 5°C and strong winds were frequent. Operative temperatures (T_e) for adults ranged from –8 to 28°C, averaging 5–6°C, for the period from courtship to fledging of chicks. The average energy cost of thermoregulation (C_th) for adult penguins was equivalent to 10–16% of basal metabolism. C_th comprised about 15% of the estimated daily energy budget (DEB) of incubating adults, but only about 1% of the DEB of adults feeding chicks. The T_e's for chicks older than 14 days ranged from 0 to 31°C, averaging 8.0 C. The C_th for downy chicks ranged from about 31% of minimal metabolic rate (MMR) in 1 kg chicks to about 10% of MMR in 3 kg chicks. Between initial thermal independence (age 12–14 days) and the cessation of parental feeding (age 35–40 days), chicks use about 10–11% of assimilated energy for thermoregulation. C_th is equivalent to about 17% of the MMR of fledglings during their 2–3 week fast. We observed no indication of thermal stress (i.e., conditions in which birds cannot maintain stable T_b) in adults and no indication of cold stress in any age class. However, on clear, calm days when air temperature exceeds 7–10°C for several hours, downy chicks are vulnerable to lethal hyperthermia.     

Biomarkers for early detection of breast cancer: what, when, and where?

Early detection of breast cancer reduces the suffering and cost to society associated with the disease. A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. The earlier and more accurate the diagnostic biomarker can predict disease onset, the more valuable it becomes. Here, a brief review of existing and emerging approaches for breast cancer biomarker identification and analysis is presented. Those biomarkers found in biological fluids, blood in particular, apparently hold the best promise for fast development of screening assays. Autoantibodies and abnormal tumor-specific DNA methylation found in cell-free plasma DNA may provide the best opportunity for constructing multiplexed and highly redundant tests, which will be sufficiently specific and sensitive for early detection of breast cancer. It is expected that technologies developed for breast cancer detection will be useful for other types of cancer.     

Biocatalytic,one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes – with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes.By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli.The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes.