BodyMap incorporated PCR-based expression profiling data and a gene ranking system

BodyMap is a human and mouse gene expression database that is based on site-directed 3′-expressed sequence tags generated at Osaka University. To date, it contains more than 300 000 tag sequences from 64 human and 39 mouse tissues. For the recent release, the precise anatomical expression patterns for more than half of the human gene entries were generated by introduced amplified fragment length polymorphism (iAFLP), which is a PCR-based high-throughput expression profiling method. The iAFLP data incorporated into BodyMap describe the relative contents of more than 12 000 transcripts across 30 tissue RNAs. In addition, a newly developed gene ranking system helps users obtain lists of genes that have desired expression patterns according to their significance. BodyMap supports complete transfer of unique data sets and provides analysis that is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp.

Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed “supernatant protein factor (SPF),” which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921–930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as α-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer

Although the systemic administration of a number of different gene products has been shown to result in the inhibition of angiogenesis and tumor growth in different animal tumor models, the relative potency of those gene products has not been studied rigorously. To address this issue, recombinant adenoviruses encoding angiostatin, endostatin, and the ligand-binding ectodomains of the vascular endothelial growth factor receptors Flk1, Flt1, and neuropilin were generated and used to systemically deliver the different gene products in several different preexisting murine tumor models. Single i.v. injections of viruses encoding soluble forms of Flk1 or Flt1 resulted in ≈80% inhibition of preexisting tumor growth in murine models involving both murine (Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic carcinoma) tumors. In contrast, adenoviruses encoding angiostatin, endostatin, or neuropilin were significantly less effective. A strong correlation was observed between the effects of the different viruses on tumor growth and the activity of the viruses in the inhibition of corneal micropocket angiogenesis. These data underscore the need for comparative analyses of different therapeutic approaches that target tumor angiogenesis and provide a rationale for the selection of specific antiangiogenic gene products as lead candidates for use in gene therapy approaches aimed at the treatment of malignant and ocular disorders.

Neural crest-directed gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development

Wnt1 signaling has been implicated as one factor involved in neural crest-derived melanocyte (NC-M) development. Mice deficient for both Wnt1 and Wnt3a have a marked deficiency in trunk neural crest derivatives including NC-Ms. We have used cell lineage-directed gene targeting of Wnt signaling genes to examine the effects of Wnt signaling in mouse neural crest development. Gene expression was directed to cell lineages by infection with subgroup A avian leukosis virus vectors in lines of transgenic mice that express the retrovirus receptor tv-a. Transgenic mice with tva in either nestin-expressing neural precursor cells (line Ntva) or dopachrome tautomerase (DCT)-expressing melanoblasts (line DCTtva) were analyzed. We overstimulated Wnt signaling in two ways: directed gene transfer of Wnt1 to Ntva+ cells and transfer of β-catenin to DCTtva+ NC-M precursor cells. In both methods, NC-M expansion and differentiation were effected. Significant increases were observed in the number of NC-Ms [melanin+ and tyrosinase-related protein 1 (TYRP1)+ cells], the differentiation of melanin− TYRP1+ cells to melanin+ TYRP1+ NC-Ms, and the intensity of pigmentation per NC-M. These data are consistent with Wnt1 signaling being involved in both expansion and differentiation of migrating NC-Ms in the developing mouse embryo. The use of lineage-directed gene targeting will allow the dissection of signaling molecules involved in NC development and is adaptable to other mammalian developmental systems.

Three Drought-Responsive Members of the Nonspecific Lipid-Transfer Protein Gene Family in Lycopersicon pennellii Show Different Developmental Patterns of Expression1

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2.

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

THE DESIGN OF A POWER SYSTEM FOR A WIRELESS SENSOR NETWORK FOR LONG-TERM OPERATION

Wireless sensor networks (WSNs) have shown wide applicability to many fields including monitoring of environmental, civil, and industrial settings. WSNs however are resource constrained by many competing factors that span their hardware, software, and networking. One of the central resource constrains is the charge consumption of WSN nodes. With finite energy supplies, low charge consumption is needed to ensure long lifetimes and success of WSNs. This thesis details the design of a power system to support long-term operation of WSNs. The power system’s development occurs in parallel with a custom WSN from the Queen’s MEMS Lab (QML-WSN), with the goal of supporting a 1+ year lifetime without sacrificing functionality. The final power system design utilizes a TPS62740 DC-DC converter with AA alkaline batteries to efficiently supply the nodes while providing battery monitoring functionality and an expansion slot for future development. Testing tools for measuring current draw and charge consumption were created along with analysis and processing software. Through their use charge consumption of the power system was drastically lowered and issues in QML-WSN were identified and resolved including the proper shutdown of accelerometers, and incorrect microcontroller unit (MCU) power pin connection. Controlled current profiling revealed unexpected behaviour of nodes and detailed current-voltage relationships. These relationships were utilized with a lifetime projection model to estimate a lifetime between 521-551 days, depending on the mode of operation. The power system and QML-WSN were tested over a long term trial lasting 272+ days in an industrial testbed to monitor an air compressor pump. Environmental factors were found to influence the behaviour of nodes leading to increased charge consumption, while a node in an office setting was still operating at the conclusion of the trail. This agrees with the lifetime projection and gives a strong indication that a 1+ year lifetime is achievable. Additionally, a light-weight charge consumption model was developed which allows charge consumption information of nodes in a distributed WSN to be monitored. This model was tested in a laboratory setting demonstrating +95% accuracy for high packet reception rate WSNs across varying data rates, battery supply capacities, and runtimes up to full battery depletion.

The Dilemmas of Diffusion: Institutional Transfer & the Remaking of Vocational Training Practices in Eastern Germany, CES Germany & Europe Working Papers, No. 05.10, 1996

Through a case study of the diffusion of the celebrated West Gennan "dual system" of vocational training to the territory of the fonner German Democratic Republic, we develop the argument that local sociopolitical relations matter crucially for the successful transfer and implementation of institutional arrangements. Notwithstanding massive levels of government funding, the presence of complementary supports, and the concerted efforts of Ger­many's social partners, the dual system is experiencing significant difficulties in the new federal states of the East. These difficulties are not due simply to the particular politics of unification (the wholesale transfer of West German institutions whether or not they were appropriate to Eastern Germany) nor even simply to the paucity of dynamic private firms capable of and willing to train new apprentices. The difficulties stem also from the under­ lying weaknesses of the East German sociopolitical infrastructure on which the entire dual system rests. This. hy­ pothesis is elaborated and substantiated through a range of data on training in the East and especially through the use of detailed case studies of Leipzig and Chemrutz.

Safe Harbour or into the storm? EU-US data transfers after the Schrems judgment. Liberty and security in Europe No. 85/November 2015

In its recent Schrems judgment the Luxembourg Court annulled Commission Decision 2000/520 according to which US data protection rules are sufficient to satisfy EU privacy rules regarding EU-US transfers of personal data, otherwise known as the ‘Safe Harbour’ framework. What does this judgment mean and what are its main implications for EU-US data transfers? In this paper the authors find that this landmark judgment sends a strong message to EU and US policy-makers about the need to ensure clear rules governing data transfers, so that people whose personal data is transferred to third countries have sufficient legal guarantees. Without such rules there is legal uncertainty and mistrust. Any future arrangement for the transatlantic transfer of data will therefore need to be firmly anchored in a framework of protection commensurate to the EU Charter of Fundamental Rights and the EU data protection architecture.

Measuring Dependency Ratios using National Transfer Accounts. CEPS Working Document No. 420/April 2016

It is now widely recognised that the socio-economic changes that ageing societies will bring about are poorly captured by the traditional demographic dependency ratios (DDRs), such as the old-age dependency ratio that relates the number of people aged 65+ to the working-age population. Future older generations will have increasingly better health and are likely to work longer. By combining population projections and National Transfer Accounts (NTA) data for seven European countries, we project the quantitative impact of ageing on public finances until 2040 and compare it to projected DDRs. We then simulate the public finance impact of changes in three key indicators related to the policy responses to population ageing: net immigration, healthy ageing and longer working lives. We do this by linking age-specific public health transfers and labour market participation rates to changes in mortality. Four main findings emerge: first, the simple old-age dependency ratio overestimates the future public finance challenges faced by the countries studied – significantly so for some countries, e.g. Austria, Finland and Hungary. Second, healthy ageing has a modest effect (on public finances) except in the case of Sweden, where it is substantial. Third, the long-run effect of immigration is well captured by the simple DDR measure if immigrants are similar to the native population. Finally, increasing the length of working lives is central to addressing the public finance challenge of ageing. Extending the length of working lives by three to four years over the next 25 years – equivalent to the increase in life expectancy – severely limits the impact of ageing on public transfers.

Sinking rates of particles measured from deployments in the Atlantic Ocean based on seasonal data

The flux of materials to the deep sea is dominated by larger, organic-rich particles with sinking rates varying between a few meters and several hundred meters per day. Mineral ballast may regulate the transfer of organic matter and other components by determining the sinking rates, e.g. via particle density. We calculated particle sinking rates from mass flux patterns and alkenone measurements applying the results of sediment trap experiments from the Atlantic Ocean. We have indication for higher particle sinking rates in carbonate-dominated production systems when considering both regional and seasonal data. During a summer coccolithophorid bloom in the Cape Blanc coastal upwelling off Mauritania, particle sinking rates reached almost 570 m per day, most probably due the fast sedimentation of densely packed zooplankton fecal pellets, which transport high amounts of organic carbon associated with coccoliths to the deep ocean despite rather low production. During the recurring winter-spring blooms off NW Africa and in opal-rich production systems of the Southern Ocean, sinking rates of larger particles, most probably diatom aggregates, showed a tendency to lower values. However, there is no straightforward relationship between carbonate content and particle sinking rates. This could be due to the unknown composition of carbonate and/or the influence of particle size and shape on sinking rates. It also remains noticeable that the highest sinking rates occurred in dust-rich ocean regions off NW Africa, but this issue deserves further detailed field and laboratory investigations. We obtained increasing sinking rates with depth. By using a seven-compartment biogeochemical model, it was shown that the deep ocean organic carbon flux at a mesotrophic sediment trap site off Cape Blanc can be captured fairly well using seasonal variable particle sinking rates. Our model provides a total organic carbon flux of 0.29 Tg per year down to 3000 m off the NW African upwelling region between 5 and 35° N. Simple parameterisations of remineralisation and sinking rates in such models, however, limit their capability in reproducing the flux variation in the water column.