Development and validation of a new method to measure walking speed in free-living environments using the actibelt® platform

Walking speed is a fundamental indicator for human well-being. In a clinical setting, walking speed is typically measured by means of walking tests using different protocols. However, walking speed obtained in this way is unlikely to be representative of the conditions in a free-living environment. Recently, mobile accelerometry has opened up the possibility to extract walking speed from long-time observations in free-living individuals, but the validity of these measurements needs to be determined. In this investigation, we have developed algorithms for walking speed prediction based on 3D accelerometry data (actibelt?) and created a framework using a standardized data set with gold standard annotations to facilitate the validation and comparison of these algorithms. For this purpose 17 healthy subjects operated a newly developed mobile gold standard while walking/running on an indoor track. Subsequently, the validity of 12 candidate algorithms for walking speed prediction ranging from well-known simple approaches like combining step length with frequency to more sophisticated algorithms such as linear and non-linear models was assessed using statistical measures. As a result, a novel algorithm employing support vector regression was found to perform best with a concordance correlation coefficient of 0.93 (95%CI 0.92-0.94) and a coverage probability CP1 of 0.46 (95%CI 0.12-0.70) for a deviation of 0.1 m/s (CP2 0.78, CP3 0.94) when compared to the mobile gold standard while walking indoors. A smaller outdoor experiment confirmed those results with even better coverage probability. We conclude that walking speed thus obtained has the potential to help establish walking speed in free-living environments as a patient-oriented outcome measure.     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Expression of POX2 gene and disruption of POX3 genes in the industrial Yarrowia lipolytica on the γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate can produce γ-decalactone, the worthy aroma compound, which can exhibit fruity and creamy sensorial notes, and recognized internationally as a safe food additive. Unfortunately, the yield is poor because of lactone degradation by enzyme Aox3 (POX3 gene encoded), which was responsible for continuation of oxidation after C(10) level and lactone reconsumption. In this paper, we chose the industrial Y. lipolytica (CGMCC accession number 2.1405), which is the diploid strain as the starting strain and constructed the recombinant strain Tp-12 by targeting the POX3 locus of the wild type, one copy of POX3 was deleted by CRF1+POX2 insertion. The other recombinant strain Tpp-11, which was a null mutant possessing multiple copies of POX2 and disrupted POX3 genes on two chromosomes, was constructed by inserting XPR2+hpt into the other copy of POX3 of Tp-12. The growth ability of the recombinants was changed after genetic modification in the fermentation medium. The production of γ-decalactone was increased, resulting from blocking β-oxidation at the C(10) Aox level and POX2 overexpression. The recombinant strain Tpp-11 was stable. Because there was no reconsumption of γ-decalactone, the mutant strain could be grown in continuous fermentation of methyl ricinoleate to produce γ-decalactone.     

What can whole genome expression data tell us about the ecology and evolution of personality?

Consistent individual differences in behaviour, aka personality, pose several evolutionary questions. For example, it is difficult to explain within-individual consistency in behaviour because behavioural plasticity is often advantageous. In addition, selection erodes heritable behavioural variation that is related to fitness, therefore we wish to know the mechanisms that can maintain between-individual variation in behaviour. In this paper, we argue that whole genome expression data can reveal new insights into the proximate mechanisms underlying personality, as well as its evolutionary consequences. After introducing the basics of whole genome expression analysis, we show how whole genome expression data can be used to understand whether behaviours in different contexts are affected by the same molecular mechanisms. We suggest strategies for using the power of genomics to understand what maintains behavioural variation, to study the evolution of behavioural correlations and to compare personality traits across diverse organisms.     

Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: the effects of gene insertion, tissue culture and breeding

The Bacillus thuringiensis ??-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC?CMS) based metabolic profiling method was employed to determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue culture. Based on the GC?CMS metabolic profiles, the principal component analysis and significant differences determined by Student??s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes. By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent. A GC?CMS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during transformation processes.     

Assessment of Schwanniomyces occidentalis as a host for protein production using the wide-range Xplor®2 expression platform

The wide-range transformation/expression platform, Xplor®2, was employed for the assessment of Schwanniomyces occidentalis as a potential producer of the recombinant proteins human IFNα2a (IFNα2a) and S. occidentalis fructofuranosidase (SFfase), and its efficiency was compared to that of Arxula adeninivorans. ADE2 and URA3 genes from both yeast species were isolated, characterized and used as selection markers in combination with the IFNα2a and SFfase expression modules, which used the strong constitutive A. adeninivorans-derived TEF1 promoter. Yeast rDNA integrative expression cassettes and yeast integrative expression cassettes equipped with a selection marker and expression modules were transformed into auxotrophic S. occidentalis and A. adeninivorans strains and a quantitative comparison of the expression efficiency was made. Whilst IFNα2a was mainly accumulated extracellularly (>95 %) in A. adeninivorans, extracellular SFfase (>90 %) was detected in both yeast species. The DNA composition of the selection marker modules and expression modules, especially their open reading frame codon usage, affects auxotrophy recovery as well as protein expression. Auxotrophy recovery was only achieved with selection marker modules of the homologous gene donor yeast. The concentration of recombinant IFNα2a was fivefold higher in A. adeninivorans (1 mg?L^?1), whereas S. occidentalis accumulated 1.5- to 2-fold more SFfase (0.5 Units ml^?1). These results demonstrate the extension of the use of the wide-range expression platform Xplor®2 to another yeast species of biotechnological interest.     

RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Background  

Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.     

Localization of the α2-macroglobulin gene and Lpm gene family on mink chromosome 9

Summary Using cloned cDNA for human ₂-macroglobulin (A2M) as a probe, mink-Chinese hamster hybrid cells were analysed. The results allowed us to assign a gene for A2M to mink chromosome 9. Breeding tests demonstrated that the Lpm-locus coding for other related -macroglobulin protein and the gene for peptidase B (PEPB) are linked 11±3 cm apart. The PEPB gene is located on mink chromosome 9, and hence, the Lpw-locus is on the same mink chromosome. The relationship of the genetic systems controlling the isotypically different -macroglobulins in mink serum are discussed.     

Effects of Phoslock® treatment and chironomids on the exchange of nutrients between sediment and water

Effects of chironomids on sediment–water exchange of nutrients and their impact on the efficiency of Phoslock^® (a lanthanum (La) modified clay for phosphorus (P) removal in freshwater systems) were tested during a 35 days incubation experiment with sediment cores from a Danish eutrophic Lake. Four different sediment treatments with increased or natural densities of chironomids in combination with Phoslock^® were used: (1) Control + (2) Chironomids + (3) Phoslock + (4) Chironomids & Phoslock. Nutrients in the overlying water were followed during the incubation period. The treatments with Phoslock reduced P in the overlying water significantly compared to the control treatment. In addition, the chironomids significantly increased sediment nitrate uptake as well as sediment ammonium release. After the incubation period, a sequential extraction of P and La was conducted. The Phoslock treatment led to a reduction of the iron-bound P pool in the sediment and a higher HCl-extractable P pool. Also, most La was recovered in the HCl extract, indicating that P became strongly bound to La in the Phoslock matrix. Sequential extraction of pure Phoslock demonstrated that the bentonite matrix of Phoslock contained redox sensitive iron, and that ammonium might be released from Phoslock, when dispersed in water.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Structure of the mouse 3-Methyladenine DNA Glycosylase gene and exact localization upstream of the α-globin gene cluster on Chromosome 11

In this paper we describe the genomic organization of the mouse 3-Methyladenine DNA Glycosylase (MPG) gene and localize three putative regulatory elements around this gene. The MPG gene plays a key role in the excision repair of methylated adenine residues and has been localized upstream of the -globin gene cluster in human and mouse. The human MPG gene has been fully characterized, whereas up to now only the cDNA sequence of the mouse MPG gene had been published. Here, we describe a detailed restriction map, the intron/exon structure, the CpG-rich putative promoter sequence, and the exact localization of the mouse MPG gene with respect to the murine -globin gene cluster. Our analysis reveals a remarkable different exon/intron structure of the mouse MPG gene compared with its human homolog. Two prominent DNase hypersensitive sites (HSS) were found 0.1 and 1.5 kb upstream of the coding sequence. In addition to these elements, an erythroid prominent HSS was mapped at the intron/exon boundary of the last exon. The characterization and localization of the MPG gene in mouse makes it now possible to carry out transgenic and gene targeting experiments and are essential to understand the control of gene expression of the MPG gene in particular and of the whole region in general.The nucleotide sequence data reported in this paper will be submitted to Genbank.     

'Natural selection merely modified while redundancy created' – Susumu Ohno's idea of the evolutionary importance of gene and genome duplications

Susumo Ohno's influential book Evolution by gene duplication dealt with the idea that gene and genome duplication events are the principal forces by which the genetic raw material is provided for increasing complexity during evolution. In 1970, the evidence for this hypothesis consisted mostly of karyotypic information, crude information by today's standard genetic data, DNA sequences. Nonetheless, although the type of data are outdated, the idea remained current and is still debated today in the age of complete genome sequences. Even more than thirty years after the initial publication more research than ever is being carried out on the evolutionary significance of gene and genome duplications and the contribution of these mechanisms to the advances in genomic and organismal evolution.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.