Poor trail making test performance is directly associated with altered dual task prioritization in the elderly--baseline results from the TREND study

Background

Deterioration of executive functions in the elderly has been associated with impairments in walking performance. This may be caused by limited cognitive flexibility and working memory, but could also be caused by altered prioritization of simultaneously performed tasks. To disentangle these options we investigated the associations between Trail Making Test performance—which specifically measures cognitive flexibility and working memory—and dual task costs, a measure of prioritization.

Methodology and Principal Findings

Out of the TREND study (Tuebinger evaluation of Risk factors for Early detection of Neurodegenerative Disorders), 686 neurodegeneratively healthy, non-demented elderly aged 50 to 80 years were classified according to their Trail Making Test performance (delta TMT; TMT-B minus TMT-A). The subjects performed 20 m walks with habitual and maximum speed. Dual tasking performance was tested with walking at maximum speed, in combination with checking boxes on a clipboard, and subtracting serial 7 s at maximum speeds. As expected, the poor TMT group performed worse when subtracting serial 7 s under single and dual task conditions, and they walked more slowly when simultaneously subtracting serial 7 s, compared to the good TMT performers. In the walking when subtracting serial 7 s condition but not in the other 3 conditions, dual task costs were higher in the poor TMT performers (median 20%; range −6 to 58%) compared to the good performers (17%; −16 to 43%; p<0.001). To the contrary, the proportion of the poor TMT performance group that made calculation errors under the dual tasking situation was lower than under the single task situation, but higher in the good TMT performance group (poor performers, −1.6%; good performers, +3%; p = 0.035).

Conclusion

Under most challenging conditions, the elderly with poor TMT performance prioritize the cognitive task at the expense of walking velocity. This indicates that poor cognitive flexibility and working memory are directly associated with altered prioritization.     

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction

(S(C5'), R(P)) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.     

Cellulosomics, a gene-centric approach to investigating the intraspecific diversity and adaptation of Ruminococcus flavefaciens within the rumen

Background

The bovine rumen maintains a diverse microbial community that serves to break down indigestible plant substrates. However, those bacteria specifically adapted to degrade cellulose, the major structural component of plant biomass, represent a fraction of the rumen microbiome. Previously, we proposed scaC as a candidate for phylotyping Ruminococcus flavefaciens, one of three major cellulolytic bacterial species isolated from the rumen. In the present report we examine the dynamics and diversity of scaC-types both within and between cattle temporally, following a dietary switch from corn-silage to grass-legume hay. These results were placed in the context of the overall bacterial population dynamics measured using the 16S rRNA.

Principal Findings

As many as 117 scaC-types were estimated, although just nineteen were detected in each of three rumens tested, and these collectively accounted for the majority of all types present. Variation in scaC populations was observed between cattle, between planktonic and fiber-associated fractions and temporally over the six-week survey, and appeared related to scaC phylogeny. However, by the sixth week no significant separation of scaC populations was seen between animals, suggesting enrichment of a constrained set of scaC-types. Comparing the amino-acid translation of each scaC-type revealed sequence variation within part of the predicted dockerin module but strong conservation in the N-terminus, where the cohesin module is located.

Conclusions

The R. flavefaciens species comprises a multiplicity of scaC-types in-vivo. Enrichment of particular scaC-types temporally, following a dietary switch, and between fractions along with the phylogenetic congruence suggests that functional differences exist between types. Observed differences in dockerin modules suggest at least part of the functional heterogeneity may be conferred by scaC. The polymorphic nature of scaC enables the relative distribution of R. flavefaciens strains to be examined and represents a gene-centric approach to investigating the intraspecific adaptation of an important specialist population.     

Circulating endothelial progenitor cells in kidney transplant patients

Background

Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs.

Methods

We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry.

Results

Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1 – a cytokine responsible for EPC mobilization – is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1.

Conclusions

We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.     

Altered dopamine and serotonin metabolism in motorically asymptomatic R6/2 mice

The pattern of cerebral dopamine (DA) abnormalities in Huntington disease (HD) is complex, as reflected by the variable clinical benefit of both DA antagonists and agonists in treating HD symptoms. In addition, little is known about serotonin metabolism despite the early occurrence of anxiety and depression in HD. Post-mortem enzymatic changes are likely to interfere with the in vivo profile of biogenic amines. Hence, in order to reliably characterize the regional and chronological profile of brain neurotransmitters in a HD mouse model, we used a microwave fixation system that preserves in vivo concentrations of dopaminergic and serotoninergic amines. DA was decreased in the striatum of R6/2 mice at 8 and 12 weeks of age while DA metabolites, 3-methoxytyramine and homovanillic acid, were already significantly reduced in 4-week-old motorically asymptomatic R6/2 mice. In the striatum, hippocampus and frontal cortex of 4, 8 and 12-week-old R6/2 mice, serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly decreased in association with a decreased turnover of serotonin. In addition, automated high-resolution behavioural analyses displayed stress-like behaviours such as jumping and grooming and altered spatial learning in R6/2 mice at age 4 and 6 weeks respectively. Therefore, we describe the earliest alterations of DA and serotonin metabolism in a HD murine model. Our findings likely underpin the neuropsychological symptoms at time of disease onset in HD.     

Heightened stress response in primary fibroblasts expressing mutant eIF2B genes from CACH/VWM leukodystrophy patients

Childhood ataxia with central nervous system hypomyelination (CACH), also called vanishing white matter (VWM) leukoencephalopathy, is a fatal genetic disease caused by mutations in eukaryotic initiation factor 2B (eIF2B) genes. The five subunits eIF2B factor is critical for translation initiation under normal conditions and regulates protein synthesis in response to cellular stresses. Primary fibroblasts from CACH/VWM patients and normal individuals were used to measure basal eIF2B activity as well as global protein synthesis and ATF4 induction in response to stress in the endoplasmic reticulum. We show that although the cells expressing mutant eIF2B genes respond normally to stress conditions by reduced global translation rates, they exhibit significantly greater increase in ATF4 induction compared to normal controls despite equal levels of stress and activity of the upstream eIF2α kinase. This heightened stress response observed in primary fibroblasts that suffer from minor loss of basal eIF2B activity may be employed as an initial screening tool for CACH/VWM leukodystrophy.     

Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

Ischemic preconditioning decreases the reperfusion-related formation of hydroxyl radicals in a rabbit model of regional myocardial ischemia and reperfusion: the role of K(ATP) channels

The objective of this study was to assess the effects of ischemic preconditioning (IP) on hydroxyl free radical production in an in vivo rabbit model of regional ischemia and reperfusion. Another goal was to determine whether K_ATP channels are involved in these effects.

The hearts of anesthetized and mechanically ventilated New Zealand White rabbits were exposed through a left thoracotomy. After IV salicylate (100 mg/kg) administration, all animals underwent a 30-min stabilization period followed by 40 min of regional ischemia and 2 h of reperfusion. In the IP group, IP was elicited by 5 min of ischemia followed by 10 min of reperfusion (prior to the 40-min ischemia period). Glibenclamide, a K_ATP channel blocker, was administered prior to the preconditioning stimulus. Infarct size was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. We quantified the hydroxyl-mediated conversion of salicylate to its 2,3 and 2,5-dihydroxybenzoate derivatives during reperfusion by high performance liquid chromatography coupled with electro-chemical detection.

IP was evidenced by reduced infarct size compared to control animals: 22% vs. 58%, respectively. Glibenclamide inhibited this cardioprotective effect and infarct size was 53%. IP limited the increase in 2,3 and 2,5-dihydroxybenzoic acid to 24.3 and 23.8% above baseline, respectively. Glibenclamide abrogated this effect and the increase in 2,3 and 2,5-dihydroxybenzoic acid was 94.3 and 85% above baseline levels, respectively, similar to the increase in the control group. We demonstrated that IP decreased the formation of hydroxyl radicals during reperfusion. The fact that glibenclamide inhibited this effect, indicates that K_ATP channels play a key role in this cardioprotective effect of IP.     

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.