Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry.

To measure the average length of telomere repeats at chromosome ends in individual cells we developed a flow cytometry method using fluorescence in situ hybridization (flow FISH) with labeled peptide nucleic acid (PNA) probes. Results of flow FISH measurements correlated with results of conventional telomere length measurements by Southern blot analysis (R = 0.9). Consistent differences in telomere length in CD8+ T-cell subsets were identified. Naive and memory CD4+ T lymphocytes in normal adults differed by around 2.5 kb in telomere length, in agreement with known replicative shortening of telomeres in lymphocytes in vivo. T-cell clones grown in vitro showed stabilization of telomere length after an initial decline and rare clones capable of growing beyond 100 population doublings showed variable telomere length. These results show that flow FISH can be used to measure specific nucleotide repeat sequences in single cells and indicate that the very large replicative potential of lymphocytes is only indirectly related to telomere length.

Increasing the carbon flux toward synthesis of short-chain-length--medium-chain-length polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae through modification of the beta-oxidation cycle.

Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the beta-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes. The quantity of polymer produced was increased by using a mutant of the beta-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward R-3-hydroxybutyryl coenzyme A.

Limits on the validity of infinite length assumptions for modelling shallow landslides

The infinite slope method is widely used as the geotechnical component of geomorphic and landscape evolution models. Its assumption that shallow landslides are infinitely long (in a downslope direction) is usually considered valid for natural landslides on the basis that they are generally long relative to their depth. However, this is rarely justified, because the critical length/depth (L/H) ratio below which edge effects become important is unknown. We establish this critical L/H ratio by benchmarking infinite slope stability predictions against finite element predictions for a set of synthetic two-dimensional slopes, assuming that the difference between the predictions is due to error in the infinite slope method. We test the infinite slope method for six different L/H ratios to find the critical ratio at which its predictions fall within 5% of those from the finite element method. We repeat these tests for 5000 synthetic slopes with a range of failure plane depths, pore water pressures, friction angles, soil cohesions, soil unit weights and slope angles characteristic of natural slopes. We find that: (1) infinite slope stability predictions are consistently too conservative for small L/H ratios; (2) the predictions always converge to within 5% of the finite element benchmarks by a L/H ratio of 25 (i.e. the infinite slope assumption is reasonable for landslides 25 times longer than they are deep); but (3) they can converge at much lower ratios depending on slope properties, particularly for low cohesion soils. The implication for catchment scale stability models is that the infinite length assumption is reasonable if their grid resolution is coarse (e.g. >25?m). However, it may also be valid even at much finer grid resolutions (e.g. 1?m), because spatial organization in the predicted pore water pressure field reduces the probability of short landslides and minimizes the risk that predicted landslides will have L/H ratios less than 25. Copyright (c) 2012 John Wiley & Sons, Ltd.

Italy's Path to Very Low Fertility : The Adequacy of Economic and Second Demographic Transition Theories

The deep drop of the fertility rate in Italy to among the lowest in the world challenges contemporary theories of childbearing and family building. Among high-income countries, Italy was presumed to have characteristics of family values and female labor force participation that would favor higher fertility than its European neighbors to the north. We test competing economic and cultural explanations, drawing on new nationally representative, longitudinal data to examine first union, first birth, and second birth. Our event history analysis finds some support for economic determinants of family formation and fertility, but the clear importance of regional differences and of secularization suggests that such an explanation is at best incomplete and that cultural and ideational factors must be considered.

Exploring models for the length of stay distribution.

Diagnosis Related Groups (DRG) are frequently used to standardize the comparison of consumption variables, such as length of stay (LOS). In order to be reliable, this comparison must control for the presence of outliers, i.e. values far removed from the pattern set by the majority of the data. Indeed, outliers can distort the usual statistical summaries, such as means and variances. A common practice is to trim LOS values according to various empirical rules, but there is little theoretical support for choosing between alternative procedures. This pilot study explores the possibility of describing LOS distributions with parametric models which provide the necessary framework for the use of robust methods.

Relative and absolute reliability of the clinical version of the Narrow Path Walking Test (NPWT) under single and dual task conditions.

Decline in gait stability has been associated with increased fall risk in older adults. Reliable and clinically feasible methods of gait instability assessment are needed. This study evaluated the relative and absolute reliability and concurrent validity of the testing procedure of the clinical version of the Narrow Path Walking Test (NPWT) under single task (ST) and dual task (DT) conditions. Thirty independent community-dwelling older adults (65-87 years) were tested twice. Participants were instructed to walk within the 6-m narrow path without stepping out. Trial time, number of steps, trial velocity, number of step errors, and number of cognitive task errors were determined. Intraclass correlation coefficients (ICCs) were calculated as indices of agreement, and a graphic approach called "mountain plot" was applied to help interpret the direction and magnitude of disagreements between testing procedures. Smallest detectable change and smallest real difference (SRD) were computed to determine clinically relevant improvement at group and individual levels, respectively. Concurrent validity was assessed using Performance Oriented Mobility Assessment Tool (POMA) and the Short Physical Performance Battery (SPPB). Test-retest agreement (ICC1,2) varied from 0.77 to 0.92 in ST and from 0.78 to 0.92 in DT conditions, with no apparent systematic differences between testing procedures demonstrated by the mountain plot graphs. Smallest detectable change and smallest real change were small for motor task performance and larger for cognitive errors. Significant correlations were observed for trial velocity and trial time with POMA and SPPB. The present results indicate that the NPWT testing procedure is highly reliable and reproducible.

Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Length of activity season drives geographic variation in body size of a widely distributed lizard

Understanding the factors that drive geographic variation in life history is an important challenge in evolutionary ecology. Here, we analyze what predicts geographic variation in life-history traits of the common lizard, Zootoca vivipara, which has the globally largest distribution range of all terrestrial reptile species. Variation in body size was predicted by differences in the length of activity season, while we found no effects of environmental temperature per se. Females experiencing relatively short activity season mature at a larger size and remain larger on average than females in populations with relatively long activity seasons. Interpopulation variation in fecundity was largely explained by mean body size of females and reproductive mode, with viviparous populations having larger clutch size than oviparous populations. Finally, body size-fecundity relationship differs between viviparous and oviparous populations, with relatively lower reproductive investment for a given body size in oviparous populations. While the phylogenetic signal was weak overall, the patterns of variation showed spatial effects, perhaps reflecting genetic divergence or geographic variation in additional biotic and abiotic factors. Our findings emphasize that time constraints imposed by the environment rather than ambient temperature play a major role in shaping life histories in the common lizard. This might be attributed to the fact that lizards can attain their preferred body temperature via behavioral thermoregulation across different thermal environments. Length of activity season, defining the maximum time available for lizards to maintain optimal performance, is thus the main environmental factor constraining growth rate and annual rates of mortality. Our results suggest that this factor may partly explain variation in the extent to which different taxa follow ecogeographic rules.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage.

To test the hypotheses that mutant huntingtin protein length and wild-type huntingtin dosage have important effects on disease-related transcriptional dysfunction, we compared the changes in mRNA in seven genetic mouse models of Huntington's disease (HD) and postmortem human HD caudate. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in and full-length transgenic models of HD took longer to appear, 15- and 22-month CHL2(Q150/Q150), 18-month Hdh(Q92/Q92) and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. Whereas it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared with those caused by the expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. In addition, very high correlations between the signatures of mice expressing normal levels of wild-type huntingtin and mice in which the wild-type protein is absent suggest a limited effect of the wild-type protein to change basal gene expression or to influence the qualitative disease-related effect of mutant huntingtin. The combined analysis of mouse and human HD transcriptomes provides important temporal and mechanistic insights into the process by which mutant huntingtin kills striatal neurons. In addition, the discovery that several available lines of HD mice faithfully recapitulate the gene expression signature of the human disorder provides a novel aspect of validation with respect to their use in preclinical therapeutic trials.

Head-to-head comparison of length of stay, patients' outcome and satisfaction in Switzerland before and after SwissDRG-Implementation in 2012 in 2012: an observational study in two tertiary university centers.

On 1 January 2012 Swiss Diagnosis Related Groups (DRG), a new uniform payment system for in-patients was introduced in Switzerland with the intention to replace a "cost-based" with a "case-based" reimbursement system to increase efficiency. With the introduction of the new payment system we aim to answer questions raised regarding length of stay as well as patients' outcome and satisfaction. This is a prospective, two-centre observational cohort study with data from University Hospital Basel and the Cantonal Hospital Aarau, Switzerland, from January to June 2011 and 2012, respectively. Consecutive in-patients with the main diagnosis of either community-acquired pneumonia, exacerbation of COPD, acute heart failure or hip fracture were included. A questionnaire survey was sent out after discharge investigating changes before and after SwissDRG implementation. Our primary endpoint was LOS. Of 1,983 eligible patients 841 returned the questionnaire and were included into the analysis (429 in 2011, 412 in 2012). The median age was 76.7 years (50.8% male). Patients in the two years were well balanced in regard to main diagnoses and co-morbidities. Mean LOS in the overall patient population was 10.0 days and comparable between the 2011 cohort and the 2012 cohort (9.7 vs 10.3; p = 0.43). Overall satisfaction with care changed only slightly after introduction of SwissDRG and remained high (89.0% vs 87.8%; p = 0.429). Investigating the influence of the implementation of SwissDRG in 2012 regarding LOS patients' outcome and satisfaction, we found no significant changes. However, we observed some noteworthy trends, which should be monitored closely.