Tools for T-RFLP data analysis using Excel

Background

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed.

Results

The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data.

Conclusions

The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0361-7) contains supplementary material, which is available to authorized users.     

Incidence of ADL Disability in Older Persons,Physical Activities as a Protective Factor and the Need for Informal and Formal Care – Results from the SNAC-N Project

Background

The aim of the study was to examine 1) the incidence of disability in Activities of Daily Living (ADL), in persons 78 years and older 2) explore whether being physical active earlier is a significant predictor of being disability free at follow-up and 3) describe the amount of informal and formal care in relation to ADL-disability.

Methods

Data were used from a longitudinal community-based study in Nordanstig (SNAC-N), a part of the Swedish National Study on Aging and Care (SNAC). To study objectives 1) and 2) all ADL-independent participants at baseline (N = 307) were included; for objective 3) all participants 78 years and older were included (N = 316). Data were collected at baseline and at 3- and 6-year follow-ups. ADL-disability was defined as a need for assistance in one or more activities. Informal and formal care were measured using the Resource utilization in Dementia (RUD)-instrument.

Results

The incidence rates for men were similar in the age groups 78-81and 84 years and older, 42.3 vs. 42.5/1000 person-years. For women the incidence rate for ADL-disability increased significantly from the age group 78–81 to the age group 84 years and older, 20.8 vs.118.3/1000 person-years. In the age group 78–81 years, being physically active earlier (aOR 6.2) and during the past 12 month (aOR 2.9) were both significant preventive factors for ADL-disability. Both informal and formal care increased with ADL-disability and the amount of informal care was greater than formal care. The incidence rate for ADL-disability increases with age for women and being physically active is a protective factor for ADL-disability.

Conclusion

The incidence rate for ADL-disability increases with age for women, and being physical active is a protective factor for ADL-disability.     

Comparing Scots pine tree-ring proxies and detrending methods among sites in Jämtland,west-central Scandinavia

Scots pine tree-ring width (TRW) data from Jämtland in the Central Scandinavian Mountains has been used to reconstruct summer temperatures back to 1630 BC. However, it was recently shown that this reconstruction was of limited spatial importance. In this paper, we aim to explain this limitation in the TRW data as a temperature proxy, as well as assess the temperature information from new maximum latewood density (MXD) data. Furthermore, the effect of two standardization methods is evaluated: regional curve standardization (RCS) and a more traditional standardization, termed “non-RCS” standardization. Three TRW and two MXD sites were analyzed. Our results showed that despite the proximity to the Norwegian Sea, the MXD data is a powerful temperature proxy. Difference among sites in TRW data, especially on decadal timescales, together with a lower temperature association, suggests that other factors, such as changes in the local climate regimes, weakens the temperature signal. In general the RCS method overestimates pine growth trends in the latter half of the twentieth century, a feature not seen when using “non-RCS” standardization. This is likely due to an age-bias of older trees in most recent parts of the tree-ring chronologies. This effect will have consequences when reconstructing climate with tree-ring data. To overcome this problem, all age-classes should be represented throughout a chronology. If this is not possible, the use of “non-RCS” standardization is recommended, although this method results in a loss of low-frequency variability.     

A comparison of growing season indices for the Greater Baltic Area

Predictions of the effects of global warming suggest that climate change may have large impacts on ecosystems. The length of the growing season is predicted to increase in response to increasing global temperatures. The object of this study was to evaluate different indices used for calculating the thermal growing season for the Greater Baltic Area (GBA). We included established indices of growing season start, end and length, as well as new and modified indices. Based on the results, the GBA can be divided into a maritime western part and a more continental eastern part, with the western part reacting more sensitively to the use of different indices. The eastern part is more stable, but even here the index-to-index differences are large. It was found that including or excluding a frost criterion had a significant influence on the initiation of the growing season in the western, maritime, parts of the GBA. Frost has not the same importance for the end of the growing season. However, some end indices can result in a “never ending” growing season. When looking at twentieth century trends in growing season parameters, it was found that, when averaged over the whole GBA, there was little difference in trends depending on the indices used. The general mean trend in the GBA for the twentieth century discloses an earlier onset of c. 12 days, a delayed end of c. 8 days and consequently a lengthening of the growing season of about 20 days.     

Design and synthesis of 2'-anilino-4,4'-bipyridines as selective inhibitors of c-Jun N-terminal kinase-3

The design and synthesis of a new series of c-Jun N-terminal kinase-3 (JNK3) inhibitors with selectivity against JNK1 are reported. The novel series of substituted 2'-anilino-4,4'-bipyridines were designed based on a combination of hits from high throughput screening and X-ray crystal structure information of compounds crystallized into the JNK3 ATP binding active site.     

Cryptic contamination and phylogenetic nonsense

Ancient human DNA has been treated cautiously ever since the problems related to this type of material were exposed in the early 1990s, but as sequential genetic data from ancient specimens have been key components in several evolutionary and ecological studies, interest in ancient human DNA is on the increase again. It is especially tempting to approach archaeological and anthropological questions through this type of material, but DNA from ancient human tissue is notoriously complicated to work with due to the risk of contamination with modern human DNA. Various ways of authenticating results based on ancient human DNA have been developed to circumvent the problems. One commonly used method is to predict what the contamination is expected to look like and then test whether the ancient human DNA fulfils this prediction. If it does, the results are rejected as contamination, while if it does not, they are often considered authentic. We show here that human contamination in ancient material may well deviate from local allele frequencies or the distributions to be found among the laboratory workers and archaeologists. We conclude that it is not reliable to authenticate ancient human DNA solely by showing that it is different from what would be expected from people who have handled the material.     

The conserved active site asparagine in class I ribonucleotide reductase is essential for catalysis.

The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate. To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover but promoted rapid protein R2 tyrosyl radical decay in the presence of the k(cat) inhibitor 2'-azido-2'-deoxy-CDP with similar decay rate constants as the wild-type R1. These results show that all Asn-437 mutants can perform 3'-H abstraction, the first substrate-related step in the reaction mechanism. The most interesting observation was that three of the mutant proteins (N437A/S/D) behaved as suicidal enzymes by catalyzing a rapid tyrosyl radical decay also in reaction mixtures containing the natural substrate CDP. The suicidal CDP-dependent reaction was interpreted to suggest elimination of the substrate's protonated 2'-OH group in the form of water, a step that has been proposed to drive the 3'-H abstraction step. A furanone-related chromophore was formed in the N437D reaction, which is indicative of stalling of the reaction mechanism at the reduction step. We conclude that Asn-437 is essential for catalysis but not for 3'-H abstraction. We propose that the suicidal N437A, N437S, and N437D mutants can also catalyze the water elimination step, whereas the inert N437Q mutant cannot. Our results suggest that Asn-437, apart from hydrogen bonding to the substrate, also participates in the reduction steps of catalysis by class I ribonucleotide reductase.     

Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition

We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.     

Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.