Terminal Restriction Fragment Analysis Research Articles

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization.

The human telomere system is highly dynamic. Both short and long leucocyte average telomere lengths (aTL) are associated with an increased risk of cancer and early death, illustrating the complex relationship between TL and human health and the importance of assessing TL distributions with single TL analysis. A DNA microarray and telomere fluorescent in situ hybridization (DNA-array-FISH) approach was developed to measure the base-pair (bp) lengths of single telomeres. On average 32000 telomeres were measured per DNA sample with one microarray chip assaying 96 test DNA samples. Various telomere parameters, i.e. aTL and the frequency of short/long telomeres, were computed to delineate TL distribution. The intra-assay and inter-assay coefficient of variations of aTL ranged from 1.37% to 3.98%. The correlation coefficient (r) of aTL in repeated measurements ranged from 0.91 to 1.00, demonstrating high measurement precision. aTLs measured by DNA-array-FISH predicted aTLs measured by terminal restriction fragment (TRF) analysis with r ranging 0.87-0.99. A new accurate and high-throughput method has been developed to measure the bp lengths of single telomeres. The large number of single TL data provides an opportunity for an in-depth analysis of telomere dynamics and the complex relationship between telomere and age-related diseases.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Profile of Gut Bacteria in Hypertensive Patients Based on Terminal Restriction Fragment Length Polymorphism Analysis

Hypertension is a severe public health problem due to its high prevalence worldwide. About 7.5 million deaths or 12.8% of all annual deaths worldwide occur due to high blood pressure. The hypertensive disease is also associated with the intestinal bacteria. To our knowledge, the diversity of the gut bacteria in hypertensive patients has not been reported yet in Indonesia. Thus, the aims of this study were to analyze profile of gut bacteria in hypertensive patients compared to non-hypertensive based on metagenomic analysis, Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results of the affiliation analysis of the entire Terminal Restriction Fragments (TRF) contained 6 groups of bacteria from 26 TRFs in hypertensive and non-hypertensive respondents. Cutting the 16S rRNA gene with the BslI restriction enzyme successfully detected intestinal bacterial groups, namely Clostridium subcluster XIVa, Prevotella, Clostridium cluster IV, Clostridium cluster XI, Bacteroides and others. In hypertensive patients, a higher relative abundance of bacterial groups showed in Clostridium cluster XI, Clostridium cluster IV and Clostridium subcluster XIVa. The abundance of Bacteroides and Prevotella groups in hypertensive patients were lower than non-hypertensive. The abundance of enterotype I and enterotype II was lower in hypertensive patients than non-hypertensive. Contrarily to that enterotype III cluster. It is worth noting that the F/B ratio was higher in hypertensive patients than that in non-hypertensive. Our data suggest that the gut bacteria profile of hypertensive patients differs to that non-hypertensive.

Correlation between reduced telomere length and behavioural and emotional problems in left-behind children in a rural area in China

Evidence shows that being left behind experience (LBE) during childhood may increase the risks of poor psychopathological outcomes. However, it is unclear to what extent the mental health is affected by the LBE. Telomere length (TL), one of the most extensively studied biological markers of cellular ageing, provides a valuable tool for exploring the potential effects of parent-child separation on psychological problems by integrating genetic and environmental factors. In this study, a total of 613 children (mean age = 10.77, SD = 1.92) were recruited from the rural area of Deyang, Sichuan Province, China. We used a self-designed questionnaire to assess LBE, and collected psychopathological outcomes by using the Piers-Harris Children's Self-concept Scale, the Teacher's Report Form 6/18 and the Youth Self-Report 11/18. Terminal restriction fragment analysis was used to measure TL in peripheral blood leukocytes. Analyses revealed that 342 out of 613 participants (55.79%) were Left-behind children. LBE was observed to associated with shorter TL, lower self-esteem, and increased behavioural and emotional problems. The cumulative effects of LBE may be reflected by greater altered telomere homeostasis, decreased self-esteem, and worsened behavioural and emotional problems. The association of the total time of being left behind with self-esteem and behavioural and emotional problems was significantly mediated by altered telomere homeostasis, with estimated effects of 14.19%, 47.95% and 45.13%, respectively. The LBE in childhood, especially prolonged parent-child separation, increases the risk of mental health problems in childhood and adolescence.

Telomere length in dromedary camels (Camelus dromedarius) produced by somatic cell nuclear transfer (SCNT) and their age-matched naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

Increased telomere length in patients with frontotemporal dementia syndrome

BackgroundTelomeres are repetitive DNA sequences of TTAGGG at the ends of chromosomes. Many studies have shown that telomere shortening is associated with aging-related diseases, such as cardiovascular diseases, hypertension, diabetes, cancer, and various neurodegenerative diseases, including Alzheimer's disease, vascular dementia, Parkinson's disease, and dementia with Lewy bodies. However, changes in telomere length (TL) in patients with frontotemporal dementia (FTD) syndrome are unclear. Accordingly, in this study, we assessed TL in blood samples from patients with FTD syndrome. MethodsAbsolute TL was measured in peripheral blood leukocytes from 53 patients with FTD syndromes (25 with behavioral variant FTD, 19 with semantic variant primary progressive aphasia [PPA], six with nonfluent/agrammatic variant PPA, and three with amyotrophic lateral sclerosis [ALS] plus) and 28 cognitively unimpaired (CU) controls using terminal restriction fragment analysis. ResultsTL was significantly longer in the FTD group than in the CU group. All FTD subtypes had significantly longer TL than controls. There were no significant differences in TL among FTD syndromes. No significant correlations were found between TL and demographic factors in the FTD group. ConclusionsLonger telomeres were associated with FTD syndrome, consistent with a recent report demonstrating that longer telomeres are related to ALS. Therefore, our results may support a shared biology between FTD and ALS. More studies with larger sample sizes are needed.

Continuous reference intervals for leukocyte telomere length in children: the method matters.

Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement. Healthy children aged 30days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyteTL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performedto generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined. A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=-0.28 and r=-0.38, p<0.001). In contrast, Flow-FISH showed no change in TL with age (r=-0.08, p=0.23). Gender did not have significant influence on TL in children. This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change.

14 Telomere length in cloned camels produced by somatic cell nuclear transfer is not different from that in their naturally produced counterparts

There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, and their age-matched naturally produced counterparts by terminal restriction fragment (TRF) analysis. Genomic DNA was extracted from venous blood collected from 6 cloned animals (aged 3, 12, and 24 months), and their age-matched counterparts by a conventional phenol/chloroform protocol. Denatured and neutralized DNA was blotted onto a positively charged nylon membrane and fixed by baking at 80°C for 3h. After washing in a prewarmed digoxigenin (DIG) easy hybridization solution at 42°C for 1h, DNA hybridization was carried out using a telomere (TTAGGG)n-specific, DIG-labelled hybridization probe (Roche Diagnostics, Germany) at 42°C for 4h. Stringent washes were carried out at the same temperature, followed by chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. Molecular weights of the unknown TRF bands on the gel were calculated using known molecular weight marker provided by Telo TAGGG Telomere Length Assay Kit (Roche Diagnostics). A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated 3 times, and the data, presented as mean±s.e.m., were analysed using a 2-sample t-test (Minitab statistical software). No difference (P&amp;gt;0.05) was found in the mean telomere length of cloned camels compared with their naturally produced age-matched counterparts (21.7±0.3 vs. 22.1±0.3; 21.9±0.3 vs. 22.1±0.3; 22.2±0.5 vs. 22.0±0.1; 20.5±0.5 vs. 22.5±0.7; 20.1±0.1 vs. 22.5±0.7; 21.7±1.1 vs. 22.6±0.2), respectively. In conclusion, this is the first study where telomere length has been reported in naturally produced and cloned dromedary camels produced by SCNT. We found that telomere lengths in cloned camels were similar to those of their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.

An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length.

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease.

Telomere length is repeatable, shortens with age and reproductive success, and predicts remaining lifespan in a long-lived seabird.

Telomeres are protective caps at the end of chromosomes, and their length is positively correlated with individual health and lifespan across taxa. Longitudinal studies have provided mixed results regarding the within-individual repeatability of telomere length. While some studies suggest telomere length to be highly dynamic and sensitive to resource-demanding or stressful conditions, others suggest that between-individual differences are mostly present from birth and relatively little affected by the later environment. This dichotomy could arise from differences between species, but also from methodological issues. In our study, we used the highly reliable Terminal Restriction Fragment analysis method to measure telomeres over a 10-year period in adults of a long-lived seabird, the common tern (Sterna hirundo). Telomeres shortened with age within individuals. The individual repeatability of age-dependent telomere length was high (>0.53), and independent of the measurement interval (i.e., one vs. six years). A small (R2 =.01), but significant part of the between-individual variation in telomere length was, however, explained by the number of fledglings produced in the previous year, while reproduction in years prior to the previous year had no effect. We confirmed that age-dependent telomere length predicted an individual's remaining lifespan. Overall, our study suggests that the majority of between-individual variation in adult telomere length is consistent across adult life, and that a smaller part of the variation can be explained by dynamic factors, such as reproduction.

TPP1 OB-fold domain protein suppresses cell proliferation and induces cell apoptosis by inhibiting telomerase recruitment to telomeres in human lung cancer cells.

Maintaining telomeres by recruiting telomerase-to-chromosome ends is essential for cancer cell survival. Inhibiting telomerase recruitment to telomeres represents a novel strategy for telomere-based lung cancer therapy. However, approaches for interrupting telomerase recruitment for cancer therapy still need to be explored. The telomere-binding protein TPP1 is responsible for recruiting telomerase to telomeres and synthesizing telomeres through the association between the oligosaccharide/oligonucleotide-binding (OB)-fold domain of TPP1 and telomerase reverse transcriptase. We overexpressed the TPP1 OB domain (TPP1-OB) by lentivirus infection in lung cancer cells. Telomere length was examined by Southern blot analysis of terminal restriction fragments. The effects of TPP1-OB on cell proliferation, the cell cycle, apoptosis, chemosensitivity, and tumor growth were evaluated in vitro and in vivo. TPP1-OB inhibited the recruitment of telomerase to telomeres and shortened telomere length by acting as a dominant-negative mutant of TPP1. TPP1-OB resulted in reduced cell proliferation, G1 cell cycle arrest, and increased cell apoptosis in lung cancer cells. Cell apoptosis occurred mainly through the caspase-3-dependent signaling pathway. TPP1-OB also suppressed anchorage-independent growth and tumor growth in vivo. Moreover, we demonstrated that TPP1-OB enhances the sensitivity of lung cancer cells to the chemotherapeutic drug paclitaxel. Our results suggest that inhibiting TPP1-mediated telomerase recruitment by expressing the TPP1-OB domain is a potential novel strategy for telomere-targeted lung cancer therapy.

Association between antidiabetic agents use and leukocyte telomere shortening rates in patients with type 2 diabetes.

Telomere length and telomere shortening rate (TSR) are accepted indicators of aging in cross-sectional population studies. This study aimed to investigate the potential influence of common antidiabetic agents on telomere length and TSR in patients with type 2 diabetes mellitus (T2DM). Leukocyte telomere length was measured through terminal restriction fragment analysis, and TSR was calculated in 388 T2DM patients. Depending on whether or not they received antidiabetic medication, patients were first divided into a treatment group and a nontreatment group. Treated patients were further subdivided into an acarbose-free group (patients taking antidiabetic agents without acarbose) and an acarbose group (patients using acarbose for more than 3 months). Results showed that untreated patients had higher TSRs than patients on antidiabetic drugs. Interestingly, patients in the acarbose group had significantly higher TSRs than patients in the acarbose-free group. Compared to the nontreatment group, the acarbose group showed better glycemic control of HbA1c, but the TSR was also higher. Our results suggest that antidiabetic treatments without acarbose can slow aging. By contrast, acarbose may accelerate biological aging in patients with T2DM, independently of glycemic control.

Early life adiposity and telomere length across the life course: a systematic review and meta-analysis.

Background: The relationship between adiposity at birth and in childhood, and telomere length is yet to be determined. We aimed to systematically review and meta-analyse the results of studies assessing associations between neonatal and later childhood adiposity, and telomere length. Methods: We searched Medline, EMBASE and PubMed for studies reporting associations between adiposity measured in the neonatal period or later childhood/adolescence, and leucocyte telomere length, measured at any age via quantitative polymerase chain reaction, or terminal restriction fragment analysis, either cross-sectionally, or longitudinally. Papers published before April 2017 were included. Results: Out of 230 abstracts assessed, 23 papers (32 estimates) were retained, from which 19 estimates were meta-analysed (15 cross-sectional, four longitudinal). Of the 15 cross-sectional estimates, seven reported on neonates: four used binary exposures of small-for-gestational-age vs. appropriate-for-gestational age (or appropriate- and large-for-gestational age), and three studied birth weight continuously. Eight estimates reported on later childhood or adolescent measures; five estimates were from studies of binary exposures (overweight/obese vs. non-obese children), and three studies used continuous measures of body mass index. All four longitudinal estimates were of neonatal adiposity, with two estimates for small-for-gestational-age vs. appropriate-for-gestational age neonates, and two estimates of birth weight studied continuously, in relation to adult telomere (49-61 years). There was no strong evidence of an association between neonatal or later childhood/adolescent adiposity, and telomere length. However, between study heterogeneity was high, and there were few combinable studies. Conclusions: Our systematic review and meta-analysis found no strong evidence of an association between neonatal or later childhood or adolescent adiposity and telomere length.

Early life adiposity and telomere length across the life course: a systematic review and meta-analysis.

Background: The relationship between adiposity at birth and in childhood, and telomere length is yet to be determined. We aimed to systematically review and meta-analyse the results of studies assessing associations between neonatal and childhood adiposity, and telomere length. Methods: We searched Medline, EMBASE and PubMed for studies reporting associations between adiposity measured in the neonatal period or childhood, and leucocyte telomere length, measured at any age via quantitative polymerase chain reaction, or terminal restriction fragment analysis, either cross-sectionally, or longitudinally. Papers published before April 2017 were included. Results: Out of 230 abstracts assessed, 23 papers (32 estimates) were retained, from which 19 estimates were meta-analysed (15 cross-sectional, four longitudinal). Of the 15 cross-sectional estimates, seven reported on neonates: four used binary exposures of small-for-gestational-age vs. appropriate-for-gestational age (or appropriate- and large-for-gestational age), and three studied birth weight continuously. Eight estimates reported on childhood measures; five estimates were from studies of binary exposures (overweight/obese vs. non-obese children), and three studies used continuous measures of body mass index. All four longitudinal estimates were of neonatal adiposity, with two estimates for small-for-gestational-age vs. appropriate-for-gestational age neonates, and two estimates of birth weight studied continuously, in relation to adult telomere (49-61 years). There was no strong evidence of an association between neonatal or childhood adiposity, and telomere length. However, between study heterogeneity was high, and there were few combinable studies. Conclusions: Our systematic review and meta-analysis found no strong evidence of an association between neonatal or childhood adiposity and telomere length.

Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review.

Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility? DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations. The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations. Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes. Two DNA isolation methods were compared: Genomic Tips which isolate 'High Molecular Weight' (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates 'Total' genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis). HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis. Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences. This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning. This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest. N/A.

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization.

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern1,2. Here, we present a detailed description of this procedure, with some modifications.

Diversity of bacteria and archaea in the rhizosphere of bioenergy crop Jatropha curcas.

Plant-microbial interaction in rhizosphere plays vital role in shaping plant’s growth and ecosystem function. Most of the rhizospheric microbial diversity studies are restricted to bacteria. In natural ecosystem, archaea also constitutes a major component of the microbial population. However, their diversity is less known compared to bacteria. Experiments were carried out to examine diversity of bacteria and archaea in the rhizosphere of bioenergy crop Jatropha curcas (J. curcas). Samples were collected from three locations varying widely in the soil physico-chemical properties. Diversity was estimated by terminal restriction fragment length polymorphism (TRFLP) targeting 16S rRNA gene of bacteria and archaea. Fifteen bacterial and 17 archaeal terminal restriction fragments (TRFs) were retrieved from J. curcas rhizosphere. Bacterial indicative TRFs were Actinobacteria, Firmicutes, Acidobacteria, Verrumicrobiaceae, and Chlroflexi. Major archaeal TRFs were crenarchaeota, and euryarchaeota. In case of bacteria, relative fluorescence was low for TRF160 and high for TRF51, TRF 420. Similarly, for archaea relative fluorescence of TRF 218, and TRF 282 was low and high for TRF 278, TRF468 and TRF93. Principal component analysis (PCA) of bacterial TRFs designated PC 1 with 46.83% of variation and PC2 with 31.07% variation. Archaeal TRFs designated 90.94% of variation by PC1 and 9.05% by PC2. Simpson index varied from 0.530 to 0.880 and Shannon index from 1.462 to 3.139 for bacteria. For archaea, Simpson index varied from 0.855 to 0.897 and Shannon index varied from 3.027 to 3.155. Study concluded that rhizosphere of J. curcas constituted of diverse set of both bacteria and archaea, which might have promising plant growth promoting activities.

DNA methylation changes in response to active smoking exposure are associated with leukocyte telomere length among older adults.

Telomere length (TL) is associated with an increased risk of aging-related diseases. As a preventable environmental hazard of morbidity and mortality, smoking has been reported to promote TL attrition by producing a variety of oxidants and free radicals. Since DNA methylation has been demonstrated to play an important role in the pathways of smoking and smoking-induced diseases, this study aimed to address whether the smoking-associated DNA methylation changes could be associated with accelerated TL shortening. We obtained DNA methylation profiles in whole blood samples by Illumina Infinium Human Methylation 450 Beadchip array in two independent subsamples of the ESTHER study and measured their relative TL by quantitative PCR. Terminal Restriction Fragment analysis was additionally performed in a subsample to obtain absolute TL in base pairs. TL measurements across panels were standardized by z-transformation. After correction for multiple testing, we successfully confirmed that seven out of 151 smoking-related CpG sites were associated with TL (FDR <0.05). A smoking index based on the seven loci showed monotonic associations with TL, cumulative smoking exposure and time after smoking cessation. In conclusion, our study supports suggestions that epigenetic alterations could play a role in smoking-associated disproportionate aging as reflected by TL. Further research is required to examine whether the identified epigenetic signatures of smoking can be of value in clinical practice to assess individual aging across the lifespan.

Characterization of telomeres and telomerase from the single-celled eukaryote Giardia intestinalis

The ends of linear chromosomes, telomeres, are most commonly maintained by the enzyme telomerase. Our study presents the characteristics of telomeres and telomerase from the single-celled parasitic eukaryote Giardia intestinalis. Using fluorescence in situ hybridization, we localized telomeres during all stages of the trophozoite cell cycle and demonstrated differences in the observed number of telomeric foci, indicating telomere clustering. The length of Giardia telomeres was determined in different cell lines derived from WB clinical isolate using terminal restriction fragment analysis and ranged from 0.5 to 2.5kb; moreover, a BAL-31 digestion experiment did not reveal any long interstitial telomeric sequences in the genome. Despite the absence of the specific T motif in the telomerase catalytic subunit, the presence of an active telomerase enzyme synthesising telomeric repeats in Giardia was proved by a Telomere repeat amplification protocol assay, and its localization in nuclei was determined by the expression of recombinant GiTERT. Except for the Giardia-type TAGGG telomeric repeat, Giardia telomerase was proved to synthesize in vitro also other repeat variants, TAAGG and TAAGGG. In summary, despite its unusual characteristics, including a structurally divergent but active telomerase, unique terminal sequences and relatively short telomeres, the present data support the view that the chromosomal termini in Giardia are maintained in a conservative manner that is common to other eukaryotes.