Terminal Restriction Fragment Analysis Research Articles

Microbial Communities in Pre-Columbian Coprolites

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures.

Alternative lengthening of telomeres is induced by telomerase inhibitors in Barrett’s esophageal cells

A crucial step in the path to the malignant transformation of cells and tumor formation is immortalization, which essentially depends on telomere maintenance. The aim of this study was to investigate the role of telomerase in the progression of Barrett's esophagus. Telomerase activity was measured in Barrett's cells using terminal restriction fragment(TRF) analysis. Telomere length was measured using Q-FISH analysis. Furthermore, the telomere recombination events were detected between sister chromatids using chromosome orientation FISH(CO-FISH). There was a reduction in telomerase activity in the CP-A cells transduced with MT-hTER/47A+siRNA, which led to an almost complete disappearance of telomerase activity. The telomere length of the CP-A cells transduced with MT-hTER/47A+siRNA was slightly shorter compared to that of the untransduced cells. The telomerase-inhibited cells were morphologically indistinguishable from those untransduced and WT-hter-transduced cells. In the control cells, the growth rate was between 0.9 to 1.1 with the population doubling per day. Although the transduction of the telomerase inhibitors in the CP-A cells did not cause a significant reduction in cell growth, these transduced cells grew generally slower compared with the control cells. The heterogeneous telomere length was also be detected in the telomerase-inhibited CP-A cells. However, the telomere length remained homogeneous in the control cells. The metaphase of the CP-A cells transduced with MT-hTER/47A+siRNA demonstrated 70% heterogeneous telomeres. In addition, no increased recombination was observed between sister chromatids in the transduced CP-A cells compared with the control cells. Our findings suggest that an alternative lengthening of telomeres (ALT) may be induced by telomerase inhibitors in CP-A cells. Therefore, telomerase inhibitors may exhibit high potency in the treatment of esophageal adenocarcinoma arising from Barrett's esophagus.

A Chemistry-Based Method To Detect Individual Telomere Length at a Single Chromosome Terminus

The understanding of telomeres is expected to provide major insights into genome stability, cancer, and telomere-related diseases. In recent years, there have been considerable improvements in the technologies available to determine the length of telomeres of human chromosomes; however, the present methods for measuring telomere length are fraught with shortcomings that have limited their use. Here we describe a method for detection of individual telomere lengths (DITL) that uses a chemistry-based approach that accurately measures the telomere lengths from individual chromosomes. The method was successfully used to determine telomere DNA by breaking in the target sequence and producing a "real telomere fragment." The DITL approach involves cleavage of the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. Comparison of the DITL method and the traditional terminal restriction fragment (TRF) analysis indicates that the DITL approach appears to be promising for the quantification of telomere repeats in each chromosome and the detection of accurate telomere lengths that can be missed using TRF analysis.

Telomere Length Plays No Role in Clinical Unit Dominance After Double-Unit Cord Blood Transplantation

Abstract 4661 Background:Umbilical cord blood (CB) is an alternative source of allogeneic hematopoietic stem cells for transplantation and has the advantage of a reduced stringency of the required human leukocyte antigen-match. It is limited, however, by low total nucleated cell (TNC) dose and a low progenitor number in single CB units, thus restricting the use of CB transplantation (CBT) in larger children and adults. One strategy to augment engraftment is to combine 2 units from 2 different donors in a double-unit graft. We have previously shown that in most cases, one unit emerges as the sole source of hematopoiesis long-term, but as yet the mechanism of unit dominance remains unknown (Blood, 2010, 116(19):3999–4006). CB units are known to have an inherent biological variation in telomere length, the repeat sequence capping the ends of chromosomes. Telomere length variation and progressive shortening of telomere could later hematopoietic potential. Methods:We evaluated if telomere length has a role in unit dominance and how telomere length progresses over time post-transplant. We purified mononuclear cells from small aliquots of each unit of double-unit grafts and post-transplant peripheral blood (days +28, 100, 180 and 1 year) in 12 adult double-unit CBT recipients transplanted for hematologic malignancies at MSKCC. Average telomere length was measured using the TeloTTAGGG Telomere Length Assay (Roche) which utilizes Southern analysis of terminal restriction fragments (TRF) that are obtained by the digestion of isolated genomic DNA. Results:All 12 patients engrafted with one unit showing predominance. While there was a range of telomere length (5.57–12.09 kb) in the 24 units evaluated on the day of transplant, when comparing the telomere length of engrafting to non-engrafting units, there was no association between telomere length at day 0 and subsequent unit dominance. In 5 patients the engrafting unit had longer telomeres and in 7 patients the non-engrafting unit had longer telomeres. In serial assays, 7 of 12 patients demonstrated telomere length stabilization post-transplant in the dominant unit with a mean percentage loss of telomere length of 7.15% ± 2.33%. A second group (n = 5) demonstrated a decrease in telomere length with a mean percentage loss of 29.48% ± 5.12%. While this difference is significant (p = 0.0015) the clinical significance of this finding is uncertain. We are currently following these patients in order to correlate telomere length (stabilization versus decrease) with clinical outcomes. Conclusions:This data suggests that unit dominance is not influenced by telomere length. It is likely, based on emerging data from our laboratory as well as others, that unit dominance is immune mediated. However, the influence of telomere length on the quality of engraftment is of interest and analysis of this is ongoing with larger numbers of patients required to also consider the infused cell dose. It is notable that 7/12 patients had telomere length stabilization which correlates with the high levels of telomerase activity previously reported in the in vitro expansion of CB (Blood, 2004, 103(12):4440-8). Disclosures:No relevant conflicts of interest to declare.

Absolute standards as a useful addition to the avian quantitative PCR telomere assay

Bird populations provide excellent systems to investigate variation in longevity in the wild since individuals can often be monitored over their lifetime. A number of recent studies suggest that the dynamics of protective telomere chromosome caps (telomere length and rate of loss) are indicative of biological state and potentially useful as indicators of future longevity. Currently, Terminal Restriction Fragment (TRF) analysis and relative quantitative PCR (qPCR) are used to measure telomeres in birds, but with limitations. TRF analysis is time consuming, while relative qPCR gives a within‐study relative value making it difficult to compare across experiments. Utilising an approach first developed in humans of using synthetic oligomer telomeric (TTAGGG)n and normaliser gene standards of known length to calibrate qPCR values, we describe a methodological adaptation to the avian qPCR telomere assay to make results comparable within, and potentially between, bird species. We evaluate this absolute qPCR method in the Seychelles warbler Acrocephalus sechellensis against relative qPCR measurements on the same samples. Telomere estimates from both methods showed an age‐related decline in telomere length, and were highly correlated (r = 0.99). Absolute qPCR avian telomere analysis may prove a useful means of estimating telomere lengths in a calibrated, sensitive, and efficient way using small amounts of archived bird sample.

Correlation of short telomeres (ST) with vulnerability, toxicity, and early death in elderly AOC patients receiving carboplatin: A multicenter GINECO trial.

9011 Background: Age induces a progressive decline in the functional reserve and interferes with cancer treatments. As aging is heterogeneous, this decline has to be assessed individually. Telomere attrition leads to tissue senescence. We tested the hypothesis that telomere lenght (TL) could predict pts vulnerability and outcome during cancer treatment. Methods: This study was performed in the “Elderly women” GINECO trial designed to evaluate the impact of geriatric covariates on survival in AOC pts over 70 receiving 6 courses of carboplatin. TL was estimated in duplicate using standard Terminal Restriction Fragment analysis from peripheral blood cells at inclusion and tested for its correlation with geriatric covariates and pts outcomes (TC and tolerance, overall survival: OS). Results: TL (in base pair) was estimated for 109/111 pts (median 5997; extremes [4517-8333]). No significant correlation was found with any pts characteristics. With a cutoff of 5770 bp, TL discriminated two groups with significantly different Treatment Completion (TC) rates: 0.80 (95CI[0.71-0.89]) and 0.59 (95CI[0.41-0.76]), OR=2.8, p=0.02 for long telomere (LT) and short telomere groups, respectively . ST pts were at higher risk of severe adverse events (SAE, OR=2.7; p<0.02) and tended to have more unplanned hospital admissions (OR=2.1; p<0.08). Considering OS, after adjustment on FIGO stage, TL shorter than the median was a nearly significant risk factor of premature death (HR=1.57; p=0.06. Finally, we addressed if TL correlated with our previously validated geriatric vulnerability score (GVS)c including ADL score<6, IADL score<25, albuminemia<35g/l, lymphopenia<1G/L, HADS score£15 as risk factors of poorer survival. Despite no significant correlation with any of these factors, GVS³3) and ST tended to be correlated (OR=2.1; p=0.08). Conclusions: This exploratory study identifies TL as predictive factor of decreased TC, SAE risk, unplanned hospital admissions and OS after adjustment on FIGO stage.

Effects of Disturbance Intensity and Frequency on Bacterial Community Composition and Function

Disturbances influence community structure and ecosystem functioning. Bacteria are key players in ecosystems and it is therefore crucial to understand the effect of disturbances on bacterial communities and how they respond to them, both compositionally and functionally. The main aim of this study was to test the effect of differences in disturbance strength on bacterial communities. For this, we implemented two independent short-term experiments with dialysis bags containing natural bacterial communities, which were transplanted between ambient and ‘disturbed’ incubation tanks, manipulating either the intensity or the frequency of a salinity disturbance. We followed changes in community composition by terminal restriction fragment analysis (T-RFLP) and measured various community functions (bacterial production, carbon substrate utilization profiles and rates) directly after and after a short period of recovery under ambient conditions. Increases in disturbance strength resulted in gradually stronger changes in bacterial community composition and functions. In the disturbance intensity experiment, the sensitivity to the disturbance and the ability of recovery differed between different functions. In the disturbance frequency experiment, effects on the different functions were more consistent and recovery was not observed. Moreover, in case of the intensity experiment, there was also a time lag in the responses of community composition and functions, with functional responses being faster than compositional ones. To summarize, our study shows that disturbance strength has the potential to change the functional performance and composition of bacterial communities. It further highlights that the overall effects, rates of recovery and the degree of congruence in the response patterns of community composition and functioning along disturbance gradients depend on the type of function and the character of the disturbance.

Microbial community structure in sand on two olive ridley arribada nesting beaches, Playa La Flor, Nicaragua and Playa Nancite, Costa Rica

Microbial relative abundance, diversity and richness were assessed based on 16S rRNA gene-based TRF community analysis via terminal restriction fragment (TRF) analysis on two olive ridley turtle arribada nesting beaches in Central America, Playa La Flor in Nicaragua and Playa Nancite in Costa Rica. Arribada beaches have very high densities of egg clutches in small areas, and the sand contains numerous broken eggs due to turtles inadvertently disturbing previously laid clutches. The high organic content (broken eggs) in the sand causes microbial build up in clutches, which in return may affect hatching success. Phylotype relative abundance (fragment abundance) differed with nest density and distance from the water at both Playa Nancite and Playa La Flor. Higher relative abundance of certain bacterial species in high nest density areas may have contributed to lower hatching success. Bacterial diversity and richness increased with nest density and were higher in the zones closer to vegetation suggesting that bacterial diversity and richness may be important factors affecting hatching success of olive ridley eggs on these nesting beaches. To better conserve olive ridley sea turtles and manage egg harvest on arribada nesting beaches further studies should focus on identifying bacterial species that are pathogenic to turtle eggs and their effects on hatching success.

Diet-related telomere shortening and chromosome stability

Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.

Reduced dosage of the modifiers of epigenetic reprogramming Dnmt1, Dnmt3L, SmcHD1 and Foxo3a has no detectable effect on mouse telomere length in vivo

Studies carried out in cultured cells have implicated modifiers of epigenetic reprogramming in the regulation of telomere length, reporting elongation in cells that were null for DNA methyltransferase DNA methyltransferase 1 (Dnmt1), both de novo DNA methyltransferases, Dnmt3a and Dnmt3b or various histone methyltransferases. To investigate this further, we assayed telomere length in whole embryos or adult tissue from mice carrying mutations in four different modifiers of epigenetic reprogramming: Dnmt1, DNA methyltransferase 3-like, structural maintenance of chromosomes hinge domain containing 1, and forkhead box O3a. Terminal restriction fragment analysis was used to compare telomere length in homozygous mutants, heterozygous mutants and wild-type littermates. Contrary to expectation, we did not detect overall lengthening in the mutants, raising questions about the role of epigenetic processes in telomere length in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-011-0318-9) contains supplementary material, which is available to authorized users.

Telomerase activity in pleural malignant mesotheliomas

New treatments are needed for malignant pleural mesothelioma (MPM), which currently has a poor prognosis. Cellular immortalisation, one of the hallmarks of cancer, depends on the activity of a telomere length maintenance mechanism (TMM) – either telomerase or alternative lengthening of telomeres (ALT). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. The aim of this study was to determine what proportion of MPMs use ALT and/or telomerase. Forty-three MPMs from 42 patients were examined for telomerase and ALT activity. Telomerase activity was detected by immunoaffinity purification followed by the telomere repeat amplification protocol (TRAP), and ALT activity was determined by the C-circle assay and by assessing telomere lengths using terminal restriction fragment analyses. We found that 43 of 43 MPMs were telomerase-positive[+] and ALT-negative[−]. Therefore, to investigate whether pleural mesothelial cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalisation, in which normal pleural mesothelial cells were transduced with simian virus 40 (SV40) oncogenes. We found that normal mesothelial cells were TMM-negative, and that expression of the SV40 oncogenes did not directly activate telomerase or ALT. Immortalisation, which in this experimental system results from additional genetic changes that have not yet been identified, was accompanied by activation of either TMM. Therefore, pleural mesothelial cells are capable of activating either TMM in vitro, and the observation that 100% of MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumour type. We conclude that MPM is a tumour that could be considered for anti-telomerase therapy.

Telomeres and Telomerase Activity in Scleractinian Corals and Symbiodinium spp.

Telomeres are the repetitive sequences of DNA and associated proteins that cap the ends of eukaryotic chromosomes and play an essential role in maintaining chromosome stability. Compromised telomeres can lead to cell cycle arrest, senescence, apoptosis, or genetic instability, whereas maintenance of telomeres can endow cells with the capacity for indefinite self-renewal. Telomere integrity is maintained in most cells by the activity of telomerase, a ribonucleoprotein that can catalyze the addition of repeat sequences onto chromosome ends. Using the telomeric repeat amplification protocol (TRAP) assay, we detected telomerase activity in host nuclear extracts prepared from two scleractinian corals, Madracis auretenra and Madracis decactis, and also in cultured Symbiodinium, the symbiotic algae that live within corals. Sequencing the TRAP reaction products indicated that the telomeric DNA repeat sequence was TTAGGG for coral and TTTAGGG for Symbiodinium. Using this sequence information, we estimated telomere lengths by terminal restriction fragment (TRF) analysis to be greater than 19 kb for several species of coral and their associated Symbiodinium. Maintenance of coral telomeres by telomerase activity may be a mechanism that confers continuous growth and reproductive plasticity to these long-lived organisms.

Biogeography of wetland rice methanotrophs

We focused on the functional guild of methane oxidizing bacteria (MOB) as model organisms to get deeper insights into microbial biogeography. The pmoA gene was used as a functional and phylogenetic marker for MOB in two approaches: (i) a pmoA database (> 4000 sequences) was evaluated to obtain insights into MOB diversity in Italian rice paddies, and paddy fields worldwide. The results show a wide geographical distribution of pmoA genotypes that seem to be specifically adapted to paddy fields (e.g. Rice Paddy Cluster 1 and Rice Paddy Cluster 2). (ii) On the smaller geographical scale, we designed a factorial experiment including three different locations, two rice varieties and two habitats (soil and roots) within each of three rice fields. Multivariate analysis of terminal restriction fragment analysis profiles revealed different community patterns at the three field sites, located 10-20 km apart. Root samples were characterized by high abundance of type I MOB whereas the rice variety had no effect. With the agronomical practice being nearly identical, historical contingencies might be responsible for the field site differences. Considering a large reservoir of viable yet inactive MOB cells acting as a microbial seed bank, environmental conditions might have selected and activated a different subset at a time thereby shaping the community.

Methane dynamics across wetland plant species

We examined patterns of methane flux, plant biomass, and microbial methanogenic populations in nine wetland plant species. Methane dynamics varied across plant functional groupings, with patterns distinctive among forbs, clonal dominants, and tussock/clump-forming graminoids. Carex stricta and Scirpus atrovirens showed the highest emissions (31.7 and 20.6 mg CH 4-C m −2 h −1), followed by other tussock- or clump-forming graminoids that averaged 11.0 mg CH 4-C m −2 h −1 ( Scirpus cyperinus, Glyceria striata, and Juncus effusus). The clonal dominants ( Phalaris arundinacea and Typha angustifolia) had the lowest methane emissions (1.3 and 3.4 mg CH 4-C m −2 h −1) of all seven graminoid species, and the forbs ( Mimulus ringens and Verbena hastata) emitted no detectable methane flux from their leaves. In general, methane emissions decreased with greater plant biomass. Terminal restriction fragment analysis (T-RFLP) of archaeal 16S rRNA revealed that the structure of the soil methanogen communities isolated from plant rhizospheres had no effect on methane flux. The relative proportions of the different terminal fragments were not correlated with either methane emissions or plant biomass. Methanogen populations from J. effusus soils were dominated by acetoclastic archaea of the Methanosarcinaceae and Methanosaetaceae families, while all other graminoid soils were colonized primarily by hydrogenotrophic archaea of the Methanobacteriaceae family. The results indicate that plant functional groups and plant biomass are useful in predicting methane flux differences across plant species, while soil methanogen community structure showed no distinguishable patterns.

OsKu70 Is Associated with Developmental Growth and Genome Stability in Rice

The cellular functions of Ku70 in repair of DNA double-stranded breaks and telomere regulation have been described in a wide range of organisms. In this study, we identified the rice (Oryza sativa) Ku70 homolog (OsKu70) from the rice genome database. OsKu70 transcript was detected constitutively in every tissue and developmental stage examined and also in undifferentiated callus cells in rice. Yeast two-hybrid and in vitro pull-down experiments revealed that OsKu70 physically interacts with OsKu80. We obtained loss-of-function osku70 T-DNA knockout mutant lines and constructed transgenic rice plants that overexpress the OsKu70 gene in the sense (35S:OsKu70) or antisense (35S:anti-OsKu70) orientation. The homozygous G2 osku70 mutant lines were more sensitive than wild-type plants to a DNA-damaging agent (0.01%-0.05% methyl-methane sulfonate), consistent with the notion that OsKu70 participates in the DNA repair mechanism. Terminal restriction fragment analysis revealed that telomeres in homozygous G2 osku70 mutants were markedly longer (10-20 kb) than those in wild-type plants (5-10 kb), whereas telomere length in heterozygous G2 osku70 mutant and T2 OsKu70-overexpressing transgenic (35S:OsKu70) rice resembled that of the wild-type plant. In contrast to what was observed in Arabidopsis (Arabidopsis thaliana) atku70 mutants, homozygous G2 osku70 rice plants displayed severe developmental defects in both vegetative and reproductive organs under normal growth conditions, resulting in sterile flowers. Analysis of meiotic progression in pollen mother cells demonstrated that up to 11.1% (seven of 63) of G2 mutant anaphase cells displayed one or more chromosomal fusions. These results suggest that OsKu70 is required for the maintenance of chromosome stability and normal developmental growth in rice plants.

노화촉진마우스의 텔로미어 함량 분석

Telomeres, comprised of tandem repeats of TTAGGG sequences, are special nucleoprotein structures that protect and stabilize chromosome ends. These structures form the crux of the telomere concept of aging, senescence and genomic instability. The classic terminal restriction fragment (TRF) analysis to quantify the amount of telomeric DNA is disadvantageous in species containing ultra long telomeres like in mice (100Kb). In this study, we used a more sensitive quantitative fluorescence in situ hybridization (Q FISH) technique to quantify telomeric DNA, and used it as a biological aging marker in mice. 12 litters each of Senescence-Resistant (SAMR1) and -Prone (SAMP1) known as senescence accelerated mouse strains were purchased from Central Lab, Animal Inc. We quantified the amount of telomeric DNA using telomere specific DNA probes on the two strains of male mice at 8 weeks, 18 weeks and 26 weeks of age. The amount of telomeric DNA correlated with aging and age associated changes in body and organ weight between SAMR1 and SAMP1 strains of mice. These data suggest the usefulness of the amount of telomeric DNA as a biological aging marker in human aging studies.

Telomere shortening may be associated with human keloids

BackgroundKeloids are benign skin tumors that are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are inherited with an autosomal dominant mode with incomplete clinical penetrance and variable expression. Keloids are characterized by formation of excess scar tissue beyond the boundaries of the wound. The exact etiology is still unknown and there is currently no appropriate treatment for keloid disease.MethodsWe analyzed sample tissues were obtained from 20 patients with keloid skin lesions and normal skin was obtained from 20 healthy donors. The telomeres were measured by Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay. Quantitative Real-Time RT-PCR analysis of hTERT gene expression was performed and intracellular ROS generation was measured.ResultsIn this study, we determined whether telomeric shortening and the expression of human telomerase reverse transcriptase (hTERT) occurs in keloid patients. Using Terminal Restriction Fragment (TRF) analysis and Real-Time PCR assay, we detected a significant telomere shortening of 30% in keloid specimens compared to normal skin. Using quantitative Real-Time RT-PCR, telomerase activity was found absent in the keloid tissues. Moreover, an increase in ROS generation was detected in fibroblasts cell cultures from keloid specimens as more time elapsed compared to fibroblasts from normal skin.ConclusionTelomere shortening has been reported in several metabolic and cardiovascular diseases. We found that telomere shortening can also be associated with human keloids. Chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases. Here we found increased ROS generation in fibroblasts from keloid fibroblasts cell cultures when compared to normal skin fibroblasts. Hence we conclude that oxidative stress might be an important modulator of telomere loss in keloid because of the absence of active telomerase that counteracts telomere shortening.

Arabidopsis Replication Protein A 70a is Required for DNA Damage Response and Telomere Length Homeostasis

Replication protein A1 (RPA1/RPA70) forms a heterotrimeric complex together with RPA2/RPA32 and RPA3/RPA14 subunits which plays essential roles in various aspects of DNA metabolism including replication, repair, recombination and telomere maintenance. Compared with RPA70 in yeast and mammals, limited information is available about the factor in plants. In this study, we analyzed the functions of AtRPA70a, which is most similar to human RPA70 among four paralogs in Arabidopsis thaliana. RNA blot analysis showed that AtRPA70a is expressed ubiquitously in plant organs containing differentiated and meristematic tissues, while its expression was up-regulated in response to DNA damage stress. Yeast two-hybrid and co-immunoprecipitation analyses showed that AtRPA70a interacted preferentially with Arabidopsis RPA32a, one of two paralogs. Inactivation of AtRPA70a by T-DNA insertion did not affect growth under normal conditions, but resulted in increased sensitivity to genotoxic agents such as methylmethane sulfonate, bleomycin and hydroxyurea. Terminal restriction fragment analysis revealed that telomere lengths in an AtRPA70a-deficient line were significantly larger than in the wild type, whereas those in the mutant expressing antisense AtTERT (telomerase catalytic subunit gene) were shortened during successive generations. These results demonstrate that AtRPA70a is involved in repair of double-strand DNA breaks and possibly contributes to telomerase-dependent telomere length regulation.

Bacterial Associates of Arboreal Ants and Their Putative Functions in an Obligate Ant-Plant Mutualism

Bacterial communities are highly diverse and have great ecological importance. In the present study, we used an in silico analysis of terminal restriction fragments (tRF) to characterize the bacterial community of the plant ant Pseudomyrmex ferrugineus. This species is an obligate inhabitant of Acacia myrmecophytes and feeds exclusively on plant-derived food sources. Ants are the dominant insect group in tropical rain forests. Associations of ants with microbes, which contribute particularly to the ants' nitrogen nutrition, could allow these insects to live on mostly or entirely plant-based diets and could thus contribute to the explanation of the high abundances that are reached by tropical ants. We found tRF patterns representing at least 30 prokaryotic taxa, of which the Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria, and Spirochaetes comprised 93%. Because most bacterial taxa were found in all ant-derived samples studied and because the bacteria detected on the ants' host plant revealed little overlap with this community, we regard our results as reliably representing the bacterial community that is associated with P. ferrugineus. Genera with a likely function as ant symbionts were Burkholderia, Pantoea, Weissella, and several members of the Enterobacteriaceae. The presence of these and various other groups was confirmed via independent PCR and cultivation approaches. Many of the bacteria that we detected belong to purportedly N-fixing taxa. Bacteria may represent important further partners in ant-plant mutualisms, and their influences on ant nutrition can contribute to the extraordinary abundance and evolutionary success of tropical arboreal ants.

Spatial and temporal variability in a stratified hypersaline microbial mat community

Hypersaline microbial mat communities have recently been shown to be more diverse than once thought. The variability in community composition of hypersaline mats, both in terms of spatial and temporal dimensions, is still poorly understood. Because this information is essential to understanding the complex biotic and abiotic interactions within these communities, terminal restriction fragment analysis and 16S rRNA gene sequencing were used to characterize the near-surface community of a hypersaline microbial mat in Guerrero Negro, Mexico. Core samples were analyzed to assay community variability over large regional scales (centimeter to kilometer) and to track depth-related changes in population distribution at 250-microm intervals over a diel period. Significant changes in total species diversity were observed at increasing distances across the mat surface; however, key species (e.g. Microcoleus sp.) were identified throughout the mat. The vertical position and abundance of >50% of the 60 peaks detected varied dramatically over a diel cycle, including Beggiatoa sp., cyanobacteria, Chloroflexus sp., Halochromatium sp., Bacteroidetes sp. and several as-yet-identified bacteria. Many of these migrations correlated strongly with diel changes in redox conditions within the mat, contributing to strong day-night community structure differences.