Concerted action of ataxin-2 and PABPC1-bound mRNA poly(A) tail in the formation of stress granules.

Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.

Age-related telomere attrition in the human putamen.

Age is a major risk factor for neurodegenerative diseases. Shortening of leucocyte telomeres with advancing age, arguably a measure of "biological" age, is a known phenomenon and epidemiologically correlated with age-related disease. The main mechanism of telomere shortening is cell division, rendering telomere length in post-mitotic cells presumably stable. Longitudinal measurement of human brain telomere length is not feasible, and cross-sectional cortical brain samples so far indicated no attrition with age. Hence, age-related changes in telomere length in the brain and the association between telomere length and neurodegenerative diseases remain unknown. Here, we demonstrate that mean telomere length in the putamen, a part of the basal ganglia, physiologically shortens with age, like leukocyte telomeres. This was achieved by using matched brain and leukocyte-rich spleen samples from 98 post-mortem healthy human donors. Using spleen telomeres as a reference, we further found that mean telomere length was brain region-specific, as telomeres in the putamen were significantly shorter than in the cerebellum. Expression analyses of genes involved in telomere length regulation and oxidative phosphorylation revealed that both region- and age-dependent expression pattern corresponded with region-dependent telomere length dynamics. Collectively, our results indicate that mean telomere length in the human putamen physiologically shortens with advancing age and that both local and temporal gene expression dynamics correlate with this, pointing at a potential mechanism for the selective, age-related vulnerability of the nigro-striatal network.

Increased fidelity of protein synthesis extends lifespan.

Loss of proteostasis is a fundamental process driving aging. Proteostasis is affected by the accuracy of translation, yet the physiological consequence of having fewer protein synthesis errors during multi-cellular organismal aging is poorly understood. Our phylogenetic analysis of RPS23, a key protein in the ribosomal decoding center, uncovered a lysine residue almost universally conserved across all domains of life, which is replaced by an arginine in a small number of hyperthermophilic archaea. When introduced into eukaryotic RPS23 homologs, this mutation leads to accurate translation, as well as heat shock resistance and longer life, in yeast, worms, and flies. Furthermore, we show that anti-aging drugs such as rapamycin, Torin1, and trametinib reduce translation errors, and that rapamycin extends further organismal longevity in RPS23 hyperaccuracy mutants. This implies a unified mode of action for diverse pharmacological anti-aging therapies. These findings pave the way for identifying novel translation accuracy interventions to improve aging.

Selective Elimination of Osteosarcoma Cell Lines with Short Telomeres by Ataxia Telangiectasia and Rad3-Related Inhibitors.

To avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of 17 osteosarcoma-derived cell lines, defining three separate groups according to TMM and the length of telomeres maintained. Eight were ALT-positive, including the previously uncharacterized lines, KPD and LM7. While ALT-positive lines all showed excessive telomere length, ALT-negative cell lines fell into two groups according to their telomere length: HOS-MNNG, OHSN, SJSA-1, HAL, 143b, and HOS displayed subnormally short telomere length, while MG-63, MHM, and HuO-3N1 displayed long telomeres. Hence, we further subcategorized ALT-negative TMM into long-telomere (LT) and short-telomere (ST) maintenance groups. Importantly, subnormally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the protein kinase ataxia telangiectasia and Rad3-related (ATR) (AZD-6738/Ceralasertib, VE-822/Berzoserib, and BAY-1895344) compared to long telomeres maintained via ALT or telomerase. Within 24 h of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between the mode of telomere maintenance and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR inhibitor sensitivity in cancer.

How long does telomerase extend telomeres? Regulation of telomerase release and telomere length homeostasis.

Telomerase, the enzyme that replenishes telomeres, is essential for most eukaryotes to maintain their generations. Telomere length homeostasis is achieved via a balance between telomere lengthening by telomerase, and erosion over successive cell divisions. Impaired telomerase regulation leads to shortened telomeres and can cause defects in tissue maintenance. Telomeric DNA is composed of a repetitive sequence, which recruits the protective protein complex, shelterin. Shelterin, together with chromatin remodelling proteins, shapes the heterochromatic structure at the telomere and protects chromosome ends. Shelterin also provides a foothold for telomerase to be recruited and facilitates telomere extension. Such mechanisms of telomere recruitment and activation are conserved from unicellular eukaryotes to humans, with the rate of telomere extension playing an important role in determining the length maintained. Telomerase can be processive, adding multiple telomeric repeats before dissociating. However, a question remains: how does telomerase determine the number of repeats to add? In this review, I will discuss about how telomerase can monitor telomere extension using fission yeast as a model. I propose a model whereby the accumulation of the Pot1 complex on the synthesised telomere single-strand counteracts retention of telomerase via chromatin proteins and the similar system may be conserved in mammals.

LARP7 family proteins have conserved function in telomerase assembly.

Understanding the intricacies of telomerase regulation is crucial due to the potential health benefits of modifying its activity. Telomerase is composed of an RNA component and reverse transcriptase. However, additional factors required during biogenesis vary between species. Here we have identified fission yeast Lar7 as a member of the conserved LARP7 family, which includes the Tetrahymena telomerase-binding protein p65 and human LARP7. We show that Lar7 has conserved RNA-recognition motifs, which bind telomerase RNA to protect it from exosomal degradation. In addition, Lar7 is required to stabilise the association of telomerase RNA with the protective complex LSm2-8, and telomerase reverse transcriptase. Lar7 remains a component of the mature telomerase complex and is required for telomerase localisation to the telomere. Collectively, we demonstrate that Lar7 is a crucial player in fission yeast telomerase biogenesis, similarly to p65 in Tetrahymena, and highlight the LARP7 family as a conserved factor in telomere maintenance.

Fission yeast Ccq1 is a modulator of telomerase activity.

Shelterin, the telomeric protein complex, plays a crucial role in telomere homeostasis. In fission yeast, telomerase is recruited to chromosome ends by the shelterin component Tpz1 and its binding partner Ccq1, where telomerase binds to the 3' overhang to add telomeric repeats. Recruitment is initiated by the interaction of Ccq1 with the telomerase subunit Est1. However, how telomerase is released following elongation remains to be established. Here, we show that Ccq1 also has a role in the suppression of telomere elongation, when coupled with the Clr4 histone H3 methyl-transferase complex and the Clr3 histone deacetylase and nucleosome remodelling complex, SHREC. We have dissected the functions of Ccq1 by establishing a Ccq1-Est1 fusion system, which bypasses the telomerase recruitment step. We demonstrate that Ccq1 forms two distinct complexes for positive and negative telomerase regulation, with Est1 and Clr3 respectively. The negative form of Ccq1 promotes dissociation of Ccq1-telomerase from Tpz1, thereby restricting local telomerase activity. The Clr4 complex also has a negative regulation activity with Ccq1, independently of SHREC. Thus, we propose a model in which Ccq1-Est1 recruits telomerase to mediate telomere extension, whilst elongated telomeric DNA recruits Ccq1 with the chromatin-remodelling complexes, which in turn releases telomerase from the telomere.

The telomere bouquet facilitates meiotic prophase progression and exit in fission yeast.

During meiotic prophase, chromosome arrangement and oscillation promote the pairing of homologous chromosomes for meiotic recombination. This dramatic movement involves clustering of telomeres at the nuclear membrane to form the so-called telomere bouquet. In fission yeast, the telomere bouquet is formed near the spindle pole body (SPB), which is the microtubule organising centre, functionally equivalent to the metazoan centrosome. Disruption of bouquet configuration impedes homologous chromosome pairing, meiotic recombination and spindle formation. Here, we demonstrate that the bouquet is maintained throughout meiotic prophase and promotes timely prophase exit in fission yeast. Persistent DNA damages, induced during meiotic recombination, activate the Rad3 and Chk1 DNA damage checkpoint kinases and extend the bouquet stage beyond the chromosome oscillation period. The auxin-inducible degron system demonstrated that premature termination of the bouquet stage leads to severe extension of prophase and consequently spindle formation defects. However, this delayed exit from meiotic prophase was not caused by residual DNA damage. Rather, loss of chromosome contact with the SPB caused delayed accumulation of CDK1-cyclin B at the SPB, which correlated with impaired SPB separation. In the absence of the bouquet, CDK1-cyclin B localised near the telomeres but not at the SPB at the later stage of meiotic prophase. Thus, bouquet configuration is maintained throughout meiotic prophase, by which this spatial organisation may facilitate local and timely activation of CDK1 near the SPB. Our findings illustrate that chromosome contact with the nuclear membrane synchronises meiotic progression of the nucleoplasmic chromosomes with that of the cytoplasmic SPB.

Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.

Association between the body mass index and the clinical findings in patients with acute heart failure: evaluation of the obesity paradox in patients with severely decompensated acute heart failure.

Obesity is known to be associated with the development of heart failure (HF). However, the relationship between the body mass index (BMI) and acute HF (AHF) remains to be elucidated. Eight hundred and eight AHF patients were enrolled in this study. The patients were assigned to four groups according to their BMI values: severely thin (n = 11, BMI <16), normal/underweight (n = 579, 16 ≤ BMI <25), overweight (n = 178, 25 ≤ BMI <30) and obese (n = 40, BMI ≥30). The patients in the severely thin group were more likely to be female, have systolic blood pressure (SBP) <100 mmHg and have valvular disease than normal/underweight patients. The patients in the overweight group were significantly younger than those in the normal/underweight, and those in the overweight group were more likely to have SBP ≥140 mmHg and hypertensive heart disease and less likely to have valvular disease than the patients in the normal/underweight group. The prognosis, including all-cause death, was significantly poorer among patients who were severely thin than those who were normal/underweight, overweight and significantly better among those who were overweight than those who were normal/underweight, severely thin and obese patients. A multivariate Cox regression model identified that severely thin [HR: 3.372, 95% confidence interval (CI) 1.362-8.351] and overweight (HR: 0.615, 95% CI 0.391-0.966) were independent predictors of 910-day mortality as the reference of normal/underweight. Overweight patients tended to have SBP ≥140 mmHg and be relatively young, while severely thin patients tended to have SBP <100 mmHg and be female. These factors were associated with a better prognosis of overweight patients and adverse outcomes in severely thin patients. These factors may contribute to the "obesity paradox" in severely decompensated AHF patients.

Sequential and counter-selectable cassettes for fission yeast.

BACKGROUND: Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes. RESULTS: Here we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2'-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method. CONCLUSIONS: The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study.

The serum heart-type fatty acid-binding protein (HFABP) levels can be used to detect the presence of acute kidney injury on admission in patients admitted to the non-surgical intensive care unit.

BACKGROUND: No cardiac biomarkers for detecting acute kidney injury (AKI) on admission in non-surgical intensive care patients have been reported. The aim of the present study is to elucidate the role of cardiac biomarkers for quickly identifying the presence of AKI on admission. METHODS: Data for 1183 patients who underwent the measurement of cardiac biomarkers, including the serum heart-type fatty acid-binding protein (s-HFABP) level, in the emergency department were screened, and 494 non-surgical intensive care patients were enrolled in this study. Based on the RIFLE classification, which was the ratio of the serum creatinine value recorded on admission to the baseline creatinine value, the patients were assigned to a no-AKI (n = 349) or AKI (Class R [n = 83], Class I [n = 36] and Class F [n = 26]) group on admission. We evaluated the diagnostic value of the s-H-FABP level for detecting AKI and Class I/F. The mid-term prognosis, as all-cause death within 180 days, was also evaluated. RESULTS: The s-H-FABP levels were significantly higher in the Class F (79.2 [29.9 to 200.3] ng/mL) than in the Class I (41.5 [16.7 to 71.6] ng/mL), the Class R (21.1 [10.2 to 47.9] ng/mL), and no-AKI patients (8.8 [5.4 to 17.7] ng/mL). The most predictive values for detecting AKI were Q2 (odds ratio [OR]: 3.743; 95 % confidence interval [CI]: 1.693-8.274), Q3 (OR: 9.427; 95 % CI: 4.124-21.548), and Q4 (OR: 28.000; 95 % CI: 11.245-69.720), while those for Class I/F were Q3 (OR: 5.155; 95 % CI: 1.030-25.790) and Q4 (OR: 22.978; 95 % CI: 4.814-109.668). The s-HFABP level demonstrating an optimal balance between sensitivity and specificity (70.3 and 72.8 %, respectively; area under the curve: 0.774; 95 % CI: 0.728-0.819) was 15.7 ng/mL for AKI and 20.7 ng/mL for Class I/F (71.0 and 83.1 %, respectively; area under the curve: 0.818; 95 % CI: 0.763-0.873). The prognosis was significantly poorer in the high serum HFABP with AKI group than in the other groups. CONCLUSIONS: The s-H-FABP level is an effective biomarker for detecting AKI in non-surgical intensive care patients.

Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.

Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Telomere protein Rap1 is a charge resistant scaffolding protein in chromosomal bouquet formation.

BACKGROUND: Chromosomes reorganize in early meiotic prophase to form the so-called telomere bouquet. In fission yeast, telomeres localize to the nuclear periphery via interaction of the telomeric protein Rap1 with the membrane protein Bqt4. During meiotic prophase, the meiotic proteins Bqt1-2 bind Rap1 and tether to the spindle pole body to form the bouquet. Although it is known that this polarized chromosomal arrangement plays a crucial role in meiotic progression, the molecular mechanisms of telomere bouquet regulation are poorly understood. RESULTS: Here, we detected high levels of Rap1 phospho-modification throughout meiotic prophase, and identified a maximum of 35 phosphorylation sites. Concomitant phosphomimetic mutation of the modification sites suggests that Rap1 hyper-phosphorylation does not directly regulate telomere bouquet formation or dissociation. Despite the negative charge conferred by its highly phosphorylated state, Rap1 maintains interactions with its binding partners. Interestingly, mutations that change the charge of negatively charged residues within the Bqt1-2 binding site of Rap1 abolished the affinity to the Bqt1-2 complex, suggesting that the intrinsic negative charge of Rap1 is crucial for telomere bouquet formation. CONCLUSIONS: Whereas Rap1 hyper-phosphorylation observed in meiotic prophase does not have an apparent role in bouquet formation, the intrinsic negative charge of Rap1 is important for forming interactions with its binding partners. Thus, Rap1 is able to retain bouquet formation under heavily phosphorylated status.

Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation.

Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere-centrosome contact instead of telomere-centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks.

Serum heart-type fatty acid-binding protein level can be used to detect acute kidney injury on admission and predict an adverse outcome in patients with acute heart failure.

BACKGROUND: Different mechanisms of acute kidney injury (AKI) may exist for acute heart failure (AHF) patients compared with other patients. METHODS AND RESULTS: We analyzed data from 282 patients with AHF. The biomarkers were measured within 30 min of admission. Patients were assigned to a no-AKI (n=213) or AKI group (Class R (n=49), Class I (n=15) or Class F (n=5)) using the RIFLE classifications on admission. We evaluated the relationships between the biomarkers and AKI, in-hospital mortality, all-cause death and HF events (HF re-admission, all-cause death) within 90 days. The serum heart-type fatty acid-binding protein (s-HFABP) levels were significantly higher in the AKI than in the no-AKI group, and the predictive biomarker for AKI was s-HFABP (odds ratio: 6.709; 95% confidence interval: 3.362-13.391). s-HFABP demonstrated an optimal balance between sensitivity and specificity (71.0%, 79.3%; area under the receiver-operating characteristic curve [AUC]=0.790) at 22.8 ng/ml for AKI, at 22.8 ng/ml for Class I/F (90.0%, 71.4%; AUC=0.836) and at 21.0 ng/ml for in-hospital mortality (74.3%, 70.0%; AUC=0.726). The Kaplan-Meier survival curves showed a significantly poorer prognosis in the high s-HFABP group (≥22.9 ng/ml) than in other groups. CONCLUSIONS: The s-HFABP level can indicate AKI on admission, and a high s-HFABP level is associated with a poorer prognosis for AHF patients.

Seasonal variation in patients with acute heart failure: prognostic impact of admission in the summer.

There have been few reports discussing the clinical significance of the season of admission of acute heart failure (AHF) patients. The data of 661 patients with AHF admitted to the intensive care unit were analyzed. Patients were assigned to a summer admission (Group-S, n = 113, between July and September), a winter admission (Group-W, n = 214, between December and February), or to the other seasons admission group (Group-O, n = 334). We evaluated the relationships between the seasonal variations and the clinical profiles, and the long-term prognosis. There were significantly more patients with cardiomyopathy and New York Heart Association class 4, and the serum levels of total bilirubin were significantly higher in Group-S (85.8, 24.8 %, and 0.60 [0.50-0.90]) than in Group-W (75.2, 15.4 %, and 0.60 [0.40-0.78]). The left ventricular ejection fraction on admission was significantly reduced and intravenous administration of dobutamine was used more frequently in Group-S (30.0 [25.0-46.0], 31.9 %) than in Group-W (34.4 [25.2-48.0], 20.6 %) and Group-O (35.0 [25.0-46.0], 19.8 %). The multivariate Cox regression model found that summer admission was independently associated with cardiovascular death (HR: 1.58, 95 % CI 1.01-2.48; p = 0.044) and heart failure (HF) events (HR: 1.55, 95 % CI 1.05-2.28; p = 0.028). The Kaplan-Meier curves showed that the cardiovascular death rate was significantly higher in Group-S than in Group-W and Group-O, and the HF events were significantly higher in Group-S than in Group-O. The summer admission AHF patients included sicker patients, and the prognosis in these patients was worse than in the patients admitted at other times.

Telomerase activation after recruitment in fission yeast.

Current models depict that telomerase recruitment equates to activation. Telomeric DNA-binding proteins and the telomerase accessory proteins coordinate the recruitment of telomerase to the ends of chromosomes in a telomere length- and cell-cycle-dependent manner [1-4]. Recent studies have demonstrated that the telomeric protein TPP1 and its binding protein TIN2 are key proteins for both telomerase recruitment and processivity in mammalian cells [5-7]. Although the precise molecular mechanism of telomerase recruitment has not yet been established, targeted point mutations within the oligonucleotide/oligosaccharide-binding (OB)-fold domain of TPP1 have been shown to impair telomerase association and processivity [8-10]. In fission yeast, telomerase is recruited through an interaction between the telomerase subunit Est1 and Ccq1, a component of the Pot1-Tpz1 telomere complex (POT1-TPP1 orthologs) [11-15]. Here, we demonstrate that association of telomerase with telomeres does not engage activity. We describe a mutation of Tpz1 that causes critical telomere shortening despite telomeric accumulation of the telomerase catalytic subunit, Trt1. Furthermore, Est1-directed telomerase association with Ccq1 is transient, and the Est1-Ccq1 interaction does not remain the bridge between telomeres and telomerase. Rather, direct interaction of Trt1 with Tpz1 is critical for telomere elongation. Moreover, Ccq1, which has been well characterized as a telomerase recruiter, is also required for the activation of telomere-associated telomerase. Our findings reveal a layer of telomerase regulation that controls activity after recruitment.

Prognostic impact of the serum heart-type fatty acid-binding protein (H-FABP) levels in patients admitted to the non-surgical intensive care unit.

BACKGROUND: Biomarkers predicting adverse outcomes in non-surgical intensive care patients have not been reported. METHODS AND RESULTS: Data for 1,006 emergency department patients were prospectively analyzed. The serum heart-type fatty acid-binding protein (s-H-FABP) level was measured within 10 min of admission. The patients were assigned to intensive care (n = 835) or other departments (n = 171). The intensive care patients were divided into survivors (n = 745) and non-survivors (n = 90) according to the in-hospital mortality and assigned to four groups according to the quartiles of s-H-FABP (Q1, Q2, Q3 and Q4). The s-H-FABP levels were significantly higher in the intensive care patients (12.7 [6.1-38.8] ng/ml versus 5.3 [3.1-9.4] ng/ml) and in the non-survivors (44.9 [23.2-87.6] ng/ml versus 11.5 [5.6-32.6] ng/ml). A Kaplan-Meier curve showed a significantly higher survival rate in Q3 than in Q1 and Q2 and in Q4 than in the other groups. The multivariate Cox regression model identified Q3 (HR 4.646, 95 % CI 1.526-14.146) and Q4 (HR 9.483, 95 % CI 3.152-28.525) as independent predictors of 90-day mortality. The sensitivity and specificity of H-FABP for in-hospital mortality were 81.1 and 66.0 % (AUC 0.775) at 20.95 ng/ml. The in-hospitality rate was significantly higher in the high s-H-FABP patients than in the low s-H-FABP patients in each etiology group. CONCLUSIONS: The s-H-FABP level is an effective biomarker for risk stratification in non-surgical intensive care patients.