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Background: Hospitalization presents an opportunity to begin people with opioid use disorder (OUD) on medications for opioid use disorder (MOUD) and link them to care after discharge; regrettably, people admitted to the hospital with an underlying OUD typically do not receive MOUD and are not connected with subsequent treatment for their condition. To address this gap, we launched a multi-site randomized controlled trial to test the effectiveness of a hospital-based addiction consultation team (the Substance Use Treatment and Recovery Team (START)) consisting of an addiction medicine specialist and care manager team that provide collaborative care and a specified intervention to people with OUD during the inpatient stay. Successful implementation of new practices can be impacted by organizational context, though no previous studies have examined context prior to implementation of addiction consultation services (ACS). This study assessed pre-implementation context for implementing a specialized ACS and tailoring it accordingly., Methods: We conducted semi-structured interviews with hospital administrators, physicians, physician assistants, nurses, and social workers at the three study sites between April and August 2021 before the launch of the pragmatic trial. Using an analytical framework based on the Consolidated Framework for Implementation Research, we completed a thematic analysis of interview data to understand potential barriers or enablers and perceptions about acceptability and feasibility., Results: We interviewed 28 participants across three sites. The following themes emerged across sites: (1) START is an urgently needed model for people with OUD; (2) Intervention adaptations are recommended to meet local and cultural needs; (3) Linking people with OUD to community clinicians is a highly needed component of START; (4) It is important to engage stakeholders across departments and roles throughout implementation. Across sites, participants generally saw a need for change from usual care to support people with OUD, and thought the START was acceptable and feasible to implement. Differences among sites included tailoring the START to support the needs of varying patient populations and different perceptions of the prevalence of OUD., Conclusions: Hospitals planning to implement an ACS in the inpatient setting may wish to engage in a systematic pre-implementation contextual assessment using a similar framework to understand and address potential barriers and contextual factors that may impact implementation. Pre-implementation work can help ensure the ACS and other new practices fit within each unique hospital context., (© 2024. The Author(s).)

The foraging activity of honey bees used to pollinate almonds was examined in relation to their hive entrance orientation and colony strength. Twenty-four colonies of honey bees, twelve in each group, were situated with their entrances facing east and west cardinal points. Bee out counts were recorded continuously and hive weight data at ∼10 min intervals from 17 February to 15 March 2023. Colony strength was assessed using the frames of adult bees (FOB) metric. East-facing hives started flight 44.2 min earlier than west-facing hives. The hive direction did not affect the timing of the cessation of foraging activity. The hive strength played a significant role: hives assessed as weak (≤3.0 FOB) commenced foraging activity 45 min later than strong hives (>3.0 FOB) and ceased foraging activity 38.3 min earlier. Hive weight data did not detect effects of either the hive direction or colony strength on the commencement and cessation of foraging activity, as determined using piecewise regression on 24 h datasets. However, the hive weight loss due to foraging activity at the start of foraging activity was significantly affected by both direction (East > West) and colony strength (Strong > Weak). Our study showed that, during almond pollination, both hive entrance exposure and hive strength have quantifiable effects on colony foraging behaviour and that these effects combine to regulate the overall foraging activity of the pollinating colonies.

Primary care is an opportune setting to deliver treatments for co-occurring substance use and mental health disorders; however, treatment delivery can be challenging due multi-level implementation barriers. Documenting organizational context can provide insight into implementation barriers and the adaptation of new processes into usual care workflows. This study surveyed primary care and behavioral health staff from 13 clinics implementing a collaborative care intervention for opioid use disorders co-occurring with PTSD and/or depression as part of a multisite randomized controlled trial. A total of 323 completed an online survey for a 60% response rate. The Consolidated Framework for Implementation Research guided this assessment of multi-level factors that influence implementation. Most areas for improvement focused on inner setting (organizational level) constructs whereas individual-level constructs tended to be strengths. This work addresses a research gap regarding how organizational analyses can be used prior to implementation and provides practical implications for researchers and clinic leaders., (© 2023. The Author(s).)

Introduction: The utility of cell block (CB) preparation is well established in cytopathology. Despite 23.3% of College of American Pathologists-accredited laboratories using CB with liquid-based preparations on urine cytology (UC) cases, there are very few studies on their performance. To determine their usefulness, we conducted a retrospective review of UC cases that received CB., Materials and Methods: We identified 27 UC cases with ThinPrep (TP) and CB preparation between 2016 and 2020 at our institution. Clinical history and follow-up data were compiled. A blinded review of TP alone, and TP together with CB, was performed by 2 pathologists and 2 cytotechnologists. Diagnoses were rendered in accordance with The Paris System for Reporting Urine Cytology., Results: Blood and acute inflammation were common background elements in cases that received CB preparation. In total, CB upgraded the diagnosis in 7 of 27 cases (26%). The maximum utility of CB preparation was seen in indeterminate cases where 60% (6 of 10) were upgraded, including 71% (5 of 7) of atypical urothelial cells (AUC) and 30% (1 of 3) of suspicious for high-grade urothelial carcinoma (HGUC). One case (1 of 12, 8%) diagnosed as negative for HGUC on TP was diagnosed as low-grade urothelial neoplasia on CB., Conclusions: Our results demonstrate that adjunct use of CB preparation aids in a definitive diagnosis on AUC category and may be helpful in cases with cell clusters or tissue fragments, or cases suspicious for HGUC. Further correlation studies are warranted in this area to expand our knowledge about the utility of CBs in urine cytology., (Copyright © 2020 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Methods: Develop a new version of the Activity Card Sort-Advancing Inclusive Participation to include occupations experienced by the homeless population, including nonsanctioned occupations. This study occurred in two phases: (1) tool development (item selection, content expert review, line development drawing, and assessment of content validity) and (2) tool use to determine face validity. Participants were selected through a convenience sample at a local homeless shelter and academic institution. Participants experiencing homelessness (phase 1: N = 13, phase 2: N = 10) were required to be seeking services at the homeless shelter, while nonhomeless participants (phase 2: N = 30) worked full-time, resided with a significant other, and had personal transportation., Results: An assessment of 76 occupations, corresponding line drawings, and follow-up questions was created. An initial construct validity study demonstrated differences between occupational participation of those who are homeless and nonhomeless in the areas of social engagement, nonsanctioned occupations, work and education, and home management. Both groups reported previous, current, or desired engagement in the occupations identified in the assessment. Conclusions and relevance. The purpose of this study was to create an inclusive assessment for use in the homeless population and complete a construct validity study of the assessment tool. Although the results indicated some differences in the frequency with which occupations were performed, the results demonstrated that all individuals participate in occupations that many not contribute to their health and wellness. This initial work supports the future development of a tool that is inclusive of all occupations to obtain a holistic picture of an individual's participation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Quinn P. Tyminski et al.)

Serine integrases have been shown to be efficient tools for metabolic pathway assembly. To further improve the flexibility and efficiency of pathway engineering via serine integrases, we explored how multiple orthogonally active serine integrases can be applied for use in vitro for the heterologous expression of complex biosynthesis pathways in Streptomyces spp., the major producers of useful bioactive natural products. The results show that multiple orthogonal serine integrases efficiently assemble the genes from a complex biosynthesis pathway in a single in vitro recombination reaction, potentially permitting a versatile combinatorial assembly approach. Furthermore, the assembly strategy also permitted the incorporation of a well-characterised promoter upstream of each gene for expression in a heterologous host. The results demonstrate how site-specific recombination based on orthogonal serine integrases can be applied in Streptomyces spp., (© 2020 KeAi Communications Co.(+) Ltd.)

Neuroblastoma represents approximately 6 to 10 percent of childhood cancers, yet is one of the most common solid tumors observed in neonates; approximately 700 cases are reported in the United States each year. Neuroblastoma occurs secondary to oncogene mutations that cause abnormal proliferation of neural crest cells and tumor formation anywhere along the spinal cord. Visible manifestations include a blueberry rash and subcutaneous skin nodules. Common histologic findings include multifocal, small, round, blue cell tumors. Cytogenetics testing differentiates aggressive versus nonaggressive forms of neuroblastoma. Treatment ranges from supportive care to surgery and chemotherapy; targeted molecular therapies and immunotherapy offer opportunity to individualize treatment. Morbidity and mortality are contingent upon age at diagnosis and genetic abnormalities. Neonatal clinicians must establish and maintain active knowledge of the current science pertaining to this neoplasm to assist in early identification and timely initiation of medical management. This article presents a case report and comprehensive discussion of the state of the science on metastatic familial (congenital) neuroblastoma., (© Copyright 2019 Springer Publishing Company, LLC.)

Agro-wastes such as sugar cane bagasse can be explored for use in different aspects. Its applicability as a source of cellulose has attracted much interests especially in biomedical field among various applications. In the current work chemically purified cellulose (CPC) and cellulose nanocrystals (CNCs) were effectively extracted from sugarcane bagasse (SCB). The cellulose was obtained by chemical treatment of SCB using HNO 3 , NaOH and a bleaching agent. Nanocrystals were further prepared from the extracted cellulose using H 2 SO 4 hydrolysis followed by washing with deionized water and acetone. The obtained materials were characterized for surface morphological using Fourier transform infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) analysis. The thermal properties were evaluated using TGA/DTG. The FTIR showed the disappearance of the peaks responsible for the hemicelluloses and lignin. These results were confirmed by TGA which proved gradual elimination of non-cellulosic constituents. X-ray Diffractometer depicted an increase in crystallinity occasioned by sequential treatments to get the cellulose nanocrystals. Cellulose nanocrystals had a spherical shape with a diameter of 38nm as compared to the chemically purified cellulose which had a diameter of 76nm. The CNCs prepared with this method were seen to be less agglomerated and more crystalline thus possess a higher potential as bionanocomposite either for biomedical applications or for wastewater treatment among other industrial application. This approach also provides an opportunity for the sugar companies to effectively manage their waste product., (© 2019 The Authors.)

Background: Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify more integrating vectors, using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569., Results: Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from Escherichia coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing., Conclusions: The homologous TG1 and R4 attB sites within the genus Amycolatopsis have been identified. The results indicate that vectors based on TG1 and R4 integrases could be widely applicable in this genus.

Purpose: Iron deficiency is prevalent in idiopathic pulmonary arterial hypertension (IPAH), but whether iron deficiency or ferritin levels are associated with pulmonary hypertension (PH) in the general population is unknown., Methods: We performed a cross-sectional analysis of data on iron deficiency (exposure), and PH (pulmonary artery systolic pressure>40mmHg on echocardiogram) (outcome) on subjects with complete data on exposures and outcomes as well as covariates (n = 2,800) enrolled in the Jackson Heart Study, a longitudinal prospective observational cohort study of heart disease in African-Americans from Jackson, Mississippi. Iron deficiency was defined as a serum ferritin level < 15ng/mL (females); < 30ng/mL (males). We determined crude prevalence ratios (PRs) for PH in iron deficient versus non-iron deficient groups using modified Poisson regression modeling. We also analyzed the prevalence of PH by sex-specific quartiles of ferritin (Females ≤ 47ng/mL; > 47ng/mL- 95ng/mL; > 95ng/mL- 171ng/mL; > 171ng/mL; Males ≤ 110ng/mL; > 110ng/mL- 182ng/mL; > 182ng/mL- 294ng/mL; > 294ng/mL), using the same modeling technique with the lowest quartile as the referent., Results: Median pulmonary artery systolic pressure was 27mmHg (interquartile range 23-31mmHg) in the study cohort. 147 subjects (5.2%) had PH and 140 (5.0%) had iron deficiency. However, of the 147 subjects with PH, only 4 were also iron deficient. The crude PH PR was 0.5 (95% CI 0.2-1.4) in iron-deficiency compared to non-deficient. In analysis by quartiles of ferritin, adjusting for age and sex, there was no evidence of association with PH in quartiles 2 (PR 1.1, 95% CI 0.7-1.6), 3 (PR 0.8, 95% CI 0.5-1.3), or 4 (PR 0.8, 95% CI 0.5-1.2) compared with quartile 1 (referent group, PR 1). Further analyses of the relationship between PH and ferritin as a log-transformed continuous variable or by quartiles of serum iron showed similar results., Conclusions: In the Jackson Heart Study, the prevalence of PH was similar in iron-deficient and non-iron deficient subjects. There was no evidence of association between ferritin (or serum iron) levels and PH., Clinical Implications: Iron deficiency has been associated with IPAH, a rare disorder. However, in a large community-based sample of African-Americans, there was no evidence that iron deficiency or low iron levels were associated with PH., Competing Interests: The authors have declared that no competing interests exist.

Cocaine is one of the most commonly abused drugs in the United States. Ingestion of cocaine may result in a wide array of disease processes due to its stimulant properties, contaminants, or to downstream effects, such as myo- cardial infarction, stroke, or cardiac arrest. Pulmonary complaints are common in patients seeking treatment for cocaine-associated medical problems and include acute eosinophilic pneumonia, pneumothorax, pneumomediastium, diffuse alveolar hemorrhage (DAH), pulmonary hypertension and granulomatosis. We present a case of DAH due to cocaine abuse and rapid resolution with mechanical ventilation and supportive care. [Full article available at http://rimed.org/rimedicaljournal-2016-08.asp, free with no login].

Introduction: The kidneys play a crucial role in the regulation of electrolytes and acid-base homeostasis. Urinary Strong Ion Difference (SIDu = NaU + KU-ClU) represents an important aspect of renal acid-base regulation. We evaluated the role of SIDu as a marker of renal dysfunction in critically ill patients., Materials and Methods: Patients admitted to the Medical Intensive Care Unit with a diagnosis of AKI for whom concomitant urinary samples available for SIDu calculation were retrospectively reviewed and staged according to KDIGO criteria for 3 days from inclusion. Patients were classified as Recovered (R-AKI) or Persistent-AKI (P-AKI) whether they exited KDIGO criteria within the 3-day observation period or not. A control group with normal renal function and normal serum acid-base and electrolytes was prospectively recruited in order to identify reference SIDu values., Results: One-hundred-and-forty-three patients with a diagnosis of AKI were included: 77 with R-AKI, and 66 with P-AKI. Thirty-six controls were recruited. Patients with P-AKI had more severe renal dysfunction and higher mortality than patients with R-AKI (SCr 2.23(IQR:1.68-3.45) and 1.81(IQR1.5-2.5) mg/dl respectively, p<0.001; 24-h UO 1297(950) and 2100(1094) ml respectively, p = 0.003); 30-d mortality, 39% and 13% respectively; p<0.001). SIDu significantly differed between groups, with rising values from controls to P-AKI groups (16.4(12), 30(24) and 47.3(21.5) mEq/l respectively, p<0.001)., Discussion: SIDu may be a simple and inexpensive tool in AKI patients' evaluation. Further research is needed to evaluate the ability of SIDu to identify patients with renal dysfunction before derangements in serum creatinine or urine output are observed.

The target of rapamycin complex 1 (TORC1) is a key conserved regulator of eukaryotic cell growth. The xenobiotic rapamycin is a potent inhibitor of the yeast complex. Surprisingly, the EGO complex, a nonessential in vivo activator of TORC1, is somehow required for yeast cells to recover efficiently from a period of treatment with rapamycin. Why? Here, we found that rapamycin is only a partial inhibitor of TORC1. We confirmed that saturating amounts of rapamycin do not fully inhibit proliferation of wild-type cells, and we found that the residual proliferation in the presence of the drug is dependent on the EGO complex and on the activity of TORC1. We found that this residual TORC1-dependent proliferation is key to recovery from rapamycin treatment. First, the residual proliferation rate correlates with the ability of cells to recover from treatment. Second, the residual proliferation rate persists long after washout of the drug and until cells recover. Third, the total observable pool of cell-associated rapamycin is extremely stable and decreases only with increasing cell number after washout of the drug. Finally, consideration of the residual proliferation rate alone accurately and quantitatively accounts for the kinetics of recovery of wild-type cells and for the nature and severity of the ego- mutant defect. Overall, our results revealed that rapamycin is a partial inhibitor of yeast TORC1, that persistence of the drug limits recovery, and that rapamycin is not detoxified by yeast but is passively diluted among progeny cells because of residual proliferation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose: Totally implantable venous access devices (Ports) represent the mainstay for infusion therapy in patients undergoing chemotherapy, total parenteral nutrition and/or long-term antibiotic treatment. Amongst mechanical complications, lesions of the catheter wall represent a rare but potentially severe condition. We report our experience with the accidental detection of catheter ruptures in a series of ports removed for complication or for end of use., Methods: All ports removed from January 2011 to June 2013 were considered. All removed ports had been inserted according to a standardized protocol including ultrasound-guided percutaneous venipuncture (out-of-plane or in-plane approaches) and electrocardiogram-guided positioning of the tip. Once removed, each catheter was checked by inspection and saline instillation in order to evaluate the integrity of the device itself and rule out possible ruptures., Results: In over 338 removed ports, 12 Groshong catheters out of 65 (18.5%) had evidence of partial rupture of the catheter wall. Amongst considered variables, "out-of-plane" approach and type of port (silicon, closed tip with Groshong valve) were the only ones significantly associated with catheter ruptures (p=0.0003 and 0.0008, respectively). We could detect no evidence of rupture in any silicon open-ended catheter (Celsite ports) or in any catheter inserted by "in-plane" approach to the vein., Conclusions: The actual advantage of using port connected with Groshong silicon catheters should be questioned, since apparently they are more fragile than standard catheters. Furthermore, ultrasound-guided "out-of-plane" puncture of the internal jugular vein should be discouraged.

Purpose: The impact of a focused education initiative to increase pharmacists' documentation of clinical interventions is described., Summary: A focused education initiative was developed to increase the consistency of pharmacists' documentation of clinical interventions in order to achieve pharmacy goals and to demonstrate the value of pharmacy services at Carolinas Medical Center in Charlotte, North Carolina. Education was provided through weekly pharmacy newsletter publications, weekly huddles, and monthly staff meetings. Pharmacy clinical specialists were tasked with providing examples of activities that should be documented as interventions, reviewing the selection of intervention categories to best capture the specific activity, and emphasizing the need for appropriate documentation. Monthly progress was monitored, and results were posted publicly to incentivize staff and assist with accountability. Increases in the number of clinical interventions was reported at monthly pharmacy staff meetings to reinforce the value of this process. The total number of pharmacy clinical interventions increased from an average of 12,493 per month in the first quarter of 2012 to an average of 27,978 per month in the second quarter of 2013, representing a 120% improvement. Associated cost-avoidance dollars also increased proportionally, such that the average cost-avoidance dollars in the second quarter of 2013 was $1.5 million per month. In addition, the pharmacy department far exceeded the health system's division of pharmacy targets for established quality indicators. Effects were sustained during the 12 months after completion of the education initiative., Conclusion: Implementation of a focused pharmacist education initiative led to increased numbers of clinical interventions reported and increased documentation of costs avoided., (Copyright © 2014 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Genes that are highly expressed in cancer cells and are essential for their viability are attractive targets for the development of novel cancer therapeutics. Activating transcription factor 5 (ATF5) is an anti-apoptotic protein that is highly expressed in malignant glioma but not normal brain tissues, and is essential for glioma cell survival. Recent work has revealed an essential survival pathway mediated by ATF5 in malignant glioma; pharmacological inhibition of this pathway leads to tumor regression in mice. ATF5 is also highly expressed in a variety of other cancers, and preliminary studies have shown that the ATF5-mediated survival pathway is active in diverse human cancer cell lines. Targeting this pathway may therefore have therapeutic implications for the treatment of a wide range of cancers. In this perspective, we summarize recent advances in ATF5 research, focusing on its role in promoting cancer and its potential as a target for cancer therapy.

Acetophenones were screened for activity against positive phototaxis of Chlamydomonas cells, a process that requires co-ordinated flagellar motility. The structure-activity relationships of a series of acetophenones are reported, including acetophenones that affect flagellar motility and cell viability. Notably, 4-methoxyacetophenone, 3,4-dimethoxyacetophenone, and 4-hydroxyacetophenone induced negative phototaxis in Chlamydomonas, suggesting interference with activity of flagellar proteins and control of flagellar dominance.

The p53 tumor suppressor protein has long been recognized as the central factor protecting humans from cancer. It has been famously dubbed "the guardian of the genome" due to its ability to respond to genotoxic stress, such as DNA damage and other stress signals, and to protect the genome by inducing a variety of biological responses including DNA repair, cell cycle arrest, and apoptosis. However, the tumor suppressive effects of p53 go far beyond its roles in mediating these three processes. There is growing evidence that p53 also exerts its effects on multiple aspects of tumor formation, including suppression of metastasis and, as summarized in this review, inhibition of new blood vessel development (angiogenesis). The p53 protein has been shown to limit angiogenesis by at least three mechanisms: (1) interfering with central regulators of hypoxia that mediate angiogenesis, (2) inhibiting production of proangiogenic factors, and (3) directly increasing the production of endogenous angiogenesis inhibitors. The combination of these effects allows p53 to efficiently shut down the angiogenic potential of cancer cells. Inactivation of p53, which occurs in approximately half of all tumors, reverses these effects; as a consequence, tumors carrying p53 mutations appear more vascularized and are often more aggressive and correlate with poor prognosis for treatment. Thus, the loss of functional p53 during tumorigenesis likely represents an essential step in the switch to an angiogenic phenotype that is displayed by aggressive tumors.

The faithful execution of biological processes requires a precise and carefully orchestrated set of steps that depend on the proper spatial and temporal expression of genes. Here we review the various classes of transcriptional regulatory elements (core promoters, proximal promoters, distal enhancers, silencers, insulators/boundary elements, and locus control regions) and the molecular machinery (general transcription factors, activators, and coactivators) that interacts with the regulatory elements to mediate precisely controlled patterns of gene expression. The biological importance of transcriptional regulation is highlighted by examples of how alterations in these transcriptional components can lead to disease. Finally, we discuss the methods currently used to identify transcriptional regulatory elements, and the ability of these methods to be scaled up for the purpose of annotating the entire human genome.

The first step in transcriptional activation of protein-coding genes involves the assembly on the promoter of a large PIC (pre-initiation complex) comprising RNA polymerase II and a suite of general transcription factors. Transcription is greatly enhanced by the action of promoter-specific activator proteins (activators) that function, at least in part, by increasing PIC formation. Activator-mediated stimulation of PIC assembly is thought to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the 'target'. The unambiguous identification of direct, physiologically relevant in vivo targets of activators has been a considerable challenge in the transcription field. The major obstacle has been the lack appropriate experimental methods to measure direct interactions with activators in vivo. The development of spectral variants of green fluorescent protein has made it possible to perform FRET (fluorescence resonance energy transfer) analysis in living cells, thereby allowing the detection of direct protein-protein interactions in vivo. Here we discuss how FRET can be used to identify activator targets and to dissect in vivo mechanisms of transcriptional activation.

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.

The protective caps on chromosome ends - known as telomeres - consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.

EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

Cdc13 and Est1 are single-strand telomeric DNA binding proteins that contribute to telomere replication in the yeast Saccharomyces cerevisiae. Here it is shown that fusion of Cdc13 to the telomerase-associated Est1 protein results in greatly elongated telomeres. Fusion proteins consisting of mutant versions of Cdc13 or Est1 confer similar telomere elongation, indicating that close physical proximity can bypass telomerase-defective mutations in either protein. Fusing Cdc13 directly to the catalytic core of telomerase allows stable telomere maintenance in the absence of Est1, consistent with a role for Est1 in mediating telomerase access. Telomere length homeostasis therefore is maintained in part by restricting access of telomerase to chromosome termini, but this limiting situation can be overcome by directly tethering telomerase to the telomere.

Telomeres comprise a specialized chromosome end structure distinct from the standard nucleosomal architecture of the remainder of the genome. Telomere maintenance and chromosome stability require both replication of telomeric sequences by telomerase and telomeric end protection through binding of proteins. We have shown that Cdc13p and the heterodimer Ku are required, along with telomerase, for full telomere function, and we have proposed that Ku and Cdc13p contribute distinct roles in end protection. Ku has recently been shown to exhibit defects in transcriptional repression of telomere-proximal genes, known as telomere position effect (TPE), or telomeric silencing. We investigate here whether alterations in genes involved in the telomerase pathway also exhibit TPE defects and find that deletion or overexpression of EST1 or EST2 does not significantly affect telomeric silencing. However, telomeric silencing is derepressed upon overexpression of certain nonfunctional alleles of each. In addition, we determined that overproduction of telomerase pathway components partially alleviates the TPE defect in hdf1Delta cells. This indicates that there is genetic crosstalk between these two telomere maintenance pathways, and suggests that overproduction of telomerase pathway components may at least partially compensate for the loss of Ku in maintaining telomeric silencing.

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.