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RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We useRNA:RNAbinding bySHAPE (RABS) to investigatemicroRNA-34a(miR-34a) binding its mRNA target, thesilent information regulator 1(mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the nakedmiR-34ais able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

The humanFactor VIII(F8) protein is essential for the blood coagulation cascade and specificF8mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations onF8pre-mRNA splicing. We found that 14/97 (∼14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 (∼36.4%)F8exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3′ end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3′ splice site strength. Using exon-16 of theF8gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing ofF8exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3’ splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specificF8mutations and provide a framework for precision RNA-based HA therapies.

New atomic-resolution structures provide detailed insights into the molecular mechanism of telomerase, ushering in a new era of rational drug design to target telomerase in human genetic disorders and cancer.

Background\ud \ud Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown.\ud \ud Methods\ud \ud To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model.\ud \ud Results\ud \ud In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control.\ud \ud Conclusion\ud \ud Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

The serine and arginine-rich splicing factor SRSF1 is an evolutionarily conserved, essential pre-mRNA splicing factor. Through a global protein-RNA interaction survey we discovered SRSF1 binding sites 25-50nt upstream from hundreds of pre-miRNAs. Using primary miRNA-10b as a model we demonstrate that SRSF1 directly regulates microRNA biogenesis both in vitro and in vivo. Selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) defined a structured RNA element located upstream of the precursor miRNA-10b stem loop. Our data support a model where SRSF1 promotes initial steps of microRNA biogenesis by relieving the repressive effects of cis-regulatory elements within the leader sequence.

The expression of the neural cell adhesion molecule (NCAM) in 6 normal human pituitaries and 25 pituitary adenomas was investigated by immunohistochemistry and in situ hybridization. NCAM protein and mRNA were present in all normal and neoplastic human pituitary tissues. There were tumor type-specific differences in the distribution of NCAM in various pituitary adenomas. Growth hormone adenomas and prolactin-producing adenomas usually expressed lower levels of NCAM, compared to null cell and gonadotroph adenomas. Adreno-corticotropic hormone adenomas expressed the highest levels of NCAM mRNA. Six freshly dissociated pituitary adenomas were cultured in serum-free medium for 7 days to analyze the regulation of NCAM mRNA by in situ hybridization. The lower levels of NCAM expression in growth hormone and prolactin adenomas were not present in cells cultured for 7 days in serum-free medium on extracellular matrix. Phorbol 12-myristate 13-acetate (PMA) stimulated NCAM mRNA expression in 5 of 6 tumors. Gonadotropin-releasing hormone and growth hormone-releasing hormone increased NCAM expression in some adenomas. This study demonstrates that there is a variable expression of NCAM in pituitary adenomas and that hypotha-lamic hormones and PMA can regulate NCAM mRNA levels in neoplastic pituitary cells.

Telomerase is a specialized enzyme that maintains telomere length by adding DNA repeats to chromosome ends. The catalytic protein subunit of telomerase utilizes the integral telomerase RNA to direct telomere DNA synthesis. The telomerase essential N-terminal (TEN) domain is required for enzyme function; however, the precise mechanism of the TEN domain during catalysis is not known. We report a single-molecule study of dynamic TEN-induced conformational changes in its nucleic acid substrates. The TEN domain from the yeast Candida parapsilosis (Cp) exhibits a strong binding preference for double-stranded nucleic acids, with particularly high affinity for an RNA–DNA hybrid mimicking the template–product complex. Surprisingly, the telomere DNA repeat sequence from C. parapsilosis forms a DNA hairpin that also binds CpTEN with high affinity. Mutations to several residues in a putative nucleic acid-binding patch of CpTEN significantly reduced its affinity to the RNA–DNA hybrid and telomere DNA hairpin. Substitution of comparable residues in the related Candida albicans TEN domain caused telomere maintenance defects in vivo and decreased primer extension activity in vitro. Collectively, our results support a working model in which dynamic interactions with telomere DNA and the template–product hybrid underlie the functional requirement for the TEN domain during the telomerase catalytic cycle.

Telomeres safeguard the genome by suppressing illicit DNA damage responses at chromosome termini. In order to compensate for incomplete DNA replication at telomeres, most continually dividing cells, including many cancers, express the telomerase ribonucleoprotein (RNP) complex. Telomerase maintains telomere length by catalyzing de novo synthesis of short DNA repeats using an internal telomerase RNA (TR) template. TRs from diverse species harbor structurally conserved domains that contribute to RNP biogenesis and function. In vertebrate TRs, the conserved regions 4 and 5 (CR4/5) fold into a three-way junction (3WJ) that binds directly to the telomerase catalytic protein subunit and is required for telomerase function. We have analyzed the structural properties of the human TR (hTR) CR4/5 domain using a combination of in vitro chemical mapping, endogenous RNP assembly assays, and single-molecule structural analysis. Our data suggest that a functionally essential stem loop within CR4/5 is not stably folded in the absence of the telomerase reverse transcriptase protein subunit in vitro. Rather, the hTR CR4/5 domain adopts a heterogeneous ensemble of conformations. RNA structural engineering intended to bias the folding landscape of the hTR CR4/5 demonstrates that a stably folded 3WJ motif is necessary but not sufficient to promote assembly of a functional RNP complex. Finally, single-molecule measurements on the hTR CR4/5 domain show that RNP assembly selects for a conformation that is not the major population in the heterogeneous free RNA ensemble, suggesting that non-canonical hTR folds may be required during telomerase biogenesis.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K(+) versus GQ-destabilizing Li(+) salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li(+) cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K(+) but not Li(+), supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Telomerase restores chromosome-capping telomeric repeats lost with each round of genome replication by DNA-templated DNA polymerases. The telomerase reverse transcriptase (TERT) N-terminal (TEN) domain is a peripheral, telomerase-specific, processivity-stimulatory addition to more conserved domains that encircle the active site cavity. Reports of ciliate, yeast, and mammalian telomerase TEN domain associations with the telomerase RNA subunit (TR) describe low affinity interactions of uncertain specificity. Unfortunately two cryo-EM structures of synthesis-paused telomerase holoenzymes lack sufficient resolution to discriminate molecular specificity of possible TR contact(s) with the TEN domain, and there is no assigned density for the TEN domain termini implicated in RNA binding. Furthermore, studies have revealed alternative secondary structures for TR regions that could interact with TERT prior to TR folding into active conformation. Informed by recent advances in knowledge of telomerase structure, we returned to the investigation of Tetrahymena thermophila TERT TEN domain interaction with TR. Instead of finding specificity for a particular TR sequence or structure, we discovered that the tagged TEN domain used in previous characterizations has trace contamination with a bacterial RNA-interacting protein not detectable by SDS-PAGE. By resolving this interference, we show that the TEN domain binds RNAs dependent on RNA length rather than sequence.

Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.

Telomerase maintains telomere length by reverse transcribing short G-rich DNA repeat sequences from its internal RNA template. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using K+ versus Li+ salts yield changes in DNA product profiles consistent with formation of GQ structure within the telomerase-DNA complex. Single-molecule FRET experiments reveal complex DNA structural dynamics during real-time catalysis, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we fit telomerase time series data to a global kinetic model that converges to a unique set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis and provide a detailed characterization of GQ modulation of polymerase function.SIGNIFICANCETelomeres protect the ends of linear chromosomes from illicit DNA processing events that can threaten genome stability. Telomere structure is built upon repetitive G-rich DNA repeat sequences that have the ability to fold into stable secondary structures called G-quadruplexes (GQs). In rapidly dividing cells, including the majority of human cancers, telomeres are maintained by the specialized telomerase enzyme. Thus, telomerase and its telomere DNA substrates represent important targets for developing novel cancer drugs. In this work, we provide evidence for GQ folding within the newly synthesized DNA product of an actively extending telomerase enzyme. Our results highlight the delicate interplay between the structural properties of telomere DNA and telomerase function.

An evidence-based clinical guideline for the diagnosis and management of Paget's disease of bone (PDB) was developed using GRADE methodology, by a Guideline Development Group (GDG) led by the Paget's Association (UK). A systematic review of diagnostic tests and pharmacological and nonpharmacological treatment options was conducted that sought to address several key questions of clinical relevance. Twelve recommendations and five conditional recommendations were made, but there was insufficient evidence to address eight of the questions posed. The following recommendations were identified as the most important: 1) Radionuclide bone scans, in addition to targeted radiographs, are recommended as a means of fully and accurately defining the extent of metabolically active disease in patients with PDB. 2) Serum total alkaline phosphatase (ALP) is recommended as a first-line biochemical screening test in combination with liver function tests in screening for the presence of metabolically active PDB. 3) Bisphosphonates are recommended for the treatment of bone pain associated with PDB. Zoledronic acid is recommended as the bisphosphonate most likely to give a favorable pain response. 4) Treatment aimed at improving symptoms is recommended over a treat-to-target strategy aimed at normalizing total ALP in PDB. 5) Total hip or knee replacements are recommended for patients with PDB who develop osteoarthritis in whom medical treatment is inadequate. There is insufficient information to recommend one type of surgical approach over another. The guideline was endorsed by the European Calcified Tissues Society, the International Osteoporosis Foundation, the American Society of Bone and Mineral Research, the Bone Research Society (UK), and the British Geriatric Society. The GDG noted that there had been a lack of research on patient-focused clinical outcomes in PDB and identified several areas where further research was needed. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

To examine the relationship between pre-operative vitamin D status and post-thyroidectomy hypocalcemia.Retrospective study examining 264 total and completion thyroidectomies conducted between 2007 and 2011. Subjects included had a recorded 25-hydroxyvitamin D (25[OH]D) level within 21 days prior to or 1 day following surgery, did not have a primary parathyroid gland disorder, and were not taking 1,25-dihydroxyvitamin D3 (calcitriol) prior to surgery. Some subjects were repleted with vitamin D pre-operatively if a low 25(OH)D level (typically below 20 ng/mL) was identified. Pre-operative 25(OH)D, concurrent neck dissection, integrity of parathyroid glands, final pathology, postoperative parathyroid hormone (PTH), calcium nadir and repletion, and length of stay were examined.The mean pre-operative 25(OH)D for all subjects was 25 ng/mL, and the overall rate of post-operative hypocalcemia was 37.5%. Lower pre-operative 25(OH)D did not predict postoperative hypocalcemia (P = .96); however, it did predict the need for postoperative 1,25-dihydroxyvitamin D3 administration (P = .01). Lower postoperative PTH levels (P = .001) were associated with postoperative hypocalcemia.Pre-operative 25(OH)D did not predict a postoperative decrease in serum calcium, although it did predict the need for 1,25-dihydroxyvitamin D3 therapy in hypocalcemic subjects. We recommend that 25(OH)D be assessed and, if indicated, repleted pre-operatively in patients undergoing total thyroidectomy.

Telomerase is an ancient ribonucleoprotein (RNP) that protects the ends of linear chromosomes from the loss of critical coding sequences through repetitive addition of short DNA sequences. These repeats comprise the telomere, which together with many accessory proteins, protect chromosomal ends from degradation and unwanted DNA repair. Telomerase is a unique reverse transcriptase (RT) that carries its own RNA to use as a template for repeat addition. Over decades of research, it has become clear that there are many diverse, crucial functions played by telomerase RNA beyond simply acting as a template. In this review, we highlight recent findings in three model systems: ciliates, yeast and vertebrates, that have shifted the way the field views the structural and mechanistic role(s) of RNA within the functional telomerase RNP complex. Viewed in this light, we hope to demonstrate that while telomerase RNA is just one example of the myriad functional RNA in the cell, insights into its structure and mechanism have wide-ranging impacts. WIREs RNA 2018, 9:e1456. doi: 10.1002/wrna.1456 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

Telomeres are specialized chromatin structures that protect chromosome ends from dangerous processing events. In most tissues, telomeres shorten with each round of cell division, placing a finite limit on cell growth. In rapidly dividing cells, including the majority of human cancers, cells bypass this growth limit through telomerase-catalyzed maintenance of telomere length. The dynamic properties of telomeres and telomerase render them difficult to study using ensemble biochemical and structural techniques. This review describes single-molecule approaches to studying how individual components of telomeres and telomerase contribute to function. Single-molecule methods provide a window into the complex nature of telomeres and telomerase by permitting researchers to directly visualize and manipulate the individual protein, DNA, and RNA molecules required for telomere function. The work reviewed in this article highlights how single-molecule techniques have been utilized to investigate the function of telomeres and telomerase.

The original discovery of enzymes that synthesize DNA using an RNA template appeared to contradict the central dogma of biology, in which information is transferred, in a unidirectional way, from DNA genes into RNA molecules. The paradigm-shifting discovery of RNA-dependent DNA polymerases, also called reverse transcriptases (RTs), reshaped existing views for how cells function; however, the scope of the impact RTs impose on biology had yet to be realized. In the decades of research since the early 1970s, the biomedical and biotechnological significance of retroviral RTs, as well as the evolutionarily related telomerase enzyme, has become exceedingly clear. One common theme that has emerged in the course of RT-related research is the central role of nucleic acid binding and dynamics during enzyme function. However, directly interrogating these dynamic properties is challenging because of the stochastic properties of biological macromolecules. In this review, we describe how the development of single-molecule biophysical techniques has opened new windows through which to observe the dynamic behavior of this remarkable class of enzymes. Specifically, we focus on how the powerful single-molecule Förster resonance energy transfer (FRET) method has been exploited to study the structure and function of the human immunodeficiency virus (HIV) RT and telomerase ribonucleoprotein (RNP) enzymes. These exciting studies have refined our understanding of RT catalysis, have revealed unforeseen structural rearrangements between RTs and their nucleic acid substrates, and have helped to characterize the mode of action of RT-inhibiting drugs. We conclude with a discussion of how the ongoing development of single-molecule technologies will continue to empower researchers to probe RT mechanisms in new and exciting ways.

The ends of linear chromosomes are extended by telomerase, a ribonucleoprotein complex minimally consisting of a protein subunit called telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). TERT functions by reverse transcribing a short template region of TER into telomeric DNA. Proper assembly of TERT and TER is essential for telomerase activity; however, a detailed understanding of how TERT interacts with TER is lacking. Previous studies have identified an RNA binding domain (RBD) within TERT, which includes three evolutionarily conserved sequence motifs: CP2, CP, and T. Here, we used site-directed hydroxyl radical probing to directly identify sites of interaction between the TERT RBD and TER, revealing that the CP2 motif is in close proximity to a conserved region of TER known as the template boundary element (TBE). Gel shift assays on CP2 mutants confirmed that the CP2 motif is an RNA binding determinant. Our results explain previous work that established that mutations to the CP2 motif of TERT and to the TBE of TER both permit misincorporation of nucleotides into the growing DNA strand beyond the canonical template. Taken together, these results suggest a model in which the CP2 motif binds the TBE to strictly define which TER nucleotides can be reverse transcribed.

The purpose of this study was to evaluate the rate of change (RoC) in the size of the lumpectomy cavity (LC) before and during breast radiotherapy (RT) using cone‐beam computed tomography (CBCT), relative to the initial LC volume at CT simulation (CTVLC) and timing from surgery. A prospective institutional review board‐approved study included 26 patients undergoing breast RT: 20 whole breast irradiation (WBI) patients and six partial breast irradiation (PBI) patients, with surgical clips outlining the LC. The patients underwent CT simulation (CTsim) followed by five CBCTs during RT, once daily for PBI and once weekly for WBI. The distance between surgical clips and their centroid (D) acted as a surrogate for LC size. The RoC of the LC size, defined as the percentage change of D between two scans divided by the time interval in days between the scans, was calculated before (CTsim to CBCT1) and during RT (CBCT1 to CBCT5). The mean RoC of D for all patients before starting RT was −0.25%/day (range, −1.3 to 1.4) and for WBI patients during RT was −0.15%/day (range, −0.45 to 0.40). Stratified by median CTVLC, the RoC before RT for large CTVLC group (≥25.7cc) was 15 times higher (−0.47%/day) than for small CTVLC group (

Telomerase catalyzed synthesis of telomere DNA provides the foundation for DNA-protein structures called telomeres. Telomeres are required to evade DNA processing which may result in nucleolytic degradation and fusion of linear chromosomes. Human telomerase contains a protein reverse transcriptase (hTERT), telomerase RNA (hTR), and several additional proteins.[1] hTR provides the template sequence for hTERT during a novel reverse transcription reaction that produces short telomere DNA repeat sequences.[2] Additionally, hTR provides a flexible scaffold for the assembly and function of telomerase associated proteins[3] and has recently been suggested to contribute to enzyme catalysis.[4] Many pathogenic mutations have been mapped to hTR;[5] however the precise mechanisms of these mutations remain unclear.

The telomerase ribonucleoprotein is a specialized reverse transcriptase required to maintain protective chromosome end-capping structures called telomeres. In most cells, telomerase is not active and the natural shortening of telomeres with each round of DNA replication ultimately triggers cell growth arrest. In contrast, the presence of telomerase confers a high level of renewal capacity upon rapidly dividing cells. Telomerase is aberrantly activated in 90% of human cancers and thus represents an important target for anticancer therapeutics. However, the naturally low abundance of telomerase has hampered efforts to obtain high-resolution models for telomerase structure and function. To circumvent these challenges, single molecule techniques have recently been employed to investigate telomerase assembly, structure, and catalysis.

Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3' end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Forster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation.

Exercise is known to facilitate physical and emotional adjustment among women treated for breast cancer, and exercise exerts a profound effect on clinical depression. However, the effect of exercise on reducing clinical depression among breast cancer patients has not been demonstrated, especially among ethnic minority women who have a higher incidence of depression and higher mortality following breast cancer. First, literature is presented to assess exercise effects on depression among women with breast cancer. Second, we present the results of a randomized controlled trial assessing the effect of a structured exercise intervention on depression and exercise behavior in a multiethnic sample of women with early stage breast cancer enrolled prior to the start of adjuvant treatment. Results suggest that, in comparison to population norms, the rate of depression was higher in breast cancer patients. Analyses further showed that the intervention significantly increased self‐reported exercise and redu...

Disodium pamidronate (APD) is a potent inhibitor of bone resorption, with less risk of defective mineralization than earlier bisphosphonates. We assessed the response to six spaced low-dose intravenous infusions of APD given at intervals of approximately 6 weeks followed by weekly infusions if bone turnover remained abnormal. Three groups of 10 patients were studied, each group receiving infusions of 15, 30, or 45 mg. Hydroxyproline excretion fell by 62% and alkaline phosphatase was reduced by 72%, with no difference between the dose levels. A total of 21 patients (70%) achieved normal levels of bone turnover, indicating that low-dose infusions of APD are a safe and effective treatment for Paget's disease.

The bisphosphonate space (BPS) is a quantitative measurement of skeletal uptake of 99mTc-HMDP. We measured BPS in 36 patients with Paget's disease of bone, both before and 6 months after treatment with intravenous APD (disodium pamidronate) infusions. BPS fell after treatment, but proportionally less than serum alkaline phosphatase (ALP) and fasting urinary hydroxyproline/creatinine (HYPRO). There was no dose-response relationship between the dose of APD given and the percentage reduction in ALP and HYPRO at 6 months. Log dose of APD/pretreatment BPS, however, predicted the percentage reduction in ALP and HYPRO very well, and from the respective regression equations it was possible to predict the dose of APD needed to achieve normal values of ALP and HYPRO. In the 10 patients who achieved a normal ALP and 9 patients a normal HYPRO after more than 6 months treatment with APD (range 7-18 months), the predicted dose of APD agreed closely with the actual dose. In conclusion, our data support the idea that log dose APD/pretreatment BPS is a valid predictor of biochemical response in Paget's disease.

Proliferative breast lesions, such as simple ductal hyperplasia (SH) and atypical ductal hyperplasia (ADH), are candidate precursors to ductal carcinoma in situ (DCIS) and invasive cancer. To better understand the relationship of breast lesions to more advanced disease, we used microdissection and DNA microarrays to profile the gene expression of patient-matched histologically normal (HN), ADH, and DCIS from 12 patients with estrogen receptor positive sporadic breast cancer. SH were profiled from a subset of cases. We found 837 differentially expressed genes between DCIS-HN and 447 between ADH-HN, with >90% of the ADH-HN genes also present among the DCIS-HN genes. Only 61 genes were identified between ADH-DCIS. Expression differences were reproduced in an independent cohort of patient-matched lesions by quantitative real-time PCR. Many breast cancer-related genes and pathways were dysregulated in ADH and maintained in DCIS. Particularly, cell adhesion and extracellular matrix interactions were overrepresented. Focal adhesion was the top pathway in each gene set. We conclude that ADH and DCIS share highly similar gene expression and are distinct from HN. In contrast, SH appear more similar to HN. These data provide genetic evidence that ADH (but not SH) are often precursors to cancer and suggest cancer-related genetic changes, particularly adhesion and extracellular matrix pathways, are dysregulated before invasion and even before malignancy is apparent. These findings could lead to novel risk stratification, prevention, and treatment approaches.

Telomerase is required to maintain repetitive G-rich telomeric DNA sequences at chromosome ends. To do so, the telomerase reverse transcriptase (TERT) subunit reiteratively uses a small region of the integral telomerase RNA (TER) as a template. An essential feature of telomerase catalysis is the strict definition of the template boundary to determine the precise TER nucleotides to be reverse transcribed by TERT. We report the 3-A crystal structure of the Tetrahymena TERT RNA-binding domain (tTRBD) bound to the template boundary element (TBE) of TER. tTRBD is wedged into the base of the TBE RNA stem-loop, and each of the flanking RNA strands wraps around opposite sides of the protein domain. The structure illustrates how the tTRBD establishes the template boundary by positioning the TBE at the correct distance from the TERT active site to prohibit copying of nontemplate nucleotides.

BI-RADS-3 is a category in mammography for probably benign lesions and for which periodic follow-up with repeat imaging is recommended. At our institution repeated mammograms are performed at 6, 12, 18, and 24 months. The purpose of this study was to assess the significance of 18-month mammogram for evaluation of BI-RADS-3 lesions. Following IRB approval, electronic medical records and picture archiving and communications system were used to review 121,862 consecutive mammograms between February, 2002-May, 2009. A total of 8,400 patients with BI-RADS-3 mammograms were identified. Of these, 7,632 patients were followed until completion of 24 month mammogram or biopsy following an upgrade in their BI-RADS status. Over the follow-up, 197 patients received an upgrade in their BI-RADS status of which 179 were biopsied. Histopathologic results were reviewed. The majority of the BI-RADS-3 lesions were upgraded at 6-month mammogram (n = 150, 76.1%) followed by 32 (16.2%), 11 (5.6%), and 4 (2.0%) at 12, 18, and 24 month mammograms respectively. Thirty-four of 179 upgraded and biopsied lesions were found to be malignant. From these 27 (79.4%), 3 (8.8%), 3 (8.8%), and 1 (2.9%) lesions were identified at 6, 12, 18, and 24 month mammograms respectively. At the 18-month mammogram 3/7,632 lesions (0.04%) were found to be malignant. The vast majority of malignant lesions (88.2%) were detected within the first 12-months of follow-up. Only three of 179 biopsies (1.7%) were malignant at 18-month follow-up. Based on those results a 6-, 12-, and 24-month follow-up protocol for BI-RADS-3 lesions is sufficient.

Objectives. Genome wide association studies identified TNFSF member TNF-like protein 1A (TL1A, TNFSF15) as a potential modulator of ankylosing spondylitis (AS). TL1A is the only confirmed TNFSF ligand of death receptor 3 (DR3, TNFRSF25); however, its role in disease pathology is not characterised. We evaluated DR3’s role in controlling osteoblast- (OB-) dependent bone formation in vitro and in vivo. Methods. Osteoprogenitor cells and OB were cultured from male DR3-deficient (DR3ko) and wild-type (DR3wt) DBA/1 mice. DR3 and RANKL expression were tested by flow cytometry. Alkaline phosphatase and mineralization were quantified. Osteopontin, osteoprotegerin, and pro MMP-9 were measured by ELISA. A fluorescent probe (BoneTag) was used to measure in vivo mineralization in 10-month-old mice. Results. DR3 was expressed on osteoprogenitors and OB from DR3wt mice. Alkaline phosphatase, osteopontin, and mineral apposition were significantly elevated in DR3wt cultures. Levels of RANKL were comparable whilst osteoprotegerin was significantly increased in DR3wt cultures. In vivo incorporation of BoneTag was significantly lower in the thoracic vertebrae of 10-month-old DR3ko mice. Conclusions. These data identify new roles for DR3 in regulating OB-dependent bone mineral apposition. They potentially begin to explain the atypical pattern of new bone formation observed in the axial skeleton of grouped, aging DBA/1 mice.

Thyroid nodules are common, with an estimated incidence of 5%-10% in the United States. The current gold standard for diagnosis is fine needle aspiration biopsy (FNAB). The incidence of indeterminate diagnoses varies from 10% to 25%. Surgical resection is usually indicated to exclude the diagnosis of cancer in these patients. However, only a minority (about 20%) of indeterminate thyroid nodules actually harbor a malignancy, resulting in surgery for diagnostic purposes alone in many patients. The increased detection of benign nodules and microcarcinomas reinforces the need for improved non-operative methods to differentiate benign from malignant disease and discriminate low-risk from high-risk cancers. In this article we present a current, rational diagnostic approach to the patient with a thyroid nodule, evaluate new advances including thyroid genomic and predictor models, and propose the development of prospective trials to incorporate these new additions into clinical decision making. Given how many questions still exist for patients with thyroid nodules, partnership and collaboration, or the "bench to bedside" concept should find its way into most every thyroid surgeon and endocrinologist's lexicon.

Previous studies have been unable to identify risk factors for prevalent vertebral fractures (VF), which are suitable for use in selection strategies intended to target high-risk sub-groups for diagnostic assessment. However, these studies generally consisted of large epidemiology surveys based on questionnaires and were only able to evaluate a limited number of risk factors. Here, we investigated whether a stronger relationship exists with prevalent VF when conventional risk factors are combined with additional information obtained from detailed one-to-one assessment. Women aged 65–75 registered at four geographically distinct GP practices were invited to participate (n=1,518), of whom 540 attended for assessment as follows: a questionnaire asking about risk factors for osteoporosis such as height loss compared to age 25 and history of non-vertebral fracture (NVF), the get-up-and-go test, Margolis back pain score, measurement of wall-tragus and rib-pelvis distances, and BMD as measured by the distal forearm BMD. A lateral thoraco-lumbar spine X-ray was obtained, which was subsequently scored for the presence of significant vertebral deformities. Of the 509 subjects who underwent spinal radiographs, 37 (7.3%) were found to have one or more VF. Following logistic regression analysis, the four most predictive clinical risk factors for prevalent VF were: height loss (P=0.006), past NVF (P=0.004), history of back pain (P=0.075) and age (P=0.05). BMD was also significantly associated with prevalent VF (P=0.002), but its inclusion did not affect associations with other variables. Factors elicited from detailed one-to-one assessment were not related to the risk of one or more prevalent VFs. The area under ROC curves derived from these regressions, which suggested that models for prevalent VF had modest predictive accuracy, were as follows: 0.68 (BMD), 0.74 (four clinical risk factors above) and 0.78 (clinical risk factors + BMD). Analyses were repeated in relation to the subgroup of 13 patients with two or more VFs, which revealed that in this instance, the Margolis back pain score and rib-pelvis distance were associated with the presence of multiple VFs (P=0.022 and 0.026, respectively). Moreover, the predictive value as reflected by the ROC curve area was improved: 0.80 (BMD), 0.88 (the four most predictive clinical risk factors consisting of the height loss, past NVF, Margolis back pain score and rib-pelvis distance) and 0.91 (clinical risk factors + BMD). Evaluation of additional risk factors from detailed one-to-one assessment does not improve the predictive value of risk factors for one or more prevalent vertebral deformities in postmenopausal women. However, the use of factors such as the Margolis back pain score and rib-pelvis distance may be helpful in identifying postmenopausal women at high risk of multiple prevalent VFs.

Knowledge of the elastic properties of DNA is required to understand the structural dynamics of cellular processes such as replication and transcription. Measurements of force and extension on single molecules of DNA 1‐3 have allowed direct determination of the molecule’s mechanical properties, provided rigorous tests of theories of polymer elasticity 4 , revealed unforeseen structural transitions induced by mechanical stresses 3,5‐7 , and established an experimental and conceptual framework for mechanical assays of enzymes that act on DNA 8 . However, a complete description of DNA mechanics must also consider the effects of torque, a quantity that has hitherto not been directly measured in micromanipulation experiments. We have measured torque as a function of twist for stretched DNA—torsional strain in over- or underwound molecules was used to power the rotation of submicrometre beads serving as calibrated loads. Here we report tests of the linearity of DNA’s twist elasticity, direct measurements of the torsional modulus (finding a value ,40% higher than generally accepted), characterization of torque-induced structural transitions, and the establishment of a framework for future assays of torque and twist generation by DNA-dependent enzymes. We also show that cooperative structural transitions in DNA can be exploited to construct constanttorque wind-up motors and force‐torque converters. Previous investigations of the force‐extension behaviour of supercoiled DNA have found that, under low tension, DNA behaves like an isotropic flexible rod 2,9 : as turns are added to the molecule, its

Objectives. Genome wide association studies identified TNFSF member TNF-like protein 1A (TL1A, TNFSF15) as a potential modulator of ankylosing spondylitis (AS). TL1A is the only confirmed TNFSF ligand of death receptor 3 (DR3, TNFRSF25); however, its role in disease pathology is not characterised. We evaluated DR3's role in controlling osteoblast- (OB-) dependent bone formation in vitro and in vivo. Methods. Osteoprogenitor cells and OB were cultured from male DR3-deficient (DR3(ko)) and wild-type (DR3(wt)) DBA/1 mice. DR3 and RANKL expression were tested by flow cytometry. Alkaline phosphatase and mineralization were quantified. Osteopontin, osteoprotegerin, and pro MMP-9 were measured by ELISA. A fluorescent probe (BoneTag) was used to measure in vivo mineralization in 10-month-old mice. Results. DR3 was expressed on osteoprogenitors and OB from DR3(wt) mice. Alkaline phosphatase, osteopontin, and mineral apposition were significantly elevated in DR3(wt) cultures. Levels of RANKL were comparable whilst osteoprotegerin was significantly increased in DR3(wt) cultures. In vivo incorporation of BoneTag was significantly lower in the thoracic vertebrae of 10-month-old DR3(ko) mice. Conclusions. These data identify new roles for DR3 in regulating OB-dependent bone mineral apposition. They potentially begin to explain the atypical pattern of new bone formation observed in the axial skeleton of grouped, aging DBA/1 mice.

Background. Long work hours that result in fatigue may adversely affect cognitive function. Chronically sleep-deprived surgical residents fear that being on call the night before sitting for a standardized test puts them at a potential disadvantage. We examined American Board of Surgery In-Training Examination (ABSITE) scores to assess the effect of call status on exam performance. Methods. Results of the 1994 ABSITE for 424 residents in 15 New England general surgery programs were collected. We compared standard scores of residents for the total test, clinical management, and basic science components with resident call status (on/off) for the night before the exam. Results. Differences were apparent in total test scores (mean: off, 496.1; on, 466.0; P

Background We hypothesized that the method of breast cancer margin assessment may be associated with different rates of positive margins and residual carcinoma. Methods A total of 178 breast cancer specimens were divided into 2 groups (A and B) based on the margin assessment method used. Rates of positive margins, re-excision, and residual carcinoma at re-excision were compared and analyzed statistically. Results At least 1 margin was positive in 64.7% in group A and in 65.2% in group B. At directed re-excision 54% in group A and 51% in group B had residual carcinoma. The lateral margin was positive in 44% in group A compared with 26% in group B ( P = .06). The posterior margin was positive in 19% in group A and in 51% in group B ( P = .001). Conclusions Two different breast cancer specimen margin assessment methods had comparable rates of positive margins and residual carcinoma at re-excision. Different patterns of specific margin positivity suggest that the method of margin assessment may alter results.

Proliferative myositis (PM) along with proliferative fasciitis and nodular fasciitis are a group of pseudosarcomatous myofibroblastic proliferations. Although the histologic presentation of each is almost identical, the magnetic resonance imaging (MRI) appearance of proliferative myositis is closer to that of inflammatory myopathies. We report a case of PM in which the imaging and histologic features combine typical findings of PM with unusual imaging features, suggesting of reactive (or nodular) fasciitis.

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the 7 proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labeling. Fitting with high-resolution structures reveals the organization of TERT, TER, and p65 in the RNP catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

Surgical education and practice are inextricably linked: Each is essential to the other's existence, and what happens in one arena inevitably affects the other. Both are subject to the governmental and market forces that are revolutionizing the delivery of health care in the United States today. Public debate over national health care reform has focused on the issues of access and cost, but while much lip service has been given to the issue of access, most attention has centered not on health care delivery but its financing. 23 In the absence of governmental reform, economic market forces predominate as the agent of change. Increasing consolidation of the nation's health care resources in the hands of for-profit companies has led to the promotion and adoption of managed care plans. Enrollment in managed care plans (HMOs and PPOs) is growing at more than 11% a year, 1 totaling 50,000,000 by the end of 1994, 16 or roughly one out of four insured Americans. In today's market, relatively few, large corporations dominate health care and do so in the primary interest of profits, not the health of the nation's people. 1,29 The basic tenet of managed care, and the mechanism for increasing profits, is cost reduction achieved through a shift of financial risk to physicians and hospital organizations, with capitation as the mature market model. 7 Efforts to reduce costs are having a profound impact on surgical practice, including decreased utilization of services and changing practice patterns (Figs. 1 and 2). Some changes have had a positive effect on surgical practice: Increased emphasis on efficiency, for example, forces practitioners to cast off diagnostic and treatment routines based on tradition rather than scientific thinking. These changes will have significant impact on surgical education because much of it takes place within surgical practice. In the managed care schema, education is no longer a priority but a cost center. Surgical educators need to adapt to many changes in surgical practice while opposing some potential changes that may be harmful to the learning process. A balance must be struck: Whereas government, business and hospital administrators will need to recognize that "the quality of American medicine is dependent on the quality of medical education," 24 surgical faculty need to recognize that "unless we train students to provide demonstrably effective care at a competitive cost, we will train them for obsolescence." 22 We will suggest changes in medical education beginning in the preclinical years and continuing through clinical surgical training during and after medical school. We will describe the effects of the changing health care environment on surgical practice, and consequently on surgical education, at both the medical school and residency levels. Specifically, we will suggest changes in curriculum and educational methods necessary for surgical training to adapt to changes in health care financing, educational venue, and availability of resources (faculty and patients).

The unique cellular activity of the telomerase reverse transcriptase ribonucleoprotein (RNP) requires proper assembly of protein and RNA components into a functional complex. In the ciliate model organism Tetrahymena thermophila, the La-domain protein p65 is required for in vivo assembly of telomerase. Single-molecule and biochemical studies have shown that p65 promotes efficient RNA assembly with the telomerase reverse transcriptase (TERT) protein, in part by inducing a bend in the conserved stem IV region of telomerase RNA (TER). The domain architecture of p65 consists of an N-terminal domain, a La-RRM motif, and a C-terminal domain (CTD). Using single-molecule Förster resonance energy transfer (smFRET), we demonstrate the p65CTD is necessary for the RNA remodeling activity of the protein and is sufficient to induce a substantial conformational change in stem IV of TER. Moreover, nuclease protection assays directly map the site of p65CTD interaction to stem IV and reveal that, in addition to bending stem IV, p65 binding reorganizes nucleotides that comprise the low-affinity TERT binding site within stem–loop IV.

Telomerase is a cellular ribonucleoprotein (RNP) complex comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The activity of the telomerase enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report the development and application of a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from individual enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. In particular, we have established that the phylogenetically conserved pseudoknot motif within telomerase RNA is properly folded in catalytically active enzymes. Interestingly, the pseudoknot motif is misfolded in the absence of proteins, and the protein subunits of the telomerase holoenzyme counteract RNA misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of various telomerase RNA regions such as the pseudoknot but also establish a direct method for elucidating how the structure of telomerase enables its function.

We administered risedronate, a potent oral bisphosphonate, to patients with mild primary hyperparathyroidism in order to 1) determine if we could normalize the serum calcium concentration in the short term, and 2) analyze changes in the homeostatic mechanisms responsible for maintaining hypercalcemia in this patient population. When administered for 7 days, risedronate reduced fasting serum calcium concentrations without significant toxicity in patients with primary hyperparathyroidism. The decrease in serum calcium was accompanied by evidence of inhibition of bone resorption, as assessed by measurement of urinary hydroxyproline, increased serum immunoreactive PTH concentrations, enhanced renal tubular reabsorption of calcium, and a progressive decrease in serum alkaline phosphatase. Serum PTH was partially suppressed by an oral calcium load in untreated patients as well as in patients treated with risedronate. Although patients treated with risedronate had normal fasting serum calcium levels, serum calcium values in these normocalcemic patients were labile after oral ingestion of calcium. After daily calcium intake of 2 g, serum calcium levels in risedronate-treated patients were similar to those in untreated patients with primary hyperparathyroidism, suggesting that there are likely to be fluctuations in serum calcium in risedronate-treated patients with normal fasting serum calcium during postprandial periods. These studies show that risedronate lowers fasting serum calcium during short term treatment. However, further studies are required to determine whether the lability in serum calcium in these patients after an oral calcium load has clinical significance, and whether longer term treatment would maintain serum calcium in the normal range.

Mechanistic studies of RNA enzymes (ribozymes) and ribonucleoprotein (RNP) complexes such as the ribosome and telomerase, often seek to characterize RNA structural features, either dynamic or static, and relate these properties to specific catalytic functions. Many experimental techniques that probe RNA structure-function relationships rely upon site-specific incorporation of chemically modified ribonucleotides into the RNA of interest, often in the form of chemical cross-linkers to probe for sites of protein-RNA interaction or small organic fluorophores to measure dynamic structural properties of RNAs. The ability to arbitrarily modify any RNA molecule has been greatly enabled by modern RNA synthesis techniques; however, there remains a practical size limitation (~70 bases). Consequently, experimental approaches involving specific chemical modifications of larger RNAs require the use of RNA ligation methods. The aim of this chapter is to describe a general approach for covalently joining multiple site-specifically modified RNA fragments, drawing from our fluorescence-based structural studies of telomerase RNA as an example.