Infected stent graft and severe aortitis after transcaval glue embolization of type II endoleak: a case report.

Background: Type II endoleaks are common and embolization is often performed if treatment is necessary. Although transarterial embolization is common, other methods including trans-caval embolization are also utilized. Complications can occur and we report a case of infection that was challenging to diagnose and treat. There is no data regarding the risk of aortic stent graft infection after trans-caval embolization with n-butyl 2-cyanoacrylate (n-BCA) glue of a type II endoleak. Case Description: We report a rare case of infected, Gore Excluder infrarenal stent graft after transcaval embolization with coil and n-BCA glue to treat a type II endoleak in a 71-year-old male. The endoleak caused a rapid sac enlargement. The stent graft was placed 5 years earlier electively. Soon after the endoleak embolization, the patient experienced abdominal pain and malaise. There was an intense inflammatory reaction involving the aneurysm wall and the adjacent bowel mesentery. Our differential included normal inflammation after embolization vs. infection and this was difficult to distinguish. The infection was confirmed by positron emission tomography scan and tissue biopsy. The patient was deemed high-risk for surgery because of his extensive cardiac history, status post coronary bypass and tissue mitral valve replacement, congestive heart failure with residual left ventricular ejection fraction of 36%. He was optimized by correcting fluid status, administration of intravenous antibiotic, and nutrition consultation with dietary supplementation before surgery over the course of 2 weeks. The graft was explanted through a transabdominal approach, and the aorta was reconstructed with cryopreserved allograft. Interestingly, the small and large intestine with their mesentery were found to be plastered to the aneurysm sac. The post-operative course was unremarkable except for a transient acute kidney injury that resolved within 1 week. Follow-up computed tomography scan at 6 months showed widely patent bypass. Conclusions: Glue embolization induces inflammation promoting thrombus formation inside the aneurysm sac. With a transcaval approach to the sac, there is the risk of extravasation of glue outside the sac as well as contamination of the graft with instrumentation. Differentiating between inflammation and infection can be difficult, and tissue biopsy provided the most conclusive diagnosis. Risk minimization considerations include, pre-operative optimization, a transabdominal approach, ureteral stenting, and tissue buttressing of anastomosis.

Amplification and Quantitation of Telomeric Extrachromosomal Circles.

Telomeres are structures that cap the ends of linear chromosomes and play critical roles in maintaining genome integrity and establishing the replicative lifespan of cells. In stem and cancer cells, telomeres are actively elongated by either telomerase or the alternative lengthening of telomeres (ALT) pathway. This pathway is characterized by several hallmark features, including extrachromosomal C-rich circular DNAs that can be probed to assess ALT activity. These so-called C-circles are the product of ALT-associated DNA damage repair processes and simultaneously serve as potential templates for iterative telomere extension. This bifunctional nature makes C-circles highly sensitive and specific markers of ALT. Here, we describe a C-circle assay, adapted from previous reports, that enables the quantitation of C-circle abundance in mammalian cells subjected to a wide range of experimental perturbations. This protocol combines the Quick C-circle Preparation (QCP) method for DNA isolation with fluorometry-based DNA quantification, rolling circle amplification (RCA), and detection of C-circles using quantitative PCR. Moreover, the inclusion of internal standards with well-characterized telomere maintenance mechanisms (TMMs) allows for the reliable benchmarking of cells with unknown TMM status. Overall, our work builds upon existing protocols to create a generalizable workflow for in vitro C-circle quantitation and ascertainment of TMM identity.

Telomerase in Cancer: Function, Regulation, and Clinical Translation.

During the process of malignant transformation, cells undergo a series of genetic, epigenetic, and phenotypic alterations, including the acquisition and propagation of genomic aberrations that impart survival and proliferative advantages. These changes are mediated in part by the induction of replicative immortality that is accompanied by active telomere elongation. Indeed, telomeres undergo dynamic changes to their lengths and higher-order structures throughout tumor formation and progression, processes overseen in most cancers by telomerase. Telomerase is a multimeric enzyme whose function is exquisitely regulated through diverse transcriptional, post-transcriptional, and post-translational mechanisms to facilitate telomere extension. In turn, telomerase function depends not only on its core components, but also on a suite of binding partners, transcription factors, and intra- and extracellular signaling effectors. Additionally, telomerase exhibits telomere-independent regulation of cancer cell growth by participating directly in cellular metabolism, signal transduction, and the regulation of gene expression in ways that are critical for tumorigenesis. In this review, we summarize the complex mechanisms underlying telomere maintenance, with a particular focus on both the telomeric and extratelomeric functions of telomerase. We also explore the clinical utility of telomeres and telomerase in the diagnosis, prognosis, and development of targeted therapies for primary, metastatic, and recurrent cancers.

The role of RNA processing and regulation in metastatic dormancy.

Tumor dormancy is a major contributor to the lethality of metastatic disease, especially for cancer patients who develop metastases years-to-decades after initial diagnosis. Indeed, tumor cells can disseminate during early disease stages and persist in new microenvironments at distal sites for months, years, or even decades before initiating metastatic outgrowth. This delay between primary tumor remission and metastatic relapse is known as "dormancy," during which disseminated tumor cells (DTCs) acquire quiescent states in response to intrinsic (i.e., cellular) and extrinsic (i.e., microenvironmental) signals. Maintaining dormancy-associated phenotypes requires DTCs to activate transcriptional, translational, and post-translational mechanisms that engender cellular plasticity. RNA processing is emerging as an essential facet of cellular plasticity, particularly with respect to the initiation, maintenance, and reversal of dormancy-associated phenotypes. Moreover, dysregulated RNA processing, particularly that associated with alternative RNA splicing and expression of noncoding RNAs (ncRNAs), can occur in DTCs to mediate intrinsic and extrinsic metastatic dormancy. Here we review the pathophysiological impact of alternative RNA splicing and ncRNAs in promoting metastatic dormancy and disease recurrence in human cancers.

SLX4IP promotes RAP1 SUMOylation by PIAS1 to coordinate telomere maintenance through NF-κB and Notch signaling.

The maintenance of telomere length supports repetitive cell division and therefore plays a central role in cancer development and progression. Telomeres are extended by either the enzyme telomerase or the alternative lengthening of telomeres (ALT) pathway. Here, we found that the telomere-associated protein SLX4IP dictates telomere proteome composition by recruiting and activating the E3 SUMO ligase PIAS1 to the SLX4 complex. PIAS1 SUMOylated the telomere-binding protein RAP1, which disrupted its interaction with the telomere-binding protein TRF2 and facilitated its nucleocytoplasmic shuttling. In the cytosol, RAP1 bound to IκB kinase (IKK), resulting in activation of the transcription factor NF-κB and its induction of Jagged-1 expression, which promoted Notch signaling and the institution of ALT. This axis could be targeted therapeutically in ALT-driven cancers and in tumor cells that develop resistance to antitelomerase therapies. Our results illuminate the mechanisms underlying SLX4IP-dependent telomere plasticity and demonstrate the role of telomere proteins in directly coordinating intracellular signaling and telomere maintenance dynamics.

Epigenetic plasticity in metastatic dormancy: mechanisms and therapeutic implications.

The overwhelming majority of cancer-associated morbidity and mortality can be ascribed to metastasis. Metastatic disease frequently presents in a delayed fashion following initial diagnosis and treatment, requiring that disseminated cancer cells (DCCs) spread early in tumor progression and persist in a dormant state at metastatic sites. To accomplish this feat, DCCs exhibit substantial phenotypic plasticity that is mediated by the epigenetic regulation of dormancy programs in response to intrinsic (i.e., cellular) and extrinsic (i.e., microenvironmental) cues. The epigenome is a dynamic landscape that encompasses transcriptional regulation via alteration of chromatin architecture, posttranscriptional RNA processing, and the diverse functions carried out by noncoding RNAs. Signals converging on DCCs are transduced through epigenetic effectors. Conversely, epigenetic regulation of gene expression controls the crosstalk between DCCs and cells of the metastatic niche, a phenomenon that is essential for the institution of dormant phenotypes. Importantly, epigenetic effectors can be targeted therapeutically, and the development of novel epigenetic therapies may provide new inroads to combating recurrent metastatic disease. Here we provide an overview of the dynamics of metastatic dormancy and summarize our current understanding of the intersections between dormancy and the epigenome, both mechanistically and therapeutically.

SLX4IP and telomere dynamics dictate breast cancer metastasis and therapeutic responsiveness.

Metastasis is the leading cause of breast cancer-related death and poses a substantial clinical burden owing to a paucity of targeted treatment options. The clinical manifestations of metastasis occur years-to-decades after initial diagnosis and treatment because disseminated tumor cells readily evade detection and resist therapy, ultimately giving rise to recurrent disease. Using an unbiased genetic screen, we identified SLX4-interacting protein (SLX4IP) as a regulator of metastatic recurrence and established its relationship in governing telomere maintenance mechanisms (TMMs). Inactivation of SLX4IP suppressed alternative lengthening of telomeres (ALT), coinciding with activation of telomerase. Importantly, TMM selection dramatically influenced metastatic progression and survival of patients with genetically distinct breast cancer subtypes. Notably, pharmacologic and genetic modulation of TMMs elicited telomere-dependent cell death and prevented disease recurrence by disseminated tumor cells. This study illuminates SLX4IP as a potential predictive biomarker for breast cancer progression and metastatic relapse. SLX4IP expression correlates with TMM identity, which also carries prognostic value and informs treatment selection, thereby revealing new inroads into combating metastatic breast cancers.

Stem cells, immortality, and the evolution of metastatic properties in breast cancer: telomere maintenance mechanisms and metastatic evolution.

Breast cancer is the most significant cause of cancer-related death in women around the world. The vast majority of breast cancer-associated mortality stems from metastasis, which remains an incurable disease state. Metastasis results from evolution of clones that possess the insidious properties required for dissemination and colonization of distant organs. These clonal populations are descended from breast cancer stem cells (CSCs), which are also responsible for their prolonged maintenance and continued evolution. Telomeres impose a lifespan on cells that can be extended when they are actively elongated, as occurs in CSCs. Thus, changes in telomere structure serve to promote the survival of CSCs and subsequent metastatic evolution. The selection of telomere maintenance mechanism (TMM) has important consequences not only for CSC survival and evolution, but also for their coordination of various signaling pathways that choreograph the metastatic cascade. Targeting the telomere maintenance machinery may therefore provide a boon to the treatment of metastatic breast cancer. Here we review the two major TMMs and the roles they play in the development of stem and metastatic breast cancer cells. We also highlight current and future approaches to targeting these mechanisms in clinical settings to alleviate metastatic breast cancers.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay.

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.

Exercise performance and physiological responses: the potential role of redox imbalance.

Increases in oxidative stress or decreases in antioxidant capacity, or redox imbalance, are known to alter physiological function and has been suggested to influence performance. To date, no study has sought to manipulate this balance in the same participants and observe the impact on physiological function and performance. Using a single-blind, placebo-controlled, and counterbalanced design, this study examined the effects of increasing free radicals, via hyperoxic exposure (FiO2 = 1.0), and/or increasing antioxidant capacity, through consuming an antioxidant cocktail (AOC; vitamin-C, vitamin-E, α-lipoic acid), on 5-kilometer (km) cycling time-trial performance, and the physiological and fatigue responses in healthy college-aged males. Hyperoxic exposure prior to the 5 km TT had no effect on performance, fatigue, or the physiological responses to exercise. The AOC significantly reduced average power output (222 ± 11 vs. 214 ± 12 W), increased 5 km time (516 ± 17 vs. 533 ± 18 sec), suppressed ventilation (VE; 116 ± 5 vs. 109 ± 13 L/min), despite similar oxygen consumption (VO2; 43.1 ± 0.8 vs. 44.9 ± 0.2 mL/kg per min), decreased VE/VO2 (35.9 ± 2.0 vs. 32.3 ± 1.5 L/min), reduced economy (VO2/W; 0.20 ± 0.01 vs. 0.22 ± 0.01), increased blood lactate (10 ± 0.7 vs. 11 ± 0.7 mmol), and perception of fatigue (RPE; 7.39 ± 0.4 vs. 7.60 ± 0.3) at the end of the TT, as compared to placebo (main effect, placebo vs. AOC, respectively). Our data demonstrate that prior to exercise, ingesting an AOC, but not exposure to hyperoxia, likely disrupts the delicate balance between pro- and antioxidant forces, which negatively impacts ventilation, blood lactate, economy, perception of fatigue, and performance (power output and 5 km time) in young healthy males. Thus, caution is warranted in athletes taking excess exogenous antioxidants.

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Means to the ends: The role of telomeres and telomere processing machinery in metastasis.

Despite significant clinical advancements, cancer remains a leading cause of mortality throughout the world due largely to the process of metastasis and the dissemination of cancer cells from their primary tumor of origin to distant secondary sites. The clinical burden imposed by metastasis is further compounded by a paucity of information regarding the factors that mediate metastatic progression. Linear chromosomes are capped by structures known as telomeres, which dictate cellular lifespan in humans by shortening progressively during successive cell divisions. Although telomere shortening occurs in nearly all somatic cells, telomeres may be elongated via two seemingly disjoint pathways: (i) telomerase-mediated extension, and (ii) homologous recombination-based alternative lengthening of telomeres (ALT). Both telomerase and ALT are activated in various human cancers, with more recent evidence implicating both pathways as potential mediators of metastasis. Here we review the known roles of telomere homeostasis in metastasis and posit a mechanism whereby metastatic activity is determined by a dynamic fluctuation between ALT and telomerase, as opposed to the mere activation of a generic telomere elongation program. Additionally, the pleiotropic nature of the telomere processing machinery makes it an attractive therapeutic target for metastasis, and as such, we also explore the therapeutic implications of our proposed mechanism.

Transoral robotic medial hypopharyngectomy: Surgical technique.

BACKGROUND: Transoral robotic surgery (TORS) has been shown to be both feasible and oncologically sound for use in early T classification hypopharyngeal cancers. The purpose of this study was to present our surgical technique for performing a transoral robotic hypopharyngectomy. METHODS: A 48-year-old man with a T2, N2a, M0 squamous cell carcinoma of the hypopharynx underwent a transoral robotic hypopharyngectomy and a left select neck dissection of levels II to IV. RESULTS: The hypopharyngeal tumor was removed en bloc after circumferential cuts were made until the tumor was finally amputated from the apex of the piriform sinus. Negative margins were achieved. A left select neck dissection was performed. The patient was tolerating an oral diet and his tracheostomy tube was decannulated before discharge. CONCLUSION: TORS offers improved manual dexterity and tumor manipulation over conventional transoral approaches to hypopharyngeal cancers. It is both a feasible and effective method for certain early-stage hypopharyngeal cancers. © 2015 Wiley Periodicals, Inc. Head Neck 38: E2127-E2129, 2016.

(R)-ß-lysine-modified elongation factor P functions in translation elongation.

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-ß-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Investigation of hydroxyl-(ß)-lysyl-EF-P showed 30% increased puromycin reactivity but no differences in dipeptide synthesis rates when compared with the ß-lysylated form. Unlike disruption of the other genes required for EF-P modification, deletion of yfcM had no phenotypic consequences in Salmonella. Taken together, our findings indicate that EF-P functions in translation elongation, a role critically dependent on post-translational ß-lysylation but not hydroxylation.