The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

YM155 Induces DNA Damage and Cell Death in Anaplastic Thyroid Cancer Cells by Inhibiting DNA Topoisomerase IIα at the ATP-Binding Site.

Anaplastic thyroid cancer (ATC) is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing ATC cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, ATC patient samples show overexpression of topoisomerase Top2α compared with benign thyroid samples and to differentiated thyroid cancers. ATC cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which shows decreased expression of Top2α and is resensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP-binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of ATC.

Metal Allergy in Tracheostomy Tube Placement Resulting in Complete Subglottic Stenosis: A Case Report.

OBJECTIVES: Metal hypersensitivity reaction to surgical implants is a well- known phenomenon that is associated with pain, swelling, inflammation, and decreased efficacy of the implant. We present a unique case of a patient with placement a metal Jackson tracheostomy tube that led to expeditious total subglottic stenosis. METHODS: The patient was a 33-year old, severely atopic woman with history of asthma exacerbations requiring several intubations for acute respiratory failure with several subsequent tracheal dilations with steroid injections, and eventual tracheostomy placement with a metal Jackson tracheostomy tube that led to expeditious total subglottic stenosis. RESULTS: Initial intervention included performing an airway evaluation, CO2 laser, and steroid injection of the area of complete subglottic stenosis. Follow up several months later revealed little improvement in level of tracheal narrowing proximal to the tracheostomy tube. Patient did not have shortness of breath but continued to be aphonic. Cricotracheal versus tracheal resection have been proposed but surgical morbidity was deemed too high due to patient's obesity. CONCLUSIONS: Metal hypersensitivity reactions are well known phenomena as it relates to surgical implants in other surgical specialties but are seldom reported within the ear, nose and throat literature. Oftentimes, it takes astute observation to diagnose and establish a connection. Prompt recognition and treatment can be acquired from interdisciplinary collaboration with allergy.

Pilot Study: Pneumatic Compression Garment Therapy for Postradiotherapy Laryngopharyngeal Edema.

OBJECTIVES: Pneumatic compression garment therapy (PCGT) has been established as treatment for postradiotherapy lymphedema, and its use in head and neck patients is becoming more common. Although effects on interstitial edema of the cervical soft tissues have been studied, effects on internal laryngopharyngeal edema, as well as associated symptoms of dysphagia and dysphonia, have yet to be published. METHODS: We surveyed 7 patients treated with radiation for head and neck cancer (HNC) who had also been prescribed PCGT for cervical lymphedema. Patients were asked about subjective experience with the device, and also administered the Eating Assessment Tool-10 (EAT-10) and Voice Handicap Index-10 (VHI-10) surveys regarding their symptoms after using PCGT. Laryngoscopy videos from these same periods were also reviewed and scored using a validated tool for assessing laryngopharyngeal edema. RESULTS: 85% of patients reported at least some improvement in dysphagia and dysphonia following PCGT. Average EAT-10 score after PCGT was 11.4 and average VHI-10 score after PCGT was 8.7. These compare more favorably to historical scores for the same questionnaires in similar patient populations. Laryngeal edema scores on endoscopic examination were not significantly different after at least 3 months of therapy (pre: 20.15, post: 20.21, P = .975); however, the utility of this result is limited by a low inter-rater reliability (Krippendorff α = .513). CONCLUSIONS: While we are unable to show any difference in objective assessment of laryngopharyngeal edema on endoscopic examination in this small pilot study, patients report substantial subjective improvement in postradiotherapy dysphagia and dysphonia following cervical PCGT that warrants more formal investigation.

Ym155 Induces Oxidative Stress-Mediated DNA Damage and Cell Cycle Arrest, and Causes Programmed Cell Death in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

Steroid eluting biocompatible stent for subglottic stenosis.

OBJECTIVE: Develop a prototype steroid eluting stent suitable for endoscopic treatment of subglottic stenosis. METHODS: Rectangular-shaped spoke design stents thermally molded into horseshoe-shaped stents were developed using AutoCAD program, and printed on a Lulzbot 3D printer with polycaprolactone (PCL). Kenalog saturated AEROSIL 200 was embedded in the PCL filament. Horizontal radial force measurements were measured at baseline, 1 day, and 1 month when deformation switched from bending to compression. Amount of Kenalog eluted after 1 day, 1 week and 1 month were measured using HPLC. RESULTS: Horizontal pressure applied to the PCL stent corresponding to a 5-0 ET were 1.27 ± 0.38 lb. at baseline, 1.79 ± 0.045 lb. at 1 day, 1.94 ± - 0.22 lb. at 1 week and 2.07 ± 0.11 lb. at 1 month. The horizontal pressure applied to PCL stent corresponding to an 8-0 ET tube were 0.82 ± 0.018 lb. at baseline, 1.008 ± 0.045 lb. at 1 day, 0.95 ± - 0.064 lb. at 1 week and 1.078 ± 0.021 lb. at 1 month. The amount of Kenalog eluted increased from 5.78 µg/mL at 1 day to 15.01 µg/mL at 1 week to 19.35 µg/mL at 1 month. CONCLUSION: This proof-of-concept project is an initial step to demonstrate and create a novel stent in the treatment of subglottic stenosis that applies expansile force on the trachea, elutes steroids and dissolves. Over time the expansile force along the trachea increases allowing the PCL to mucosalize, while it dissolves and continues to elute steroids. The limitations of this in vitro study necessitate experiments on animal models, such as rabbit tracheas to observe for complications and histologic changes. LEVEL OF EVIDENCE: This proof-of-concept project is a Level 5 mechanism-based reasoning study.

R-Loop Physiology and Pathology: A Brief Review.

Physiological and pathological roles for R-loop structures continue to be discovered, and studies suggest that R-loops could contribute to human disease. R-loops are nucleic acid structures characterized by a DNA:RNA hybrid and displaced single-stranded DNA that occur in connection with transcription. R-loops form naturally and have been shown to be important for a number of physiological processes such as mitochondrial replication initiation, class switch recombination, DNA repair, modulating DNA topology, and regulation of gene expression. However, subsets of R-loops or persistent R-loops lead to DNA breaks, chromosome rearrangement, and genome instability. In addition, R-loops have been linked to human diseases, specifically neurological disorders and cancer. Of the large amount of research produced recently on R-loops, this review covers evidence for R-loop involvement in normal cellular physiology and pathophysiology, as well as describing factors that contribute to R-loop regulation.

Frequent HPV-independent p16/INK4A overexpression in head and neck cancer.

OBJECTIVES: p16INK4A (p16) is the most widely used clinical biomarker for Human Papillomavirus (HPV) in head and neck squamous cell cancer (HNSCC). HPV is a favourable prognostic marker in HNSCC and is used for patient stratification. While p16 is a relatively accurate marker for HPV within the oropharynx, recent reports suggest it may be unsuitable for use in other HNSCC subsites, where a smaller proportion of tumors are HPV-driven. MATERIALS AND METHODS: We integrated reverse phase protein array (RPPA) data for p16 with HPV status based on detection of viral transcripts by RNA-seq in a set of 210 HNSCCs profiled by The Cancer Genome Atlas project. Samples were queried for alterations in CDKN2A, and other pathway genes to investigate possible drivers of p16 expression. RESULTS: While p16 levels as measured by RPPA were significantly different by HPV status, there were multiple HPV (-) samples with similar expression levels of p16 to HPV (+) samples, particularly at non-oropharyngeal subsites. In many cases, p16 overexpression in HPV (-) tumors could not be explained by mutation or amplification of CDKN2A or by RB1 mutation. Instead, we observed enrichment for inactivating mutations in the histone H3 lysine 36 methyltransferase, NSD1 in HPV (-)/p16-high tumors. CONCLUSIONS: RPPA data suggest high p16 protein expression in many HPV (-) non-oropharyngeal HNSCCs, limiting its potential utility as an HPV biomarker outside of the oropharynx. HPV-independent overexpression of wild-type p16 in non-oropharyngeal HNSCC may be linked to global deregulation of chromatin state by inactivating mutations in NSD1.

Cytokeratin-8 in Anaplastic Thyroid Carcinoma: More Than a Simple Structural Cytoskeletal Protein.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

The feasibility of gamma radiation sterilization for decellularized tracheal grafts.

OBJECTIVES/HYPOTHESIS: The most promising stem cell-derived tracheal transplantation approach is dependent upon the use of decellularized tracheal grafts. It has been assumed that a sterilization step, such as gamma radiation, would damage the delicate extracellular matrix of the graft, thus rendering it less viable for cellular repopulation, although this has not been thoroughly investigated. STUDY DESIGN: Laboratory-based comparative analysis. METHODS: Fifteen murine tracheas of strain C57/B-6 mice were obtained. Thirteen were subjected to a detergent-enzymatic decellularization process. Of these decellularized tracheas (DT), eight were irradiated, exposing five tracheas to a radiation level of 25 kGy (DT25) and three to 5 kGy (DT5). Two were left untreated. The two untreated tracheas, two DTs, and two DT25s were prepared and examined using both scanning and transmission electron microscopy. Bioburden calculations were obtained from three DTs, three DT25s, and three DT5s by homogenization, serial dilution, and streak plating. RESULTS: Electron microscopy of untreated fresh tracheas and DTs showed a slight qualitative degradation of cartilage ultrastructure due to the decellularization process. In contrast, examination of DT25 shows significant degradation including poor overall preservation of cartilage architecture with disorganized collagen fibers. The nonirradiated DTs had a calculated bacterial bioburden of 7.8 × 107 to 3.4 × 108 colony-forming units per gram. Both the DT25 and DT5 specimens were found to have a bioburden of zero. CONCLUSIONS: Gamma radiation at 25 kGy degrades the architecture of decellularized tracheal grafts. These ultrastructural changes may prove detrimental to graft viability; however, bioburden calculations suggest that a 5 kGy radiation dose may be sufficient for sterilization. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E258-E264, 2017.

YM155 inhibits topoisomerase function.

YM155 (sepantronium bromide) has been evaluated in clinical trials as a survivin suppressant, but despite positive signals from early work, later studies were negative. Clarification of the mechanism of action of YM155 is important for its further development. YM155 affects cells in a cell cycle-specific manner. When cells are in G1, YM155 prevented their progression through the S phase, leaving the cells at G1/S when exposed to YM155. Passage through mitosis from G2 is also defective following YM155 exposure. In this study, YM155 did not behave like a typical DNA intercalator in viscosity, circular dichroism, and absorption spectroscopy studies. In addition, molecular modeling experiments ruled out YM155 DNA interaction to produce DNA intercalation. We show that YM155 inhibited topoisomerase 2α decatenation and topoisomerase 1-mediated cleavage of DNA, suggesting that YM155 inhibits the enzyme function. Consistent with these findings, DNA double-strand break repair was also inhibited by YM155.

Cell Cycle M-Phase Genes Are Highly Upregulated in Anaplastic Thyroid Carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) accounts for only 3% of thyroid cancers, yet strikingly, it accounts for almost 40% of thyroid cancer deaths. Currently, no effective therapies exist. In an effort to identify ATC-specific therapeutic targets, we analyzed global gene expression data from multiple studies to identify ATC-specific dysregulated genes. METHODS: The National Center for Biotechnology Information Gene Expression Omnibus database was searched for high-throughput gene expression microarray studies from human ATC tissue along with normal thyroid and/or papillary thyroid cancer (PTC) tissue. Gene expression levels in ATC were compared with normal thyroid or PTC using seven separate comparisons, and an ATC-specific gene set common in all seven comparisons was identified. We investigated these genes for their biological functions and pathways. RESULTS: There were three studies meeting inclusion criteria, (including 32 ATC patients, 69 PTC, and 75 normal). There were 259 upregulated genes and 286 downregulated genes in ATC with at least two-fold change in all seven comparisons. Using a five-fold filter, 36 genes were upregulated in ATC, while 40 genes were downregulated. Of the 10 top globally upregulated genes in ATC, 4/10 (MMP1, ANLN, CEP55, and TFPI2) are known to play a role in ATC progression; however, 6/10 genes (TMEM158, CXCL5, E2F7, DLGAP5, MME, and ASPM) had not been specifically implicated in ATC. Similarly, 3/10 (SFTA3, LMO3, and C2orf40) of the most globally downregulated genes were novel in this context, while 7/10 genes (SLC26A7, TG, TSHR, DUOX2, CDH1, PDE8B, and FOXE1) have been previously identified in ATC. We experimentally validated a significant correlation for seven transcription factors (KLF16, SP3, ETV6, FOXC1, SP1, EGFR1, and MAFK) with the ATC-specific genes using microarray analysis of ATC cell lines. Ontology clustering of globally altered genes revealed that "mitotic cell cycle" is highly enriched in the globally upregulated gene set (44% of top upregulated genes, p-value <10-30). CONCLUSIONS: By focusing on globally altered genes, we have identified a set of consistently altered biological processes and pathways in ATC. Our data are consistent with an important role for M-phase cell cycle genes in ATC, and may provide direction for future studies to identify novel therapeutic targets for this disease.

In vitro characterization of design and compressive properties of 3D-biofabricated/decellularized hybrid grafts for tracheal tissue engineering.

Infection or damage to the trachea, a thin walled and cartilage reinforced conduit that connects the pharynx and larynx to the lungs, leads to serious respiratory medical conditions which can often prove fatal. Current clinical strategies for complex tracheal reconstruction are of limited availability and efficacy, but tissue engineering and regenerative medicine approaches may provide viable alternatives. In this study, we have developed a new "hybrid graft" approach that utilizes decellularized tracheal tissue along with a resorbable polymer scaffold, and holds promise for potential clinical applications. First, we evaluated the effect of our decellularization process on the compression properties of porcine tracheal segments, and noted approximately 63% decrease in resistance to compression following decellularization. Next we developed four C-shape scaffold designs by varying the base geometry and thickness, and fabricated polycaprolactone scaffolds using a combination of 3D-Bioplotting and thermally-assisted forming. All scaffolds designs were evaluated in vitro under three different environmental testing conditions to determine the design that offered the best resistance to compression. These were further studied to determine the effect of gamma radiation sterilization and cyclic compression loading. Finally, hybrid grafts were developed by securing these optimal design scaffolds to decellularized tracheal segments and evaluated in vitro under physiological testing conditions. Results show that the resistance to compression offered by the hybrid grafts created using gamma radiation sterilized scaffolds was comparable to that of fresh tracheal segments. Given that current clinical attempts at tracheal transplantation using decellularized tissue have been fraught with luminal collapse and complications, our data support the possibility that future embodiments using a hybrid graft approach may reduce the need for intraluminal stenting in tracheal transplant recipients.

Transgelin increases metastatic potential of colorectal cancer cells in vivo and alters expression of genes involved in cell motility.

BACKGROUND: Transgelin is an actin-binding protein that promotes motility in normal cells. Although the role of transgelin in cancer is controversial, a number of studies have shown that elevated levels correlate with aggressive tumor behavior, advanced stage, and poor prognosis. Here we sought to determine the role of transgelin more directly by determining whether experimental manipulation of transgelin levels in colorectal cancer (CRC) cells led to changes in metastatic potential in vivo. METHODS: Isogenic CRC cell lines that differ in transgelin expression were characterized using in vitro assays of growth and invasiveness and a mouse tail vein assay of experimental metastasis. Downstream effects of transgelin overexpression were investigated by gene expression profiling and quantitative PCR. RESULTS: Stable overexpression of transgelin in RKO cells, which have low endogenous levels, led to increased invasiveness, growth at low density, and growth in soft agar. Overexpression also led to an increase in the number and size of lung metastases in the mouse tail vein injection model. Similarly, attenuation of transgelin expression in HCT116 cells, which have high endogenous levels, decreased metastases in the same model. Investigation of mRNA expression patterns showed that transgelin overexpression altered the levels of approximately 250 other transcripts, with over-representation of genes that affect function of actin or other cytoskeletal proteins. Changes included increases in HOOK1, SDCCAG8, ENAH/Mena, and TNS1 and decreases in EMB, BCL11B, and PTPRD. CONCLUSIONS: Increases or decreases in transgelin levels have reciprocal effects on tumor cell behavior, with higher expression promoting metastasis. Chronic overexpression influences steady-state levels of mRNAs for metastasis-related genes.

Why sinonasal disease spares the inferior turbinate: An immunohistochemical analysis.

OBJECTIVES/HYPOTHESIS: Clinically, inflammatory polyps are found in the middle turbinate (MT) in patients with chronic rhinosinusitis (CRS) but not in the inferior turbinate (IT). The purpose of this study was to investigate differences in protein expression between IT and MT tissue in patients with CRS. STUDY DESIGN: Prospective cohort. METHODS: Pathologic specimens obtained from patients with CRS undergoing functional endoscopic sinus surgery with IT reduction were evaluated by immunohistochemical analysis of inflammatory markers cysteinyl leukotriene 1 receptor (CysLT1R), toll-like receptor 2 (TLR2), and vascular cell adhesion molecule 1 (VCAM1). Protein expression was quantified with nuance multispectral analysis and results compared between MT and IT tissue. RESULTS: The total expression of VCAM1 and CysLT1R was decreased in the IT compared to the MT. There was no difference in total TLR2 expression between the IT and MT. When comparing patients with eosinophilic CRS to noneosinophilic CRS (neCRS), there was decreased expression of VCAM1 in the IT of patients with neCRS. When comparing patients with nasal polyposis to those without polyps, there was decreased expression of VCAM1 in the IT of patients without polyps. CONCLUSIONS: There is a difference in protein receptor expression of VCAM1 and CysLT1R in MT compared to IT tissue. Although the leukotrienes are a well-known target for treatment of chronic sinusitis, this is the first study demonstrating an upregulation of VCAM1 expression in the MT and could be a potential future target for the treatment of CRS. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E179-E183, 2016.

Mechanical Modeling of the Human Cricoid Cartilage Using Computer-Aided Design: Applications in Airway Balloon Dilation Research.

OBJECTIVES: Balloon dilation is generally considered first-line treatment for airway stenosis. Some dilation systems utilize a compliant balloon that can conform around rigid structures. Others use a noncompliant balloon that does not conform, allowing for dilation of more rigid stenoses. We hypothesized that subglottic dilation with a noncompliant balloon increases the likelihood of fracture of the cricoid when compared to a compliant balloon. METHODS: Three fresh human cricoid cartilages were placed in a universal testing system to determine the expansile force necessary for cricoid fracture. Using these data, a 3D printer was used to construct a synthetic cricoid model possessing near identical physical characteristics to the human cricoid. Simulated dilation was then performed on the model using a compliant and a noncompliant balloon. RESULTS: Human cricoid fracture occurred at 97.25 N (SD = 8.34), and the synthetic cricoid model fractured at 100.10 N (SD = 7.32). Both balloons fractured the model in every replicate experiment. Mean balloon internal pressure at fracture was 7.67 ATM (SD = 1.21) for the compliant balloon and 11.34 ATM (SD = 1.29) for the noncompliant balloon. CONCLUSIONS: These data show that fracture of the cricoid is a valid concern in balloon dilation procedures where the balloon spans the subglottis. Furthermore, the hypothesis was rejected in that the compliant balloon system was at least as likely to fracture the cricoid model as the noncompliant.

A Novel Temporal Bone Simulation Model Using 3D Printing Techniques.

HYPOTHESIS: An inexpensive temporal bone model for use in a temporal bone dissection laboratory setting can be made using a commercially available, consumer-grade 3D printer. BACKGROUND: Several models for a simulated temporal bone have been described but use commercial-grade printers and materials to produce these models. The goal of this project was to produce a plastic simulated temporal bone on an inexpensive 3D printer that recreates the visual and haptic experience associated with drilling a human temporal bone. METHODS: Images from a high-resolution CT of a normal temporal bone were converted into stereolithography files via commercially available software, with image conversion and print settings adjusted to achieve optimal print quality. The temporal bone model was printed using acrylonitrile butadiene styrene (ABS) plastic filament on a MakerBot 2x 3D printer. Simulated temporal bones were drilled by seven expert temporal bone surgeons, assessing the fidelity of the model as compared with a human cadaveric temporal bone. Using a four-point scale, the simulated bones were assessed for haptic experience and recreation of the temporal bone anatomy. RESULTS: The created model was felt to be an accurate representation of a human temporal bone. All raters felt strongly this would be a good training model for junior residents or to simulate difficult surgical anatomy. Material cost for each model was $1.92. CONCLUSIONS: A realistic, inexpensive, and easily reproducible temporal bone model can be created on a consumer-grade desktop 3D printer.

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Biomechanical changes from long-term freezer storage and cellular reduction of tracheal scaffoldings.

OBJECTIVES/HYPOTHESIS: To determine structural biomechanical changes in tracheal scaffolds resulting from cellular reduction and storage at -80(o) C. STUDY DESIGN: Laboratory-based study. METHODS: Forty-four rabbit tracheal segments were separated into four treatment groups: untreated (group A, control), cellular-reduced (group B), storage at -80(o) C followed by cellular reduction (group C), and cellular-reduced followed by storage at -80(o) C (group D). Tracheal segments were subjected to uniaxial tension (n = 21) or compression (n = 23) using a universal testing machine to determine sutured tensile yield load and radial compressive strengths at 50% lumen occlusion. Mean differences among groups for tension and compression were compared by analysis of variance with post-hoc Tukey-Kramer test. RESULTS: The untreated trachea (group A) demonstrated mean yield strength of 5.93 (± 1.65) N and compressive strength of 2.10 (± 0.51) N. Following treatment/storage, the tensile yield strength was not impaired (group B = 6.79 [± 1.58] N, C = 6.21 [± 1.40] N, D = 6.26 [± 1.18]; P > 0.10 each). Following cellular reduction, there was a significant reduction in compressive strength (group B = 0.44 N [± 0.13], P < 0.0001), but no further reduction due to storage (group C = 0.39 N [± 0.10]; P = 0.97 compared to group B). CONCLUSION: The data suggest cellular reduction leads to loss of compressive strength. Freezing at -80°C (either before, or subsequent to cellular reduction) may be a viable storage method for tracheal grafts.

Comparison of tensile strength of fibrin glue and suture in microflap closure.

OBJECTIVES/HYPOTHESIS: Suture closure and fibrin glue placement have been advocated as alternatives to healing by secondary intention. The aim of this study was to examine the tensile strength of these microflap closure techniques. STUDY DESIGN: Basic research. METHODS: Three pairs of excised bovine true vocal folds underwent microflap creation and closure by either single 6-0 polyglactin suture or fibrin glue. Vocal folds were distracted to failure on a universal testing system. Excised porcine true vocal folds underwent microflap creation and were closed with either single 6-0 polyglactin suture or fibrin glue, or were left without closure. Tensile strength testing was performed with a universal testing system measuring load at 1 mm, 5 mm, and 10 mm of distraction. RESULTS: The bovine vocal fold model failed after an average extension of 22.6 mm (range, 21.4-23.9 mm) corresponding to 11.61 N (range, 8.04-13.47 N), with no failure of the suture prior to model failure. Fibrin glue did not demonstrate any measureable resistance to tension application. In the porcine vocal fold model, there was a significant difference between the median tensile load of suture closure (2.91 N) and no closure (1.16 N) at 10 mm of distraction (P = .01). There was no significant difference in median load of vocal folds undergoing fibrin glue closure or no closure. CONCLUSIONS: There is no significant difference in tensile strength of a microflap closed with fibrin glue or not closed. Suture closure of a microflap provides a significantly stronger mechanical closure than no closure. This suggests that use of fibrin glue is of little benefit on the vocal folds.