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Feeding is a complex process that involves an integrated response of multiple functional systems. Animals evolve phenotypic integration of complex morphological traits to covary and maximize performance of feeding behaviors. Specialization, such as feeding on dangerous prey, can further shape the integration of behavior and morphology as traits are expected to evolve and maintain function in parallel. Feeding on centipedes, with their powerful forcipules that pinch and inject venom, has evolved multiple times within snakes, including the genus Tantilla. However, the behavioral and morphological adaptations used to consume this dangerous prey are poorly understood. By studying snakes with varying degrees of dietary specialization we can test the integration of diet, morphology, and behavior to better understand the evolution of consuming difficult prey. We studied prey preference and feeding behavior of Tantilla using the flat-headed snake (T. gracilis) and the crowned snake (T. coronata) which differ in the percentage of centipedes in their diet. We then quantified cranial anatomy using geometric morphometric data from CT scans. To test prey preference, we offered multiple types of prey and recorded snake behavior. Both species of snakes showed interest in multiple prey types, but only struck or consumed centipedes. To subdue centipedes, crowned snakes used coiling and holding (envenomation) immediately after striking, while flat-headed snakes used the novel behavior of pausing and holding onto centipedes for a prolonged time prior to completion of swallowing. Each skull element differed in shape after removing the effects of size, position, and orientation. The rear fang was larger in crowned snakes, but the mechanical advantage of the lower jaw was greater in flat-headed snakes. Our results suggest that the integration of behavioral and morphological adaptations is important for the success of subduing and consuming dangerous prey.

XLSX file - 15K, Supplementary Table 1. Clinical characteristics of discovery cohort. Supplementary Table 2. Clinical characteristics of mRNA expression array cohort. Supplementary Table 3. Clinical characteristics of validation cohort.

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PDF file - 133K, Supplementary Figure 1. miRNA-21 is upregulated in HNSCC tumors. Supplementary Figure 2. RT-qPCR validation of miRNA-21 overexpression by transient transfection. (A) pre-miRNA-21 and (B) miRNA-21 expression level. Supplementary Figure 3. miRNA-21 overexpression stimulates cell proliferation. All measurements were obtained with the Cell Counting Kit -8 cell proliferation assay. ∗p

XLSX file - 137K, Supplementary Table 7. has-mir-21 gene target prediction (miRanda). Supplementary Table 8. has-mir-21 gene target prediction (TargetScan 4.2). Supplementary Table 9. has-mir-21 gene target prediction (PicTar).

XLSX file - 841K, Supplementary Table 4. miRNA array analysis of discovery cohort. Supplementary Table 5. miRNA array significant call signals. Supplementary Table 6. RT-qPCR validation of miRNA array.

XLSX file - 886K, Differentially expressed mRNAs in HNSCC tumors.

Purpose: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21–specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology.Experimental Design: miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets.Results: miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3′-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression.Conclusions: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21. Clin Cancer Res; 20(4); 868–77. ©2013 AACR.

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NOTCH1 mutations have been reported to occur in 10% to 15% of head and neck squamous cell carcinomas (HNSCC). To determine the significance of these mutations, we embarked upon a comprehensive study of NOTCH signaling in a cohort of 44 HNSCC tumors and 25 normal mucosal samples through a set of expression, copy number, methylation, and mutation analyses. Copy number increases were identified in NOTCH pathway genes, including the NOTCH ligand JAG1. Gene set analysis defined a differential expression of the NOTCH signaling pathway in HNSCC relative to normal tissues. Analysis of individual pathway-related genes revealed overexpression of ligands JAG1 and JAG2 and receptor NOTCH3. In 32% of the HNSCC examined, activation of the downstream NOTCH effectors HES1/HEY1 was documented. Notably, exomic sequencing identified 5 novel inactivating NOTCH1 mutations in 4 of the 37 tumors analyzed, with none of these tumors exhibiting HES1/HEY1 overexpression. Our results revealed a bimodal pattern of NOTCH pathway alterations in HNSCC, with a smaller subset exhibiting inactivating NOTCH1 receptor mutations but a larger subset exhibiting other NOTCH1 pathway alterations, including increases in expression or gene copy number of the receptor or ligands as well as downstream pathway activation. Our results imply that therapies that target the NOTCH pathway may be more widely suitable for HNSCC treatment than appreciated currently. Cancer Res; 74(4); 1091–104. ©2013 AACR.

PDF file - 82K, Growth effects of NOTCH1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. NOTCH1 was downregulated in UPCI-SCC090 (090, top, NOTCH1 wt) and SCC61 (bottom, NOTCH1 wt) by siRNA as described in methods. The effect of NOTCH1 downregulation on cell proliferation for these two cell lines is shown in the middle. The effect of siNOTCH1 on NOTCH1 expression (left) or expression of its targets (HES1 and HEY1, right) is also shown. The mRNA expression levels were calculated relative to GAPDH and were normalized to 1 in the control experiments with scrambled siRNA sequences. Experiments were performed in pentaplicates, mean plus-minus SEM were shown. *, p-value < 0.05.

PDF file - 73K, Significant overexpression of JAG1 (A) and PSEN1 (B) in the tumors with increased copy number gains in comparison to that in the normal tissues in the cohort of 44 HNSCC tumors and 25 normal mucosa. N, normal mucosa; T, HNSCC tumors; -, without JAG1(A) or PSEN1 (B) copy number gain; +, with JAG1 (A) or PSEN1 (B) copy number gain. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

PDF file - 169K, NOTCH pathway activation in HNSCC. A, Immunohistochemical analysis of NOTCH1, HES1 and HEY1 protein expression in HNSCC. Tissue microarray arrays with quadruplicates of samples from each of 56 HNSCC tumor and 11 non-tumor patients were stained with anti-NOTCH1, anti-HES1 or anti-HEY1 antibody, as described in methods. Top, the heat-map of immunohistochemical analysis for tissue microarray slides. Rows, genes; column, each individual sample. The average staining for four tissues was categorized to Strong (brown), Moderate (grey) or Weak/Negative (blue) staining. Samples are ranked by the pathology ID numbers in tumor (left) and normal (right) groups. Bottom: quantification of immunohistochemical data. The number and percentage of tumor (Tu) and normal (No) patient in strong, moderate and weak/negative category was quantified. P-values were calculated by comparing numeric scores in tumor vs normal sample by independent t test. The samples 57964, 58602, 22146, 5931, 26514 represent tumor samples X6, X7, X11, X28 and X34 from the original discovery cohort of 44 HNSCC tumors. B, Representative immunohistochemical results. The representative slides of positive or moderate staining are shown for tumors and compared to normal tissue. Bar, 100 um equivalent. Tumor (T) and normal (N) tissues on each sample are labeled. ID 22146 represented X11 samples, which were used both in array and in immunohistochemical analysis, where protein and RNA expression strongly correlate.

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PDF file - 54K, Effect of gamma-secretase inhibitor on the NOTCH1 pathway genes and cell growth in HNSCC cells with NOTCH1 wildtype status. UPCI-SCC090 cells were growing on the media supplemented with 0.2 uM gamma-secretase inhibitor XXI (GSI-XXI) for up to 72 hours. The effect of GSI-XXI on the NOTCH1 targets HES1 and HEY1 is shown on the left for UPSI-SCC090 cell line.

PDF file - 68K, Growth effects of HEY1 siRNA inhibition in HNSCC cells with wildtype NOTCH1 status. HEY1 was downregulated in UPCI-SCC090 and SCC61 by siRNA as described in methods. The effect of siHEY1 on the expression of HEY1 is shown on the left. The effect of HEY1 downregulation on cell proliferation for these two cell lines is shown on the right. Note, there is over 90% transfection efficiency of HEY1 downregulation for each cell line, however the strong growth inhibition effect was shown only for NOTCH1 wildtype 090 cell line.

PDF file - 108K, Validation of JAG1, JAG2, NOTCH1 and PSEN1 overexpression in TCGA HNSCC cohort. A, Overexpression of JAG1, JAG2 and NOTCH1 in tumors in comparison to that in normal tissues in the TCGA HNSCC cohort. B, Overexpression of JAG1 and PSEN1 in the tumors with increased copy number gains in comparison to that in the normal tissues as well as to that in the tumors without copy number gains in the TCGA HNSCC cohort. N, normal mucosa; T, HNSCC tumors; -, without JAG1 or PSEN1 copy number gain; +, with JAG1 or PSEN1 copy number gain. Dots, relative expression levels in different tissue samples. Boxes represent the interquartile range (25th-75th percentile) and horizontal lines inside the boxes indicate median. Whiskers indicate the minimum and maximum values. P value was calculated by using t test.

PDF file - 88K, Supplementary Table I. Baseline characteristics of the HNSCC patients for microarray analysis. Supplementary Table II. Short Tandem Repeat Profile of the 2 HNSCC Cell Lines. Supplementary Table III. NOTCH Signaling Pathway Genes assigned in KEGG pathway database and shown on expression microarrays. Supplementary Table IV. Summary of the mRNA levels of NOTCH signaling pathway genes in the cohort of 44 HNSCC tumors and 25 normal mucosas measured by Affymetrix expression array.

Continuum models of two-phase flow have been employed to investigate a wide variety of physical phenomena, ranging from bubbly-gas and solid-loaded liquid flows to detonation waves in granular expl...

Gene set analysis provides a method to generate statistical inferences across sets of linked genes, primarily using high-throughput expression data. Common gene sets include biological pathways, operons, and targets of transcriptional regulators. In higher eukaryotes, especially when dealing with diseases with strong genetic and epigenetic components such as cancer, copy number loss and gene silencing through promoter methylation can eliminate the possibility that a gene is transcribed. This, in turn, can adversely affect the estimation of transcription factor or pathway activity from a set of target genes, since some of the targets may not be responsive to transcriptional regulation. Here we introduce a simple filtering approach that removes genes from consideration if they show copy number loss or promoter methylation and demonstrate the improvement in inference of transcription factor activity in a simulated data set based on the background expression observed in normal head and neck tissue.

OBJECTIVE: This study objectively characterized the microenvironment of indolent, chronic wounds by developing a method by which minute quantities of cytokines could be extracted from chronic wounds and separately identified. SUMMARY BACKGROUND DATA: Recombinant DNA technology and the ability to clone compounds such as cytokines allow new management schemes for the treatment of acute and chronic wounds. Before treatment with an exogenous cytokine is started, it would be helpful to know the endogenous level of that cytokine in the wound. Although various methods of extracting cytokines from acute wounds have been reported, no techniques have existed to reliably measure endogenous levels of cytokines in chronic wounds. METHODS: Porous, inert hydrophilic dextranomer beads were tested for their ability to absorb or adsorb protein and cytokines in vitro with either albumin or albumin laced with various known amounts of cytokines, and then from chronic human pressure ulcers. The Bradford protein assay was used to determine protein levels. Enzyme-linked immunosorbent assay (ELISA) techniques were used to determine levels of platelet-derived growth factor (PDGF)-AB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor-Beta (TGF-beta) extracted by the beads. RESULTS: Between 88% and 98% of known amounts of albumin could be recovered. Similarly, more than 90% of the laced cytokines could be recovered. In 20 grade III/IV pressure ulcers, although protein concentrations were remarkably similar, endogenous levels of cytokine growth factors varied tremendously. Platelet-derived growth factor-AB ranged from 49 to 867 pg/mL; bFGF from 47 to 697 pg/mL; and EGF from nondetectable to 247.5 pg/mL. TGF-B was not detected in 17 of the 20 pressure ulcers. CONCLUSIONS: This new technique appears useful for measuring endogenous levels of cytokines. Levels of cytokines found in these chronic wounds are much lower than those reported from acute wounds. The marked variation found among the 20 wounds may help to explain the differences reported in recent wound healing trials with exogenous cytokines.

Half a million new cases of Head and Neck Squamous Cell Carcinoma (HNSCC) are diagnosed annually worldwide. Given the advanced stage at diagnosis, 5 year survival after initial diagnosis is only 50% for HNSCC patients despite the current therapeutic strategies. Recently, high throughput technologies have accelerated the genome-wide characterization of HNSCC, but given the high sensitivity of this methodology, these data require careful evaluation. To improve the detection of cancer-related changes in HNSCC we applied outlier statistics to high throughput DNA methylation and gene expression data, and identified 76 top-scoring candidate proto-oncogenes and tumor suppressor genes with significantly changed expression in tumors compared to normal tissues. We identified epigenetically regulated candidates with potential role in HNSCC by selecting genes with strong negative correlation between gene expression and DNA methylation of their promoters. Differential expression and methylation of selected candidates were confirmed with assays on an independent HNSCC cohort and on public domain high throughput data on TCGA-HNSCC cohort (The Cancer Genome Atlas). We further performed functional evaluation of a lead candidate - DTX1 and demonstrated that DTX1 is a negative regulator of cell migration. We confirmed that epigenetic-dependent downregulation of DTX1 enhanced invasiveness of head and neck cancer cells. Outlier analysis integrated with high throughput expression and methylation can be used to identify an epigenetically- regulated tumor suppressor genes, including DTX1. Citation Format: Daria A. Gaykalova, Veronika Zizkova, Ilse Tiscareno, Yingying Wei, Rajita Vatapalli, Patrick T. Hennessey, Julie Ahn, Ludmila V. Danilova, Zubair Khan, Justin A. Bishop, Wayne M. Koch, William H. Westra, Michael F. Ochs, Joseph A. Califano. DTX1 is an epigenetically regulated tumor suppressor gene discovered by integrative analysis of epigenetic and transcriptional alterations in HNSCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 815. doi:10.1158/1538-7445.AM2015-815

Received August 17, 1999; revised December 27, 1999; accepted January 12, 2000. From the Department of Psychiatry, Westmead Hospital, University of Sydney; the Department of Psychiatry, College of Physicians and Surgeons Columbia University, New York; the New York State Psychiatric Institute; and the Department of Psychiatry, The New York Hospital-Cornell University Medical Center. Address reprint requests to Dr. de Moore, Department of Psychiatry, Westmead Hospital 2145, Sydney, NSW Australia. Copyright 2000 The Academy of Psychosomatic Medicine. Let her cover the mark as she will, the pang of it will be always in her heart. The Scarlet Letter, 1850 — Nathaniel Hawthorne

Fourteen variables were tested for their ability to predict visceral or bony metastases in 177 patients with clinical Stage I melanoma of intermediate thickness (1.51 - 3.39 mm). A Cox multivariate analysis yielded a combination of four variables that best predicted bony or visceral metastases for these patients: 1) mitoses greater than 6/min 2 (p = 0.0007), 2) location other than the forearm of leg) p = 0.009, 3) ulceration width greater than 3 mm (p = 0.04), 4) microscopic satellites (p = 0.05). The overall prognostic model chi square was 32.40 with 4 degrees of freedom (p less than 10 (-5). Combinations of the above variables were used to separate these patients into at least two risk groups. The high risk patients had at least a 35% or greater chance of developing visceral metastases within five years, while the low risk group had greater than an 85% chance of being disease free at five years. Criteria for the high risk group were as follows: 1) mitoses greater than 6/mm 2 in at least one area of the tumor, irrespective of primary tumor location, or 2) a melanoma located at some site other than the forearm or leg and histologic evidence in the primary tumor of either ulceration greater than 3 mm wide or microscopic satellites. The low risk group was defined as follows: 1) mitoses less than or equal to 6/mm 2 and a location on the leg or forearm, or 2) mitoses less than or equal to 6/mm 2 and the absence in histologic sections of the primary tumor of both microscopic satellites and ulceration greater then 3 mm wide. The number of patients in this series who did not undergo elective regional node dissection (N = 47) was probably too small to detect any benefit from this procedure. Based on survival rates from this and other studies, it is estimated that approximately 1500 patients with clinical Stage I melanoma of intermediate thickness in each arm of a randomized clinical trial would be needed to detect an increase in survival rates from elective regional node dissection.

A multivariate analysis was performed on 20 clinical and histologic variables from 327 Stage I prospectively studied melanoma patients who underwent elective regional lymph node dissection (ERLD). Primary tumor thickness, microscopic satellites, and the elapsed interval between diagnosis and ERLD, were selected as the combination of variables that were most highly associated with clinically occult regional lymph node metastases (P = 10(-15), model chi-square). Microscopic satellites were defined as tumor nests, greater than 0.05 mm in diameter, in the reticular dermis, panniculus, or vessels beneath the principal invasive tumor mass but separated from it by normal tissue on the section in which the Breslow measurement was taken. The probability of finding nodal metastases for melanomas less than 0.75 mm thick was 0% (0/41 patients); for those 0.76-1.50 mm, 4% (4/108); 1.51-3.0 mm, 14% (14/102); and greater than 3.0 mm, 39.5% (30/76). Primary melanomas greater than 1.50 mm thick with microscopic satellites were more often associated with nodal metastases than those of similar thickness without satellites (30/57 (53%) versus 14/121 (12%), P = 0.01). Some satellites probably represent intraspecimen metastases, while others do not. Any predictive model for occult regional lymph node metastases based on data from ERLD done less than 50 days after diagnosis may underestimate the prevalence of metastases.

Fourteen prognostic factors were examined in 79 patients with clinical Stage I melanoma greater than or equal to 3.65 mm in thickness. All nine patients with melanoma of the hands or feet died of melanoma. A Cox proportional hazards (multivariate) analysis of the remaining 70 patients showed that a combination of the following four variables best predicted bony or visceral metastases: 1) a nearly absent or minimal lymphocyte response at the base of the tumor, 2) histologic type other than superficial spreading melanoma, 3) location on the trunk, and 4) positive nodes or no initial node dissection. Ulceration and/or ulceration width were not useful in predicting outcome either singly or in combination with other variables. Patients with negative lymph nodes and primary tumors of the trunk, hands, and feet did not do better than patients with positive nodes at those sites. Conversely, non of 16 patients with negative lymph nodes and extremity melanomas (excluding the hands and feet) or head and neck melanomas developed visceral or bony metastases (i.e., five-year disease-free survival rate 100%).

Fourteen variables were tested for their prognostic usefulness in 203 patients with clinical Stage I melanoma and primary tumor 0.76-169 mm thick. Only two variables, primary tumor location and level of invasion, were useful in predicting death from melanoma for these patients. Of the 12 deaths from melanoma, 11 occurred in patients with primary tumors located on the upper back, posterior arm, posterior neck, and posterior scalp (=BANS). There has been only one death from melanoma in 136 patients with melanoma located at other sites (11/67 vs 1/136, p less than 0.0001 Fisher's Exact Test). Of the 67 BANS patients, 51 had level II or level III lesions and five (10%0 died of melanoma. This compared with six deaths from melanoma in 16 patients (37.5%) with level IV BANS lesions (5/51 vs 6/16, p = 0.01 Fisher's Exact Test). The relatively high incidence of both melanoma deaths and regional node metastases for the BANS group merits consideration for testing the efficacy of elective regional node dissection for these patients.

Thirteen variables were studied for their relative usefulness in predicting recurrent disease in 107 patients with clinical Stage I melanoma of the upper extremity. After a mean follow-up period of 54 months, the only patents who have had recurrent disease to date are those who primary lesions were located either on the hand or posterior upper arm. The five-year disease-free survival role for 44 patients with melanoma at these sites was 68%. None of 63 patients with melanoma located on the forearm of anterior upper arm have had recurrent disease (i.e., the five-year, disease-free survival rate was 100% (p = 0.00004), compared with the hand or posterior arm group). A Cox proportional hazards (multivariate) analysis demonstrated that two primary tumor histologic variable, thickness in millimeters and ulceration, interacted to produce the best prognostic model for those 44 patients with melanoma of the hand or posterior upper arm. Twenty-one patients with primary lesions at these sites had primary tumors less than 2.25 mm in thickness and no evidence of ulceration histologically. Their five-year, disease-free survival role was 95%. For the remaining 23 patients with primary tumors on the hand or posterior upper arm who had either histologic evidence of ulceration or primary tumors greater than or equal to 2.25 mm, the five-year disease-free survival rate was 37% (p = 0.002, compared with group nonulcerated, thin lesions). The excellent survival rate for patients with melanomas on the forearm or anterior upper arm was not completely explained by pathologic stage, by primary tumor thickness, or by histologic ulceration of the primary tumor.