bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Diagnosis of Activating EGFR Mutations in Non–Small Cell Lung Cancer Using Mutation-Specific Antibodies for Immunohistochemical Analysis
Authors: Kawahara Akihiko , Yamamoto Chizuko , Nakashima Kazutaka , Azuma Koichi , Hattori Satoshi , Kashihara Masaki , Aizawa Hisamichi , Basaki Yuji , Kuwano Michihiko , Kage Masayoshi , Mitsudomi Tetsuya , Ono Mayumi
Published: April 2010

Purpose: Therapeutic responses of non–small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)–targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for ∼90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of ∼90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC.

Experimental Design: We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing.

Results: We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti–delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry.

Conclusions: This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFR-targeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics. Clin Cancer Res; 16(12); 3163–70. ©2010 AACR.
Inhibition of the Insulin-Like Growth Factor I Receptor by Epigallocatechin Gallate Blocks Proliferation and Induces the Death of Ewing Tumor Cells
Authors: Kang Hyung-Gyoo , Jenabi Jasmine M. , Liu Xian Fang , Reynolds C. Patrick , Triche Timothy J. , Sorensen Poul H.B.
Published: April 2010

The insulin-like growth factor I receptor (IGFIR) has emerged as a key therapeutic target in many human malignancies, including childhood cancers such as Ewing family tumors (EFT). In this study, we show that IGFIR is constitutively activated in EFTs and that the major catechin derivative found in green tea, (−)-epigallocatechin gallate (EGCG), can inhibit cell proliferation and survival of EFT cells through the inhibition of IGFIR activity. Treatment of EFT cell lines with EGCG blocked the autophosphorylation of IGFIR tyrosine residues and inhibited its downstream pathways including phosphoinositide 3-kinase-Akt, Ras-Erk, and Jak-Stat cascades. EGCG treatment was associated with dose- and time-dependent inhibition of cellular proliferation, viability, and anchorage-independent growth, as well as with the induction of cell cycle arrest and apoptosis. Apoptosis in EFT cells by EGCG correlated with altered expression of Bcl-2 family proteins, including increased expression of proapoptotic Bax and decreased expression of prosurvival Bcl2, Bcl-XL, and Mcl-1 proteins. Our results provide further evidence that IGFIR is an attractive therapeutic target in EFTs and that EGCG is an effective inhibitor of this receptor tyrosine kinase. EGCG may be a useful agent for targeting IGFIR, either alone or in combination, with other potentially more toxic IGFIR inhibitors for the management of EFTs. Mol Cancer Ther; 9(5); 1396–407. ©2010 AACR.
AT7867 Is a Potent and Oral Inhibitor of AKT and p70 S6 Kinase That Induces Pharmacodynamic Changes and Inhibits Human Tumor Xenograft Growth
Authors: Grimshaw Kyla M. , Hunter Lisa-Jane K. , Yap Timothy A. , Heaton Simon P. , Walton Mike I. , Woodhead Steven J. , Fazal Lynsey , Reule Matthias , Davies Thomas G. , Seavers Lisa C. , Lock Victoria , Lyons John F. , Thompson Neil T. , Workman Paul , Garrett Michelle D.
Published: April 2010

The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a single-agent anticancer strategy. Mol Cancer Ther; 9(5); 1100–10. ©2010 AACR.
Targeting p70S6K Prevented Lung Metastasis in a Breast Cancer Xenograft Model
Authors: Akar Ugur , Ozpolat Bulent , Mehta Kapil , Lopez-Berestein Gabriel , Zhang Dongwei , Ueno Naoto T. , Hortobagyi Gabriel N. , Arun Banu
Published: April 2010

Overexpression of p70S6K in breast cancer patients is associated with aggressive disease and poor prognosis. Recent studies showed that patients with breast cancer with increased p70S6K phosphorylation had poor survival and increased metastasis. The purpose of our study was to determine whether knockdown of p70S6K would inhibit cell growth, invasion, and metastasis in breast cancer. We therefore stably knocked down p70S6K expression in MDA-231, a highly metastatic breast cancer cell line, using a lentiviral short hairpin RNA (shRNA) based approach. Inhibition of p70S6K led to inhibition of cell growth, migration, and invasion in vitro. To determine the role of p70S6K in breast cancer tumorigenesis and metastasis, we used an MDA-231 orthotopic and metastatic animal model. In the orthotopic model, mice injected with MDA-231–p70S6K shRNA cells developed significantly smaller tumors than control mice injected with MDA-231 control shRNA cells (P < 0.01). No metastasis was observed in the p70S6K downregulated group, whereas lung metastasis was detected in all mice in the control group. To determine the role of p70S6K on growth and invasion, we tested downstream signaling targets by Western blot analysis. Knockdown of p70S6K inhibited phosphorylation of focal adhesion kinase, tissue transglutaminase 2, and cyclin D1 proteins, which promote cell growth, survival, and invasion. In addition, downregulation of p70S6K induced expression of PDCD4, a tumor-suppressor protein. In conclusion, we showed that p70S6K plays an important role in metastasis by regulating key proteins like cyclin D1, PDCD4, focal adhesion kinase, E-cadherin, β-catenin, and tissue transglutaminase 2, which are essential for cell attachment, survival, invasion, and metastasis in breast cancer. Mol Cancer Ther; 9(5); 1180–7. ©2010 AACR.
Transcriptional Analysis of an E2F Gene Signature as a Biomarker of Activity of the Cyclin-Dependent Kinase Inhibitor PHA-793887 in Tumor and Skin Biopsies from a Phase I Clinical Study
Authors: Locatelli Giuseppe , Bosotti Roberta , Ciomei Marina , Brasca Maria G. , Calogero Raffaele , Mercurio Ciro , Fiorentini Francesco , Bertolotti Matteo , Scacheri Emanuela , Scaburri Angela , Galvani Arturo , Pesenti Enrico , De Baere Thierry , Soria Jean-Charles , Lazar Vladimir , Isacchi Antonella
Published: April 2010

A transcriptional signature of the pan–cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors. Mol Cancer Ther; 9(5); 1265–73. ©2010 AACR.
A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells
Authors: Sylvester Juliesta E. , Kron Stephen J.
Published: April 2010

Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing. Mol Cancer Ther; 9(5); 1469–81. ©2010 AACR.
Extract of Oregano, Coffee, Thyme, Clove, and Walnuts Inhibits NF-κB in Monocytes and in Transgenic Reporter Mice
Authors: Paur Ingvild , Balstad Trude R. , Kolberg Marit , Pedersen Marit K. , Austenaa Liv M. , Jacobs David R. , Blomhoff Rune
Published: April 2010

The transcription factor NF-κB is a promising target for chemoprevention. Several dietary plants are efficient inhibitors of NF-κB activation in vitro and could act synergistically on the NF-κB signaling pathway. In this study, we tested whether dietary plant extracts could inhibit NF-κB activation in a synergistic manner in vitro. Second, we investigated the potency of the same dietary plant extracts in the inhibition of NF-κB activation in vivo. A combined extract of clove, oregano, thyme, walnuts, and coffee synergistically inhibited lipopolysaccaride (LPS)-induced NF-κB activation in a monocytic cell line, compared with the sum of effects from the single extracts. Transgenic NF-κB luciferase reporter mice were given a single dose of the combined extract and subsequently challenged with LPS. NF-κB activation was monitored by in vivo imaging for 6 hours. In addition, NF-κB activity in organs and the expression of immune-related genes in liver were investigated. Based on the area under the curve, the extract decreased whole body LPS-induced NF-κB activity the first 6 hours by 35% compared with control mice. Organ-specific NF-κB activation was inhibited in intestine, liver, testis, and epididymis of the mice receiving the combination extract. In addition, dietary plants reduced the expression of genes related to inflammation, cell migration, and proliferation in liver. This study shows that dietary plants may be potent modulators of NF-κB signaling both in vitro and in vivo, and thus support further investigation of consumption of these plant foods as part of a healthy diet or as a mode of chemoprevention. Cancer Prev Res; 3(5); 653–63. ©2010 AACR.
Incorporation of a Genetic Factor into an Epidemiologic Model for Prediction of Individual Risk of Lung Cancer: The Liverpool Lung Project
Authors: Raji Olaide Y. , Agbaje Olorunsola F. , Duffy Stephen W. , Cassidy Adrian , Field John K.
Published: April 2010

The Liverpool Lung Project (LLP) has previously developed a risk model for prediction of 5-year absolute risk of lung cancer based on five epidemiologic risk factors. SEZ6L, a Met430IIe polymorphic variant found on 22q12.2 region, has been previously linked with an increased risk of lung cancer in a case-control population. In this article, we quantify the improvement in risk prediction with addition of SEZ6L to the LLP risk model. Data from 388 LLP subjects genotyped for SEZ6L single-nucleotide polymorphism (SNP) were combined with epidemiologic risk factors. Multivariable conditional logistic regression was used to predict 5-year absolute risk of lung cancer with and without this SNP. The improvement in the model associated with the SEZ6L SNP was assessed through pairwise comparison of the area under the receiver operating characteristic curve and the net reclassification improvements (NRI). The extended model showed better calibration compared with the baseline model. There was a statistically significant modest increase in the area under the receiver operating characteristic curve when SEZ6L was added into the baseline model. The NRI also revealed a statistically significant improvement of around 12% for the extended model; this improvement was better for subjects classified into the two intermediate-risk categories by the baseline model (NRI, 27%). Our results suggest that the addition of SEZ6L improved the performance of the LLP risk model, particularly for subjects whose initial absolute risks were unable to discriminate into “low-risk” or “high-risk” group. This work shows an approach to incorporate genetic biomarkers in risk models for predicting an individual's lung cancer risk. Cancer Prev Res; 3(5); 664–9. ©2010 AACR.
Methylselenocysteine Resets the Rhythmic Expression of Circadian and Growth-Regulatory Genes Disrupted by Nitrosomethylurea In vivo
Authors: Fang Ming Zhu , Zhang Xun , Zarbl Helmut
Published: April 2010

Epidemiologic and animal studies indicate that disruption of circadian rhythm increases breast cancer risk. Previously, we showed that methylselenocysteine reduced the incidence of N-nitroso-N-methylurea (NMU)–induced mammary carcinomas in Fischer 344 rats by 63%. Methylselenocysteine also increased the expression of Period 2 (Per2) and D-binding protein (DBP), providing evidence for a link between circadian rhythm and chemoprevention. Here, we report that NMU disrupted the expression of core circadian genes (Per1, Per2, Cry1, Cry2, and RevErbAα) and circadian-controlled genes, including melatonin receptor 1α (MTNR1A), estrogen receptors (ERα and ERβ), and growth-regulatory genes (Trp53, p21, Gadd45α, and c-Myc) in mammary glands of Fischer 344 rats. By contrast, dietary methylselenocysteine (3 ppm selenium) given for 30 days, significantly enhanced the circadian expression of these genes (except for Cry1 and Cry2). The largest effect was on the levels of the Per2, MTNR1A, and ERβ mRNAs, which showed 16.5-fold, 4.7-fold, and 9.5-fold increases in their rhythm-adjusted means, respectively, and 44.5-fold, 6.5-fold, and 9.7-fold increases in amplitude as compared with the control diet, respectively. Methylselenocysteine also shifted the peak expression times of these genes to Zeitgeber time 12 (ZT12; lights off). Methylselenocysteine also induced rhythmic expression of Trp53, p21, and Gadd45α mRNAs with peak levels at ZT12, when c-Myc expression was at its lowest level. However, methylselenocysteine had no significant effect on the circadian expression of these genes in liver. These results suggest that dietary methylselenocysteine counteracted the disruptive effect of NMU on circadian expression of genes essential to normal mammary cell growth and differentiation. Cancer Prev Res; 3(5); 640–52. ©2010 AACR.
A Large Prospective Study of SEP15 Genetic Variation, Interaction with Plasma Selenium Levels, and Prostate Cancer Risk and Survival
Authors: Penney Kathryn L. , Schumacher Fredrick R. , Li Haojie , Kraft Peter , Morris J. Steven , Kurth Tobias , Mucci Lorelei A. , Hunter David J. , Kantoff Philip W. , Stampfer Meir J. , Ma Jing
Published: April 2010

The role of selenium in prostate cancer (PCa) risk remains controversial, but many epidemiologic studies suggest an inverse association with more aggressive disease. A recently discovered selenoprotein, SEP15, which is highly expressed in the prostate, may play a role either independently or by modifying the effects of selenium. We genotyped four common single-nucleotide polymorphisms capturing common variation (frequency >5%; R2 > 0.8) within SEP15, as well as rs5859 in the 3′ untranslated region, previously reported to reduce the efficiency of selenium incorporation into SEP15. We examined the association of these single-nucleotide polymorphisms with PCa risk and PCa-specific mortality, as well as their interactions with plasma selenium levels, in the Physicians' Health Study. In this nested case-control study (1,286 cases and 1,267 controls), SEP15 polymorphisms were not significantly associated with PCa risk. However, among the cases, three variants were significantly associated with PCa-specific mortality [rs479341 hazard ratio (HR), 1.94; 95% confidence interval (95% CI), 1.15-3.25; rs1407131 HR, 2.85; 95% CI, 1.45-5.59; rs561104 HR, 1.54; 95% CI, 1.12-2.11] with a recessive model. Additionally, rs561104 significantly modified the association of plasma selenium with PCa survival (Pinteraction = 0.02); an inverse relationship of high levels of selenium with PCa mortality was apparent only among those without the increased risk genotype. This study provides evidence that SEP15 genetic variation may influence PCa mortality. Additionally, the association of selenium with PCa mortality was modified by a variant, suggesting the possibility that some men with PCa may benefit more from selenium than others, depending on their genotype. Cancer Prev Res; 3(5); 604–10. ©2010 AACR.
Social Isolation Reduces Mammary Development, Tumor Incidence, and Expression of Epigenetic Regulators in Wild-type and p53-Heterozygotic Mice
Authors: Hasen Nina S. , O'Leary Kathleen A. , Auger Anthony P. , Schuler Linda A.
Published: April 2010

Chronic stress is associated with more rapid tumor progression, and recent evidence suggests that stress may contribute to social and ethnic disparities in the incidence and mortality of breast cancer. We evaluated the p53+/− FVB/N mouse as a model to investigate effects of chronic social stress on mammary gland development, gene expression, and tumorigenesis. We individually housed (IH) wild-type and p53+/− female FVB/N mice, starting at weaning. At 14 weeks of age, both wild-type and p53+/− IH mice showed strikingly reduced mammary development compared with group-housed (GH) controls, with IH mice having significantly fewer preterminal end buds. This morphologic difference was not reflected in levels of mammary transcripts for estrogen receptor-α or progestin receptor. However, IH increased levels of mRNA for the kisspeptin receptor in the medial preoptic area of the hypothalamus, associated with reduced duration of estrous cycles. Furthermore, IH altered mammary transcripts of genes associated with DNA methylation; transcripts for methyl-binding protein 2 and DNA methyltransferase 3b (DNMT3b), but not DNMT1 and DNMT3a, were reduced in IH compared with GH females. Interestingly, the glands of p53+/− females showed reduced expression of all these mediators compared with wild-type females. However, contrary to our initial hypothesis, IH did not increase mammary tumorigenesis. Rather, p53+/− GH females developed significantly more mammary tumors than IH mice. Together, these data suggest that social isolation initiated at puberty might confound studies of tumorigenesis by altering mammary development in mouse models. Cancer Prev Res; 3(5); 620–9. ©2010 AACR.
Premalignant Breast Neoplasia: A Paradigm of Interlesional and Intralesional Molecular Heterogeneity and Its Biological and Clinical Ramifications
Authors: Berman Hal K. , Gauthier Mona L. , Tlsty Thea D.
Published: April 2010

As is well established in invasive breast disease, it is becoming increasingly clear that molecular heterogeneity, both between and within lesions, is a prevalent, distinct phenotype of premalignant lesions of the breast. Key pathways of tumorigenesis modulate critical features of premalignant lesions such as proliferation, differentiation, stress response, and even the generation of diversity. Current studies show that evaluation of these lesions may provide clinically useful information on future tumor formation as well as biological insights into the origin and functional significance of this distinct phenotype. Cancer Prev Res; 3(5); 579–87. ©2010 AACR.
Pseudohypoaldosteronism Type 1 due to a Novel Mutation in the Mineralocorticoid Receptor Gene
Authors: Loomba-Albrecht Lindsey A. , Nagel Mato , Bremer Andrew A.
Published: April 2010

Background/Aims: Autosomal dominant pseudohypoaldosteronism type 1 is caused by mutations in the mineralocorticoid receptor (NR3C2) gene, often leading to life-threatening hyponatremia and hyperkalemia in the newborn period. We report a novel mutation in the NR3C2 gene, and report, for the first time, the association of well-treated pseudohypoaldosteronism with failure to thrive. This report additionally highlights the importance of aldosterone-sensitive sodium transport in the neonatal period. Patient and Methods: The patient presented with salt loss, hyperkalemia and a mild metabolic acidosis in the neonatal period (day of life 8). Further evaluation revealed significantly elevated levels of 18-hydroxycorticosterone, aldosterone and plasma renin activity, suggesting the diagnosis of pseudohypoaldosteronism. Results: Analysis of the patient’s NR3C2 gene revealed a novel missense mutation (c.1817G>C), which was subsequently analyzed in his parents and sister. Interestingly, the patient’s mother was found to have an identical mutation. Conclusion: We report a novel mutation in the gene for the mineralocorticoid receptor and an unusual clinical course of pseudohypoaldosteronism type 1 in an adequately treated patient.
Evo-Devo of Child Growth III: Premature Juvenility as an Evolutionary Trade-Off
Authors: Hochberg Ze’ev
Published: April 2010

Juvenility was previously defined as a distinct clinical life history stage, characterized by adrenarche, decelerating growth and accelerating adiposity. This review presents the theory of evolutionary predictive adaptive strategies for premature juvenility in response to (mostly) energy supply, but also to other environmental cues. In the absence of longitudinal adrenal androgen levels, premature juvenility can be diagnosed by auxological criteria. Syndromes of short stature that are associated with premature juvenility include Prader-Willi syndrome, Silver-Russell syndrome and Noonan syndrome, and it is part of the pygmy adaptive shortness of stature. The review elucidates in evolutionary terms the association of premature juvenility with SGA and PCOS.
Re-Thinking Diagnostic Classification of the Dysarthrias: A Developmental Perspective
Authors: Morgan A.T. , Liégeois F.
Published: April 2010

Acquired childhood dysarthria (ACD) receives little attention in the research literature in contrast with the adult correlate of the disorder. Speech language pathologists working in this field find diagnosis and management challenging, arguably because there is no child-based dysarthria diagnostic classification. Clinicians are either dependent upon developmental speech models that are not specific to dysarthria and that ignore the neural basis of the disorder, or on adult-based neurobehavioural classification systems. Here we consider the necessary elements for developing a clinically useful and empirically driven diagnostic classification system for ACD. The paper is divided into 2 parts. First, we question whether an adult diagnostic model can be validly applied to children. Second, we propose a methodological approach to develop a classification system for ACD. Specifically, we propose that advancing knowledge in neurobehavioural correlations of ACD is contingent upon large-scale studies, likely requiring international collaboration, which pool brain and speech outcome data. Ideally, researchers across centres would apply standard protocols to: (1) characterize speech behaviour, and (2) brain structure, function and connectivity. When enough data is available to achieve statistical power, analysis could determine subgroups of dysarthria defined by speech behaviour. The commonalities of neural profiles of subgroups could then be examined to create an empirically driven theory of brain-behaviour relationships in ACD to underpin the classification system. Clinical diagnosis for children with ACD will remain limited until such data become available.
Effects of Online Augmented Kinematic and Perceptual Feedback on Treatment of Speech Movements in Apraxia of Speech
Authors: McNeil M.R. , Katz W.F. , Fossett T.R.D. , Garst D.M. , Szuminsky N.J. , Carter G. , Lim K.Y.
Published: April 2010

Apraxia of speech (AOS) is a motor speech disorder characterized by disturbed spatial and temporal parameters of movement. Research on motor learning suggests that augmented feedback may provide a beneficial effect for training movement. This study examined the effects of the presence and frequency of online augmented visual kinematic feedback (AVKF) and clinician-provided perceptual feedback on speech accuracy in 2 adults with acquired AOS. Within a single-subject multiple-baseline design, AVKF was provided using electromagnetic midsagittal articulography (EMA) in 2 feedback conditions (50 or 100%). Articulator placement was specified for speech motor targets (SMTs). Treated and baselined SMTs were in the initial or final position of single-syllable words, in varying consonant-vowel or vowel-consonant contexts. SMTs were selected based on each participant’s pre-assessed erred productions. Productions were digitally recorded and online perceptual judgments of accuracy (including segment and intersegment distortions) were made. Inter- and intra-judge reliability for perceptual accuracy was high. Results measured by visual inspection and effect size revealed positive acquisition and generalization effects for both participants. Generalization occurred across vowel contexts and to untreated probes. Results of the frequency manipulation were confounded by presentation order. Maintenance of learned and generalized effects were demonstrated for 1 participant. These data provide support for the role of augmented feedback in treating speech movements that result in perceptually accurate speech production. Future investigations will explore the independent contributions of each feedback type (i.e. kinematic and perceptual) in producing efficient and effective training of SMTs in persons with AOS.
Variability in Sex-Determining Mechanisms Influences Genome Complexity in Reptilia
Authors: Janes D.E. , Organ C.L. , Edwards S.V.
Published: April 2010

In this review, we describe the history of amniote sex determination as a classic example of Darwinian evolution. We suggest that evolutionary changes in sex determination provide a foundation for understanding important aspects of chromosome and genome organization that otherwise appear haphazard in their origins and contents. Species with genotypic sex determination often possess heteromorphic sex chromosomes, whereas species with environmental sex determination lack them. Through a series of mutations followed by selection at key genes, sex-determining mechanisms have turned over many times throughout the amniote lineage. As a consequence, amniote genomes have undergone gains or losses of sex chromosomes. We review the genomic and ecological contexts in which either temperature-dependent or genotypic sex determination has evolved. Once genotypic sex determination emerges in a lineage, viviparity and heteromorphic sex chromosomes become more likely to evolve. For example, in extinct marine reptiles, genotypic sex determination apparently led to viviparity, which in turn facilitated their pelagic radiation. Sex chromosomes comprise genome regions that differ from autosomes in recombination rate, mutation rate, levels of polymorphism, and the presence of sex-determining and sexually antagonistic genes. In short, many aspects of amniote genome complexity, life history, and adaptive radiation appear contingent on evolutionary changes in sex-determining mechanisms.
Chromosome Evolution and Diversification in North American Spiny Lizards (Genus Sceloporus)
Authors: Leaché A.D. , Sites, Jr. J.W.
Published: April 2010

The genus Sceloporus is a diverse clade of lizards that exhibits substantial variation in chromosome numbers and sex chromosome heteromorphisms, 2 features of the genome that are static among most other pleurodont iguanian lizards. Evolutionary changes to the fundamental number of chromosomes are hypothesized to be a primary factor responsible for driving the diversification of Sceloporus. We explore the patterns of chromosome evolution in Sceloporus using a combination of ancestral state estimations and species diversification tests. Phylogenetic relationships and divergence times within Sceloporus (53 species representing all 19 species groups) are estimated using 4 nuclear genes (>3.3 kb) and relaxed-clock analyses that incorporate a fossil calibration on the root of the tree. We test the hypothesis that chromosome evolution is correlated with shifts in species diversification using cross-validation predictive densities, a new Bayesian approach for modeling the number of species that are predicted to have evolved in the absence of a certain historical event (e.g., a change in chromosome numbers). Results of the cross-validation predictive densities approach indicate that chromosomal evolution is correlated with significantly higher species diversity than predicted under the background rate of diversification in Sceloporus. We conclude by discussing the future of comparative cytogenetic investigations in Sceloporus.
Standardized Reference Ideogram for Physical Mapping in the Saltwater Crocodile (Crocodylus porosus)
Authors: Dalzell P. , Miles L.G. , Isberg S.R. , Glenn T.C. , King C. , Murtagh V. , Moran C.
Published: April 2010

Basic cytogenetic data, such as diploid number and general chromosome morphology, are available for many reptilian species. Here we present a detailed cytogenetic examination of the saltwater crocodile (Crocodylus porosus) karyotype, including the creation of the first fully annotated G-band standard ideogram for any crocodilian species. The C. porosus karyotype contains macrochromosomes and has a diploid number of 34. This study presents a detailed description of each chromosome, permitting unambiguous chromosome identification. The fully annotated standardized C. porosus ideogram provides the backbone to a standard nomenclature system which can be used to accurately identify specific band locations. Seven microsatellite containing fosmid clones were fluorescently labeled and used as fluorescent in situ hybridization (FISH) probes for physical localization. Chromosome locations for each of these FISH probes were successfully assigned, demonstrating the utility of the fully annotated ideogram for genome mapping.
Skinks (Reptilia: Scincidae) Have Highly Conserved Karyotypes as Revealed by Chromosome Painting
Authors: Giovannotti M. , Caputo V. , O’Brien P.C.M. , Lovell F.L. , Trifonov V. , Nisi Cerioni P. , Olmo E. , Ferguson-Smith M.A. , Rens W.
Published: April 2010

Skinks represent the most diversified squamate reptiles with a great variation in body size and form, and are found worldwide in a variety of habitats. Their remarkable diversification has been accompanied by only a few chromosome rearrangements, resulting in highly-conservative chromosomal complements of these lizards. In this study cross-species chromosome painting using Scincus scincus (2n = 32) as the source genome, was used to detect the chromosomal rearrangements and homologies between the following skinks: Chalcides chalcides (2n = 28), C. ocellatus (2n = 28), Eumeces schneideri (2n = 32), Lepidothyris fernandi (2n = 30), Mabuya quinquetaeniata (2n = 32). The results of this study confirmed a high degree of chromosome conservation between these species. The main rearrangements in the studied skinks involve chromosomes 3, 5, 6 and 7 of S. scincus. These subtelocentric chromosomes are homologous to the p and q arms of metacentric pair 3 and 4 in C. chalcides, C. ocellatus, L. fernandi, and M. quinquetaeniata, while they are entirely conserved in E. schneideri. Other rearrangements involve S. scincus 11 in L. fernandi and M. quinquetaeniata, supporting the monophyly of Lygosominae, and one of the chromosomes S. scincus 12–16, in M. quinquetaeniata. In conclusion, our data support the monophyly of Scincidae and confirm that Scincus-Eumeces plus Chalcides do not form a monophyletic clade, suggesting that the Scincus-Eumeces clade is basal to other members of this family. This study represents the first time the whole genome of any reptile species has been used for cross-species chromosome painting to assess chromosomal evolution in this group of vertebrates.
Cytogenetics and Molecular Data in Snakes: A Phylogenetic Approach
Authors: Oguiura N. , Ferrarezzi H. , Batistic R.F.
Published: April 2010

Snakes are among the most successful groups of reptiles, numbering about 3,000 extant species. In spite of centuries of comparative anatomical and morphological studies, many aspects of snake systematics remain unsolved. To better understand the evolution and diversity of genomic characteristics in Serpentes, we analyzed online sequence data of mitochondrial and nuclear genes, as well as cytogenetic data and reviewed other genomic characteristics such as toxin genes. After the analysis of the whole-genome and chromosomal organization, we find that: (1) cytogenetic comparisons could provide a useful tool to investigate intergeneric and tribal relationships within the extremely diverse neotropical xenodontine snakes; (2) toxin genes could also help to understand snake evolution if special care is taken to choose the sequences because of the difficulty in avoiding paralogs; (3) snake phylogeny based on mitochondrial genome sequences is largely consistent with the relationship obtained using nuclear genes.
Co-Evolution of Genomic Structure and Selective Forces Underlying Sexual Development and Reproduction
Authors: Valenzuela N.
Published: April 2010

Genomic structure affects the expression and evolution of phenotypes via its effect on genomic function. One example is the presence of sex chromosomes and the development of the sexual phenotype. Multiple forces account for the evolution of genomic structure and function, some acting synergistically and some in an antagonistic manner, in a co-evolutionary process between genomic architecture and environmental effects at different time scales. Here I review the evolution of sex chromosomes and how they affect sex determination, sex ratios, sexual selection, and sexual conflict, at the same time that these phenomena influence the evolution of genome organization. Because of this reciprocal interaction, the need to correctly identify sex chromosome systems in order to understand the causes and consequences of their evolution is emphasized. Importantly, it is argued that the existence of taxa lacking sex chromosomes poses a challenge to current evolutionary models and a unique opportunity to test them empirically using reptiles as a model system.
From Reptilian Phylogenomics to Reptilian Genomes: Analyses of c-Jun and DJ-1 Proto-Oncogenes
Authors: Katsu Y. , Braun E.L. , Guillette, Jr. L.J. , Iguchi T.
Published: April 2010

Genome projects have revolutionized our understanding of both molecular biology and evolution, but there has been a limited collection of genomic data from reptiles. This is surprising given the pivotal position of reptiles in vertebrate phylogeny and the potential utility of information from reptiles for understanding a number of biological phenomena, such as sex determination. Although there are many potential uses for genomic data, one important and useful approach is phylogenomics. Here we report cDNA sequences for the c-Jun(JUN) and DJ-1(PARK7) proto-oncogenes from 3 reptiles (the American alligator, Nile crocodile, and Florida red-belly turtle), show that both genes are expressed in the alligator, and integrate them into analyses of their homologs from other organisms. With these taxa it was possible to conduct analyses that include all major vertebrate lineages. Analyses of c-Jun revealed an unexpected but well-supported frog-turtle clade while analyses of DJ-1 revealed a topology largely congruent with expectation based upon other data. The conflict between the c-Jun topology and expectation appears to reflect the overlap between c-Jun and a CpG island in most taxa, including crocodilians. This CpG island is absent in the frog and turtle, and convergence in base composition appears to be at least partially responsible for the signal uniting these taxa. Noise reduction approaches can eliminate the unexpected frog-turtle clade, demonstrating that multiple signals are present in the c-Jun alignment. We used phylogenetic methods to visualize these signals; we suggest that examining both historical and non-historical signals will prove important for phylogenomic analyses.
Diploid-Triploid Mosaicism and Tissue Ploidy Diversity within Platemys platycephala from Suriname
Authors: Bickham J.W. , Hanks B.G.
Published: April 2010

The twist-necked turtle, Platemys platycephala, is 1 of only 2 known species to possess sexual reproduction and diploid-triploid mosaicism. Previous studies have shown that mosaics occur in Suriname and French Guiana but only diploids are known from Bolivia and Brazil. In this paper, the frequency of ploidy mosaicism was studied in a large sample of P. platycephala from Suriname to more fully explore the diversity of ploidy levels within and among individuals. Flow-cytometric analysis of blood revealed a wide diversity of conditions including diploids, diploid-triploid mosaics, triploids, and triploid-tetraploid mosaics. The largest frequency class was 100% diploid, and the second largest was 100% triploid. However, mosaic individuals were observed from the entire spectrum of mixtures ranging from nearly all-diploid to nearly all-triploid and 2 individuals were triploid-tetraploid mosaics. It appears likely that diploids, triploids and mosaics do not represent distinct biotypes, but simply different conditions within a spectrum of possible ploidy mixtures. Studies of multiple tissues from 5 individuals showed blood alone is a good indicator of ploidy, but subtle differences were found among tissues for some individuals, and some individuals that were all-diploid or all-triploid in blood were found to be mosaic in other tissues. Triploidy was statistically associated with males, and we hypothesize that genome size plays a role in sex determination in this species.
Sex Chromosome Evolution in Lizards: Independent Origins and Rapid Transitions
Authors: Ezaz T. , Sarre S.D. , O’Meally D. , Marshall Graves J.A. , Georges A.
Published: April 2010

Reptiles epitomize the variability of reproductive and sex determining modes and mechanisms among amniotes. These modes include gonochorism (separate sexes) and parthenogenesis, oviparity, viviparity, and ovoviviparity, genotypic sex determination (GSD) with male (XX/XY) and female (ZZ/ZW) heterogamety and temperature-dependent sex determination (TSD). Lizards (order Squamata, suborder Sauria) are particularly fascinating because the distribution of sex-determining mechanisms shows no clear phylogenetic segregation. This implies that there have been multiple transitions between TSD and GSD, and between XY and ZW sex chromosome systems. Approximately 1,000 species of lizards have been karyotyped and among those, fewer than 200 species have sex chromosomes, yet they display remarkable diversity in morphology and degree of degeneration. The high diversity of sex chromosomes as well as the presence of species with TSD, imply multiple and independent origins of sex chromosomes, and suggest that the mechanisms of sex determination are extremely labile in lizards. In this paper, we review the current state of knowledge of sex chromosomes in lizards and the distribution of sex determining mechanisms and sex chromosome forms within and among families. We establish for the first time an association between the occurrence of female heterogamety and TSD within lizard families, and propose mechanisms by which female heterogamety and TSD may have co-evolved. We suggest that lizard sex determination may be much more the result of an interplay between sex chromosomes and temperature than previously thought, such that the sex determination mode is influenced by the nature of heterogamety as well as temperature sensitivity and the stage of sex chromosome degeneration.
Cytogenetic and Genetic Trends in the Evolution of Unisexual Lizards
Authors: Kupriyanova L.
Published: April 2010

Extensive karyological, allozyme and molecular genetic analyses of many parthenogenetic lizards confirm the origin of these species by hybridization. The concept of reticulate (hybridogeneous) speciation based on 3 interrelated phenomena such as hybridization-unisexuality-polyploidy has been elaborated. Cytogenetic and genetic variability and some regular trends in the evolution of hybrid unisexual species have been demonstrated. Cytogenetic investigations of unisexual-bisexual complex lizards of the genus Darevskia (formerly Lacerta) in the family Lacertidae suggest that some characteristics of their karyotypes play a role in the evolution of parthenogenesis. The maternal species with advanced w sex microchromosomes (Zw type) appeared to be successful in hybridization and produced the diploid unisexual species. The polyploid back-cross hybrids between said diploids and bisexual parent species exhibited many chromosome aberrations which were associated with their complete or partial sterility. In addition, the karyotype and the structure of mitotic and meiotic lampbrush chromosomes in 2 species and in some back-cross hybrids of this unisexual-bisexual complex have been examined here using molecular cytogenetic techniques. The genomic and functional disturbances, genomic interactions between homeologous chromosomes, and possibly both genomic imprinting and the presence of mobile elements are some cytogenetic mechanisms that increase genetic diversity in hybrid parthenogenetic species.
Chromosome Evolution in the Lizard Genus Gekko (Gekkonidae, Squamata, Reptilia) in the East Asian Islands
Authors: Shibaike Y. , Takahashi Y. , Arikura I. , Iiizumi R. , Kitakawa S. , Sakai M , Imaoka C. , Shiro H. , Tanaka H. , Akakubo N. , Nakano M. , Watanabe M. , Ohne K. , Kubota S. , Kohno S. , Ota H.
Published: April 2010

The lizard genus Gekko consists of over 30 species distributed in Asia and Oceania. From the insular region of East Asia including Japan and Taiwan, 9 species (G. hokouensis, G. japonicus, G. shibatai, G. tawaensis, G. vertebralis,G. yakuensis, and 3 undescribed species) are currently recognized. We made karyological analyses for all these species. Their karyotypes invariably consisted of 2N = 38 chromosomes, but exhibited considerable variation in fundamental number (ranging from 56–62). Substantial chromosomal variation was detected even among populations of a morphologically relatively uniform species, G. hokouensis. Populations of G. hokouensis from the central and northern Ryukyus exhibited prominent female heteromorphic (i.e., ZW type) sex chromosomes. Populations of the southern Ryukyus exclusive of Yonagunijima also had ZW sex chromosomes, whose heteromorphisms were, however, much less prominent. The other G. hokouensis populations including the topotypic continental representatives and the population from Yonagunijima of the southern Ryukyus exhibited no sex chromosome heteromorphism at all. These results strongly suggest that G. hokouensis in the current taxonomic definition actually includes more than 2 species. The process of chromosomal evolution in the East Asian Gekko is hypothesized.
Cytogenetic Characterization and Fluorescence in situ Hybridization of (GATA)10 Repeats on Established Primary Cell Cultures from Indian Water Snake (Natrix piscator) and Indian Mugger (Crocodylus palustris) Embryos
Authors: Rao L. , Turlapati R. , Patel M. , Panda B. , Tosh D. , Mangalipalli S. , Tiwari A. , Orunganti V.P. , Rose D. , Anand A. , Kulashekaran M.K. , Priya S.R. , Mishra R.K. , Majumdar K. , Aggarwal R.K. , Singh L.
Published: April 2010

Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from various species of reptiles would help in our understanding of the mechanisms of evolution and differentiation of sex chromosomes. Therefore, in the present study, we have established for the first time primary cell cultures from Indian water snake (Natrix piscator) and Indian mugger (Crocodylus palustris) embryos. In the preliminary growth stage, 2 types of cells, fibroblast- and epithelial-like, were found to be attached and proliferating in vitro. These fibroblast-like cell cultures were later overtaken by epithelial cells. The cell lines were grown in minimal essential medium supplemented with fetal bovine serum and subcultured for a period of 8–10 months. The morphology of cell types was kept under constant observation microscopically. Interestingly, at a subsequent passage of the cells sporadically scattered neuronal-like and beating cells were observed. The suitable temperature for growth of these cell cultures was 28–30°C. Chromosome analysis was performed from the actively proliferating cells, which revealed 5 pairs of macrochromosomes and 15 pairs of microchromosomes in Natrix piscator, and 15 pairs of only macrochromosomes in Crocodylus palustris. (GATA)n repeats are well known to be associated with sex chromosomes. Fluorescence in situ hybridization performed with (GATA)10 repeats delineated the W chromosome in the cells of Natrix piscator which has so far not been reported. This cell culture method has presently only been applied to water snakes and crocodile embryos in the current study, but it will be employed in other reptilian species and could go a long way to being a sustainable source of primary cells. This would eventually serve as an important tool for molecular studies in reptiles and other species in the future.