Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

© 2018 The Author(s). Background: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. Methods: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. Results: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by >25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Conclusions: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy

A Signature Inferred from Drosophila Mitotic Genes Predicts Survival of Breast Cancer Patients

Introduction: The classification of breast cancer patients into risk groups provides a powerful tool for the identification of patients who will benefit from aggressive systemic therapy. The analysis of microarray data has generated several gene expression signatures that improve diagnosis and allow risk assessment. There is also evidence that cell proliferation-related genes have a high predictive power within these signatures. Methods: We thus constructed a gene expression signature (the DM signature) using the human orthologues of 108 Drosophila melanogaster genes required for either the maintenance of chromosome integrity (36 genes) or mitotic division (72 genes). Results: The DM signature has minimal overlap with the extant signatures and is highly predictive of survival in 5 large breast cancer datasets. In addition, we show that the DM signature outperforms many widely used breast cancer signatures in predictive power, and performs comparably to other proliferation-based signatures. For most genes of the DM signature, an increased expression is negatively correlated with patient survival. The genes that provide the highest contribution to the predictive power of the DM signature are those involved in cytokinesis. Conclusion: This finding highlights cytokinesis as an important marker in breast cancer prognosis and as a possible targe

Abatacept in individuals at high risk of rheumatoid arthritis (APIPPRA): a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial

\ua9 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Background: Individuals with serum antibodies to citrullinated protein antigens (ACPA), rheumatoid factor, and symptoms, such as inflammatory joint pain, are at high risk of developing rheumatoid arthritis. In the arthritis prevention in the pre-clinical phase of rheumatoid arthritis with abatacept (APIPPRA) trial, we aimed to evaluate the feasibility, efficacy, and acceptability of treating high risk individuals with the T-cell co-stimulation modulator abatacept. Methods: The APIPPRA study was a randomised, double-blind, multicentre, parallel, placebo-controlled, phase 2b clinical trial done in 28 hospital-based early arthritis clinics in the UK and three in the Netherlands. Participants (aged ≥18 years) at risk of rheumatoid arthritis positive for ACPA and rheumatoid factor with inflammatory joint pain were recruited. Exclusion criteria included previous episodes of clinical synovitis and previous use of corticosteroids or disease-modifying antirheumatic drugs. Participants were randomly assigned (1:1) using a computer-generated permuted block randomisation (block sizes of 2 and 4) stratified by sex, smoking, and country, to 125 mg abatacept subcutaneous injections weekly or placebo for 12 months, and then followed up for 12 months. Masking was achieved by providing four kits (identical in appearance and packaging) with pre-filled syringes with coded labels of abatacept or placebo every 3 months. The primary endpoint was the time to development of clinical synovitis in three or more joints or rheumatoid arthritis according to American College of Rheumatology and European Alliance of Associations for Rheumatology 2010 criteria, whichever was met first. Synovitis was confirmed by ultrasonography. Follow-up was completed on Jan 13, 2021. All participants meeting the intention-to-treat principle were included in the analysis. This trial was registered with EudraCT (2013–003413–18). Findings: Between Dec 22, 2014, and Jan 14, 2019, 280 individuals were evaluated for eligibility and, of 213 participants, 110 were randomly assigned to abatacept and 103 to placebo. During the treatment period, seven (6%) of 110 participants in the abatacept group and 30 (29%) of 103 participants in the placebo group met the primary endpoint. At 24 months, 27 (25%) of 110 participants in the abatacept group had progressed to rheumatoid arthritis, compared with 38 (37%) of 103 in the placebo group. The estimated proportion of participants remaining arthritis-free at 12 months was 92\ub78% (SE 2\ub76) in the abatacept group and 69\ub72% (4\ub77) in the placebo group. Kaplan–Meier arthritis-free survival plots over 24 months favoured abatacept (log-rank test p=0\ub7044). The difference in restricted mean survival time between groups was 53 days (95% CI 28–78; p<0\ub70001) at 12 months and 99 days (95% CI 38–161; p=0\ub70016) at 24 months in favour of abatacept. During treatment, abatacept was associated with improvements in pain scores, functional wellbeing, and quality-of-life measurements, as well as low scores of subclinical synovitis by ultrasonography, compared with placebo. However, the effects were not sustained at 24 months. Seven serious adverse events occurred in the abatacept group and 11 in the placebo group, including one death in each group deemed unrelated to treatment. Interpretation: Therapeutic intervention during the at-risk phase of rheumatoid arthritis is feasible, with acceptable safety profiles. T-cell co-stimulation modulation with abatacept for 12 months reduces progression to rheumatoid arthritis, with evidence of sustained efficacy beyond the treatment period, and with no new safety signals. Funding: Bristol Myers Squibb

Ultrasound-assisted extraction of natural products

Ultrasound-assisted extraction (USAE) is an interesting process to obtain high valuable compounds and could contribute to the increase in the value of some food by-products when used as sources of natural compounds. The main benefits will be a more effective extraction, thus saving energy, and also the use of moderate temperatures, which is beneficial for heat-sensitive compounds. For a successful application of the USAE, it is necessary to consider the influence of several process variables, the main ones being the applied ultrasonic power, the frequency, the extraction temperature, the reactor characteristics, and the solvent-sample interaction. The highest extraction rate is usually achieved in the first few minutes, which is the most profitable period. To optimize the process, rate equations and unambiguous process characterization are needed, aspects that have often been lacking. © 2011 Springer Science+Business Media, LLC.The authors thank the Generalitat Valenciana for their financial support in project PROMETEO/2010/062 and the Caja de Ahorros del Mediterraneo for M.D. Esclapez's pre-doctoral grant.Esclapez Vicente, MD.; García Pérez, JV.; Mulet Pons, A.; Cárcel Carrión, JA.; Esclapez, MD. (2011). Ultrasound-assisted extraction of natural products. Food Engineering Reviews. 3(2):108-120. https://doi.org/10.1007/s12393-011-9036-6S10812032Abad Romero B, Bou-Maroun E, Reparet JM, Blanquet J, Cayot N (2010) Impact of lipid extraction on the dearomatisation of an Eisenia foetida protein powder. Food Chem 119:459–466Adewuyi YG (2001) Sonochemistry: environmental science and engineering applications. Ind Eng Chem Res 40:4681–4715Atchley AA, Crum LA (1988) Acoustic cavitation and bubble dynamics. In: Suslick KS (ed) Ultrasound, its chemical, physical, and biological effects. VHS Publishers, Weinheim, pp 1–64Arnold G, Leiteritz L, Zahn S, Rohm H (2009) Ultrasonic cutting of cheese: composition affects cutting work reduction and energy demand. Int Dairy J 19:314–320Barbero GF, Liazid A, Palma M, Barroso CG (2008) Ultrasound-assisted extraction of capsaicinoids from peppers. Talanta 75:1332–1337Benedito J, Carcel JA, Sanjuan N, Mulet A (2000) Use of ultrasound to assess Cheddar cheese characteristics. Ultrasonics 38:727–730Benedito J, Carcel JA, Rossello C, Mulet A (2001) Composition assessment of raw meat mixtures using ultrasonics. Meat Sci 57:365–370Bhaskaracharya RK, Kentish S, Ashokkumar M (2009) Selected applications of ultrasonics in food processing. Food Eng Rev 1:31–49Boonkird S, Phisalaphong C, Phisalaphong M (2008) Ultrasound-assisted extraction of capsaicinoids from Capsicum frutescens on a lab- and pilot-plant scale. Ultrason Sonochem 15:1075–1079Cárcel JA, Benedito J, Bon J, Mulet A (2007) High intensity ultrasound effects on meat brining. Meat Sci 76:611–619Cárcel JA, Benedito J, Rosselló C, Mulet A (2007) Influence of ultrasound intensity on mass transfer in apple immersed in a sucrose solution. J Food Eng 78:472–479Cavitus (2009) Grape colour and flavour extraction (Pat. Pend.) for red must extraction http://www.cavitus.com . Crafers. Accessed 10 Jan 2011Chea Chua S, Ping Tan C, Mirhosseini H, Ming Lai O, Long K, Sham Baharin B (2009) Optimization of ultrasound extraction condition of phospholipids from palm-pressed fiber. J Food Eng 92:403–409Chena R, Menga F, Zhang S, Liu Z (2009) Effects of ultrahigh pressure extraction conditions on yields and antioxidant activity of ginsenoside from ginseng. Sep Purif Technol 66:340–346Chivate MM, Pandit AB (1995) Quantification of cavitation intensity in fluid bulk. Ultrason Sonochem 2:19–25Da Porto C, Decorti D (2009) Ultrasound-assisted extraction coupled with under vacuum distillation of flavour compounds from spearmint (carvone-rich) plants: comparison with conventional hydrodistillation. Ultrason Sonochem 16:795–799Da Porto C, Decorti D, Kikic I (2009) Flavour compounds of Lavandula angustifolia L. to use in food manufacturing: Comparison of three different extraction methods. Food Chem 112:1072–1078Domínguez H, Núñez MJ, Lema JM (1994) Enzymatic pretreatment to enhance oil extraction from fruits and oilseeds: a review. Food Chem 49:271–286Dong J, Liu Y, Liang Z, Wanga W (2010) Investigation on ultrasound-assisted extraction of salvianolic acid B from Salvia miltiorrhiza root. Ultrason Sonochem 17:61–65Entezari MH, Kruus P (1994) Effect of frequency on sonochemical reactions. I: oxidation of iodide. Ultrason Sonochem 1:75–79Esclapez MD, Sáez V, Milán-Yáñez D, Tudela I, Louisnard O, González-García J (2010) Sonoelectrochemical treatment of water polluted with trichloroacetic acid: from sonovoltammetry to pre-pilot plant scale. Ultrason Sonochem 17:1010–1020Ferraro V, Cruz IB, Ferreira R, Malcata JFX, Pintado ME, Castro PML (2010) Valorisation of natural extracts from marine source focused on marine by-products: review. Food Res Int 43:2221–2233Fischer CH, Hart EJ, Henglein AJ (1986) Hydrogen/deuterium isotope exchange in the hydrogen deuteride-water system under the influence of ultrasound. Phys Chem 90:3059–3060Garcia-Noguera J, Weller CL, Oliveira FIP, Rodrigues S, Fernandes FAN (2010) Dual-stage sugar substitution in strawberries with a Stevia-based sweetener. Innovative Food Sci Emerg Technol 11:225–230García-Pérez JV, Cárcel JA, de la Fuente-Blanco S, Riera-Franco de Sarabia E (2006) Ultrasonic drying of foodstuff in a fluidized bed: parametric study. Ultrasonics 44:539–543García-Pérez JV, García-Alvarado MA, Carcel JA, Mulet A (2010) Extraction kinetics modeling of antioxidants from grape stalk (Vitis vinifera var. Bobal): Influence of drying conditions. J Food Eng 101:49–58González-García J, Sáez V, Tudela I, Díez-Garcia MI, Esclapez MD, Louisnard O (2010) Sonochemical treatment of water polluted by chlorinated organocompounds. A review. Water 2:28–74Handa SS, Preet S, Khanuja S, Longo G, Rakesh DD (2008) Extraction Technologies for Medicinal and Aromatic Plants. United Nations Industrial Development Organization and the International Centre for Science and High Technology, TriesteHemwimol S, Pavasant P, Shotipruk A (2006) Ultrasound-assisted extraction of anthraquinones from roots of Morinda citrifolia. Ultrason Sonochem 13:543–548Hielscher (2011) Teltow http:// www.hielscher.com . Accessed 10 Jan 2011Hu Y, Wang T, Wang M, Han S, Wan P, Fan M (2008) Extraction of isoflavonoids from Pueraria by combining ultrasound with microwave vacuum. Chem Engin Process 47:2256–2261Ince NH, Tezcanli G, Belen RK, Apikyan PG (2001) Ultrasound as a catalyzer of aqueous reaction systems: the state of the art and environmental applications. Appl Catal B 29:167–176Jadhav D, Rekha BN, Gogate PR, Rathod VK (2009) Extraction of vanillin from vanilla pods: a comparison study of conventional soxhlet and ultrasound assisted extraction. J Food Eng 93:421–426Ji J-b, Lu X-h, Cai M-q, Xu C-c (2006) Improvement of leaching process of Geniposide with ultrasound. Ultrason Sonochem 13:455–462Kanthale PM, Gogate PR, Pandit AB, Wilhelm AM (2003) Mapping of an ultrasonic horn: link primary and secondary effects of ultrasound. Ultrason Sonochem 10:331–335Karki B, Lamsal BP, Jung S, van Leeuwen JH, Pometto AL III, Grewell D, Khanal SK (2010) Enhancing protein and sugar release from defatted soy flakes using ultrasound technology. J Food Eng 96:270–278Kardos N, Luche J-L (2001) Sonochemistry of carbohydrate compounds. Carbohydr Res 332:115–131Kotronarou A, Mills G, Hoffmann MR (1991) Ultrasonic Irradiation of para-Nitrophenol in Aqueous Solution. J Phys Chem 95:3630–3638Kuijpers MWA, Kemmere MF, Keurentjes JTF (2002) Calorimetric study of the energy efficiency for ultrasound-induced radical formation. Ultrasonics 40:675–678Leighton TG (2007) What is ultrasound? Prog Biophys Mol Biol 93:3–83Leonelli C, Mason TJ (2010) Microwave and ultrasonic processing: now a realistic option for industry. Chem Eng Process 49:885–900Li H, Pordesimo L, Weiss J (2004) High intensity ultrasound-assisted extraction of oil from soybeans. Food Res Int 37:731–738Liu J, Li J-W, Tang J (2010) Ultrasonically assisted extraction of total carbohydrates from Stevia rebaudiana Bertoni and identification of extracts. Food Bioprod Process 88:215–221Lianfu Z, Zelong L (2008) Optimization and comparison of ultrasound/microwave assisted extraction (UMAE) and ultrasonic assisted extraction (UAE) of lycopene from tomatoes. Ultrason Sonochem 15:731–737Liazid A, Schwarz M, Varela RM, Palma M, Guillén DA, Brigui J, Macías FA, Barroso CG (2010) Evaluation of various extraction techniques for obtaining bioactive extracts from pine seeds. Food Bioprod Process 88:247–252Londoño-Londoño J, Rodrigues de Lima V, Lara O, Gil A, Crecsynski Pasa TB, Arango GJ, Ramirez Pineda JR (2010) Clean recovery of antioxidant flavonoids from citrus peel: optimizing an aqueous ultrasound-assisted extraction method. Food Chem 119:81–87Lou Z, Wang H, Zhang M, Wang Z (2010) Improved extraction of oil from chickpea under ultrasound in a dynamic system. J Food Eng 98:13–18Louisnard O, González-García J, Tudela I, Klima J, Sáez V, Vargas-Hernández Y (2009) FEM simulation of a sono-reactor accounting for vibrations of the boundaries. Ultrason Sonochem 16:250–259Luque de Castro MD, Priego-Capote F (2007) Analytical Applications of Ultrasound, Vol. 26, Techniques and Instrumentation in Analytical Chemistry. Elsevier Science, AmsterdamMa Y, Ye X, Hao Y, Xu G, Xu G, Liu D (2008) Ultrasound-assisted extraction of hesperidin from Penggan (Citrus reticulata) peel. Ultrason Sonochem 15:227–232Ma Y, Chen J-C, Liu Dong-Hong, Ye X-Q (2009) Simultaneous extraction of phenolic compounds of citrus peel extracts: effect of ultrasound. Ultrason Sonochem 16:57–62Makino K, Mossoba MM, Riesz P (1982) Chemical effects of ultrasound on aqueous solutions. Evidence for hydroxyl and hydrogen free radicals (.cntdot. OH and. cntdot. H) by spin trapping. J Chem Soc 104:3537–3539Margulis MA, Margulis IM (2003) Calorimetric method for measurement of acoustic power absorbed in a volume of liquid. Ultrason Sonochem 10:343–345Martin CJ, Law ANR (1983) Design of thermistor probes for measurement of ultrasound intensity distributions. Ultrasonics 21:85–90Mason TJ, Lorimer JP, Bates DM, Zhao Y (1994) Dosimetry in sonochemistry: the use of aqueous terephthalate ion as a fluorescence monitor. Ultrason Sonochem 1:91–95Meinhardt (2011) Leipzig. http://www.meinhardt-ultraschall.de . Accessed 10 Jan 2011Montalbo-Lomboy M, Khanal SK, van Leeuwen JH, Raman DR, Dunn L Jr, Grewell D Jr (2010) Ultrasonic pretreatment of corn slurry for saccharification: a comparison of batch and continuous Systems. Ultrason Sonochem 17:939–946Mulet A, Cárcel JA, Sanjuán N, Bon J (2003) New food drying technologies. Use of ultrasound. Food Sci Technol Int 9:215–221Naguleswaran S, Vasanthan T (2010) Dry milling of field pea (Pisum sativum L.) groats prior to wet fractionation influences the starch yield and purity. Food Chem 118:627–633Orozco-Solano M, Ruiz-Jiménez J, Luque de Castro MD (2010) Ultrasound-assisted extraction and derivatization of sterols and fatty alcohols from olive leaves and drupes prior to determination by gas chromatography–tandem mass spectrometry. J Chromatogr A 1217:1227–1235Patist A, Bates D (2008) Ultrasonic innovations in the food industry: from the laboratory to commercial production. Innovative Food Sci Emerg Technol 9:147–154Price GJ (1990) The use of ultrasound for the controlled degradation of polymer solutions. In: Mason TJ (ed) Advances in sonochemistry, vol 1. Jai Press, Cambridge, pp 231–287Riener J, Noci G, Cronin DA, Morgan DJ, Lyng JG (2010) A comparison of selected quality characteristics of yoghurts prepared from thermosonicated and conventionally heated milks. Food Chem 119:1108–1113Riera E, Golás Y, Blanco A, Gallego JA, Blasco M, Mulet A (2004) Mass transfer enhancement in supercritical fluids extraction by means of power ultrasound. Ultrason Sonochem 11:241–244Riera E, Blanco A, García J, Benedito J, Mulet A, Gallego-Juárez JA, Blasco M (2010) High-power ultrasonic system for the enhancement of mass transfer in supercritical CO2 extraction processes. Physics Procedia 3:141–146Roldán-Gutiérrez JM, Ruiz-Jiménez J, Luque de Castro MD (2008) Ultrasound-assisted dynamic extraction of valuable compounds from aromatic plants and flowers as compared with steam distillation and superheated liquid extraction. Talanta 75:1369–1375Romdhane M, Gourdon C (2002) Investigation in solid–liquid extraction: influence of ultrasound. Chem Eng J 87:11–19Rong L, Kojima Y, Koda S, Nomura H (2008) Simple quantification of ultrasonic intensity using aqueous solution of phenolphthalein. Ultrason Sonochem 8:11–15Sáez V, Frias-Ferrer A, Iniesta J, Gonzalez-Garcıa J, Aldaz A, Riera E (2005) Chacterization of a 20 kHz sonoreactor. Part I: analysis of mechanical effects by classical and numerical methods. Ultrason Sonochem 12:59–65Sáez V, Frias-Ferrer A, Iniesta J, Gonzalez-Garcıa J, Aldaz A, Riera E (2005) Characterization of a 20 kHz sonoreactor. Part II: analysis of chemical effects by classical and electrochemical methods. Ultrason Sonochem 12:67–72Sahena F, Zaidul ISM, Jinap S, Karim AA, Abbas KA, Norulaini NAN, Omar AKM (2009) Application of supercritical CO2 in lipid extraction–A review. J Food Eng 95:240–253Science Direct Database (2011) www.sciencedirect.com (Data of consulting: February 2011)Soria AC, Villamiel M (2010) Effect of ultrasound on the technological properties and bioactivity of food: a review. Trends Food Sci Technol 21:323–331Starmans DAJ, Nijhuis HH (1996) Extraction of secondary metabolites from plant material: a review. 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Anthropogenic disturbance of deep-sea megabenthic assemblages: a study with Remotely-Operated Vehicles in the Faroe-Shetland Channel, NE Atlantic

The effects of local-scale anthropogenic disturbance from active drilling platforms on epibenthic megafaunal abundance, diversity and assemblage pattern were examined in two West of Shetland hydrocarbon fields at 420 m and 508 m water depth. These areas were selected to include a range of disturbance regimes and contrasting faunal assemblages associated with different temperature regimes. Remotely Operated Vehicle (ROV) video provided high-resolution megafaunal abundance and diversity data, which were related to the extent of visible disturbance from drilling spoil. These data, in conjunction with a study deeper in the Faroe-Shetland Channel, have allowed comparison of the effects of disturbance on megabenthos across a range of sites. Disturbance to megafaunal assemblages was found to be high within 50 m of the source of drill spoil and in areas where spoil was clearly visible on the seabed, with depressed abundances (Foinaven 1900 individuals ha-1; Schiehallion 2178 individuals ha-1) and diversity (H´ = 1.75 Foinaven; 1.12 Schiehallion) as a result of smothering effects. These effects extended to around 100 m from the source of disturbance, although this was variable, particularly with current regime and nature of drilling activity. Further from the source of disturbance, megafaunal assemblages became more typical of the background area with increased diversity (H´ = 2.02 Foinaven; 1.77 Schiehallion) and abundance (Foinaven 16484 individuals ha-1; Schiehallion 5477 individuals ha-1). Visible effects on megafaunal assemblages as a result of seabed drilling were limited in extent although assemblage responses were complex, being controlled by differing effects to individual species often based on their motility

Robust prognostic value of a knowledge-based proliferation signature across large patient microarray studies spanning different cancer types.

Tumour proliferation is one of the main biological phenotypes limiting cure in oncology. Extensive research is being performed to unravel the key players in this process. To exploit the potential of published gene expression data, creation of a signature for proliferation can provide valuable information on tumour status, prognosis and prediction. This will help individualizing treatment and should result in better tumour control, and more rapid and cost-effective research and development. From in vitro published microarray studies, two proliferation signatures were compiled. The prognostic value of these signatures was tested in five large clinical microarray data sets. More than 1000 patients with breast, renal or lung cancer were included. One of the signatures (110 genes) had significant prognostic value in all data sets. Stratifying patients in groups resulted in a clear difference in survival (P-values <0.05). Multivariate Cox-regression analyses showed that this signature added substantial value to the clinical factors used for prognosis. Further patient stratification was compared to patient stratification with several well-known published signatures. Contingency tables and Cramer's V statistics indicated that these primarily identify the same patients as the proliferation signature does. The proliferation signature is a strong prognostic factor, with the potential to be converted into a predictive test. Furthermore, evidence is provided that supports the idea that many published signatures track the same biological processes and that proliferation is one of them