The use of the comet assay in the study of human nutrition and cancer.

The influence of diet on carcinogenesis is a hugely complex area; not only is the consumption of major dietary factors such as meat, fat and fruits and vegetables associated with increased or decreased risk of a range of cancers but also an increasing number of specific nutrients such as vitamins, minerals and phytochemicals are being proposed as the next 'superfoods' to combat the development of cancer. As well as epidemiological studies to determine the association of these dietary factors with cancer risk, it is also essential to investigate the underlying mechanisms through which these factors may causally influence carcinogenesis. The comet assay provides a relatively simple, cheap and rapid method to examine DNA damage and repair and is, therefore, an ideal biomarker for the study of the effects of nutrition on cancer. This review focuses on the use of the comet assay in studies involving human subjects or human cell lines, which investigate the effects of various nutrients on biomarkers relevant to carcinogenesis, and discusses the potential of the comet assay and its various modifications for use as cancer-related biomarkers suitable for use in nutritional studies.

[1]  G. Eisenbrand,et al.  Modulation of oxidative cell damage by reconstituted mixtures of phenolic apple juice extracts in human colon cell lines. , 2006, Molecular nutrition & food research.

[2]  A. Collins.  Assays for oxidative stress and antioxidant status: applications to research into the biological effectiveness of polyphenols. , 2005, The American journal of clinical nutrition.

[3]  S. Duthie,et al.  The effect of dietary flavonoids on DNA damage (strand breaks and oxidised pyrimdines) and growth in human cells. , 1997, Mutation research.

[4]  Mi Young Lee,et al.  Electrolyzed-reduced water protects against oxidative damage to DNA, RNA, and protein , 2006, Applied biochemistry and biotechnology.

[5]  J. Kleinjans,et al.  GSTM1 and GSTT1 polymorphism influences protection against induced oxidative DNA damage by quercetin and ascorbic acid in human lymphocytes in vitro. , 2007, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[6]  K. White,et al.  Dietary antioxidants and DNA damage in patients on long-term acid-suppression therapy: a randomized controlled study , 2002, British Journal of Nutrition.

[7]  A. Azmi,et al.  Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: A putative mechanism for anticancer properties , 2006, FEBS letters.

[8]  Iris F. F. Benzie,et al.  Vitamins C and E: acute interactive effects on biomarkers of antioxidant defence and oxidative stress. , 2004, Mutation research.

[9]  G. Ma,et al.  A Pilot Study on the Effects of Almond Consumption on DNA Damage and Oxidative Stress in Smokers , 2006, Nutrition and cancer.

[10]  A. Collins,et al.  Quercetin and myricetin protect against hydrogen peroxide-induced DNA damage (strand breaks and oxidised pyrimidines) in human lymphocytes. , 1997, Mutation research.

[11]  M. Wolters,et al.  Folic acid and colorectal cancer prevention: molecular mechanisms and epidemiological evidence (Review). , 2005, International journal of oncology.

[12]  J. Cadet,et al.  Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture. , 1998, Free radical research.

[13]  A. Rao,et al.  Carotenoids and human health. , 2007, Pharmacological research.

[14]  I. Rowland,et al.  Shift from a dairy product-rich to a dairy product-free diet: influence on cytotoxicity and genotoxicity of fecal water--potential risk factors for colon cancer. , 1997, The American journal of clinical nutrition.

[15]  I. Rowland,et al.  Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress. , 2006, Carcinogenesis.

[16]  A. Collins,et al.  Kiwifruit Protects Against Oxidative DNA Damage in Human Cells and In Vitro , 2001, Nutrition and cancer.

[17]  M. Neuhouser.  Review: Dietary Flavonoids and Cancer Risk: Evidence From Human Population Studies , 2004, Nutrition and cancer.

[18]  Y. Jeon,et al.  Antioxidant activities of enzymatic extracts from brown seaweeds. , 2003, Bioresource technology.

[19]  M. Glei,et al.  The main catechin of green tea, (-)-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[20]  S. Loft,et al.  Antioxidant intervention studies related to DNA damage, DNA repair and gene expression. , 2000, Free radical research.

[21]  D. Phillips,et al.  Genotoxicity of human milk extracts and detection of DNA damage in exfoliated cells recovered from breast milk. , 1999, Biochemical and biophysical research communications.

[22]  P. McMillan,et al.  Uracil misincorporation in human DNA detected using single cell gel electrophoresis. , 1997, Carcinogenesis.

[23]  N. Rajab,et al.  Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. , 1998, Mutagenesis.

[24]  H. McNulty,et al.  Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation. , 2006, The Journal of nutrition.

[25]  N. Başaran,et al.  The effects of thyme volatiles on the induction of DNA damage by the heterocyclic amine IQ and mitomycin C. , 2006, Mutation research.

[26]  U. Vogel,et al.  No effect of 600 grams fruit and vegetables per day on oxidative DNA damage and repair in healthy nonsmokers. , 2003, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[27]  A. Vachálková,et al.  Protective effect of quercetin and luteolin in human melanoma HMB-2 cells. , 2005, Mutation research.

[28]  J. Crowell,et al.  Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer. , 2003, The American journal of clinical nutrition.

[29]  P. Møller,et al.  Vitamin C supplementation decreases oxidative DNA damage in mononuclear blood cells of smokers , 2004, European journal of nutrition.

[30]  C. Gardana,et al.  Effect of a tomato-based drink on markers of inflammation, immunomodulation, and oxidative stress. , 2006, Journal of agricultural and food chemistry.

[31]  H. Adlercreutz,et al.  Isoflavonoids and lignans have different potentials to modulate oxidative genetic damage in human colon cells. , 2000, Carcinogenesis.

[32]  V. Steenkamp,et al.  Antioxidant and genotoxic properties of South African herbal extracts. , 2005, Mutation research.

[33]  M. Kundi,et al.  Effects of mustard sprouts and allylisothiocyanate on benzo(a)pyrene-induced DNA damage in human-derived cells: a model study with the single cell gel electrophoresis/Hep G2 assay. , 2003, Teratogenesis, carcinogenesis, and mutagenesis.

[34]  A. Collins,et al.  Detection of alkylation damage in human lymphocyte DNA with the comet assay. , 2001, Acta biochimica Polonica.

[35]  Kanupriya,et al.  Flavonoids protect U-937 macrophages against tert-butylhydroperoxide induced oxidative injury. , 2006, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[36]  D. Fedeli,et al.  Influence of astaxanthin, zeaxanthin and lutein on DNA damage and repair in UVA-irradiated cells. , 2006, Journal of photochemistry and photobiology. B, Biology.

[37]  N. Habermann,et al.  Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model , 2005, Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals.

[38]  B. Jiang,et al.  Antioxidant properties of apple juice and its protection against Cr(VI)-induced cellular injury. , 2002, Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer.

[39]  T. McMillan,et al.  Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the Comet assay. , 2002, Mutagenesis.

[40]  S. Duthie,et al.  DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[41]  W. Berger,et al.  Green tea extract and (−)‐epigallocatechin‐3‐gallate, the major tea catechin, exert oxidant but lack antioxidant activities , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[42]  J. Kerry,et al.  Modulatory effects of resveratrol, citroflavan-3-ol, and plant-derived extracts on oxidative stress in U937 cells. , 2006, Journal of medicinal food.

[43]  R. Russell,et al.  β-Carotene and Other Carotenoids as Antioxidants , 1999 .

[44]  N. Zhou,et al.  DNA Damage-mediated Apoptosis Induced by Selenium Compounds* , 2003, Journal of Biological Chemistry.

[45]  A. Collins,et al.  THE COMET ASSAY: a biomonitoring tool for nutraceutical research , 2005 .

[46]  D. Phillips,et al.  DNA damage in human breast milk cells and its induction by 'early' and 'late' milk extracts. , 2000, Carcinogenesis.

[47]  A. Azmi,et al.  Resveratrol–Cu(II) induced DNA breakage in human peripheral lymphocytes: Implications for anticancer properties , 2005, FEBS letters.

[48]  I. Rowland,et al.  Equol: A Comparison of the Effects of the Racemic Compound With That of the Purified S-Enantiomer on the Growth, Invasion, and DNA Integrity of Breast and Prostate Cells In Vitro , 2006, Nutrition and cancer.

[49]  J. Gama-Rodrigues,et al.  Diet and colorectal cancer: current evidence for etiology and prevention. , 2005, Nutricion hospitalaria.

[50]  R. Marcos,et al.  Analysis of glutathione and vitamin C effects , 2006, TheScientificWorldJournal.

[51]  H. Vrolijk,et al.  The Combined Effects of Extracts Containing Carotenoids and Vitamins E and C on Growth and Pigmentation of Cultured Human Melanocytes , 2004, Skin Pharmacology and Physiology.

[52]  T. Güngör,et al.  Analysis of DNA Damage Using the Comet Assay in Infants Fed Cow’s Milk , 2003, Neonatology.

[53]  S. Campbell,et al.  The green tea polyphenol (−)‐epigallocatechin gallate and green tea can protect human cellular DNA from ultraviolet and visible radiation‐induced damage , 2005, Photodermatology, photoimmunology & photomedicine.

[54]  Peter Møller,et al.  The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. , 2006, Basic & clinical pharmacology & toxicology.

[55]  L. Kupper,et al.  Associations of Antioxidant Nutrients and Oxidative DNA Damage in Healthy African-American and White Adults , 2007, Cancer Epidemiology Biomarkers & Prevention.

[56]  P. Riso,et al.  Supplementation of Jurkat T cells with green tea extract decreases oxidative damage due to iron treatment. , 1999, The Journal of nutrition.

[57]  I. Benzie,et al.  Antioxidants in Fruits and Vegetables: a Study of Cellular Availability and Direct Effects on Human DNA , 2006, Bioscience, biotechnology, and biochemistry.

[58]  J. Kleinjans,et al.  Protection by quercetin and quercetin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes. , 2005, Mutation research.

[59]  I. Rowland,et al.  In vitro antioxidant activity and antigenotoxicity of 5-n-alkylresorcinols. , 2006, Journal of agricultural and food chemistry.

[60]  P. Riso,et al.  Daily intake of a formulated tomato drink affects carotenoid plasma and lymphocyte concentrations and improves cellular antioxidant protection , 2005, British Journal of Nutrition.

[61]  H. McNulty,et al.  Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay , 2003, British journal of cancer.

[62]  G. Davison,et al.  Exercise and mononuclear cell DNA damage: the effects of antioxidant supplementation. , 2005, International journal of sport nutrition and exercise metabolism.

[63]  P. Riso,et al.  Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. , 2000, The Journal of nutrition.

[64]  Y. Park,et al.  Green vegetable drink consumption protects peripheral lymphocytes DNA damage in Korean smokers , 2004, BioFactors.

[65]  S. Venitt,et al.  Heterocyclic aromatic amines induce DNA strand breaks and cell transformation. , 1999, Carcinogenesis.

[66]  R. Fuller,et al.  Probiotics in man and animals. , 1989, The Journal of applied bacteriology.

[67]  G. Rechkemmer,et al.  Dietary intervention with the probiotics Lactobacillus acidophilus 145 and Bifidobacterium longum 913 modulates the potential of human faecal water to induce damage in HT29clone19A cells. , 2004, The British journal of nutrition.

[68]  D. Phillips,et al.  Genotoxicity of human breast milk from different countries. , 2001, Mutagenesis.

[69]  P. Riso,et al.  Lycopene and vitamin C concentrations increase in plasma and lymphocytes after tomato intake. Effects on cellular antioxidant protection , 2004, European Journal of Clinical Nutrition.

[70]  M. Renis,et al.  Hydroxytyrosol lipophilic analogues: enzymatic synthesis, radical scavenging activity and DNA oxidative damage protection. , 2007, Bioorganic chemistry.

[71]  Xinhan Zhao,et al.  Epigallocatechin gallate, the main ingredient of green tea induces apoptosis in breast cancer cells. , 2006, Frontiers in bioscience : a journal and virtual library.

[72]  C. Ong,et al.  Sodium selenite‐induced oxidative stress and apoptosis in human hepatoma HepG2 cells , 1999, International journal of cancer.

[73]  T. Milewicz,et al.  Uracil misincorporation into DNA of leukocytes of young women with positive folate balance depends on plasma vitamin B12 concentrations and methylenetetrahydrofolate reductase polymorphisms. A pilot study. , 2005, The Journal of nutritional biochemistry.

[74]  I R Rowland,et al.  Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro. , 2007, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[75]  G. Muir,et al.  The influence of dietary and environmental factors on prostate cancer risk , 2000, Prostate Cancer and Prostatic Diseases.

[76]  A. Collins,et al.  Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay , 1996 .

[77]  Andrew R Collins,et al.  Nutritional modulation of DNA repair in a human intervention study. , 2003, Carcinogenesis.

[78]  B. Halliwell,et al.  The measurement of oxidative damage to DNA by HPLC and GC/MS techniques. , 1992, Free radical research communications.

[79]  M. Lean,et al.  Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. , 1998, The American journal of clinical nutrition.

[80]  R. Doll,et al.  The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. , 1981, Journal of the National Cancer Institute.

[81]  Jun Cao,et al.  Mitochondrial and nuclear DNA damage induced by curcumin in human hepatoma G2 cells. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[82]  E. Hwang,et al.  The effect of lycopene on cell growth and oxidative DNA damage of Hep3B human hepatoma cells , 2005, BioFactors.

[83]  Y. Jeon,et al.  Antioxidant activity of far-infrared radiated rice hull extracts on reactive oxygen species scavenging and oxidative DNA damage in human lymphocytes. , 2006, Journal of medicinal food.

[84]  P. Guiraud,et al.  Study of antimutagenic and antioxidant activities of gallic acid and 1,2,3,4,6-pentagalloylglucose from Pistacia lentiscus. Confirmation by microarray expression profiling. , 2007, Chemico-biological interactions.

[85]  M. Kundi,et al.  Coffee consumption induces GSTP in plasma and protects lymphocytes against (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide induced DNA-damage: results of controlled human intervention trials. , 2005, Mutation research.

[86]  B. Davis,et al.  Dietary carotenoids and genetic instability modify bladder cancer risk. , 2004, The Journal of nutrition.

[87]  J. Kerry,et al.  Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells , 2007, British Journal of Nutrition.

[88]  Yiwei Li,et al.  Soy Isoflavones and Cancer Prevention , 2003, Cancer investigation.

[89]  C. Belloir,et al.  Protective effects of garlic sulfur compounds against DNA damage induced by direct- and indirect-acting genotoxic agents in HepG2 cells. , 2006, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[90]  D. Galaris,et al.  DNA protecting and genotoxic effects of olive oil related components in cells exposed to hydrogen peroxide , 2005, Free radical research.

[91]  G. Loo,et al.  Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA. , 2000, Mutation research.

[92]  Seung-Cheol Lee,et al.  Antioxidative and Anticancer Activity of Extracts of Cherry (Prunus serrulata var. spontanea) Blossoms , 2007, Plant foods for human nutrition.

[93]  P. Bowen,et al.  DNA Damage, a Biomarker of Carcinogenesis: Its Measurement and Modulation by Diet and Environment , 2007, Critical reviews in food science and nutrition.

[94]  I. Rowland,et al.  The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro and in vivo. , 2004, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[95]  D. Cooper.  Carotenoids in health and disease: recent scientific evaluations, research recommendations and the consumer. , 2004, The Journal of nutrition.

[96]  P. Andrade,et al.  Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage. , 2007, Chemico-biological interactions.

[97]  P. Møller,et al.  Oxidative DNA damage in human white blood cells in dietary antioxidant intervention studies. , 2002, The American journal of clinical nutrition.

[98]  G. Rechkemmer,et al.  A diet high in fat and meat but low in dietary fibre increases the genotoxic potential of 'faecal water'. , 1999, Carcinogenesis.

[99]  L. Ferguson,et al.  DNA stability and serum selenium levels in a high-risk group for prostate cancer. , 2004, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[100]  M. Knize,et al.  Formation and human risk of carcinogenic heterocyclic amines formed from natural precursors in meat. , 2005, Nutrition reviews.

[101]  E. Cemeli,et al.  Antigenotoxic properties of selenium compounds on potassium dichromate and hydrogen peroxide. , 2003, Teratogenesis, carcinogenesis, and mutagenesis.

[102]  A. Collins,et al.  Absorption and DNA protective effects of flavonoid glycosides from an onion meal , 2000, European journal of nutrition.

[103]  C. Pereira-Wilson,et al.  Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. , 2006, Life sciences.

[104]  R. Fimmers,et al.  Single and repeated moderate consumption of native or dealcoholized red wine show different effects on antioxidant parameters in blood and DNA strand breaks in peripheral leukocytes in healthy volunteers: a randomized controlled trial [ISRCTN68505294] , 2005, Nutrition journal.

[105]  N. Başaran,et al.  The effects of thyme volatiles on the induction of DNA damage by the heterocyclic amine IQ and mitomycin C. , 2005, Mutation research.

[106]  A. Azmi,et al.  Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties. , 2006, The international journal of biochemistry & cell biology.

[107]  T. Skorski,et al.  Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine. , 2002, Chemico-biological interactions.

[108]  Diallyl sulfide inhibits PhIP-induced DNA strand breaks in normal human breast epithelial cells. , 2007, Oncology reports.

[109]  M. Green,et al.  Effects of selenium compounds on induction of DNA damage by broadband ultraviolet radiation in human keratinocytes , 2003, The British journal of dermatology.

[110]  Jack A. Taylor,et al.  Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair. , 2007, Mutation research.

[111]  A. Azmi,et al.  Prooxidant DNA breakage induced by caffeic acid in human peripheral lymphocytes: involvement of endogenous copper and a putative mechanism for anticancer properties. , 2007, Toxicology and applied pharmacology.

[112]  I. Rowland,et al.  Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults. , 2007, The American journal of clinical nutrition.

[113]  H. McNulty,et al.  Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients , 2005, British Journal of Cancer.

[114]  B. Popkin.  Understanding global nutrition dynamics as a step towards controlling cancer incidence , 2007, Nature Reviews Cancer.

[115]  M. Dobrzyńska,et al.  Flavonoids modulate comet assay responses to food mutagens in human lymphocytes and sperm. , 1998, Mutation research.

[116]  I R Rowland,et al.  Genotoxic activity in human faecal water and the role of bile acids: a study using the alkaline comet assay. , 1997, Carcinogenesis.

[117]  G R Gibson,et al.  Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. , 1995, The Journal of nutrition.

[118]  Y. Seo,et al.  Selenomethionine induction of DNA repair response in human fibroblasts , 2002, Oncogene.

[119]  R. Russell,et al.  Beta-carotene and other carotenoids as antioxidants. , 1999, Journal of the American College of Nutrition.

[120]  B. Ames,et al.  Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison , 2004, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[121]  K. Riganakos,et al.  Protection against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide: the role of iron chelation. , 2005, Free radical biology & medicine.

[122]  C. Matthias,et al.  The influence of high doses of vitamin C and zinc on oxidative DNA damage. , 2005, Anticancer research.

[123]  G. McKerr,et al.  The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells. , 1999, Cancer research.

[124]  S. Mayne,et al.  Antioxidant nutrients and chronic disease: use of biomarkers of exposure and oxidative stress status in epidemiologic research. , 2003, The Journal of nutrition.

[125]  K. Kikugawa,et al.  Effective inhibition by beta-carotene of cellular DNA breaking induced by peroxynitrous acid. , 1999, Free radical research.

[126]  S. Bingham,et al.  Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N‐Nitroso compound formation attributed to red meat consumption , 2006, Environmental and molecular mutagenesis.

[127]  M. Dusinska,et al.  DNA damage and antioxidants; fluctuations through the year in a central European population group. , 2002, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[128]  I. Rowland,et al.  Antigenotoxicity of probiotics and prebiotics on faecal water-induced DNA damage in human colon adenocarcinoma cells. , 2004, Mutation research.

[129]  G. McKerr,et al.  Use of the Comet-FISH Assay to Demonstrate Repair of the TP53 Gene Region in Two Human Bladder Carcinoma Cell Lines , 2003, Radiation research.

[130]  P. Riso,et al.  Orange juice vs vitamin C: effect on hydrogen peroxide-induced DNA damage in mononuclear blood cells. , 2007, The British journal of nutrition.

[131]  G. Eisenbrand,et al.  An anthocyanin/polyphenolic‐rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands , 2006, Biotechnology journal.