Modulation of mitochondrial function by the microbiome metabolite propionic acid in autism and control cell lines

Translational Psychiatry

Volumn 6, Issue 10, 2016, Pages

  Frye, R E ^a,b   Rose, S ^a,b   Chacko, J ^a   Wynne, R ^a,b   Bennuri, S C ^a,b   Slattery, J C ^a,b   Tippett, M ^a,b   Delhey, L ^a,b   Melnyk, S ^a,b   Kahler, S G ^a,b   MacFabe, D F ^c  

^a UNIVERSITY OF ARKANSAS FOR MEDICAL SCIENCES   (United States)

^b ARKANSAS CHILDREN S HOSPITAL RESEARCH INSTITUTE   (United States)

^c UNIVERSITY OF WESTERN ONTARIO   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

3 NITROPROPIONIC ACID; PROPIONIC ACID; REACTIVE OXYGEN METABOLITE; PROPIONIC ACID DERIVATIVE;

ARTICLE; AUTISM; CHILD; CONTROLLED STUDY; DIABETES MELLITUS; ENTEROBACTERIACEAE; HUMAN; HUMAN CELL; IN VITRO STUDY; LYMPHOBLASTOID CELL LINE; MALE; METABOLITE; MITOCHONDRIAL RESPIRATION; MITOCHONDRION; OBESITY; OXIDATION REDUCTION STATE; OXIDATIVE STRESS; SCHOOL CHILD; TUMOR MICROENVIRONMENT; CASE CONTROL STUDY; CELL LINE; DISORDERS OF MITOCHONDRIAL FUNCTIONS; INTESTINE FLORA; METABOLISM; PATHOPHYSIOLOGY; PHYSIOLOGY; PROPIONIC ACIDEMIA;

AUTISM SPECTRUM DISORDER; CASE-CONTROL STUDIES; CELL LINE; CHILD; GASTROINTESTINAL MICROBIOME; HUMANS; MALE; MITOCHONDRIA; MITOCHONDRIAL DISEASES; PROPIONATES; PROPIONIC ACIDEMIA;

EID: 85029857267     PISSN: None     EISSN: 21583188     Source Type: Journal    
DOI: 10.1038/tp.2016.189     Document Type: Article

References (102)

1. Diaz Heijtz R, Wang S, Anuar F, Qian Y, Bjorkholm B, Samuelsson A et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci USA 2011; 108: 3047-3052.
2. Sherwin E, Rea K, Dinan TG, Cryan JF. A gut (microbiome) feeling about the brain. Curr Opin Gastroenterol 2016; 32: 96-102.
3. Johnson CC, Ownby DR. Allergies and asthma: do atopic disorders result from inadequate immune homeostasis arising from infant gut dysbiosis? Expert Rev Clin Immunol 2016; 12: 379-388.
4. Borre YE, Moloney RD, Clarke G, Dinan TG, Cryan JF. The impact of microbiota on brain and behavior: mechanisms & therapeutic potential. Adv Exp Med Biol 2014; 817:373-403.
5. Bull MJ, Plummer NT. Part 2: treatments for chronic gastrointestinal disease and gut dysbiosis. Integr Med (Encinitas) 2015; 14: 25-33.
6. Dolpady J, Sorini C, Di Pietro C, Cosorich I, Ferrarese R, Saita D et al. Oral Probiotic VSL#3 prevents autoimmune diabetes by modulating microbiota and promoting indoleamine 2,3-dioxygenase-enriched tolerogenic intestinal environment. J Diabetes Res 2016; 2016: 7569431.
7. Lambeth SM, Carson T, Lowe J, Ramaraj T, Leff JW, Luo L et al. Composition, diversity and abundance of gut microbiome in prediabetes and type 2 diabetes. J Diabetes Obes 2015; 2: 1-7.
8. Dugas LR, Fuller M, Gilbert J, Layden BT. The obese gut microbiome across the epidemiologic transition. Emerg Themes Epidemiol 2016; 13: 2.
9. Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W et al. Host-gut microbiota metabolic interactions. Science 2012; 336: 1262-1267.
10. Alenghat T. Epigenomics and the microbiota. Toxicol Pathol 2015; 43: 101-106.
11. Shenderov BA, Midtvedt T. Epigenomic programing: a future way to health? Microb Ecol Health Dis 2014; 25: 24145, (doi:http://dx.doi.org/10.3402/mehd.v25.24145).
12. Brock M, Buckel W. On the mechanism of action of the antifungal agent pro-pionate. Eur J Biochem 2004; 271: 3227-3241.
13. Coblentz WK, Bertram MG. Effects of a propionic acid-based preservative on storage characteristics, nutritive value, and energy content for alfalfa hays packaged in large round bales.. J Dairy Sci 2012; 95: 340-352.
14. Coblentz WK, Coffey KP, Young AN, Bertram MG. Storage characteristics, nutritive value, energy content, and in vivo digestibility of moist, large rectangular bales of alfalfa-orchardgrass hay treated with a propionic acid-based preservative.. J Dairy Sci 2013; 96: 2521-2535.
15. Couallier EM, Payot T, Bertin AP, Lameloise ML. Recycling of distillery effluents in alcoholic fermentation: role in inhibition of 10 organic molecules. Appl Biochem Biotechnol 2006; 133: 217-238.
16. Darzi J, Frost GS, Robertson MD. Effects of a novel propionate-rich sourdough bread on appetite and food intake. Eur J Clin Nutr 2012; 66: 789-794.
17. Fernandez U, Vodovotz Y, Courtney P, Pascall MA. Extended shelf life of soy bread using modified atmosphere packaging. J Food Prot 2006; 69: 693-698.
18. Quitmann H, Fan R, Czermak P. Acidic organic compounds in beverage, food, and feed production. Adv Biochem Eng Biotechnol 2014; 143: 91-141.
19. Al-Lahham SH, Peppelenbosch MP, Roelofsen H, Vonk RJ, Venema K. Biological effects of propionic acid in humans; metabolism, potential applications and underlying mechanisms. Biochim Biophys Acta 2010; 1801: 1175-1183.
20. Scotter MJ, Thorpe SA, Reynolds SL, Wilson LA, Strutt PR. Survey of baked goods for propionic acid and propionates. Food Addit Contam 1996; 13: 133-139.
21. Lind H, Jonsson H, Schnurer J. Antifungal effect of dairy propionibacteria-contribution of organic acids. Int J Food Microbiol 2005; 98: 157-165.
22. Al-Lahham S, Roelofsen H, Rezaee F, Weening D, Hoek A, Vonk R et al. Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects. Eur J Clin Invest 2012; 42: 357-364.
23. Kasubuchi M, Hasegawa S, Hiramatsu T, Ichimura A, Kimura I. Dietary gut microbial metabolites, short-chain fatty acids, and host metabolic regulation. Nutrients 2015; 7: 2839-2849.
24. Macfarlane S, Macfarlane GT. Regulation of short-chain fatty acid production. Proc Nutr Soc 2003; 62: 67-72.
25. Haska L, Andersson R, Nyman M. A water-soluble fraction from a by-product of wheat increases the formation of propionic acid in rats compared with diets based on other by-product fractions and oligofructose.. Food Nutr Res 2011; 55: 6397, (doi: 10.3402/fnr.v55i0.6397).
26. Anderson RC, Majak W, Rassmussen MA, Callaway TR, Beier RC, Nisbet DJ et al. Toxicity and metabolism of the conjugates of 3-nitropropanol and 3-nitropropionic acid in forages poisonous to livestock. J Agric Food Chem 2005; 53: 2344-2350.
27. Dannhardt G, Lehr M. Nonsteroidal antiinflammatory agents, XVII: inhibition of bovine cyclooxygenase and 5-lipoxygenase by N-alkyldiphenyl-pyrrolyl acetic and propionic acid derivatives.. Arch Pharm (Weinheim) 1993; 326: 157-162.
28. Levison ME. Effect of colon flora and short-chain fatty acids on growth in vitro of Pseudomonas aeruginsoa and Enterobacteriaceae. Infect Immun 1973; 8: 30-35.
29. Bolognini D, Tobin AB, Milligan G, Moss CE. The pharmacology and function of short chain fatty acid receptors. Mol Pharmacol 2015; 89: 388-398.
30. Nankova BB, Agarwal R, MacFabe DF, La Gamma EF. Enteric bacterial metabolites propionic and butyric acid modulate gene expression, including CREB-dependent catecholaminergic neurotransmission, in PC12 cells-possible relevance to autism spectrum disorders.. PLoS One 2014; 9: e103740.
31. Wajner M, Latini A, Wyse AT, Dutra-Filho CS. The role of oxidative damage in the neuropathology of organic acidurias: insights from animal studies. J Inherit Metab Dis 2004; 27: 427-448.
32. Coulter DL. Carnitine, valproate, and toxicity. J Child Neurol 1991; 6: 7-14.
33. Calabrese V, Rizza V. Formation of propionate after short-term ethanol treatment and its interaction with the carnitine pool in rat. Alcohol 1999; 19: 169-176.
34. Desviat LR, Perez B, Perez-Cerda C, Rodriguez-Pombo P, Clavero S, Ugarte M. Propionic acidemia: mutation update and functional and structural effects of the variant alleles. Mol Genet Metab 2004; 83: 28-37.
35. MacFabe DF. Enteric short-chain fatty acids: microbial messengers of metabolism, mitochondria, and mind: implications in autism spectrum disorders. Microb Ecol Health Dis 2015; 26: 28177.
36. Frye RE, Rose S, Slattery J, MacFabe DF. Gastrointestinal dysfunction in autism spectrum disorder: the role of the mitochondria and the enteric microbiome. Microb Ecol Health Dis 2015; 26: 27458.
37. Frye RE, Slattery J, MacFabe DF, Allen-Vercoe E, Parker W, Rodakis J et al. Approaches to studying and manipulating the enteric microbiome to improve autism symptoms. Microb Ecol Health Dis 2015; 26: 26878.
38. Developmental Disabilities Monitoring Network Surveillance Year Principal ICenters for Disease C, Prevention. Prevalence of autism spectrum disorder among children aged 8 years - autism and developmental disabilities monitoring network, 11 sites, United States, 2010. MMWR Surveill Summ 2014; 63: 1-21.
39. Hallmayer J, Cleveland S, Torres A, Phillips J, Cohen B, Torigoe T et al. Genetic heritability and shared environmental factors among twin pairs with autism. Arch Gen Psychiatry 2011; 68: 1095-1102.
40. Schaefer GB, Mendelsohn NJ, Professional P, Guidelines C. Clinical genetics evaluation in identifying the etiology of autism spectrum disorders: 2013 guideline revisions. Genet Med 2013; 15: 399-407.
41. Rossignol DA, Genuis SJ, Frye RE. Environmental toxicants and autism spectrum disorders: a systematic review. Transl Psychiatry 2014; 4: e360.
42. Hsiao EY, McBride SW, Hsien S, Sharon G, Hyde ER, McCue T et al. Microbiota modulate behavioral and physiological abnormalities associated with neurode-velopmental disorders. Cell 2013; 155: 1451-1463.
43. Macfabe D. Autism: metabolism, mitochondria, and the microbiome. Glob Adv Health Med 2013; 2: 52-66.
44. Macfabe DF. Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders. Microb Ecol Health Dis 2012; 23: (doi:10.3402/mehd.v23i0.19260).
45. De Angelis M, Piccolo M, Vannini L, Siragusa S, De Giacomo A, Serrazzanetti DI et al. Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified. PLoS One 2013; 8: e76993.
46. Kesli R, Gokcen C, Bulug U, Terzi Y. Investigation of the relation between anaerobic bacteria genus clostridium and late-onset autism etiology in children. J Immunoassay Immun 2014; 35: 101-109.
47. Finegold SM. Therapy and epidemiology of autism-clostridial spores as key elements. Med Hypotheses 2008; 70: 508-511.
48. Parracho HM, Bingham MO, Gibson GR, McCartney AL. Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. J Med Microbiol 2005; 54(Pt 10): 987-991.
49. Song Y, Liu C, Finegold SM, Real-time PCR. quantitation of clostridia in feces of autistic children. Appl Environ Microbiol 2004; 70: 6459-6465.
50. Finegold SM, Downes J, Summanen PH. Microbiology of regressive autism. Anaerobe 2012; 18: 260-262.
51. Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E et al. Gastrointestinal microflora studies in late-onset autism. Clin Infect Dis 2002; 35(SUPPL. 1): S6-s16.
52. Williams BL, Hornig M, Buie T, Bauman ML, Cho Paik M, Wick I et al. Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.. PLoS One 2011; 6: e24585.
53. Shultz SR, MacFabe DF, Ossenkopp KP, Scratch S, Whelan J, Taylor R et al. Intracerebroventricular injection of propionic acid, an enteric bacterial metabolic end-product, impairs social behavior in the rat: implications for an animal model of autism.. Neuropharmacology 2008; 54: 901-911.
54. Thomas RH, Meeking MM, Mepham JR, Tichenoff L, Possmayer F, Liu S et al. The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders.. J Neuroinflammation 2012; 9: 153.
55. MacFabe DF, Cain DP, Rodriguez-Capote K, Franklin AE, Hoffman JE, Boon F et al. Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders.. Behav Brain Res 2007; 176: 149-169.
56. Shultz SR, Macfabe DF, Martin S, Jackson J, Taylor R, Boon F et al. Intracer-ebroventricular injections of the enteric bacterial metabolic product propionic acid impair cognition and sensorimotor ability in the Long-Evans rat: further development of a rodent model of autism.. Behav Brain Res 2009; 200: 33-41.
57. MacFabe DF, Cain NE, Boon F, Ossenkopp KP, Cain DP. Effects of the enteric bacterial metabolic product propionic acid on object-directed behavior, social behavior, cognition, and neuroinflammation in adolescent rats: relevance to autism spectrum disorder.. Behav Brain Res 2011; 217: 47-54.
58. Thomas RH, Foley KA, Mepham JR, Tichenoff LJ, Possmayer F, MacFabe DF. Altered brain phospholipid and acylcarnitine profiles in propionic acid infused rodents: further development of a potential model of autism spectrum disorders.. J Neurochem 2010; 113: 515-529.
59. Foley KA, MacFabe DF, Kavaliers M, Ossenkopp KP. Sexually dimorphic effects of prenatal exposure to lipopolysaccharide, and prenatal and postnatal exposure to propionic acid, on acoustic startle response and prepulse inhibition in adolescent rats: relevance to autism spectrum disorders.. Behav Brain Res 2014; 278: 244-256.
60. Foley KA, Ossenkopp KP, Kavaliers M, Macfabe DF. Pre- and neonatal exposure to lipopolysaccharide or the enteric metabolite, propionic acid, alters development and behavior in adolescent rats in a sexually dimorphic manner.. PLoS One 2014; 9: e87072.
61. Foley KA, MacFabe DF, Vaz A, Ossenkopp KP, Kavaliers M. Sexually dimorphic effects of prenatal exposure to propionic acid and lipopolysaccharide on social behavior in neonatal, adolescent, and adult rats: implications for autism spectrum disorders.. Int J Dev Neurosci 2014; 39: 68-78.
62. Celestino-Soper PB, Violante S, Crawford EL, Luo R, Lionel AC, Delaby E et al. A common X-linked inborn error of carnitine biosynthesis may be a risk factor for nondysmorphic autism. Proc Natl Acad Sci USA 2012; 109: 7974-7981.
63. Frye RE, Rossignol DA. Treatments for biomedical abnormalities associated with autism spectrum disorder. Front Pediatr 2014; 2: 66.
64. Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE et al. Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Transl Psychiatry 2012; 2: e134.
65. Frye RE, Melnyk S, Macfabe DF. Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder. Transl Psychiatry 2013; 3: e220.
66. Francis K, Smitherman C, Nishino SF, Spain JC, Gadda G. The biochemistry of the metabolic poison propionate 3-nitronate and its conjugate acid, 3-nitropropionate. IUBMB Life 2013; 65: 759-768.
67. Rose S, Frye RE, Slattery J, Wynne R, Tippett M, Melnyk S et al. Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines. Transl Psychiatry 2014; 4: e377.
68. Rose S, Frye RE, Slattery J, Wynne R, Tippett M, Pavliv O et al. Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort. PLoS One 2014; 9: e85436.
69. Oh JH, Kim YJ, Moon S, Nam HY, Jeon JP, Lee JH et al. Genotype instability during long-term subculture of lymphoblastoid cell lines. J Hum Genet 2013; 58: 16-20.
70. Nickles D, Madireddy L, Yang S, Khankhanian P, Lincoln S, Hauser SL et al. In depth comparison of an individual's DNA and its lymphoblastoid cell line using whole genome sequencing. BMC Genomics 2012; 13: 477.
71. Karuri AR, Dobrowsky E, Tannock IF. Selective cellular acidification and toxicity of weak organic acids in an acidic microenvironment. Br J Cancer 1993; 68: 1080-1087.
72. Laird NM, Ware JH. Random-effects models for longitudinal data. Biometrics 1982; 38: 963-974.
73. Muir AD, Majak W. Quantitative determination of 3-nitropropionic acid and 3-nitropropanol in plasma by HPLC. Toxicol Lett 1984; 20: 133-136.
74. Cummings JH, Pomare EW, Branch WJ, Naylor CP, Macfarlane GT. Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 1987; 28: 1221-1227.
75. Gallego-Villar L, Perez-Cerda C, Perez B, Abia D, Ugarte M, Richard E et al. Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia. J Inherit Metab Dis 2013; 36: 731-740.
76. Halloran M, Parakh S, Atkin JD. The role of s-nitrosylation and s-glutathionylation of protein disulphide isomerase in protein misfolding and neurodegeneration. Int J Cell Biol 2013; 2013: 797914.
77. Wang L, Christophersen CT, Sorich MJ, Gerber JP, Angley MT, Conlon MA. Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder. Digest Dis Sci 2012; 57: 2096-2102.
78. Whiteley P, Haracopos D, Knivsberg AM, Reichelt KL, Parlar S, Jacobsen J et al. The ScanBrit randomised, controlled, single-blind study of a gluten- and casein-free dietary intervention for children with autism spectrum disorders.. Nutr Neurosci 2010; 13: 87-100.
79. Desler C, Hansen TL, Frederiksen JB, Marcker ML, Singh KK, Juel Rasmussen L. Is there a link between mitochondrial reserve respiratory capacity and aging? J Aging Res 2012; 2012: 192503.
80. Sansbury BE, Jones SP, Riggs DW, Darley-Usmar VM, Hill BG. Bioenergetic function in cardiovascular cells: the importance of the reserve capacity and its biological regulation. Chemico-biol Interact 2011; 191: 288-295.
81. Nicholls DG. Spare respiratory capacity, oxidative stress and excitotoxicity. Biochem Soc Trans 2009; 37(Pt 6): 1385-1388.
82. Yadava N, Nicholls DG. Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mito-chondrial complex I with rotenone.. J Neurosci 2007; 27: 7310-7317.
83. Hill BG, Dranka BP, Zou L, Chatham JC, Darley-Usmar VM. Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal. Biochem J 2010; 424: 99-107.
84. Melnyk S, Fuchs GJ, Schulz E, Lopez M, Kahler SG, Fussell JJ et al. Metabolic imbalance associated with methylation dysregulation and oxidative damage in children with autism. J Autism Dev Disord 2012; 42: 367-377.
85. Sajdel-Sulkowska EM, Xu M, McGinnis W, Koibuchi N. Brain region-specific changes in oxidative stress and neurotrophin levels in autism spectrum disorders (ASD). Cerebellum 2011; 10: 43-48.
86. Rossignol DA, Frye RE. Evidence linking oxidative stress, mitochondrial dysfunction, and inflammation in the brain of individuals with autism. Front Physiol 2014; 5: 150.
87. Frye RE. Central tetrahydrobiopterin concentration in neurodevelopmental disorders. Front Neurosci 2010; 4: 52.
88. Frye RE, DeLatorre R, Taylor HB, Slattery J, Melnyk S, Chowdhury N et al. Metabolic effects of sapropterin treatment in autism spectrum disorder: a preliminary study. Transl Psychiatry 2013; 3: e237.
89. Frye RE, Huffman LC, Elliott GR. Tetrahydrobiopterin as a novel therapeutic intervention for autism. Neurotherapeutics 2010; 7: 241-249.
90. Haska L, Andersson R, Nyman M. The effect of dietary fiber from wheat processing streams on the formation of carboxylic acids and microbiota in the hindgut of rats. J Agric Food Chem 2011; 59: 3406-3413.
91. Nakao S, Moriya Y, Furuyama S, Niederman R, Sugiya H. Propionic acid stimulates superoxide generation in human neutrophils. Cell Biol Int 1998; 22: 331-337.
92. Rorig B, Klausa G, Sutor B. Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones. J Physiol 1996; 490, Pt 1, 31-49.
93. Nguyen NH, Morland C, Gonzalez SV, Rise F, Storm-Mathisen J, Gundersen V et al. Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia. J Neurochem 2007; 101: 806-814.
94. Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME et al. Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. J Biol Chem 2003; 278: 25481-25489.
95. DeCastro M, Nankova BB, Shah P, Patel P, Mally PV, Mishra R et al. Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway. Brain Res 2005; 142: 28-38.
96. Hara H, Haga S, Aoyama Y, Kiriyama S. Short-chain fatty acids suppress cholesterol synthesis in rat liver and intestine. J Nutr 1999; 129: 942-948.
97. Sandler RH, Finegold SM, Bolte ER, Buchanan CP, Maxwell AP, Vaisanen ML et al. Short-term benefit from oral vancomycin treatment of regressive-onset autism. J Child Neurol 2000; 15: 429-435.
98. Kalghatgi S, Spina CS, Costello JC, Liesa M, Morones-Ramirez JR, Slomovic S et al. Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells. Sci Transl Med 2013; 5: 192ra185.
99. Tonazzi A, Giangregorio N, Console L, Indiveri C. Mitochondrial carnitine/acyl-carnitine translocase: insights in structure/function relationships. Basis for drug therapy and side effects prediction. Mini Rev Med Chem 2015; 15: 396-405.
100. Frohlich EE, Mayerhofer R, Holzer P. Reevaluating the hype: four bacterial metabolites under scrutiny. Eur J Microbiol Immunol 2015; 5: 1-13.
101. Toker L, Agam G. Mitochondrial dysfunction in psychiatric morbidity: current evidence and therapeutic prospects. Neuropsychiatr Dis Treat 2015; 11: 2441-2447.
102. Frye RE, Macfabe D. The gut and mitochondrial function. Microbiome Health Dis 2014.