
B.Sc., Ph.D.
Emeritus Professor
- About
-
- Office Address
Rowett Institute, University of Aberdeen, Ashgrove Road West, Aberdeen AB25 2ZD
- School/Department
- School of Medicine, Medical Sciences and Nutrition
Biography
Prof John Wallace obtained a 1st class Honours B.Sc. in Biochemistry from the University of Glasgow in 1972. In 1973 and 1974, he worked at Distillers Company Ltd research labs, Menstrie. Shortly after completing his Ph.D. at the University of Glasgow in 1975 ‘Maintenance energy and molar growth yields of Escherichia coli’, he joined the Microbiology Department of the Rowett Research institute, at that time headed by P.N. Hobson, with Sir Kenneth Blaxter as Institute Director. He has remained at the Rowett since 1976, developing interests in rumen and intestinal microbiology and ruminant nutrition. Following the merger of the Rowett with the University of Aberdeen in 2008, he was appointed to a Personal Chair at the University, and upon his retirement in 2016 was awarded emeritus status. At various times during his career, he has undertaken research sojourns in Australia, Japan and Ethiopia, and has served on the Editorial Boards of Microbiology, British Journal of Nutrition (Deputy Editor-in-Chief), FEMS Microbiology Letters, Animal Feed Science and Technology and Applied and Environmental Microbiology. He has served on the research committees of BBSRC, the FEEDAP (feed additives) panel of the European Food Safety Authority and curr ently chairs the Feed and Feed Additives group at the UK's Food Standards Agency. Career highlights include being invited to speak at the Pasteur Institute, Papeete, Tahiti, in ‘The Year of Louis Pasteur’ Symposium’, Microbes, Environment , Biotechnology, 8-12 May 1995, and being recognised via the DSM Nutrition Innovation Award in 2007 'in recognition of his pioneering research in animal nutrition.'
External Memberships
Member of the FEEDAP panel of the European Food Safety Authority, 2012-2018, and external adviser (2018 - present).
Member of Editorial Boards, Applied and Environmental Microbiology, Animal Feed Science and Technology.
Member of the International Science Foundation assessment committee.
Member of the Stakeholder Advisory Group, 'HoloRuminant', Horizon 2020
Project advisor, 'Influence of tannins on fatty acid biohydrogenation' Faculdade de Medicina Veterinária (FMV/UTL), Lisboa;Instituto de Ciências e Tecnologias Agrárias e Agro-Alimentares - Porto (ICETA-Porto/UP)
Project reviewer, Australian National Livestock Methane Program, Livestock Production Innovation, Meat & Livestock Australia.
Assessor and rapporteur, COST Association, European Commission.
Latest Publications
The rumen microbiome: balancing food security and environmental impacts
Nature reviews. Microbiology, vol. 19, pp. 553–566Contributions to Journals: Review articles- [ONLINE] DOI: https://doi.org/10.1038/s41579-021-00543-6
- [ONLINE] Springer SharedIt link
Postmortem observations on rumen wall histology and gene expression and ruminal and caecal content of beef cattle fattened on barley-based rations
Animal , vol. 14, no. 7, pp. 1447-1460Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1017/S1751731119002878
- [ONLINE] View publication in Scopus
Effect of dietary fish oil supplements alone or in combination with sunflower and linseed oil on ruminal lipid metabolism and bacterial populations in lactating cows
Journal of Dairy Science, vol. 101, no. 4, pp. 3021-3035Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.3168/jds.2017-13776
- [ONLINE] View publication in Scopus
Adipose specific disruption of seipin causes early-onset generalised lipodystrophy and altered fuel utilisation without severe metabolic disease
Molecular Metabolism, vol. 10, pp. 55-65Contributions to Journals: ArticlesCultivation and sequencing of rumen microbiome members from the Hungate1000 Collection
Nature Biotechnology, vol. 36, no. 4, pp. 359-367Contributions to Journals: Review articles
Prizes and Awards
DSM Nutrition Innovation Award in 2007 'in recognition of his pioneering research in animal nutrition.'
1998, 2003, 2005, 2007 - BBSRC Exceptional Performance Award
2007, 2008 - City of Aberdeen Ambassador awards
2008 - BBSRC Outstanding Performance Award
Plos Genetics Research Prize 2017.
- Research
-
Research Overview
Research interests encompass microbial physiology, biochemistry and ecology that govern gut function, particularly in ruminants. Aspects of gut function of particular interest include: protein metabolism – which leads to inefficient protein retention by ruminants and the formation of toxic products in the human intestine; fatty acid biohydrogenation – which causes ruminant products to be low in most health-promoting PUFA; methane formation – a significant contributor to climate change; and ruminal acidosis – a major welfare issue for ruminants. Coupled with these issues are means by which manipulation of ruminal fermentation can be achieved, such as probiotics, phytochemicals and antimicrobials.
Current Research
Methane is a greenhouse gas (GHG), 28 times as potent as carbon dioxide. Ruminants are major methane emitters, contributing 3-4% of global GHG emissions. The methane is derived from microbial fermentation in the rumen, being produced by microbes known as archaea. In collaboration with Bob Mayes of the James Hutton Institute and Rainer Roehe and John Rooke of the Scotland’s Rural College (SRUC), Edinburgh, we have established that the abundance of the archaea in ruminal digesta can be used to predict methane emissions of beef cattle, even using post-mortem samples. Furthermore, the Framework 7 project which I coordinated, ‘RuminOmics’ involving 12 European and Israeli partners, made the groundbreaking discovery that methane emissions in cattle vary according to the microbiome that is present in the rumen and that the microbiome itself is regulated by the host animal's genetics. This enables the intriguing capability that cattle may be bred according to their rumen microbiome, leading to progeny that persistently produce lower methane emissions.
Hydrogen is also utilized by fatty acid biohydrogenation in the rumen, which leads to a high proportion of health-threatening saturated fatty acids in foods derived from ruminants and to the formation, followed by the destruction, of health-promoting conjugated linoleic acids (CLA), and to the destruction of n-3, health-promoting fatty acids. The primary aim of this part of our research has been to improve the fatty acid composition of ruminant milk and meat for human health. In order to achieve this aim, the microorganisms responsible for fatty acid transformations in the rumen were identified, the fluxes through pathways of biohydrogenation and desaturation measured, the population sizes of the most significant microbial species evaluated, and ways of altering these fluxes and populations were investigated. Currently, the research is being continued via the National Buffalo Research Institute, Nanning, China.
Sub-acute ruminal acidosis (SARA) is a disorder that can afflict all ruminant species, but one that is particularly prevalent in intensively produced dairy and beef cattle. It is thought to result from dysfunction of ruminal microbial ecology. SARA might be regarded as mainly an unseen, and most certainly under-researched, disorder, because its symptoms are those of ill-thrift rather than illness. The pathology is only found clearly upon post mortem inspection. However, next to the ill-thrift, SARA is associated with reduced fibre degradation, reduced feed intake, laminitis and diarrhoea and thereby also increased involuntary culling. Its economic impact is therefore hard to determine exactly. The loss to the UK economy could easily exceed £200M p.a. The welfare justification for investigating SARA is also compelling, as SARA-related illnesses are painful and detrimental to health, particularly laminitis.
The Scottish SARA Consortium, funded by BBSRC, to investigated:
- If on-farm management practices can be identified that predispose beef and dairy cattle to SARA.
- If remote motion sensing devices mounted around the animal’s neck can give early warning of SARA.
- What is the precise pathology of black patches on the rumen wall?
- If soluble LPS in SARA-susceptible cattle differs from soluble LPS in SARA-resistant cattle.
- The relative importance of the rumen and the hindgut in SARA.
- The precise pathotypes of E. coli present in SARA-susceptible cattle.
- If probiotic bacteria can be isolated from individual SARA-resistant cattle.
Past Research
Protein nutrition of ruminants is compromised by the intervention of ruminal microorganisms, which break down dietary protein, via peptides and amino acids, to ammonia. Mechanisms and microorganisms at each stage of the process were identified, with the highlight being the discovery of a two-step breakdown mechanism for peptides. Major efforts to control protein and other rumen functions using plants and p[lant extracts resulted in the discovery of >20 useful plants which, if incorporated into the ruminant diet, would modify rumen microbial activity to the benefit of animal nutrition and health. Essential oils proved particularly beneficial, and the microbiological explanation of their effects led to commercial exploitation.
Human gut microbiology is much simpler than that of ruminants, but shares some similarities. The unexpected biphasic breakdown of peptides, for example, occurs in the human intestine as well, with implications for human health.
Collaborations
Kevin Shingfield, Aberystwyth University (fatty acid metabolism, methane); Veerle Fievez, University of Gent (fatty acid metabolism); Rui Bessa, University of Lisbon (fatty acid metabolism); Margarida Maia, University of Porto (fatty acid metabolism); Mick Watson, University of Edinburgh (metagenomics); Nick Jonsson, University of Glasgow (SARA); Ivan Andonovic, Strathclyde University (SARA); Pekka Huhtanen, Sveriges lantbruksuniversitet, Umeå (methane); Phil Garnsworthy, Nottingham University (methane); Paolo Bani, Catholic University, Piacenza (methane); Sinead Waters, Teagasc, Dublin (methane); Tim McAllister, Lethbridge, Canada; Pierre Taberlet, Grenoble University, France; Chris McSweeney, CSIRO, Brisbane.
Supervision
I supervised 23 Ph.D. and 7 Masters students, mainly from overseas, and >50 international postdoctoral visiting scientists.
Funding and Grants
2011 TSB Genomics Competition, with Ingenza Ltd. £513,000
2011 EC FP7, FOOD-SEG partner, €23,219 euros (of total 999,915 euros)
2012 Studentship, Commonwealth Scholarship Commission
2012 Framework 7 project RuminOmics, coordinator, €7.7M, 2012-2015
2012 BBSRC Industrial Partnership Award: Sub-acute ruminal acidosis (SARA). Consortium leader. £1.2M, 2012-2015
2012 Technology Strategy Board SPARK, methane £5k
2012 EBLEX (with SRUC) SafeBeef. £280k
2013 BBSRC Studentship with SRUC, methane
- Teaching
-
Teaching Responsibilities
No undergraduate teaching responsibilities
- Publications
-
Page 2 of 6 Results 51 to 100 of 257
Metabolism of conjugated linoleic acids and 18:1 fatty acids by ruminal bacteria: products and mechanisms
Microbiology , vol. 156, no. 2, pp. 579-588Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1099/mic.0.036442-0
Screening for anti-proteolytic compounds
In vitro screening of plant resources for extra-nutritional attributes in ruminants: nuclear and related methodologies. Vercoe, P. E., Makkar, H. P. S., Schlink, A. C. (eds.). Springer: International Atomic Energy Agency, pp. 55-86, 32 pagesChapters in Books, Reports and Conference Proceedings: Chapters- [ONLINE] DOI: https://doi.org/10.1007/978-90-481-3297-3_5
Gastrointestinal tract: fat metabolism in the colon
Handbook of Hydrocarbon and Lipid Microbiology:. Timmis, K. N. (ed.). Springer Verlag, pp. 3111-3118, 8 pagesChapters in Books, Reports and Conference Proceedings: Chapters- [ONLINE] DOI: https://doi.org/10.1007/978-3-540-77587-4_233
Gastrointestinal tract: intestinal fatty acid metabolism and implications for health
Handbook of Hydrocarbon and Lipid Microbiology. Timmis, K. N. (ed.). Springer Verlag, pp. 3119-3132, 14 pagesChapters in Books, Reports and Conference Proceedings: Chapters- [ONLINE] DOI: https://doi.org/10.1007/978-3-540-77587-4_234
Effect of high-protein diets on faecal N-nitrosocompounds and genotoxicity of faecal water
Mutagenesis, vol. 24, no. 6, pp. 528Contributions to Journals: Abstracts- [ONLINE] DOI: https://doi.org/10.1093/mutage/gep046
Effect of protein and carbohydrate intake on intestinal toxicity of human feces
Annals of Nutrition & Metabolism, vol. 55, no. Suppl. 1, pp. 80Contributions to Journals: Abstracts- [ONLINE] DOI: https://doi.org/10.1159/000248277
Rumen protozoa are rich in polyunsaturated fatty acids due to the ingestion of chloroplasts
FEMS Microbiology Ecology, vol. 69, no. 3, pp. 461-471Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1574-6941.2009.00717.x
- [ONLINE] View publication in Scopus
Encapsulated fumaric acid as a feed ingredient to decrease ruminal methane emissions
Animal Feed Science and Technology, vol. 152, no. 1-2, pp. 62-71Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.anifeedsci.2009.03.006
Physiological changes in rumen fermentation during acidosis induction and its control using a multivalent polyclonal antibody preparation in heifers
Journal of Animal Science, vol. 87, no. 5, pp. 1722-1730Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.2527/jas.2008-1184
High protein diets impact on microbial metabolites and toxicity in the human large intestine
Microbial Ecology, vol. 57, no. 3, pp. 572-573Contributions to Journals: Abstracts- [ONLINE] DOI: https://doi.org/10.1007/s00248-009-9496-x
Differences between human subjects in the composition of the faecal bacterial community and faecal metabolism of linoleic acid
Microbiology , vol. 155, no. 2, pp. 513-520Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1099/mic.0.023416-0
Mechanism of conjugated linoleic acid and vaccenic acid formation in human faecal suspensions and pure cultures of intestinal bacteria
Microbiology , vol. 155, no. 1, pp. 285-294Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1099/mic.0.022921-0
Australian plants with potential to inhibit bacteria and processes involved in ruminal biohydrogenation of fatty acids
Animal Feed Science and Technology, vol. 145, no. 1-4, pp. 271-284Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.anifeedsci.2007.05.052
Enzymes, direct-fed microbials and plant extracts in ruminant nutrition
Animal Feed Science and Technology, vol. 145, no. 1-4, pp. 1-4Contributions to Journals: Editorials- [ONLINE] DOI: https://doi.org/10.1016/j.anifeedsci.2007.07.006
In vitro screening of the potential of numerous plant species as antimethanogenic feed additives for ruminants
Animal Feed Science and Technology, vol. 145, no. 1-4, pp. 245-258Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.anifeedsci.2007.04.015
Adaptation to flavomycin in the ruminal bacterium, Prevotella bryantii
Journal of Applied Microbiology, vol. 104, no. 6, pp. 1617-1623Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1365-2672.2007.03689.x
Fish oil increases the duodenal flow of long chain polyunsaturated fatty acids and trans-11 18:1 and decreases 18:0 in steers via changes in the rumen bacterial community
The Journal of Nutrition, vol. 138, no. 5, pp. 889-896Contributions to Journals: ArticlesGut microbiology: broad genetic diversity, yet specific metabolic niches
Animal , vol. 2, no. 5, pp. 661-668Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1017/S1751731108001687
- [ONLINE] View publication in Scopus
Plant extracts as antimicrobials in ruminants
Recent Advances in Animal Nutrition - 2007. Garnsworthy, P. C., Wiseman, J. (eds.). Nottingham University Press, pp. 139-153, 15 pagesChapters in Books, Reports and Conference Proceedings: ChaptersBoard-invited review: recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem
Journal of Animal Science, vol. 86, no. 2, pp. 397-412Contributions to Journals: Literature Reviews- [ONLINE] DOI: https://doi.org/10.2527/jas.2007-0588
Effect of a blend of essential oil compounds on the colonization of starch-rich substrates by bacteria in the rumen
Journal of Applied Microbiology, vol. 103, no. 6, pp. 2132-2141Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1365-2672.2007.03455.x
A meta-analysis of fumarate effects on methane production in ruminal batch cultures
Journal of Animal Science, vol. 85, no. 10, pp. 2556-2563Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.2527/jas.2006-674
Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communis
Journal of Applied Microbiology, vol. 103, no. 4, pp. 924-929Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1365-2672.2007.03327.x
Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria
Journal of Lipid Research, vol. 48, no. 10, pp. 2247-2254Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1194/jlr.M700271-JLR200
Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach
Journal of Applied Microbiology, vol. 103, no. 4, pp. 1251-1261Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1365-2672.2007.03349.x
Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation
British Journal of Nutrition, vol. 98, no. 1, pp. 45-53Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1017/S0007114507472506
Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen
ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, vol. 91, no. 4, pp. 303-314Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1007/s10482-006-9118-2
Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen
Antonie van Leeuwenhoek , vol. 91, no. 4, pp. 417-422Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1007/s10482-006-9121-7
Plants and plant extracts for manipulating ruminal fermentation
South African Journal of Botany, vol. 73, no. 2, pp. 275Contributions to Journals: Abstracts- [ONLINE] DOI: https://doi.org/10.1016/j.sajb.2007.02.007
Metabolism of linoleic acid by human gut bacteria: different routes for biosynthesis of conjugated linoleic acid
Journal of Bacteriology, vol. 189, no. 6, pp. 2566-2570Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1128/JB.01359-06
Protein metabolism in the herbivore gut: old and new perspectives
Herbivore Nutrition for the Development of Efficient, Safe and Sustainable Livestock Production: Proceedings of the 7th International Symposium on the Nutrition of Herbivores. Meng, Q. X., Ren, L. P., Cao, Z. J. (eds.). China Agricultural University Press, pp. 159-191, 33 pagesChapters in Books, Reports and Conference Proceedings: ChaptersClostridium proteoclasticum: a ruminal bacterium that forms stearic acid from linoleic acid
FEMS Microbiology Letters, vol. 265, no. 2, pp. 195-201Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1574-6968.2006.00487.x
Rumen ciliate protozoa contain high concentrations of conjugated linoleic acids and vaccenic acid, yet do not hydrogenate linoleic acid or desaturate stearic acid
British Journal of Nutrition, vol. 96, no. 4, pp. 697-704Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/BJN20061884
Increased expression of a molecular chaperone GroEL in response to unsaturated fatty acids by the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens
FEMS Microbiology Letters, vol. 262, no. 2, pp. 244-248Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1111/j.1574-6968.2006.00399.x
Encapsulated fumaric acid as a means of decreasing ruminal methane emissions
International Congress Series, vol. 1293, pp. 148-151Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.ics.2006.02.018
Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids
British Journal of Nutrition, vol. 95, no. 6, pp. 1199-1211Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/BJN20061783
Differing objectives and key target microbes for manipulation of ruminal fermentation
Journal of Animal Science, vol. 84, no. Supplement 1, pp. 424Contributions to Journals: AbstractsThe influence of selenium on the level of linoleic acid isomers in incubated ruminal fluid
Journal of Animal and Feed Sciences, vol. 15, no. Supplement 1, pp. 97-100Contributions to Journals: ArticlesInfluence of flavomycin on microbial numbers, microbial metabolism and gut tissue protein turnover in the digestive tract of sheep
British Journal of Nutrition, vol. 94, no. 1, pp. 64-70Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/BJN20051444
Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro
British Journal of Nutrition, vol. 94, no. 1, pp. 27-35Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/BJN20051445
An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum
FEMS Microbiology Letters, vol. 247, no. 2, pp. 113-121Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.femsle.2005.04.034
Assessment of amino acid requirements for optimum fermentation of xylan by mixed micro-organisms from the sheep rumen
Animal Science, vol. 80, no. 3, pp. 353-360Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/ASC41730353
Influence of flavomycin on ruminal fermentation and microbial populations in sheep
Microbiology , vol. 151, no. 3, pp. 717-725Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1099/mic.0.27602-0
A pepD-like peptidase from the ruminal bacterium, Prevotella albensis
FEMS Microbiology Letters, vol. 243, no. 2, pp. 399-404Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1016/j.femsle.2004.12.032
Nitrogen metabolism in the rumen
Nitrogen and Phosphorus Nutrition of Cattle. Pfeffer, E., Hristov, A. (eds.). CAB International, pp. 71-115, 45 pagesChapters in Books, Reports and Conference Proceedings: ChaptersAntimicrobial properties of plant secondary metabolites
Proceedings of the Nutrition Society, vol. 63, no. 4, pp. 621-629Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1079/PNS2004393
16S rDNA library-based analysis of ruminal bacterial diversity
Antonie van Leeuwenhoek , vol. 86, no. 3, pp. 263-281Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1023/B:ANTO.0000047942.69033.24
Metabolic properties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing' anaerobe with metabolic properties analogous to those of Clostridium kluyveri
Microbiology , vol. 150, no. 9, pp. 2921-2930Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1099/mic.0.27190-0
Purification and properties of glutamate-phenylpyruvate aminotransferase from the ruminal protozoan Entodinium caudatum
Australian Journal of Agricultural Research, vol. 55, no. 9, pp. 991-997Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1071/AR04050
Influence of dipeptidyl peptidase inhibitors on growth, peptidase activity, and ammonia production by ruminal microorganisms
Current Microbiology, vol. 49, no. 2, pp. 115-122Contributions to Journals: Articles- [ONLINE] DOI: https://doi.org/10.1007/s00284-004-4295-6