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BiotecVisions 2012, September

2012; Wiley; Volume: 7; Issue: 9 Linguagem: Inglês

10.1002/biot.201200056

1860-7314

Anja Gaugel,

Gene expression and cancer classification

Biotechnology JournalVolume 7, Issue 9 p. A1-A8 BiotecVisionsFree Access BiotecVisions 2012, September First published: 05 September 2012 https://doi.org/10.1002/biot.201200056Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Editors: Judy Peng /jp; Uta Göbel /ug; Lucie Kalvodova /lk E-mail: biotecvisions@wiley.com www.biotecvisions.com Contributors: Anja Gaugel /ag; Bill Mullen /bm; Danny Asling /da; Fran Harding /fh; Gillian van Beest /gvb; Joanna Cipolla /jc; Kerstin Brachhold /kb; Lotte Nielsen /ln; Meghana Hemphill /mh; Melissa McCulloch /mmc; Mia Ricci /mr; Peter W. Bass /pwb; Sarah Andrus /sa; Sarah Brett /sb; Susan Vice /sv; Tiffany McKerahan /tmk; Vera Köster /vk September 2012 Journal spotlight Microbial Biotechnology is going Open Access! In January, 2013, Microbial Biotechnology will become part of the Wiley Open Access program, bringing you research articles that are immediately free to read, download and share. Microbial Biotechnology is a peer-reviewed journal edited by Kenneth N. Timmis, Juan Luis Ramos, Willem de Vos, Willy Verstraete, and Martin Rosenberg. With an impact factor of 2.534, Microbial Biotechnology publishes papers of original research reporting significant advances in any aspect of microbial applications to biotechnologies. /mmc Published jointly with the Society for Applied Microbiology, Microbial Biotechnology covers areas of research including: • Biotechnologies related to chemicals • Pharmaceuticals • Energy • Mining • Agriculture • Food • Environmental protection Microbial Biotechnology is indexed in PubMed, Scopus and Thomson Reuters (ISI). /mmc www.microbialbiotech.com Special issue: Microbial Resource Management (May 2012), Guest Editors: Nico Boon and Willy Verstraete http://goo.gl/qTpMW Top 5 most downloaded papers for 2011 • Crystal ball – 2011 http://goo.gl/Q9nd3 • Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters http://goo.gl/OthKV • Marine genomics: At the interface of marine microbial ecology and biodiscovery http://goo.gl/Kzzow • Cross-talk of global nutritional regulators in the control of primary and secondary metabolism in Streptomyces http://goo.gl/KMNza • Genome-wide gene expression changes in an industrial clavulanic acid overproduction strain of Streptomyces clavuligerus http://goo.gl/jlgNe Getting published B&B – An interview with Editor-in-Chief Douglas S. Clark Q: Please describe your role in Biotechnology & Bioengineering A: As Editor-in-Chief, I oversee handling of all manuscripts submitted to the journal, which involves deciding which papers are sent for review (and which are rejected without review), and assigning papers to Associate Editors or processing them for review myself. Ultimately I am responsible for the journal's overall scope and content. Q: What are the publication criteria of your journal? A: Novelty and impact within one or more of the many subject areas encompassed by the broad scope of the journal. As stated on our website, we “will consider papers for publication based on novelty, their immediate or future impact on biotechnological processes, and their contribution to the advancement of biochemical engineering science.” Q: There are several “Dos” and “Don'ts” when it comes to publishing, what would you say to the authors as “Dos”? A: Know the style and standards of the journal you're writing for, and prepare the manuscript accordingly. Know the background and nature of the audience you're writing for – is the paper likely to be read mainly by specialists or is the aim to reach a more general audience? Be sure the abstract or summary clearly conveys the most important results and why they're significant (it may be as far as the reader gets). Carefully consider how best to present the results and data, weighing how much should be described in the text versus shown in figures, tables, and graphs. If several people are working together to write the paper, make sure someone serves as chief editor. Q: And “Don'ts”? A: Don't lose sight of your target audience, and don't confuse the issue (and risk confusing readers) by including extraneous information or data that detract from the key results and logical flow of the paper. Don't overstate the significance and importance of your results, and don't present them out of their proper context. Don't neglect to cite the pertinent literature, or rely too heavily on out-dated references. Don't overuse self-citations. Don't include anything – text, figures, tables, captions – that isn't crystal clear. Don't underestimate how hard it is or how long it takes to write a good paper. www.biotechbioeng.com First computational model of a whole organism A team led by Markus Covert (Stanford University, Stanford, CA, USA) has reported a whole-cell computational model of Mycoplasma genitalium including all of its 525 annotated genes. The model incorporates data from more than 900 scientific papers including 1900 experimentally observed parameters. This first virtual cell does not only allow to re-examine experimental data but also opens up opportunities to study and predict complex cellular behaviors, which are difficult to investigate in living organisms. The article presents insights into previously unobserved cellular behaviors, such as DNA-binding protein dynamics, and identifies new gene functions. Following the first synthetic genome reported by the J. Craig Venter Institute in 2008, this model will be instrumental in creating minimal cells first in silico and then ex vivo. /ug [1] http://dx.doi.org/10.1016/j.cell.2012.05.044 Also of interest: Computational modeling of mammalian signaling networks [2] http://dx.doi.org/10.1002/wsbm.52 The virus as metabolic engineer [3] http://dx.doi.org/10.1002/biot.201000080 Detecting life on Mars? Nucleic acid extraction methods for the detection of life on Mars, but also in low-biomass environments on Earth, should be robust and produce high quality nucleic acids, with minimal loss during extraction. This article in FEMS Microbiology Ecology aims at the recovery of nucleic acids from relevant minerals on Earth and Mars, the testing of the tolerance of DNA to various physical and chemical conditions that are applied to dissolve mineral matrices or to extract organic compounds from solid matrices, and the optimization of sensitive DNA extraction methods. /ln [4] http://dx.doi.org/10.1111/j.1574-6941.2012.01325.x Red wine myth: Is it really cardioprotective? It has been suggested that although the negative impact of alcohol consumption varies from person to person, on a global level the adverse effect of alcohol on cardiovascular disease outweighs any protective effect by between two- and three-fold. This is inaccurate. There is a proven positive relationship between alcohol consumption and cardiovascular disease that is acknowledged by the World Health Organization. This article in the Journal of the Science of Food and Agriculture examines the suggestions and evidence surrounding the relationship between light to moderate alcohol consumption and benefits to human health. /sb [5] http://dx.doi.org/10.1002/jsfa.5696 Microorganisms to improve our daily meals Several popular fermented foods, such as salami, sourdough bread, and fermented milks, are produced by acidification via lactic acid bacteria, frequently in collaboration with other microorganisms. The microbial species diversity of these foods can be vast, in particular in artisan-type products that are based on spontaneous fermentation. In contract, most industrial production processes are using defined starter cultures, which are narrowing down species diversity and the associated microbial metabolic activities that contribute to food quality. In Engineering in Life Sciences, Frédéric Leroy and colleagues review the microbial species diversity for fermented food production. Making use of the microbial treasure chest of traditional fermented foods may lead to considerable starter culture innovations and thus to further improvements of flavor, color, texture, healthiness, and safety of the end-products. [6] http://dx.doi.org/10.1002/elsc.201100119 Storage impact on free and encapsulated probiotics Viable beneficial probiotic bacteria are currently used to produce functional foods; however, often technological challenges arise, when probiotics are incorporated into foods with very different conditions to their usual environment. In Engineering in Life Sciences, Sousa et al. (Porto and Viseu, Portugal) present their study on the effect of storage conditions on free and encapsulated probiotics. Free and probiotic alginate microcapsules have been stored at four different temperatures (from freezing to non-refrigerated temperatures). Four different strains of different sensitivities to harsh environmental conditions were assayed. This is the first report showing the impact of storage temperature and on the effectiveness of encapsulation in alginate systems. Encapsulation in alginate protects probiotic strains mainly at negative storage temperatures. [7] http://dx.doi.org/10.1002/elsc.201200007 Also of interest: Special issue: Biotechnology of fermented food systems in Engineering in Life Sciences http://onlinelibrary.wiley.com/doi/10.1002/elsc.v12.4/issuetoc Personalized health care “Personalized Health Care” utilizes personalized health planning in conjunction with coordinated delivery systems to provide personalized, predictive, preventive and participatory (P4) care. This strategic approach to care is built on the concepts of systems biology and enables personalized medicine, which should be applied broadly. In Biotechnology Journal, Ralph Snyderman reviews the transformation of medical practice with its current focus on the treatment of disease to an emphasis on enhancing health, preventing disease and personalizing care to meet each individual's specific health needs. He points out that personalized medicine is more than genomic medicine and discusses the available tools. These new concepts are likely to contribute to the reduction of preventable chronic diseases, which are not effectively addressed by our current approach to care. /ug [8] http://dx.doi.org/10.1002/biot.201100297 Quantifying your body The digital transformation of healthcare is contributing to Leroy Hood's vision of P4 – predictive, preventive, personalized, and participatory – medicine. In Biotechnology Journal, Larry Smarr (California Institute for Telecommunications and Information Technology – Calit2, La Jolla, CA, USA) shares his decade-long personal experience in quantifying his own body. Larry employs an excellent metaphor: the car analogy, in which car maintenance has been transformed from the approach in the 60's of only dealing with symptoms once they occur, to the current approach of identifying potential problems before the occurrence of irreversible damage. Larry Smarr has tracked his own blood and stool biomarkers and has put this into perspective with the genetics of his cellular and gut microbial DNA. This Perspective article exemplifies how one can take a proactive approach to his or her own healthcare. /ug [9] http://dx.doi.org/10.1002/biot.201100495 Molecular signatures: From chaos to consensus “Omics” technologies have made it possible to obtain high-resolution molecular snapshots of organisms, tissues and even individual cells. These data can be used for promising applications, such as disease-specific diagnostics and accurate phenotype classification. Many of these “molecular signatures”, however, suffer from limited reproducibility in independent datasets, insufficient sensitivity or specificity to meet clinical needs. In Biotechnology Journal, Nathan D. Price and colleagues review the process of molecular signature discovery on the basis of omics data. They highlight potential pitfalls in the discovery process, as well as strategies for overcoming these issues. Despite the difficulties, the authors provide a basis of how these tools can be used to contribute to personalized medicine. /ug [10] http://dx.doi.org/10.1002/biot.201100305 Biomarker profiling of circulating tumor cells The advent of molecular targeted anticancer drugs warrants the development of research tools for monitoring their pharmacodynamic effects. The standard method of obtaining tumor biopsies is too invasive to allow repeated sampling during treatment. Therefore, Pluim and coworkers have developed and validated a robust, sensitive, and simple flow cytometry (FACS) method for the enumeration and biomarker profiling of phosphorylated extracellular regulated kinases and DNA in circulating tumor cells (CTCs), which is reported in Cytometry A. The new essay has a lower limit of quantitation of only two CTCs per 8 ml of peripheral blood. The method is based on standard research protocols and FACS equipment available to most clinical research labs. The applicability is demonstrated in peripheral blood from patients with advanced metastatic breast, colon, lung, ovarian, and urothelial cancer. The authors believe that their method will be widely applicable to the study of other phospho-epitope expressing biomarkers in CTCs. /mr [11] http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22049/abstract Open source software platform for high-throughput cell analysis A multidisciplinary consortium comprised of researchers from Finland and Germany has announced the development of a new image processing software, BioImage XD, one of three open source microscopy analysis software packages described in the latest issue of Nature Methods. Designed to be applicable as a general 2D microscopy data processing tool, it is also capable of analysing large data sets generated in high-throughput, multidimensional and timelapse studies. The software architecture is modular and developed to be extendable. BioImage XD generally performed favorably in comparison to both proprietary and open source image processing software (such as Fiji/ImageJ and Olympus Fluoview), particularly in terms of operation speed. /fh [12] http://dx.doi.org/ 10.1038/nmeth.2047 μFIC platform expanded to MTT assay Microfluidic image cytometry (?????FIC) is a novel approach for in situ cytotoxicity assessment of the cells cultured and treated within microfluidic channels under a precisely controlled chemical environment. Following their recent publication on morphology-based μFIC (Lab Chip 2010, 10,415–417), Lim and coworkers demonstrate in this article in Cytometry A expanded capabilities of μFIC to the MTT assay. In this MTT-based μFIC, all the treatment, measurement, and analysis of adherent cells are performed in a precisely controlled microenvironment with minimum disturbances. Then, combinatorial analysis of cell death events, such as mitochondrial enzyme activity and cellular morphology changes, are performed. By providing a more efficient and objective way to evaluate the cell death process, this ?????FIC platform may have significant implications as a future high-throughput–high-content screening (HT-HCS) platform for cytotoxicity assays and drug screening. /mr [13] http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22079/abstract Reprogramming organelle size as therapeutic strategy? In Bioessays Wallace Marshall discusses the mechanisms governing organellar size control and hypothesizes that altering organelle size may reprogram cellular function. Organelles act as reaction vessels, and their size influences the rate of the biochemical pathways within them. Their surface area limits the rate of import of substrates and efflux of products, whereas their volume dictates the quantity of intermediates that can build up. Cancer cells show certain alterations of organelle size such as enlarged nuclei, and there are two possible scenarios for this. Firstly, organelle size may be adjusted as a response to pathological changes of a cell's metabolism. Alternatively, an altered signaling pathway may impinge on the organellar size control system in which case alterations in metabolism and cell behavior would be a downstream effect. Both of these scenarios might be amenable to organelle-directed medical approaches; however, as yet, no small molecule (or genetic) modulators of size have been identified. /kb [14] http://dx.doi.org/10.1002/bies.201200043 Proteoglycans control stem cell fate The presence of proteoglycans at prominent locations within the stem cell niche implies that such molecules play an integral role in regulating stem cell behaviour. A recent review published in Biotechnology and Applied Biochemistry examines how proteoglycans function to control stem cell interaction with chemokines, growth factors and signalling molecules. After reviewing signalling pathways involved in the cell instrinsic and cell extrinsic regulation of stem cell fate, the work considers the role proteoglycans play within this framework. Glycosaminoglycan chain composition is highlighted as the crucial determinant of the interaction between proteoglycan molecules and stem cells. The authors hypothesise that modification of proteoglycan domains during development might be a viable mechanism for the cell to interact with various growth factors and morphogens at different stages of development. /fh [15] http://dx.doi.org/10.1002/bab.1002 Genista – a viable mouse model of neutropenia Neutrophils contribute to host defense but are also responsible for tissue damage during inflammation. Mice lacking the growth factor independence 1 (Gfi1) transcriptional repressor have been used as a model of neutropenia, i.e. this means they are presenting a low number of neutrophils. Due to the high mortality rate of these mice, it has been difficult to determine whether their many phenotypic abnormalities are cell-intrinsic or result indirectly from morbidity associated with the lack of Gfi1. In the European Journal of Immunology, Ordoñez-Rueda et al. use N-ethyl-N-nitrosourea-induced mutagenesis to establish a viable mouse model of neutropenia – called Genista – that results from a point mutation in Gfi1. The Genista mutation has a very limited impact on lymphopoiesis and blocks terminal granulopoiesis just after the metamyelocytic stage. A few atypical neutrophils are generated which induce mild forms of autoantibody-induced pathologies but fail to provide resistance to acute bacterial infection and to contribute to NK-cell maturation. These data reveal the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses. /cp [16] http://dx.doi.org/10.1002/eji.201242589 Engineering biomaterials to integrate and heal This review article in Biotechnology and Bioengineering focuses on one of the major failure routes of implanted medical devices, the foreign body reaction (FBR)–that is, the phagocytic attack and encapsulation by the body of the so-called “biocompatible” biomaterials comprising the devices. Bryers and coworkers present strategies currently under development that might lead to biomaterial constructs that will harmoniously heal and integrate into the body. The authors discuss in detail emerging strategies to inhibit the FBR by engineering biomaterials that elicit more biologically pertinent responses. /mr [17] http://onlinelibrary.wiley.com/doi/10.1002/bit.24559/abstract Staphylococcus aureus and its food poisoning toxins Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. This review in FEMS Microbiology Reviews is divided into four parts and focuses on: (1) the worldwide story of SFP outbreaks, (2) the characteristics and behaviour of S. aureus in food environment, (3) the toxinogenic conditions and characteristics of SEs, and (4) SFP outbreaks including symptomatology, occurrence in the European Union and currently available methods used to characterize staphylococcal outbreaks. /gvb [18] http://dx.doi.org/10.1111/j.1574-6976.2011.00311.x Non-enzymatic palladium bio-recovery The use of microorganisms as support for reduction of dissolved Pd(II) to immobilized Pd(0)-nanoparticles is an environmentally friendly approach for Pd recovery from waste. In this Editor's Choice paper in Biotechnology and Bioengineering, Meyer and co-workers propose a non-enzymatic mechanism for bio-supported Pd(II)-reduction on microbial surfaces, since microorganisms stimulated Pd(II)-reduction independent of being alive or dead. A comparable effect has been observed for amine-functionalized microparticles, which could be hampered if amine-groups were blocked by acetylation. Infrared-spectroscopy corroborate the role of amine-groups in Pd(II) sorption and reduction on microbial and synthetic surfaces. /mr [19] http://onlinelibrary.wiley.com/doi/10.1002/bit.24500/abstract Organizing a European multilevel biorefinery project The EUROpean multilevel integrated BIOREFinery design for sustainable biomass processing or EuroBioRef project is a four-year program launched in 2010. It deals with the entire process of transformation of biomass, from fields to final commercial products. Professor Franck Dumeignil, Unit of Catalysis and Solid State Chemistry (UCCS), Université des Sciences et Technologies de Lille, France, coordinates the program supported by a €23m grant from the EU 7th Framework Program (FP7). In an interview Dumeignil talks about how he started this huge, highly collaborative network and the challenges of coordinating 29 partners from industry, Small and Medium-sized Enterprises (SMEs), and academics from 15 different countries. Read more about it in ChemistryViews.org. /vk [20] http://dx.doi.org/10.1002/chemv.201200068 Virtual issue Bioprocessing for Biopharmaceuticals Biotechnology Journal and Engineering in Life Sciences are pleased to announce a joint virtual issue, “Bioprocessing of Biopharmaceuticals”, covering the latest and most important topics, such as siRNA therapeutics, single-use bioreactors, PEGylation and much more. All articles are freely available in August and September./jp http://goo.gl/PNaCL Current protocols Use of Zinc Fingers A limitation of the traditional Caco-2 cell assay for measuring transporter-mediated efflux of a given substrate is that it is not possible to determine which specific transporter is involved. An alternate approach can generate specific transporter knockout cell lines that can be used to test efflux with any desired substrates. In this approach, human C2BBe1 cells are nucleofected with specific zinc finger nucleases (ZFN), which can target any gene of interest and generate a double-stranded break. The cell's normal repair mechanisms can then generate targeted deletions (or integrations). A single ZFN can be used to generate a single transporter knockout, or multiple ZFNs can be used to knock out more than one transporter. /bm [21] www.currentprotocols.com/WileyCDA/CPUnit/refId-tx2302.html Axonal tracing using rAAV Many commonly used anterograde tracers are not amenable to the new genetic tools available. Recombinant adeno-associated virus (rAAV)-mediated expression of fluorescent proteins in neurons provides the required flexibility for both anatomical tract tracing and genetic strategies to characterize projections from defined cell types. Use in mice will result in expression of fluorescent proteins in cell bodies, dendrites, and axons in targeted neurons, and can be easily adapted to address various experimental questions. /bm [22] www.currentprotocols.com/WileyCDA/CPUnit/refId-ns0120.html BiotecEvents Advertorial: Systems Biology Europe Systems Biology Europe (Oct. 16–17, 2012, Madrid, Spain) aims to integrate the understanding of normal cellular processes with that of pathological proteomics by exploring various ground-breaking techniques. There are, of course, numerous technical challenges that must be overcome in order for proteomic techniques to be used in the clinic, but this conference will provide insight to the details of using this procedure to identify and diagnose cancer in the lab. With a host of international speakers and a choice of four exclusive tracks, this meeting will provide delegates with translational information on exosomes, informatics and metabolic profiling. http://selectbiosciences.com/conferences/index.aspx?conf=SBE2012 Industry news Scripps and Bristol-Myers Squibb The Scripps Research Institute announced it has entered into a five-year collaboration with Bristol-Myers Squibb focused on applying novel chemistry to drug discovery and synthesis. In broad terms, the research will utilize Scripps Research investigators' expertise in applying chemistry methodologies to prepare novel synthetic intermediates and analogs for biological evaluation against Bristol-Myers Squibb's targets. /ag www.scripps.edu Colorectal cancer research The Fortis-IBN TissueBank (FIT) is the result of a S$4.5 million investment from a partnership between the Institute of Bioengineering and Nanotechnology (IBN) and Fortis Colorectal Hospital (FCH) in Singapore. The two will work on the development of non-invasive technologies to offer a viable alternative to conventional biopsy. The research areas of interest are cancer biomarkers, advanced diagnostics such as circulating tumor cells, nanoparticle and exosome-based diagnostics, as well as colorectal cancer metabolism. /ag www.ibn.a-star.edu.sg Read the latest R&D and business-specific news at G.I.T. Laboratory Journal Europe. http://www.laboratory-journal.com BiotecCareers Effective networking strategies In a previous article in our BiotecCareers column, Barbara Janssens mentioned the importance of networking in getting the job. Expanding on the topic of networking, Wiley Job Network recently hosted a webinar on “Effective Networking Strategies”, presented by Alaina Levine. Alaina Levine is the president of Quantum Success Solutions, a public speaking and leadership training enterprise that provides consultation and workshops to corporate clients. In addition to this, Alaina Levine has written over 100 articles in local, regional and national newspapers on the topic of careers and career development. In her latest Wiley webinar, Alaina shares with the audience her insights on effective networking. While some scientist may think of “networking” as a dirty word and beneath their lofty aspirations, Alaina points out an excellent motivation behind networking – not only is networking important for getting the job, but it is also important for advancing science and engineering, as new and novel ideas are most likely to be generated as you discuss your work with others. In the webinar, Alaina highlights, with vivid real-life examples, several key notions of networking, some of these include: • Successful people network all the time out of curiosity for other people and the world around them • Networking leads to opportunities that are otherwise not available • Networking is about creating win-win opportunities for both parties • Reputation is your most important asset – create a positive image of your own unique brand • Maintaining your network is as important as creating new ones Networking notions alone are not enough, where should you go about looking for contacts? While most of the attendees in the webinar stated conferences as their main choice, Alaina also broadens the scope of locations where one should be looking for contacts and how one can effective network on each occasion. /jp To find out more, listen to a recorded version of the webinar at: http://www.wiley.com//college/wli/WJNEvent/ See also: Getting the job: But how? http://onlinelibrary.wiley.com/doi/10.1002/biot.201200051/full#sec1-26 Wiley Job Network www.wileyjobnetwork.com Special issues Plant Biotechnology Journal: Next-generation sequencing technologies The past 5 years have seen a revolution in next-generation DNA sequencing, with plant researchers readily adopting the technology to decipher the structure and function of plant genomes. This Special issue reports the latest developments in plant biotechnology driven by advances in DNA-sequencing technology. As next-generation DNA sequencing continues to develop, we can expect to see many more examples of gene identification and trait association with valuable outcomes, increasing our understanding of plant growth and development and accelerating crop improvement. /jc http://onlinelibrary.wiley.com/doi/10.1111/pbi.2012.10.issue-6/issuetoc Cytoskeleton: Emerging concepts in neuronal cytoskeleton In April 2011, over fifty scientists and students met in Chile, bringing together scientists from South American countries with colleagues from the USA and the global community. The content of this free Cytoskeleton Special Issue “Emerging concepts in neuronal cytoskeleton,” attempts to capture the spirit of the meeting. All of the studies presented in this Special issue involve cutting-edge biotechniques such as the n