Transcription factors are frequently involved in the process of cellular transformation, and many malignancies are characterized by a distinct genetic event affecting a specific transcription factor. This probably reflects a tissue specific ability of transcription factors to contribute to the generation of cancer but very little is known about the precise mechanisms that governs these restricted effects.To investigate this selectivity in target gene activation we compared the overall gene expression patterns by micro-array analysis and expression of target genes for the transcription factor EBF in lymphoma and neuroblastoma cells by RT-PCR. The presence of transcription factors in the different model cell lines was further investigated by EMSA analysis.In pre-B cells mb-1 and CD19 are regulate by EBF-1 in collaboration with Pax-5 and E-proteins. We here show that neuroblastoma cells express these three, for B cell development crucial transcription factors, but nevertheless fail to express detectable levels of their known target genes. Expression of mb-1 could, however, be induced in neuroblastoma cells after disruption of the chromatin structure by treatment with 5-azacytidine and Trichostatin A.These data suggest that transcription factors are able to selectively activate target genes in different tissues and that chromatin structure plays a key role in the regulation of this activity. This factor is, however, broadly expressed and directly involved in cell cycle regulation and apoptosis explaining the common involvement of the protein in tumor development. Many malignancies are characterized by specific chromosomal translocations that frequently affects the expression or structure of transcription factors with a more tissue specific expression pattern, often with an important function during development  labeled oligonucleotide for 12 hours at 57°C in the same solution. Membranes were washed at room temperature 2 times in 2XSSC supplemented with 0.1% SDS for 15 minutes.RNA was prepared from cells using Trizol (Life Technology) and cDNA was generated by annealing 1 μg of total RNA to 0,5 μg of random hexamers in 10 μl DEPC-treated water. Reverse transcriptase reactions were performed with 200 units of SuperScript Reverse Transcriptase (Life Technologies) in the manufacturers' buffer supplemented with 0,5 mM dNTP, 10 mM DTT and 20 units RNase inhibitor  in a total volume of 20 μl, at 37°C for 1 hr. One-twentieth of the RT reactions were used in the PCR assays. PCR reactions were performed with 1 unit of Taq-polymerase (Life Technologies) in the manufacturers' buffer supplemented with 0.2 mM dNTP, in a total volume of 25 μl. GADPH was amplified by 25 cycles  while 30 cycles were used to amplify CGA, SCG10, mb-1 and CD19 message . Primers were added to a final concentration of 1 mM. PCR products were blotted onto Hybond NOligonucleotides used for RT-PCR were:GADPH sense; 5'-CCACCCATGGCAAATTCCATGGCA;GADPH antisense; 5'-TCTAGACGGCAGGTCAGGTCCACC;CGA sense: 5'-GAAGATGAACTCTCAGAGGTTCCGA antisense: 5'-GGATCTCCTTGTAGCCAAGGCTCGCD19 sense: 5'-AGTCATTGCTGAGCCTAGAGCTGCD19 antisense: 5'-CTCGGAGTCCTCCTCACTGTCAGmb-1 sense; 5'-CCAGCATCATTGATGGTGAGCCmb-1 antisense: 5'-GACATCTCCTATGTTGAGGCTGCmb-1 hybridization; 5'-CCCGCACAATAGCAGCAACAACGCCAACGTSCG10 sense; 5'-ATGCTGTCACTGATCTGCTCTTGCSCG10 antisense; 5'-CAGGTTGAACTGTCTGGCTGAAG32P] ATP by incubation with T4 polynucleotide kinase , annealed and purified on a 5% polyacrylamide Tris-borate-EDTA (TBE) gel. Nuclear extract [in vitro transcribed-translated protein, was incubated with labeled probe  for 30 min at room temperature in binding buffer  with 0.75 μg Poly(dI/dC) (Amersham Pharmacia). Antibodies  were added 10 min before the addition of the DNA probe. The samples were separated on a 6% polyacrylamide TBE gel, which was dried and subjected to autoradiography.DNA probes were labeled with γ[ extract , or in vOligonucleotides used for EMSA were the following:mb-1 sense 5'-AGCCACCTCTCAGGGGAATTGTGG;mb-1 antisense 5'-CCACAATTCCCCTGAGAGGTGGCT;CD19-BSAP sense 5'-GCAGACACCCATGGTTGAGTGCCCTCCAGG;CD19-BSAP antisense 5'-CCTGGAGGGCACTCAACCATGGGTGTCTGC;E5 sense: 5'-GGCCAGAACACCTGCAGACG;μE5 antisense: 5'-CGTCTGCAGGTGTTCTGGCC;μOct binding site sense 5'-CATCTCAAGTGATTTGCATCGCATGAGACG;Oct binding site antisense 5'-CGTCTCATGCGATGCAAATCACTTGAGATC;Lambda B (Pu.1 site) sense: 5'-GAAAAAGAGAAATAAAAGGAAGTGAAACCA AG;Lambda B antisense: 5'-CTTGGTTTCACTTCCTTTTATTTCTCTTTTTC;CRE (ATF5 site) sense: TCA TGG TAA AAA TGA CGT CAT GGT AAT TACRE antisense: TAA TTA CCA TGA CGT CAT TTT TAC CAT GARNA from Nalm6, SK-N-BE(2)c, SH-SY5Y, KM3, THP-1 and HeLa cells was extracted using Trizol™ . A common RNA control obtained by mixing a variety of cell lines was used for all hybridizations. RNA was concentrated to 50 μg total RNA (25 μg sample RNA and 25 μg control RNA) to generate aminoallyl-modified cDNA. Sample cDNA was labeled with Cy3-dCTP and control RNA was labeled with Cy5-dCTP using CyScribe Post-Labeling Kit (Amersham Pharmacia Biosciences). A hybridization solution was made by combining labeled cDNA with 20 μl Cot-1 DNA (1 mg/ml), 3 μl Poly dA (4 mg/ml) and 1.5 μl yeast t-RNA (4 mg/ml), dry down by speed-vac and resuspended in 40 μl Pronto! Universal Hybridization Solution™ . The hybridization solution was added to a pre-hybridized microarray slide . The arrays were hybridized at 42°C for 18 hrs, washed according to the manufacturers recommendations , dried by centrifugation and scanned on Agilent microarray scanner. Scans were analyzed using GenePix Pro versions 4.0.1.9 and 4.1.1.4. BioArray Software Environment (BASE) :software0003.1–0003.6). The settings for the analysis presented were, Background Correlation: Mean FG – Mean BG, Spot filter: (Raw) SNR ch1 mean > = 2, (Raw) SNR ch2 mean > = 2, (Raw) Flags = 0, (Raw) Spot diameter > = 40, Normalization: Lowess, Reporter filter: in # of assays = 16, Analysis: Hierarchical clustering (reporter)N-myc gene, which in the clinic is correlated to adverse outcome of the disease, while the SH-SY5Y cell line carry an N-myc gene in germ line configuration [Knowing that neuroblastoma and pre-B cells share the expression of EBF ,13,14, wguration .CD19 [mb-1 [SCG10 and CGA [SCG10 and CGA message they did not express either CD19 or mb-1 message. The opposite pattern was observed in the pre-B cell lines where CD19 and mb-1 but not SCG10 or CGA message could be detected. This show that even though neuroblastoma and pre-B lymphoma cells to some extent share gene expression patterns, they do not appear to share the expression of specific EBF target genes.The finding that these two different types of tumors did display a degree of similarity in overall gene expression patterns opened the possibility that the previously identified EBF target genes would be expressed in both cell types. To investigate this possibility we extracted RNA from pre-B cell lines as well as neuroblastoma cells and investigated the expression of the pre-B cell EBF target genes 19 [mb-1  as well  and CGA  by RT-PCmb-1 and CD19 genes are genetic targets for both EBF and the paired domain protein Pax-5 (BSAP) [CD19 promoter [CD19 Pax-5 binding site. No such binding activity was detected in extracts from epithelial cells (Data not shown). To verify that the binding activity was due to the presence of Pax-5 we performed a super-shift experiment using a Pax-5 specific antibody, a control antibody or no antibody in the binding reaction  ,19-21. Tpromoter . These en Figure . While tn Figure . Since tn Figure . This inIn addition to provide information of overall relationships between different types of tumors, the micro-array analysis also yield preliminary information about differential gene expression patterns of individual genes (Appendix A). The data obtained in our experiments suggested differential expression of genes such as myb, CBFβ and HMG 1, 3 and 4 proteins with high expression restricted to the pre-B cells while Bcl-6 appeared to be over-expressed specifically in neuroblastoma cells (Appendix A). To further investigate other potential differences and similarities between pre-B and neuroblastoma cells with regard to expression of gene regulatory proteins we extracted expression data regarding additional genes encoding transcription factors from our data set Figure . SeveralTo further investigate the E-box binding activities in SH-SY5Y cells as compared to Nalm6 cells we performed super-shift assays using antibodies against E2-2, E47 or c-myc Figure . The majThe micro-array analysis did also suggest a difference in the expression of activator of transcription 5 (ATF5/ATF7/ATFX) ,26. ATF5EBF has also been suggested to share pre-B cell restricted target genes with Ets proteins and one factor belonging to this family of proteins suggested to contribute to B lineage identity is the Ets protein Pu.1 ,29. In omb-1 gene in neuroblastoma cells after disruption of the chromatin structure by culturing the cells in the presence of the DNA de-methylating agent 5-azaC and the histone deacetylase inhibitor TSA. RT-PCR analysis suggested that while the untreated control cells did not express any detectable amount of mb-1 transcripts the 5-azaC/TSA treated cells expressed significant amounts of mb-1 transcripts c cells treated with 5-azaC/TSA was about one twentieth of that observed in a pre-B cell, the SH-SY5Y cells were induced to express levels comparable to that observed in the pre-B cells. In contrast we were unable to observe an induction of CD19 expression (Data not shown). Thus, we conclude that the lineage specific regulation of the mb-1 gene may be directly dependent on chromatin status, while expression of other pre-B cell specific EBF target genes, such as CD19, must be explained by other mechanisms.These experiments suggested that there indeed are specific differences in transcription factor expression between the two cell types, but also that they share expression of several key regulatory molecules. This indicates that additional components of gene regulation may be of crucial importance for target gene selection. One potential barrier to gene activation could be the chromatin structure, that is, the epigenetic setting of the cells directs the transcription factors to their tissue specific target genes. To investigate this possibility we analyzed the expression of the s Figure . To obtain vitro data [We here report that neuroblastoma and pre-B lymphoma cells share the expression of transcription factors believed to be essential for normal B cell development while the expression of their B lineage target genes remains tissue specific. The finding that neuroblastoma cells expressed Pax-5 was somewhat surprising even though this factor has been shown to be involved in midbrain development. Mice carrying a homologous disruption of the Pax-5 gene lack both B-lymphoid cells and a fully developed central nervous system ,31. In ttro data  as well tro data -40 indicIn contrast to the stable expression of Pax-5 there appeared to be large differences in E-protein expression in the neuroblastoma cell lines investigated. In neuronal tissues a number of tissue specific bHLH proteins have been defined. Importantly, these pro-neuronal bHLH proteins require heterodimerization with E-proteins for the formation of DNA binding complexes. In the developing sympathetic nervous system HASH-1 is transiently expressed and is of pivotal importance for the formation of the autonomic and olfactory nervous system, neuroendocrine cells of the lung and specific regions of the telencephalon (reviewed in ). We havmb-1 promoter is has been shown that methylation of specific C residues in the promoter prevents the complex formation between Pax-5 and Ets proteins, thereby reducing functional activity of the promoter [mb-1 transcription in the SH-SY5Y cells then in the SK-N-BE(2)c cells  declare that they have no competing interests.Anna Lagergren has performed the EMSAs, the micro-array experiments and the PCR experiments under the supervision of Mikael Sigvardsson while Christina Manetopoulus has performed the cell culture work as well as 5-aza-Cytidine treatments and RNA purifications under the supervision of Håkan Axelson. All contributors have been involved in the writing of the manuscript.The pre-publication history for this paper can be accessed here:The data shows hierarchical clustering of the microarray data displaying differential gene expression patterns of individual genes in the different cell lines used in this study.Click here for file


The 4-day course was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the course was to collectively explore how to best harness genomics to improve health in the region. This article presents the course findings and recommendations for genomics policy in EMR.While innovations in medicine, science and technology have resulted in improved health and quality of life for many people, the benefits of modern medicine continue to elude millions of people in many parts of the world. To assess the potential of genomics to address health needs in EMR, the World Health Organization's Eastern Mediterranean Regional Office and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20The course brought together senior representatives from academia, biotechnology companies, regulatory bodies, media, voluntary, and legal organizations to engage in discussion. Topics covered included scientific advances in genomics, followed by innovations in business models, public sector perspectives, ethics, legal issues and national innovation systems.A set of recommendations, summarized below, was formulated for the Regional Office, the Member States and for individuals.• Advocacy for genomics and biotechnology for political leadership;• Networking between member states to share information, expertise, training, and regional cooperation in biotechnology; coordination of national surveys for assessment of health biotechnology innovation systems, science capacity, government policies, legislation and regulations, intellectual property policies, private sector activity;• Creation in each member country of an effective National Body on genomics, biotechnology and health to:- formulate national biotechnology strategies- raise biotechnology awareness- encourage teaching and training of biotechnology- devise integration of biotechnology within national health systems.The recommendations provide the basis for a road map for EMR to take steps to harness biotechnology for better and more equitable health. As a result of these recommendations, health ministers from the region, at the 50th Regional Committee Meeting held in October 2003, have urged Member States to establish national bodies of biotechnology to formulate a strategic vision for developing biotechnology in the service of the region's health. These efforts promise to raise the profile of genomics in EMR and increase regional cooperation in this exciting new field. In a recent study, University of Toronto researchers identified the "Top 10 Biotechnologies to Improve Health in Developing Countries" . Its misThere is a wide range of scientific capacity and health system development across the Eastern Mediterranean Region (EMR) , which cth–23rd, 2003, in Muscat, Oman. This 4-day course/workshop was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health.In order to assess the potential of genomics to address health needs in the Region, the World Health Organization-Eastern Mediterranean Regional Office (WHO-EMRO) and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20The overall objective of the Genomics Policy Executive Course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs and to collectively address the question of how best to harness genomics to improve health in the region table .We based the program and designed the sessions and lecture topics of this course on prior courses held in Nairobi, Kenya in March 2002; in Toronto, Canada in May 2002; and in Kumarakom, India in January 2003. The sessions and presenters of the course are shown in table "How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?" The groups met frequently to discuss the presentations, and each participant was also provided a course reader with additional literature on the lecture topics (table 3). The overall task of these study groups was to draw upon the course material and their own experiences and propose a set of recommendations for genomics and biotechnology policy in the region. On the last day of the meeting each group presented these recommendations.The sessions dealt with a wide range of relevant topics, starting with recent scientific advances in genomics and stem cell research, followed by discussions on business models in genomics and biotechnology, intellectual property rights and regulatory frameworks, public engagement and an internet-based opinion leaders' network. The presentations were designed to be interactive and foster active discussion from, and among, the participants, and each presentation was followed by a moderated discussion period. Early in the course, the attendees were placed into one of five study groups – these groups were carefully designed to capitalize on the diverse backgrounds of the participants. Each group was assigned the task to on the deliberate the key question In order to assess the level of interest in the formation of an email-based opinion leaders' network to continue discussion among the participants following the course, a brief survey was conducted on the participants' internet access and their expectations of the network.Dr Peter A. Singer, Director of the University of Toronto Joint Centre for Bioethics, described the overall aim of the course "How to best harness genomics to improve health in the region", as well as the 4-day program. Dr Ali Jaffer Suleiman of the Ministry of Health in Oman acted as chairperson of the meeting.Professor Riad Bayoumi of Sultan Qaboos University highlighted new scientific developments that have resulted from the genomics revolution, such as proteomics; mapping of single nucleotide polymorphisms (SNPs) to understand human genetic variation and its relationship with disease; gene expression chips to monitor differential gene expression and identify drug targets; and bioinformatics as a new field that combines biology, mathematics, statistics and computer programming to mine large-scale biological data.Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights at WHO, described the 2002 report of the World Health Organization "Genomics and World Health" . He callProfessor Abdallah Daar described a recent study, conducted by the University of Toronto Joint Centre for Bioethics' Canadian Program on Genomics and Global Health, to identify the ten most promising biotechnologies for improving health in developing countries in the next five to ten years. These technologies offer guidance to those who can influence the direction of R&D in developing countries and challenge common assumptions about the relevance of biotechnology for these countries, as shown by the mapping of the Top 10 Biotechnologies onto the UN Millennium Development Goals [Professor Al Bar of King Fahad University, Saudi Arabia, illustrated Islamic perspectives on genetic testing, cloning, recombinant DNA technology and other genomics-related technologies. He showed that while Islam and science have always been aligned through history, today's religious leaders in the region must take the initiative to develop and formulate recommendations for genomics.Dr. Peter Singer described the concept of innovation systems and presented some observations of other countries' innovation systems and their successes. One definition of a national system of innovation (NSI) is the "network of institutions in the public and private sector whose activities initiate, import, modify and diffuse new technologies" -13. The Mr. Khalil Ahmed, Managing Director of Shantha Biotechnics, described the successes of this biotechnology company based in Hyderabad, India. Shantha was established in 1993, in part with seed money from the Government of Oman. Today, it is the first Indian company to receive WHO certification for a recombinant hepatitis-B vaccine Shanvac-B™, paving the way for UNICEF to buy 8.5 million doses for distribution globally. Hepatitis B vaccine is currently priced internationally as high as $8–10 per dose, while Shantha is selling it at $2 per dose ,16.Professor Richard Gold of McGill University's Centre for Intellectual Property Policy described the basics of intellectual property rights, patents and copyright issues, as well as policy options for developing countries in the international context. He outlined the characteristics of international agreements, which offer considerable flexibility to countries on how to apply patent laws to genomics and biotechnology. Developing countries have a number of options, such as compulsory licensing and research exemptions, to deal with property rights.Dr Jayasuriya, Director of UNAIDS in Pakistan described ways in which legal systems can facilitate best use of biotechnology. It is essential that legal reform keep up with the rapidly evolving science of genomics. Health law can facilitate best use of biotechnology – by providing for fast-track approval of biotechnology products; reducing import duties on health interventions; and allowing multiple channels of procurement and distribution to improve access and optimize prices.Dr Anwar Nasim, Chairman of Pakistan's National Council of Biotechnology, described the role of regulations both to promote useful biotechnologies and limit their risks for human health and the environment. A national-level regulatory body could provide guidelines for the use and release of biotechnology products, conduct biosafety reviews and risk assessments and formulate feedback mechanisms to improve the system through experience. The National Commission of Biotechnology of Pakistan, established in November 2001, focuses on biotechnology regulatory issues in health, agriculture, environment and industry. Similar commissions could be set up in other countries of the region and their interaction could further regional cooperation in biotechnology.Mr. Ehsan Masood of Scidev.net pointed out that active public engagement based upon knowledge can stimulate action to improve public health. Public engagement is far greater in today's world than it has ever been in the past. This is because of several factors including; a) the growing implications of research on public health, b) the increased awareness in civil societies to invest in health care and research, and to influence policy change and action, c) development and access of information technologies."How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?", were invited to present their findings. The main points of these group presentations are summarized in the recommendations that emerged from the course.On the last day of the course the five participant groups, who had been assigned the task to on the deliberate the key question The session discussions and group presentations underscored the urgent need for action to create the enabling environments  for research and development in genomics and biotechnology.The issue of awareness of biotechnology among political leaders to garner support at the highest level was raised early on in the course, and was reinforced throughout the course discussions. The participants felt strongly that political commitment is crucial to the advancement of biotechnology in the region. Political leadership is a critical factor in raising the profile of science in developing countries. A prominent example in the Eastern-Mediterranean Region is that of the Sultan Bin Mohammed Al-Qassimi, Shaikh of the Arab emirate of Sharjah. He has made a dedicated effort to change the face of science in the Gulf . For exaThe course attendees recommended that WHO-EMRO should take the responsibility to engage political leaders in the region's national governments. The participants shared experiences of the importance of government support for biotechnology – in Egypt there appears to be relatively strong support for biotechnology by the government, with biotechnology activity in academic and other research institutions as well as in the private sector. In Iran, considerable advances have been made in private and academic research centres, leading to a number of biotechnology products that are soon to reach the market, as well as well-respected scientific journals such as the Iranian Journal of Biotechnology . Other cIt will also be crucial to involve, inform and engage religious leaders in the region in order to promote genomics and biotechnology for improving public health. In doing so, it is worthwhile to note that Islam and science have always been aligned through history and it is well-established that Islam has historically contributed tremendous achievements to the advancement of science.  A recentThe active participation and involvement of organizations like COMSTECH was recommended to give support and guidance to WHO-EMRO's efforts. COMSTECH was established by the Islamic Summit in 1981 and includes among its objectives the building of indigenous capabilities in the fields of science and technology, promotion and continuing cooperation and coordination in scientific and technological areas of is member states and creation of effective institutional structure for planning research, development and monitoring of scientific and technological activities. COMSTECH has already launched networks across the region for exchange of information, and has valuable experience in the promotion of cooperation and coordination amongst the member states in science and technology activities in high technology areas .Bacillus thuringiensis [A major discussion point at the workshop was the effective assessment of existing capacity in the region. Several participants shared disappointment over the relative lack of participation of the region in global science and technology advances, citing, for instance, poor representation of the region at international scientific conferences. Many felt the need to evaluate and assess the level of scientific activity and capacity in individual countries in the region, in order to identify, among other things, strengths and weaknesses, entry points for countries, opportunities for regional collaboration, and areas for improvement. The workshop attendees felt it important to carry out this type of survey of each country's innovation system as a prerequisite to revising and revamping national and regional genomics policy. The factors identified by the participants as important for assessment in this survey – scientific capacity within public and private sector, private enterprise, religious and political leadership and intellectual property rights – can be considered to be part of the National System of Innovation (NSI). This proposed survey of NSI in the region can help to identify factors for successful growth of biotechnology sectors. Similarly, the recently completed study by the Canadian Program on Genomics and Global Health of the health biotechnology innovation systems of Brazil, China, Cuba, Egypt, India, South Africa and South Korea may also help to identify some of these factors . Workshongiensis . In IranAs highlighted by the 2004 report of the UN Commission on Private Sector and Development report , the proThe attendees observed that the key to effective participation in the genomics revolution is building scientific capacity, and expressed serious concern over the region's limited capacity to absorb and utilize genomics and biotechnology. Critical to capacity-building is access not just to technology but, more importantly, to scientific knowledge. One point-of-entry that was greeted with enthusiasm by the participants was bioinformatics, which is typically less-resource intensive compared with other genomics-related sciences, such as sequencing and proteomics. According to the report on the "Top 10 technologies to improve health in developing countries" countries of EMR can take advantage of genomic data and apply the power of bioinformatics to local health problems without having to invest heavily in the technologies used to produce them, A dedicated Genomics and Health Research Fund for the region may help to break down financial barriers to encourage scientific research and development in the region.The participants expressed enthusiasm for setting up National Biotechnology Commissions to address genomics policies at the national level and to contribute to the development of the above-mentioned national biotechnology strategy. A National Commission on Biotechnology (NCB) has been set up in Pakistan. The overall goal would be to help devise regulations to facilitate innovation in biotechnology. The NCB could help coordinate national biotechnology activities and policy, with activities including evaluation of biotechnology capacity (see above) to priority setting and public engagement. Accordingly, the NCB should have broad cross-sectoral representation in order to minimize the negative effects of inter-institutional rivalry. One of the main goals of the NCB , following the above-mentioned national biotechnology survey, will be to help develop and adopt a national biotechnology strategy, perhaps as part of a long-term science and technology policy. Other important objectives are discussed below.public awareness) of biotechnology and genomics in the region, and that this lack of awareness may be the biggest barrier to advances in genomics. Public awareness and engagement can help change the pace of research, leading to increased opportunities for greater societal involvement for improved health care. Media can be used as a catalyst to raise community awareness for social beneficence, equity and justice in health care. Recently, in May 2004, science journalists from across the region met in Cairo to discuss the hurdles they face in science reporting. The hurdles identified at this meeting included bureaucracy and poor access to scientific research taking place in the region [At the outset of the conference, participants voiced their concern about the low level of awareness  Request regional governments and policy makers at highest level to give priority to genomics for health and health biotechnologyb) Develop linkages with Organization of Islamic Conference Standing Committee for Science and Technology (COMSTECH) and other international partners to build Regional networking and cooperation for developing and utilizing health biotechnology.c) In collaboration with Member States undertake a national survey/situation analysis/needs assessment of health biotechnology innovation systems including resource capacities, government policies  In collaboration with COMSTECH initiate a research grant for applied  genomics and biotechnologyThe participants agreed that each member state would benefit from creating effective National Commissions on Biotechnology (NCB) to develop national biotechnology strategies. NCB should; a) develop national priorities, programmes and guidelines aimed at raising public education and awareness and b) collaborate with civil sectors to develop plans to integrate genetic and genomic products  build capacities for utilization and access of health biotechnology to the needy and e) ensure ethical safeguards against unwanted harm and exploitation and improve equity to improve health of the poor. The participants also felt that there was a need for strong personal commitment at individual level by experts and key actors to engage actively in the implementation of the recommendations to strengthen the initiative on genomics and biotechnology to improve the health and well-being of people in EMR. One concrete outcome of the workshop that may contribute to strengthening capacity in the region is the joint agreement between WHO/EMRO and the University of Toronto Joint Centre for Bioethics to provide scholarships to the Master of Health Sciences Program at the JCB. Furthermore, there seems to be growing need to establish a Regional Health Biotechnology Network for EMR, and WHO-EMRO is now planning to hold a Regional meeting to discuss this in Iran in July 2004.th Regional Committee Meeting held in October 2003, urged Member States to establish national bodies for genomics and biotechnology to formulate strategic vision for creating public awareness and for developing biotechnology for equitable health care in the region.WHO-EMRO has initiated follow-up of these recommendations. Ministers of Health from the region, at the 50Recently, a paper on harnessing genomics and biotechnology for public health was presented at the EM 28th Regional Consultative Committee Meeting (RCC) held in Cairo in April of this year. The recommendations in the paper were derived from those developed at the workshop. The paper will be presented at this year's Regional Committee Meeting to be held in October 2004.The methods and recommendations outlined in this paper demonstrate progress in bringing genomics and biotechnology to the forefront of science policy in developing countries. Our procedure has now led to the formation of three regional networks – the two previous ones being the African Genome Policy Forum, which encompasses participants from 10 African nations, the Indian Genome Policy Forum. We have also now held a course for Latin America and the Caribbean in association with PAHO and the UN University in Venezuela May 23–26 2004, and a similar network is being created of participants of that course. The next one will be held in the Southeast Asian region, most likely based in Hong Kong. These regional genome policy networks will provide models to establish a Global Genome Policy Forum.The author(s) declare that they have no competing interests.TA drafted the manuscript; PAS and ASD conceived of the course, and all authors participated in its design and coordination. All authors revised the manuscript for critical content and approved the final draft.


Duhuo Jisheng Wan (DJW) is perhaps the best known and most widely used Chinese herbal recipe for arthralgia, but the clinical study to verify its efficacy is lacking. The purpose of this study was to compare the efficacy of DJW versus diclofenac in symptomatic treatment of osteoarthritis (OA) of the knee.This study was a randomized, double-blind, double-dummy, controlled trial. The 200 patients suffering from OA of the knee, were randomized into the DJW and diclofenac group. The patients were evaluated after a run-in period of one week (week 0) and then weekly during 4 weeks of treatment. The clinical assessments included visual analog scale (VAS) score that assessed pain and stiffness, Lequesne's functional index, time for climbing up 10 steps, as well as physician's and patients' overall opinions on improvement.Ninety four patients in each group completed the study. In the first few weeks of treatment, the mean changes in some variables  of the DJW group were significantly lower than those of the diclofenac group. Afterwards, these mean changes became no different throughout the study. Most of the physician's and patients' overall opinions on improvement at each time point did not significantly differ between the two groups. Approximately 30% of patients in both groups experienced mild adverse events.DJW demonstrates clinically comparable efficacy to diclofenac after 4 weeks of treatment. However, the slow onset of action as well as approximately equal rate of adverse events to diclofenac might limit its alternative role in treatment of OA of the knee. Osteoarthritis (OA) is the most prevalent joint disorder characterized by articular cartilage degradation with an accompanying peri-articular bone response . OA affeThe use of Chinese and other foreign patent herbal medicines (pills and tablets) in arthralgia treatment is highly prevalent and increasing in Thailand, but importing these medicines from the People's Republic of China and other foreign countries is usually rather expensive. However, the cost of similar preparations can be minimized by using imported dried herbs available in Thailand as raw materials in the manufacturing process coupled with simple and inexpensive traditional drug manufacturing techniques. Thus, if clinical studies suggest that these herbal medicines are as effective and/or less toxic than conventional treatment, promotion of self-produced recipes in each community will lead to community-directed osteoarthritic treatment in Thailand.Bei Ji Qian Jing Yao Fang compiled by Sun Simiao in the Tang Dynasty (652 A.D.) [The herbal recipe used in this study was "Duhuo Jisheng Wan (DJW)", which means pill of Pubescent angelica root and Mulberry mistletoe combination, and it was quoted from the book 52 A.D.) ,12. Alth52 A.D.) , the cliThis randomized, double-blind, double-dummy, controlled trial was approved by the Medical Ethics Committee of the Faculty of Medicine, Chiang Mai University and was in compliance with the Helsinki Declaration.Two hundred out-patients of either sex were recruited. They were aged over 40 years, and had been suffering from unilateral or bilateral OA of the knee according to the criteria of the American College of Rheumatology  for moreDuring a run-in period of 1 week (week 0), patients considered eligible for the study were informed to discontinue all analgesics, anti-inflammatory drugs, and other modalities for the treatment of arthralgia and arthritis. At the beginning of week 1, patients who still met the eligible criteria were randomized into 2 groups (DJW and diclofenac group) and treated for 4 weeks (Table ®) were provided by Novartis (Thailand) Co., Ltd. In order to completely blind the patients, each diclofenac tablet was packed into a capsule with an appearance identical to its placebo. Either diclofenac or placebo was prescribed at 1 capsule, 3 times a day, immediately after meals.Twenty five mg film-coated tablets of commercially marketed diclofenac sodium , Radix Gentianae Macrophyllae (Qinjiao), Cortex Eucommiae (Duzhong), Radix Achyranthis Bidentatae (Niuxi), Radix Angelicae Sinensis (Danggui), Herba Taxilli (Sangjisheng), Radix Rehmanniae Preparata (Shudihuang), Rhizoma Chuanxiong (Chuanxiong), Cortex Cinnamomi (Rougui) and Radix Ledebouriellae (Fangfeng), 5% each of Radix Paeoniae Alba (Baishao), Radix Codonopsis (Dangshen), Radix Glycyrrhizae (Gancao) and Poria (Fuling), as well as 2.5% of Herba Asari (Xixin).Xixin, Niuxi, Shudihuang and Rougui were imported from the Shantou Traditional Chinese Medicine Factory, the People's Republic of China (PRC). The remaining herbs were imported from the Qixin Co., Ltd. (Hebei Province), PRC. Each pulverized ingredient was mixed thoroughly together according to the formula mentioned above and prepared into honeyed pills, which were baked in a hot air oven until completely dry, and then pulverized. The pulverized powder was finally filled into capsules of 500 mg per capsule. The quality control and standardization of DJW  were conducted by using guidelines recommended by the Food and Drug Administration of Thailand . DJW andClinical assessments were evaluated for base-line data at the end of a run-in period (week 0) and then weekly for 4 weeks. These assessments included 100-mm VAS that assessed pain , 100-mm VAS that assessed stiffness , Lequesne's functional index that assessed the patient's daily activities (score ranging from 0–24) , and timAt the end of week 1–4, 100-mm VAS that assessed the physician's and patients' overall opinions on improvement were also evaluated. The assessment forms were designed so that the patients and evaluator could view their own previously recorded scores, but they were not allowed to view each other's VAS. Clinical assessments were evaluated by the same physician who had been blinded to the treatment. Complete physical examination and non-directive questioning for adverse events were also performed weekly for 4 weeks in order to acquire a safety assessment.In within the group analysis, the mean VAS and Lequesne's functional index between base-line and the following weeks were compared by a non-parametric Wilcoxon's signed-rank test, whereas, the average time for climbing up 10 steps was compared by the paired t-test.In the analysis between the groups, a non-parametric Wilcoxon's rank-sum test was used to determine whether the two groups differed in the physician's and patients' overall opinions on improvement. In addition, the mean changes in VAS that assessed pain and stiffness, as well as Lequesne's functional index were compared by the same test. The student's t-test was used to compare the mean changes in the time for climbing up 10 steps.A total of 429 patients were recruited into this study, of whom 229 were excluded Figure . The remThe VAS that assessed pain and stiffness at the end of week 1–4 decreased significantly when compared to their own base-line values , as did Lequesne's functional index and time for climbing up 10 steps . All adverse events reported were mild in intensity in both groups. The most common adverse events occurring in the DJW and diclofenac group were raised blood pressure (16% vs. 19%), central nervous system symptoms including dizziness, somnolence and drowsiness (16% vs. 11%), and gastrointestinal symptoms including nausea/vomiting, dyspepsia, diarrhea and constipation (12% vs. 5%). The least common adverse events were increased appetite, cramp, rash and flu. More than one adverse events might be occurred in some patients. However, the percentages of patients who experienced each adverse event in both groups were not significantly different.In summary, the VAS that assessed pain and stiffness, Lequesne's functional index and time for climbing up 10 steps at each time point decreased significantly in the DJW and diclofenac group when compared to their own base-line values. The mean changes in all VAS that assessed pain, except those for walking and standing, did not differ significantly between both groups. The differences in mean changes in the VAS that assessed walking pain, standing pain and stiffness were found only during week 0–1, whereas, those in Lequesne's functional index were found during week 0–1 and 0–2.Since the preparations and dosages of DJW and diclofenac were different, this study was designed as a randomized, double dummy, controlled trial in order to completely blind both patients and physician (double-blind). Therefore, the placebo of DJW was also prescribed for the patients in the diclofenac group, and vice versa, the placebo of diclofenac was prescribed for the patients in the DJW group.Among the 15 herbs used as raw materials in DJW, Xixin (Herba Asari) seemed to be the most toxic, due to its pungent taste and warm property . GeneralIn an ITT analysis , the mean changes in some variables between the two groups were significantly different after the first few weeks of treatment, and became no different afterwards. These differences suggest that the onset of DJW is significantly slower than diclofenac for at least 2 weeks  or 3 weeks . The reason why DJW needs a few weeks to exert its effect may be due to 3 possibilities. Firstly, from the pharmacokinetic point of view, the elimination half-life of the active ingredients in DJW might be too long, and therefore needs weeks to accumulate until a steady state concentration is reached  and its maximal therapeutic effect is evident. Secondly, from the pharmacodynamic point of view, DJW may exert its effects via several probable mechanisms (similar to many novel biologic treatments of arthropathy) involved modifications of cartilage metabolism, normalized viscosity and elasticity of synovial fluid, etc. These mechanisms of action might resemble many symptomatic slow acting drugs in osteoarthritis (SYSADOA) such as glucosamine sulfate, intra-artricular hyaluronan, and others. These interventions always need a period of time to exert their therapeutic action. Thirdly, the major effect of DJW might be the result from placebo effect and/or natural fluctuation of the OA symptoms. It could be simply that diclofenac worked quickly, but patients in both groups got better anyway by 2–3 weeks. Although the last possibility cannot be entirely ruled out, but it seems unlikely because even there is a tendency of OA symptoms to improve after placebo treatment, it has been reported that diclofenac was significantly superior to placebo in relieving pain, improving stiffness, and improving physical function after 4 weeks of treatment . FurtherSince this study demonstrated that approximately 30% of study subjects in each group experienced adverse events, this data suggest that the toxicity profiles of DJW are similar to diclofenac. Therefore, cautious use of DJW should be considered in the same manner as using diclofenac including other NSAIDs. However, the gastrointestinal adverse effects in the diclofenac group were quite low when compared to other short-term NSAIDs studies ,19. ThisDJW demonstrates clinically comparable efficacy to diclofenac after 4 weeks of treatment. However, the slow onset of action as well as approximately equal rate of adverse events to diclofenac might limit its alternative role in treatment of OA of the knee.The author(s) declare that they have no competing interests.ST carried out the randomization, supervised data collection and analysis, and drafted the manuscript. PK participated in the design of the study and performed the statistical analysis. NR participated in the selection of patients eligible for the study. KS carried out the outcome assessments. SP participated in the report of radiographic findings of knee. SL participated in the preparation of DJW and its placebo. SP carried out the screening for microorganism contamination in DJW and its placebo. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


This study was planned to collect data about causes, prevalence and responses to work-related musculoskeletal disorders reported by physiotherapists employed in Izmir, Turkey.A two-page survey with closed ended questions was used as the data collected method. This survey was distributed to 205 physiotherapists working in Izmir, Turkey, and 120 physiotherapists answered. Questions included occupational history of physiotherapists and musculoskeletal symptoms, special areas, tasks, job-related risk factors, injury prevention strategies, and responses to injury.Eighty-five percent of the physiotherapists have had a musculoskeletal injury once or more in their lifetime. Injuries have been occurred mostly in low back (26 %), hand-wrist (18 %), shoulders (14 %) and neck (12 %). The highest risk factor in causing the injury was transferring the patient at 15%. Sixty-nine percent of physiotherapists visited a physician for their injury and sixty-seven percent of the respondents indicated that they had not limited their patient contact time as a result to their injuryAccording to the results of this study, the rate of musculoskeletal disorders in physiotherapists in Izmir-Turkey has been found to be high due to their profession. Respondents felt that a change in work habits was required in order to decrease the risk of another injury. A work-related musculoskeletal disorder (WRMD) is defined as a musculoskeletal injury that results from a work-related event. This may result in lost work time, work restriction, or transfer to another job -4. TheseUnderstanding the issues related to musculoskeletal disorders in physical therapists requires some awareness of the context in which these professionals work. In Turkey, physiotherapists cannot treat patients directly. According to law, direct accesses to physiotherapists are not possible . FurtherThe purpose of this study was to investigate the prevalence and features of WRMDs in a group of Turkish physiotherapists working in various capacities in a large Turkish city, and to compare the findings to reports from other countries.The study was approved by the Ethics Committee of Dokuz Eylül University. The research population was selected physiotherapists who were employed in a broad spectrum of practice settings in İzmir, Turkey. All data were collected by a mailed questionnaire (Appendix 1 see ) that coThe questionnaire was based on another published survey ,6 and si2 were used to analyze influence personal characteristics  to WRMDs.Data were analyzed using SPSS 10.0 for Windows. Results for the general information items were expressed as mean ± standard deviation, and results for items in the occupational portion were expressed as percentages. χThe 120 respondents included 92 females and 28 males of mean age 30.4 ± 6.9 years . Total response rate was 59%, for women 59%, for men 58%. General information about the group is given in Table 2=.234, P=.629), age , number of years in physiotherapy practice , or number of hours per week in direct patient care  (Table Frequency of WRMDs was not correlated with sex (χOne hundred and two respondents (85%) reported that they had experienced work-related musculoskeletal pain or discomfort at some time in their occupational lives. Seventy-eight (65%) reported that they had sustained more than one WRMD.The lower back was the body part with the highest frequency of occupational injury (26%), and wrist-hand (18%), shoulder 14%), and neck (12%) were other sites frequently affected Table . The mai%, and neThe respondents who had experienced a WRMD indicated that lifting (18%), maintaining a position for prolonged period of time (17%), performing repetitive tasks 16%), and transferring patients (16%) were the activities that most often exacerbated their symptoms during clinical practice Table . Improve%, and trSixty-nine percent of the respondents with WRMDs had visited a physician for the problem, and 46% of whom stated that they had officially reported the injury to their employer. The respondents who had suffered WRMDs reported they used their own occupational knowledge, rest, medications, and exercise to treat the problem Table . Sixty-sMusculoskeletal system problems connected to occupational conditions are common among health care workers. The costs of these are substantial, both in terms of money and in terms of work time lost ,9,17,18.The results from studies on WRMDs in physiotherapists have generally been similar, though some have differed according to country. Such variations are linked to level of development, the status of the profession of physiotherapy in a given country, psychosocial, and epidemiological factors ,11,13,14In this study, we collected demographic and WRMD data from 120 physiotherapists in Izmir, Turkey, and analyzed rates of injury, risk factors, injury types and sites, and post-injury management. We asked to complete the self-administered questionnaire if they had more than 2 years of experience in practice. Thus, response rate of questionnaire was low 58.5 %) in our study. The survey answers revealed that 85% of the respondents had experienced WRMDs. Cromie .5 % in o investigConcerning sites of musculoskeletal injury during professional activities, the highest incidence is in the low-back region. Biomechanical studies have shown that physical loading factors, such as body flexion, rotation and weight loading, play a role in this . In a stStudies of WRMDs in health care of professionals have identified the lower back as the most commonly involved area of the body, followed by neck and upper extremities ,13,19-21According to the literature the work-related activities that most commonly lead to injury in health professionals are lifting heavy equipment and patients, transferring patient, maintaining the same posture for a long period, manual therapy practices, responding to patients' sudden movements, and repeated movements ,18,22-24Holder et al.  identifiIn Turkey, most physiotherapists work in general hospitals. In these facilities, the daily treated patient number is too high, and this number far exceeds the number of physiotherapists on staff. In addition, the majority of patients in these hospitals are seriously ill .etc.) is not ergonomic, and consequently the physiotherapist cannot follow ergonomic principles while doing his or her job.People who suffer injuries on the job may be treated with medication, rest and exercise. Physiotherapists have fundamental knowledge about ergonomics and biomechanics, and using this knowledge may vary depending on professional knowledge and skills. In Turkey, physiotherapists are trained in ergonomic working principles at the undergraduate level. However, in many working environments the equipment used (treatment table, chair, A study conducted by Cromie  in 2002 et al. [Previous research has shown that physiotherapists who have suffered a WRMD tend to change their professional attitudes to avoid additional injuries. Holder et al.  found thMierzejewski  reportedSome professions have very high WRMD prevalence, and this has led to more intensive research in recent years. These types of injury have major consequences for society, workers, employers, and the insurance sector due to loss of labor force, long-term disability and delay in returning to work, decreased productivity, and psychological effects on employees. Therefore, minimizing and preventing WRMDs has significant potential social and economic benefits. It is important to invest in studies aimed at reducing these types of injuries in physiotherapists.Our study has three main limitations. A physiotherapist's working time in a day is not long, but he or she treats a very large number of patients during this time. Our survey would have been more informative if we collected data for the number of daily treated patients. We also did not inquire about the physiotherapists' activity levels. This would have been valuable information, as sports and recreational activity may affect WRMD frequency. Another limitation of our study was comparing various areas of practice and techniques used by physiotherapists and investigating their relationship to WRMDs.Our survey reveals that the WRMDs in physiotherapists in Turkey are similar to rates reported in other countries. Physiotherapists in our country suffer similar work-related injuries as their counterparts elsewhere, despite different legal working conditions and cultural differences. This study provides data related to WRMDs in physiotherapists in Turkey. Further studies can be very useful if it research prevalence of WRMDs in physiotherapists who have employed different working conditions.None declared.YS participated in the design, managed the data collection and performed statistical analysis.AO participated in designed the study protocol, managed the coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 1, Table 2. Questionnaire on occupational injuries in physical therapists. Turkish version of questionnaire on occupational injuries in physical therapistsClick here for file


Cholesterol ester transfer protein (CETP) plays a major role in regulating the levels of LDL- and HDL-cholesterol. We previously observed a fish-oil-induced elevation of low-density lipoprotein (LDL)-and very-low-density lipoprotein (VLDL)-cholesterol concentrations and a decrease in high-density lipoprotein (HDL)-cholesterol concentration in F1B hamsters. The molecular mechanism/s by which fish oil induces hyperlipidaemic effect was investigated in this study. We examined whether the effects of dietary fish oil on plasma lipoprotein concentrations are due to fish-oil-induced alterations in plasma CETP activity. MIX diet, a diet supplemented with a mixture of lard and safflower oil, was used as the control diet.We found that fish oil feeding in hamsters reduced CETP mass as well as CETP activity. Increasing the dietary fat level of fish-oil from 5% to 20% (w/w) led to a further decrease in CETP mass. Supplementation with dietary cholesterol increased both CETP mass and CETP activity in fish-oil and MIX-diet fed hamsters. However, there was no correlation between CETP mass as well as CETP activity and LDL-cholesterol concentrations.These findings suggest that cholesterol ester transfer between HDL and LDL is not likely to play a major role in determining fish-oil-induced changes in LDL- and HDL-cholesterol concentrations in F1B hamsters. A possible role of reduced clearance of LDL-particles as well as dietary fat level and dietary cholesterol dependent changes in LDL-lipid composition have been discussed. Fish oil, rich in n-3 polyunsaturated fatty acids (PUFA), is considered beneficial in lowering the risk of coronary heart disease ,2. The bOne of the factors that determine LDL concentrations is reverse cholesterol transport pathway, which removes cholesterol from peripheral tissues and returns to the liver. Cholesterol ester transfer protein (CETP) plays a major role in this pathway to transfer cholesterol esters from high-density lipoproteins (HDL) to VLDL and LDL, in exchange for triacylglycerols ,12. ThusCETP mediated exchange of cholesterol esters for triacylglycerols occurs mainly through an equimolar heteroexchange mechanism, which leads to alterations in cholesterol ester: triacylglycerol ratio. In addition to plasma CETP concentration, fatty acid composition of lipoproteins also modifies reverse cholesterol transfer. Cholesterol esters with n-3 polyunsaturated fatty acyl groups are more likely to transfer compared to cholesterol esters with saturated and monounsaturated fatty acyl groups . This efWe previously observed fish-oil-induced elevation of VLDL-and LDL-cholesterol concentrations and decrease in HDL-cholesterol concentration in F1B hamsters . In thisThe LDL-cholesterol, free cholesterol, cholesterol ester, phospholipid and triacylglycerol concentrations were measured in Bio F1B hamsters treated with fish-oil or MIX diet  to core lipids (cholesterol esters and triacylglycerols) ratio was higher in MIX diet fed hamsters compared to hamsters fed the fish oil diet, at both low and high fat levels Table . IncreasIt has been shown previously that there is a correlation between the level of CETP and that of LDL-cholesterol. To further investigate our observation of fish-oil-induced elevation of LDL-cholesterol concentrations and a decrease in HDL-cholesterol concentration in F1B hamsters, we measured CETP mass as well as CETP activity. The changes in CETP mass and CETP activity are shown in Figure CETP mass was significantly and positively correlated with CETP activity in low fat fish oil  and MIX diet (r = 0.96 p < 0.0003) fed hamsters Figure  and highThe correlations between CETP mass, CETP activity and LDL-cholesterol are shown in Figure CETP plays an important role in maintaining the levels of plasma LDL and HDL . IncreasCholesterol esters are more liable to transfer in fish-oil fed subjects since the core of the lipoprotein particles from these subjects have lower transition temperature  due to aIncreasing dietary fat level of fish oil decreased cholesterol ester transfer as reflected by decrease in CETP mass Figure . Thus, dHDL-total cholesterol and cholesterol ester concentrations were significantly lower in fish-oil diet fed hamsters compared to MIX-diet fed hamsters, while there was no difference in HDL-triacylglycerol concentrations Table . PreviouIncreasing the dietary fat level from low fat to high fat caused a decrease in HDL-total cholesterol and cholesterol ester levels Table , which mThe interactive effect of dietary cholesterol and n-3 PUFA on plasma cholesterol ester transfer is not yet known. Cholesterol supplementation caused an increase in plasma CETP activity in both fish-oil and MIX-diet fed hamsters at low fat levels  were obtained from Bio Breeders Inc.  and kept on chow diet for one week prior to feeding specific diets. After this equilibration period, hamsters were divided into 8 groups (n = 12) and each group was fed with one of the specified diets. The specified diets consisted of fat free semi-purified diet  that was supplemented with either fish oil  or a mixture of lard and safflower oil in 1.5:1 ratio (MIX diet) . The fatThe animals were maintained on specific diets for 2 weeks ad libitum. The food intake was measured daily during the study period, and the body weight was checked at the beginning of the study period, one week later and at the conclusion of the study. There was no difference in food intake and body weight gain between different diet groups. All animals were housed in individual cages in a single room with enriched environment. The Institutional Animal Care use Committee (IACC) approved all experimental procedures, which are in accordance with the principles and guidelines of the Canadian Council on Animal Care. After two weeks on specified diets the animals were sacrificed after 14 hrs of fasting. Blood was collected by cardiac puncture into tubes containing EDTA and centrifuged immediately to separate plasma. Plasma samples were stored at 4°C on ice until further use.g for 20 min at 12°C [Plasma was centrifuged at 15,500  at 12°C  to separ at 12°C . IsolateTotal cholesterol concentration was assayed in all lipoprotein fractions using cholesterol assay kit # 402 . Total triacylglycerol concentration of lipoprotein fractions was assayed using triglyceride assay kit # 344 . Free cholesterol in plasma and individual lipoprotein fractions was assayed using free cholesterol assay kit . Cholesterol ester concentration was determined by subtracting the free cholesterol concentration from total cholesterol concentration. Phospholipids were analyzed using the method of Bartlett [14C-cholesterol oleate [CETP in the plasma samples was assayed by a radioactive method  that wasl oleate . Plasma CETP mass in plasma samples was quantitatively assayed using CETP ELISA- DAIICHI kit  as previously published . ConcentP value was <0.05 [The effect of diet type, dietary fat level and dietary cholesterol was determined using 3-way analysis of variance, and a Tukey's post hoc test was used to test significant differences revealed by the ANOVA. Values are group means ± SD, n = 12; Differences were considered to be statistically significant if the associated as <0.05 .The author(s) declare that they have no competing interests.PPD conducted all the experiments in this study. PPD and AAM analyzed and interpreted the data, as well as drafted the manuscript. PJD is a collaborator on this project. SKC is the Principal investigator, has conceived the study, participated in its design and final approval of the version to be published. All Authors read and approved the final manuscript.


The speech signal contains both information about phonological features such as place of articulation and non-phonological features such as speaker identity. These are different aspects of the 'what'-processing stream (speaker vs. speech content), and here we show that they can be further segregated as they may occur in parallel but within different neural substrates. Subjects listened to two different vowels, each spoken by two different speakers. During one block, they were asked to identify a given vowel irrespectively of the speaker , while during the other block the speaker had to be identified irrespectively of the vowel (speaker categorization). Auditory evoked fields were recorded using 148-channel magnetoencephalography (MEG), and magnetic source imaging was obtained for 17 subjects.During phonological categorization, a vowel-dependent difference of N100m source location perpendicular to the main tonotopic gradient replicated previous findings. In speaker categorization, the relative mapping of vowels remained unchanged but sources were shifted towards more posterior and more superior locations.These results imply that the N100m reflects the extraction of abstract invariants from the speech signal. This part of the processing is accomplished in auditory areas anterior to AI, which are part of the auditory 'what' system. This network seems to include spatially separable modules for identifying the phonological information and for associating it with a particular speaker that are activated in synchrony but within different regions, suggesting that the 'what' processing can be more adequately modeled by a stream of parallel stages. The relative activation of the parallel processing stages can be modulated by attentional or task demands. This study explores attentional modulation within the 'what'-stream of the auditory modality during phoneme processing. Knowledge of speech sound representation in the auditory domain is still sparse. However, parallels to the extensively studied visual modality and also to the somatosensory domain are becoming evident. For example, columnar mapping of several stimulus properties  has been revealed in human and animal research: acoustic parameters like spectral bandwidth, periodicity, stimulus intensity ,2 or – f1-F2 distance) were located more posterior in auditory association cortex than responses to CORONAL vowels , and a topographical shift between these classes of vowels even when embedded in non-words has been reported  and [ø], both produced by a male and a female speaker. Subject's attention was either on the vowel or on the speaker difference, in a counterbalanced order. How would a controlled shift of attention from specific phonological features of speech to features of speaker identity affect the speech sound mapping in timing and topography of the brain response? Two concurrent outcomes are conceivable here: First, from the numerous parallels between the auditory and other sensory domains, one might expect a blurring of differences of the phonological map in auditory cortex when features such as the speaker identity rather than phonological differences are attended over minutes. Second, phonological processing could be the default process needed in all speech-listening situations and should therefore activate phonological feature maps irrespectively of attentional demands. We would then expect that the separate mapping of DORSAL and CORONAL vowels described previously  is unaffIn 21 of 22 subjects, a clear waveform deflection around 100 ms post vowel onset was observed . However, the effect was significant in source space that is not influenced by varying head-to-sensor positions: The [ø] dipole source strength, an estimate for the amount of massed neuronal activity, was larger for the [ø] vowel than for the [o] by 25 % or 6 nAm . No hemispheric differences in signal power between vowel categories or tasks were apparent.Analysis of the N100m root mean square (RMS) peak latency revealed foremost a main effect of vowel , that is, the auditory processing in the DORSAL vowel [o] was reflected by a more posterior ECD location , a more vertical orientation  than the [ø] source, and from an angular difference between the two vowel categories in the sagittal plane  and in the axial plane . None of these effects showed an interaction with hemisphere, but data gained further validity as the right-hemispheric sources were all located more posterior , more inferior  and were tilted more vertically  than their left-hemispheric counterpart. Such a difference is to be expected from previously reported N100 asymmetries between cerebral hemispheres  source and the CORONAL vowel [ø] ,17 couldThat is, a shift of subjects' attention to a non-phonological acoustic feature, the speaker identity, did not blur the spatial segregation within the speech sound map. In contrast, the [ø] and [o] generators were slightly displaced towards more posterior and more superior locations when subjects focused on speaker identity.In most situations, a listener may automatically extract the phonological invariants from the speech signal in order to access lexical information, for example the meaning of the information inherent in speech. Speaker-dependent features such as pitch and periodicity should not play a crucial role in this phonological decoding process. This is what we mimicked by asking our subjects to detect a certain vowel in a stream of varying speech sounds. However, in cocktail-party-like situations there is the additional demand to attend acoustic properties of certain speech streams or speakers, and we implemented it by asking our subjects to detect a certain voice in a stream of varying speakers. Speaker identification comprises an important but not necessarily orthogonal process to phonological decoding in speech perception: areas in the upper bank of the superior temporal sulcus (STS) have been identified previously  to be voData presented here suggest that the systematic mapping of speech sounds within the auditory cortex is robust under changing attentional demands and not tied to phonological awareness. However, the general shift of activity when a non-phonological speaker categorization must be accomplished shows that speech sound representations are modulated in their locations in a context-dependent manner. Situational demands obviously influence the differential but time-synchronous involvement of specialized neuronal assemblies that contribute to speech sound decoding in a top-down fashion. Hence, the spectrally high-resolving analysis of the incoming speech stream is performed at the same time but in different locations, i.e. in a different mix of cell assemblies than the analysis of speaker-dependent features .Further spatially high-resolution brain imaging studies are needed to quantify as to which extent voice-selective areas in the upper bank of the STS  become i22 subjects  participated in the procedure. All subjects were monolingual native speakers of German. Only right-handers as ascertained by the Edinburgh Handedness Questionnaire  were incIn an auditory target detection task, subjects listened to randomized sequences of four German natural vowel exemplars: The DORSAL rounded vowel [o] in two exemplars, in one spoken by a male voice and in the other by a female voice, and the CORONAL rounded vowel [ø], also produced by both voices Fig. . 200 ms In a test sequence, subjects repeated vowels aloud and recognized all stimuli correctly, i.e. they distinguished between both vowel categories and voices without difficulty. Binaural loudness was slightly re-adjusted where necessary to ensure perception in the head midline.phonological categorization), subjects had to press a button with their right index finger whenever a given vowel  occurred, irrespective of the speaking voice. In a task B , subjects had to press a button whenever a given voice  uttered a vowel, irrespective of the uttered vowel category. Fig.In the actual measurement, vowel exemplars were presented in two randomized sequences with equal probability and a randomized stimulus onset asynchrony of 1.6 – 2 s. All subjects performed – in a counterbalanced order – two different tasks during these two sequences: In a task A  –,37 –15,3Data acquisition and analysis, including source modeling, closely followed the procedure described in : AuditorWe analyzed up to 150 artifact-free vowel responses that remained for both vowel categories [o] and [ø] after off-line noise correction, and averaged them separately for vowel category but across speaker voice. Splitting up vowel conditions into male and female speaker sub-conditions was not possible due to a resulting small number of averages. However, we also performed separate averages and analyses of male and female speaker across vowel categories. In any case, the resulting averages thus contained brain responses to two acoustically variant exemplars which makes results more comparable to our previous studies . A 20 HzThe N100m component was defined as the prominent waveform deflection in the time range between 90 and 160 ms Fig. . IsofielN100m peak latency was defined as the sampling point in this latency range by which the first derivative of the Root Mean Square (RMS) amplitude reached its minimum and second derivative was smaller than zero. RMS was calculated across 34 magnetometer channels selected to include the field extrema over the left and the right hemisphere, respectively.Prior to statistical analyses, all brain response latencies were corrected for a constant sound conductance delay of 19 ms in the delivery system. Using the same sets of channels, an equivalent current dipole (ECD) in a spherical volume conductor  was modeled at every sampling point separately for the left and the right hemisphere . The N10As source location displacements do not appear exactly and exclusively along the Cartesian axes of the source space (cf. ), we addJ.O., T.E. and C.E. conceived the experiment and drafted the manuscript. J.O. and C.E. prepared the exact experimental setup. J.O. supervised data acquisition, and performed all data and statistical analyses. All authors read and approved the final manuscript.


Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations.Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium.Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer. Gene expression profiling, along with other methods to evaluate the global changes in genomes, provides the opportunity to understand whole scale changes present in human biology. Yet the sheer mass of data presented by these techniques often makes subsequent analysis difficult. Techniques such as gene expression profiling may result in the identification of hundreds if not thousands of differentially expressed genes that may be associated with the biological process, but may also represent noise related to the biological and technical variation. In an economic environment where limited resources are available for the follow-up and validation of potential target genes methods must be provided for the prioritization and sorting of data. Previous methods have relied heavily on the mapping of metabolic pathways or transcription factor binding sites -5. TheseSequence validated gene libraries for both the rat and human DNAs were obtained from Research Genetics , and were supplemented with additional DNA samples obtained from the University of Iowa rat clone sequencing program . The majWe have sought to compare the rat and human prostate cancer transcriptomes in an effort to judge the degree of similarity between the two cell types. Because the use of differentially expressed genes would bias the comparison by eliminating the majority of genes that do not show any difference, we used the absolute level of expression for each gene and compared the rat and human genes for significant differences in absolute expression levels. In order to derive the absolute level of expression for individual genes in human or rat prostate cancer cells we used expression values derived from the associated self-self hybridizations performed for each cell line. The experiments were facilitated by the use of slides that have been quality controlled for the quantity of spotted target DNA through the use of a FITC label third dye . These sThe presence of a large quantity of genes whose expression may be related to general cellular functions, as opposed to prostate specific metabolism, could infuse a significant amount of homogeneity to the data. In the presence of such homogeneity it may be impossible to identify the true differences that are related to prostate cellular function, and thus the perceived similarities may be artifactual. To address this issue we sought to repeat the analysis using only prostate related genes. To generate a list of such genes we used cDNAs in eight normal human prostate cDNA libraries present in the NCI Cancer Genome Anatomy Project . Generatin-vitro studies of prostate cancer cells) a Program,. AdditioProtein extracts were prepared and immunoprecipitations and/or western blots made from five day twenty-five micromolar Selenium treated or control PC3 or PAIII prostate cancer cell lines as described previously. In brieAfter expedited institutional review board approval normal prostate tissues and prostate cancer samples were obtained from de-identified discarded patient specimens. The formalin-fixed paraffin embedded specimens were prepared as 5 micron sections. Tissue microarrays were prepared from donor tissue blocks as 0.6 mm cores in 12 (4 × 4) or (5 × 5) grids with between 192 to 300 samples and used in the preparation of 5 micron sections. Immunohistochemistry was performed using primary rabbit polyclonal antibodies to the insulin-like growth factor binding protein 3 , or retinoic-X-receptor alpha  using methods previously described ,49. In bNone declared.M.W.D. was responsible for the conception and implementation of this project in association with P.J.T., M.S., and M.P. H.L., X.W., and G.Z. were actively involved in the programming, database construction, and testing of the software. M.S. and M.H. were responsible for spotted cDNA construction, hybridization, and experimental analysis along with M.W.D. Cell culture, western blots, immunoprecipitations, and selenium treatments were performed by M.S. with assistance by B.M. Tissue microarray staining and analysis was performed by M.W.D., R.D., T.B., and B.M. All the authors reviewed and accepted the final version of the paper.Table 1, Word document, Table of the genes identified in the selenium gene expression studies.Click here for file