Fires are increasing in severity and incidence around the globe and affect many different ecosystems; will the new generation of fire science tools help managers retain biodiversity? From Bob Clark's snug office in Boise, Idaho, where he manages the United States government's Joint Fire Science Program (JFSP), he figures his computer provides fingertip reach to just about everybody who's anybody in wildfire research. This points to a primary need of nations worldwide in combating the scourge of recurrent wildfires: tools and technology suited to the job. It's no small order in places as economically, socially, and ecologically varied as, say, Brazil, South Africa, Australia, Indonesia, and the United States, which are among the countries where wildfire creates the greatest havoc.More than 750,000 acres  were burned in southern California alone during last year's wildfires. The 2000 season was one of the country's worst on record, destroying 8.4 million acres (3.4 million hectares), more than double the decade's 10-year average. Australia's summer months around the turn of 2002–2003 brought perhaps the worst drought in a century to the populous southeast and the biggest fire season for two decades. Mountain forests were extensively burned and more than 500 houses were lost. In 2002, Brazil suffered 217,000 wildfires, a number that is almost certainly too low because remote imaging cannot detect many fires under the forest canopy. In Indonesia, wildfires that burned for months during 1997–1998 were later estimated to have released the equivalent to 13%–40% of annual global carbon emissions from fossil fuels, inflicting smoke-related ailments on thousands.Where wildfire is concerned, the many differences between such countries can perhaps be pinned down to two essentials. The first is whether a blaze occurs in temperate or tropical forest, and the second is whether the nation is developed or developing.“The science can be rock solid, but it can only go so far before social, economic, and political pressures take over,” Clark says. “That's what a forest service manager's job is, picking the best option based on all those considerations.”Unfortunately, having science-based options that are applicable to local conditions is largely a luxury for developed countries. Managers there can choose to let a fire burn under hopefully contained conditions, a policy known in the United States as “wildland fire use.” They can set experimental crown fires to study their effects, as was done recently in"journal" Canada . And theThe two main fuel-reduction methods are mechanical removal of combustible materials and controlled or “prescribed” burning . During Fire hazard reduction methods must be tailored to an understanding of fuel characteristics in a given area, says David Peterson of the Forest Service's Pacific Wildland Fire Sciences Laboratory in Seattle, Washington. “There's no uniform way of doing it, partly because, as scientists, we haven't given the management folks any quantitative guidelines.” Working with other ecologists, social scientists, and economists, he's currently producing just such guidelines for the dry interior forests of the Pacific Northwest. “One thing we don't want to do is take choices out of the hands of field managers working at the local level.”For those choices to be meaningful, managers need reliable information on the risk of wildfire outbreaks and on the future behavior of existing fires. This requires models and simulations that incorporate climatic conditions, particularly wind . At the “Two or three years ago, we couldn't have done this simulation on a single-processor laptop,” says one of its developers, physical engineer Bret Butler. “It would have taken two or three days. Now we can do it in a matter of hours.”The ability of these maps to show varying wind flow in valleys, at midslope and on ridgetops, is just the beginning. The next step is to feed these data into models that predict wildfire spread. Butler and colleagues have coupled their gridded wind technology to a fire growth model and tested it against the actual spread of several wildfires, including in Southern California last summer. Maps of actual and predicted surface winds showed strong similarities, encouraging Butler to foresee an ideal scenario in which fire fighting teams enter wind flow data online or by telephone to a central base where gridded wind maps and fire growth simulations are generated within hours, before operational decisions are made.Yet he admits that challenges remain, including the current inability of fire behavior simulation to account for diurnal winds in addition to cold front-driven flow. In mountainous terrain, for example, winds often move up-canyon in the morning and down-canyon in the evening. Moreover, the effect of vegetation on wind is not yet included in such models.Those issues and others are being addressed by researchers working on improvements to the regional weather forecasts of so-called mesoscale models. At the Forest Service's Rocky Mountain Research Center in Fort Collins, Colorado, meteorologist Karl Zeller and colleagues are contributing calculations of biological processes to mesoscale weather models. Their algorithms not only can account for diurnal winds but can predict the effects on local weather when vegetation takes in carbon dioxide and releases water vapor. This process can produce different fluxes of carbon dioxide drawn into the canopy and water vapor coming out, depending largely on the type of vegetation and its canopy density.Zeller's group has analyzed current mesoscale forecasts in the Rocky Mountains and found that in the daytime, they often are too hot in the high country and too cold in the plains. Water vapor estimates are too low in the mountains and too high in the plains, which Zeller thinks is because the models feed off soil moisture estimates, not off vegetation. In coupling his team's new biophysical interface to gridded wind and mesoscale forecast models, Zeller says “point forecasts” are being developed that can focus on a prescribed burn area or even a single house.Fitting the appropriate mix of strategies to a given situation is an issue that has also received close attention in Australia. After the bushfires of 2002–2003, media commentators called for increased “hazard reduction burning” in national parks, prompting ecologists around the country to distribute a joint statement declaring that such a strategy would not further reduce bushfire risk, but would actually threaten biodiversity. Australian species are often well-adapted to fire, and researchers have learned that different fire regimes—meaning the type of fire, its intensity, severity, extent, season, and frequency—favor different species . In the “A generic problem or conundrum seems to be that species which do not prosper under relatively frequent fires can be found in most fire-prone environments,” notes Ross Bradstock, principal research scientist in the New South Wales Department of Environment and Conservation. He says it's very difficult to determine how human interventions in various habitats can foster the coexistence of species that have different fire regime requirements.As tough as such questions are to answer in developed countries, they pale compared to the problems of tropical forest wildfire researchers and managers in developing countries. In these countries, a destructive cycle of human behavior begins with land-clearing and burning for farming, logging, mining, road-building, and other uses that open gaps in the rainforest's canopy cover. This lets in sunlight and air, reducing the forest's ability to smother fire by trapping moisture, and it encourages the growth of smaller, more fire-prone plants. The first wildfires that occur are bad, but successive ones can eventually transform tropical forest to scrub savanna . Of courIn a recent paper in Science, Michigan State University Amazon expert Mark Cochrane pointed out that prescribed burning is ineffective in tropical forests, because the collateral damage outweighs any benefits. Indeed, tools and technologies employed in temperate conditions can seldom be applied usefully to tropical forests without significant alterations.“One of the main issues in fire science is that the U.S. has no capacity to develop new tools,” charges Ernesto Alvarado, a research scientist at the University of Washington in Seattle. He's been working for several years with United States Forest Service and Brazilian scientists on field studies in Mata Grosso, the southernmost state of the Brazilian Amazon. He says that fire prediction simulations developed decades ago have not yet been replaced by ones that account for tropical wildfire extremes, including either large-scale crown fires or surface fires, which often reach only 10 centimeters (3.9 inches) in height and move slowly but can burn for weeks and kill many trees. Fire behavior models don't work for tropical surface fires because the physics are different from those in temperate forests, he explains. A slow wind generated from the unburned forest blows toward the fire, forcing the small flames to advance against, rather than with, the wind. Another difference is that the fuel is mostly leaf litter, not conifer needles or sticks.Alvarado and colleagues light experimental fires in clear-cuts to determine factors limiting ignition and spread. Such experimental work is rare in tropical forests, where observation and description still predominate. But the team also monitors surface wildfires, measuring fire length, spread, and heat release.“We're trying to find applications that people can use to control fires or to explain implications of fire policy,” he says. Most wildfires originate from deliberately set burns. For example, many farmers still clear land by the ancient method of slash-and-burn, in which forest is chopped, left to dry, and then burned. These farmers are now banned by Brazilian federal law from burning at the height of the dry season, mid-July to mid-September. They cut in May, but if the rains come early in September, they can't burn after the ban ends and must wait until the next season, with nowhere to grow their crops in the meantime. Alvarado thinks a more flexible burning schedule is a solution.The challenge is to pass on technological understanding to decision-makers. For example, even ranchers in Mata Grosso's economic elite usually haven't heard of fire management techniques, says Amazonian ecologist Carlos Peres at the University of East Anglia in the United Kingdom. Educational projects from nongovernmental organizations have helped to turn some farmers away from heavy reliance on slash-and-burn techniques, but fire suppression information remains to be distributed on the frontiers.“What we really need are very large areas of primary forest that effectively serve as fire breaks,” he says. Conservation plans have been made by the federal government in collaboration with international agencies, but implementation remains a question, particularly given the high level of economic pressure from multinational resource developers eager to enter the Amazon. Major roads through the jungle are also on the drawing board. “Different categories of conservation units can be gazetted on paper, but in practice they're a long way from working. Someone draws lines on a map high in an office in Brasilia, but when you go out to that place in the forest, no one knows it's a conservation zone.”Information transfer faces similar barriers in much of Southeast Asia, as Canadian forestry researchers discovered during a five-year project (now winding up) to create a computerized early warning tool for wildfire outbreaks. The program was instigated after the 1997–1998 fires created a regional haze hazard, largely because of peat deposits up to 20 meters (21.8 yards) thick that had become susceptible to burning in swampy forests drained and cleared for development.Michael Brady, who managed the Canadian project in Jakarta, points out that headmen in remote communities are still likely to believe that wildfires start spontaneously, by grasses rubbing together or even by magic. A fire scientist whose doctorate is in tropical forest peat dynamics, Brady sees the project as a medium to strengthen regional fire ecology in general. “In some ways, that's more important to me than the tool itself.”The tool is a variation of the Fire Danger Rating System used in Canada and, with various permutations, in many other countries. The Canadian system has two components, one for indexing fire weather and another predicting fire behavior. The weather component models moisture input and output in fuels generically classed as fine, moderate, and heavy. Brady and Indonesian university scientists grouped grasslands in the fine fuel category, fallen leaves and litter as medium, and peat and woody materials as heavy. They spent three years calibrating these fuels to local weather conditions, examining moisture dynamics and performing ignition tests. In developed countries, fuels are further specified in numerous classes for fire behavior prediction, but that requires decades of field work. Brady's team concentrated instead on helping key agencies in seven Southeast Asian countries, especially Indonesia and Malaysia, to obtain and use the appropriate computing tools.Brady doesn't expect immediate results in terms of reducing acreage burned. “Canada and the U.S. still have huge fire problems after working on it for a century.” But he does hope for a change of thinking, away from a current fascination in the region with satellite imaging of “hot spots” where fires are likely to be occurring. Fire danger rating concentrates on where fires are most likely to begin. “It allows you to add prevention into your management program.”In South Africa, “retention” is a conservation buzzword referring to strategies that, in a sense, go beyond prevention of problems. What ecologists hope to retain is biodiversity in the midst of changes that can't be stopped, and their methods are producing major repercussions throughout government. The work is centered on the Cape Floristic Region of Africa's southwestern tip. Almost 90,000 square kilometers  in area, it's the world's smallest floral kingdom. A conservation plan was launched in 1998 that has drawn cooperation from tourism, mining, water use, agricultural, and land use planning groups.The project has the ambitious goal of protecting not only the usual biodiversity patterns of conservation areas but also the spatial components of evolutionary processes that enable species to adapt to potentially harmful changes. This entails a complex effort to determine which parts of developed and undeveloped lands are most necessary to such processes, including rivers, sand movement corridors, gradients from uplands to coastal lowlands, and major wilderness areas. University of Port Elizabeth botanist Richard Cowling, one of the scheme's principal architects, estimates that it might require 60%–70% of the region's landscape.As in Australia, fires are important to the Cape's biodiversity, but too-frequent burns are a problem. Cowling thinks that by consolidating mountainous megawilderness under the project's plan and protecting spatial transitions between fire-prone areas and those that resist fire, managers could move toward allowance of natural fire regimes. The current problem, he says, is that protected areas usually stop short of the transition to semidesert areas that are privately owned. When fire spreads from public to private land, the government often gets sued. Under the evolving Cape plan, landowners will sit on governing boards, and property that they contract for conservation will be tax-exempt.The Cape plan has attracted millions of dollars in support from the World Bank and other international sources, but Cowling regards that achievement as much less important than the progress made in gaining support from various interest groups. “The key issue is the extent to which you can get biodiversity concerns mainstreamed to other sectors,” he says. Threats to habitat retention, not least of which is wildfire, endanger every species. “It's about making people realize that biodiversity is the basis upon which all other things will succeed.”Plants and animals of many countries evolved for millennia with wildfire as a natural occurrence, but when human interventions increase the frequency of fire, species suffer. African fire lilies and Australian “grass trees” are among plants that are stimulated to flower by smoke constituents such as ethylene. Plant seeds in fire-prone landscapes of Australia and South Africa often require fire to stimulate their germination, but it can take more than a decade for new seed banks to mature in some species. If a second fire arrives before then, the species could die out.Animals can be similarly affected. For example, a threatened marsupial called the potoroo is capable of surviving a high-intensity wildfire, but cannot tolerate the habitat changes caused by frequent, low-intensity fires. Likewise, some species of Australian honeyeaters are threatened with extinction because too-frequent fires have changed the proportion of mature and immature nectar plants. On the other hand, ecosystems can also be transformed by fire suppression. In southern Africa, decades of such activity have encouraged forests to replace grasslands. Yet the lovely marsh rose almost disappeared from the Cape before land managers realized that fire suppression was preventing its seeds from germinating. In such ways, biodiversity must find its place among the goals and tradeoffs of human intervention.


Ancel Keys, Ph.D., who died in November, 2004, at the age of 100, was among the first scientists to recognize that human atherosclerosis is not an inevitable consequence of aging, and that a high-fat diet can be a major risk factor for coronary heart disease. During World War II, he and a group of talented co-workers at the University of Minnesota conducted a large-scale study of experimentally-induced human starvation. The data generated by this study – which was immediately recognized to be a classic – continue to be of inestimable value to nutrition scientists. In his later years, Keys spent more time at his home in Naples, Italy, where he had the opportunity to continue his personal study of the beneficial effects on health and longevity of a Mediterranean diet. New York Times obituary put it, "remained intellectually active through his 97th year." His latter years were spent mostly at his home in Naples, ItaIy. I never had the privilege of knowing him well, but encountered him occasionally at scientific meetings where we were both speakers. He was friendly but, I thought, reserved. What struck me about Ancel was his remarkable absence from the counsels of the nutrition establishment. Despite his acknowledged expertise and importance in the field, he was not a member of AMA's Council on Foods & Nutrition (at least not during the many years I served on that organization). I never saw him at any of the NIH advisory committees on which I served. He did not play a role in the deliberations of the Food and Nutrition Board of the National Research Council. He was not involved in the American Society for Clinical Nutrition during its heyday. Why was this? Perhaps the fact that he was a physiologist (later an epidemiologist) and not a physician played some role. Also, I think he preferred to go his own way, and – to some extent – he remained aloof from "academic nutrition." Yet he was willing to lecture to many audiences and was not considered to be a scientific eccentric; to the contrary, his epidemiological work was frequently cited and praised, and his monumental study of experimentally-induced semistarvation in human subjects [Ancel Keys, who died in November, 2004, was an excellent testimonial to the health-promoting effects of his beloved Mediterranean diet. He lived to be 100 and, as the Keys and his capable associates conducted careful physiological and psychological studies of 32 initially healthy conscientious objectors (to World War II) through 6 months of experimentally induced semistarvation, followed by a year or more of rehabilitation. These studies generated a cornucopia of data – data that are all the more valuable now because such an experiment would not have a chance of being approved by today's Institutional Review Boards. Protein-calorie malnutrition (PCM) – in effect, famine – remains endemic in many parts of the world; moreover, PCM is the most common nutritional problem encountered in U.S. hospitals and nursing homes. The studies carried out by Keys and his co-workers make it possible for us to distinguish the effects of semistarvation on the body's strength, composition, physiological status, and mood from the confounding effects of such underlying diseases as cancer, intestinal malabsorption, renal insufficiency, emphysema, etc. – illnesses that often give rise to conditioned PCM. The Minnesota group showed clearly that semistarvation can be independently responsible for an array of psychological problems such as anxiety, depression, and hypochondria. From their studies, it is possible to demonstrate a clear relationship between a decline in fat-free mass and PCM-associated morbidity.New York Times obituary, dated November 23, 2004. For those of us who worked for so many years to call attention to the relationship of serum total cholesterol to risk of coronary heart disease (CHD), and to the cholesterol-raising effects of certain saturated fats, Keys will always be one of the major prophets who provided the early evidence that atherosclerosis is not an inevitable concomitant of aging, and that a diet high in saturated fat content can be a major risk factor for CHD. The practical outcome of the work in this field – to which Ancel contributed so much – is the extraordinary decrease in mortality from coronary heart disease that has occurred during the past half-century. Cancer has finally replaced heart disease as America's number one killer. Ancel had his well-deserved reward – a long, productive life unencumbered by an excess of committee meetings, and the opportunity to contemplate the Tyrrhenian sea while enjoying the benefits of a Mediterranean diet.Keys's major scientific achievements are enumerated in some detail by Jane E. Brody in her


On February 11, 2005, the New York City Department of Health and Mental Hygiene announced that a city resident had recently been infected with a multi-drug resistant form of HIV and rapidly progressed to AIDS. The Health Commissioner, Thomas R. Frieden, called for increased vigilance against this new strain. Is this situation an emerging crisis or simply an unusual case report of rapid HIV progression? Shortly after his diagnosis, testing, at the Aaron Diamond AIDS Research Center in Manhattan, revealed that his virus was resistant to almost all anti-HIV therapeutics. Further, despite being infected for only 2–20 months, the man had developed AIDS. NYC Health Commissioner Thomas R. Frieden, MD, MPH, stated, "This case is a wake-up call. First, it's a wake-up call to men who have sex with men, particularly those who may use crystal methamphetamine...now, we've identified this strain of HIV that is difficult or impossible to treat and which appears to progress rapidly to AIDS." Dr. Frieden called on this community to help stop the spread of this and other drug resistant strains of HIV. He also called on NYC doctors and the public health system to improve HIV prevention counseling, to perform HIV drug resistance testing among treatment naïve, HIVth Conference on Retroviruses and Opportunistic Infections (CROI) in Boston, Drs. David Ho and Martin Markowitz of the Aaron Diamond AIDS Research Center in Manhattan presented clinical and laboratory data regarding the NYC resident[+ T-cell count was 80 cells/mm3 and it has since fallen to less than 50. The NYC resident meets one criterion for the diagnosis of AIDS; his CD4+ T-cell count is less than 200 cells/mm3. His viral load has varied from ~100 K to 650 K/ml. The NYC resident's virus was tested and found to be resistant to all but two anti-HIV drugs, efavirenz (Sustiva®) and enfuvirtide . This high degree of drug resistance existed before the NYC resident was treated with any anti-HIV compound.At the 12Is this case a harbinger of a new epidemic with this superbug or is it just an isolated, forme fruste of HIV infection? No one knows the answer to this question yet, but we do have plenty of data to suggest that the latter is the case. In people naïve to drug therapy, bone fide antiviral resistance is uncommon. A recent USA based study of treatment-naïve patients found that the prevalence of mutations associated with drug resistance was 8.8%. This meA commonly held view on why the level of drug resistance is low is that most mutations associated with drug resistance, also decrease viral fitness. Except the K103N reverse transcriptase mutation, which confers resistance to the three approved non-nucleoside inhibitors (NNRTIs), other mutations, associated with high level drug resistance, are thought to significantly decrease viral fitness. Theoretically, in the absence of drug pressure in a newly infected individual, wild-type virus is either selected for during transmission or the transmitted, resistant virus mutates back towards the fitter wild type. The current observation is that the vast majority of viruses in treatment-naïve patients are sensitive to almost all drugs.+ T-cell counts less than 300 cells/mm3 at the time of diagnosis. It is unclear why these individuals became infected, while the vast majority of Δ32 homozygotes remain uninfected. Possibly, these 8 individuals have some other aberration, which allows them to become infected with an X4 virus and, in turn, leads to an accelerated disease course. Perhaps, the NYC resident has a similar abnormality, which has lead to an increased rate of CD4+ T-cell depletion.In addition to CD4, the virus, isolated from the NYC resident, uses CXCR4 or CCR5 to enter cells. Such vi+ T-cell count existing at the first visit after diagnosis (within the first 9 months of infection). Their model predicts that 7 in 10,000 patients develop clinical AIDS within 6 months of infection. This number increases to 45 in 10,000 after 12 months. Similarly, 10 in 10,000 HIV infected individuals have a CD4+ T-cell count less than 200 cells/mm3 after 4.5–9 months of infection.Also at CROI this year, Drs. Stephen Gange and Alvarez Muňoz from Johns Hopkins University Bloomberg School of Public Health presented models of rapid HIV progression probability, based on two, large prospective cohorts. These s+ T-cell depletion. Therefore, it is highly probable that the NYC resident has a genetic predisposition, which led to rapid progression, rather than a new strain of HIV-1, which is simultaneously super-aggressive and multi-drug resistant.Several reports of rapidly progressing HIV infection have been published. The rapid disease course of the NYC resident is rare, but hardly unique. To date, no cluster of rapid progressors has been described. All rapid progressors have been unrelated, either genetically or virologically. While multi-drug resistant (MDR) viruses may be overall less fit compared with wild-type, drug sensitive strains, MDR HIV still causes steady CD4Our experience at a large, inner city HIV clinic is in agreement with the above data. We do not see rapidly progressing, newly diagnosed individuals. We also do not see MDR HIV in our treatment-naïve patients. Review of our data does not reveal any evidence of MDR virus in persons, who have never been on therapy.+ patients is prudent, especially when the person is thought to have been infected within the past year. Of course, most patients do not know when they were infected, so we are testing each new patient.To determine whether this "superbug" has spread to others, the NYC Department of Health is appropriately and aggressively investigating the sexual contacts of the NYC resident. The reason for the NYC Department of Health press release at this early point in the investigation is unclear. In the absence of a documented cluster of patients, should the entire health system react? No, we should wait for more information. I do agree that genotypic resistance testing for treatment-naïve HIVGiven the availability of free, rapid testing for HIV in New Jersey, we are strongly encouraging any one with current or previous high-risk behavior to get tested and determine his/her HIV status. The best way to fight this disease is with knowledge: knowledge on one's infection status, knowledge on how to avoid becoming infected, and knowledge on how not to infect some one else. HIV is not the common cold. It is transmitted through well-described behaviors, predominantly sex, especially receptive anal intercourse, and intravenous drug use with shared needles. These behaviors can be modified to reduce or eliminate the risk of contracting HIV. Two recent studies conclude that universal testing for HIV is a cost effective way to combat this infection in the USA -9. Outre


In this issue, Keith Yamamoto and colleagues show that nhr-49 controls two different aspects of fat metabolism, which interact to form a feedback system controlling the consumption and composition of fats in the nematode.Regulating metabolism of fat is an important challenge for any animal, from nematodes to humans. Central players in the regulatory network are the nuclear hormone receptors (NHRs), which are transcription factors that turn on or off a set of target genes when bound by specific lipid molecules. NHR genes number 48 in mammals, and a surprising 248 in nematodes. Despite the difference in quantity, there are some structural similarities between NHRs in these two groups, in particular, between the nematode gene nhr-49 was absent, the lifespan of the nematode was reduced by more than 50%, and the animal displayed numerous gross abnormalities in the gut and gonad. This was accompanied by unusually high fat content in the larvae. By using quantitative PCR to measure output of fat and glucose metabolism genes, the researchers showed that deletion of nhr-49 changed expression of 13 of these genes, with the most dramatic effects occurring within two metabolic pathways: mitochondrial lipid oxidation and fatty acid desaturation.Using RNAi to suppress gene expression, the researchers discovered that when nhr-49-suppressed larvae. One of nhr-49's normal functions is to increase expression of the mitochondrial acyl–Coenzyme A (CoA) synthetase gene acs-2. A principal role for mitochondrial acyl-CoA synthetases is to “activate” free fatty acids for transport into mitochondria, where they are oxidized. This process involves attaching a CoA group to a free fatty acid, and often serves as a rate-limiting step in lipid oxidation. Indeed, the authors found that suppression of acs-2 alone was sufficient to reproduce the highfat phenotype, while overexpression of acs-2 rescued the phenotype even in the absence of nhr-49.Oxidation degrades lipids to release energy, explaining the build-up of fat in nhr-49 increases expression of several desaturases, most importantly fat-7, which converts stearic acid to oleic acid; deletion of nhr-49 more than doubled the proportion of stearic acid compared to oleic acid.Fatty acid desaturation is the process of converting saturated fats into unsaturated ones, by forming one or more double bonds between adjacent carbons in the tail. This process is catalyzed by fatty acid desaturase enzymes. fat-7 alone produced two interesting results. First, it shortened the nematode life span, suggesting this was the primary pathway through which nhr-49 suppression exerted that same effect. Second, it produced some effects that were opposite those of nhr-49 suppression: specifically, it reduced rather than increased fat content, and it increased rather than reduced expression of acs-2.RNAi interference of nhr-49 sets in motion two opposing pathways: it increases acs-2, which leads to reduction of fat content, and it increases fat-7, which, by reducing acs-2, increases fat content. Surprisingly, this behavior links nhr-49 most closely not to HNF4, with which it shares the most structural similarity, but to another type of mammalian NHR, called peroxisome proliferator-activated receptors (PPARs). Further investigation of this link may lead to better understanding of the functions of PPARs, and provide opportunities for altering their function for treatment of fat metabolism disorders such as diabetes and obesity.These results show that in its normal actions,


To measure the prevalence of and associations among impairments, activity limitations and participation restrictions in persons living with HIV in British Columbia to inform support and care programs, policy and research.A cross-sectional population-based sample of persons living with HIV in British Columbia was obtained through an anonymous survey sent to members of the British Columbia Persons With AIDS Society. The survey addressed the experience of physical and mental impairments, and the experience and level of activity limitations and participation restrictions. Associations were measured in three ways: 1) impact of types of impairment on social restriction; 2) impact of specific limitations on social restriction; and 3) independent association of overall impairments and limitations on restriction levels. Logistic regression was used to measure associations with social restriction, while ordinal logistic regression was used to measure associations with a three-category measure of restriction level.The survey was returned by 762 (50.5%) of the BCPWA participants. Over ninety percent of the population experienced one or more impairments, with one-third reporting over ten. Prevalence of activity limitations and participation restrictions was 80.4% and 93.2%, respectively. The presence of social restrictions was most closely associated with mental function impairments . All limitations were associated with social restriction. Among those with ≤ 200 CD4 cells/mm3, odds of being at a higher restriction level were lower among those on antiretrovirals , while odds of higher restriction were increased with higher limitation . Among those with > 200 CD4 cells/mm3, the odds of higher restriction were increased with higher limitation , as well as by additional number of impairments .This population-based sample of people living with HIV has been experiencing extremely high rates of impairments, activity limitations and participation restrictions. Furthermore, the complex inter-relationships identified amongst the levels reveal lessons for programming, policy and research in terms of the factors that contribute most to a higher quality of life. For most people who are able to access and tolerate highly active antiretroviral therapy (HAART), HIV/AIDS has become a chronic condition characterized by cycles of illness and wellness. People live longer lives, but with physical, psychological and social challenges that affect quality of life -3. EvideFor instance, Brashers et al 1999) identified four categories of "uncertainties" resulting from the experience of "revival" brought about by HAART, including (a) renegotiating feelings of hope and future orientation in the face of questionable durability of immune restoration; (b) fear about social roles and identities, in the transition from a person who is dying to a person living with a chronic illness; (c) concerns with interpersonal relations, including the potential of stigmatizing reactions from employers and co-workers; and, (d) reconsidering the quality of their lives, captured in this quote from one participant, 'The good news is you're going to live, the bad news is you're not going to enjoy the rest of your life' ow well can you manage these typical daily activities?" with an indication to circle the response which best describes their experience in the past month. A fifteen-item list including ability to walk one block, eat, shower, and dress followed. For each item, participants indicated whether they were (a) completely able, (b) somewhat limited or (c) unable to perform the activity. Overall prevalence of activity limitations was calculated by including anyone indicating (b) or (c) for any one of the fifteen items.In the same way, participants were asked " [h]as your health limited your usual [role/participation]" in any of a number of categorical activities and functions. Participants were indicated to choose the response that came closest to the way they had been feeling during the past month. A ten-item list was used to assess levels of restriction in social, student, and cultural roles. Participants indicated whether they were (a) not limited, (b) somewhat limited or (c) very limited with respect to their ability to function in these roles. Overall prevalence of participation restrictions was calculated by including anyone indicating (b) or (c) for any one of the ten items.Rates of impairments, activity limitations and participation restrictions among the participants were compared across three categories of CD4 cell counts  using a chi-squared test for categorical variables and the Kruskal-Wallis test for continuous variables. Bonferroni corrections for multiple comparisons were done for each item and those which remained significant are indicated in bold.To test the hypothesis that social role restrictions would be more strongly associated with mental function impairments and personal care and mobility limitations, a series of logistic regression models were tested with each category of impairment and limitation. A dichotomous outcome was used, collapsing "somewhat" and "very much" social role restriction into any social restriction. Likewise, specific activity limitations were dichotomized into "no limitations" vs. "some effort" required or "unable" to accomplish the activity. Associations of social restriction with impairment categories and specific activity limitations were examined univariately and in adjusted models accounting for age, sex, income, depression, pain, risk category  and number of symptoms for activity limitation models.A scoring system was then used to develop categories of activity limitation and participation restriction. If a participant indicated an activity limitation item at the highest level ("unable" to accomplish) or a participatory role restriction at the highest level ("very much" restricted), two points were received, while participants indicating an activity limitation item at moderate level (requiring "effort" to accomplish) or a participation restriction item at a moderate level ("somewhat" restricted), one point was received. Overall scores for participation restriction and activity limitation were therefore dependent on both the severity and total number of challenges in activities or participatory roles.The participation restriction score, with an overall maximum of 20, was then categorized into three levels: 0 to 5 points, 6 to 10 points and > 10 points, based on the population distribution of the score. Likewise, the activity limitation score, with an overall maximum of 28, was also categorized based on distribution as follows: 0, 1 to 5 points, and > 5 points. The higher the score, the greater the disablement.An overall model examined the associations of increasing participation restriction level with number of impairments and activity limitation scores, testing the hypothesis that impairments may account for some of the associations seen between activity limitations and participation restrictions, but that both of the former would have independent associations with the latter. Ordinal logistic regression was implemented, using the three-level participation restriction outcome and testing number of symptoms, categorical limited activity score, pain and mental diagnoses as explanatory variables. All models were stratified on CD4 levels, with separate models built for individuals with counts below 200 cells/mm3, and adjusted for age, gender, employment, years since diagnosis and risk category.Of the 762 people living with HIV who completed the survey, 614 provided information about their CD4 levels and were included in this analysis. The population answering the BCPWA survey was comprised mainly of white (88.7%), sexual-minority males (76.6%) between the ages of 30 to 49 (63.9%). The 148 respondents who were not included in the analysis because they did not provide CD4 information were in a lower income bracket , were more likely to be current IDUs  and more likely to be First Nations/Inuit/Metis .A comparison of all BCPWA members who received the survey and the subset who responded found a similar distribution of age and a similar proportion identifying as Aboriginal (7.1% vs. 8.7%). The proportion of females was higher among the total BCPWA population than among the subset of respondents .Table The presence of multiple impairments among the participants was also high, with a median of 7  impairments and approximately one third of the participants experiencing more than ten impairments. At least one impairment was reported by 91.5% (N = 697). There was a significant difference in the distribution of impairments across CD4 categories, which remained after Bonferroni correction . Mental impairment was reported by 78.2% (N = 596), sensory impairment by 71.9% (N = 548), neuromuscular impairment by 49.5% (N = 377), and internal impairment by 81.0% (N = 617) of the participants.Pain was reported by over half of the participants, and by over three quarters of the participants with CD4 ≤ 200 cells/ml. Approximately one-third reported little or mild pain and 37.1% reported moderate or severe pain. For participants with lower CD4 counts, more people reported moderate and severe pain , although comparisons of each CD4 category to the others showed no significant differences.Activity limitations were reported by 80.6% (N = 607) of the participants. The median number of activity limitations reported by an individual was 3 . Six hundred and ninety-nine individuals (93.2%) reported some level of participation restriction. The median number of participatory roles in which individuals felt somewhat or highly restricted was 7 . Although distributions of activity limitations and participation restrictions were significantly different, adjustment for multiple comparisons across the CD4 categories resulted in no significant difference in prevalence.Figures Table Adjusted odds ratios stratified by CD4 counts remained significant for getting groceries, doing laundry, household chores, and mental functioning, regardless of CD4 levels, although the estimates were higher for participants with counts under 200 cells/mm3. For those with CD4 counts above 200 cells/mm3, difficulties with eating, public transportation, moderate or vigorous activities, sexual activities, neuromuscular functioning and sensory functioning also remained significantly associated with social restrictions. Adjusted odds ratios for using the toilet and getting dressed were unable to be estimated as these were co-linear with the outcome. Stratified, unadjusted estimates of 9-fold and 37-fold increases in social restriction were seen with limitations in toileting.Stratification by CD4 levels indicated a general effect modification across activity limitations and impairment categories, with greater, although more unstable, associations with social restrictions being found among participants with < 200 cells/mm3.Table Among participants with CD4 counts above 200 cells/mm3, being in a higher category of participation restriction was associated with increasing levels of limited activity [ and ]. A higher participation restriction category was also significantly associated with increasing number of impairments, with a 19% increase in the odds with additional impairment. Increased participation restriction level was only marginally significantly associated with moderate or severe pain; however, point estimates for the pain categories suggested a dose response relationship, as did the inclusion of pain as a continuous variable .This study has demonstrated that a population-based sample of people living with HIV in British Columbia have been experiencing strikingly high levels of depression, body impairments, activity limitations and participation restrictions. The latter two categories were higher among this population than a national survey of HIV positive persons in the United States. HoweverThe level of depression among this population was extremely high. Nearly 60.0% of the participants reported ever having been diagnosed with depression by a doctor. Levels of depression among HIV positive persons reported in the literature range from 5.0% to 40.0%, although among HIV positive women, 60.0% prevalence has been reported ,22. DeprThe prevalence of impairments was also high, with diarrhea at the top of the list, followed by problems with fatigue and endurance. Furthermore, challenges with daily activities and social roles were extremely common, at greater than 80.0 and 90.0%, respectively. The high proportion of individuals experiencing impairments, activity limitations and participation restrictions sheds light on the spectrum of challenges related to living with HIV. Even among those with relatively high CD4 counts, the impact of HIV on disability and health is not trivial.Of note, the differences experienced between people according to categories of CD4 levels were less and less apparent going from impairments (problems at the level of organ or body part) to activity limitations to participation restrictions . This draws attention to the variety of influences affecting a person's ability to perform daily tasks and participate in regular societal roles above and beyond his/her clinical measures of disease status.All types of activity limitation were associated with the experiencing of social role restrictions. After accounting for impairments, depression and pain levels, there remained significant associations between household upkeep, including laundry and groceries, and social role participation. Although it was hypothesized that personal care issues would have stronger associations, this was not the case. This may be because the severity of personal care limitations  is such that among those experiencing these limitations, the presence of pain or numerous impairments overshadows any independent association between the limitation and social restriction.Household chores, getting groceries and doing laundry as well as moderate and vigorous activities had significant associations with social role restrictions and had the highest prevalence in this population. Therefore, interventions that target these types of limitations may provide the most benefit at a population level. Whether or not these interventions would have any impact on an individual's feelings of participatory restriction remains to be seen; however, coordinating these types of simple interventions might offer contact with people in need of social support.Mental impairments were the most prevalent of the four impairment categories and were found to have a significant association with participation restrictions. These results mirror a study describing disability among a national sample of people living with HIV in the United States which reported a correlation between general fatigue and increased limitations in both physical and role functions . Other rThe adjusted models Table  indicateLimitations of the study include the somewhat homogeneous nature of the participants, which affects the generalizability of these findings to other populations. The participants were mainly white, sexual-minority males with moderate yearly incomes and stable housing. The under-representation of people who are homeless, injection drug users, female and Aboriginals becomes apparent when comparing the low proportions seen amongst the BCPWA membership to the higher proportions seen in incident cases reported by the British Columbia Centre for Disease Control .The survey was sent to BCPWA members consenting to receive mail. Individuals who did not consent were more likely to reside in the Greater Vancouver region, suggesting a greater geographical representation from outside of this urban area. Non-consenting BCPWA members were also more likely to be female (15.8% vs 11.9%) and more likely to be First Nations, Inuit or Metis (27.1% vs 8.4%). Furthermore, because the survey was anonymous and self-reported, there are issues with missing data and incomplete records. For example, almost 20.0% of the sample, again representing a high proportion of women and First Nations, were excluded because of missing CD4 information. While the exclusion of this population may have affected the power and generalizability of the study, one may argue that challenges reported in this study may be an underestimation of the restrictions in this population due to compounding social inequity issues.Lastly, there are limitations in the nature of self-reported diagnoses. Participants may have trouble recalling the presence or absence of impairments, limitations or restrictions over the past month. Although there was no direct incentive, participants may be biased towards increased reporting of problems as they may feel that this would be beneficial for program funding and support.Despite these limitations, this survey represents a large provincial sample and is one of few attempts to collect information from a population-based sample on this scale. Furthermore, this is one of the first studies to systematically quantify levels of disablement among persons living with HIV.This study revealed a strikingly high prevalence of impairments, activity limitations and participation restrictions among a population-based sample of people living with HIV in British Columbia. The complicated interplay among these categories requires further study, but it is clear that interventions designed to help overcome activity limitations and social support programs are required, especially those addressing mental impairments and depression. While impairments and limitations are not always reversible, innovative programs that help people living with HIV address these challenges may help to decrease the subsequent high rates of participatory restrictions experienced. Antiretroviral treatments have enabled the prolongation of the lives of people who are HIV-infected; now we need to give due attention to optimizing the quality of these extended lives.MR and KC carried out the statistical analyses; SN and AS participated in the design of the study and the development of the study instrument; PB participated in the conceptualization of the study and the interpretation of the results; RH participated in the conceptualization and design of the study. All authors read and approved the final manuscript.


Prostate cancer follow up forms a substantial part of the urology outpatient workload. Nurse led prostate cancer follow up clinics are becoming more common. Routine follow-up may involve performing DRE, which may require training.The aim of this audit was to assess the factors that influenced the change in the management of prostate cancer patients during follow up. This would allow us to pave the way towards a protocol driven follow up clinic led by nurse specialists without formal training in DRE.194 prostate cancer patients were seen over a period of two months and all the patients had DRE performed on at least one occasion. The management was changed in 47 patients. The most common factor influencing this change was PSA trend. A change in DRE findings influenced advancement of the clinic visit in 2 patients.PSA is the most common factor influencing change in the management of these patients. Nurse specialists can run prostate cancer follow-up clinics in parallel to existing consultant clinics and reserve DRE only for those patients who have a PSA change or have onset of new symptoms. However larger studies are required involving all the subgroups of patients to identify the subgroups of patients who will require DRE routinely. Prostate cancer ranks first amongst all male urological cancers . In the Widespread PSA testing and increased awareness has led to the detection of early prostate cancer in many patients . This haFaithfull et al studied the use of telephone follow up of prostate cancer patients by nurse specialists. They found that this method of follow-up at 3, 6 and 12 weeks post radiotherapy was effective and economical .The EAU guidelines  suggest The aim of this audit was to prospectively assess the various factors that influence a change in the management of the prostate cancer patients on follow up and to highlight the feasibility of nurse led clinics for the follow up of prostate cancer patients.Over a two-month period (Dec 2002–Jan 2003) all the prostate cancer patients being followed up in the Urology outpatient clinics at our institution were audited prospectively. The patients were seen by a Consultant, Specialist Registrar or Senior House Officer. The period of follow-up, initial stage of the disease, management modality, consecutive PSA values and consecutive DRE findings (if available) were recorded on specifically designed data collection forms. All the patients had DRE done on at least one occasion. The change in the management was defined as any alteration in the follow-up pattern; either as an advancement or postponement of a future appointment, the need for further investigation or treatment, the admission of a patient and the referral to a different specialist, for example an Oncologist or Palliative Care specialistThe attending physicians were requested to record whether there was any change in the management and which factors influenced the change. They were specifically requested to record whether DRE influenced a change.During the period studied 194 patients being followed up for treated prostate cancer were included. The mean age was 74.8 years and the stages at initial diagnosis were: T1 (n = 73), T2 (n = 63), T3 (n = 44), T4 (n = 14). Ten patients had metastatic disease. The management modalities that these patients had undergone included: hormonal manipulation (68), orchidectomy (8), radical radiotherapy with hormonal manipulation (15), radical radiotherapy (48), radical prostatectomy (21), brachytherapy (1) and active surveillance (33) (Table 2b disease to T3 disease as observed by the assessor). This only resulted in the subsequent visit being sooner than planned.In this audit PSA trend was the most common factor that resulted in a management change. In the two patients there was a change in DRE findings  that influence a change in the management of these patients have not been reported.The present audit shows that PSA trend is the most common factor influencing a change in management whilst DRE plays a very limited role. Further, there are other factors that influence a change in the management of these patients' e.g. Bone pain and LUTS.Although the numbers of patients involved in this audit are moderate it would suggest that Nurse Specialists could deliver the optimum care in following up treated prostate cancer patients. Such Nurse led clinics could be carried out in parallel to the existing Consultant clinics thereby allowing the availability of medical personnel to perform DRE where deemed necessary. A protocol to perform DRE when there is an increase in PSA, onset of new symptoms or worsening of existing symptoms would be suitable for such a clinic. This audit suggests that Nurse Specialists need not be trained to perform DRE before the establishment of such clinics. However larger studies are required to identify subgroups of treated prostate cancer patients who may require a DRE on a regular basis. Alternatively nurses could be taught to undertake DRE thereby further reducing clinician workload. This would require a standardised and validated teaching method, which currently does not exist. In our hospital this audit has influenced the initiation of Nurse led prostate cancer follow up clinics conducted in parallel to the consultant clinics.The author(s) declare that they have no competing interests.NR – Along with VKS conceived the study, collected the data and jointly prepared the text with VKS – Along with NR conceived the study, assessed the data and prepared the text, SG – Participated in collecting patients details and in the preparation of the text, JH – helped in approaching the patients and data collection, SSM – Advised regarding the design of the study and contributed to the text, MEW – Advised regarding the design of the study and contributed to the text, RAB – Overall supervision of the project with periodic assessment on progress and preparation of textAll authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Investigation of bioheat transfer problems requires the evaluation of temporal and spatial distributions of temperature. This class of problems has been traditionally addressed using the Pennes bioheat equation. Transport of heat by conduction, and by temperature-dependent, spatially heterogeneous blood perfusion is modeled here using a transport lattice approach.We represent heat transport processes by using a lattice that represents the Pennes bioheat equation in perfused tissues, and diffusion in nonperfused regions. The three layer skin model has a nonperfused viable epidermis, and deeper regions of dermis and subcutaneous tissue with perfusion that is constant or temperature-dependent. Two cases are considered: (1) surface contact heating and (2) spatially distributed heating. The model is relevant to the prediction of the transient and steady state temperature rise for different methods of power deposition within the skin. Accumulated thermal damage is estimated by using an Arrhenius type rate equation at locations where viable tissue temperature exceeds 42°C. Prediction of spatial temperature distributions is also illustrated with a two-dimensional model of skin created from a histological image.The transport lattice approach was validated by comparison with an analytical solution for a slab with homogeneous thermal properties and spatially distributed uniform sink held at constant temperatures at the ends. For typical transcutaneous blood gas sensing conditions the estimated damage is small, even with prolonged skin contact to a 45°C surface. Spatial heterogeneity in skin thermal properties leads to a non-uniform temperature distribution during a 10 GHz electromagnetic field exposure. A realistic two-dimensional model of the skin shows that tissue heterogeneity does not lead to a significant local temperature increase when heated by a hot wire tip.The heat transport system model of the skin was solved by exploiting the mathematical analogy between local thermal models and local electrical (charge transport) models, thereby allowing robust, circuit simulation software to obtain solutions to Kirchhoff's laws for the system model. Transport lattices allow systematic introduction of realistic geometry and spatially heterogeneous heat transport mechanisms. Local representations for both simple, passive functions and more complex local models can be easily and intuitively included into the system model of a tissue. Heat transfer in biological systems is relevant in many diagnostic and therapeutic applications that involve changes in temperature. For example, in hyperthermia the tissue temperature is elevated to 42–43°C using microwave ,2, ultraContact heating is used in transcutaneous blood gas monitoring, in which oxygen is transported out of the vasodilated capillary bed to a surface mounted oxygen sensor. Heating is used to achieve vasodilation. In 1851 it was already known that "skin breathing" occurs, in which oxygen diffuses out of ambient air into the body, supplying of order 1% of the body's oxygen uptake . TypicalInitial clinical demonstration with neonates occurred in 1969 when a polarographic electrode placed on the head was used to measure oxygen partial pressure . Since tSpatially distributed heating of skin and deeper tissue by electromagnetic fields and ultrasound is also of established interest -20. MicrIn hyperthermia, tissue is heated to enhance the effect of conventional radio- or chemotherapy. By delivering thermal energy, the tissue is stimulated to increase the blood flow by thermoregulation in order to remove the excess heat. The common method to produce local heating in the human body is the use of electromagnetic waves.ω . Alternatively, ω can be replaced by ωm, the nondirectional mass flow associated with perfusion. Perfusion is valid on the spatial scale of ~100 μm. The contributions of heat conduction and perfusion are combined in the Pennes bioheat equation  exposure ).-2 10 GHz pulse for 3 s. The layer of air farthest from the skin was set at 25°C and the core (2 cm below the surface) was set to 37°C. This resulted in the skin/air interface having a steady-state temperature of 34°C before the microwave exposure. The skin/air interface has a power transmission coefficient (|Tsa|2Re{Za/Ze}) of 0.49 at 10 GHz. Applying 10 GHz microwave results in an essentially linear rise in temperature, in agreement with prediction using other methods. When the input power level is less than 5 W cm-2, the peak surface temperature is less than 42°C. When the microwave exposure is turned off, relaxation of the skin temperature occurs over a time scale of several seconds. Onset of tissue damage occurs when the local tissue temperature reaches 42°C. The distribution of tissue damage with depth is shown for different power densities  with a hot tip that is inserted into the epidermis is also modeled. The model assumes that the tip of the metal wire  is enclosed in a thermally insulating material . The skin model contains stratum corneum, epidermis and dermis. As before, the core temperature (37°C) is fixed at 2 cm from the skin surface by extending the subcutaneous layer. Before heating the wire conducts heat outwardly to the air, consistent with the isotherms. The temperature at the hot wire tip is increased to 45°C at t = 10 s. The temperature contours at different time points is shown in Fig. The use of transport lattice approach for predicting heat transport in spatially heterogeneous structures is further illustrated with a two-dimensional model of the skin. The model is derived from an image of a histological section of skin Fig. . The temin vivo and in vitro studies have shown that the tissue response to heat stress is strongly temperature-dependent [Both ependent ,64,65. Wependent . In ordeAs shown in Fig. et al. [et al. [A more comprehensive non-linear temperature dependent perfusion model has been applied in modeling hyperthermia. Tompkins et al.  used tem [et al.  employedWe present a modular approach to modeling in which the skin is represented by three homogeneous layers, each with many interconnected local, steady state models that account for the local heat storage (heat capacity), local heat dissipation  and local transport by both conduction and perfusion Fig. . The exiAs shown in Fig. Prolonged exposure to elevated temperatures can cause tissue damage by, for example, protein alteration or denaturation, often followed by recognizable changes in the optical properties of tissue . The ratWhen skin is exposed to a 10 GHz pulse of 3 s duration, the tissue damage indicator near the skin surface may be as high as 0.08, which suggests some damage at high power levels Fig. . This exTransport of heat by conduction, and by temperature-dependent, spatially heterogeneous blood perfusion, is predicted using a transport lattice model. This approach uses interconnected, local, steady state models for transport and storage, to together represent the Pennes bioheat equation. The thermal system model of the skin was solved by exploiting the mathematical analogy between local thermal models and local electrical (charge transport) models, thereby allowing robust, circuit simulation software to obtain solutions to Kirchhoff's laws for the system model. The skin model has a nonperfused viable epidermis, and deeper regions of dermis and subcutaneous tissue with perfusion that was constant or temperature-dependent. Spatially distributed heating and surface heating cases were considered. Accumulated thermal damage was estimated by using an Arrhenius type relation at locations where viable tissue temperature exceeds 42°C. Prediction of spatial temperature distributions was also illustrated with a two-dimensional model of skin created from an image. Validation of the transport lattice approach using experimental data is necessary for practical application of this method.TRG constructed and solved the several transport lattice models and wrote much of the manuscript. DAS computed the reflected and transmitted power in the skin layers, contributed to construction and solution of the models, and to writing of the manuscript. GTM provided guidance and advice with respect to thermal modeling, and helped write the manuscript. JCW conceived the local transport lattice model for solving the bioheat equation, provided overall guidance and helped with interpretation of results and writing the manuscript. All authors read the final manuscript.


Tpl2/Cot oncogene has been identified in murine T-cell lymphomas as a target of MoMuLV insertion. Animal and tissue culture studies have shown that Tpl2/Cot is involved in interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) production by T-cells contributing to T-cell proliferation. In the present report we examined a series of 12 adult patients with various T-cell malignancies, all with predominant leukemic expression in the periphery, for the expression of Tpl2/Cot oncogene in order to determine a possible involvement of Tpl2/Cot in the pathogenesis of these neoplasms.Our results showed that Tpl2/Cot was overexpressed in all four patients with Large Granular Lymphocyte proliferative disorders (LGL-PDs) but in none of the remaining eight patients with other T-cell neoplasias. Interestingly, three of the LGL-PD patients displayed neutropenia, one in association with sarcoidosis. Serum TNF-α levels were increased in all Tpl2/Cot overexpressing patients while serum IL-2 was undetectable in all subjects studied. Genomic DNA analysis revealed no DNA amplification at the Tpl2/Cot locus in any of the samples analyzed.We conclude that Tpl2/Cot, a gene extensively studied in animal and tissue culture T-cell models may be also involved in the development of human LGL-PD and may have a role in the pathogenesis of immune manifestations associated with these diseases. This is the first report implicating Tpl2/Cot in human T-cell neoplasias and provides a novel molecular event in the development of LGL-PDs. Cells may transform to a malignant phenotype following accumulation of distinct genetic events that result in altered protein expression pattern, thus facilitating uncontrolled proliferation. Such genetic events target specific oncogenes that act in concert to provide the malignant phenotype.lck promoter in mice results in rapid development of T-cell lymphomas [Tpl2/Cot oncogene was initially cloned as a MoMuLV proviral integration locus in murine T-cell lymphoma cells, resulting in its carboxy-terminal truncation,2. Expreymphomas . Expressymphomas -7. In thymphomas . Tpl2/CoStudies in human tumor specimens have shown that Tpl2/Cot is overexpressed in early stage breast cancer , in EBV-Given the compelling evidence of the importance of Tpl2/Cot in experimental and tissue culture models of T-cell neoplasias, we designed a study to investigate possible involvement of Tpl2/Cot in the pathogenesis of human T-cell neoplasias. Specifically, we studied 12 adults with various T-cell neoplasias to obtain a broad spectrum of T-cell malignancies, all with predominant leukemic expression, and examined whether Tpl2/Cot expression is deregulated in the transformed cells. The expression levels of Tpl2/Cot were quantitated by SybrGreen real-time RT-PCR using three different quantitation approaches  E = 10to calculate the mean efficiency of Tpl2/Cot and GAPDH primers. The slopes were almost equal (from -3.59 to -3.62) for both primers which showed that we could use the Pfaffl method  without where Etarget and Eref are the Efficiencies of the target (Tpl2) and reference genes(GAPDH) respectively which both were equal to a mean of 1.89 and ÄCt is the difference between the mean Ct of control cDNA(CTR) and patient cDNA (Sample). To confirm our results we also tried the Absolute Fluorescence Increase method using the LinRegPCR software v.7.5 which measures the actual efficiency of each amplification curve by fitting its linear part in a simulation plot of the Log (fluorescence) versus Cycle and calculates the efficiency from the slope of a linear regression model of the simulation curve . As contA total of 12 adult samples with T- and NK-cell neoplasias were analyzed according to the described method. They all had leukemic expression in the periphery. Morphology was assessed by light microscopy on peripheral blood smears, including measurement of absolute LGL number. Four out of twelve 33%) patients tested markedly overexpressed Tpl2/Cot (p = 0.034), as determined by either conventional RT-PCR or real-time qPCR .tpl2 genomic locus [tpl2 locus. For this purpose genomic DNA was isolated from PBMCs of the same patients and part of the genomic tpl2 locus was amplified using multiplex PCR. As reference gene we used the IFN-γ gene. The results showed a similar tpl2/IFN-γ ratio in all cases  are relatively rare and not well defined disorders, frequently associated with autoimmune diseases or immune mediated manifestations such as rheumatoid arthritis (pseudo-Felty syndrome ), RF posThere is accumulating evidence suggesting that patients with LGL-PDs display frequently immune manifestations and increased TNFα production by the neoplastic T-cells has been reported to play a role in their pathogenesis,25. InteIn conclusion, data from the present study suggest that Tpl2/Cot overexpression may have a role in the development of certain types of human T-cell neoplasms thus confirming experimental data on animal and tissue culture models for the role of Tpl2/Cot in T-cell malignancies.Peripheral blood samples from 12 adults aged 16–88 years (median age 64 years) with various T- and NK-cell neoplasias with peripheral blood leukemic expression were collected during a two-year time period. Patients with signs of infection or recently subjected to cytotoxic therapy were excluded from the study. Diagnosis was based on morphological, immunophenotypic and genomic studies and histological findings of bone marrow and/or lymph node biopsies and disease was classified according to the WHO classification . There w3 anti-coagulated peripheral blood samples by Lymphoprep density centrifugation . PBMCs were immediately lyzed in suitable volume of Trizol LS reagent  and mRNA was isolated according to the manufacturers' protocol. An aliquot of 1 μg of total RNA was treated with 1 IU DNase I, Amplification Grade  to eliminate any traces of genomic DNA.Peripheral blood mononuclear cells (PBMC) were isolated from 9 ml of fresh EDTA-KFirst strand cDNA was synthesized by reverse transcription of 1 μg total RNA using the Thermoscript™ RT kit . 0.8 μl of cDNA were amplified in a 20 μL PCR reaction containing 250 nM of each primer, 200 nM dNTPs, 0.5 IU Taq polymerase and 2 μl of 10X reaction buffer . Reactions were first optimized for annealing temperature, Mg and primer concentration (data not shown). Primers for Tpl2/Cot detection were derived from exons 3 and 4 of the human Tpl2/Cot gene (Genbank accession no: AL547407), spanning an 8.5 kb intron to prevent co-current genomic DNA amplification and their sequences were: forward 5'-CAG TAA TCA AAA CGA TGA GCG TTC TAA-3', reverse 5'-GAA CAT CGG AAT CTA TTT GGT AAC GTC-3' producing a 228 bp-length amplicon. For normalization of mRNA input differences human beta actin mRNA (Genbank accession no: BC013835) was detected using the following primers: forward 5'-CCG GCC AGC CAG GTC CAG A-3', reverse 5'-CAA GGC CAA CCG CGA GAA GAT G-3', amplifying a 214 bp cDNA fragment. In each reaction two negative controls were included by either omitting reverse transcriptase at the RT step or cDNA template respectively.PCR reactions were performed on a thermal cycler (PTC-200 MJ Reasearch with heated lid) and repeated 3 times with different cDNAs from the same mRNA. Expression of Tpl2/Cot was determined by semi-quantitative, relative RT-PCR. An amplification curve for each gene was acquired by performing the reaction with increasing number of PCR annealing cycles to identify the exponential phase of the reaction. The thermocycling parameters were as follows: initial denaturation at 94°C for 5 min followed by 34 for Tpl2 or 23 for actin cycles at 94°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec and a final extension at 72°C for 7 min.The RT-PCR products were analysed by electrophoresis in a 2.5% agarose gel, stained with 0.2 μg/ml ethidium bromide and visualized in a UVP transiluminator (Gel Doc 1000 Bio Rad). The band intensity was analysed by a densitometric image analysis system (TINA scan v2.07) and the results were expressed as a ratio between Tpl2/Cot and β-actin band intensity.Primers for real-time PCR were designed with the Primer Express Software v.2 (Applied Biosystems) and selected so that they amplify a region of no more than 150 bp, they have similar GC content, same Tm, no more than 3 G or C's at the 3' end and no secondary structure formation. To exclude primers with more than 3 consecutive complementary bases between them we used Qiagen Oligo Analysis & Plotting Tool . Primers forTpl2/Cot annealed to exons 6–7 and their sequences were: forward 5'-TCC TAA GGA CCT CCG AGG AAC-3', reverse 5'-CCC AGG CTG TAG ATG TCT GCT-3', amplifying a 93 bp region. GAPDH was used as a reference gene to compensate for mRNA input differences. Primers for GAPDH derived from exons 2–3 (Genbank accession no: BC023632) and their sequences were: forward 5'-GGA AGG TGA AGG TCG GAG TCA-3', reverse 5'-GTC ATT GAT GGC AAC AAT ATC CAC T-3', amplifying a 101 bp region.2 and 500 nM of the Tpl2/Cot primers or 100 nM of GAPDH primers. Reactions were carried out using an ABI Prism 7000 sequence detector  according to manufacturer's instructions. The thermal profile used consisted of 2 min at 50°C,10 min at 95°C and 40 repeats of denaturation at 95°C for 15 sec and annealing-extension-fluorescence data acquisition at 60°C for 1 min. A post-PCR Melting Curve Analysis was performed by a 20-min slow ramp from 60° to 95°C to confirm that there were no by-products. Samples were run in a 3% agarose gel after the end of reaction to confirm specificity. All samples were run in triplicates and two negative controls with either no reverse transcriptase or no cDNA template were included. Reaction was repeated twice in different days to estimate inter-assay variation. Results were analyzed using the ABI Prism 7000 SDS software  and Excel for further quantitative study.Primer concentration, cDNA dilution, Mg concentration and annealing temperature were optimized so that a maximum fluorescent signal with no inhibition from RT components and a similar reaction yield from both primer sets could be obtained. For the real-time PCR study cDNA from first strand synthesis treated with RNase H for 20' at 37°C was diluted 1:20 with DNAse-free water and 5 μl were used in a 20-μl reaction mixture consisting of 10.4 μl 2x SybrGreen PCR Master mix, 6 mM final concentration of MgClTo detect possible gene amplification in cases with Tpl2/Cot overexpression we used a multiplex DNA PCR protocol with interferon-γ (IFN-γ) as a reference gene amplified at the same reaction tube. High molecular weight DNA was isolated from PBMC by proteinase K digestion and phenol chloroform extraction with ethanol precipitation and diluted in TE buffer. The primers for Tpl2/Cot were: forward 5'-GCG ACG GAT TGA GGT TTG-3', reverse 5'-GCG TTT CAG GCG TAT GGA-3' amplifying a 139 bp region of intron 1(Genbank accession no.AY309013) and the primers for IFN-γ were: forward 5'-ATG CAG GTC ATT CAG ATG TAG C-3', reverse 5'-TTG GAT GCT CTG GTC ATC TTT A-3' amplifying a 250 bp fragment containing intron and exon sequences between exons 2–3 (Genbank accession no: J00219). 50 ng of DNA were used in a 20 μl reaction containing 10 μl Platinum qPCR Supermix-UDG , 250 nM Tpl2/Cot primers and 100 nM IFNγ primers. The number of amplification cycles was adjusted so that the reaction terminated at the middle of the exponential phase of both products (data not shown). The thermal profile consisted of a denaturation step at 95° for 10 min, followed by 29 repeats at 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec and a final extension at 72°C for 7 min. PCR products were analysed in a 3% agarose gel, visualized and scanned as described earlier and the Cot/IFNγ ratio was determined.Two-color flow cytometry was used for the analysis of peripheral blood lymphocyte subpopulations. In brief, 100 μL aliquots of EDTA-anticoagulated peripheral blood samples were surface stained with each of the following PE- or FITC-conjugated mouse antihuman monoclonal antibodies: anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD5, anti-CD7, anti-CD16, anti-CD56, anti-CD57, anti-CD19, anti-CD25, anti-CD11b, anti-CD79a, anti-FMC7 and anti-HLA-DR . Cells were also stained for intracellular Terminal deoxy-transferase (TdT) (Beckman Coulter) and CD3 using the IntraPrep intracellular staining kit (Beckman Coulter). PE- or FITC-conjugated mouse IgG of appropriate isotype served as negative controls. Following 30 min incubation at room temperature and two washes with phosphate buffer saline (PBS)-1% fetal bovine serum (FBS)-0.05% azide, contaminating red cells were lysed with 0.12% formic acid and samples were fixed in 0.2% parafolmadeyde using the Q-prep reagent system . Analysis on 10,000 events was performed in an Epics Elite model flow cytometer (Coulter) in the lymphocyte gate.+ cells by means of flow cytometry using the IOTest Beta Mark kit (Beckman Coulter), according to the manufacturer's instructions [Clonality assessment of peripheral blood T-cells was performed by analysing quantitatively different variable regions of the T-cell receptor (TCR) β chain (Vβ repertoire) of CD3ructions . T-cell TNF-α concentration was determined using the High Sensitivity human TNF-α ELISA kit  or the IL-2 ELISA kit  according to the manufacturer's instruction. According to the manufacturer, the sensitivity of these assays are 0.12 pg/ml and 7 pg/ml respectively.The comparison of Tpl2/Cot mRNA expression between patient and control samples was performed by means of the nonparametric Mann-Whitney U test using as variables the mean normalized Tpl-2 expression of each sample and control, described by the equation-(mean CtTpl2-meanCtGAPDH)(3) Tpl-2 norm = (E)where E is the mean common efficiency of the reference and target gene that we calculated . The ManAVC collected specimens and patient data and performed the major body of the experimental work as well as the data analysis and the preparation of the manuscript. HAP was involved in flow cytometric analysis, patient selection and manuscript preparation. ANM and GDE were involved in the study design, the interpretation and evaluation of the clinical data and manuscript preparation. CT conceived the project, performed the ELISAs, was responsible for the coordination of the experimental procedures and was involved in the preparation of the manuscript.


Exponential growth in the number of patients suffering from diseases caused by the consumption of sugar has become a threat to mankind's health. Artificial low calorie sweeteners available in the market may have severe side effects. It takes time to figure out the long term side effects and by the time these are established, they are replaced by a new low calorie sweetener. Saccharine has been used for centuries to sweeten foods and beverages without calories or carbohydrate. It was also used on a large scale during the sugar shortage of the two world wars but was abandoned as soon as it was linked with development of bladder cancer. Naturally occurring sweet and taste modifying proteins are being seen as potential replacements for the currently available artificial low calorie sweeteners. Interaction aspects of sweet proteins and the human sweet taste receptor are being investigated. The prevalence of obesity and diabetes has increased dramatically in recent years in the United States, with similar patterns seen in several other countries including India  as well.In humans, the sweet taste is mainly due to the recently discovered T1R2-T1R3 receptor -10, two The potential industrial applications of these proteins are the low calorie sweetener industry and the cola, snacks, food and chocolate industries.Pentadiplandra brazzeana Baillon [Brazzein is the smallest, most heat-stable  and pH-s Baillon . Heat an Baillon . Second, Baillon .Thaumatococcus danielli. The protein crystallizes in a hexagonal lattice after a temperature shift from 293 to 277 K. The structure has been solved at 1.6 Å resolution. Its fold was found to be identical to that found in three other crystal forms grown in the presence of crystallizing agents of differing chemical natures [The thaumatins are a class of intensely sweet proteins isolated from the fruit of the tropical plant  natures . It cons natures ,27. The  natures . It is a natures . Thaumat natures .Dioscoreophyllum cumminsii grown in West Africa and is approximately 100,000 times sweeter than sugar on a molar basis and several thousand times sweeter on a weight basis [B7 of protein was replaced by AbuB7 (L-2-Aminobutylicacid), showed similar 3-dimensional structures of these two proteins, indicating that the lack of the beta-carboxyl group in the AbuB7 analog is responsible for the loss of sweetness [Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, an A chain of 44 amino acid residues and a B chain of 50 amino acid residues . The proht basis . Single-ht basis . Native ht basis . One NMRweetness . Recent weetness .Curculigo latifolia acts as a good low calorie sweetener. Its maximum sweetness is equal to 0.35 M of sucrose. It has taste modifying abilities since water and sour substances elicit a sweet taste after consumption of curculin [Curculin which is extracted from curculin . There icurculin . The tascurculin .Galanthus nivalis) [The molecular weight of Curculin was determined by low angle laser light scattering and was found to be 27800 . Its thrnivalis) . The thrnivalis) . The pronivalis) .Capparis masaikai and its sweetness was estimated to be around 400 times that of sucrose on weight basis. It consists of an A chain with 33 amino acid residues and a B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges [Mabinlin is a sweet protein with the highest known thermostablility . It is d bridges . Its hea bridges . The swe bridges and of Ma bridges .Richadella dulcifica an evergreen shrub native of West Africa. The protein is a single polypeptide with 191 amino acid residues [Miraculin is a taste-modifying protein that belongs to the class of sweet proteins. It is extracted from residues . It modiresidues . Thus, mresidues .Taste-modifying protein modifies the sweet taste receptor on binding and this behavior of these proteins is responsible for modification in taste of sour substance ,47. All Pentadiplandra brazzeana, a shrub found in tropical forests of a few African countries. Not much information is available about the protein despite its isolation several years ago, in 1989 [Pentadin is a sweet protein extracted from the plant  in 1989 . The pro in 1989 .Humans detect taste with taste receptor cells. These are clustered in taste buds. Each taste bud has a pore that opens out to the surface of the tongue enabling molecules and ions taken into the mouth to reach the receptor cells inside. There are five primary taste sensations salty, sour, sweet, bitter and umami. Sweet and umami (the taste of monosodium glutamate) are the main pleasant tastes in humans. T1Rs are mammalian taste receptors that assemble two heteromeric G-protein-coupled receptor complexes T1R1+T1R3, an umami sensor, and T1R2+T1R3, a sweet receptor .Sweet and taste-modifying proteins interact with the T1R2-T1R3 receptor with a different mechanism compared to small molecular weight compounds . RecentlThe major work by Kunishima et al.  solving The exact mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated . Low molRecently it has been found that T1R3-independent sweet- and umami-responsive receptors and/or pathways also exist in taste cells .As it has been found that sweet proteins are thousands of times sweeter than sucrose and are of low calorie value, these proteins can be used as natural low calorie sweeteners by people suffering from diseases linked to consumption of sugar e.g. obesity, diabetes and hyperlipemia.Candidate proteins can be checked for biological activity with the human taste receptor. Also mutations can be induced in candidate sweet proteins to analyze changes in their physical, chemical and biological properties. The work can be taken forward by solving the structures of the proteins and taste receptors with a view to increasing the efficiency of these sweeteners.


In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs.In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different 1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours.RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.The phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT In addition, we examined whether these NSCs express receptors for the pineal hormone melatonin, which can induce GDNF mRNA and protein expression [1 receptors.Neural stem cells are multipotent cells which are capable of self-replication and differentiation into neurons, astrocytes or oligodendrocytes in the central nervous system . Becausepression ,7 and whpression -10. InitFollowing two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance Fig.  and high1 receptor mRNA was detected by RT-PCR in NSCs maintained for two days, especially in cells cultured in 1% FBS, as shown in Fig. 1 receptor protein, as revealed by western analysis  for 24 hours. Following RT-PCR analysis, GDNF mRNA levels were converted to optical density (OD) values and normalized to GAPDH OD levels, as reported previously [3,7 = 7.03, p < 0.04). A Neuman-Keuls test indicated a significant increase in relative GDNF mRNA expression in cells treated with 0.05, 0.1 and 1 nM melatonin as shown in Figure In order to assess the potential functionality of the MTeviously . After cThe expression of nestin in undifferentiated C17.2 cells is consistent with the presence of this intermediate filament protein in stem and progenitor cells in the mammalian CNS . Howeverin vitro [The expression of diverse neurotrophins by NSCs is consistent with the role of these factors in the differentiation and development of the CNS. Presumably, the robust mRNA expression observed, particularly in cells maintained in 10% FBS + 5% HS, is driven by the serum-enriched milieu of potential inducers including neurotransmitters, hormones and growth factors, such as basic fibroblast growth factor and epidermal growth factor, which can stimulate C17.2 cell growth in vitro . In contin vitro . Activatin vitro ,16, wherin vitro . The cAMin vitro . Therefo1 receptor mRNA in C17.2 cells after 2 days but not after 7 days, presumably involves downregulation of this receptor. There is considerable evidence that many G protein-coupled receptors are downregulated by their agonists [1 transcription in vitro [1 immunoreactivity within C17.2 cell bodies and extensions, as shown in Figure 1 receptors. In accordance with this view, the MT1 protein detected in short-term (2-day) cultures is about 30 kDa, which is less than the approximately 37–45 kDa molecular weight observed in various mammalian tissues [1 receptor of about 40–45 kDa was detected, as shown in Fig. 1 contains two glycosylation sites in its N-terminal [1 receptors,which have yet to undergo posttranslational modification and translocation to the plasma membrane, were detected in cells cultured for 2 days in 1% FBS, whereas a mature glycosylated receptor was present in cells grown for longer periods. Although the MT2 receptor transcript was not detected under any of the conditions used in this study, additional studies are required before the possibility of its expression in these cells can be ruled out. It is possible that MT2 mRNA may undergo rapid turnover/degradation, while a functional protein may still be present. This is the first evidence that melatonin receptors are expressed in neural stem or progenitor cells and raises the obvious question of whether this hormone plays a role in neuronal development. Although studies in this field are limited, there is increasing evidence that melatonin is involved in the early development of vertebrates. For example, melatonin is produced in chick embryos as early as the 7th day of embryonic development [in vitro [in vitro, there was a significant increase in the percentage of embryonic survival [i protein-coupled melatonin receptors, which mediate inhibition of the adenylate-cyclase-cAMP pathway, are present in the embryonic (day 8) neural retina [i protein-coupled receptor subtypes have been found in 24 hr-old embryos from Japanese quail [in situ hybridization studies indicate that the melatonin MT1 receptor is expressed in diverse areas of the human fetal brain [1 receptors in NSCs is in keeping with the foregoing, and supports the view that melatonin is involved in neurodevelopment. Colocalization evidence that the MT1 receptor is present in both neural and glial progenitor cells is consistent with a neurodevelopmental role for melatonin, and suggests that in addition to the presence of the MT1 in mammalian neurons [1 receptor is consistent with its presence not only in neural progenitor cells but also in GFAP positive glial progenitors [While this work was in progress, it was reported that C17.2 neural stem cells constitutively secrete BDNF, GDNF and NGF, but do not label for GFAP or neuronal markers like β-tubulin III . Our finagonists . More imin vitro . Interes tissues -24. Moreterminal ,26. Thuselopment , and a pin vitro . Similarsurvival . Other sl retina . Melatonse quail ,32 and pse quail . Moreoveal brain . Thus, t neurons , it may  neurons . The detgenitors . Prelimigenitors . GDNF algenitors ,40. Ther1 receptors adds to the increasing evidence that NSCs can respond to diverse modulators [in vivo modulation of this and/or other neurotrophic factors, via its G protein-coupled receptors in the brain or on transplanted NSCs, could have important implications for optimizing therapeutic strategies in neurodegenerative disorders such as Parkinson's disease.In summary, the NSCs utilized in this study exhibited an intrinsic ability to express neurotrophins under various cell culture conditions. This ability was not affected by their morphological state, except in the case of GDNF mRNA expression which was lower in cells undergoing differentiation in FBS-supplemented media. Novel evidence that neural stem/progenitor cells express MTdulators , and sugv-myc) transduction of cells from the external germinal layer of neonatal mouse cerebellum [2 – 95% air incubator at 37°C and routinely split at approximately 90% confluency [The C17.2 cell line was derived by retrovirus-mediated oncogene (1 and MT2 receptors) of the RT product and the HotStarTaq master mix kit , together with appropriate primers (Table 1 and MT2) at 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by a final incubation at 72°C for 10 min.Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier . After DNase treatment, cDNA was synthesized from 1–2 μg of total RNA using the Omniscript reverse transcriptase kit  and oligo dT primers. PCR was carried out using 1.5 μl , was amplified with intron-spanning primers [For semi-quantitative examination of the effects of melatonin on GDNF mRNA expression, cells were grown in 10% FBS + 5% horse serum (HS) for 1 week. After subculture, cells were kept in 10% FBS + 5% HS for 2 days followed by another subculture to 1% FBS for 2 or 3 days. The cells were then treated with vehicle (0.001% DMSO) or melatonin  for 24 hours. Following RNA extraction, RT-PCR was performed as described above, except that an annealing T primers , in orde1 receptor serum . Cells were washed three times with PBS and then incubated with a fluorescein (FITC)-conjugated donkey anti- rabbit IgG . In some experiments, the primary antibody was omitted or it was preincubated with the corresponding peptide immunogen , before use. In order to examine cell marker expression, mouse monoclonal antibodies against nestin (1: 500), β-tubulin III (1:200) or GFAP  were used together with a FITC-conjugated donkey anti-mouse IgG . For double- labeling studies of the MT1 and cell markers, a rhodamine (TRITC)-conjugated donkey anti-mouse IgG  was used to detect nestin, GFAP and β-tubulin III. Digital images were recorded on a Zeiss confocal microscope.After fixation for 15 minutes in 4% paraformaldehyde on poly L-ornithine-coated glass cover slips, cells were incubated overnight at 4°C with anti-melatonin MT1 antibody  at 4°C. After washing, membranes were incubated with a horseradish peroxidase-conjugated second antibody  for 1 hour. Following washing and exposure to enhanced chemiluminescence (ECL) reagents  for about 5 minutes, proteins were detected by autoradiography, as described previously [C17.2 NSCs were grown as described in Figure eviously . Buffer LPN conceived and planned the study, designed PCR primers, supervised all aspects of the study and wrote the manuscript. KJA cultured cells and performed initial RT-PCR experiments. LMRC treated cultured cells and carried out immunocytochemistry experiments. CVD cultured cells and carried out western blotting. RS treated cultured cells and performed RT-PCR experiments. CRM performed double- labeling of cultured cells. LCD provided the C17.2 cells, and collaborated on immunocytochemical studies. DLK assisted with initial cell culture. All authors read and approved the final manuscript.