Thanks to the tools of molecular biology, our understanding of the 100-plus diseases known collectively as cancer has increased dramatically over the past decades. While each of these cancers exhibits unique characteristics reflecting the particular cell or tissue it springs from, the disease follows a similar arc in nearly all its forms. Cancer is a multistep disease that begins when genetic damage—initiated by a multitude of agents—unleashes a single cell from the normal constraints on cellular proliferation. This single transformed cell generates a colony of similarly abnormal progeny that can take decades to develop into malignancies.While events that stimulate uncontrolled cell division can promote cancer, mutations in tumor suppressor genes figure prominently in tumor progression. Disruptions in the pRb (retinoblastoma 1) tumor suppressor, for example, are often seen early in cancer development, sensitizing cells to tumorigenesis. pRb, along with other “pocket proteins”—so-called because they share an amino acid domain called the Rb pocket—regulate cell cycle progression, apoptosis (programmed cell death), and cellular differentiation. Some tumor suppressors, such as p53, can trigger apoptosis, ultimately sacrificing cells that have sustained DNA damage or other types of cellular stress.Mutations in both the pRb and p53 tumor suppressor pathways are commonly seen in human cancers, though their interactions appear to vary depending on the tissue. In mouse brain epithelial cells, for example, loss of p53 function coupled with loss of pRb results in reduced apoptosis and increased tumor growth, while p53 loss in mouse brain astrocytes (cells that support neurons) does not affect tumor growth. Building on this work, Terry Van Dyke and colleagues report that loss of the pRb tumor suppressor in mammary tissue has the same effect—predisposition to tumor formation—seen in these other cell types. Despite the different environment inherent in each cell type, the initial events following loss of the pRb pathway were the same: increased proliferation and apoptosis, followed by tumorigenesis. But, surprisingly, pRb and p53 interactions varied in different cell types.Like most cancers, mammary gland cancer has a long latency period, prompting the researchers to ask what events engineer tumor progression. To investigate the relative contribution of pRb and p53 in tumorigenesis, the researchers generated a novel mouse model with a dysfunctional pRb pathway and various levels of p53 function in several cell types. This is a significant achievement in itself, as many agents that inactivate the pRb pathway also disrupt the p53 pathway. pRb inactivation, they show, causes abnormalities in mammary cell proliferation, apoptosis, and tissue morphology. In these mammary-specific pRb-deficient mice, p53 was responsible for most of the apoptotic response—decreased levels of p53 resulted in reduced apoptosis and accelerated tumorigenesis, but had no effect on proliferation. Interestingly, in other mouse models where aberrant proliferation is caused by disabling other pathways, loss of p53 was associated with increased proliferation—rather than reduced apoptosis—and early tumor formation. And while p53 is the main effector of apoptosis in brain and mammary epithelial cells, this is not the case in all tissues: in astrocytes, for example, the tumor suppressor Pten regulates apoptosis in response to pRb inactivation. Together these results indicate that specific cellular responses to a cancer-causing stimulus vary depending on the nature of the initial genetic injury and the cell type and that pRb and p53 interact in different ways in different tissues. And p53, it appears, contributes to tumor suppression—and thus progression—through multiple mechanisms.BRCA1 and BRCA2 genes, to shed light on the complex series of events that ultimately cause breast cancer.By creating a mouse model that disentangles the pRb and p53 pathways, Van Dyke and colleagues have added a valuable resource for studying breast cancer. This model, they propose, will facilitate further investigations into the relative contributions of these overlapping pathways to cancer progression. What's more, the model offers a vehicle for examining how pRb interacts with other breast cancer mutations, like the inherited mutations in the human


The administration of granulocyte colony-stimulating factor (G-CSF) to peripheral blood progenitor cell (PBPC) donors causes spleen length to increase, but the duration of enlargement is not known. Eighteen healthy subjects were given 10 μg/kg of G-CSF for 5 days and a PBSC concentrate was collected by apheresis. Ultrasound scans were used to assess craniocaudal spleen length before and after G-CSF administration. Mean spleen length increased from a baseline length of 10.7 ± 1.3 cm to 12.1 ± 1.2 cm on the apheresis day (p < 0.001). Ten days after apheresis, spleen length fell to 10.5 ± 1.2 cm and did not differ from baseline levels (p = 0.57), but in 3 subjects remained 0.5 cm greater than baseline length. Increases in spleen length in PBPC donors are transient and reversible. Peripheral blood progenitor cell (PBPC) concentrates donors are routinely given granulocyte colony-stimulating factor (G-CSF) to increase the concentration of circulating PBPCs and hence the number of progenitors that can be collected by apheresis. Typically 10 to 16 μg/kg of G-CSF are given subcutaneously daily for 4 to 6 days prior to the collection . The admWhile spontaneous rupture of the spleen in PBSC donors given G-CSF is rare, the administration of G-CSF for five days causes spleen length to increase in almost all healthy donors ,9. The iSince allogeneic PBPC donors may be at risk for splenic rupture while the spleen is enlarged, it is important to determine when spleen size returns to baseline levels. The purpose of this study was to determine if spleen length returns to baseline 10 days after G-CSF-mobilized PBPC concentrates are collected by apheresis from healthy subjects.All of the subjects were in good health and were donating G-CSF-mobilized PBPC concentrates for laboratory investigations. The donors were given 10 μg/kg of G-CSF  daily for 5 days, and a PBSC concentrate was collected approximately 2 hours after the last G-CSF dose was given. PBPC concentrates were collected with a CS3000 blood cell separator . Spleen length was evaluated by ultrasound examination three times: prior to the administration of the first dose of G-CSF, on the day of apheresis, and 10 or 11 days after apheresis. This study was approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.Craniocaudal spleen length was assessed using ultrasound  with a sector transducer . The intra-observer error for measuring spleen length using ultrasound is 4.9 mm when healthy subjects are evaluated at separate settings .Complete blood counts were performed with an automated cell counter . CD34+ cell counts were performed using a flow cytometer .Spleen lengths measured before and after the G-CSF course were compared using 2-tailed paired t-tests. Spleen length changes were also compared among males and females and Caucasians and non-Caucasians using 2-tailed t-tests. The percent change in spleen length was compared with blood counts, CD34+ cell counts, and donor age using linear regression.The median age of the 18 healthy subjects was 34 years old and ranged from 22 to 55 years of age. Eight of the subjects were male, 13 of the donors were Caucasian, 3 were African American, and 2 Asian. Apheresis day spleen length increased above baseline length in 17 of 18 donors . There was no difference between the 10-day post-apheresis and pre-G-CSF spleen length (p = 0.57). The spleen length 10 days after apheresis was less than the apheresis day length in all 17 donors whose spleen length increased. However, the spleen length 10 days after apheresis remained more than 0.5 cm greater than baseline spleen length in 3 subjects  or between Caucasians and non-Caucasians . Spleen length increase was not related to donor age (r = 0.13). In addition, spleen length increase was not related to preapheresis CD34+ (r = 0.04), WBC (r = 0.05), neutrophil (r = 0.07), lymphocyte (r = -0.14), monocyte (r = -0.04), and platelet counts (r = 0.19) or hemoglobin level (r = -0.04).Healthy PBPC concentrate donors given G-CSF should be warned that their spleens will be enlarged for a brief time and that they may be at risk of splenic rupture. Most donors are likely at risk for splenic rupture only during the time of G-CSF administration and for about 10 days after the completion of the G-CSF course. Since splenic enlargement may persist for longer periods in some donor, until more data are available it may be worthwhile to counsel PBPC donors to avoid activities that could lead to abdominal and splenic trauma for 2 to 3 weeks after the last dose of G-CSF.


One of the most common and most serious complications of both type 1 and type 2 diabetes is diabetic nephropathy. It occurs in around 30% of patients with type 1 diabetes and 10% to 40% of patients with type 2 diabetes. Diabetic nephropathy is the leading cause of renal failure in the developed world. The main effect of diabetic nephropathy is proteinuria, initially in very small amounts but which increases, leading to nephrotic syndrome and end-stage renal disease in most cases.Various risk factors in individuals with diabetes are known to increase the chance of developing diabetic nephropathy, including South Asian or African background, male sex, long history of diabetes, poor blood sugar control, high blood pressure, and smoking. One early change associated with diabetic nephropathy is degeneration of the renal tubular epithelium, but the exact cause of this at the cellular level is unclear. Erwin Böttinger and colleagues have dissected out one key point in the progression to diabetic nephropathy. They looked at cell lines of renal tubular cells from humans and mice and kidney biopsies from patients with diabetic nephropathy, patients with non-diabetic renal disease, and mice with genetic and induced diabetes. In the human cell lines they showed that glucose induced the expression of CD36, a receptor known to have a role in adhesion and signal transduction . They then went on to show that apoptosis of these cells occurred in the presence of glycated (glucose-modified) albumins or free fatty acids, which are present in increased amounts in patients with diabetes, and that CD36 was essential for the apoptosis to occur. They then examined how CD36 triggered apoptosis and found that it involved src kinase, p38 MAP kinase, and caspase 3.Comparing mice and humans, the researchers found that the two species are not alike: diabetic mice did not show an increase in tubular expression of CD36—even though the gene is present in mice—and had normal tubular epithelium and no tubular apoptosis. They confirmed this difference between humans and mice by showing that normal mouse epithelial cell lines were resistant to apoptosis caused by the glycated albumins; however, artificially expressing CD36 in these lines made them susceptible to apoptosis by these modified albumins.These results provide insight into one of the crucial steps in diabetic nephropathy and, in humans at least, might help to explain why high blood glucose is so damaging to the kidney, hence providing a good reason—if another is needed—for encouraging patients to control blood glucose as tightly as possible.


The Drugs for Neglected Diseases Initiative is a new, public- sector organization dedicated to drug discovery In wealthy countries, state-funded research has yielded breakthroughs in molecular biology, chemistry, and engineering. These advances have been taken up by the pharmaceutical industry and applied to drug development for a growing range of illnesses and conditions. As a result, patients have access to new drugs that are better tolerated, more specific, and more effective than old ones.In poor countries, however, millions of people have yet to experience the benefits wrought by science. The deadly infectious diseases that plague them, such as sleeping sickness, Chagas disease, and visceral leishmaniasis, fail to arouse the interest of drug developers.The Drugs for Neglected Diseases Initiative (DNDi) is a new, not-for-profit organisation set up to correct this fatal imbalance by developing new drugs for these forgotten patients.Most of the drugs still used to treat ‘neglected diseases’ were developed in colonial times. These are often expensive, difficult to administer, and hard to tolerate; several of them are also becoming ineffective because of increasing parasite resistance. Very few new alternatives have been developed in the past decades: between 1975 and 1999, 1,393 new drugs were made available to the public, but only 16 of these were meant for neglected diseases [What makes the lack of drugs more difficult to accept is that scientists know an enormous amount about kinetoplastids, the organisms responsible for sleeping sickness, Chagas disease, and leishmaniasis . The weaIt is dangerous to oversimplify the causes of this situation. What share of responsibility for the world's health is borne by the pharmaceutical industry, which has the know-how and the resources for innovation? Aren't international organisations also partly responsible? After all, they are the ones who allocate major funding for health programmes and encourage research programmes. And what about public research institutions in rich countries that generate the knowledge used by industry? Governments have the power to influence their research priorities and drug development decisions, either through funding or direct involvement. Unfortunately drugs for neglected diseases are low priority for governments . They teAll is not doom and gloom. In the past few years, there has been some movement on research and development (R&D) for neglected diseases. Despite its broad mandate and limited resources, the Special Programme for Research and Training in Tropical Diseases (TDR)—established and funded by the World Health Organization, the World Bank, and the United Nations Development Programme—can be credited with several important successes in the fight against malaria and leishmaniasis. The Medicines for Malaria Venture and the Global Alliance for TB Drug Development were set up as public–private partnerships to tackle malaria and tuberculosis. These partnerships were made possible by the fact that malaria and tuberculosis are global diseases, affecting patients in the North and South, so there was enough of a market to persuade industry to develop new drugs for these diseases.A different solution, however, was needed for diseases that are limited to tropical countries, are of no military or strategic interest to wealthy countries, and are not supported by markets or patients' organisations capable of attracting the attention of politicians. This is the kind of solution put forward by the DNDi.DNDi is a not-for-profit organisation designed to mobilise resources for R&D of new drugs for neglected diseases.Many people and organisations around the world share an ambition to redress the lack of new treatments for neglected diseases, and bring the benefits of science to forgotten patients. Several of them came together to create DNDi: one humanitarian organisation—Médecins Sans Frontières; five research institutions—the Oswaldo Cruz Foundation from Brazil, the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health Malaysia, and the Pasteur Institute from France; and the TDR .DNDi:http://www.dndi.orgTDR:http://www.who.int/tdrMedicines for Malaria Venture:http://www.mmv.orgGlobal Alliance for TB Drug Development:http://www.tballiance.orgOswaldo Cruz Foundation:http://www.fiocruz.brIndian Council for Medical Research:http://icmr.nic.in/home.htmKenya Medical Research Institute:http://www.kemri.orgMinistry of Health Malaysia:http://dph.gov.my/Pasteur Institute:http://www.pasteur.fr/externeMédecins Sans Frontières:http://www.msf.orgThe initiative is a virtual organisation with a growing network of academic and R&D expertise at its disposal. The different players involved in DNDi are bringing their knowhow in parasitology and clinical trials, their experience treating neglected patients, and their drug manufacturing capacity. They are pooling these resources to move drugs stuck in the pipeline all the way to the patients themselves. Pharmaceutical companies have a particularly important role to play: they possess vast repositories of molecules, the means to move from development to industrial production, and highly specialised teams of researchers. Their contribution will be crucial to the success of DNDi.DNDi is a needs-driven initiative—in other words, the needs of patients suffering from neglected diseases are paramount in its search for new drugs to treat them. The organisations that make up DNDi have firsthand knowledge of these needs because they work with patients in disease-endemic countries throughout the developing world. The initiative is taking this knowledge of patient needs, matching it with opportunities in R&D, and pushing the most relevant projects through the pipeline. Ultimately, neglected patients will have access to drugs targeting their specific diseases, drugs that were designed with them specifically in mind—such as short-course, low-toxicity treatments that don't require hospitalisation, or tablets to swallow rather than injections.http://www.dndi.org). These have already pinpointed several promising projects. DNDi is also proactively contacting scientists working on infectious diseases, and surveying published literature for research of interest.To identify opportunities in R&D that are both relevant to patient needs and that meet required criteria of scientific merit, DNDi is sending out calls for letters of interest to the scientific community via advertisements in journals and posted on the DNDi website (DNDi's project portfolio currently holds nine projects at different stages of development to address identified needs for the treatment of visceral leishmaniasis, sleeping sickness , Chagas Only a few drugs exist to treat sleeping sickness, and they are toxic or difficult to administer. Melarsoprol kills one in 20 patients. Eflornithine requires four daily infusions over 14 days. Given these limited options, DNDi is focusing on identifying new compounds that can cross the blood–brain barrier to treat second stage sleeping sickness.DNDi is using high throughput screening on whole cell trypanosomes to discover novel lead compounds, and is working to identify and optimise inhibitors of the enzyme protein farnesyltransferase. The initiative is working on validating the kinetoplastid enzyme dihydrofolate reductase as a drug target. Identifying trypanothione inhibitors is also relevant to other trypanosome parasites. These are long term projects.Nifurtimox, a drug for Chagas disease, has been used to treat sleeping sickness since the 1970s in some isolated places. It has never been extended to more people because no one has studied its safety or effectiveness. DNDi will assess its short-term usefulness by conducting clinical trials on a treatment combination of eflornithine and nifurtimox. DNDi will continue to explore other short- and medium-term projects.The R&D of new drugs is time-consuming and expensive if the process starts at the early discovery stage, because of the associated risk of project attrition along the way. DNDi is therefore investing resources in several pre-development and development projects as well. These include developing fixed dose combinations of artesunate/amodiaquine and artesunate/mefloquine for use against chloroquine-resistant malaria in Africa and Asia, respectively; pushing for registration of paromomycin for use against visceral leishmaniasis in Africa; assessing combinations of existing drugs for visceral leishmaniasis; and evaluating the usefulness of nifurtimox in combination with eflornithine in the treatment of sleeping sickness.Governments can—some might say should—influence drug development choices. DNDi strongly believes that governments in both developed and developing countries should take an active interest in the R&D of new drugs for neglected diseases. In parallel to its own drug development activities, DNDi is working to raise awareness of the neglected disease crisis among key policy- and decision-makers, for instance the European Commission and the National Institutes for Health in the United States.In the poorer countries in the world, over 350 million people are at risk from neglected diseases. Currently available treatments are inadequate or nonexistent, and new solutions are urgently needed. DNDi is working to ensure that the advances of science that have brought health and comfort to wealthy nations also benefit these neglected populations.


Because "laws of nature" do not exist in ecology, much of the foundations of community ecology rely on broad statistical generalisations. One of the strongest generalisations is the positive relationship between density and distribution within a given taxonomic assemblage; that is, locally abundant species are more widespread than locally sparse species. Several mechanisms have been proposed to create this positive relationship, and the testing of these mechanisms is attracting increasing attention.We report a strong, but counterintuitive, negative relationship between density and distribution in the butterfly fauna of Finland. With an exceptionally comprehensive data set , we have been able to submit several of the mechanisms to powerful direct empirical testing. Without exception, we failed to find evidence for the proposed mechanisms creating a positive density-distribution relationship. On the contrary, we found that many of the mechanisms are equally able to generate a negative relationship.We suggest that one important determinant of density-distribution relationships is the geographical location of the study: on the edge of a distribution range, suitable habitat patches are likely to be more isolated than in the core of the range. In such a situation, only the largest and best quality patches are likely to be occupied, and these by definition can support a relatively dense population leading to a negative density-distribution relationship. Finally, we conclude that generalizations about the positive density-distribution relationship should be made more cautiously. Species that are locally abundant tend to be more widespread than species that are locally rare ,2. This Nine mechanisms have been proposed to explain the positive relationship between density and distribution ,14,16-25Since low density species are less likely to be detected in surveys, a positive density-distribution relationship may result from systematic under-recording of the distribution of species that occur at low density ,3,20. ItBoth positive and negative density-distribution relationship may result from related species being considered as independent data points ,26,27. HA positive density-distribution relationship may be generated as a result of an underlying theoretical spatial distribution of individuals. For a given level of aggregation, a species with more individuals in a given area is expected to occur in more locations than a species with fewer individuals in the same area ,24. HoweEmpirical observations suggest that habitat occupancy and the density of individuals decline when moving along a gradient from the centre of the species geographical distribution range toward its edge ,30-32. TBrown  hypothesOn the assumption that density and distribution of resources determine the density and distribution of the species utilizing them, a positive or a negative density-distribution relationship in the resource will generate the same relationship in the consumer ,22. MostIf a species tends to inhabit more habitats when density is high and fewer when density is low, then locally abundant species will tend to occupy more habitats and have wider distributions . At presMetapopulation theory may explain both positive and negative density-distribution relationships . A positIf a set of species differs only in respect to its mortality rate, then species with higher mortality will have a lower density. Moreover, the species will inhabit fewer patches than a species with low mortality, leading to a positive density-distribution . Under rHere, we will examine the relationship between density and distribution of Finnish butterflies (Hesperioidea and Papilionoidea), and test the possible mechanisms affecting this relationship. The density-distribution patterns of butterflies have been examined in many previous studies, and if a relationship was found, it was generally positive ,38,45-471,93 = 229.97, P < 0.001, r2 = 0.71) , and both of the predictor variables had an independent positive effect on the distribution of the butterflies , and thus the interaction term was removed from the analysis. However, both larval specificity and habitat breadth had a significant effect on butterfly distribution . A significant difference was found in the distribution of butterfly species with different habitat breadths. Specialist butterfly species had smaller distribution than generalist species , and intermediate species had smaller distribution than generalist species . Specialist species tended to have smaller distributions than intermediate species, but the difference was not significant  and Tukey's multiple comparisons test. There was no significant interaction between larval specificity and habitat breadth on butterfly distribution , and both the predictor variables had independently negative effects on butterfly density , and thus the interaction term was removed. However, habitat breadth had a significant main effect on butterfly density . Specialist species had a greater density than intermediate  or generalist species , but there was no significant difference between intermediate and generalist species . Monophagous species had significantly greater density than polyphagous species . However, Tukey test detected no differences between monophagous and oligophagous, or between oligophagous and polyphagous species   verified that none of the results reported above were artefacts of treating species as independent data points. The results with the phylogenetically independent contrasts have been tabulated in Table A positive relationship between the density and distribution of species is expected to be an almost universal pattern in ecology -6. In coAccording to the range position hypothesis, species are expected to inhabit increasingly fewer localities when moving along a gradient from the centre of the species' geographical distribution range toward the edges of the range ,31,32. IThe niche breadth hypothesis predicts that a positive density-distribution relationship arises because species that are able to use a broader range of resources are widespread and also have high density . There iAccording to the metapopulation theory, differences in dispersal ability of species may generate negative density-distribution relationships ,21,33. IWe conclude that many of the ecological mechanisms proposed to create a positive density-distribution relationship are also able to generate a negative relationship. Indeed, it is intriguing that many studies, which have found a positive density – distribution relationship have failed to find an ecological mechanism that would explain the observed pattern. This may not be surprising if the ecological mechanisms are more likely to generate a negative rather than positive density – distribution relationship. In support of this view we found a strong negative density-distribution relationship but what is more important also empirical support for ecological mechanisms that are able to explain the observed pattern.Our study area (Finland) is a long, northern country extending more than 1100 km from south to north. Many of the studied butterfly species meet the edge of their distributional range in Finland. On the edge of a distribution range suitable habitat patches are likely to be more isolated than in the core of the range. In such a case, only the largest and best quality patches are likely to be occupied, but because these are the best quality patches, they may support a relatively dense population , leadingOur data is predominantly based on published literature ,50 and tThe distribution of butterfly species is based on the "Atlas of Finnish Macrolepidoptera" . This atThe National Butterfly Scheme in Finland (NAFI) is a formal study that was established in 1991 for the purpose of monitoring the population trends of the butterflies across the country ,52. MoniTo obtain density for each butterfly species per 10 km × 10 km grid square, we divided the total number of individuals of each butterfly species by the number of 10 km × 10 km squares occupied by the species. Some rare butterfly species with known occurrence sites may face proportionally higher sampling effort than common species . To remo1,93 = 0.04; P = 0.835, r2 = 0.00 and F 1,93 = 0.99; P = 0.987, r2 = 0.00, respectively).It is possible that size confounds the density and distribution data if larger species are more visible and thus reported more often than smaller species. We analysed the possibility that the size of the butterfly species is a confounding factor. There is practically no dimorphism between the sizes of male and female butterflies. Therefore, as a size measure we used only the female wing span reported in Marttila et al. , in whicLycaena helle and Clossiana thore) which do not range to southern Finland.To determine the range position, we measured the distance (km) between the northernmost distribution record and the southernmost point (Hankoniemi) of Finland from the maps in Huldén et al. (2000). Note that the longest possible range was 1155 km. In analysis including range position, we only included species, the distribution range of which begins from southern Finland . Thus, wWe describe the niche breadth for each butterfly species using two different measures: 1) larval host plant specificity, and 2) the number of habitat types occupied by the adults. We excluded from this analysis the species which occur only in Northern Finland . This wThe data on larval host plant specificity in Finland are based on Huldén et al.  and WahlPieris napi and Gonepteryx rhamni) that occupied all four main habitat types, habitat types three and four were combined. Value one represents specialist species that are limited to one habitat type, value two represents intermediate species that occur in two habitat types, and value three represents generalist species that occur in three or four habitat types.The main habitats that Finnish butterflies occupy have been categorised into four types : (1) uncMetapopulation theory predicts that species with greater dispersal ability are likely to occupy more habitat patches than species with lower dispersal ability . To desc3,27 = 8.74, r = 0.567, P < 0.001, Pearson correlation; r = 0.703, n = 31, P < 0.001 and Pearson correlation r = 0.602, n = 11, P = 0.050, respectively), indicating that our dispersal ability is in line with other independent estimates and thus reliable .Lack of statistical independence among species for the traits of interest was tested using the method of phylogenetically independent contrasts  as impleJP, VK, AK and JSK compiled and analysed most of the data, and wrote the manuscript. NW constructed the phylogeny and AG conducted the phylogenetic analyses. KS provided the density data.


Bacillus licheniformis ATCC 14580 genome was determined, revealing 4,208 predicted protein-coding genes, 7 rRNA operons and 72 tRNA genes.The complete sequence of the  Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.We determined the complete nucleotide sequence of the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.Despite the unmistakable organizational similarities between the  Bacillus licheniformis is a Gram-positive, spore-forming bacterium widely distributed as a saprophytic organism in the environment. This species is a close relative of Bacillus subtilis, an organism that is second only to Escherichia coli in the level of detail at which it has been studied. Unlike most other bacilli, which are predominantly aerobic, B. licheniformis is a facultative anaerobe, which may allow it to grow in additional ecological niches. Certain B. licheniformis isolates are capable of denitrification; the relevance of this characteristic to environmental denitrification may be small, however, as the species generally persists in soil as endospores [dospores .B. licheniformis and its extracellular products. The species has been used for decades in the manufacture of industrial enzymes including several proteases, α-amylase, penicillinase, pentosanase, cycloglucosyltransferase, β-mannanase and several pectinolytic enzymes. The proteases from B. licheniformis are used in the detergent industry as well as for dehairing and bating of leather [B. licheniformis are deployed for the hydrolysis of starch, desizing of textiles and sizing of paper [B. licheniformis strains are also used to produce peptide antibiotics such as bacitracin and proticin in addition to a number of specialty chemicals such as citric acid, inosine, inosinic acid and poly-γ-glutamic acid [B. licheniformis isolates can mitigate the affects of fungal pathogens on maize, grasses and vegetable crops [There are numerous commercial and agricultural uses for  leather ,3. Amylamic acid . Some B.le crops . As an eB. licheniformis can be differentiated from other bacilli on the basis of metabolic and physiological tests [B. licheniformis is closely related to B. subtilis and Bacillus amyloliquefaciens on the basis of comparisons of 16S rDNA and 16S-23S internal transcribed spacer (ITS) nucleotide sequences [et al. [B. licheniformis chromosome using a PCR approach, and established a number of regions of colinearity where gene content and organization were conserved with the B. subtilis genome.al tests ,7; howevequences . Lapidus [et al.  recentlyB. licheniformis is an industrial organism used for the manufacture of enzymes, antibiotics, and chemicals, important in nutrient cycling in the environment, and a species that is taxonomically related to B. subtilis, perhaps the best studied of all Gram-positive bacteria, we derived the complete nucleotide sequence of the B. licheniformis type strain (ATCC 14580) genome. With this data in hand, functional and comparative genomics studies can be initiated that may ultimately lead to new strategies for improving industrial strains as well as better understanding of genome evolution among the species within the subtilis-licheniformis group.Given that B. licheniformis ATCC 14580 consists of a circular chromosome of 4,222,336 base-pairs (bp) with an average G+C content of 46.2% . Among the list of hypothetical and conserved hypothetical gene products, 683 (52%) have protein motifs contained in PFAM . There are 72 tRNA genes representing all 20 amino acids and seven rRNA operons.The genome of B. subtilis and other bacteria. These included co-localization of four genes  near the origin, GC nucleotide skew ((G-C)/(G+C)) analysis, and the presence of multiple dnaA-boxes and AT-rich sequences immediately upstream of the dnaA gene [BstBI restriction site between the rpmH and dnaA genes to be the first nucleotide of the B. licheniformis genome. The replication termination site was localized near 2.02 megabases (Mb) by GC skew analysis. This region lies roughly opposite the origin of replication  in B. licheniformis. The Bacillus halodurans genome also lacks an obvious rtp function [B. subtilis acquired the rtp gene following its divergence from B. halodurans and B. licheniformis.The likely origin of replication Figure  was idennaA gene -12. On tfunction ; therefoB. licheniformis ATCC 14580 contains nine identical copies of a 1,285 bp insertion sequence element termed IS3Bli1 [IS3 family elements [IS3Bli1 encodes two predicted overlapping CDSs, designated orfA and orfB in relative translational reading frames of 0 and -1. The presence of a 'slippery heptamer' motif, AAAAAAG, before the stop codon in orfA may indicate that programmed translational frameshifting occurs between these two coding sequences, resulting in a single gene product [orfB gene product harbors the DD(35)E(7)K motif, a highly conserved pattern among insertion sequences. Eight of these IS3Bli1 elements lie in intergenic regions, and one interrupts the comP gene as noted previously [-11) to one identified in the Thermoanaerobacter tengcongensis genome [B. halodurans [Oceanobacillus iheyensis [Streptococcus agalactiae [Streptococcus pyogenes [The genome of  IS3Bli1 . This seelements  includineviously . In addis genome ; howeverlodurans , Oceanobheyensis , Streptoalactiae  and Strepyogenes .B. subtilis prophages PBSX and φ-105), were discovered by noting the presence of nearby genes that code for the large subunit of terminase, a signature protein that is highly conserved among prophages [B. subtilis phage SPP1); however, its absence may be the result of genome deterioration during evolution. Interestingly, we observed that regions in which the G+C content is less than 39% usually encoded proteins that have no B. subtilis ortholog and share identity only with hypothetical and conserved hypothetical genes. Two of these AT-rich segments correspond to the NZP2 and NZP3 prophages.The presence of several bacteriophage lysogens or prophage-like elements was revealed by Smith-Waterman comparisons to other bacterial genomes and by their AT-rich signatures Figure , Table 2rophages . InteresB. licheniformis. The first gene encodes a protein of 1,277 amino acids for which Interpro predicts 16 collagen triple-helix repeats, and the amino acid pattern TGATGPT is repeated 75 times within the polypeptide. The second CDS is smaller, and encodes a protein with 11 collagen triple-helix repeats; the same TGATGPT motif recurs 56 times. The primary translation products from these genes do not contain canonical signal peptides for secretion, and they do not contain motifs for the twin-arginine or sortase-mediated translocation pathways. Therefore, it is not likely that they are exported to the cell surface or the extracellular medium. Interestingly, the chromosomal region (19 kb) adjacent to these genes is clearly non-colinear with the B. subtilis genome, and virtually all of the predicted genes encode hypothetical or conserved hypothetical proteins. There are a number of bacterial proteins listed in PIR that also contain collagen triple-helix repeat regions, including two from Mesorhizobium loti (accession numbers NF00607049 and NF00607035) and three from B. cereus . These putative orthologs share 53-76% amino-acid sequence identity with their counterparts in B. licheniformis, and their functions are unknown.An isochore plot Figure  also revBacillus licheniformis genome, 689 of the 4,208 gene models have signal peptides forecast by SignalP [B. licheniformis ATCC 14580 genome that are not found in B. subtilis 168. In accordance with its saprophytic lifestyle, the secretome of B. licheniformis encodes numerous secreted enzymes that hydrolyze polysaccharides, proteins, lipids and other nutrients.In the  SignalP  and 134 B. licheniformis, and there are no counterparts in B. subtilis 168. The enzymes encoded by the first gene cluster include two putative endoglucanases belonging to glycoside hydrolase families GH9 and GH5, a probable cellulose-1,4-β-cellobiosidase of family GH48, and a putative β-mannanase of family GH5. The β-mannanase (GH5) and endoglucanase (GH9) both harbor carbohydrate-binding motifs. With the exception of the cellulose-1,4-β-cellobiosidase (GH48), all of the gene products encoded in this cluster have secretory signal peptides, and all have homologs in Bacillus species other than B. subtilis. The overall G+C content of this cluster (48%) does not appear to differ appreciably from that of the genome average (46%). The second gene cluster encodes a putative β-glucosidase (GH1) and three components of a cellobiose-specific PTS transport complex. A second β-glucosidase (GH3) gene is present at an unlinked locus in the genome. Collectively, the genes in these two clusters should enable B. licheniformis to utilize cellulose as a carbon and energy source, converting it to cellobiose and ultimately glucose. In this regard, we have confirmed that B. licheniformis ATCC 14580 is capable of growth on carboxymethyl cellulose as a sole carbon source (not shown). The chromosome of B. licheniformis ATCC 14580 encodes a number of additional carbohydrase activities that may allow the organism to grow on a broad range of polysaccharides. These include xylanase, endo-arabinase and pectate lyase that may be involved in degradation of hemicellulose, α-amylase and α-glucosidase for starch hydrolysis, chitinases for the breakdown of chitooligosaccharides from fungi and insects, and levanase for utilization of β-D-fructans (levans). Several of these activities are marketed as industrial enzymes.Cellulose is the most abundant polysaccharide on Earth, and microorganisms that hydrolyze cellulose contribute to the global carbon cycle. Interestingly, two gene clusters involved in cellulose degradation and utilization were discovered in B. licheniformis ATCC 14580 possesses the ability to acquire nitrogen from exogenous proteins, peptides, amino acids, ammonia, nitrate and nitrite. Like B. subtilis, the repertoire of extracellular proteases produced by B. licheniformis includes serine proteases , metalloprotease (mpr), and an assortment of endo- and exopeptidases , yfxM, yuiE, yusX, ywaD, pepT). However, B. licheniformis also has the capacity to produce a number of additional proteases and peptidases that are not encoded in the B. subtilis genome. These include a clostripain-like protease, a zinc-metallopeptidase, a probable glutamyl endopeptidase, an aminopeptidase C homolog, two putative dipeptidases and a zinc-carboxypeptidase.Saprophytic organisms must utilize a variety of nitrogenous compounds as nutrients for growth and metabolism. On the basis of the information encoded in its genome, B. licheniformis also has the ability to utilize amino and imino nitrogen from arginine, asparagine and glutamine via arginine deiminase, arginase, asparaginase and glutaminase activities. Interestingly, there appear to be two genes each for arginase, asparaginase and glutaminase. Presumably, the arginine deiminase activity is expressed during anaerobic growth on arginine, whereas the arginase activities are predominant during aerobic growth. The occurrence of putative arginase genes is somewhat of an enigma in B. licheniformis, because there are no genes encoding urease activity for the hydrolysis of urea that is generated by the arginase reaction. In addition to the absence of urease gene homologs (ureABC) in B. licheniformis, the glutamine ABC transporters  are also lacking.B. licheniformis and B. subtilis owing to the presence of key genes such as glnA, glnR, tnrA and nrgA in both species. Likewise, the pathways for nitrate/nitrite transport and metabolism in B. licheniformis appear to be analogous to the corresponding pathways in B. subtilis as suggested by the presence of nasABC (nitrate transport), narGHIJ (respiratory nitrate reductase), and nasDEF (NADH-dependent nitrite reductase) genes. Unlike B. subtilis, B. licheniformis evidently possesses the capability for anaerobic respiration using nitric oxide reductase. Moreover, the gene encoding this activity lies in a cluster that includes CDSs for narK (nitrite extrusion protein), two putative fnr proteins , and a dnrN-like gene product (nitric oxide-dependent regulator). These observations are consistent with previous findings that certain B. licheniformis isolates are capable of denitrification [B. licheniformis may be small as the species exists predominantly as endospores in soil [It appears that nitrogen assimilation and transport pathways may be coordinated similarly in fication . While d in soil .N-carbamoyl amino acids. Hydantoinase activities have been detected in a variety of bacterial genera, and a cluster of six genes in B. licheniformis appears to confer a similar capability. This gene cluster encodes N-methylhydantoinase (ATP-hydrolyzing), hydantoin utilization proteins A and B (hyuAB homologs), a possible transcriptional regulator (TetR/AcrR family), a putative pyrimidine permease, and a hypothetical protein that contains an Interpro domain (IPR004399) for phosphomethylpyrimidine kinase.Microbial D-hydantoinase enzymes have been applied to the industrial production of optically pure D-amino acids for synthesis of antibiotics, pesticides, sweeteners and therapeutic amino acids . This enet al. [B. subtilis 168. This list includes several chaperonins, signal peptidases, components of the signal recognition particle and protein translocase complexes. All members of this list have B. licheniformis counterparts. In addition to the Sec pathway, some B. subtilis proteins are directed into the twin-arginine (Tat) export pathway, possibly in a Sec-independent manner. Curiously, the B. licheniformis genome encodes three tat gene orthologs , but two others (tatAC and tatAD) are conspicuously absent. Furthermore, specific proteins may be exported to the cell surface via lipoprotein signal peptides or sortase factors. Lipoprotein signal peptides are cleaved with a specific signal peptidase (Lsp) encoded by the lspA gene in B. subtilis. An lspA homolog can be found in B. licheniformis as well, suggesting that this species may possess the ability to export lipoproteins via a similar mechanism. Lastly, surface proteins in Gram-positive bacteria are frequently attached to the cell wall by sortase enzymes, and genome analyses have revealed that more than one sortase is often produced by a given species. In this regard, three possible sortase gene homologs were detected in the genome of B. licheniformis ATCC 14580. Together these observations suggest that the central features of the protein export machinery are principally conserved in B. subtilis and B. licheniformis.Kunst et al.  listed 1et al. [B. licheniformis. These six exceptions (spsABCEFG) comprise an operon involved in synthesis of a spore coat polysaccharide in B. subtilis. In addition, the response regulator gene family (phrACEFGI) appears to have a low level of sequence conservation between B. subtilis and B. licheniformis.From the list of 139 sporulation genes tabulated by Kunst et al. , all butB. licheniformis strains [B. licheniformis genome encodes all of the late competence functions ascribed in B. subtilis , it lacks an obvious comS gene, and the comP gene is punctuated by an insertion sequence element (IS3Bli1), suggesting that the early stages of competence development have been pre-empted in B. licheniformis ATCC 14580. Whether these early functions can be restored by introducing the corresponding genes from B. subtilis is unknown. In addition to an apparent deficiency in DNA uptake, two type I restriction-modification systems were discovered that may also contribute to diminished transformation efficiencies. These are distinct from the ydiOPS genes of B. subtilis, and could participate in degradation of improperly modified DNA from heterologous hosts used during construction of recombinant expression vectors. Each of these loci in B. licheniformis (designated as BliI and BliII) encode putative HsdS, HsdM and HsdR subunits that share significant amino-acid sequence identity to type I restriction-modification proteins in other bacteria. Curiously, the G+C-content for these loci (37%) is substantially lower than the overall genome average (46%) which may hint that they are the result of gene acquisitions. Lastly, the synthesis of a glutamyl polypeptide capsule has also been implicated as a potential barrier to transformation of B. licheniformis strains [B. subtilis usually do not produce significant capsular material, the genome sequence of B. subtilis 168 indicates that they may harbor the genes required for synthesis of polyglutamic acid. In contrast, many B. licheniformis isolates produce copious amounts of capsular material, giving rise to colonies with a wet or slimy appearance. Six genes were predicted (ywtABDEF and ywsC orthologs) that may be involved in the synthesis of polyglutamic acid capsular material in B. licheniformis.Natural competence (the ability to take up and process exogenous DNA in specific growth conditions) is a feature of few  strains . The rea strains . While lB. licheniformis isolates [bac operon [bac operon is not present in the type strain (ATCC 14580) genome. Seemingly, the only non-ribosomal peptide synthase operon encoded by the B. licheniformis type strain genome is that responsible for lichenysin biosynthesis. Lichenysin structurally resembles surfactin from B. subtilis [B. licheniformis counterparts for the pps (plipastatin synthase) and polyketide synthase (pks) operons of B. subtilis. Collectively, these two regions represent sizeable portions  of the chromosome in B. subtilis, although they are reportedly dispensable [Bacitracin is a cyclic peptide antibiotic that is synthesized non-ribosomally by some isolates . While tc operon . Interessubtilis , and thepensable .Bacillus sp. strain HIL-Y85/54728 [B. licheniformis may be an attractive candidate for the development of such an expression system.Unexpectedly, a cluster of 11 genes was found encoding a lantibiotic, with its associated modification and transport functions. We designated this peptide of 75 amino acids as lichenicidin, and its closest homolog is mersacidin from 85/54728 . Lantibi85/54728 . Like me85/54728 . HoweverB. subtilis 168, the B. licheniformis ATCC 14580 chromosome harbors a siderophore biosynthesis gene cluster (dhbABCEF), and the organization of the cluster is similar to the corresponding chromosomal segment in B. subtilis. In addition, the B. licheniformis genome contains a second gene cluster of four genes (iucABCD) that show significant similarity to proteins involved in aerobactin biosynthesis in E. coli. Surprisingly, a gene encoding the receptor protein (iutA homolog) was not found in B. licheniformis. The B. halodurans genome also contains genes that are homologous to iucABCD, but like B. licheniformis, no iutA homolog could be found using BLAST or Smith-Waterman searches.Like B. licheniformis ATCC 14580 gene models were compared to the list of essential genes in B. subtilis [B. subtilis have orthologs in B. licheniformis, and most are present in a wide range of bacterial taxa. In pairwise BLAST comparisons, 66% of the predicted B. licheniformis genes have orthologs in B. subtilis, and 55% of the gene models are represented by orthologous sequences in B. halodurans .The subtilis . Predictet al. [B. licheniformis and B. subtilis .The organism ; however. cereus . Cereuli. cereus . ImportaB. licheniformis, Manachini et al. [B. licheniformis. Similarly, De Clerck and De Vos [B. licheniformis isolates.In a comparison of the genotypic and phenotypic characteristics among 182 soil isolates of i et al.  observedd De Vos  proposedB. licheniformis ATCC 14580 was sequenced by a combination of the whole-genome shotgun method [MboI-digested genomic DNA that was enriched for fragments of 2-3 kb by preparative agarose gel electrophoresis. Approximately 49,000 random clones were sequenced using dye-terminator chemistry (Applied Biosystems) with ABI 377 and ABI 3700 automated sequencers yielding approximately 6× coverage of the genome. A combination of methods was used for gap closure, including sequencing on fosmids [The genome of n method  and fosmn method . Plasmid fosmids  and prim fosmids .AAGGAG . This motif concurs with the consensus Shine-Dalgarno sequence for B. subtilis (AAAGGAGG) [B. subtilis homologs were also used to determine the incidence of specific genes. During the gene-finding process, possible errors and frameshifts were detected by both visual inspection of the CDSs to look for interrupted or truncated genes and by deploying FrameD software [IS3Bli1 elements). It is not known at present whether these represent pseudogenes or instances of programmed translational frameshifting. The positions of rRNA operons in the genome assembly were confirmed by long-range PCR amplification using primers that annealed to genes flanking the rRNA genes. These PCR fragments were sequenced to high redundancy and the consensus sequences were manually inserted into the assembly. Among the seven rRNA operons, the nucleotide sequences of 16S and 23S genes are at least 99% identical, differing by only one to three nucleotides in pairwise comparisons. Protein-coding sequences were annotated in an automated fashion with the following software applications. Predicted proteins were compared to the nonredundant database PIR-NREF [B. subtilis genome [-5. InterProScan was used to predict putative function [B. licheniformis genes that shared significant homology with B. subtilis counterparts were named using the nomenclature in the SubtiList database [Protein-coding regions in the assembled genome sequence were identified using a combination of previously described software tools including EasyGene , GlimmerAAGGAGG) . RBSFINDsoftware . FrameshPIR-NREF  and the s genome  using BLfunction . The Intfunction , TIGRFAMfunction , Interprfunction  signal pfunction  and tranfunction . These Cfunction  with manfunction ,55. Phagfunction . B. lichdatabase  with upddatabase  and UniPdatabase  databaseB. licheniformis, B. subtilis and B. halodurans genomes with precomputed BLAST results stored in a local database.VisualGenome software  was used for comparisons of ortholog distribution among B. licheniformis ATCC 14580 genome is CP000002. An interactive web portal for viewing and searching the assembled genome based on the generic genome browser developed by Stein et al. [The GenBank accession number for the n et al.  is availn et al. .


Study group I) and 2) future self-reported sick leave due to NSBD in nursing aides/assistant nurses (Study group II).Sick leave due to neck, shoulder and back disorders (NSBD) is higher among health-care workers, especially nursing aides/assistant nurses, compared with employees in other occupations. More information is needed about predictors of sick leave among health care workers. The aim of the study was to assess whether self-reported factors related to health, work and leisure time could predict: 1) future certified sick leave due to any cause, in nursing aides/assistant nurses  and previous sick leave , were associated with a higher risk of future sick leave due to any cause. Factors related to health, work and leisure time, i.e. previous low back disorders  previous sick leave, not due to NSBD , high strain work  and high perceived physical exertion in domestic work  were associated with a higher risk of future sick leave due to NSBD. In the final analyses, previous low back disorders and previous sick leave remained significant in both study groups.The results suggest a focus on previous low back disorders and previous sick leave for the design of early prevention programmes aiming at reducing future sick leave due to any cause, as well as due to NSBD, among nursing aides/assistant nurses. A multifactorial approach may be of importance in the early prevention of sick leave due to NSBD. Over the past century, sick leave, mainly attributed to musculoskeletal disorders (MSD), has increased in Sweden, especially among women . In 2003Many studies have focused on the association between risk factors and the reporting of neck, shoulder and back pain -8. HowevStudies have pointed to different factors in different populations to relate with future sick leave. Demographic factors such as age may be associated with sick leave mainly in individuals with chronic neck-or-back disorders . In someMental problems, such as, for example, anxiety, also increased the risk of future sick leave among nurses . MoreoveThe effects of physical exercising on future sick leave are not clear. Sedentary activities in leisure time were, however, associated with a higher prevalence of back symptoms and sick leave . EriksenTo summarise, a large number of factors, demographic, medical, physical, psychosocial, psychological as well as socioeconomic, have been documented as risk factors for future sick leave in different working populations .It has been concluded that there is a lack of studies on neck-and-back disorders among women working within special groups as for example the health-care sector . BecauseIt is of interest to analyse whether the above-mentioned factors associated with future sick leave in different populations, can also predict future sick leave among nursing aides/assistant nurses working within the home-care service.Working as a nursing aide/assistant nurse within the home-care service is generally physically heavy work, requiring repeated transfers and lifts of patients. The physical load on the spine depends on several factors, for example, the weight of the patient, work place design, work organisation, work technique, work equipment, the cooperation of the patient etc . Even trThe aim of the present study was to assess whether a selection of self-reported measures of factors related to health, work and leisure time could predictStudy group I).1. future, certified sick leave after 18 months, due to any cause, in a group of working nursing aides/assistant nurses (Study group II).2. future, self-reported sick leave after 18 months, due to neck, shoulder and/or back disorders, in a group of working nursing aides/assistant nurses . All participants gave their written consent before participation.The municipal home-care services were organised in six units situated in different geographically defined areas of a medium-sized town in the south of Sweden. Initially, all of the 659 women working in five of the six units were invited to participate in the prevention project. (One unit was excluded due to its participation in another study). The inclusion criteria were: Swedish speaking, permanently employed, not pregnant, in work and working at least 50% of full time. Participation was accepted by 534 81%) of the women. The main reasons not to participate were: the opinion that the project was important only for younger staff, dissatisfaction with the work situation, participation in compulsory, further education for nursing aides to qualify as assistant nurse, lack of time or family reasons. As we wanted to elucidate predictors of future sick leave at an 18 month follow-up, women who at that time had retired from work, had left work or were off duty, were excluded from the analyses. Two women were deceased (n = 91). Thus, the final sample consisted of 443 nursing aides/assistant nurses  or to a Control group. Intervention groups and the Control group were evaluated and compared at the 18-month follow-up. No significant differences between the groups could be shown . Thus, iStudy group I comprised 443 women who were not on sick leave when they completed the baseline questionnaire. Information on certified sick leave due to any cause during the six months preceding the 18-month follow-up was obtained from the National Social Insurance Board. The number of women who participated in intervention was 241 (54%). Demographic data, disorders, sick leave and participation in intervention for Study group I are presented in Table Study group II comprised 274 women. At baseline, 383 women had reported no sick-leave due to symptoms from the neck, shoulder and/or back during the preceding 12 months. At the 18-month follow-up, 109 of these women did not fill in the questionnaire . At the 18-month follow-up, questionnaires were sent out to a contact person at each work place, who was made responsible for the distribution to the participants in the study. Each respondent received a stamped envelope in which she returned her own questionnaire to the project nurse (Study group II).All participants were asked to fill in a questionnaire at the start of the study and after 6, 12 and 18 months. At baseline, the questionnaires were administered by the project nurse and filled in at the work place .Study group II at the 18-month follow-up. The validity of the responses was checked for all follow-up questionnaires against the general Nordic Musculoskeletal Questionnaire (NMQ) [At all follow-ups the nursing aides/assistant nurses were asked: "Have you been on sick leave any time during the previous six months due to neck, shoulder and/or back disorders?". Response options were yes/no and constituted the dependent variable for re (NMQ)  i.e. theAnxiety and depression were assessed by the Hospital Anxiety and Depression Scale (HAD)  which coThe prevalence of musculoskeletal disorders from the neck, shoulders and back was assessed by the general Nordic Musculoskeletal Questionnaire (NMQ) . ParticiInformation on sick leave was obtained from the National Social Insurance Board.Study group II, who had been on sick leave, but who in the questionnaires had reported no sick leave due to symptoms from the neck, shoulder and/or back were assumed to be on sick leave due to other reasons. Work loss was recorded as whole working days. As all data on sick leave were extremely skewed, we chose to dichotomise the material. The third quartile was used as cut-off point, which for Study group I was 0 days/≥1 days and for Study group II <9 days/≥9 days.Individuals in The participants were asked: "What degree of physical exertion do you usually perceive in your present job?" . The quePsychosocial factors at work, such as social support, decision latitude and psychological load were assessed by a questionnaire developed by Rubenowitz ,35. The The participants were asked: "To what extent have you performed physical activities or fitness training during the previous six months?" . The queThe participants were asked about physical exertion in domestic work in the same way as for physical exertion at work: "What degree of physical exertion do you usually perceive in your daily domestic work?" . ResponsThis question, originally developed for the Malmo Diet and Cancer study on 53000 Swedish men and women, was defined by "Have you lately felt mentally stressed or been under psychological pressure due to problems outside work?" Response options were yes/no .Perceived satisfaction with her own economic situation was assessed by a seven-point scale where the first point represented "very bad" and the seventh point "Excellent, could not be better" .All logistic regression analyses were adjusted for age and intervention.Study group II. There were minimal differences between the quartiles of the two study groups. Thus, for the purpose of comparing the two groups, the same cut-off points were used. However, for sick-leave before baseline we chose two different cut-off points, as the third quartile differed between the two study groups, being 0 days for Study group I and 9 days for Study group II.Three models were tested, namely, for factors related to health, to work and to leisure time. The associations between the independent variables and the outcome variables were first analysed for each of the three models by univariate logistic regression. Secondly, each model was analysed with all variables included in a backward stepwise multivariate regression analysis with a likelihood ratio test. Finally, a multivariate model including all the variables from the three models together was tested. The criteria for inclusion and exclusion were p = 0.05. The logistic regression analyses were tested for goodness of fit by means of the Hosmer and Lemeshow method. Factors were dichotomized by using cut-off points described in the literature. When no such literature was found, cut-off points were taken as the first or the third quartile for frequencies for The sample size may vary in the different analyses due to missing values. Comparisons between drop-outs and participants were analysed by t-test or the chi-square test. Correlations between the exposure variables were calculated with Pearson correlation coefficients (r). All statistical calculations were performed using the SPSS 11.5.1 Software for Windows .In the univariate logistic regression analysis, there were minimal differences between odds ratios adjusted and not adjusted for age and intervention. Thus, in Tables Age or participation in intervention (presented under section Methods-Subjects) did not have any effect on future sick leave due to any cause.Perceived disorders of the low back and sick leave during the 12 months preceding baseline predicted future sick leave due to any cause in the unadjusted and the adjusted univariate analyses as well as in the multivariate analysis Table . PerceivFactors related to work did not predict future sick leave due to any cause Table  and 5.Factors related to leisure time did not predict future sick leave in the univariate or in the multivariate analysis Tables  and 5.When all variables from the three models were entered into the final model, disorders of the low back during the preceding 12 months  and sick leave during the 12 months preceding baseline  were the only factors predicting sick leave due to any cause after 18 months.A tendency towards a reduced risk of future sick leave due to neck, shoulder and/or back disorders was found for the women who participated in some intervention . Age did not have an impact on sick leave .Anxiety, low back disorders during the 12 months preceding baseline as well as sick leave due to reasons other than disorders of the neck, shoulders and/or back were significant predictors of sick leave after 18 months due to disorders of the neck, shoulders and/or back p < 0.05) before and after adjustment for age and intervention . However, after adjustments, only high-strain work demonstrated significant association with future sick leave . Only high-strain work predicted future sick leave due to disorders in the neck, shoulder and/or back Table .High perceived physical exertion in domestic work and perceived psychological stress outside work were associated with future sick leave due to neck, shoulder and/or back disorders both before and after adjustments for age and intervention Table . In the In the final model, where all variables from the three models were entered into the logistic regression analysis, perceived low back disorders some time during the 12 months preceding baseline and sick leave of > 9 days in the year before baseline remained significant predictors of sick leave due to neck, shoulder and/or back disorders at 18 months after baseline .Study group I and Study group II. Thus, the hypothesis that risk factors for future sick leave due to disorders in the neck, shoulders and/or back are different from sick leave due to any cause was only partly demonstrated. This is in concordance with other studies, which have also documented earlier experience of back pain and sick leave preceding baseline as predictors of future sick leave due to low back pain among nursing personnel [Study group I as well as in Study group II. One explanation for this finding may be the content of the work performed by a nursing aide/assistant nurse, with repeated repositioning, transfers and lifts of patients, which result in high spinal loads mainly on the low back [Study group I and Study group II, including all variables from the three models, only low back disorders and previous sick leave remained significant. However, in Study group II, due to the small number of women on sick leave due to neck, shoulder and/or back disorders at the 18-month follow-up, results of this final analysis should be interpreted with caution.The results of the present study indicated that only factors related to health were associated with future sick leave due to any cause in a group of nursing aides/assistant nurses. On the other hand, factors related to health, the workplace as well as leisure time were associated with future sick leave due to neck, shoulder and/or back disorders, indicating a multifactorial background for these disorders. Low back disorders and sick leave during the 12 months preceding baseline, were significantly associated with future sick leave in both ersonnel  as well ersonnel . Natvig ersonnel , showed low back ,26. ThusGenerally, most studies on predictors of future sick leave are made on men and few studies focusing on nursing personnel have been published. Thus, comparisons of the results from the current study with similar studies are limited.The present study was performed during a period with frequent reorganisations and reductions of staff within the home-care services in the city studied, as in general in Sweden. A greater amount of work was performed faster than before and the category of patients, for whom the community home-care organisation took responsibility for, were more handicapped than previously. Thus, the physical and psychological stresses were generally exacerbated during the period studied. In the present study, among the factors related to work, only high-strain work  predicted future sick leave due to neck, shoulder and/or back. Marras et al.  showed tWomen still do more unpaid work than men. In a 23-year perspective study, the physical work load decreased among men but not among women . In the We could not verify inactivity to be a predictor of future sick leave. The cut-off point was based on the assumption that a sedentary life style would predict future sick leave as shown in the study by Hildebrandt et al. . In the Study group II, the conclusions of this analysis are limited. However, neither in the Study group I, was social support related to sick leave. This finding is contradictory to the results of the study by Eriksen et al. on 5563 Norwegian nursing aides [Moreover, in the present study, social support did not predict future sick leave. Since there was a significant initial difference in perceived social support between participants and non-participants in ng aides . These ang aides  could alStudy group I were obtained from the National Social Insurance Board and for Study group II were taken from the questionnaires. Systematically recorded, certified sick leave is assumed to be more valid than data on sick leave based on questionnaires [Study group II, questionnaires were filled in at the 6, 12 and 18 month follow-ups. In order to strengthen the validity of the question "Have you been on sick leave any time during the preceding six months due to neck, shoulder and/or back disorders", answers at the follow-ups were checked against the NMQ [Data on the dependent variable sick leave, for onnaires . For Stu the NMQ . Partici the NMQ .Age was not related to any reason for sick leave, which is in contrast with the results of a review study by Turner et al.  on peoplStudy group II, a large number of participants dropped out at the 18-month follow-up, which constitutes a risk of selection bias. The number of drop-outs is comparable with that found in other studies on nursing personnel [Study group II to be representative as a working group of nursing aides/assistant nurses.In ersonnel ,30,44. IStudy group I to other women working as nursing aides/nursing assistants should be satisfactory, as all women participating at baseline were analysed at18 months and the data of the dependent variable, certified sick leave, were provided by the National Social Insurance Board.The feasibility to generalise the results from Study group I, 54% and in Study group II, 64% of the nursing aides/assistant nurses had participated in some intervention. The main purpose of this intervention project was not to reduce sick leave. The participants were informed that the aim of the project was to prevent or reduce pain and discomfort of the neck, shoulder and back. Sick leave was not mentioned. Thus, in our opinion, participation in intervention groups should not have biased the reporting of sick leave due to neck, shoulder and back disorders.In The present study indicated that previous disorders of the low back and previous sick leave were the strongest predictors of future sick leave due to any cause as well as of future sick leave due to neck, shoulder and/or back disorders in a group of nursing aides/assistant nurses, who at baseline were at work. Previous neck or shoulder disorders did not predict future sick leave.Moreover, factors related to health, work and leisure time were all related to sick leave due to neck, shoulder and/or back disorders, while factors related only to health predicted sick leave due to any cause.The results point to the importance of a primary focus on previous low back disorders and previous sick leave when designing early prevention programmes for future sick leave among this working population. The results might also point to the importance of a multifactorial approach when customising early prevention programmes with the purpose to decrease future sick leave due to neck, shoulders and/or back disorders in a group of women working as nursing aides/assistant nurses.The author(s) declare that they have no competing interests.EHj participated in the design of the study, participated in collecting the data, performed the statistical analyses, and drafted the manuscript. IJ and EHm participated in the design of the study and in the progress and revision of the manuscript. CE participated in the progress and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Previous studies suggest that the cyclooxygenase-2 (COX-2) inhibitor nimesulide has a remarkable protective effect against different types of brain injury including ischemia. Since there are no reports on the effects of nimesulide on permanent ischemic stroke and because most cases of human stroke are caused by permanent occlusion of cerebral arteries, the present study was conducted to assess the neuroprotective efficacy of nimesulide on the cerebral infarction and neurological deficits induced by permanent middle cerebral artery occlusion (pMCAO) in the rat.via surgical insertion of a nylon filament into the internal carotid artery. Infarct volumes  and functional recovery, assessed by neurological score evaluation and rotarod performance test, were performed 24 h after pMCAO. In initial experiments, different doses of nimesulide  or vehicle were administered 30 min before pMCAO and again at 6, 12 and 18 h after stroke. In later experiments we investigated the therapeutic time window of protection of nimesulide by delaying its first administration 0.5–4 h after the ischemic insult.Ischemia was induced by permanent occlusion of the middle cerebral artery in rats, Repeated treatments with nimesulide dose-dependently reduced cortical, subcortical and total infarct volumes as well as the neurological deficits and motor impairment resulting from permanent ischemic stroke, but only the administration of the highest dose (12 mg/kg) was able to significantly (P < 0.01) diminish infarct volume. The lower doses failed to significantly reduce infarction but showed a beneficial effect on neurological function. Nimesulide (12 mg/kg) not only reduced infarct volume but also enhanced functional recovery when the first treatment was given up to 2 h after stroke.These data show that nimesulide protects against permanent focal cerebral ischemia, even with a 2 h post-treatment delay. These findings have important implications for the therapeutic potential of using COX-2 inhibitors in the treatment of stroke. The brain is highly sensitive to disturbance of its blood supply. Stroke is a devastating disease and is the third most common cause of death, and the most common cause of motor and mental disability in adults, in developing countries . Complex2+-related activation of intracellular second messenger systems, the increase in reactive oxygen species formation, as well as hypoxia itself triggers the expression of a large number of pro-inflammatory genes following cerebral ischemia. Thus, mediators of inflammation such as platelet-activating factor (PAF), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), chemokines  and other pro-inflammatory factors are produced by the ischemic brain tissue [A number of interacting and sequentially evoked events tend to reinforce the initial ischemic insult. A key role in these processes is played by post-ischemic inflammation. The Can tissue . In addin tissue -12.50 selectivity ratio of 0.06 (whole blood assay) [Nimesulide (N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide) is a non-steroidal anti-inflammatory drug with potent effects. It shows a high affinity and selectivity for COX-2 with a COX-2/COX-1 ICd assay) . Nimesuld assay) ,14. Seved assay) , kainated assay) , quisquad assay) , diffused assay) ,19, glutd assay)  and indud assay) .Recently, we have found a significant neuroprotective effect of nimesulide both in global cerebral ischemia ,22, a tySince most cases of human ischemic stroke are caused by permanent occlusion of cerebral arteries -26, the Male Sprague-Dawley rats  weighing 280–340 g at the time of surgery were used in the present study. Our institutional animal care and use committee approved the experimental protocol (No. 02/67). The animals were quarantined for at least 7 days before the experiment. Animals were housed in groups in a room whose environment was maintained at 21–25°C, 45–50 % humidity and 12-h light/dark cycle. They had free access to pellet chow and water. Animal housing, care, and application of experimental procedures were in accordance with institutional guidelines under approved protocols.2 and pCO2). The effects observed with nimesulide in the present study were not related to modification of physiological variables since these parameters did not differ between nimesulide-treated and vehicle-treated rats (data not shown). These findings are in agreement with our previous results [Rats were anesthetized with chloral hydrate . Once surgical levels of anesthesia were attained , ischemia was induced by using an occluding intraluminal suture as described previously -29. Brie results ,12, suggAn unaware independent observer performed the neurological evaluations prior to the sacrifice of the animals according to a six-point scale: 0= no neurological deficits, 1= failure to extend left forepaw fully, 2= circling to the left, 3= falling to left, 4= no spontaneous walking with a depressed level of consciousness, 5= death ,31.Motor impairment in this study was assessed with the use of the accelerating rotarod . Rats were given 2 training sessions 10 minutes apart before surgery. Rats were first habituated to the stationary rod. After habituation they were exposed to the rotating rod. The rod was started at 2 rpm and accelerated linearly to 20 rpm within 300 sec. Latency to fall off the rotarod was then determined before ischemia (presurgery) and before sacrificing the animals. Animals were required to stay on the accelerating rod for a minimum of 30 sec. If they were unable to reach this criterion, the trial was repeated for a maximum of five times. The two best (largest) fall latency values a rat could achieve then were averaged and used for data analysis. Rats not falling off within 5 min were given a maximum score of 300 seconds ,33. A shThe method for quantification of infarct volume was performed exactly as reported by others ,35. BrieAt various times after pMCAO  the animals were sacrificed and the brains were quickly removed, sectioned and stained as previously described in order to calculate the infarct volume.In order to evaluate the effect of nimesulide administration on rat focal cerebral ischemia, three different doses of nimesulide 3, 6 and 12 mg/kg) were given to rats by intraperitoneal administration 30 min before the onset of pMCAO . Additional doses were given at 6, 12 and 18 h after stroke. This treatment schedule and dosage range was based on the pharmacokinetic profile of nimesulide  and on o mg/kg weAfter investigating the dose-response relationship, we studied the effect of nimesulide  when administered 0.5, 1, 2, 3 or 4 h after ischemia . The corresponding vehicle-treated groups were included as controls (n = 7–10 rats per group). Three additional doses were given every 6 h after the first treatment with nimesulide or vehicle exactly as described before for the repeated treatment schedule in the dose-response experiment. After completing the neurological evaluation and rotarod performance at 24 h after permanent focal cerebral ischemia, animals were sacrificed and the brains were removed to calculate the infarct size.post-hoc Student-Newman-Keuls test (multiple comparison). Neurological deficit scores were analyzed by Kruskal-Wallis non-parametric ANOVA followed by the Dunn test (multiple comparison) or Mann-Whitney test for analysis of individual differences. Rotarod performance was expressed as a percentage of pre-surgery values for each rat and analyzed by ANOVA for repeated measures followed by the Student-Newman-Keuls test. Differences were considered significant when p < 0.05.Data are presented as means ± S.D. Values were compared using t-test (two groups) or one-way ANOVA with The temporal evolution of the lesion volumes is presented in Fig. With regard to the neurological deficits and motor impairment induced by pMCAO , it is important to emphasize the fact that these parameters were maximal by 12 h after stroke and the animals did not show any further increase in the neurological deficits or motor impairment after 24 or 48 h of the occlusion, as depicted in Fig. Repeated treatments with nimesulide dose-dependently reduced cortical, subcortical and total infarct volumes in the permanent model of stroke, although only the administration of the highest dose 12 mg/kg) was able to significantly (P < 0.01) diminish brain damage  in a situation in which its first administration was delayed for 0.5–4 h after the ischemic challenge. A significant reduction in subcortical infarct volume was observed when the treatment was delayed until 0.5–1 h after pMCAO, but this protective effect of nimesulide was not evident when administered after 2–4 h of the onset of permanent occlusion Fig. . In the Of interest is the finding that nimesulide not only reduced infarct volume but also enhanced functional recovery when the first treatment is given 2 h after permanent ischemic stroke. Post-ischemic treatment with nimesulide significantly reduced neurological deficits and increased the fall latencies to remain on the accelerating rotarod as compared to those rats given only the vehicle Table . HoweverThe present study was prompted by our previous encouraging results with nimesulide in a model of transient focal cerebral ischemia, which show that this COX-2 inhibitor is able to potently reduce infarct volume and improve functional recovery . These nThe core findings of this study are: (i) administration of clinically relevant doses of nimesulide confers protection against the damage induced by permanent focal cerebral ischemia in two modalities  and (ii) nimesulide's neuroprotection is still evident when the first administration is delayed until 2 h after the onset of stroke.Depending on the experimental conditions, the temporal evolution of ischemic damage may vary considerably ,40,41. TRepeated treatments with nimesulide afforded a more remarkable neuroprotection than the administration of a single dose given before the insult Tables  and 2. TUnfortunately, a large number of promising neuroprotective compounds identified from preclinical experiments have failed in clinical trials in stroke patients ,45-47. ATherefore, our next experiments were conducted to evaluate the effects of nimesulide when administered in a delayed treatment schedule in order to establish the therapeutic time window of protection of this COX-2 inhibitor in pMCAO, thus increasing predictive outcome in the clinic. Interestingly, reduction in infarct size and neurological deficits and improvement of rotarod performance were still observed when nimesulide treatment was delayed until 2 h after ischemia Fig. , Table 3It is important to compare our present results in pMCAO with those previously obtained in transient ischemia . In the However, our present results suggest that in permanent stroke, COX-2 inhibition by nimesulide is not as protective as in transient models (39 % of infarct reduction with pre-treatment in pMCAO vs. 60 % lesion reduction in transient ischemia with immediate treatment) and the therapeutic time window is narrower as compared to temporary occlusion models (2 h in pMCAO vs. 24 h in transient ischemia) as the present results  declare that they have no competing interests.ECJ carried out the surgical procedures to induce stroke, participated in the design of the study and in the statistical analysis, reviewed the data and drafted the manuscript. NHM performed the evaluation of neurological deficits and rotarod performance. AGF and MGC performed the calculation of the infarct volumes and participated in the statistical analysis of the data. OSL, EM and BLF participated in the design and coordination of the study, reviewed the data, provided consultation and helped to draft the manuscript. OSL and BLF share senior authorship. All authors read and approved the final manuscript.