Chronic obstructive pulmonary disease (COPD) is a major global health problem and is predicted to become the third most common cause of death by 2020. Apart from the important preventive steps of smoking cessation, there are no other specific treatments for COPD that are as effective in reversing the condition, and therefore there is a need to understand the pathophysiological mechanisms that could lead to new therapeutic strategies. The development of experimental models will help to dissect these mechanisms at the cellular and molecular level. COPD is a disease characterized by progressive airflow obstruction of the peripheral airways, associated with lung inflammation, emphysema and mucus hypersecretion. Different approaches to mimic COPD have been developed but are limited in comparison to models of allergic asthma. COPD models usually do not mimic the major features of human COPD and are commonly based on the induction of COPD-like lesions in the lungs and airways using noxious inhalants such as tobacco smoke, nitrogen dioxide, or sulfur dioxide. Depending on the duration and intensity of exposure, these noxious stimuli induce signs of chronic inflammation and airway remodelling. Emphysema can be achieved by combining such exposure with instillation of tissue-degrading enzymes. Other approaches are based on genetically-targeted mice which develop COPD-like lesions with emphysema, and such mice provide deep insights into pathophysiological mechanisms. Future approaches should aim to mimic irreversible airflow obstruction, associated with cough and sputum production, with the possibility of inducing exacerbations. The global burden of disease studies point to an alarming increase in the prevalence of chronic obstructive pulmonary disease (COPD)  which isIn contrast to the large amount of experimental studies on allergic asthma and the detailed knowledge that exists on mediators of allergic airway inflammation ,6, much in vivo animal models to examine more closely pathogenesis, functional changes and the effects of new compounds or treatments. However, animal models have limitations since there is no spontaneous model, and models do not necessarily mimic the entire COPD phenotype. The best model remains chronic exposure to cigarette smoke, since this is the environmental toxic substance(s) that cause COPD in man. However, other substances are also implicated such as environmental pollution due to car exhaust fumes. The present review draws attention to specific aspects of functional and structural features of COPD that need to be realized when interpreting molecular mechanisms identified in animal models of COPD. It identifies important issues related to the ongoing experimental COPD research which may in the future provide optimized COPD diagnosis and treatment.Studying the molecular pathways in human subjects is restricted to the use of morphological and molecular assessment of lung tissues obtained at surgery or performing limited in vitro studies at one single point in time . There iBefore characterizing and discussing the different animal models of COPD which have been established so far, it is crucial to reflect that within COPD, different disease stages exist and that only some of them may be mimicked in animal models. The diagnosis of COPD largely relies on a history of exposure to noxious stimuli (mainly tobacco smoke) and abnormal lung function tests. Since COPD has a variable pathology and the molecular mechanisms are only understood to a minor extent, a simple disease definition has been difficult to establish. However, the diagnosis of COPD relies on the presence of persistent airflow obstruction in a cigarette smoker .A classification of disease severity into four stages has been proposed by the GOLD guidelines based primarily on FEV1 . The staThe presence of airflow obstruction which has a small reversible component, but which is largely irreversible is a major feature of COPD as indicated by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines . It is pIn addition to pulmonary alterations, other organ systems may be affected in COPD . SystemiAn important feature of COPD is the ongoing chronic inflammatory process in the airways as indicated by the current GOLD definition of COPD . There a1 and sputum neutrophilia is more prevalent in subjects with chronic cough and sputum production [Neutrophils play a prominent role in the pathophysiology of COPD as they release a multitude of mediators and tissue-degrading enzymes such as elastases which can orchestrate tissue destruction and chronic inflammation ,23. Neutoduction .The second major cell type involved in cellular mechanisms are macrophages . They caLymphocytes are also involved in cellular mechanisms underlying COPD ,35. IncrDifferent inflammatory cell types have also been characterized in airway tissues. Epithelial neutrophilia has been seen in proximal and distal airways of patients with COPD ,43. The On the basis of the different pathophysiological mechanisms illustrated in Fig. An imbalance between protease and antiprotease enzymes has been hypothesized with respect to the pathogenesis of emphysema . This coOxidative stress arising from inhaled noxious stimuli such as tobacco smoke or nitrogen dioxide may be important cause of the inflammation and tissue damage in COPD. This potential mechanism is supported by clinical reports of increased levels of oxidative stress indicators in exhaled breath condensates of COPD patients -55. AparMany mediators have been identified which may contribute to COPD pathogenesis . As in bA Th2 cytokine that has been proposed to be implicated in the pathophysiology of COPD is IL-13. It is also overexpressed and related to the pathogenesis of the asthmatic Th2 inflammation and airway remodelling process . The effParallel to protease-based and extracellular mediator-based concepts, altered intracellular pathways may also play a role in COPD. MAPK signalling pathways i.e. p38 and c-Jun N terminal kinase (JNK) ,77 seem Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD) ,84. It mSince only a minority of smokers (approximately 15 to 20%) develop symptoms and COPD is known to cluster in families, a genetic predisposition has been hypothesized. Many candidate genes have been assessed, but the data are often unclear and systematic studies are currently performed to identify disease-associated genes. Next to alpha1-antitrypsin deficiency, several candidate genes have been suggested to be linked to COPD induction. Genetic polymorphisms in matrix metalloproteinase genes MMP1, MMP9 and MMP12 may be important in the development of COPD. In this respect, polymorphisms in the MMP1 and MMP12 genes, but not MMP9, have been suggested to be related to smoking-related lung injury or are in a linkage disequilibrium with other causative polymorphisms -93. An aThere are three major experimental approaches to mimic COPD encompassing inhalation of noxious stimuli, tracheal instillation of tissue-degrading enzymes to induce emphysema-like lesions and gene-modifying techniques leading to a COPD-like phenotype Figure . These aA variety of animal species has been exposed to tobacco smoke. Next to guinea pigs, rabbits, and dogs, and also rats and mice have been used. Guinea pigs have been reported to be a very susceptible species. They develop COPD-like lesions and emphysema-like airspace enlargement within a few months of active tobacco smoke exposure . By contThe first species to be examined in detail for COPD-like lesions due to tobacco smoke exposure was the guinea pig . DiffereMice represent the most favoured laboratory animal species with regard to immune mechanisms since they offer the opportunity to manipulate gene expression. However, it is more difficult to assess lung function and mice tolerate at least two cigarettes daily for a year with minimal effects on body weight and carboxyhemoglobin levels. Mice differ considerably in respiratory tract functions and anatomy if compared to humans: they are obligate nose breathers, they have lower numbers of cilia, fewer Clara cells and a restriction of submucosal glands to the trachea. Next to a lower filter function for tobacco smoke, mice also do not have a cough reflex and many mediators such as histamine or tachykinins have different pharmacological effects. The development of emphysema-like lesions is strain-dependent: enlarged alveolar spaces and increased alveolar duct area are found after 3–6 months of tobacco smoke exposure in susceptible strains such as B6C3F1 mice . At thesRats are also often used for models of COPD. However, they appear to be relatively resistant to the induction of emphysema-like lesions. Using morphometry and histopathology to assess and compare emphysema development in mice and rats, significant differences were demonstrated : AnimalsRecent work on cigarette exposure in rats indicate that this model also achieves a degree of corticosteroid resistance that has been observed in patients with COPD ,104. ThuGenerally, tobacco smoke exposure may be used to generate COPD features such as emphysema and airway remodelling and chronic inflammation. Although the alterations still differ from the human situation and many involved mediators may have different functional effects especially in the murine respiratory tract, these models represent useful approaches to investigate cellular and molecular mechanisms underlying the development and progression of COPD. As a considerable strain-to-strain and species-to-species variation can be found in the models used so far, the selection of a strain needs to be done with great caution. Animal models of COPD still need to be precisely evaluated as to whether they mimic features of human COPD, and their limitations must be appreciated. Findings obtained from these models may provide significant advances in terms of understanding novel mechanisms involved in COPD.2) is a gaseous irritant which can be used to induce COPD-like lesions in animal models. With daily exposure to high concentrations of SO2, chronic injury and repair of epithelial cells can be observed in species such as rat or guinea pig. The exposure to high-levels of this gas ranging from 200 to 700 ppm for 4 to 8 weeks has been demonstrated to lead to neutrophilic inflammation, morphological signs of mucus production and mucus cell metaplasia and damage of ciliated epithelial cells in rats [2 also leads to loss of cilia and exfoliation of ciliated cells as demonstrated in SO2-exposed dogs using transmission electron microscopy [in vivo and in vitro [Sulfur dioxide  is a another gas that may lead to COPD-like lesions depending on concentration, duration of exposure, and species genetic susceptibility [Nitrogen dioxide  and resistant C3H/HeJ (C3) mice, as well as F1, F2, and backcross (BX) populations derived from them, were acutely exposed to 15 parts per million NO2 for 3 h to determine differences [2 closely resembled C3 mice. Ranges of cellular responses to NO2 in B6:BX and intercross progeny were reported to overlap both progenitor and mean responses of both populations were intermediate to progenitors. Therefore, there were likely two major unlinked genes that account for differential susceptibility to acute NO2 exposure [2 exposure was recently established leading to signs of pulmonary inflammation and progressive development of airflow obstruction [There is also a significant mode of inheritance of susceptibility to NOferences . Signifiexposure . Based otruction .The administration of oxidants such as ozone also causes significant lung injury with some features related to inflammatory changes occurring in human COPD  and thisFurther animal models focus on the exposure to ultrafine particles, silica and coal dust ,139. UltExposure to diesel exhaust particles (DEP) may also lead to chronic airway inflammation in laboratory animals as it was shown to have affect various respiratory conditions including exacerbations of COPD, asthma, and respiratory tract infections . Both thA further toxin is cadmium chloride, a constituent of cigarette smoke. Administration of this substance also leads to alterations in pulmonary integrity with primarily interstitial fibrosis with tethering open of airspaces . A combiEmphysema-like lesions can also be achieved by intrapulmonary challenge with tissue-degrading enzymes and other compounds  Figure . ProteinAmong the different emphysema models, elastase-induced emphysema has also been characterized to be accompanied by pulmonary function abnormalities, hypoxemia, and secretory cell metaplasia which represent characteristic features of human COPD. Recent studies suggested that exogenous retinoic acid can induce alveolar regeneration in models of elastase-induced experimental emphysema  and thatThe mechanisms of emphysema induction by intratracheal administration of elastase encompass an initial loss of collagen and elastin. Later, glycosaminoglycan and elastin levels normalize again but collagen levels are enhanced. The extracellular matrix remains distorted in structure and diminished with resulting abnormal airway architecture . The enlIn general, intrapulmonary administration of tissue-degrading enzymes represents a useful tool especially when focusing on mechanisms to repair emphysematic features. However, the lack of proximity to the human situation needs to be realized since the mechanisms of emphysema induction are clearly not related to the human situation. An advantage of proteinase-based models is the simple exposure protocol with a single intratracheal administration leading to significant and rapid changes. However, extrapolating these findings to slowly developing features of smoking induced human COPD is very difficult since a large number of mediators may not be involved in the rapid proteinase approach. Therefore, these models may not encompass important features of human COPD which may be more closely mimicked by inhalation exposures and it is clear that tissue-degrading enzyme models always represent the picture of an "induced pathogenesis".The genetic predisposition to environmental disease is an important area of research and a number of animal strains prone to develop COPD-like lesions have been characterized -162  gene  and in mA further mechanism to induce emphysema-like lesions is to expose developmentally normal genetically-modified animals to exogenous noxious stimuli such as tobacco smoke. This also allows identifying potential molecular mechanisms involved in the pathogenesis of COPD. Using macrophage elastase (MMP-12) gene-depletion studies it was shown that in contrast to wild type mice, the lung structure of MMP-12 gene-depleted animals remains normal after long term exposure to cigarette smoke . These aIn summary, gene-targeting techniques display very useful tools to examine potential molecular mechanisms underlying human COPD. In combination with inhalation protocols they may identify important protective or pro-inflammatory mediators of the disease.Various other agents have also been characterized to induce airway inflammation injury. In this respect, administration of toxins such as endotoxin leads to a recruitment of neutrophils and macrophage activation with concomitant airspace enlargement ,178.Non-inflammatory emphysema-like lesions may also be accomplished by intravascular administration of a vascular endothelial cell growth factor receptor-2 (VEGFR-2) blocker . VEGF isIn contrast to the variable pathology and different stages of severity in human COPD, currently available animal models are restricted to mimicking a limited amount of characteristic features of COPD. Animal models need to be precisely evaluated based on whether they agree with features of human COPD in order to advance the understanding of mechanisms in human COPD.Based on inhalative exposure to noxious stimuli such as cigarette smoke, the administration of tissue-degrading enzymes or gene-targeting techniques, a number of experimental approaches to mimic acute and chronic features of COPD have been established in the past years. Due to the complexity of the disease, and species-specific differences they are all limited concerning their clinical significance.While the induction of the COPD lesions by tissue-degrading enzymes may appear artificial in many cases, it does not mean that these models are not valuable because they can be used to study many aspects of pulmonary pathophysiology of end-stage emphysema. Cellular mechanisms can be studied efficiently and underlying molecular mechanisms and potential therapeutic approaches can be revealed if the data is extrapolated cautiously.Combined models of inhalative exposure, proteinase-based tissue degradation to produce emphysema and gene-targeting techniques may provide models of COPD which encompass more features of the disease. However, one cannot assume that reproducing COPD with a high degree of fidelity in the animal necessarily means that the model simulates the human condition. In fact, a model that only produces a single pathologic COPD feature may be more useful as long as it produces this feature via a relevant mechanism that allows exploratory research. By contrast, a model producing all kinds of COPD features via irrelevant mechanisms may be less useful. In this respect, validation of models as being relevant is an extremely important issue in the early steps of model development. Animal models should not only assess histopathological features but also attempt to focus on functional features of human COPD such as airflow limitation, mucus hypersecretion, chronic cough and exacerbations, and also on pharmacological features such as corticosteroid resistance or diminished β-adrenergic bronchodilator responses. In conclusion, there are many benefits that can accrue from the development of animal models of COPD, most important of which is understanding of mechanisms and development of specific drugs for COPD.


Previous results with the planar robot MIT-MANUS demonstrated positive benefits in trials with over 250 stroke patients. Consistent with motor learning, the positive effects did not generalize to other muscle groups or limb segments. Therefore we are designing a new class of robots to exercise other muscle groups or limb segments. This paper presents basic engineering aspects of a novel robotic module that extends our approach to anti-gravity movements out of the horizontal plane and a pilot study with 10 outpatients. Patients were trained during the initial six-weeks with the planar module . This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week .Pilot study demonstrated that the protocol was safe and well tolerated with no patient presenting any adverse effect. Consistent with our past experience with persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy and we have not observed increases in muscle tone or spasticity during the anti-gravity training protocol. Pilot results showed also a reduction in shoulder-elbow impairment following planar horizontal training. Furthermore, it suggested an additional reduction in shoulder-elbow impairment following the anti-gravity training.Our clinical experiments have focused on a fundamental question of whether task specific robotic training influences brain recovery. To date several studies demonstrate that in mature and damaged nervous systems, nurture indeed has an effect on nature. The improved recovery is most pronounced in the trained limb segments. We have now embarked on experiments that test whether we can continue to influence recovery, long after the acute insult, with a novel class of spatial robotic devices. This pilot results support the pursuit of further clinical trials to test efficacy and the pursuit of optimal therapy following brain injury. Rather than using robotics as an assistive technology for a disabled individual, our research focus is on the development and application of robotics as a therapy aid, and in particular a tool for therapists. We foresee robots and computers as supporting and enhancing the productivity of clinicians in their efforts to facilitate a disabled individual's functional motor recovery. To that end, we deployed our first robot, MIT-MANUS  that nurture has a significant impact in speeding motor recovery of the paretic shoulder and elbow, and that robot therapy is effective in delivering the necessary exercise. This recovery is most pronounced in the trained muscle groups and limb segments. Encouraged by these positive results, we have expanded our project to develop a family of novel, modular robots, designed to be used independently or together to rehabilitate other muscle groups and limb segments. This paper describes two different implementations of a new module developed at MIT that expands the capabilities of MIT-MANUS to include motion in a three-dimensional workspace. We will present both implementations, the basic engineering differences between these modules, and pilot clinical results from their use with stroke patients.Volpe  reportedModularity. From the outset we believed that modularity is essential to success in robotic therapy, particularly in extending the approach to patients suffering from distinct afflictions. Consider, for example, patients undergoing surgery at the wrist  who might not require a device that manipulates the payload of all the degrees-of-freedom (DOF) of the arm. Therapy for these patients requires only the wrist robot [MIT's experience with well over 250 stroke patients has reinforced the importance of one of our core design specifications: st robot -13. ConvAll of our robot modules are specifically designed and built for clinical rehabilitation applications. Unlike most industrial robots, they are configured for safe, stable, and compliant operation in close physical contact with humans. This is achieved using backdrivable hardware and impedance control, a key feature of the robot control system. Each active module can move, guide or perturb movements of a patient's limb and can record motions and mechanical quantities such as the position, velocity, and forces applied.The most profound engineering challenge specific to this family of robots is achieving the dual goals of high force production capability and backdrivability. Each module must be capable of generating sufficient force to move a patient's limb, but it must also itself be easily movable by an elderly or frail patient. Backdrivability is essential in keeping the patient engaged in the task and in allowing him to observe his successful and unsuccessful attempts at motion. Backdrivable hardware also improves the performance of systems controlled by impedance controllers. Achieving backdrivability and high force production, together in a single machine, is often difficult and becomes more so when the robot geometry is more complex.The robot control system is an impedance controller that modulates the way the robot reacts to mechanical perturbation from a patient or clinician and ensures a gentle compliant behavior. Impedance control refers to using a control system  to impose a desired behavior at a specified port of interaction with a robot, in this case the attachment of the robot to the patient's hand. Conceived in the early 1980's by one of the co-authors , it has The MIT-MANUS project was initiated in 1989 with support from the National Science Foundation. MIT-MANUS has been in daily operation since 1994 delivering therapy to stroke patients at the Burke Rehabilitation Hospital. This robot has been extensively described in the literature [The selected design created a highly backdrivable robot capable of delivering therapy in a workspace of 15" by 18" with an end-point anisotropy of 2:1 ratio  and achievable impedances between 0 and 8 N/mm. Note that the static friction is significantly below the just noticeable difference (JNF) for force, which is 7% of the reference force. The robot maximum achievable impedance is above the human perception of 4.2 N/mm for a "virtual wall." The robot is capable of delivering forces up to 45 N although the robot target design aimed at a force of 28 N, which corresponds to the arm strength during elbow extension for a weak woman in seated position .Following the successful clinical trials of MIT-MANUS, a 1-dof module to provide vertical motion and force was conceived and built. The primary goal of this module is to bring the benefits of planar therapy on MIT-MANUS to spatial arm movements, including movement against gravity. The module can be used independently or mounted to MIT-MANUS for movement in a limited spatial workspace. The module can permit free motion of the patient's arm, or can provide partial or full assistance or resistance as the patient moves against gravity. Because the vertical module moves with the endpoint of the planar module when the two are integrated, overall module mass is an important design concern in addition to on-axis mass and friction. Two embodiments are described below. is used to hold the patient's hand in place.One prototype of the 1-dof module was completed at MIT late 2000 and is shown alongside a test stand in Figure This prototype has been fully characterized at MIT ,18 Figu and 5. IThe screw-driven prototype has proven very successful both in standalone operation and mounted at the end of the planar module enabling spatial movement therapy in the clinic with compliant and stable behavior. However recent changes in linear motor technology have created the potential to achieve similar outcomes with effective vertical endpoint inertia comparable to the planar MIT-MANUS and much lower friction, without the need for force feedback control. The main change is complete enclosure of the magnets within the motor forcer. While this does not increase the magnetic field strength, it dramatically increases the line integral and concatenates magnetic lines.The practical advantages of converting the spatial system to direct-drive linear motors would be a significant reduction of friction and elimination of backlash. This simplification would also carry through to the control system and controller, as well as affording a reduction in the system's overall dimensions and weight. To determine if the expected friction levels are realistic, we tested Copley Control ThrustTube TB2504. Figures To test the novel vertical module we conducted a pilot study to analyze whether additional anti-gravity training further improves motor outcomes for "graduates" of the planar robot-assisted protocol.Outpatients were included in the study if they met the following criteria: a) first single focal unilateral lesion with diagnosis verified by brain imaging (MRI or CT scans) that occurred at least 6 months prior; b) cognitive function sufficient to understand the experiments and follow instructions ; c) Motor Power score ≥1/5 or ≤3/5 (neither hemiplegic nor fully recovered motor function in the 14 muscles of the shoulder and elbow); d) informed written consent to participate in the study. Patients were excluded from the study if they have a fixed contracture deformity in the affected limb that limited pain-free range of motion. We have found severe tendon contractures around the rotator cuff particularly, in patients with complete hemiplegia for longer than 6 months after stroke. It is reasonable to expect that robotic training for the upper limb would not have an impact on a fixed contracture deformity. Trials commenced only after baseline assessment across three consecutive evaluations, 2 weeks apart, shows a stable condition in three motor impairment scales . Our rationale for administering multiple baseline evaluations is based on an interesting "Hawthorne effect" that we observed in previous subjects ,20. BetwTen (10) community dwelling volunteers who have suffered a single stroke at least 6 months prior to enrollment were enrolled in the pilot protocol. The mean group age was 62 ± 4.3 years old (mean ± sem) with the onset of the stroke occurring 50 ± 8.9 months (mean ± sem) prior to enrollment. Table Patients were trained during the initial six-weeks with the planar module . This training was followed by six-weeks of robotic therapy that focused on performing vertical arm movements against gravity. The 12-week protocol includes three one-hour robot therapy sessions per week  Figure .For shoulder-and-elbow planar therapy, the center of the workspace was located in front of the subject at the body midline with the shoulder elevation at 30° with the elbow slighted flexed. The point-to-point movements started at the workspace center and extended in eight different directions of the compass Figure . A one-hJust as in the planar study , the antIn this pilot, standard clinical evaluations included the upper extremity sub-test of the Fugl-Meyer Assessment  from which we derived a Fugl-Meyer score for shoulder/elbow coordination ; a more comprehensive evaluation of motor power or strength in 14 different muscle groups of the shoulder and elbow, using the MRC Motor Power score ; and the Motor Status Score which is divided into two subscales, one for shoulder and elbow movements , and a second for wrist and hand abilities  ,3,24,6,7We have completed the study with ten (10) outpatients. Nine (9) outpatients who completed the planar training protocol at the Burke Rehabilitation Hospital were enrolled for an additional six-weeks with training three sessions per week on the vertical module. One (1) naïve outpatient completed only the six-week training on the vertical module (no previous exposure to the planar training). Table Results of this pilot suggested that the vertical protocol was safe and well tolerated by the patients with no patient presenting any adverse effect . Furthermore, pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training. In fact, for these 9 patients the reduction in impairment during the vertical training phase was comparable to the reduction during the planar phase . As mentioned earlier, we reliably incorporated therapists' feedback during the design phase of the protocol and have not observed increases in muscle tone or spasticity, as indicated by the Modified Ashworth scale, during the anti-gravity training protocol . Note also that consistent with our past experience with the persons with chronic strokes, there was a statistically significant reduction in tone measurement from admission to discharge of performance-based planar robot therapy.While the benefits from the additional anti-gravity therapy did not achieve statistical significance, this is likely due to the small sample size (9 outpatients). We anticipate that a modest increase of sample size will demonstrate statistical improvement for the shoulder and elbow of the anti-gravity training across the three clinical scales and we plan to commence trials shortly . We have now embarked on experiments that will test whether we can continue to influence recovery, long after the acute insult, with a novel class of robotic devices.It is broadly accepted that outcome measures do not significantly change in persons with chronic motor impairments more than six months from stroke onset. However, recent task specific training programs have resulted in improved motor performance in persons with chronic stroke. For example, our trials with persons with chronic stroke-related impairments showed that planar robotic training contributed to statistically significant improvements in motor abilities -10. If sOur pilot results and novel robotic modules provide a proof of concept that supports our engineering efforts, as well as further clinical trials to test efficacy. We found that the protocol was safe and well tolerated by the patients with no patient presenting any adverse effect . Furthermore, the pilot results suggested an additional reduction in shoulder-elbow impairment following the anti-gravity vertical training without detrimental changes in muscle tone or spasticity. Therefore we plan to investigate in detail the effect of training each movement component in isolation versus integrated spatial movement, and study its impact on disability. We expect that this will bring us closer to our ultimate goal, efficient delivery of optimal therapy, personalized to serve the individual's needs.


Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation.cab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet.Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (BvThis work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed. Beta vulgaris L.) is a biennial plant, a member of the Chenopodiaceae family [Sugar beet  which was recently applied to sugar beet . With thA straight forward approach for the isolation of differentially expressed genes was achieved by the "Suppression Subtractive Hybridization" method (SSH) . SSH is cab genes that are both expressed. To investigate the use of the cab promoters for gene expression, the 5' regulatory sequences were isolated and linked to reporter genes. Transient reporter gene assays indicate that both promoters are expressed in sugar beet leaves. In transgenic sugar beet both promoters are expressed in green tissue. Sequence analysis revealed that the cab11 promoter, in contrast to the cab12 promoter, is void of G-box like regulatory sites with a palindromic ACGT core sequence. A leaf specific promoter in transgenic sugar beets can be employed for biotechnological applications.Here we have employed SSH for the isolation of leaf expressed sugar beet genes. Among the genes isolated was a cDNA fragment for a light-harvesting chlorophyll a/b binding protein (CAB). It is shown that sugar beet genotypes harbor either one or two To isolate cDNA fragments corresponding to leaf expressed genes, poly(A)+RNAs from leaf and taproot were isolated and subjected to cDNA synthesis. Suppression subtractive hybridization was performed as described in Methods. A total of 23 cDNA clones specific for the subtraction for leaf expressed genes were isolated and sequenced (data not shown). Fourteen out of 23 cDNAs were found to be different. Table To further confirm that the enriched cDNA fragments correspond to tissue specific genes, Northern blot hybridizations to total RNA isolated from leaves (L), taproots (R), stems (S), and inflorescences (I) were performed with 7 cDNA fragments. Figure cab gene was isolated and analysed with reporter gene fusions. Compared to the other genes, the expression profile of the gene for SSH fragment L2 showed the strongest tissue specificity during the different time points analysed (data not shown).The expression profile of the gene for SSH fragment L2 encoding a chlorophyll a/b binding protein (CAB) was analysed in more detail. RNA from different tissues and from different developmental stages were used in RNA blot experiments. RNA was isolated from different organs of sugar beets from a field 4, 6, 10, 12, 16 and 19 weeks after sowing. This interval covers the lifetime of a sugar beet in central Europe. Figure cab gene, a complete cDNA clone was isolated . A homology search with the encoded 252 amino acid long protein reveals a 87% identity to the cab11 and 85% identity to the cab12 gene from tomato encoding chlorophyll a/b binding protein [For the isolation of a sugar beet promoter corresponding to the EcoRI, HindIII) and four (PstI) hybridizing fragments are detected, while only one (EcoRI) or two  fragments are detected in genotype 4B5421. From this result it is concluded that genotype 4B5421 harbors one and genotype 1K0088 harbors two copies of the cab gene.Prior to the isolation of genomic clones, a gene copy number analysis was performed. DNA from sugar beet genotypes 1K0088 and 4B5421 was digested with different restriction enzymes and hybridized to the L2 fragment (Table Genomic clones for the two different genes were isolated (Methods). Sequence comparison between the cDNA and both genomic clones indicate a very high degree of sequence identity within the coding region. The CAB11 and CAB12 amino acid sequence differ only in one position (data not shown).cab11 promoter and the 3049 bp fragment cab12 promoter. Both fragments harbor 51 base pairs coding region of the CAB protein and 113 (cab11) and 70 (cab12) base pairs upstream untranslated sequence. Upstream of the untranslated region only about 300 bp are homologous between the two promoters while the rest of the sequence is highly divergent (data not shown). Because the cDNA clone isolated before originates from the genotype 4B5421 and corresponds to the cab11 gene, it was investigated if the second gene is also transcribed. Towards these ends 5' RACE amplifications were performed with RNA from genotype 1K0088 and sequenced. This analysis revealed that the cab12 gene is also transcribed (data not shown).From both genes the promoter regions were subcloned into plasmid vectors and sequenced (Methods). Sequence of 1148 and 3049 base pairs, respectively, containing most of the upstream region were deposited to GenBank (Acc. Nr. AX449166 and AX449167). The 1148 bp promoter fragment is designated Photinus pyralis in the vector pGEM-luc were constructed (see Methods). Table cab11 gene and the 342 bp fragment from the cab12 gene harbor all cis-regulatory sequences required for gene expression in sugar beet leaves at least under the conditions of the transient bioassay.To test whether the isolated promoters confer reporter gene expression in sugar beet leaves, a transient assay was performed. Towards these ends translational fusions of promoter fragments with the luciferase gene from cab11 and cab12 promoters from sugar beet drive reporter gene expression in transgenic plants, promoter reporter gene fusions were introduced via Agrobacterium mediated transformation in sugar beet (see Methods). The length of the cab11 promoter fragment is 1097 bp and the length of the cab12 promoter fragment 2998 bp. Leaves of transgenic lines transferred to the greenhouse were analysed for reporter gene activity. The cab11 promoter of 12 independent transformants showed a specific β-glucuronidase (GUS) activity from 9 to 40599 pmol Mu × min-1 × mg-1, respectively . Using the program Patch™ six experimentally verified binding sites for bZIP factors were detected among these twelve sites that harbor the ACGT core in the cab12 promoter  in tomato [cab11 from tomato and Bvcab11 from sugar beet are identical (data not shown).Subtractive hybridization was used to isolate leaf expressed genes from sugar beet. The goal was the identification of a promoter that drives leaf specific gene expression in transgenic sugar beet plants. Among seven analysed genes a cDNA fragment corresponding to a chlorophyll a/b binding protein gene was shown by RNA gel blot hybridization to be highly specific for green tissue Fig.  and 2. Gb12 Fig. . A compln tomato . This ann tomato . Furthercab6a and Lhca4/cab11) expressed in E. coli form a heterodimer in vitro that closely resembles the native LHCI-730 dimer from tomato leaves [LHCI can be subdivided into at least two different chlorophyll-protein complexes, one of which appears to be responsible for the 730 nm fluorescence of PSI (LHCI-730) and the other complex (LHCI-680) fluoresces at lower wavelength . In barlo leaves . Therefocab genes were isolated and linked to reporter genes. Transient gene expression studies in sugar beet indicated that 1097 bp upstream of the ATG from the Bvcab11 gene and 342 bp upstream of the ATG from the Bvcab12 gene are sufficient for leaf specific gene expression in sugar beet . In sugar beet both promoters are expressed in leaves  and 900 plants, ) in whiccab11 derived GUS expression is 40599 pmol Mu x min-1 × mg-1 which is comparable with the expression strength of the strong CaMV 35S promoter in tobacco.The highest level of cab promoters from sugar beet. Particularly, the Bvcab11 promoter may be useful to drive strong and specific gene expression in transgenic host plants. The lack of bZIP binding sites harboring the ACGT core sequence could also be advantageous for transient analysis of bZIP transcription factors when using a Bvcab11 reporter gene construct as a transformation control. Furthermore these promoters may be useful to express resistance improving genes against foliar diseases such as Cercospora leaf spot.In summary, this work presents the isolation and expression analysis of two Two different methods for RNA preparation were employed. To isolate RNA for cDNA subtraction, the procedure described below was followed. For some of the Northern blot analyses a method described earlier was employed .g, 10 min.) the nucleic acid containing pellet was dissolved in 20 ml 100 mM NaCl, 10 mM EDTA pH8, 50 mM Tris-HCl pH8, and 0.2% SDS. Afterwards, a phenol:chloroform (1:1) and a chloroform:isoamylalcohol (24:1) extraction was performed. The pH of the aqueous solution was adjusted to about 5 with acidic acid and nucleic acids were precipitated by addition of 0.6 volume isopropanol and 0.05 volume 4 M NaCl and incubation for 2 hrs at -20°C. After centrifugation  the nucleic acids containing pellet was resuspended in 10 ml H2ODEPC containing 0.1% SDS. Total RNA was precipitated by addition of 0.25 volume 8 M LiCl and incubation for at least 15 hrs at 4°C with subsequent centrifugation for 20 min at 4°C, 10000 g. Total RNA was resuspended in 400 μl H2ODEPC. After ethanol precipitation  total RNA was resuspended in a volume of 50–100 μl H2ODEPC. The isolation of poly(A)+ RNA was carried out with the Oligotex Kit according to the manufacturers protocol . Measurements of RNA yield and electrophoretic separation on formaldehyde gels were done following standard protocols [[For RNA isolation plant material was homogenized in liquid nitrogen and resuspended in a solution containing 4 M guanidinthiocyanat, 25 mM Tris-HCl, pH8 und 100 mM β-mercaptoethanol. After centrifugation  nucleic acids in the supernatant were precipitated by addition of 0.03 volume sodium acetate  and 0.75 volume ethanol (100%) and incubation over night at -20°C. After centrifugation . Prior to ligation into pCR®2.1 the subtracted PCR cDNA products were subjected to an additional incubation of 1 hour at 72°C with dATP and Taq polymerase  to ensure that the majority of the PCR fragments contain "A-overhangs" for an efficient cloning into the T/A cloning vector.The synthesis of cDNA was performed using the CLONTECH PCR-Select™ cDNA Subtraction Kit . Each synthesis was carried out with 8 μg poly(A)+ RNA from sugar beet isolated either from leaves or taproots. Subtractive hybridization was done following the user manual (PT1117-1) of the CLONTECH PCR-Select™ cDNA Subtraction Kit. After the second PCR the amplified fragments from the forward and the reverse subtraction were cloned into the PCR cloning vector pCR® database was employed [The inserts of the plasmids were sequenced with fluorescently labeled M13 reverse and forward (-20) primers using the AutoRead Sequencing Kit (Pharmacia) and the Automated Laser Fluorescent A.L.F.™ DNA Sequencer from Pharmacia LKB . The DNA sequence analysis of the genomic and full-length cDNA clones was done by the custom sequencing service of MWG Biotech AG . Sequences were subjected to data bank analysis using the BLAST algorithms  and analemployed . DNA seqemployed  and a PCRadioactive probes were generated by the method of random hexamer priming with the Amersham Multiprime DNA Labelling System . Southern and Northern hybridizations were carried out following standard protocols ,36.32-dATP .For genomic Southern blot hybridizations 10 μg of DNA from sugar beet genotypes 1K0088 and 4B5421 was digested with different restriction enzymes. Electrophoretic separation, transfer to Hybond nylon membranes , hybridization to radioactive probes, and exposure of the membrane to X-ray films were done according to standard protocols . Radioaccab cDNA clones were identified after screening of 10000 clones using the SSH fragment L2 as a probe  and cloned into the plasmid vector pCMV Sport 6.0. Screening of the library was done according to standard protocols . Seven pcab loci were isolated.A genomic library from sugar beet genotype 1K0088 was generated in the lambda vector EMBL3 SP6/T7 and screened using standard protocols . Genomiccab11 is present on a ClaI fragment that was subcloned into a plasmid vector and completely sequenced. The fragment is 6294 bp long and contains 51 bp from the coding region of the gene. The resulting plasmid was designated pC1a. Additionally, a 6026 bp large SalI/ClaI fragment was released from the phage clone and subcloned into a Bluescript plasmid and designated pC1b. The promoter for the gene cab12 is present on a PstI fragment that was also subcloned into a plasmid vector and completely sequenced. The fragment is 4002 bp long and the harboring plasmid was designated pC2.The promoter for the gene cab11 promoter and the 3049 bp fragment cab12 promoter.From both genomic clones 1148 and 3049 base pairs containing most of the upstream region were deposited in GenBank (Acc. Nr. AX449166 and AX449167). The 1148 bp promoter fragment is designated Photinus pyralis in the reporter gene vector pGEM-luc . To introduce a plant polyA addition signal into pGEM-luc the respective fragment was isolated from pBI101.3  by EcoRI digestion, followed by a Klenow fill in reaction and by redigestion with SacI. This released a 260 bp DNA fragment from the nopaline synthase (nos) gene containing the polyA addition signal. To directionally clone this fragment into pGEM-luc, this plasmid was first linearised with SfiI, treated with T4-polymerase to generate blunt ends and subsequently redigested with SacI. After inserting the nos fragment the resulting plasmid was designated pLuc-nos2. To insert the cab11 promoter fragment, a SalI(fill in)-AviII fragment was cloned into the ApaI linearised and T4-polymerase treated plasmid pLuc-nos2. This plasmid harbors 1145 bp from the cab11 promoter including the coding sequence for the first 16 amino acids of the cab11 gene. This plasmid was designated pC1L-1097. In this plasmid the luciferase gene is translationally fused with the first 16 amino acids from the cab11 gene. A second plasmid was generated which harbors additional upstream sequences. Towards these ends a 6099 bp KpnI fragment was released from the plasmid pC1b (see above) and the ends treated with T4-polymerase. The fragment was redigested with NotI and the desired fragment was directionally cloned as a KpnI(blunt end)-NotI fragment upstream of the cab11 fragment in pC1L-1097. To generate compatible ends pC1L-1097 was digested with HindIII treated with T4-polymerase and redigested with NotI. The resulting plasmid was designated pC1L-7126.For transient gene expression assays, promoter fragments were linked as translational fusions to the luciferase reporter gene from cab12 upstream to the luciferase coding region, the promoter fragment from pC2 was released by NotI/EcoRI digestion and subsequently subjected to a partial digestion with AviII. A 3100 bp long NotI/AviII fragment was purified and subcloned into pLuc-nos2. The plasmid pLuc-nos2 was digested with ApaI, the ends treated with T4-polymerase and redigested with NotI. After ligation the resulting plasmid was designated pC2L-2998. In this plasmid the luciferase gene is translationally fused with the first 16 amino acids from the cab12 gene. To generate 5' promoter deletions pC2L-2998 was (1) digested with KpnI/NotI, T4-polymerase treated, and religated to yield pC2L-1827, (2) digested with SmaI and religated to yield pC2L-989, and (3) digested with NotI and SalI , Klenow polymerase treated, and religated to yield pC2L-342.To clone the promoter for gene cab11 and cab12 promoters were cloned 5' to the β-glucuronidase gene (uidA). Towards these ends a 1.17 kb HindIII/BamHI fragment was released from pC1L-1097 and cloned into the binary vector pBI101.3 . The resulting plasmid pC1G-1097 harbors a translational fusion between the first 16 amino acids of the cab11 gene and the uidA gene. Similarly, the cab12 promoter was released as a PstI fragment from plamid pC2L-2998, treated with T4-polymerase then digested with BamHI and subcloned into pBI101.3 which was linearised with SalI, ends filled in with Klenow and redigested with BamHI. The resulting plasmid was named pC2G-2998.For stable transformation the Renilla reniformis under the control of the doupled CaMV 35S promoter was employed as a second reporter gene [Photinus and Renilla luciferase activity were measured with the dual-luciferase reporter assay system  in a Lumat 9501 luminometer (PE Biosystem) according to the protocol of the supplier.The luciferase expression from plasmids pC1L-1097, pC1L-7126, pC2L-2998, pC2L-1827, pC2L-989, and pC2L-342 were measured in sugar beet leaves after biolistic transformation . For bioter gene . For eacAgrobacterium tumefaciens strain GV2260 [Agrobacterium tumefaciens mediated transformation techniques were performed with the binary T-DNA plasmids on sugar beet  according to [-1 × mg-1.For the generation of transgenic plants pC1G-1097 and pC2G-2998 were directly transformed into n GV2260 . Agrobacrding to . Selectirding to  using 4-cis-regulatory elements. RH and DS conceived of the study, and participated in its design and coordination.DS isolated the cDNA and genomic clones, analysed the transcription during different developmental stages and tissues, generated promoter reporter gene constructs, transformed sugar beet, and performed the quantitative reporter gene assays. DUK performed the suppression subtractive hybridization and analysed the homology and tissue specificity of the cDNA fragments. RH identified


Staphylococcus aureus (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis.Methicillin resistant 9/ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group , antibiotic group , and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated.Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 × 10One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group.G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational. Staphylococcus aureus is an extremely virulent pathogen, and causes serious and deep-seated infections  [S. aureus is the leading pathogenic cause of both community-acquired and nosocomial infections, especially in bacteremia and sepsis [S. aureus sepsis is one of the most important causes of death [S. aureus (MRSA) infections is glycopeptides (vancomycin or teicoplanin). However glycopeptides are intrinsically less effective against staphylococci than are antistaphyloccal β-lactams [yelitis) . In reced sepsis -4. Despiof death . Furtherof death ,7. The e-lactams . This maAdvances in the pathophysiology of sepsis and septic shock suggest new therapeutic agents and approaches. Granulocyte-colony stimulating factor (G-CSF), a potent stimulator of neutrophil counts and functions, is one of these new strategies in sepsis. Despite a number of studies in sepsis, especially in neonatal sepsis, clinical efficacy of G-CSF is still controversial .The aim of this experimental study was to evaluate the role of G-CSF in the treatment of MRSA sepsis.Male BALB-C mice  were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water.7/ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 109/mL.The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin , diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF , diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days .Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received.Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted.Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu.The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++  . The patSurvival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group.Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant.Esherichia coli sepsis were excluded. 106 mice were evaluated. Six mice in control group, six mice in G-CSF group, 15 mice in vancomycin 25 mg, 15 mice in vancomycin 25 mg+G-CSF group, 18 mice in vancomycin 50 mg group and 14 mice in vancomycin 50 mg+G-CSF group were sacrificed at the end of seventh day. The death day and the survival rate of the mice in the groups are shown by the survival curve in figure This study included 110 mice. Three mice that developed intraabdominal hemorrhage, and one mouse that developed Culture and histopathology results are shown in Table S. aureus strains, isolated from the bloodstream infections [In recent years, MRSA has become widespread around the world, and become highly endemic in some hospitals. In the United States, the proportion of MRSA isolates increased from 2.4% in 1975 to up to 55% in recent years ,12. Simifections . Unfortufections .G-CSF is a cytokine that stimulates myeloid progenitor cell proliferation and increases the bone marrow storage pool and the number of circulating mature neutrophils, which are important component of the host defense . Also, iThe previous experimental therapeutic studies were mostly carried out in gram-negative sepsis, and in this experimental study, we investigated the effect of G-CSF in MRSA sepsis. The survival rates were not significantly different in G-CSF group compared with the control group, and also in antibiotic+G-CSF group compared with antibiotic alone group. Likewise, most of the other experimental studies in gram negative sepsis, showed that prophylactic rhG-CSF administration reduced endotoxemia and serum TNF-α levels and also improved cardiac function and survival, whereas therapeutic rhG-CSF (i.e. administered after the onset of infection) did not improve outcome and at very high dosages appeared harmful ,26. A reIn this study, we did not measure the WBC count, so not have subgroups with neutropenia. In an experimental gram-negative sepsis in rabbits, Smith and colleguaes  showed tBacteria are rapidly cleared from blood and tissues following intravenous antibiotic therapy . In our S. aureus infections are leukocytic infiltration, focal pneumonitis, edema, microabscesses, etc. In an experimental study, significant pathologic changes during and after the elimination of bacteria from the blood and tissues were noted. Also, expression of cytokines  was observed in all of the infected tissues, and correlated tissue damage after clearance of bacteria from the blood and tissues[The histopathologic findings of invasive d tissues. HoweverIn conclusion, G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. Furthermore, G-CSF treatment is very expensive, likewise glycopeptides. Because of high mortality and morbidity rates and excess costs, more interest in infection control measures, and prevent the spread of MRSA infections is more rational.This study was supported by Erciyes University Research Fund. There was no non-financial competing interest.EA and SG were the primary researchers. OC and BK are the pathologist and examined tissue samples. MD was the director of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


E. faecalis and E. faecium accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.Enterococci tend to be one of the leading causes of nosocomial infections, with The study was conducted in a tertiary care hospital in South India from July 2001 to June 2003. Isolates of enterococci were collected from various clinical specimens and speciated using extensive phenotypic and physiological tests. Antimicrobial susceptibility testing were performed and interpreted as per NCCLS guidelines. Whole cell protein (WCP) fingerprinting of enterococci were done for species validation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed computationally.E. gallinarum (6.2%), 10 E. avium (4.1%), 6 E. raffinosus (2.5%), 6 E. hirae (2.5%), 4 E. mundtii (1.7%), 3 E. casseliflavus-including the two atypical isolates (1.2%) and 2 E. durans (0.8%). The 12 atypical enterococcal species (5%) that showed aberrant sugar reactions in conventional phenotyping were confirmed as E. faecalis, E. faecium and E. casseliflavus respectively by WCP fingerprinting. The antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance among different species apart from intrinsic vancomycin resistance by some species, while all the species tested were susceptible for linezolid and teicoplanin.Our study showed the prevalence of unusual  and atypical  species of enterococci as 19% (46 isolates) and 5% (12 isolates) respectively. The 7 unusual species (46 isolates) isolated and confirmed by phenotypic characterization includes: 15 Our study reveals the emergence of multi-drug resistance among unusual species of enterococci posing a serious therapeutic challenge. Precise identification of enterococci to species level enables us to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections. Enterococcus faecalis and Enterococcus faecium accounts up to 90% of clinical isolates belonging to this genus [E. faecalis and E. faecium, and only few reports or studies of non-faecalis and non-faecium enterococci are prevalent [Enterococci, generally regarded as normal flora of gastrointestinal and genitourinary tract of humans, have emerged as the etiogen of several nosocomial as well community-acquired infections since last two decades. Globally, many studies have revealed that enterococci tend to be one of the leading causes of several nosocomial infections, with the emergence and spread of multi drug resistance among isolates -3. Sinceis genus . Neverthis genus ,6. But tis genus . Thus prrevalent ,6. HenceThe study was conducted in a 900-bedded tertiary care hospital at Pondicherry, South India from July 2001 to June 2003. Isolates of enterococci were collected over the time period from various clinical specimens such as blood, urine, wound swabs and pus , catheters, ascitic fluid, synovial fluid, by plating them on 5% Sheep Blood agar and Mac-conkey agar, as well on Bile esculin azide agar  as per nature of the specimen. Extensive phenotypic and physiological characterization was carried out by the conventional tests devised by Facklam et al . CarbohyAntibiotic susceptibility testing of the clinical isolates along with the quality control strains were performed using BHI agar instead of Muller Hinton agar by disk diffusion method , standard agar dilution  analysis of the enterococcal isolates, including atypical biochemical variants of enterococci and the reference/type strains of enterococci  were done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously with minor modifications ,11 for sE. faecalis and E. faecium-data not shown) which included 15 E. gallinarum (6.2%), 10 E. avium (4.1%), 6 E. raffinosus (2.5%), 6 E. hirae (2.5%), 4 E. mundtii (1.7%), 3 E. casseliflavus (1.2%) and 2 E. durans (0.8%). The distribution by site of isolation for the 46 unusual enterococcal species included 30 isolates- 12 E. gallinarum, 6 E. avium, 3 each of E. hirae, E. casseliflavus and E. raffinosus, 2 E. mundtii and 1 E. durans (65.2%) from bloodstream, 6 isolates- 3 E. raffinosus, 2 E. avium and 1 E. mundtii (13 %) from surgical and non-surgical wound swabs, 10 isolates- 3 each of E. hirae and E. gallinarum, 2 E. avium and 1 each of E. durans and E. mundtii (21.8%) from miscellaneous sites, including muscle tissues sent for anaerobic culture, catheter tips, peritoneal fluid, ear swab and urine. Of the 46 persons from whom unusual enterococci were obtained, 56.5% were males and 43.5% were females including newborn/neonates. The infections were polymicrobial in 6 (13%) of the 46 cases from which unusual enterococci were isolated, including 2 (6.7%) of 30 bloodstream infections. The 12 atypical enterococcal strains (5%) showing aberrant sugar reactions included 6 mannitol negative variant E. faecalis like species, 1 arginine negative variant E. faecalis like species, 3 mannitol negative variant E. faecium like species and, 2 arginine negative variant E. casseliflavus like species. The WCP analysis by SDS-PAGE confirmed the species identities of seven different species. Atypical strains showed a similar banding pattern like the reference strains from CDC  except for minor quantitative differences, with no qualitative difference. The computational analysis of the WCP gel images of atypical strains were performed by Dice coefficient, and the dendrogram constructed using unweighted pair group method using arithmetic averages (UPGMA) as shown in Figure-E. faecalis, E. faecium and E. casseliflavus respectively. The 2  isolates of arginine negative variant E. casseliflavus like species after taxonomic validation were included as an unusual species of enterococci accounting to 3 E. casseliflavus isolated overall.We isolated a total of 242 enterococci during our 2-year study period from different clinical samples. The biochemical phenotyping results revealed 46 isolates 19%) belonging to 7 different unusual species of enterococci , 2 E. durans (n = 2), 11 E. gallinarum (n = 15), 2 E. hirae (n = 6), 2 E. mundtii (n = 4) and, 3 E. raffinosus (n = 6). Only E. casseliflavus (n = 3) exhibited 100% susceptibility to ciprofloxacin. None of the 46 isolates was positive for β-lactamase, but resistance for β-lactam agents were prevalent variably among different species. The results of MICs for penicillin, ampicillin, high-level gentamicin and high-level streptomycin resistance were in accordance with the disk diffusion testing results except for vancomycin. Disk diffusion testing showed vancomycin resistance for 6 isolates , but the agar screening method exhibited vancomycin resistance for 11 isolates .The antimicrobial susceptibilities of the isolates given in Table-E. faecalis and E. faecium as the only prevalent species [E. faecalis and E. faecium) prevalence of unusual species of enterococci from catheterized patients with urinary tract infections. E. mundtii and E. durans were not reported in their study, whose prevalence was 1.7% and 0.8% respectively in our study. E. gallinarum (6.2%) and E. avium (4.1%) were the most commonly identified species, which markedly differs in isolation rate (0.3–1.2%) from other studies [E. gallinarum isolated during our study period were from pediatrics unit, while 7 of the 10 isolates exhibiting a similar antibiotype were isolated from the same ward within a span of 2 months. The remaining 3 of the 10 E. gallinarum were isolated from the same ward in the preceding 3 months, one of which showed an antibiotype similar to the cluster of 7 isolates. The same was the case of 3 E. casseliflavus isolated from the same pediatrics unit within a span of 2 months in the preceding year. Most of these  isolates were from cases of septicemia. Although molecular epidemiological studies have not been done to compare the genetic similarities of these isolates, the data depicts the nosocomial spread of these species. WCP analysis by SDS-PAGE had been proven to assist in validating the species identities as well, to identify strains that do not exhibit phenotypic characteristics identical to the type strains of each species [Our study reveals that the prevalence of unusual species of enterococci as 19% in our clinical setup in South India. Many studies and reviews show the prevalence of non-faecalis and non-faecium enterococci as 2–10% ,6,12. Pr species -16, whic species ,17. The  species  showing  studies . The incE. avium .1% were  species . We wereE. casselifalvus) which proves that it may be successful only in treating enterococcal urinary tract infections [E. gallinarum and, 1 E. casseliflavus isolates, which may go undetected by disk diffusion testing [E. durans and two E. mundtii (MIC ≤ 6 μg/ml). The genotypic basis of vancomycin resistance for these 3 isolates yet to be studied, will give us a definitive picture regarding its clinical significance, since studies have reported the prevalence of vancomycin resistance in these two species, and its transferable nature from E. durans to E. faecium [Ciprofloxacin resistance was 63% among isolates  low-level vancomycin resistance by disk diffusion testing  emphasizThe author(s) declare that they have no competing interests.PVP designed the study and carried out the experimental works and analysis, and drafted the manuscript. RSR supervised and participated in the design of the study and coordination, and helped to draft the manuscript. SCP participated in the coordination of the study and helped to draft the manuscript.The pre-publication history for this paper can be accessed here:


The discovery of cis-regulatory modules in metazoan genomes is crucial for understanding the connection between genes and organism diversity. It is important to quantify how comparative genomics can improve computational detection of such modules.We run the Stubb software on the entire D. melanogaster genome, to obtain predictions of modules involved in segmentation of the embryo. Stubb uses a probabilistic model to score sequences for clustering of transcription factor binding sites, and can exploit multiple species data within the same probabilistic framework. The predictions are evaluated using publicly available gene expression data for thousands of genes, after careful manual annotation. We demonstrate that the use of a second genome (D. pseudoobscura) for cross-species comparison significantly improves the prediction accuracy of Stubb, and is a more sensitive approach than intersecting the results of separate runs over the two genomes. The entire list of predictions is made available online.in silico. The future availability of additional fruitfly genomes therefore carries the prospect of highly specific genome-wide predictions using Stubb.Evolutionary conservation of modules serves as a filter to improve their detection  Berman et al. , Markste [et al.  and Half [et al.  predicte [et al. , called  [et al. ). Most pet al. [et al. [The Stubb algorithm  extende [et al. ). The seD. melanogaster and D. pseudoobscura) using Stubb can lead to a significant improvement in the accuracy of genome-wide CRM prediction. To our knowledge, this is the first direct evaluation of the effect of cross-species comparison on CRM prediction on a genome-wide scale. Another important contribution of this paper is to present a benchmark for evaluating genome-wide CRM prediction tools, collected from the BDGP database and the literature, and curated by manual inspection of several hundred expression patterns. Using the same benchmark, we evaluate the effect of varying how background sequence information is incorporated in the algorithm, since this is the only tunable parameter in the Stubb program, other than the module length. We are thus able to suggest the optimal parameter settings for genome-wide CRM prediction using Stubb. Finally, we report all genome-wide predictions for cis-regulatory modules involved in anterior-posterior patterning in the early fly embryo, using both single-species and two-species Stubb, many of which make a strong case for experimental validation.In this paper, we present evidence that the exploitation of cross-species comparison  axis during early -Volhard ; Rivera--Volhard ; Furriol-Volhard ). The maet al. [et al. [et al. [et al. [et al. [The segmentation paradigm has been used as a test system for the computational detection of CRMs by us and others . Here,  [et al. ), we useD. melanogaster and D. pseudobscura have been sequenced, and Stubb was used to predict CRM's in the D. melanogaster genome. This was done in two modes – (i) STUBBSS, where Stubb is run on D. melanogaster genomic sequence alone, and (ii) STUBBMS, where Stubb uses orthologous sequence data from D. pseudobscura to help predict CRM's in D. melanogaster. For each mode of execution, we obtain a separate list of predicted CRM's, sorted in order of confidence in the prediction. The ideal test for our purpose would be to compare the accuracy of these two sorted lists. However, the set of experimentally verified CRM's involved in this system is sparse compared to the size of the system – roughly 50 CRM's are known  The Berkeley Drosophila Genome Project (BDGP) has catalogued the expression patterns of a large number of genes in D. melanogaster, at various stages of development. We considered such a catalogue of 2167 genes, obtained from BDGP and from the literature.  Visualst Genes .) Hence,We note that some accuracy is lost in the translation of a list of predicted CRM's to the predicted genes it is mapped to, as per the mapping defined above. For instance, it is known that CRM's may control a gene located at large distances, i.e., further than the distance threshold of 20 Kb used in the mapping procedure. Also, it is possible that a CRM is located close to two genes, and directs the expression of both genes, or only of the farther gene, being somehow insulated from the nearer one. To address these concerns, we repeat our evaluation with a slightly different mapping from the one described above. A caveat that remains is that there may be genomic sequences that are functional, in the sense that they are capable of directing a specific blastoderm pattern in reporter gene constructs, but whose activity is 'silenced' in native genomic context and does not translate to patterning of any gene. Also, the CRM may direct expression of the gene only at post-blastodermal stages, so that the gene is not included in the "positive" test set of blastoderm patterned genes. Conversely, it may also happen that a predicted CRM lies close to a patterned gene, thereby being counted as a true positive, but the predicted CRM is not the sequence responsible for the gene's regulation. We assume that such effects are not biased against either algorithm.st order background for STUBBSS and global, 2nd order background for STUBBMS.  The x-axis is the number of unique genes that are predicted by the algorithm (by progressively decreasing its score threshold) and are in the set of 2167 genes with expression information. On the y-axis we plot how many of those predicted genes are in the "positive" set  Thus, the y-axis is the specificity of the algorithm. We observe that STUBBMS performs significantly better than STUBBSS. For instance, to predict 100 genes correctly, STUBBSS has to make 343 predictions, while STUBBMS only has to make 267 predictions. Figure Figure -12 and <10-37 respectively, Binomial Proportions test). Further examination of the top 102 gene predictions made by each algorithm revealed that 24 true positives are common to both lists. STUBBMS reports 37 true positives not discovered by STUBBSS, while the latter reports 15 true positives not found by the former. Similar results are seen for the top 311 predictions , STUBBSS reports 39 positives, while STUBBMS scores 61 hits, an improvement of over 56%. In comparison, the random expectation is ~13.5 hits. Thus the predictions of both STUBBSS and STUBBMS are significantly enriched in patterned genes (P < 10We next examined separately the following three sets of genes: (i) INTERSECTION  (ii) MS-ONLY (predicted only by STUBBMS) and (iii) SS-ONLY (predicted only by STUBBSS). Table D. melanogaster, but intersecting these predictions with a similar number of top predictions in D. pseudoobscura gives only 68 unique genes, 33 of which are patterned. Of the top 68 predictions made in D. melanogaster alone, 29 are patterned. Thus the "intersection" strategy yields only a modest improvement over the single-species search, and does so at the price of significantly reducing the total number of predictions. Similar results were obtained when intersecting modules instead of gene predictions.One possible strategy that uses two-species sequence is to make predictions using STUBBSS on each of the two genomes separately and then intersect the respective lists. We found this strategy to be very restrictive – for instance, with a particular score threshold, STUBBSS predicts 205 unique genes in et al. [et al. [We have noted above that the evaluation method is influenced by the way we map the predicted CRM's to predicted genes. To offset potential biases induced by this mapping, we repeated our analysis with a slightly different evaluation procedure, borrowing from the approach of Grad et al. . We now  [et al. , is thatet al. [et al. [The default mapping from CRM's to genes used in our evaluations predicts a gene to be patterned only if its proximal end is less than 20 Kb from the CRM. Schroeder et al.  studied  [et al.  observe Genes in the segmentation hierarchy often have multiple aspects to their expression pattern, with more than one CRM regulating them. We therefore measured how the Stubb predictions fare if we required that each predicted gene be evidenced by at least two predicted CRM's. This heuristic improves the performance of STUBBSS more prominently than that of STUBBMS, though much fewer predictions are made by either algorithm.  ubiquitously expressed at all developmental stages, (ii) not expressed at any stage, or (iii) only maternally expressed.  Incluset al. [I to I+19 were examined, with I being incremented in steps.) In each step, we computed the fraction of the 20 genes that belonged to the following three non-exclusive categories: (i) genes with dv (in addition to ap) modulation, (ii) genes with strong expression pattern, and (iii) genes in the "core" set of 48 genes. These values are reported in Figure Our annotations of the blastoderm patterned genes also include whether the gene expression is strong, weak or of intermediate strength; if it has a dorsal-ventral (dv) modulation in addition to the primary anterior-posterior pattern; and if the gene belongs to a "core" set of 48 genes that have been shown experimentally to be required for the segmentation of the embryo . We wer1. Genes with dorsal-ventral aspects to their blastoderm pattern are more frequent at lower ranks of prediction; i.e., the top predictions are enriched in genes with anterior-posterior patterns only.2. Core genes are predominantly found in the top predictions.3. Genes found at higher ranks are somewhat more likely to be strongly expressed.The first two observations imply that the genes more directly involved in the ap axis formation are recovered at better ranks, and that the lower rank genome-wide predictions are richer in derivative patterns characteristic of genes with more complex regulatory inputs . The same trends were found for the correct predictions made by STUBBSS. (Data not shown.)k for this parameter means that local correlations are assumed to be present at the level of (k+1)-mers, i.e., the random probability of seeing a particular base at a position depends on the bases seen at the previous k positions. th order background in the terminology of this paper.) We vary this parameter to take the values k = 1 and k = 2, in different runs. The other parameter is the actual sequence used by Stubb to measure background nucleotide frequencies. Here the two options are (i) to use the current sequence window as background, or (ii) to use a pre-specified sequence (or collection of sequences) as background. We call these two the "local" and "global" background models respectively. For the "global" model, we input into Stubb 150 Kb of sequence from non-coding regions of the D. melanogaster genome, collected from the five chromosome arms 2L, 2R, 3L, 3R, and X.We next evaluate the effect of varying how background sequence information is incorporated in the Stubb algorithm. This is the only configurable aspect of the program, other than the module length.  One important parameter is the "Markov order" of background. A value of y et al.  and Schrst order background, though the other parameter values produce only slightly different results. On the other hand, the effect of background parameters on two-species Stubb is more pronounced, with the best choice being a global, 2nd order background. Using a global 1st background order gives almost identical results (data not shown), hence we infer that a global background is the optimal choice for STUBBMS.Figure et al. [st and 2nd order Markov backgrounds), and comparing its performance to that of Stubb.  Figure As mentioned earlier, the STUBBSS program implements the same class of algorithm as the Ahab algorithm of Rajewsky et al. , with soAll the above runs were on genomic sequence with tandem repeats masked by the Tandem Repeats Finder program of Benson . We haveThe Stubb program is an extension of Ahab, with the important feature that it can handle two-species data within its probabilistic framework. The two programs differ in their underlying optimization method, with Stubb using an Expectation-Maximization approach in contrast to Ahab's conjugate gradient method. Performance evaluation of the two programs shows little difference between them, implying that the algorithm is robust to the actual optimization method used. Another technical difference between Ahab and Stubb is in the manner that orientation of binding sites is treated. While Stubb assumes a uniform prior on the orientation of a binding site, Ahab picks the best orientation for each site, with the caveat that probabilities are not strictly normalized.et al. [et al. [et al. [An important component of Stubb is the alignment step where the two species are aligned (using LAGAN) and blocks of high sequence similarity are extracted. (See Methods.) The parameters used in LAGAN runs were obtained from Emberly et al. , who der [et al. versus SM [et al. ) for CRMa priori how tandem repeats should affect module detection – repeats similar to binding sites of the system may improve sensitivity when they occur in CRM's; but if repeats resembling binding sites occur by chance in non-functional regions, prediction specificity may suffer. The occurrence of tandem repeats marks statistical deviation from Stubb's probabilistic model of sequence generation. In our tests, we found that repeats distract the algorithm more than they help, as manifested in better performance on repeat-masked sequence. , first  [et al. report anRegarding the recovery of patterned genes by Stubb, several observations can be made. Of the 286 genes with ap patterns, we recover roughly half at a score cut-off of 10, using STUBBMS. Why is the other half not found? While it is obvious that lowering the cut-off will detect more patterned genes, there are other reasons why a patterned gene may be missed by Stubb. Some genes are likely to be lost due to the distance filter we have imposed (CRM to nearest gene <20 kb), since the regulatory regions of some genes  are likely to be larger than that. More importantly, most of the patterned genes that are not part of the core transcriptional machinery have derivative patterns that reflect a more complex input  and thus will only be recovered to the extent their input has a solid maternal/gap component. Conversely, there are at least two reasons for reporting false positives (roughly two thirds at a score cutoff of 10). The presence of an insulator could prevent the interaction between a CRM and its nearest basal promoter. More likely is a scenario where the predicted CRM's do drive expression but at post-blastoderm stages. All gap factors are active in multiple tissues in later development and therefore CRM's with dominant or exclusive gap input may well be active in these later contexts. These caveats affect all current CRM detection algorithms, and accounting for such additional axes of information as genomic context and module composition rules will be a difficult but important challenge for the future.A very interesting observation comes from the analysis in Table changes in terms of their binding site compositions. This leads to a powerful bioinformatic tool to predict regulatory changes between the two fly species. We can thus obtain hypotheses about changes in expression patterns, which can be verified experimentally. We have examined a representative collection of CRM's, and experimentally verified several of the changes predicted by Stubb, thereby building a catalogue of the different modes of cis-regulatory evolution. The results of this study will be reported in the near future.The Stubb program not only predicts cis-regulatory modules genome-wide, it additionally outputs the binding site profile of each predicted CRM, i.e., the locations and probabilities of binding sites in the CRM. Schroeder et al  use the We have seen that the use of a second fly genome significantly improves genome-wide module prediction. Since STUBBMS uses a natural "two-species" extension of the algorithm of STUBBSS, this finding is largely a statement about the inherent potential of cross-species comparison as a paradigm for improving functional genomics. The STUBBMS program also has a natural extension to incorporate more than two genomes, and it will be very interesting to see how much of a difference a third genome makes. The genome of D. yakuba is expected to be sequenced soon, and since this species is closer to D. melanogaster, it may help better discriminate conserved regulatory modules.D. melanogaster sequences were obtained from Flybase Release 3. The analysis was limited to the five chromosome arms 2L, 2R, 3L, 3R, and X. D. pseudobscura contigs were obtained from  (February 2003 Release). Based on Blast results, we created a mapping, called "CONTIGMAP", between regions of the D. melanogaster genome and D. pseudobscura contigs, each region typically being tens of Kb long. This mapping is many to many, i.e., different regions of D. melanogaster may map to the same contig, and the same (or overlapping) region in D. melanogaster may map to two or more D. pseudobscura contigs. For each entry  in CONTIGMAP, where M is the D. melanogaster region and P is the D. pseudobscura contig, the LAGAN alignment program  was runD. melanogaster genome with a sliding window of length 500 bp, in shifts of 50 bp. The input weight matrices for the maternal and gap transcription factors Bcd, Hb, Cad, Kni, Kr, Tll, Dstat and the torRE binding factor were obtained from Rajewsky et al. [et al. [D. melanogaster sequence M, in shifts of 50 bp. Thus, STUBBMS was not run on regions of D. melanogaster that are not aligned with some D. pseudoobscura contig. The weight matrices used were the same as in STUBBSS runs. The locations of the blocks computed in the alignment step (above) were input to STUBBMS, and the input value of the neutral mutation rate was 0.5, the value being chosen due to its better performance over alternatives tested.Tandem repeats in the input sequences were masked with the Tandem Repeat Finder program of Benson , with pay et al.  and SchrEach genome-wide run of Stubb produces, for each starting position of the sliding window, a score that measures the likelihood of the sequence having a cluster of binding sites. The next step is to extract the coordinates of each window that scores better than all other windows overlapping it. Such windows correspond to local "peaks" in the score profile along the genome. All such "peak" windows with scores above a certain threshold are sorted in decreasing order of their score, to produce a sorted list of predicted CRM's. Each window in this list is annotated with useful information including the identity and relative location of its neighboring genes. The list is then filtered to retain only those predicted CRM's where Stubb predicts occurrences of at least two weight matrices. This is a heuristic that incorporates the combinatorial nature of CRM's, i.e., their tendency to have sites for multiple transcription factors (activators as well as repressors.) Finally, any predicted CRM that overlaps with an exon is removed from the list before evaluation. The predictions made by STUBBMS and STUBBSS are listed in the files "Predicted CRM's – two species"  and "Pre lists as showing expression during blastoderm (embryonic stages 4–6) were visually inspected. From this list, we removed genes with ubiquitous expression , extremely faint or irreproducible expression (31), or expression in pole cells or yolk nuclei only (64), as well as genes whose expression is modulated along the dv axis only (13). The remaining 258 genes show patterned expression in the somatic portion along the ap axis of the blastoderm embryo; 28 known segmentation genes not captured in the BDGP expression database were added to the list, for a total of 286 genes showing ap patterned blastoderm expression. These genes were further categorized by expression level  and type of pattern . ap includes gap, pair rule and segment polarity-like patterns ; ap+dv denotes ap pattern with some dv modulation ; dv+ap denotes dv pattern with some ap modulation .The 792 genes which the BDGP expression database SS and EDS worked out the details of genome-wide Stubb runs. SS performed the Stubb runs, collected all statistics from the runs, and drafted the manuscript. EDS suggested several analyses reported. MDS, UU, and UG annotated the genes for expression pattern, suggested many of the analyses in the Results and Discussion sections, and wrote parts of the manuscript. All authors read and approved the final manuscript.et al: Genome Biol 2004, 5:R61, published 20 August 2004.) also evaluates the effect of cross-species comparison on CRM prediction in Drosophila.A recently published paper (Berman Test Genes List of 2167 genes with expression information. (Source BDGP + literature.)Click here for filePatterned Genes List of 286 genes with blastoderm pattern, as determined by manual inspection of in-situ expression pattern. (Source BDGP + literature.)Click here for fileAdditional Genes List of 2065 genes that are ubiquitous, maternal only, or not expressed. (Source BDGP.)Click here for filend order background, sorted in decreasing order of Stubb score.Predicted CRM's – two species List of predicted CRM's using two-species Stubb with a global, 2Click here for filest order background, sorted in decreasing order of Stubb score. This list of predictions, as well as that in Additional File 4 (above), is meant to be as inclusive as possible; therefore, the specificity of the lowest ranked predictions may be poor.Predicted CRM's – single species List of predicted CRM's using single-species Stubb with a local, 1Click here for file