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Background: Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterization of a RV strain from a giraffe calf. Results: This report describes the identification and detailed molecular characterisation of a rotavirus strain isolated from a 14 day old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. Molecular characterisation of the RV strain isolated from the giraffe was identified as a G10P[11] type. Detailed sequence analysis of VP-4 and VP-7 revealed significant identity at the amino acid sequence level to Bovine RV. Conclusions: This study demonstrates the need for continuous surveillance of RV strains in various animal populations, which will facilitate the identification of rotavirus hosts not previously reported. Furthermore, extending typical epidemiology studies to a broader host range will contribute to the timely identification of new emerging strain types.

Background: Abyssal microorganisms have evolved particular features that enable them to grow in their extreme habitat. Genes belonging to specific functional categories are known to be particularly susceptible to high pressure; therefore, they should show some evidence of positive selection. To verify this hypothesis we computed the amino acid substitution rates between two deep sea microorganisms, Photobacterium profundum SS9 and Shewanella benthica KT99, and their respective shallow water relatives. Results: A statistical analysis of all the orthologs, led to the identification of positive selected (PS) genes, which were then used to evaluate adaptation strategies. We were able to establish "Motility" and "Transport" as two classes significantly enriched with PS genes. The prevalence of transporters led us to analyze variable amino acids (PS sites) by mapping them according to their membrane topology, the results showed a higher frequency of substitutions in the extra cellular compartment. A similar analysis was performed on soluble proteins, mapping the PS sites on the 3D structure, revealing a prevalence of substitutions on the protein surface. Finally, the presence of some flagellar proteins in the Vibrionaceae PS list confirms the importance of bacterial motility as a SS9 specific adaptation strategy. Conclusions: The approach presented in this paper is suitable for identifying molecular adaptations to particular environmental conditions. The statistical method takes into account differences in the ratio between non synonymous to synonymous substitutions, thus allowing the detection of the genes that underwent positive selection. We found that positive selection in deep sea adapted bacteria targets a wide range of functions, for example solute transport, protein translocation, DNA synthesis and motility. From these data clearly emerges an involvement of the transport and metabolism processes in the deep sea adaptation strategy of both bathytypes considered, whereas the adaptation of other biological processes seems to be specific to either one or the other. An important role is hypothesized for five PS genes belonging to the transport category that had been previously identified as differentially expressed in microarray experiments. Strikingly, structural mapping of PS sites performed independently on membrane and soluble proteins revealed that residues under positive selection tend to occur in specific protein regions.

ObjectiveA great deal of international attention has been focussed recently on how much additional funding is required to scale up health interventions to meet global targets such as the Millennium Development Goals (MDGs). Most of the cost estimates that have been made in response have assumed that unit costs of delivering services will not change as coverage increases or as more and more interventions are delivered together. This is most unlikely. The main objective of this paper is to measure the impact of patient load on the cost per visit at primary health care facilities and the extent to which this would influence estimates of the costs and financial requirements to scale up interventions. Methods: Multivariate regression analysis was used to explore the determinants of variability in unit costs using data for 44 countries with a total of 984 observations.FindingsControlling for other possible determinants, we find that the cost of an outpatient visit is very sensitive to the number of patients seen by providers each day at primary care facilities. Each 1% increase in patient through-put results, on average, in a 27% reduction in the cost per visit (p <0.0001), which can lead to a difference of up to $30 in the observed costs of an outpatient visit at primary facilities in the same setting, other factors held constant. Conclusion: Variability in capacity utilization, therefore, need to be taken into account in cost estimates, and the paper develops a method by which this can be done.

Background: Mucin alterations are a common feature of esophageal neoplasia, and alterations in MUC2 mucin have been associated with tumor progression in the esophagus. Bile acids have been linked to esophageal adenocarcinoma and mucin secretion, but their effects on mucin gene expression in human esophageal adenocarcinoma cells is unknown. Methods: Human esophageal adenocarcinoma cells were treated 18 hours with 50-300 uM deoxycholic acid, chenodeoxycholic acid, or taurocholic acid. MUC2 transcription was assayed using a MUC2 promoter reporter luciferase construct and MUC2 protein was assayed by Western blot analysis. Transcription Nuclear factor-kappaB activity was measured using a Nuclear factor-kappaB reporter construct and confirmed by Western blot analysis for Nuclear factor-kappaB p65. Results: MUC2 transcription and MUC2 protein expression were increased four to five fold by bile acids in a time and dose-dependent manner with no effect on cell viability. Nuclear factor-kappaB activity was also increased. Treatment with the putative chemopreventive agent aspirin, which decreased Nuclear factor-kappaB activity, also decreased MUC2 transcription. Nuclear factor-kappaB p65 siRNA decreased MUC2 transcription, confirming the significance of Nuclear factor-kappaB in MUC2 induction by deoxycholic acid. Calphostin C, a specific inhibitor of protein kinase C (PKC), greatly decreased bile acid induced MUC2 transcription and Nuclear factor-kappaB activity, whereas inhibitors of MAP kinase had no effect. Conclusions: Deoxycholic acid induced MUC2 overexpression in human esophageal adenocarcinoma cells by activation of Nuclear factor-kappaB transcription through a process involving PKC-dependent but not PKA, independent of activation of MAP kinase.

Background: High participation rates are needed to ensure that breast cancer screening programs effectively reduce mortality. We identified the determinants of non-participation in a public breast cancer screening program. Methods: In this case-control study, 274 women aged 50 to 64 years included in a population-based mammography screening program were personally interviewed. Socio-demographic characteristics, health beliefs, health service utilization, insurance coverage, prior mammography and other preventive activities were examined. Results: Of the 192 cases and 194 controls contacted, 101 and 173, respectively, were subsequently interviewed. Factors related to non-participation in the breast cancer screening program included higher education (odds ratio [OR] = 5.28; 95% confidence interval [CI95%]=1.57-17.68), annual dental checks-ups (OR=1.81; CI 95%1.08-3.03), prior mammography at a private health center (OR=7.27; CI95% 3.97-13.32), gynecologist recommendation of mammography (OR=2.2; CI95%1.3-3.8), number of visits to a gynecologist (median visits by cases=1.2, versus controls=0.92, P= 0.001), and supplemental private insurance (OR=5.62; CI95%=3.28-9.6). Among women who had not received a prior mammogram or who had done so at a public center, perceived barriers were the main factors related to non-participation. Among women who had previously received mammograms at a private center, supplemental private health insurance also influenced non-participation. Benign breast symptoms increased the likelihood of participation. Conclusions: Our data indicate that factors related to the type of insurance coverage (such as prior mammography at a private health center and supplemental private insurance) influenced non-participation in the screening program.

Background: Atopic dermatitis (AD) is believed to result from complex interactions between genetic and environmental factors. A main feature of AD as well as other allergic disorders is serum and tissue eosinophilia. Human eosinophils contain high amounts of cationic granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO) and major basic protein (MBP). Recently, variation in genes encoding eosinophil granule proteins has been suggested to play a role in the pathogenesis of allergic disorders. We therefore genotyped selected single nucleotide polymorphisms within the ECP, EDN, EPO and MBP genes in a cohort of 361 German AD patients and 325 healthy controls. Results: Genotype and allele frequencies did not differ between patients and controls for all polymorphisms investigated in this study. Haplotype analysis did not reveal any additional information. Conclusions: We did not find evidence to support an influence of variation in genes encoding eosinophil granule proteins for AD pathogenesis in this German cohort.

Background: Endeavors to manage obesity have been heavily reliant on controlling energy intake and expenditure equilibrium, but have failed to curtail the overweight and obesity epidemic. This dynamic equilibrium is more complex than originally postulated and is influenced by lifestyle, calorie and nutrient intake, reward cravings and satiation, energy metabolism, stress response capabilities, immune metabolism and genetics. Fat metabolism is an important indicator of how efficiently and to what extent these factors are competently integrating. We investigated whether an Irvingia gabonensis seed extract (IGOB131) would provide a more beneficial comprehensive approach influencing multiple mechanisms and specifically PPAR gamma, leptin and adiponectin gene expressions, important in anti-obesity strategies. Methods: Using murine 3T3-L1 adipocytes as a model for adipose cell biology research, the effects of IGOB131 were investigated on PPAR gamma, adiponectin, and leptin. These adipocytes were harvested 8 days after the initiation of differentiation and treated with 0 to 250 microM of IGOB131 for 12 and 24 h at 37 degree C in a humidified 5 percent CO2 incubator. The relative expression of PPAR gamma, adiponectin, and leptin in 3T3-L1 adipocytes was quantified densitometrically using the software LabWorks 4.5, and calculated according to the reference bands of beta-actin. Results: The IGOB131 significantly inhibited adipogenesis in adipocytes. The effect appears to be mediated through the down-regulated expression of adipogenic transcription factors (PPAR gamma) [P less than 0.05] and adipocyte-specific proteins (leptin) [P less than 0.05], and by up-regulated expression of adiponectin [P less than 0.05]. Conclusions: IGOB131 may play an important multifaceted role in the control of adipogenesis and have further implications in in-vivo anti obesity effects by targeting the PPAR gamma gene, a known contributory factor to obesity in humans.

Background: In vivo positioning and covalent modifications of nucleosomes play an important role in epigenetic regulation, but genome-wide studies of positioned nucleosomes and their modifications in human still remain limited. Results: This paper describes a novel computational framework to efficiently identify positioned nucleosomes and their histone modification profiles from nucleosome-resolution histone modification ChIP-Seq data. We applied the algorithm to histone methylation ChIP-Seq data in human CD4+ T cells and identified over 438,000 positioned nucleosomes, which appear predominantly at functionally important regions such as genes, promoters, DNase I hypersensitive regions, and transcription factor binding sites. Our analysis shows the identified nucleosomes play a key role in epigenetic gene regulation within those functionally important regions via their positioning and histone modifications. Conclusions: Our method provides an effective framework for studying nucleosome positioning and epigenetic marks in mammalian genomes. The algorithm is open source and available at http://liulab.dfci.harvard.edu/NPS/.

Background: Previous studies have indicated that the wide variation in intron density (the number of introns per gene) among different eukaryotes largely reflects varying degrees of intron loss during evolution. The most popular model, which suggests that organisms lose introns through a mechanism in which reverse-transcribed cDNA recombines with the genomic DNA, concerns only one mutational force. Hypothesis Using exons as the units of splicing-site recognition, exon definition constrains the length of exons. An intron-loss event results in fusion of flanking exons and thus a larger exon. The large size of the newborn exon may cause splicing errors, i.e., exon skipping, if the splicing of pre-mRNAs is initiated by exon definition. By contrast, if the splicing of pre-mRNAs is initiated by intron definition, intron loss does not matter. Exon definition may thus be a selective force against intron loss. An organism with a high frequency of exon definition is expected to experience a low rate of intron loss throughout evolution and have a high density of spliceosomal introns. Conclusion: The majority of spliceosomal introns in vertebrates may be maintained during evolution not because of potential functions because of their splicing mechanism (i.e., exon definition). Further research is required to determine whether exon definition is a negative force in maintaining the high intron density of vertebrates. Reviewers This article was reviewed by Dr. Scott W. Roy (nominated by Dr. John Logsdon), Dr. Eugene V. Koonin, and Dr. Igor B. Rogozin (nominated by Mikhail Gelfand). For the full reviews, please go to the Reviewers' comments section.

Background: Taste receptor ("Type II") cells express G protein-coupled receptors (GPCRs) for sweet, umami (T1Rs and mGluRs) or bitter (T2Rs), as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCbeta2-mediated release of stored Ca2+. Whereas Galphagus (gustducin) couples to the T2R (bitter) receptors, which Galpha-subunit couples to the sweet (T1R2 + T1R3) receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Galphaq family (alphaq, alpha11, alpha14) in mouse taste epithelium. Results: By RT-PCR, Galpha14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate), but not anterior (fungiform and palate) taste fields. Galphaq and Galpha11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Galpha14 mRNA is limited to Type II taste receptor cells. Immunocytochemistry on vallate papillae using a broad Galphaq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCbeta2). This staining persists in Galphaq knockout mice and immunostaining with a Galpha11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Galpha14 is the dominant Galphaq family member detected. Immunoreactivity for Galpha14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Galpha14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. Conclusion: Galpha14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.

Background: Gibbons has argued that science and society are in the midst of a far reaching renegotiation of the social contract between science and society with society becoming a far more active partner in the creation of knowledge. On the one hand new forms of knowledge production are emerging, and on the other both science and society are experiencing a rapid acceleration in new forms of knowledge utilization. Concomitantly since the Second World War, the science underpinning the knowledge utilization field has had exponential growth. Few in-depth examinations of this field exist and no comprehensive analyses have used bibliometric methods. Methods: Using bibliometric analysis, specifically first author co-citation analysis, our group undertook a domain analysis of the knowledge utilization field tracing its historical development between 1945 and 2004. Our purposes were: (1) to map the historical development of knowledge utilization as a field and (2) to identify the changing intellectual structure of its scientific domains. We analyzed more than 5000 articles using citation data drawn from the Web of Science. Search terms were combinations of knowledge, research, evidence, guideline, ideas, science, innovation, technology, information theory and use, utilization, and uptake. Results: We provide a bird's eye view of the intellectual structure and how it changes over six decades. The field does not become large enough to represent with a co-citation map until the mid-1960s. Our findings demonstrate vigorous growth from the mid-1960s through 2004, as well as the emergence of specialized domains reflecting distinct collectives of intellectual activity and thought. Until the mid-1980s, the major domains were focused on innovation diffusion, technology transfer, and knowledge utilization. Beginning slowly in the mid-1980s and then growing rapidly, a fourth scientific domain, evidence-based medicine, emerged. The field is dominated in all decades by one individual, Everett Rogers, and by one paradigm, innovation diffusion. Conclusions: We conclude that the received view that social science disciplines tend to be in a state where no accepted set of principles or theories guide research in the social sciences (i.e., that they are pre-paradigmatic) could not be supported for this field. Second, we document the emergence of a new domain within the knowledge utilization field, evidence-based medicine. Third, we conclude that Everett Rogers was the dominant figure in the field and, until the emergence of evidence based medicine, his general diffusion model the dominant paradigm in the field.

Isolated non-skeletal injuries of the cervical spine are rare and frequently missed. Different evaluation algorithms for C-spine injuries, such as the Canadian C-spine Rule have been proposed, however with strong emphasis on excluding osseous lesions. Discoligamentary injuries may be masked by unique clinical situations presenting to the emergency physician. We report on the case of a 28-year-old patient being admitted to our emergency department after a snowboarding accident, with an assumed hyperflexion injury of the cervical spine. During the initial clinical encounter the only clinical finding the patient demonstrated, was a burning sensation in the palms bilaterally. No neck pain could be elicited and the patient was not intoxicated and did not have distracting injuries. Since the patient described a fall prevention attempt with both arms, a peripheral nerve contusion was considered as a differential diagnosis. However, a high level of suspicion and the use of sophisticated imaging (MRI and CT) of the cervical spine, ultimately led to the diagnosis of a traumatic disc rupture at the C5/6 level. The patient was subsequently treated with a ventral microdiscectomy with cage interposition and ventral plate stabilization at the C5/C6 level and could be discharged home with clearly improving symptoms and without further complications. This case underlines how clinical presentation and extent of injury can differ and it furthermore points out, that injuries contracted during alpine snow sports need to be considered high velocity injuries, thus putting the patient at risk for cervical spine trauma. In these patients, especially when presenting with an unclear neurologic pattern, the emergency doctor needs to be alert and may have to interpret rigid guidelines according to the situation. The importance of correctly using CT and MRI according to both - standardized protocols and the patient's clinical presentation - is crucial for exclusion of C-spine trauma.

Background: Quantifying the amount of standing genetic variation in fitness represents an empirical challenge. Unfortunately, the shortage of detailed studies of the genetic architecture of fitness has hampered progress in several domains of evolutionary biology. One such area is the study of sexual selection. In particular, the evolution of adaptive female choice by indirect genetic benefits relies on the presence of genetic variation for fitness. Female choice by genetic benefits fall broadly into good genes (additive) models and compatibility (non-additive) models where the strength of selection is dictated by the genetic architecture of fitness. To characterize the genetic architecture of fitness, we employed a quantitative genetic design (the diallel cross) in a population of the seed beetle Callosobruchus maculatus, which is known to exhibit post-copulatory female choice. From reciprocal crosses of inbred lines, we assayed egg production, egg-to-adult survival, and lifetime offspring production of the outbred F1 daughters (F1 productivity). Results: We used the bio model to estimate six components of genetic and environmental variance in fitness. We found sizeable additive and non-additive genetic variance in F1 productivity, but lower genetic variance in egg-to-adult survival, which was strongly influenced by maternal and paternal effects. Conclusions: Our results show that, in order to gain a relevant understanding of the genetic architecture of fitness, measures of offspring fitness should be inclusive and should include quantifications of adult offspring reproductive success. We note that our estimate of additive genetic variance in F1 productivity (CVA = 14%) is sufficient to generate indirect selection on female choice. However, our results also show that the major determinant of offspring fitness is the genetic interaction between parental genomes, as indicated by large amounts of non-additive genetic variance (dominance and/or epistasis) for F1 productivity. We discuss the processes that may maintain additive and non-additive genetic variance for fitness and how these relate to indirect selection for female choice.

Background: Glaucoma is a major cause of blindness worldwide and an increasingly significant global health problem. Glaucoma prevention and management efforts have been challenging due to inherent difficulty in developing a simple and cost-effective screening plan, as well as limited access to health care and educational resources, poverty, and inadequate knowledge of the disease, particularly in developing countries. Starting in 2004 the Tilganga Eye Centre in Kathmandu, Nepal has provided targeted glaucoma screening, treatment, and education through a combination of clinical outreach programs and educational activities for patients. Methods: A simple, age-based glaucoma screening algorithm was incorporated into three one-day cataract screening clinics. Using this algorithm, patients who were newly diagnosed with glaucoma were referred to TEC, where medication, and surgery if indicated, was provided free of charge through private donor funding. In addition, we describe two ongoing educational programs for increasing glaucoma awareness: an annual Glaucoma Awareness Week (which includes free screening, treatment, and counseling), and a repeating lecture series which generates new counselors. Results: From 2004 to 2007 screening at the annual Glaucoma Awareness Week has resulted in the diagnosis of 120 individuals with glaucoma, or 7.6% of total registrants. Attendance has increased annually with a trend toward an increasing number of returning patients but a decreasing percentage of newly diagnosed patients, though the absolute numbers have remained relatively stable (range 21 to 38). Data from the three one-day screening clinics show a more modest percentage of newly diagnosed glaucoma patients (approximately 2-4% of patients 50 years of age or older per clinic). Conclusions: This multi-faceted approach appears to successfully identify individuals with glaucoma and provide treatment to those who would otherwise not be able to afford it. While more data is needed to validate this model, specifically regarding the effectiveness of educational activities, long-term visual outcomes, and medication compliance, it may serve as a useful framework for other developing countries with similarly limited resources.

Background: Eating away from home has increased in prevalence among US adults and now comprises about 50% of food expenditures. Calorie labeling on chain restaurant menus is one specific policy that has been proposed to help consumers make more healthful food choices at restaurants. The present review evaluates the available empirical literature on the effects of calorie information on food choices in restaurant and cafeteria settings. Methods: Computer-assisted searches were conducted using the PUBMED database and the Google Scholar world wide web search engine to identify studies published in peer-review journals that evaluated calorie labeling of cafeteria or restaurant menu items. Studies that evaluated labeling only some menu items (e.g. low calorie foods only) were excluded from the review since the influence of selective labeling may be different from that which may be expected from comprehensive labeling. Results: Six studies were identified that met the selection criteria for this review. Results from five of these studies provided some evidence consistent with the hypothesis that calorie information may influence food choices in a cafeteria or restaurant setting. However, results from most of these studies were weak or inconsistent. One study found no evidence of an effect of calorie labeling on food choices. Each of the studies had at least one major methodological shortcoming, pointing toward the need for better designed studies to more rigorously evaluate the influence of point-of-purchase calorie labeling on food choices. Conclusions: More research is needed that meets minimum standards of methodological quality. Studies need to include behavioral outcomes such as food purchase and eating behaviors. Studies implemented in realistic settings such as restaurants and cafeterias are needed.

The effects of an analgesic treatment (lidocaine patches) on brain activity in chronic low back pain (CBP) and in knee osteoarthritis (OA) were investigated using serial fMRI (contrasting fMRI between before and after two weeks of treatment). Prior to treatment brain activity was distinct between the two groups: CBP spontaneous pain was associated mainly with activity in medial prefrontal cortex, while OA painful mechanical knee stimulation was associated with bilateral activity in the thalamus, secondary somatosensory, insular, and cingulate cortices, and unilateral activity in the putamen and amygdala. After 5% lidocaine patches were applied to the painful body part for two weeks, CBP patients exhibited a significant decrease in clinical pain measures, while in OA clinical questionnaire based outcomes showed no treatment effect but stimulus evoked pain showed a borderline decrease. The lidocaine treatment resulted in significantly decreased brain activity in both patient groups with distinct brain regions responding in each group, and sub-regions within these areas were correlated with pain ratings specifically for each group (medial prefrontal cortex in CBP and thalamus in OA). We conclude that the two chronic pain conditions involve distinct brain regions, with OA pain engaging many brain regions commonly observed in acute pain. Moreover, lidocaine patch treatment modulates distinct brain circuitry in each condition, yet in OA we observe divergent results with fMRI and with questionnaire based instruments.

Background: The acute coronary syndrome diagnosis includes different classifications of myocardial infarction, which have been shown to differ in their pathology, as well as their early and late prognosis. These differences may relate to the underlying extent of infarction and/or residual myocardial ischaemia. The study aim was to compare scar and ischemia mass between acute non-ST elevation myocardial infarction (NSTEMI), ST-elevation MI with Q-wave formation (Q-STEMI) and ST-elevation MI without Q-wave formation (Non-Q STEMI) in-vivo, using cardiovascular magnetic resonance (CMR). Methods and Results: This was a prospective cohort study of twenty five consecutive patients with NSTEMI, 25 patients with thrombolysed Q-STEMI and 25 patients with thrombolysed Non-Q STEMI. Myocardial function (cine imaging), ischemia (adenosine stress first pass myocardial perfusion) and scar (late gadolinium enhancement) were assessed by CMR 2-6 days after presentation and before any invasive revascularisation procedure. All subjects gave written informed consent and ethical committee approval was obtained. Scar mass was highest in Q-STEMI, followed by Non-Q STEMI and NSTEMI (24.1%, 15.2% and 3.8% of LV mass, respectively; p<0.0001). Ischemia mass showed the reverse trend and was lowest in Q-STEMI, followed by Non-Q STEMI and NSTEMI (6.9%, 14.7% and 19.9% of LV mass, respectively; p=0.012). The combined mass of scar and ischemia was similar between the three groups (p=0.17). The ratio of scar to ischemia was 3.5, 1.0 and 0.2 for Q-STEMI, Non-Q STEMI and NSTEMI, respectively. Conclusions: Prior to revascularisation, the ratio of scar to ischemia differs between NSTEMI, Non-Q STEMI and Q-STEMI, whilst the combined scar and ischemia mass is similar between these three types of MI. These results provide in-vivo confirmation of the diverse pathophysiology of different types of acute myocardial infarction and may explain their divergent early and late prognosis.

Background: Secondary metabolites biosynthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. In view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. A number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity.Therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining. Results: In this work, we have carried out a comprehensive bioinformatics analysis of methyltransferase (MT) domains present in multi functional type I PKS and NRPS proteins encoded by PKS/NRPS gene clusters having known secondary metabolite products. Based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting MT domains present in PKS and NRPS megasynthases, delineating their correct boundaries and classifying them as N-MT, C-MT and O-MT using profile HMMs. Analysis of proteins in nr database of NCBI using these class specific profiles has revealed several interesting examples, namely, C-MT domains in NRPS modules, N-MT domains with significant homology to C-MT proteins, and presence of NRPS/PKS MTs in association with other catalytic domains. Our analysis of the chemical structures of the secondary metabolites and their site of methylation suggested that a possible evolutionary basis for the presence of a novel class of N-MT domains with significant homology to C-MT proteins could be the close resemblance of the chemical structures of the acceptor substrates, as in the case of pyochelin and yersiniabactin. These two classes of MTs recognize similar acceptor substrates, but transfer methyl groups to N and C positions on these substrates. Conclusions: We have developed a novel knowledge based computational approach for identifying MT domains present in type I PKS and NRPS multifunctional enzymes and predicting their site of methylation. Analysis of nr database using this approach has revealed presence of several novel MT domains. Our analysis has also given interesting insight into the evolutionary basis of the novel substrate specificities of these MT proteins.

Background: The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp.) and herbivores (Manduca sexta) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nathionin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000), which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nathionin directly or indirectly influences PST DC3000 resistance are unknown. Results: M. sexta larvae consumed less on WT and on Nathionin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nathionin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nathionin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants. Conclusion: These results demonstrate that Na-thionin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nathionin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Nathionin alone in resisting PST DC3000.

IntroductionMeasuring the patient satisfaction is a very important issue that will help very much in improving the service provided to patients and improve the level of satisfaction. Aim: To evaluate patient satisfaction with the cataract surgery service and identify any areas for improvement, determination of patient satisfaction with referral, out-patient consultation, pre-assessment clinic, surgery and post-operative care, also to report patients' comments relating to improvement in service provision. Methodology: A retrospective study was undertaken for 150 patients underwent cataract surgery at Barrow General Hospital, UK, the survey sample was by postal questionnaires. We collected our data from the theatre lists for a period of 4 month. Results: This study included 150 patients; the response rate was (72%) 108 patients, Most patients were referred from their general practitioner 86.1%, 93 (86.1%) patients were happy with the time interval from seeing their GP to eye clinic. In the eye out patient department many factors significantly affected the level of patient satisfaction, in general the more information provided for the patient the more the satisfaction Conclusion: Patient satisfaction is on important health outcome old understanding both the domains of satisfaction as well as their relative importance to patients is necessary to improve the overall quality of patient care. Meeting the doctor, presenting all relevant information and giving printed information are very important factors in improving the patient's satisfaction with cataract surgery.