In Denmark and many other European countries, harvest records suggest a marked decline in European brown hare numbers, a decline often attributed to the agricultural practice. In the present study, we analyse the association between agricultural land-use, predator abundance and winter severity on the number of European brown hares harvested in Denmark in the years 1955 through 2000.Winter cereals had a significant negative association with European brown hare numbers. In contrast to this, root crop area was positively related to their numbers. Remaining crop categories were not significantly associated with the European brown hare numbers, though grass out of rotation tended to be positively related. The areas of root crop production and of grass out of rotation have been reduced by approximately 80% and 50%, respectively, while the area of winter cereals has increased markedly (>70%). However, European brown hare numbers were primarily negatively associated with the number of red fox. Finally, we also found a positive association between mild winters and European brown hare numbers.The decline of Danish European brown hare populations can mainly be attributed to predation by red fox, but the development in agricultural land-use during the last 45 years have also affected the European brown hare numbers negatively. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend. Perdix perdix) , includiewed by ,8.Despite its currently declining numbers, the European brown hare is still common and one of the most important game species throughout the country . The dynVulpes vulpes) is the main predator on European brown hares, and foxes have been reported to influence the dynamics of several European brown hare populations substantially . Hence,cts [see ].Both European brown hare over-winter survival  and reprOur analyses have provided important insight into the structure of the European brown hare dynamics in Denmark, and documented important patterns within the mechanisms regulating European brown hares. Hence, we have documented that the decline of European brown hares in Denmark mainly can be attributed to predation by red fox, but also to changes in agricultural land-use. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.As indices of European brown hare and red fox density, we used the harvest records from 14 Danish counties from 1955–2000. In 1970, the geographical borders of some of the counties were changed, which in two cases lead to substantial changes in area ]. UnfortSarcoptic mange was first encountered in Denmark in the early 1980s, and is now present in seven of the 11 districts . In one . The annual crop data were grouped into seven categories  and temperature, precipitation and wind is particularly strong in Northern Europe /s(1955–2000)). Regression coefficients therefore express the rate of change in standard deviation units of the independent variable per one standard deviation unit of the dependent variable [To remove heteroscedasticity in the system, data were logvariable . To obtavariable  for detavariable  by incluFor each district, we tested for the presence of multi-collinearity among parameters prior to the analyses by means of condition indices and variance proportions , but mulX(t) vs. X(t-1) by applying the likelihood ratio test [X(t) vs. X(t-1) was not rejected λ< 7.76, P > 0.05). We analysed the data using a first-order autoregressive (AR(1)) mixed model approach with district as fixed factor:The European brown hare time series were tested for non-linearity in tio test . In all X(t)~,whereandXt-2), as inclusion of both delayed density dependence and predator abundance can be seen as redundant [where LAND_USE represents the seven crop categories (see Table edundant . All anaedundant . Model redundant .NMS designed the study, participated in data preparation, carried out the statistical analyses, and drafted the manuscript. TA participated in data preparation. MCF supported the data analyses and contributed to the writing. All authors read and approved the final manuscript.


Spontaneous T cell responses against specific tumor-associated antigens (TAA) are frequently detected in peripheral blood of tumor patients of various histiotypes. However, little is known about whether these circulating, spontaneously occurring, TAA-reactive T cells influence the clinical course of disease.neu, and CEA either by ELISPOT assay or by intracellular cytokine staining. Then, Kaplan-Meier survival analysis was performed comparing T-cell-responders and T-cell-non-responders. For comparison, a group of T-cell-non-responders was compiled stringently matched to T-cell-responders based on clinical criteria and also analyzed for survival.Fifty-four HLA-A2 positive colorectal cancer patients had been analyzed for the presence of T cell responses against epitopes derived from the TAA Ep-CAM, her-2/Sixteen out of 54 patients had a detectable T cell response against at least one of the three tested TAA. Two out of 21 patients (9.5%) with limited stage of disease (UICC I and II) and 14 out of 33 patients (42.4%) with advanced disease (UICC III and IV) were T cell response positive. Comparing all T-cell-responders (n = 16) and all T-cell-non-responders (n = 38), no survival difference was found. In an attempt to reduce the influence of confounding clinical factors, we then compared 16 responders and 16 non-responders in a matched group survival analysis; and again no survival difference was found (p = 0.7).In summary, we found no evidence that spontaneous peripheral T cell responses against HLA-A2-binding epitopes of CEA, her-2/neu and Ep-CAM are a strong prognostic factor for survival. The importance of the immune system in containing tumor growth is supported by animal studies and various observations in humans ,2. TheseIn previous studies, we have demonstrated spontaneous T cell responses against the TAA CEA, Ep-CAM, or her-2/neu in peripheral blood of approximately 25% of colorectal cancer patients ,30. Thesneu p654–662 [After institutional review board approval and informed consent, peripheral blood mononuclear cells from 132 patients with CRC in all stages of disease had been prospectively collected and frozen for T cell analysis. All analyses have been performed in compliance with the Helsinki Declaration. Fifty-four patients were tested positive for HLA-A2 and were subsequently analyzed for the presence of T cell responses against the HLA-A*0201 presented T cell epitopes Ep-CAM p263–271 , her-2/np654–662 , and CEAp654–662  either bp654–662 ,30. Posip654–662 ,30.First, we performed a Kaplan-Meier survival analysis comparing all T-cell-responders and all T-cell-non-responders. Additionally, a two-sided log rank test was used to test statistical significance. Then, in an attempt to reduce the influence of external factors, we compiled a patient group from non-responders matching them to responders according to the following criteria: UICC stage of disease, gender, presence of clinically detectable tumor at time of blood draw, duration of disease until blood draw, age at first diagnosis, and previous therapy. Survival in both groups was compared using Kaplan-Meier analysis and by a two-sided log rank test. A level of p < 0.05 was considered significant. SPSS (11.5) software was used.6 PBMC) against one of the three TAA Ep-CAM, her-2/neu, and CEA had been detected. Comparing all responders (n = 16) and all non-responders (n = 38), no survival difference was found in a two-sided log rank test  and HLA-DR ,39. ThisTaken together, no evidence was found that peripheral, spontaneous T cell responses against HLA-A*0201-binding epitopes of CEA, Ep-CAM, or her-2/neu influence survival of CRC patients. Since the low patient number limits the conclusion, further studies should investigate more patients, more detailed function and migratory pattern of spontaneous T cell responses as well as the genetic profile of the tumor; and consider a broader antigen and epitope repertoire. These studies could have implications for vaccination therapy as we learn more about why immune surveillance may fail to control tumors and if the presence of a natural T cell response may impact on the efficacy of a vaccine.CEA carcinoembryonic antigen, CRC, colorectal carcinoma; ELISPOT, enzyme-linked immunospot; IFNγ, Interferon-γ; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cells, TAA, tumor associated antigen.The author(s) declare that they have no competing interests.


Aspergillus species.Chronic granulomatous disease (CGD) is a rare disorder of phagocytes in which absence of superoxide and hydrogen peroxide production in phagocytes predisposes patients to bacterial and fungal infections. The most common fungal infections in these patients are caused by Aspergillus osteomyelitis of the ribs and hepatic abscess in a 5-year-old boy. The patient was successfully treated with Amphotericin B and INF-γ.Here, we describe With respect to the high frequency of aspergillosis in the CGD patient, immune deficiency should be investigated in patients with invasive aspergillosis. Moreover, using antifungal drugs as prophylaxis can improve the quality of life in these patients. Aspergillus spp are ubiquitous saprophytic fungi and are considered as the major causative fungal agent in these patients [Aspergillus species varies from flu-like pneumonia to life-threatening invasive aspergillosis [Aspergillus pneumonia which can be accompanied by dissemination to the ribs, chest wall and soft tissues [Chronic granulomatous disease (CGD) is a rare inherited disorder of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex of phagocytic cells resulting in failure to generate reactive oxidants and the absence of a respiratory burst . The dispatients ,3. The sgillosis . The mos tissues ,2. Here,2 every other day). After two weeks of treatment, the patient's condition improved and he was discharged with prescription of both cephalexin (100 mg/kg/day) and co-trimoxazole (10 mg/kg/day) to be taken orally as prophylaxis.A 5-year-old male patient was admitted to Children Medical Center (CMC) with inflammation and swelling in his left mandible and wrist without a history of trauma. In the past, he had suffered from several episodes of pneumonia which started at the age of seven months. On admission, laboratory findings included erythrocyte sedimentation rate (ESR) 84 mm/h, WBC count 12100/mm3 , hemoglobin 11.3 gr/dl and thrombocyte 386000/mm3. As the CRP analysis displayed 20 mg/dl, cephalexin (150 mg/kg/day) was initiated. In his roentgenogram, osteolytic lesions in the distal metaphase of hand and maxillary bone were observed. Considering history of several infections and multifocal osteomyelitis, bone biopsy was performed and his immune system function was evaluated. In the bone biopsy, non-necrotizing granulomatoid lesions were seen. The induration of purified protein derivative reaction was 10 mm diameter. Besides, HIV, hepatitis B surface antigen (HBs), rheumatoid factor and brucella agglutination tests were all negative. The serum IgG level was 1650 mg/dl . IgM and IgA were in high normal range at 250 and 175 mg/dl, respectively. Because no defect was found in his humeral and cellular immunity, the phagocytic cells function was tested with a nitroblue-tetrazolium (NBT) slide test. Based on his hematological and immunological tests (NBT = 0), CGD was considered as underlying disease in this case. Regarding his NBT test, antibiotic therapy was changed from cephalexin to co-trimoxazole  plus  interferon-γ , interferon-γ  and rifampicin (10 mg/kg/day) were administered with diagnosis of invasive aspergillosis. The only adverse event observed during treatment was hypokalemia, which was adjusted by administration of potassium chloride 15%. One month after initiation of antifungal therapy, his follow-up CT scan of the abdominal and thoracal region demonstrated relative resolution of hepatic abscess. After four weeks of intravenous treatment, the patient's clinical condition improved. He was discharged upon his parents' responsibility while continuing taking rifampicin (10 mg/kg/day) for two more weeks as a treatment in addition to co-trimoxazole (5 mg/kg/day) and itraconazole (4 mg/kg/day) as long term prophylaxis.The patient was readmitted to our center after eight months with a tender mass in his right upper quadrant (RUQ) Fig . On admiCGD is a rare inherited immune disorder whose prevalence is estimated to be about 1/1,100,000 – 1/1,300,000 individuals worldwide .In the absence of minimal oxidative metabolism in CGD which can be ascertained easily using nitro blue tetrazolium (NBT) slide test, other immune mechanisms are triggered . The relAspergillus spp, Aspergillus fumigatus is considered to be the predominant cause of invasive aspergillosis in CGD patients [Aspergillus fumigatus. As shown in this case, Aspergillus might spread from lungs to the bones of thoracic wall and cause osteomyelitis [Aspergillus fumigatus is considered to be the most isolated species, Aspergillus nidulans osteomyelitis is reported to have a higher incidence and more mortality rate in these patients [This defect is characterized by recurrent or persistent infections due to catalase-positive fungal and bacterial agents despite aggressive antibiotic therapy ,6. The ipatients ,7. Pulmomyelitis . Althougpatients .The treatment of infections in CGD patients is not easy. Since the underlying immunodeficiency is the most important factor with respect to the outcome of treatment, these patients should be treated either with immunomudulative agents such as recombinant INF-γ or with stimulating factors . Recentl


Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide modules that need advanced informatics skills to allow implementation in pipelines.Phrap contig assemblies in detail. Methods included in POSA include basecalling with quality scores (by Phred), contig assembly (by Phrap), generation of primer3 input and automated SNP annotation (by PolyPhred). Although easily implemented by users with only limited programming experience, these objects considerabily reduce hands-on analysis time compared to using the Staden package for extracting sequence information from raw sequencing files and for SNP discovery.Here we present POSA, a pair of new perl objects that describe DNA sequence traces and The POSA objects allow a flexible and easy design, implementation and usage of perl-based pipelines to handle and analyze DNA sequencing data, while requiring only minor programming skills. Today, many genetics laboratories have access to modern capillary DNA sequencing machines, such as the ABI PRISM 3100, 3700 or 3730. These machines generate vast amounts of raw sequence data with little user intervention. Consequently, the amount of data to be analyzed has expanded and the bottleneck now is the analysis capacity. Data analysis capacity can be increased by higher levels of automation. Investments in infrastructures to process the raw sequencing data in sophisticated but rigid pipelines might be justified for larger laboratories and larger projects but might be too costly for smaller laboratories. In addition, rigid pipelines are too impractical if different projects share run-time on the same machine while requiring (slightly) different analysis procedures .Nucleotide sequence analysis can be performed with a variety of software tools. Although the number of console and web-based software tools has grown rapidly, the routine use of data input, output and storage may be inconvenient. Furthermore, for performing a series of analyses with different software tools, the sequence data need to be reformatted to the required data structure. Alternatively, sophisticated software suites that provide an integrated environment often are expensive.Several of the available software solutions are designed to facilitate automated DNA sequence analysis at low cost. Well-known solutions are the Staden package and Bioperl.pregap4 and gap4, full-featured applications with an intuitive graphical user interface [The Staden Package contains nterface . These pAlternatively, Bioperl is a group of perl modules describing many genetics and genomics concepts . For exaSmaller laboratory sites, however, often need to implement versatile pipelines that can be adjusted for any research question that suits the project best; at the same time, they often also do not have dedicated programmers available.Although (semi-)automated procedures have been published by other groups ,4, thesePhrap contig in detail and are easily implemented in perl-based pipelines. Because these objects provide building blocks for sequencing data analysis pipelines and the actual pipelines are built using perl-scripts, the POSA objects can be used in very diverse settings.Here, we present POSA, a set of two new perl objects (Read.pm and Contig.pm) that describe a raw sequence and a Phred [scf) format within the object. Other methods of Read.pm use modules of the Staden Package [qclip and vector_clip . Properties of Read.pm include e.g. the DNA sequence, quality scores, template and vector names and read direction.The POSA source code is entirely coded in object-oriented Perl and consists of two objects: Read.pm and Contig.pm. In general, there are two important concepts associated with objects: methods (built-in procedures that can be performed on the object) and properties (describing some of the characteristics of the object). Most methods in the objects rely on the availability of other third-party programs (see Dependencies). Basically, POSA provides a wrapper around these programs and provides easy design and implementation of these programs in automated data analysis. The Read.pm object describes a DNA sequence trace and includes methods for data import from a variety of formats. It relies on Phred ,6 for im Package , such asPhrap program [PolyPhred [The Contig.pm object contains a method to assemble contigs of reads using the  program . The objolyPhred .Both the Read.pm and Contig.pm objects were designed with flexibility in mind. To allow a  unlimited amount of data to be processed, the perl scripts using these objects work sequence-by-sequence rather than method-by-method. Typically, these objects are called from straightforward perl scripts that outline the analysis steps to be performed. Example scripts using the objects can be accessed from the download website. An example of a script and output using the two objects to process a set of reads and annotate sequence polymorphisms from the assembled contig is given in Figure abi-files from the ABI PRISM 377 DNA Sequencer and 3100 Genetic Analyzer (Applied Biosystems). Phred, Phrap and PolyPhred versions were 0.000925.c, 0.990329 and 4.05, respectively.POSA was developed with perl 5.6.1 and tested on a SuSE linux 8.1 system for fasta, abi/ab1 or scf format retrieved from websites or from newly generated traces. In addition, new objects can be initiated from a text file or can be opened from previous stored objects. Subsequently, a variety of methods can be applied, including basecalling and assessment of quality codes (by Phred), quality clipping, vector clipping, screening for E. coli (or other) sequence, contig assembly (by Phrap) and analysis. The method asPrimer3 can automatically generate input for the primer3 program [SearchSnps method will generate output as shown in Figure PolyPhred program and uses the 'rank' argument to set the stringency.POSA provides an interface to design and implement automated sequencing data analysis. Sequencing data may be used in a variety of formats and originate from a variety of sources, e.g. data in  program  and is aexp, scf or fasta format. In addition, the data can be saved in a primer3 input file to allow automated PCR primer design, or data can be saved in MIPE format . Data on assembled contigs can be exported as a list of reads in a contig, as consensus sequence, as alignment, as putative SNPs, as SBE primers for SNP genotyping or as gff file for visualization in Gbrowse [.Finally, data can be stored in objects, or in files in either  Gbrowse . CombinaPhred, Phrap and PolyPhred.Although it represents only one of the numerous possible POSA-based pipelines, performance of POSA was validated by comparison of SNP discovery with the data after analysis using the Staden package. To do so, 5 PCR products were resequenced from a panel of 16 individuals to identify SNPs. Manual editing using the Staden Package revealed a total of 48 SNPs. Automated analysis using POSA also yielded a total of 48 SNPs with SNP ranking codes 1-3. Together, 41 SNPs were assigned with both manual editing and POSA. The remaining 7 SNPs assigned in manual editing corresponded to SNPs with ranks 4-6 in the POSA analysis. The 7 SNPs that were only assigned by POSA all originated from regions with lower quality sequence. While analysis time was reduced from several hours to a few minutes, POSA assigned SNPs in a way that was highly consistent with manual editing. This was expected because POSA provides options for an integrated analysis pipeline, but essentially is a wrapper around well-established sequence analysis tools like Phred and Phrap). Overall, this guaranties easy implementation of highly automated quality pipelines in combination with high flexibility in setup and design.POSA is a tool that provides easy and highly automated DNA sequence and contig data analysis using popular analysis tools. Automated sequence analysis reduces analysis time from several hours to a few minutes. Pipelines can easily be expanded or adapted through perl scripts. Writing or altering the perl scripts is straightforward to do for people with only basic computer skills, although more linux/unix experience might be necessary to install the required software • Perl modules: Carp; Statistics::Descriptive; Tie::File; IO::File; POSIX:: Storable.• Phred, Phrap, PolyPhred• Pregap4, gap4 )• Primer3 POSA Perl objects for DNA sequencing data analysisSNP single nucleotide polymorphismabi/ab1ABI PRISM trace file formatscf standard chromatogram format)exp experiment file format, developed by Staden (see )MIPE minimum information on PCR experiments (see BAC bacterial artificial chromosomePCR polymerase chain reactionSBE single base extensionJA programmed the Perl objects and participated in development of concept and architecture of the software; BJ participated in development of concept and architecture and wrote the manuscript; MG supervised the project. All authors read and approved the final manuscript.


Escherichia. coli by the DNA methyltransferase (Dam) and is known to be implicated in numerous cellular processes. Mutants lacking Dam are characterized by a pleiotropic phenotype. The existence of a GATC regulated network, thought to be involved in cold and oxygen shift, had been proposed and its existence has recently been confirmed. The aim of this article is to describe the components of the GATC regulated network of E. coli in detail and propose a role of this network in the light of an evolutionary advantage for the organism.The tetranucleotide GATC is methylated in E. coli's genes and the genes involved in the SOS and stress response show that the GATC network forms a group apart. The functional classes that characterize the network are the Energy metabolism (in particular respiration), Fatty acid/ Phospholipid metabolism and Nucleotide metabolism.We have classified the genes of the GATC network according to the EcoCyc functional classes. Comparisons with all of The network is thought to come into play when the cell undergoes coldshock and is likely to enter stationary phase.The respiration is almost completely under GATC control and according to our hypothesis it will be blocked at the moment of coldshock; this might give the cell a selective advantage as it increases its chances for survival when entering stationary phase under coldshock. We predict the accumulation of formate and possibly succinate, which might increase the cell's resistance, in this case to antimicrobial agents, when entering stationary phase. Escherichia. coli by the DNA methyltransferase (Dam); this enzyme methylates the adenine residue within 5'-GATC-3' sequences in double stranded DNA. GATC motifs and their methylation by Dam play an important role in E. coli; they are involved in mismatch repair (see ,,8], the E. coli in detail. In order to obtain more information about the characteristics of the network, we compare it with the genes known to be involved in the stress and SOS response. By examining the functions of the genes belonging to the GATC regulated network, we try to address the question about the possible evolutionary advantage of developing a regulatory network controlled by GATC clusters present within the coding regions.The aim of this article is to describe the GATC regulated network of E. coli to one of fifteen functional classes according to EcoCyc . Empty cells indicate genes not analyzed in their work. The column "EcoCyc genes" denotes the genes induced under various stress conditions according to EcoCyc, "Mitomycin C genes" denotes the genes induced by the stress caused by the antibiotic mitomycin C (genes involved in the SOS response and other stress response pathways) according to the recent transcriptome analysis carried out by [13], the two columns "GATC genes" denote genes containing a GATC cluster . The column "Oshima genes" denotes genes in E coli sensitive to the dam+/ dam- background. "EcoCyc functional class (modified)" gives the genes, classified according to the EcoCyc functional classes, with the modifications made by us (changes are justified in the last column). "Subclass" refers to the various groups of genes discussed in the results/ discussion section of this paper. "1": the gene is affected, "0": the gene is not affected, "N.A.": no information was available.Click here for fileDistribution of the different groups of genes according to the EcoCyc functional classification, without the class "Hypothetical" The table shows the distributions of the five groups of genes discussed in this paper,  after removal of the class "Hypothetical". The five groups are: the "GATC genes" (genes containing a GATC cluster), all of E. coli's genes, the "EcoCyc genes" (genes induced under various stress conditions according to EcoCyc), the "Mitomycin C genes" (genes induced by the stress caused by the antibiotic mitomycin C) and the "Oshima genes" (genes sensitive to the dam+/ dam- background). The distributions of the "GATC genes" and the "Oshima genes" do not differ from each other . The comparisons of the "GATC genes" with all of E. coli's, genes, the "EcoCyc genes" and the "Mitomycin C genes", however, show that the distributions continue to be significantly different, even after the removal of the class "Hypothetical".Click here for fileThe file contains the raw data used for this article and allows the reader to re-trace all the calculations madeClick here for file


Yield of hybrids parented by the pyramided genotypes was more than 50% higher than that of a control market leader variety under both wet and dry field conditions that received 10% of the irrigation water. This demonstration of the breaking of agricultural yield barriers provides the rationale for implementing similar strategies for other agricultural organisms that are important for global food security.Natural biodiversity is an underexploited sustainable resource that can enrich the genetic basis of cultivated plants with novel alleles that improve productivity and adaptation. We evaluated the progress in breeding for increased tomato  Dramatic improvements in crop yield  can be achieved by breeding selected genetic regions from wild species into domesticated varieties Plant evolution under domestication has led to increased productivity, but at the same time it has narrowed the genetic basis of crop species . A majorSolanum pennellii and introduced (through genetic crosses) onto the genetic background of the elite inbred variety M82 (Solanum). The ILs constitute an “exotic library” where the entire wild-species genome was partitioned among 76 lines each carrying a single homozygous introgressed segment. Implementation of this resource for QTL mapping is based on the nearly isogenic nature of the lines such that any phenotypic difference between M82 and an IL, or the hybrid of M82 with an IL (ILH), is attributable to the S. pennellii genomic segments . The ILs comprised marker-defined genomic regions taken from the drought-tolerant wild species segments . Similarsegments .in silico, in a search engine that displays a range of statistical and graphical outputs that describe the components of the genetic variation  and their soluble-solids content (mainly the sugars glucose and fructose), which is measured in refractometer brix (B) units (expressed as a percentage). Therefore, agricultural yield of processing tomatoes is the total sugar output per unit area ; the industry is searching for varieties that excel in BY. Our experiments were conducted in wet and dry fields, where the BY of the control variety M82 in the dry conditions was only 50% of that produced in the wet treatment, which received 10-fold greater irrigation .In “ketchup tomatoes,” which are used to produce various concentrated products, agricultural yield is made up of the total weight of the fruits harvested per unit area  in the dry fields. IL7-5-5 did not affect B, while for BY it was dominant. IL8-3 was greatly inferior to M82 for Y , but the ILH increased Y by 45% and 25% . This result indicates a strong overdominant effect for the introgression . The homozygous IL had double the effect on B relative to the ILH; this resulted in a strong overdominant effect on BY in both environments . The reduced Y of IL8-3 was caused by a pleiotropic effect of a leaf necrosis gene that was observed in all lines that were homozygous for this introgression; the necrosis was particularly severe in the dry treatment. IL9-2-5 significantly increased Y only in the homozygous condition in the wet treatment. For B and BY, ILH9-2-5 was intermediate between M82 and the homozygous IL, showing an additive mode of inheritance. The nature of the genes that improve BY in the above introgressions is unknown, with the exception of IL9-2-5, which harbors at least two QTL that affect the components of BY  that increases sugar content of the fruit as a result of a modification of enzyme functions , Chromosome 8 (IL8-3), and Chromosome 9 (IL9-2-5) that affect the components of BY. ts of BY . One of mponents .S. pennellii segments were pooled, using marker-assisted selection, into a single M82 line designated IL789 . IL789 showed a strong interaction with the environment: In the wet treatment it produced a Y similar to that produced by M82, while in the dry fields Y was reduced by 36% as a result of the pleiotropic effect of the recessive leaf necrosis gene on IL8-3. In the heterozygous condition IL789 dramatically increased Y in both field environments, and combined with the increases in B, ILH789 improved BY by 109% in the wet field and 58% in the dry fields. As described in earlier studies . BY of BOS3155 was in a range similar to that of our experimental hybrids, indicating that the experiment was conducted in elite genetic backgrounds.Our breeding program within the ermplasm A. The BYermplasm B. This iermplasm . As a reS. pennellii pyramid as the mean difference between the nearly isogenic hybrid groups: 100% (wet) and 65% (dry). These estimates for the exotic effects on BY are very close to those obtained in the uniform M82 background, indicating additivity of the exotic and cultivated effects  interactions and the stability of BY improvement associated with the heterozygous ressions . Generalressions . In the ressions . In the We have demonstrated that an IL population derived from a wild tomato species, with no yield potential, can make a wide array of previously unexplored genetic variation rapidly available to plant breeders to improve crop productivity. The effectiveness of the introgressions in diverse genetic backgrounds indicates that alleles similar to those of the wild species are not present in the cultivated tomato gene pool. The results presented here using the tomato ILs establish a genetic infrastructure to explore the molecular basis underlying yield heterosis. With the coming sequence of the tomato genome it will be easier to isolate those factors that are responsible for the strong overdominant effects, such as those observed for IL8-3 and some additional lines that are described in the IL phenotypic database Real Time QTL . Finally2. In 2003, trials were conducted in two locations: Akko and Mevo-Hama, in the Golan Heights. In Akko, experiments were both at wide-spacing planting density and in plots of 14 plants per 4 m2 (3.5 plants/m2). In Mevo-Hama all experiments were at the wide-spacing density. The seedlings were grown in a greenhouse for 35–40 d and then transplanted in the field, at the beginning of April in Akko, and at the beginning of May in Mevo-Hama. In all seasons and locations both wet and dry trials were conducted. Both the wet and dry fields started the growing season at “field capacity,” which represents the maximum amount of water that the soil could hold. For the dry treatment only 30 m3 of water was applied per 1,000 m2 of field immediately after transplanting. In the wet treatment 320 m3 of water was applied per 1,000 m2 of field throughout the growing season according to the irrigation protocols in the area. All experiments were transplanted in a randomized block design.The results presented are from three growing seasons. In 2001 and 2002 all field trials were conducted at the Western Galilee Experimental Station in Akko, Israel, at a wide-spacing planting density of 1 plant per mS. pennellii genomic segments . Lines homozygous for each of the segments and IL789 homozygous for all three introgressions were selected and verified with RFLP markers that flanked the introgressed segments from both ends (http://www.sgn.cornell.edu/). Phenotyping of the plants for Y, B, and BY was performed according to published protocols  was half of the difference between each IL and M82, and its significance level was determined by the comparison between the IL and M82. The dominance deviation (d) is the difference between ILH and the mid-value of its parents. Its significance level was calculated by contrasting the ILH (+1) with M82 (−0.5) and the appropriate IL (−0.5). The degree of dominance for each introgression (d/[a]) was calculated by dividing the mean dominance deviation by the mean additive effect. Deviation of the observed yield component values of the pyramided genotypes (IL789 and ILH789) from the expected values based on the assumption of additivity of the effects of the individual introgressions was tested using a t test at p < 0.05. G × E interaction was tested using a two-way ANOVA.Statistical analyses were performed using the JMP V.5 software package . Mean values for the parameters measured for the tested genotypes were compared using the “Fit Y by X” function and “Compare all pairs” (Tukey-Kramer). All calculations were performed with the phenotypic values, while some of the results are presented as the percent difference from M82. The additive effect


It is well known that most suicide cases meet criteria for a psychiatric disorder. However, rates of specific disorders vary considerably between studies and little information is known about gender and geographic differences. This study provides overall rates of total and specific psychiatric disorders in suicide completers and presents evidence supporting gender and geographic differences in their relative proportion.We carried out a review of studies in which psychological autopsy studies of suicide completers were performed. Studies were identified by means of MEDLINE database searches and by scanning the reference list of relevant publications. Twenty-three variables were defined, 16 of which evaluating psychiatric disorders. Mantel-Haenszel Weighted Odds Ratios were estimated for these 16 outcome variables.Twenty-seven studies comprising 3275 suicides were included, of which, 87.3% (SD 10.0%) had been diagnosed with a mental disorder prior to their death. There were major gender differences. Diagnoses of substance-related problems , personality disorders  and childhood disorders  were more common among male suicides, whereas affective disorders , including depressive disorders  were less common among males. Geographical differences are also likely to be present in the relative proportion of psychiatric diagnoses among suicides.Although psychopathology clearly mediates suicide risk, gender and geographical differences seem to exist in the relative proportion of the specific psychiatric disorders found among suicide completers. Suicide is an important public health problem that is among the leading causes of death in Western countries . Over thIt is well established that psychopathology is an important predictor of suicide completion , but theTo identify studies for this review, the National Library of Medicine (NLM) PubMed database was searched up to December 2002 using English language and human study limits. The Medical Subject Heading (MeSH) terms "suicide AND psychological autopsy", "suicide AND psychopathology", "suicide AND (postmortem diagnoses OR postmortem diagnosis)", and " AND prevalence AND  NOT suicide attempts)" were used. Finally, in order to find other articles not obtained through electronic searches, reference lists from original studies as well as from not independent studies were screened.The inclusion criteria for considering articles for this review were as follow. Studies had to: 1) be original, 2) be published in English, 3) contain information on diagnostic distribution, 4) include suicide completers unselected according to specific mental disorders, 5) use of a psychological autopsy method, which for the purpose of this review was considered as the process of reconstructing psychiatric diagnoses based either on interviews with informants (regardless of the specific diagnostic instrument methodology) or on review of multiple official records that contained interviews with informants such as general practitioners, other professionals and relatives or friends, 6) use of standard diagnostic criteria .Studies were excluded if: 1) their sample was not independent from that investigated in another study (see below for criteria on which one was included), 2) they were reports on suicide in one specific diagnostic category and 3) if diagnoses were simply extracted from medical records without review of multiple sources of information.A single reviewer (G.A.L.) made a prior screening to identify and select articles. When titles and abstracts were deemed adequate or when they remained too obscure to reach a verdict, full texts were retrieved for further evaluation in conformity with the inclusion and exclusion criteria.A total of 23 variables were defined, three of which relate to demographic information, four other concern the method of diagnosis, and 16 evaluate the presence of psychiatric diagnoses. To obtain the latter 16 variables  were reported, preference was given to the former. In four cases, secondary diagnoses were added to principal diagnoses to obtain multiple diagnoses -13. Stud2 to assess variations in the diagnostic distribution across these demographic areas. All statistical analyses were carried out using Epi Info 6, version 6.04d .Descriptive analyses and homogeneity tests were carried out before pooling the data. In order to determine the risks of having had a disorder, suicides and controls were recorded in 2 × 2 tables. These data were then stratified by the 16 outcome variables and Mantel-Haenszel Weighted Odds Ratios (OR) and 95% confidence intervals (95% CI) were estimated. Gender differences were also explored by means of Odds Ratios. Major disorders were then compared between the different demographic areas by means of χA total of 152 studies were initially identified. After selection according to inclusion/exclusion criteria, 27 studies were included in this review. The most common reasons for exclusion were that a) no diagnostic distribution was provided n = 46) [ 6,19-63-6319-63,The studies by Rich et al.  and by FAmong the 27 studies that were retained, 52% (14/27) were case-control studies. Eighty-one percent (22/27) of the studies were published after 1990. Sixty-seven percent of the studies (18/27) used DSM diagnostic criteria, whereas only 22% (6/27) and 11% (3/27) used the ICD and RDC diagnostic criteria respectively. Multiple diagnoses were investigated in 63% (17/27) of the studies, whereas principal diagnoses only were given for the other 10 studies. A description of the demographic and methodological features of these 27 studies is shown in table A total of 3275 suicides were included in our study with a mean number of 121 (standard deviation (SD) 103) suicides per study. There were 11 studies where diagnoses were given by gender for a subtotal of 933 males and 462 females ,144-150There were 14 studies ,151-154 2 : 2.27, p = 0.13).The mean percentage of suicides with a psychiatric diagnosis was 87.3 % (SD 10.0 %). However, only 14 of the 27 studies reported both axes I and II disorders  of suicide cases were diagnosed with any affective disorders (including depressive and bipolar disorders) and 25.7% (SD 14.8%) with other substance problems. In these groups, respectively, depressive disorders and alcohol problems were the most frequent. Finally, personality disorders represented 16.2% (SD 8.6%) of the suicide diagnoses and psychotic disorders, including schizophrenia accounted for 9.2% (SD 10.2%).The samples from the 14 case-control studies were found homogeneous for the 16 outcome variables according to a homogeneity test (results not shown), allowing us to pool the individual studies and determine overall risks.Table 1 see  shows thStatistically significant differences were found when male and female suicide cases were compared , and any substance disorders (present in 25.7 % of suicide cases). Recent studies on comorbidity indicate that suicide completers are more likely to have more than one psychiatric diagnosis ,161. In 2 7.29 p = 0.007), whereas affective disorders represented 59.4 % (SD 13.9 %) of the female diagnoses and 47.4 % (SD 12.7 %) of the male diagnoses (χ2 2.88 p = 0.089).The investigation of gender differences in rates of psychopathology associated to suicide should be regarded in light of the methodological limitations of this review, which are primarily related to difficulties in comparing studies carried out using different methodological procedures, diagnostic instruments and criteria, in addition to potential differences in sample characteristics, including age distribution. However, given the important effect that gender seems to have as a suicide risk moderator and the relative lack of appropriate investigation focusing on gender differences in suicide completion, the observed differences in rates of psychopathology in male and female suicides are interesting and should be considered for validation in future studies. Our results indicate that the risk of substance-related disorders, personality disorders and childhood disorders are significantly higher in male suicides, whereas, the risk of affective disorders, specifically, depressive disorders, are greater in female suicides. On average, any substance problems represented 41.8 % (SD 21.1 %) of the male diagnoses and 24.0 % (SD 16.5 %) of the female diagnoses  and they were not always in the same direction .An interesting finding of this study was precisely the absence of gender differences in schizophrenia. This is not necessarily inconsistent with suggestions that most of the suicide cases in schizophrenia are males -164, as We also found differences in rates of psychiatric disorders in studies from different geographic origins. This finding may indicate social and cultural factors influencing how one views and interprets suicide and cultural biases towards or against specific diagnoses. Alternatively, as discussed for gender-based comparisons, demographic  differences between the samples could explain some of these results. In view of that, similar limitations, as those for the analysis of gender differences, apply to the analysis of geographical differences in rates of psychopathology associated to suicide , have variation in diagnostic criteria used and have different methodological rigor. Moreover, it is possible that between-study variation in the distribution of a series of demographic variables could have had an impact on our findings. We chose not to control for these methodological differences as given the diverse sources of possible variation, doing so would have considerably limited the number of studies included in the review. Therefore, we opted to be more inclusive and consider the results of this review as preliminary and providing information to be further investigated.Over the course of this study, a report on another meta-analysis of psychological autopsy studies was published. This study, by Cavanagh et al. , revieweOur study carried out a systematic review of psychological autopsy studies of suicide and indicates that overall, 87.3% of suicide cases have a history of psychiatric disorders. We also found that male suicides have a different psychiatric profile than female suicide cases and that the relative proportion of psychiatric disorders in suicide completers tends to vary according to geographical region.The author(s) declare that they have no competing interests.GAL carried out the search, extraction of data, analysis and drafted the manuscript. CK helped with the design of the review, and the statistical analysis. GT conceived the study and participated in the design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 1 – Mantel-Haenszel Weighed Odds Ratio. This table gives Mantel-Haenszel Weighed Odds Ratio for the 14 case-control studies included in this meta-analysis for the 16 variables of psychiatric disorders.Click here for file


Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during  Escherichia coli frequently yields an inactive protein, aggregated in the form of so-called inclusion bodies.Recombinant DNA technology made available several simple techniques for transferring and efficiently expressing desired genes in a foreign cell. Thus, it was thought that unlimited and inexpensive sources of otherwise rare proteins would become accessible. It soon was observed that the host cell had a great influence on the quality and quantity of the produced recombinant protein. For example, recombinant protein production in mammalian cells yields a biologically active protein with all the required posttranslational modifications. However, mammalian cell cultivation is characterized by low volumetric yields of the recombinant protein, long cultivation times and requirements for expensive bioreactors and medium components. All these points have a great impact on the production costs. On the other hand, bacterial cultivation processes are based on inexpensive media in which fast growth and high cell concentrations can be obtained. These high cell concentrations combined with higher production rates of the bacterial expression system result in higher volumetric productivities. However, the production of recombinant proteins in bacteria such as in vivo dissolution; only recently it was shown that proteins can be resolubilized in vivo from inclusion body deposits . IncAfter their isolation, inclusion bodies are commonly solubilized by high concentrations of chaotropic agents such as guanidinium hydrochloride or urea. Although expensive, guanidinium hydrochloride is in general preferred due to its superior chaotropic properties. Moreover, urea solutions may contain and spontaneously produce cyanate , which ce.g. by dialysis) before starting the refolding procedure. As an alternative, immobilized reducing agents  could simplify reducing agent removal by centrifugation after the solubilization process. Finally, the pH must be reduced before the removal of the reducing agent from the solution containing the solubilized protein to prevent the formation of undesired disulfide bonds.In addition to the solubilizing agent, the presence of low molecular weight thiol reagents such as dithiothreitol (DTT) or 2-mercaptoethanol is generally required. These substances will reduce nonnative inter- and intramolecular disulfide bonds possibly formed by air oxidation during cell disruption and will also keep the cysteines in their reduced state ,15. Optie.g. low denaturant concentration). Moreover, appropriate redox conditions have to be established when the protein contains disulfide bonds in the native state. When proper conditions for refolding are identified, the refolding process can require a few seconds or several days. During this period, the correct refolding pathway competes, often in disadvantage, with misfolding and aggregation of the target protein  for further optimization of refolding conditions [Buffer exchange for denaturant removal can also be carried out by using size exclusion chromatography (SEC). Most frequently, the denaturant-protein solution is injected into a column previously equilibrated with the refolding buffer ,55-58. Snditions . An addinditions . Furthernditions ,61. Alsonditions .Some parameters for refolding using SEC are of key importance. For example, protein aggregation during sample injection can cause low refolding yields ; injectiClostridium thermocellum, which binds to a cellulose matrix [e.g. in the case hexahistidine-tagged proteins by elution with EDTA [Attaching the solubilized and unfolded protein to a solid support prior to changing from denaturing to native buffer conditions is another approach to avoid the unwanted intermolecular interaction between aggregation-prone folding intermediates. Binding of the solubilized and unfolded protein to the matrix requires the formation of a stable protein-matrix complex withstanding the presence of chaotropic agents. However, after changing to native buffer conditions, the detachment of the refolded target protein from the matrix should easily be accomplished. Several combinations of binding motives and matrices have been employed for binding the unfolded protein to the solid support. For example, proteins with a natural occurring charged patch in the unfolded chain, which binds to ion exchange resins ,65-67, oe matrix , have bee matrix , dialysie matrix ,73, or be matrix ,69,70,72ith EDTA ,70 or byith EDTA ,71,72. DHydrophobic interaction chromatography (HIC) has also been successfully used for protein refolding with concomitant removal of contaminating proteins during the renaturation process -78. UnfoIt has been proposed that refolding is facilitated during HIC because unfolded proteins adsorb at high salt concentrations to the hydrophobic matrix and, thus, are not prone to aggregation. Additionally, hydrophobic regions of the protein that adsorb to the HIC matrix form microdomains around which native structure elements can form. During migration through the column, the protein will pass through several steps of adsorption and desorption, controlled by the salt concentration and hydrophobicity of the intermediate(s), resulting finally in the formation of the native structure .Apart from any of the above-mentioned techniques for protein refolding, there are physical and chemical variables that have a great impact on the final yield of biologically active protein. For example, temperature as well as the composition of the refolding buffer are important variables influencing the final refolding yield.The most important physical variable influencing the refolding yield is the temperature ,45,50,51Pressure was identified as another important physical variable affecting protein structure as well as protein refolding processes . It was Certainly, L-arginine is nowadays the most commonly used refolding aiding agent . It impeDetergents ,92 and pReversed micelles, formed when an aqueous detergent solution is mixed with an organic solvent, can also facilitate protein refolding by avoiding aggregate formation . The denin vivo protein folding and protecting cellular proteins from different types of environmental stress by suppressing protein aggregation. For example, the major E. coli chaperonin GroEL is involved in the in vivo folding of 10% of all newly synthesized proteins at normal growing conditions, and of 30% under stress conditions [Chaperones are a group of proteins conserved in all kingdoms, which play a key role in assisting nditions . GroEL anditions . The relnditions .in vitro [Natural chaperones have also been applied successfully to refold various proteins in vitro . Howeverin vitro ,104. Somin vitro . Neverthin vitro .e.g. self-assembly of hydrophobized polysaccharides such as cholesterol-bearing pullulan) have been also used for the capturing step [A further development of the detergent-based micellar system mimics the two-step mechanism of chaperone-assisted protein folding. The capturing step is performed by diluting the denatured protein into a detergent solution, which prevents protein aggregation through the formation of mixed protein-detergent micelles -110. Aquing step . The reling step -112. Theing step . Also, ring step ,109 or ting step ,109. Howing step . The utiing step .In vivo, many proteins are transported through bilayered membranes in an extended and partially unfolded form either simultaneously or after their synthesis [ynthesis . A ratheynthesis . The uppIn vivo, these propeptides are known to play a key role in assisting the correct folding of the mature part of the protein [In vitro studies have demonstrated that they can facilitate the refolding in cis, when the denatured mature protein is still linked to its propeptide prior to the transfer into the refolding buffer, or in trans by including the isolated propeptide into the refolding buffer [Several proteins are synthesized in their natural environment with amino-terminal propeptides usually located between a signal sequence and the mature part of the protein.  protein . In vitrg buffer -120. Thig buffer ,122, or g buffer .Another method of template-assisted protein refolding exploits the specific binding properties of monoclonal antibodies to the target protein to reduce the time required for protein refolding and to enhance the final refolding yield ,125. Thi2+ ions [In general, solubilization of inclusion body proteins by chaotropic agents is carried out in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol to allow the disruption of nonnative disulfide bonds. Following solubilization, naturally disulfide-bonded proteins have to be refolded under conditions, which permit the formation of their native disulfide bonds. In the simplest way, free cysteine residues can be oxidized by molecular oxygen, a redox reaction catalyzed by Cu2+ ions ,127. Tho2+ ions .e.g. glutathione) in their reduced and oxidized state is added to the refolding buffer [Ka values as the aliphatic thiols thus enabling disulfide-bond formation at lower pH values [Disulfide bonds are more efficiently formed when a mixture of low molecular weight thiols  [Naturally disulfide-bonded proteins in their reduced states are often very unstable and exhibit a high tendency towards aggregation, especially during the early stages of refolding . These pathione) ,137. Theathione) ,135,136,athione) ,137.in vivo [in vitro protein refolding [in vitro refolding yields generally obtained with the common redox systems [Improvements of refolding yields of disulfide-bonded proteins have also been achieved by using protein disulfide isomerase (PDI) in combination with a redox system. PDI is a folding catalyst that assists disulfide bond formation in vivo  and was efolding ,140. In efolding . Howeverefolding . Traces efolding  and chemefolding -146 have systems ,146.e.g. cell disruption, inclusion body isolation and purification by several centrifugation and washing steps followed by a final solubilization procedure) and upstream of the renaturation process . Scale-up problems can arise when some of these steps are not transferable to larger scale processes. As an alternative to the common downstream process, inclusion bodies can directly be solubilized from chemically treated E. coli cells [E. coli host cell proteins, can be achieved by selective extraction of inclusion body proteins combined with diafiltration [e.g. by precipitation through spermidine addition [E. coli contaminants is followed directly and in the same operation unit by refolding and subsequent purification [Published refolding processes are often composed of numerous and cumbersome steps, both downstream (li cells -150 or ili cells . Even moli cells  as well li cells . A high ltration , aqueousltration  or selecltration ,149,151 ltration . Major daddition . Afterwafication ,74. Utilfication ,69-72. Ification ,64,67. Ffication ,61 with fication .After the first enthusiasm about protein production using recombinant microorganisms, it was promptly understood that obtaining an active form of the desired protein was not a simple task. Many proteins form nonnative precipitates in form of inclusion bodies when synthesized in bacteria and there is no universal refolding recipe for the generation of native protein from solubilized inclusion bodies. For any given protein, the best refolding conditions still have to be determined empirically. Among a lot of experience and "a good feeling for the best way", the use of experimental design methodologies ,155 and


Within the field of renal transplantation there is a lack of qualified and trainee surgeons and a shortage of donated organs. Any steps to tackle these issues should, in part, be aimed at future doctors.A questionnaire was distributed to final year students at a single medical school in the UK to assess their exposure to and knowledge of renal transplantation.Although 46% of responding students had examined a transplant recipient, only 14% had ever witnessed the surgery. Worryingly, 9% of students believed that xenotransplantation commonly occurs in the UK and 35% were unable to name a single drug that a recipient may need to take.This survey demonstrates a lack of exposure to, and knowledge of, the field of renal transplantation. Recommendations to address the problems with the recruitment of surgeons and donation of organs, by targeting medical students are made. With the potential for improved quality of life and increased life expectancy, renal transplantation is the first choice treatment for most patients with end-stage renal failure ,2. HowevThe field of renal transplantation also suffers from a lack of surgeons. Indeed, it is predicted that by the year 2005 there will a shortage of over twenty consultant renal transplant surgeons [Any measures to deal with these problems must include educating and attracting the doctors of tomorrow; medical students ,7. HowevIn July 2003 a PRHO job fair was held for final year medical students of Bristol University, which all 140 students within the year attended. An anonymous questionnaire was distributed to every student, after the aims of the study had been explained, at the end of a series of short lectures. . Of these responding students, thirty-two (42%) had never completed either a general medical or general surgical placement at one of the two centres for renal transplantation within the region, both of which are affiliated to the University of Bristol. National data of renal transplantation activity shows that these two centres differ in the number of transplants performed each year . Over th2, p = 0.006).Sixty-five students (86%) had never been in the operating theatre during a renal transplant. Of the eleven students that had witnessed a transplant the majority, seven, had been unscrubbed (9%). One student had been scrubbed and observing and a further three students (4%) had actually assisted with the procedure. Closer analysis of these eleven students demonstrates that only one of the eleven had been on placement at Derriford Hospital .Thirty-five (46%) of the students had examined a patient with a transplanted kidney but only thirty-three (43%) could accurately draw on a diagram the usual site of surgical incision that would be made on someone undergoing a left sided renal transplant. Interestingly, fourteen of the students that claimed to have examined a transplant recipient were unable to accurately draw the site of incision. Of the students that had examined a transplant recipient twenty-two had been on placement at one of the hospitals with transplant centres and thirteen had not (χFifty students (66%) were aware that in addition to the use of organs from brain stem dead donors, kidneys could also be transplanted from living donors. Eighteen students (24%) could not name any additional sources of organs and seven (9%) thought that xenotransplantation, using porcine kidneys, is carried out in the UK. None of the students suggested non-heart beating donation.Nineteen students (25%) were unable to name a drug that might be taken by a patient with a renal transplant. Twenty-one students (28%) were able to suggest just one.This survey highlights both a low exposure to, and a lack of knowledge about, the field of renal transplantation amongst medical students. This is cause for concern as it has implications for the future recruitment of trainees to the speciality and, potentially, to the procurement of organs.Previous work has highlighted the multiple factors that deter surgical trainees from this speciality . These iThe lack of knowledge regarding sources of organs commonly used within the UK is also of concern. In order to increase the number of kidneys available UK Transplant funds a number of non-heart beating programmes. Such initiatives can potentially increase the transplant rate by 20–40% . IdentifOne of the limitations of this study is the selection bias from a 54% response rate. From talking to some of the students who did not complete the questionnaire it became apparent that those who had no experience or knowledge of the speciality were less likely to participate. This means that the results are probably over reporting the exposure to and knowledge of renal transplantation. A further limitation is that this work only represents the situation at one medical school. We believe that this situation is not unique to Bristol University and recommend that a national study be performed to assess the true extent of the situation.If transplantation rates are to be maximised and recruitment into the speciality improved, then ideally, all doctors should have some exposure to renal transplantation during the early stages of their career. Indeed a recent study has demonstrated that increased knowledge about organ donation is associated with higher levels of medical education, an increased likelihood of holding an organ donor card and feeling more comfortable in approaching relatives of potential organ donors . Whilst This survey, carried out at a single UK medical school, has highlighted a low level of exposure to, and a lack of knowledge about, renal transplantation amongst medical students. These worrying results may influence the outcomes of any measures put in place to improve the recruitment of surgeons and the procurement of organs. If the trends within these areas are to be reversed then greater emphasis should be placed upon the promotion of renal transplantation within the undergraduate curriculum.UK United KingdomPRHO Pre-registration house officerThe author(s) declare that they have no competing interests.AGE designed the questionnaire, performed the study and was involved in the preparation of the manuscript. ARW and JDM were also involved in the design of the questionnaire and manuscript preparation.


Electroconvulsive therapy (ECT) often results in a number of short- and long-time side effects including memory impairment for past and current events, which can last for several months after ECT treatment. It has been suggested that unilateral ECT (uECT) with electrodes placed over the non-dominant  hemisphere significantly reduces side effects, especially memory disturbances. It is important to note that cerebral dominance equates to speech dominance and avoiding this area of the brain also reduces speech dysfunction after ECT. Traditionally, the routine clinical determination of cerebral dominance has been through the assessment of hand, foot and eye dominance, which is an easy and inexpensive approach that, however, does not ensure accuracy. This review of literature on different methods and techniques for determination of cerebral dominance and provides evidence that functional transcranial Doppler sonography (fTCD) represents a valid and safe alternative to invasive techniques for identifying speech lateralisation. It can be concluded that fTCD, notwithstanding its costs, could be used as a standard procedure prior to uECT treatment to determine cerebral dominance, thereby further reducing cognitive side-effects of ECT and possibly making it more acceptable to both patients and clinicians. Electroconvulsive therapy (ECT) is often regarded by the general public as a controversial procedure for the treatment of mental disorders. This is despite evidence of its safety and efficacy , and itsA further possible explanation for the reduction in ECT use may relate to the concern over adverse effects of the procedure. There are a number of short-term side effects including headache, nausea and, sometimes, brief confusion. However, the main side effect of concern is memory impairment for past events (retrograde amnesia) and for current events (anterograde amnesia) that can last for several months after a course of ECT treatment. Some of these side effects are substantially reduced by advances in safety and the introduction of controlled-current ECT machines. The utilisation of muscle relaxants, anaesthetics and resuscitation equipment, and electroencephalographic monitoring during the application of ECT are considered now considered routine. In addition, ECT guidelines issued by the UK National Institute for Clinical Excellence  restrictIt has been suggested that unilateral treatment significantly reduces side effects, especially memory disturbances ,7. DespiThe practice of determining cerebral dominance from handedness appears to mirror Broca's view that a person's handedness is opposite to hemispheric language specialisation. This, however, is incorrect, since there is no "mirror-image" cortical language organisation in left-handers. Several attempts to improve cerebral dominance assessment by introducing additional clues such as handwriting posture (i.e. inverted or hooked style versus non-inverted) and familial sinistrality have not substantially improved the prediction as to determination of cerebral dominance ,13. For A great majority of left-handers have also an ipsilateral functional specialisation for language . Although right-handers are more clearly lateralised than left-handers in this regard, a certain proportion of right-handers have language localised in the right-hemisphere. This has been confirmed by various techniques, ranging from the old and invasive procedures such as the intracarotid sodium amytal test and ECT, to the new and more sophisticated techniques such as functional magnetic resonance imaging (fMRI) and functional transcranial Doppler sonography (fTCD). The pooling of empirical data from a number of studies -27 whichFrom Table Until recently, an accurate determination of speech dominance prior to a course of ECT treatment was possible only through invasive procedures such as intracarotid sodium amytal test , also knThe advent of sophisticated and non-invasive technologies during the 1980s and 1990s has enabled a non-invasive approach to the assessment of speech dominance. One of the most elegant, mobile, and cost effective methods for determining cerebral dominance for speech is functional transcranial Doppler sonography (fTCD). fTCD is increasingly used in both clinical and research settings and is a new and robust technique based on the same principles as fMRI. Subjects in studies using this method are asked to generate as many possible words within 5-second periods after a letter presented on the computer screen cues for word generation. Basically, fTCD measures cerebral blood flow velocity which corresponds to brain activity. The physical foundation for this technique is quite old and is based on the work of the Austrian mathematician and physicist, Christian Doppler (1803–1853), who discovered that the change in pitch results from a shift in the frequency of the sound waves. This means that the speed of a physical object (i.e. blood) can be estimated by measuring the rate of change of pitch. To complete the fTCD procedure, the additional sound produced through the arteries is required.Recently, it has been argued that fTCD can reliably replace the Wada procedure in patients undergoing brain surgery . The valThis review of the literature on ECT and cerebral dominance provides evidence that fTCD represents a valid and safe alternative to invasive techniques for identifying speech lateralisation. It seems therefore, that fTCD, notwithstanding costs, could be used as a standard procedure prior to uECT treatment to determine cerebral dominance, thereby further reducing cognitive side-effects of ECT and possibly making it more acceptable to both patients and clinicians.none declared.


Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced γH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.By utilizing a panel of Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms. Type II topoisomerases are essential nuclear enzymes found in all living organisms . Their bTopoisomerase II is also a major drug target in human cancer therapy, where a number of clinically active drugs such as the epipodophyllotoxins VP-16 and VM-26, the aminoacridine m-AMSA, and antracyclines such as doxorubicin, daunorubicin and epirubicin are widely used. These drugs have collectively been called topoisomerase II poisons due to their mechanism of action on topoisomerase II. Rather than inhibiting the basic catalytic activity of the enzyme, these drugs perturb the topoisomerase II catalytic cycle resulting in an increase in the level of a transient reaction intermediate, where DNA is cleaved and covalently attached to DNA .Catalytic inhibitors of topoisomerase II have a different mode of action. These drugs exemplified by merbarone, aclarubicin, F11782 and the bisdioxopiperazines work by inhibiting topoisomerase II at other stages in the reaction cycle where DNA is not cleaved as reviewed in ,6. Amongi) Expression of bisdioxopiperazine-sensitive topoisomerase II in cells also expressing bisdioxopiperazine-resistant topoisomerase II confers dominant sensitivity to these drugs methanesulphonanilide); MMR, Mismatch Repair; NER, Nucleotide Excision Repair; NHEJ, Non-Homologous End Joining; PCR, Polymerase Chain Reaction; PRR, Post Replication Repair; SCLC, Small Cell Lung Cancer; SC-URA, Synthetic medium lacking uracil; SSA, Single Strand Anealing; YPD, Medium containing Yeast extract, Peptone and Dextrose.Lars H. Jensen: Participated in planning the experiments, performed yeast transformations and clonogenic assays, and prepared the manuscript. Marielle Dejligbjerg: Performed γH2AX western blots, primer design, real-time PCR experiments, and data quantitation. Lasse T. Hansen: Performed mammalian clonogenic assays and recombination assays. Morten Grauslund: Performed RNA purification and microarray analysis. Peter B. Jensen: Participated in planning and monitoring the study. Maxwell Sehested: Participated in the initiation and conduction of the study. All authors read and approved the final manuscript.Clonogenic sensitivity of mutant single-gene deletion yeast strains towards ICRF-187 and m-AMSA. Clonogenic sensitivity of a panel of human topoisomerase II α-transformed haploid yeast deletion strains towards equitoxic (to wt cells) concentrations of ICRF-187 and m-AMSA. Error-bars represent SEM of 3 – 10 independent experiments.Click here for filet2–4 cells were inoculated into YPD medium and grown overnight at 34°C, 150 rpm. The cultures were then diluted into 50 ml YPD medium to obtain an OD600 of 0.2. After growing the cells for 2 hours to assure exponential growth, equitoxic concentrations of ICRF-187 and m-AMSA were applied and the cells were grown for an additional 2 hours before RNA was isolated. The figure depicts the clonogenecity of drug treated and untreated cells. Exposure of the cells to the two drugs resulted in a reduction of their clonogenecity of approx. 50 %. Error bars-represent SEM of three independent experiments.Level of killing of yeast cells used in transcriptional profiling experiments. Fresh colonies of pMJ1-transformed JN362AClick here for fileTranscriptional response towards ICRF-187. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to ICRF-187.Click here for fileTranscriptional response towards m-AMSA. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to m-AMSA.Click here for fileTopoisomerase II activity levels in hamster cell lines. The levels of topoisomerase II catalytic activity in crude extracts from wt and recombination defective hamster cell lines. Error-bars represent SEM of two independent experiments. No difference in the level of topoisomerase II catalytic (DNA strand passage activity) is observed.Click here for file


Buchnera aphidicola. We show that bacteriocytes of Acyrthosiphon pisum express several gene products : Distal-less, Ultrabithorax/Abdominal-A, and Engrailed. Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo. In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development. We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B. aphidicola. Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80–150 million years. Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained. The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria. Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell.Symbiotic relationships between bacteria and insect hosts are common. Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes. We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria  Molecular markers show that bacteriocytes, the aphid cells that house the bacterial endosymbionts, are specified in a conserved two-step process that does not depend on the presence of the bacteria Buchnera aphidicola, of several species of aphids has revealed extensive gene loss  (Hemiptera: Aphididae). Using these markers, we aimed to determine the developmental origin of bacteriocytes and to what extent bacteria are required for the formation of the bacteriocytes. We also tested whether the observed patterns of bacteriocyte development are evolutionarily conserved among distantly related aphid species. We show that three transcription factors are expressed in a specific temporal order during early bacteriocyte development of the pea aphid. The final population of bacteriocytes originates from two distinct populations of nuclei recruited at different times of development. Furthermore, we experimentally demonstrate that the specification and proliferation of bacteriocytes occur independently of B. aphidicola. In distant relatives of the pea aphid, we found that the two-step determination of bacteriocytes is conserved. We also investigated two cases involving the loss of B. aphidicola. In the first case, in which the bacterial symbionts have been replaced with extracellular, eukaryotic symbionts, bacteriocyte development appears to proceed normally. In a second case, in which males do not inherit B. aphidicola, the bacteriocytes have been lost.We have identified bacteriocyte-specific markers that allow us to track the proliferation of bacteriocytes throughout the development of the pea aphid A. pisum. We infer that these antibodies are recognizing the homologues, or possibly paralogues, of their respective target proteins. The three proteins are expressed in a specific temporal order. We first observe expression of the Distal-less (Dll) protein (FlyBase ID: FBgn0000157) (We tested five cross-reacting antibodies see  for thei0000157)  in syncy0000157) C. As the0000157)   D–1F. The0000157)  that sta0000157) D′. As th0000157)  is detec0000157) G.. The nuclei of these cells are visibly smaller than those of the original bacteriocytes  approximately 80–150 million years ago (Pemphigus spyrothecae (Eriosomatinae) and Tuberaphis styraci (Hormaphidinae) (discussed below). In both cases, Dll is expressed in a small number of bacteriocyte nuclei of the blastoderm-stage embryo and additional Dll-expressing cells are recruited later. In P. spyrothecae, one or two nuclei are originally determined as bacteriocytes, as suggested by The two-step determination of bacteriocytes described in the previous sections appears to be a conserved feature of the aphids. Using the anti-Dll antibody, we examined development of the bacteriocytes in two species of aphids that diverged from ears ago : PemphigB. aphidicolahas been lost in the lineage leading to T. styraci and has been replaced by a yeast-like symbiont  or alfalfa (Medicago sativa)  . P. spyreviously .)  and mouBuchnera can be eliminated by treating animals with antibiotics (Medicago arborea in Petri-dish cultures (In the pea aphid, ibiotics . First- ibiotics . Aphids cultures . Controlcultures  were discultures .The FlyBase accession numbers discussed in this paper are FBgn0000014, FBgn0000157, FBgn0000577, FBgn0000606, FBgn0003944, and FBgn0003970.


Wall Street Journal Online poll. The respondents may not know that populations of large predatory fish, such as tuna, swordfish, and marlin, have declined 90% over the past 50 years or that less-prized species are increasingly overfished. Or that ever more fish and seafood species show rising levels of mercury contamination, rendering them unfit for human consumption—and contaminating other organisms in the ocean food chain. Humans are also affecting marine life in unexpected ways, as when large numbers of seals in Antarctica in 1955 and in Siberia in 1987 succumbed to canine distemper virus, presumably contracted from domestic dogs. In 2000, more than 10,000 Caspian seals—which also had contact with domestic dogs—died of the same virus. Such human incursions cause even more damage by exacerbating the effects of naturally occurring parasitic and pathogenic diseases that already wreak havoc as they ripple through the food chain.After the recent mad cow scare in the United States, 61% of Americans said they would start eating more fish, according to a With recent studies suggesting that disease rates have increased over the past 30 years—and are expected to increase even more, thanks to global climate change—prospects for protecting marine ecosystems depend on understanding the causes and nature of these disease outbreaks. While all indicators point to a real increase in disease rates, scientists have no baseline data to measure these increases against and so cannot directly test the hypothesis that marine diseases are increasing. Now Jessica Ward and Kevin Lafferty report a method that uses the recorded incidence of disease as a proxy for baseline data to identify disease trends in major groups of marine organisms.Ward and Lafferty conducted an online search of 5,900 journals published from 1970 to 2001 for reports of disease in nine taxonomic groups: turtles, corals, mammals, urchins, mollusks, seagrasses, decapods (crustaceans), sharks/rays, and fishes. Their approach takes into account three potentially confounding factors in determining trends in this type of search. Fluctuations in publication numbers could skew results, since an increase in the number of scientific reports published in a particular taxonomy might not reflect a true increase in the incidence of disease; a particularly prolific author could bias the search results by turning up more cases of disease in a population than actually occurred; or a single disease event reported multiple times in different papers could create the impression that disease had suddenly increased. To normalize publication rates over time, Ward and Lafferty used a proportion of disease reports from a given population relative to the total number of reports in that group. To determine whether there was an “author effect,” they removed the most prolific author in each taxonomic group and found that an author's abundant contributions did not skew the results. Finally, they confirmed that a single disease didn't bias their results by removing multiple reports of the same disease from the literature before analyzing the trends.When they analyzed the searches without adjusting for the total number of reports published, Ward and Lafferty found that reports of disease increased for all groups. But when they analyzed the normalized results, they found that trends varied. While there was a clear increase in disease among turtles, corals, mammals, urchins, and mollusks, they found no significant trends for seagrasses, decapods, and sharks/rays. And they found that disease reports actually decreased for fishes. (One explanation for this decrease could be that drastic reductions in population density present fewer opportunities for transmitting infection.) Ward and Lafferty tested the soundness of this approach by using a disease (raccoon rabies) for which baseline data exist and showing that normalized reports of raccoon rabies increased since 1970, just as the disease increased from one case reported in Virginia in 1977 to an “epizootic” outbreak, affecting eight mid-Atlantic states and Washington, D.C., by 1992.The pattern of increased reports, the authors propose, confirms scientists' perceptions about the rising distress of threatened populations and thus reflects a real underlying pattern in nature. The fact that disease did not increase in all taxonomic groups suggests that increases in disease are not simply the result of increased study and that certain stressors, such as global climate change, likely impact disease in complex ways. By demonstrating that an actual change in disease over time is accompanied by a corresponding change in published reports by scientists, Ward and Lafferty have created a powerful tool to help evaluate trends in disease in the absence of baseline data. It is only by understanding the dynamics of disease outbreaks that scientists can help develop sound methods to contain them.


Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined.Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein.We report that Dsh protein accumulates in cell nuclei when These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/β-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus. Drosophila and convergent extension cell movements and tissue separation in Xenopus [The specification of cell fates during embryonic development frequently depends on inductive interactions, which involve transmission of extracellular signals from the cell surface to the nucleus. In the transforming growth factor β (TGFβ) signal transduction pathway, Smad proteins that are initially associated with TGFβ receptors move to the nucleus to regulate target genes . Another Xenopus -12. ThusXenopus ectoderm, and this redistribution requires the DEP domain [Dsh is a common intracellular mediator of several pathways activated by Frizzled receptors and is composed of three conserved regions that are known as the DIX, PDZ and DEP domains . DiffereP domain ,18,19. TP domain ,18,20,21Xenopus ectodermal cells and mammalian cells upon inhibition of nuclear export. Dsh also accumulated in the nuclei after stimulation of mammalian cells with Wnt3a-containing culture medium. By analyzing various mutant Dsh constructs in Xenopus ectoderm, we show that the signals responsible for Dsh nuclear localization reside in a novel domain and that the nuclear translocation of Dsh is essential for its ability to activate Wnt/β-catenin signaling.The current study was based on our initial observation that a Dsh construct lacking the carboxy-terminal DEP domain was found in cell nuclei. We have now identified a nuclear export signal in the deleted region and also discovered that Dsh proteins accumulate in the nuclei of Xenopus ectodermal cells. In contrast to Dsh-GFP, which is localized in punctate structures within the cytoplasm [We studied the subcellular distribution of fusions of Dsh with green fluorescent protein (GFP) in ytoplasm ,18, the ytoplasm ,23. WhenTo examine whether inhibition of nuclear export abrogates Dsh activity, we compared the abilities of DsNESm and wild-type Dsh-GFP to induce secondary axes in frog embryos. Although the molecular mechanism operating during axis induction remains to be elucidated, this assay faithfully reflects the biological activity of Dsh in the canonical Wnt/β-catenin pathway ,16,18,24Xenopus embryonic cells under conditions in which nuclear export is blocked. When ectodermal cells expressing Dsh-GFP were incubated with N-ethylmaleimide (NEM), an inhibitor of the nuclear export receptor CRM1/exportin [Accumulation of DsNESm in the nucleus implies that the wild-type Dsh shuttles between the nucleus and the cytoplasm. We therefore studied the subcellular distribution of Dsh in exportin ,26, Dsh-exportin ,23  293 cells treated with LMB accumulated Dvl2 in the nucleus, contrasting with the cytoplasmic localization of Dvl2 in untreated cells Figure . We alsoTo identify specific amino-acid sequences that direct the transport of Dsh to the nucleus, we studied the subcellular distribution of mutated Dsh-GFP fusion constructs Figure . The remHydra to humans , a positive regulator of the β-catenin pathway ,31, or A protein   pathway, which involves Rho GTPase and JNK and controls morphogenetic movements in early embryos ,9,37-39.Xenopus, the PCP pathway involving Dsh is implicated in the control of convergent extension movements [In ovements ,41,42. Oovements ; Figure ovements ,37-39. BOur findings are consistent with a scenario in which Wnt signaling may cause nuclear translocation of Dsh followed by formation of a stable β-catenin/Tcf3 complex and transcriptional activation of target genes. In support of this hypothesis, Dsh was reported to move to the nucleus in response to Wnt signaling in primary embryonic kidney cells . In Rat-In the current view, Wnt signaling causes inactivation of the β-catenin degradation complex, leading to stabilization and nuclear translocation of β-catenin . Given tXenopus ectoderm cells overexpressing Dsh (data not shown), this process may be cell-context-dependent. On the other hand, we recently showed that Frodo, a nuclear Dsh-interacting protein, associates with Tcf3 and influences Tcf3-dependent transcription [Alternatively, nuclear localization of Dsh may affect β-catenin stability indirectly, by regulating protein interactions that sequester β-catenin in the nucleus, thereby preventing its cytoplasmic degradation . Althougcription . It is tGFP-tagged Dsh constructs were all derived from the DshGFP-RN3 plasmid that encodes the Xdsh protein fused at amino acid 724 to the first amino acid of GFP Figures , 4a. Ds1To generate these constructs, DshGFP-pRN3 was used as a template. The in-frame deletion in Ds3 was made by PCR. Other GFP fusion constructs were synthesized with specific primers and PfuI DNA polymerase followed by DpnI digestion of the template . The folIn vitro fertilization, culture and microinjections of Xenopus eggs were essentially as described previously [eviously . Stages eviously . Axis in2, 0.1 mM MgCl2, 0.5 mM Hepes, 1.2 mM sodium phosphate, (pH 7.5), 0.6 mM NaHCO3 and 0.06 mM EDTA) for one hour prior to fixation. In some experiments, nuclei were stained by addition of 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) to the final PBS wash. For membrane localization studies, Xfz8 RNA was coinjected with RNAs encoding the Dsh constructs in the animal-pole region; animal-cap explants were dissected at stage 9–9.5 and mounted for observation. Fluorescence was visualized using a Zeiss Axiophot microscope.For subcellular localization of Dsh-GFP constructs, mRNAs were injected into the animal pole region of 2–4-cell embryos. Animal cap explants were dissected at stages 9–10.5, incubated for 60 min in 10 mM N-ethylmaleimide  in 0.8 × MMR , or in control (0.8 × MMR), then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30–45 min, washed three times in PBS, and mounted in 70% glycerol, 30% PBS containing 25 mg/ml of diazabicyclo-octane (Sigma). Leptomycin B was used at 50 ng/ml in low-calcium medium  was coinjected with mRNAs encoding Xwnt3a  or Xwnt8Rat-1 fibroblasts, human embryonic kidney (HEK) 293 cells and MCF7 human breast carcinoma cells were cultured in 1 × Dulbecco's Modified Eagle Medium  supplemented with 10% fetal calf serum and 5 μg/ml gentamicin. Conditioned medium was prepared from L cells stably transfected with Wnt3a as described , with thFor immunocytochemistry, HEK293 cells were treated with 50 ng/ml LMB for 14 h while MCF7 cells were treated with Wnt3a or control conditioned medium for 1, 3, 6 or 8 h. Cells were fixed with 4% paraformaldehyde, immersed in methanol, and incubated with anti-Dvl2 antibodies and then Cy3-conjugated anti-rabbit IgG. Nuclei were stained by addition of 1 μg/ml DAPI as described for animal-cap cells. Fluorescence was observed under the Zeiss Axiophot microscope; 10–15 cells from each group were randomly picked up for measurement of the nuclear and cytoplasmic staining intensity using Image-Gauge software .2, 10 mM KCl, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Cells were swollen for 30 min, and broken open with 25 strokes in a tight fitting Dounce homogenizer. Lysates were layered into tubes containing 1 M sucrose in hypotonic lysis buffer, and spun at 1600 × g for 10 min. Supernatant remaining above the sucrose cushion was used as the cytoplasmic fraction. The pellet, containing nuclei, was resuspended in an equivalent volume of hypotonic lysis buffer.For subcellular fractionation, confluent cultures of Rat-1 cells were harvested by scraping plates and resuspended in hypotonic lysis buffer containing 1 mM EGTA, 1 mM EDTA, 2 mM MgClImmunoprecipitation and western analysis were carried out with cell and embryo lysates as described . To prepin vitro kinase assays were carried out essentially as described [Four-cell embryos were injected with 4 ng Dsh or DsNLSm RNA into four animal blastomeres. Embryo lysates were prepared at stage 10.5 and escribed , except The following is provided as an additional data file with the online version of this article. Additional data file Cloning details of Dsh mutant constructsClick here for additional data file