Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models. What is the point of single cell studies of the cell cycle? The simple answer is that they provide extra information that is not available from studies of cell populations. Without them a cell biologist can be misled.Schizosaccharomyces pombe with which I have worked since the mid-1950s. It was then a fairly obscure organism for physiological studies though it had a good genetic background found by U. Leupold in Bern [It is easiest for me to start with the theme of the extensive results on single cells of the fission yeast One the early results on its growth came from a single cell study by Bayne-Jones and Adolph . Here I Tetrahymena and Chlorella by periodic changes in their environment. Good synchronous cultures would mean that powerful biochemical techniques, often enzyme activity assays at that time, could be applied in a cell cycle context. In the next 15 years, induction synchrony was somewhat improved but the cell cycles were always and inevitably distorted. Methods were also developed to select out a fraction of an asynchronous culture in one stage of the cycle and grow it up separately (for example," membrane elution", where cells growing on a membrane come away at division). They produce less distortion but a much lower yield than induction.This period of the fifties was when attention in this field was largely focused on the successful synchronisation of Because of what can be measured in synchronous cultures, they are the natural choice for the molecular biologist. But it is as well to remember their limitations. The distortions after induction have been mentioned, but even with selection synchrony there are problems. The main one is that they are, in practice, not all that synchronous. The selected cells come from more than a very narrow region of the cycle. Some of the variation can be reduced by a correction for asynchrony  but therReturning to single cell analyses of fission yeast, volume growth was followed in finer detail by Mitchison and Nurse . One paret al. [A much more detailed analysis of populations of single cells followed on films was made by Sveiczer et al.  on fissiSingle cell studies have their problems. We have been lucky in using yeasts that are not apparently affected by growing on warm agar pads under a coverslip. They show "balanced growth", a property in which there is no change in extensive properties between successive cycles  and thatThe cells also have to keep still or be followed, a problem discussed below. We have not found ways of sticking yeast to glass (e.g. with lectins) that permit "normal" growth. Cells may also need a continuous supply of fresh medium, probably for oxygenation. Various types of microscopic mounting chambers have been described in the last 50 years or so, e.g. , but fewEscherichia coli. The limitations of synchronous cultures in hiding the fine detail of increases in volume or area could well mean that single cell studies might reveal more than an exponential increase. There might even be something like the two linear patterns that were popular models in earlier work with this bacterium [Many experimental studies on cell growth kinetics can be tedious; single cell studies are no exception. Here, however, modern automation is beginning to have very promising prospects. Anyone who has spent a day on a yeast film re-focusing the microscope every 5 min will welcome auto-focusing devices that are now available. Analysis has also become much easier with electronic imaging followed by image analysis programmes, and perhaps presentation on spreadsheets. It is now possible to have a programme that requires some hand work in the initial setting up under the microscope but will then run automatically, measuring cell length and diameter. This has been done for fifty or more single cells of fission yeast – a long way from the early days of using a ruler to measure the length of yeast cells on projected film images. Another point that should be raised here is that the new technology could profitably be applied to the growth of acterium .Amoeba proteus in an important classic paper by Prescott [Acanthamoeba and CO2 production in fission yeast , in botAnother interesting single cell method was the colorimetric enzyme assay of single yeast cells in microdrops . This miOne of the advantages of single cell work with yeasts is that they stay still on an agar pad so they can be followed for a couple of cycles before overlapping spoils the image. This is not true of many mammalian cells, which move around on the substrate. One solution to this problem comes in the work on fibroblasts (mouse L cells) described in Zetterberg , KillandWhat are needed now are techniques that combine the subtlety and precision of single cell measurements with the new techniques of automation. A promising start was made by Zicha and Dunn , and theAmoeba proteus mentioned above. The results showed that the increase of single cell "dry mass" fell in a reverse exponential, with a rapid increase at the start of the cycle falling to zero towards the end. This, of course, is lethal for anyone who believes that a rising exponential is the paradigm for the cell cycle.Organisms which tend to be forgotten about these days are those lower eukaryotes that make poor material for molecular biologists because of inadequate genetic backgrounds. One important set of results are those from the early pioneer work of Prescott  on AmoebTetrahymena pyriformis has a long and distinguished history in the cell cycle with its early induction synchrony. But in the 1960s there was a burst of studies on selected single cells or small groups. The growth patterns were often not well defined but it seems that absolute measurements of volume and of respiration rate were a better fit to linear growth (Prescott, [rescott, ). Such aPhysarum polycephalum is a myxomycete of considerable importance in some earlier work on cell cycle control. It is effectively a big single multinucleate cell with complete natural synchrony in nuclear division. It does not show exponential increase in macromolecular synthesis. For instance, there are two peaks in the rate of protein synthesis, one in the S period and the other in G2 .It would appear that there are no universal patterns of growth in these lower eukaryotes.My title makes mention of "some models". Let me be clear that there are two quite different types of cell cycle models. One type includes detailed mathematical and molecular models dealing with discrete periodic events like mitosis (e.g. ). These However, what I am concerned with here are much earlier and much simpler models, not of periodic events in the cycle like DNA synthesis, but of continuous growth. Here the two dominant models were, for simplicity, an exponential pattern of increase and a linear one. My own view  of the e


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) [Lepus europaeus) in particular has experienced a dramatic decline in many European countries [reviewed by [In most countries in Western Europe, the landscape has undergone dramatic changes during the last century due to changes in the agricultural practices. In Denmark, both the uncultivated land and the semi-cultivated land, such as permanent grass areas, have been reduced dramatically, reflecting the general intensification of agriculture . Additio perdix) . Among miewed by ], 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 [European brown hares are important prey primarily for mammalian predators. In Northern Europe, the red fox  in the European brown hare time series (G = 281.4, df = 2, P < 0.0001). Additionally, the effect of district was also significant (G = 769.0, df = 1, P < 0.0001).We found statistical significant direct density-dependence . In seven of the 11 districts the delayed density dependence was significant (P < 0.05), suggesting important inter-specific interactions [t-1 (and not yeart) most likely reflects that compared to adults, juvenile European brown hares suffer more from predation [e.g. [t result in low harvest of European brown hares in yeart, which affects the reproductive potential of the populations, and, hence, the number of European brown hares the following year (yeart+1). Also, the significant effect of district may point to differences in habitat quality, but also differences in the history of the sarcoptic mange, i.e. time since the eruption of the mange, among districts [see [In a recent study, Fox  showed tractions ,37. The on [e.g. ]. 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 [.The relationship between the winter NAO (Decembern Europe . When NAn Europe . The NAOn Europe . The NAOe-transformed, i.e. X(t) = ln(N(t) + 1), where N(t) is the number of European brown hares harvested in year(t). To allow the direct comparison of regression coefficients, variables were standardized prior to the analyses (i.e., [X(t)-µ(1955–2000)]/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)~(µ,V),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 (61% neutrophils, 39% lymphocytes), 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 (20 mg/kg/day, iv) plus  interferon-γ (50 microgram/mAspergillus fumigatus was determined as causative agent in this case. Deoxycholate Amphotericin B (1 mg/kg/day, iv), interferon-γ (50 microgeram/m2 every other day, sc) 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 (e.g. vector trimming is needed in plasmid sequencing, but needless when sequencing PCR products).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 [E. coli only) and the control of chromosome replication  and that the class Energy metabolism is particularly overrepresented in the genes sensitive to the dam genotype  Dam is a limiting factor and the DNA will be undermethylated ,10; thisAccording to the hypothesis of  and 8],,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 [E. coli containing a GATC cluster, listed in Table 1 [see E. coli's genes or not. The results are displayed in Table E. coli's genes . As the GATC network is thought to come into play when the cell suffers from stress, namely coldshock (coupled to an oxygen shift), we compared the "GATC genes" with genes induced under various stress conditions according to EcoCyc ("EcoCyc genes") as the two groups might overlap. The results of this second comparison are displayed in Table -9). A third comparison was made, between the "GATC genes" and the group of genes, whose expression is strongly affected by mitomycin C ("Mitomycin C genes") according to a recent transcriptome analysis [E. coli provokes a general stress in the bacteria, affecting almost 30 % of E. coli's genes; 7% of E. coli's genes show particularly strong changes in expression levels, including the genes in the SOS response and genes belonging to other stress response pathways [-6, see Table Firstly we assigned each gene in o EcoCyc . This alle 1 see ) follow analysis . Mitomycanalysis . Additiopathways . We haveThe three comparisons show us that the "GATC genes" form a group apart. A characteristic of the GATC network is the overrepresentation of three classes, namely Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism, indicating that the genes belonging to these three classes are the ones which characterize the network and therefore deserve a closer examination.Two distinct arguments corroborate the fact that the three classes (Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism) are at the heart of the GATC Network:E. coli in a dam+/dam- background. We have classified the genes affected by this background according to the 15 functional classes in EcoCyc and compared the distribution of these genes with the "GATC genes". At first sight the distribution (in percentage) of the genes in the fifteen classes is different in the two groups . If, however, we do not take into consideration the class "Hypothetical", the two distributions do no longer differ from each other, with a p-value of 0.03  and they follow the same distribution in the 15 EcoCyc functional classes .The second argument stems from the comparison of Twenty-three genes are in common to the two bacteria (i.e. they contain a GATC cluster in both organisms); even if we do not take these genes into account, the rest of the "GATC genes" in the two organisms still follow the same distribution .We expected the network to be evolutionarily conserved, but not to be identical in the two organisms. A particularly interesting case is given when the same pathway is affected in both organisms, but through different genes, as this represents a particularly strong argument for the evolutionary conservation of the GATC regulated network, beyond the mere conservation of the sequences themselves. The following serve as examples:prpE contains a GATC cluster in both E. coli and Salmonella. prpB, which belongs to the same catabolic pathway, is affected in E. coli only. According to our hypothesis, transcription from these two genes would be halted during coldshock. prpR codes for the positive regulator of the propionate catabolism operon, to which prpE and prpB belong to. It contains a cluster in Salmonella. According to our hypothesis, transcription of prpR would be halted under coldshock, thus inhibiting transcriptional activation of the propionate catabolism operon, effectively blocking propionate catabolism. It is interesting to note that the GATC regulated transcription block acts on two different levels: the genes involved in catabolism, as well as their operon's regulator.The first regards the propionate catabolism, where three genes contain a cluster. E. coli via selB, in Salmonella through cysN  is blocked in le 1 see  for detanlyase 2 ; by blocnarL in E. coli and narP in Salmonella. The two enzymes fulfil equivalent roles in respiration, both being involved in the (co-) regulation of a number of genes encoding oxidoreductases and dehydrogenases. The regulated oxidoreductases include a periplasmic nitrite reductase (nrfABCD) and two nitrate reductases (narGHJI and napFDAGHBC); the formate dehydrogenase-N (fdnGHI) is regulated by NarL [In both organisms, a nitrate/ nitrite response regulator is part of the GATC network:  by NarL -19.E. coli passes from a warm, nutrient rich environment, where it grows rapidly, to a cold, nutrient poor environment, where growth might be expected to be considerably slower, if not completely arrested. This process occurs naturally, when the cells pass from the intestine of their warm blooded host to the external environment. When growing rapidly, the DNA is undermethylated [According to our hypothesis, the GATC network comes into play, when thylated ,10 and pthylated .We hypothesize that when the bacterium undergoes coldshock, the transcription of genes containing a GATC cluster will be blocked at the level of the cluster because of the high stability of the hemi-methylated DNA.We will now draw the logical consequences of this hypothesis: if a gene contains a GATC cluster, its transcription will be blocked when the bacterium undergoes coldshock and the product the gene codes for will no longer be formed, affecting the biological process it is involved in.We will apply this principle systematically to the genes belonging to the three functional classes characterizing the GATC network (Fatty acid/ Phospholipid metabolism, Nucleotide metabolism and Energy metabolism), in order to try answering the following question: is there one and the same selection pressure that can explain why these three particular classes are affected by the GATC network?Two main groups of genes can be distinguished: those involved in the synthesis of Nucleotides ("Nucleotide Synthesis", see column "Subclass", Table 1 see ) and thoThe halt of the DNA repair machinery, on the other hand, might have a different reason: when the cell passes from the intestine to the outside environment, it is halted in the middle of a rapid growth phase, which means that the DNA double helix will be open in certain regions and "loose ends" of newly formed DNA will be present (like, for example, Okazaki fragments). The DNA repair machinery might interpret these fragments as damaged pieces of DNA and proceed to its elimination . An immeAgain, we will look at two classes of genes, which are particularly interesting: those involved in respiration and those involved in the metabolism of succinate ("Respiration" and "Succinate", column "Subclass", Table 1 see ).E. coli has a modular character (see [The respiratory system of ter (see  for a co1) Substrate specific dehydrogenases which oxidize their substrates and feed electrons to the mobile quinone pool.2) Quinones, which deliver reducing equivalents to the terminal oxidoreductases.3) Terminal oxidoreductases which reduce the terminal electron acceptors.E. coli  at the level of the aminolevulinic acid (ALA) production. ALA is synthesized from glutamate through the action of GltX, whose corresponding gene, gltX, contains a GATC cluster (in both organism). Hemes play a fundamental role in the energy conserving electron transport chains and are also present as cofactors in a number of enzymes [dehydrogenases poxB, sdhABCD, fdnGHI, fdoGHI, fdhF and lldD and the oxidoreductases torCAD, torYz, nirB, nrfAB, cyoABCD, cydAB and cyxAB.The heme synthesis is blocked in  enzymes . Thus, inarL, containing a GATC cluster in E. coli  which codes for a nitrate / nitrite response regulator and is involved in the (co-) regulation of a number of genes encoding oxidoreductases and dehydrogenases. The oxidoreductases regulated include a periplasmic nitrite reductase (nrfABCD) and two nitrate reductases (narGHJI and napFDAGHBC); the formate dehydrogenase-N (fdnGHI) is regulated by NarL [Respiration is further affected through  by NarL -18.As discussed in the section above, the formate dehydrogenases require selenocysteine, whose incorporation is halted. We can thus expect formate to accumulate in the cell when it undergoes coldshock, an aspect discussed further below.glpA and lldD – and oxidoreductases – dmsA and nirB- are blocked in E. coli.Two more dehydrogenases – If we now look at the respiration as a whole, at all the respiratory enzymes affected by the clusters, directly or indirectly, the following picture emerges . Applying our hypothesis we come to the conclusion that genes involved in aerobic respiration are blocked and moreover, that almost the entire respiration, aerobic as well as anaerobic, is brought to a standstill.Interestingly, Nyström  points oE. coli cultures when entering stationary phase under different conditions; they found that cell viability depends on the conditions the cell was prior to entering the stationary phase. Cells entering the stationary phase aerobically showed a very high mortality. Cells entering the stationary phase anaerobically, though, showed a much higher viability. Seen from this perspective it could be that what we predict to happen through the GATC regulation is the "attempt" of the cell to stay in an "anaerobic mode": the bacterium does not, for example, start producing enzyme complexes needed for aerobic respiration. Blocking respiration (when entering stationary phase) might be a preventive measure because all respiration, in the presence of oxygen, is a potential danger as it can lead to the production of free radicals and peroxide.A possible explanation for this can be found in the work of Dukan & Nyström . They poSalmonella, a recently discovered pathway leading from succinate to propionate is also blocked through ygfH  is not affected by GATC clusters except for the last step, leading from succinate to succinyl CoA. Thus, we expect succinate to accumulate during coldshock. (In le 1 see ), also s E. coli  might giacpS and ilvD  is present in relative abundance in the warm blooded hosts' intestine [We can make two observations regarding this group. The first is, that the synthesis of two enzymes which are essential for fatty acid synthesis are under GATC cluster control: the biosynthesis of ACP, the central coenzyme of fatty acid biosynthesis  is haltele 1 see ); furthesynthase ). The sentestine ,29, and So far we have stressed the importance of the physical property of hemi- or unmethylated GATC motifs, especially when present in clusters: the increased stability of the DNA, which comes into play when the ambient temperature of the bacterium is suddenly lowered.We hypothesize that this increased stability hinders transcription of the gene containing a GATC cluster when the cell undergoes coldshock and is likely to enter stationary phase; we have looked at the effect this might have on the cell.A question we might ask is that of the control mechanism: apart from the control exerted by the physical property of hemi- or unmethylated GATC motifs, is there something else that recognizes these motifs? The protein SeqA, for example, recognizes hemimethylated GATC sequences placed on the same face of the DNA double helix and it does so in a cooperative, histone-like manner, forming a homotetramer . It mighin silico and would require an intervention from the wet lab.This question, however, reaches beyond the abilities of the research E. coli and Salmonella possess a GATC cluster regulated network. The clusters are found within the coding sequences and their distribution is not at random. Three functional classes characterize the network: Nucleotide metabolism, Energy metabolism and Fatty acid / Phospholipid metabolism.We hypothesize that the network comes into play when the cell passes from the warm, nutrient rich environment of its warm blooded host's intestine to the external environment, i.e., when the cell undergoes coldshock and is likely to enter stationary phase.According to our theory, the transcription of genes containing a GATC cluster will be blocked at the level of the cluster when the bacterium undergoes coldshock and the product the gene codes for will no longer be formed, affecting the biological process it is involved in.We have applied this principle to the three functional classes that characterize the GATC network and come to the conclusion that respiration is almost completely under GATC control and 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 also 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.The procedure has been described elsewhere , here a We have taken a classic genomic approach, analyzing the statistical distribution of GATC along the chromosome, using a realistic model of the chromosome as theoretical reference. We thus identify local enrichments, or clusters, in the real chromosome. A GATC cluster is identified in the following manner: GATC pairs separated by less than 8 bp and triplets in regions spanning less than 62 bp are kept in a preliminary screening. In a second step we retain only those regions where there are at least four GATC motifs and where the average distance between pairs is shorter than 18 bp.E. coli is not a statistical artefact, but has a physiological role, we have carried out the same analysis on Salmonella, making the hypothesis that the genes containing a GATC clusters should be largely the same in the two bacteria. This has been indeed observed, showing that the genes containing a GATC cluster are part of a regulation networkWe confirm the presence of GATC clusters within the genes. In order to verify that the particular distribution observed in E. coli (76) and for Salmonella (57 and three pseudo genes) containing GATC clusters, displayed in Table 1 [see We thus obtain a list of genes for le 1 see . With thle 1 see . For cerle 1 see  for detaE. coli's GATC network : the ORF number and #ID of the genes according to Oshima et al. [6]. 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 (in tomatoes, in this case) 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 (brix × yield [BY], measured in g/m2); 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 (yield [Y], measured in kg/mx genotype interactions. IL7-5-5 was dominant in its effect on Y, as both the homozygous IL and heterozygous ILH increased Y in the wet fields by 30% compared to M82 and by 12% to 22% (nonsignificant) 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 (−55% and −34% for the wet and dry treatments, respectively), but the ILH increased Y by 45% and 25% (wet and dry, respectively). 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 (homozygous for IL7-5-5, IL8-3, and IL9-2-5). 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 (CT252 for IL7-5-5, CT148 for IL8-3, and GP263 for IL9-2-5). 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 (SAS Institute, Cary, North Carolina, United States). 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 (see table On average, 43.2% (SD 18.5%) 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 [E. coli can be the superior expression system compared to eukaryotic expression systems when the activity of the recombinant protein can be regained through refolding from the produced inclusion bodies. However, one needs to consider that the decision to select a specific expression system frequently is based on more trivial reasons such as staff knowledge and available equipment and facilities of the producing company/institute.Though, producing an inactive target protein in the form of inclusion bodies is an important drawback, it also has several advantages such as the high degree of purity of the target protein in the aggregate fraction and the increased protection from proteolytic degradation compared to the soluble counterpart. Inclusion bodies have long been considered completely inert towards deposits . AlthougE. coli in the form of inclusion bodies while the mammalian expression system yielded an active protein that was secreted into the culture medium. More recently, obtaining active tPA through secretion into the periplasm of E. coli was attempted [E. coli secretion system as a future potential alternative route to generate functional tPA. Other recombinant organisms such as yeast [A good example to demonstrate the diverse routes that can be used for recombinant protein production is the manufacturing of tissue-type plasminogen activator (tPA). This protein enables the dissolution of blood clots and is used therapeutically for the treatment of myocardial infarction, thrombosis, pulmonary embolism, and strokes. To assure sufficient tPA for such a widespread application, an economic production process is a necessity. From the beginning, both the mammalian as well as the microbial route were explored for the production of tPA . tPA is ttempted -5. The ettempted ,7 renderas yeast , fungi [as yeast  or insecas yeast  have notE. coli produced material was a process with a poor overall yield [®) is still produced using a mammalian expression system (Genentech: ). In addition, an amino substituted tPA produced by the mammalian expression system with increased half-life (tenecteplase) was developed. Alternatively, a non-glycosylated, truncated tPA (reteplase, Retavase®) produced in E. coli in form of inclusion bodies and afterwards refolded to its biologically active form is now on the market (Centocor: ) and apparently gains market share at the cost of the mammalian-derived product(s) (see Genentech 2004 First Quarter Report).Initially, the recombinant tPA introduced into the market was obtained from genetically engineered mammalian cells . At thatll yield . Today, E. coli produced inclusion bodies is to increase both the allowed protein concentrations during the refolding process and the final refolding yield. Recent advances in this area are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. Moreover, the different techniques are discussed regarding their applicability for large-scale production processes or high-throughput screening procedures.Thus, continuous research effort focused on developing new refolding techniques or improving existing ones by including novel refolding aiding agents can make the bacterial inclusion body system an excellent alternative to the mammalian expression system or other expression systems that can directly generate active proteins with a complex disulfide bond structure. The foremost aim in improving protein refolding from A high degree of purification of the recombinant protein can be achieved by inclusion body isolation [for recent reviews on various aspects of inclusion body formation and renaturation of inclusion body proteins please refer also to -18]. 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 (χ2, p = 0.128).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.