Hepatitis A virus (HAV) is a causative agent of acute hepatitis, which is transmitted by person-to-person contact and via the faecal-oral route. Acute HAV infection is usually confirmed by anti-HAV IgM detection. In order to detect anti-HAV IgM in the serum of patients infected with HAV, we developed a rapid assay based on immunochromatography (ICA) and evaluated the sensitivity of this assay by comparing it with a commercial microparticle enzyme immunoassay (MEIA) that is widely used for serological diagnosis.The newly developed ICA showed 100% sensitivity and specificity when used to test 150 anti-HAV IgM-positive sera collected from infected patients and 75 negative sera from healthy subjects. Also, the sensitivity of ICA is about 10 times higher than MEIA used in this study by determining end point to detect independent on infected genotype of HAV. In addition, the ICA was able to detect 1 positive sample from among 50 sera from acute hepatitis patients that had tested negative for anti-HAV IgM using the MEIA.Conclusively, ICA for the detection of anti-HAV IgM will be very effective for rapid assay to apply clinical diagnosis and epidemiological investigation on epidemics due to the simplicity, rapidity and specificity. Hepatitis A virus (HAV) is one of the common causative agents of acute hepatitis worldwide . The cliThe incubation period for HAV is from 2 to 6 weeks . Anti-HAThe anti-HAV IgG antibody increases gradually, reaching high levels during the convalescent phase  and remaIn this study, we developed an immunochromatographic assay for the detection of anti-HAV IgM and applied this to clinical specimens in order to determine the sensitivity and usefulness of this assay compared with a chemiluminescent-linked immunoassay that is widely used for the routine diagnosis of HAV infection.By using the ICA devised in this investigation, we were able detect anti-HAV IgM in 150 sera from patients with acute viral hepatitis (confirmed as recent HAV infections), who were infected by 1 of 3 different HAV genotypes. Further, anti-HAV IgM was not detected in 75 sera collected from normal subjects. In the testing of these sera, the ICA showed 100% sensitivity and specificity . The sensitivity and specificity for anti-HAV IgM were both 100% in each of the 2 assays, as determined by the assay of 150 positive and 75 negative samples. However, among the 50 serum samples shown to be negative for anti-HAV IgM using the commercial MEIA, 1 sample was confirmed positive for anti-HAV IgM using the newly developed ICA. Although this specimen was considered to be negative based on optical density readings using the reagents supplied by the manufacturer, it has significant amount anti-HAV IgM less than cut off index (COI) (COI < 1.0) by MEIA. This suggests that the inconsistency between the 2 assays is attributable to their respective sensitivities.Surprisingly, although we used only 5 μl of sera for the ICA, the sensitivity of the ICA was approximately 10 times higher than that of the MEIA used in this study based on a determination of the end-point detection limit independent of HAV genotype.Because of the long incubation periods and late appearance of anti-IgM after HAV infection, timely medical care for patients with acute hepatitis and an effective control strategy during HAV-related epidemics in the community tend to be delayed . EspeciaAlthough the MEIA using an automated system for general serological diagnosis can provide sensitive results compared other serologic assays, including the ELISA and rapid immunoassays reported previously, it involves the use of expensive equipment, and can be costly and relatively time-consuming when handling large sample sizes, as in an epidemic.Therefore, many investigators have reported rapid serological assays for the detection of anti-HAV antibodies. One such assay is the dot immunogold filtration assay (DIGFA) for the detection of IgM antibody . This asIn our study, the sensitivity of the ICA for anti-HAV IgM was demonstrated to be higher than that of the MEIA. In addition, performance of the ICA requires only a simple test kit without other detection equipment; therefore, the assay does not need expensive laboratory equipment or large amounts of space, as does DIGFA.Synthetic peptides have also been used for the detection of HAV antibodies using biosensor technology based on surface plasmon resonance . Using tEven though this assay is in the developmental stage, biosensors can be more rapid than EIAs when they are fully automated and have potential clinical value; however, their expense may prove prohibitive . In contHowever, although there was no significant difference in the specificities of the ICA and MEIA, the sensitivity of the ICA for HAV in sera was higher than that of the MEIA based on the end-point detection of antibody and the ability to detect anti-HAV IgM in samples that test negative using the MEIA. Owing to its rapidity and specificity, the ICA can be used as an effective assay for screening HAV infection. It takes less than 20 minutes to complete the assay, whereas the MEIA takes approximately 30 minutes. The shorter reaction time in the ICA does not influence its sensitivity and specificity. There is, however, a significant difference in the cost of the ICA and AxSYM HAVAB-M 2.0 systems. When assaying using the AxSYM HAVAB-M 2.0, expensive laboratory equipment, several regents, reagent vessels, matrix cells, and a sample cup are required. In addition, the AxSYM HAVAB-M 2.0 requires a certain amount of space for the necessary laboratory equipment. However, when assaying using the ICA developed in this study, we need only a simple test kit and a single reagent for diffusion. This means that the cost of detecting anti-HAV IgM using the ICA is considerably less than when using the AxSYM HAVAB-M 2.0.In conclusion, ICA can detect anti-HAV IgM effectively and use of this system without the help of extra apparatus enables to detect and screen HAV infection easily and rapidly, which can be widely used in an epidemiological survey.All samples were obtained with informed consent and approval of the KCDC Institutional Review Board (IRB). One hundred and fifty serum samples were collected from patients with acute hepatitis who had HAV infection confirmed by serological diagnostic methods and molecular analysis for the determination of the infecting HAV genotype. Among the 150 sera, we identified 70 (46.7%) as being infected with genotype IA, 77 (51.3%) infected with IIIA, and 3 (2.0%) infected with IB (data not shown). As negative controls, 75 sera were also obtained from healthy individuals confirmed as having no recent HAV infection. Additionally, 50 serum samples were collected from patients with illness compatible with HAV infection but in whom the anti-HAV IgM had not been detected. We stored serum specimens at -70°C until use, and for the serological assays we used 5 μl of serum for the ICA and 150 μl of serum for the MEIA.The ICA is designed for the detection of anti-HAV IgM in human serum or plasma. The ICA device has 2 precoated lines--"T" (an HAV IgM line coated with anti-human IgM) and "C" (a control line coated with goat anti-mouse IgG)--on the surface of the membrane. Purified HAV antigens and colloidal gold-conjugated HAV-specific monoclonal antibody were dried on a pad. The assay, based on immunochromatography, for the detection of anti-HAV IgM was performed strictly according to instructions. Briefly, 5 μl of serum or plasma drawn to black line into the square sample well marked "S" for sample inlet. Four drops of diluent containing 0.1 M phosphate buffer, 0.1% Tween 20, and 0.01% sodium azide was then introduced into the sample inlet and the test results were interpreted within 20 minutes.The results were interpreted as follows. The control line should always appear in order to obtain a valid result for diagnosis--this indicates that the procedure has been performed correctly. If a purple "T" line is visible in the result window, a positive result is confirmed--this indicates that level of the anti-HAV IgM in the sample is above the detection limit. Otherwise, we confirm a negative result.AxSYM HAVAB-M 2.0  is based on microparticle enzyme immunoassay technology. The samples and all the AxSYM HAVAB-M 2.0 reagents required for one test were pipetted with the sampling probe into wells in the reaction vessel of the sampling centre. The reaction vessel was immediately transferred to the processing centre. Further pipetting was conducted in the processing centre using the processing probe. All steps were performed automatically and the diagnostic results were reported immediately. The AxSYM HAVAB-M 2.0 system includes the following: an antibody to human IgM (goat)-coated microparticles in TRIS buffer containing protein stabilizers (0.02% solids), human hepatitis A virus in phosphate buffer containing protein stabilizers , an antibody to hepatitis A virus , alkaline phosphatase conjugate in TRIS buffer containing protein stabilizers (0.9 μg/mL), specimen diluent containing sodium chloride in TRIS buffer, sodium azide, and antimicrobial agents.-1 to 10-5. For a comparison of the detection limits, we performed both the ICA and MEIA using 5 serial dilutions of the 6 sera infected with HAV.We selected 3 sets of 6 sera with high titres of anti-HAV IgM, which were induced by infections with HAV of 3 different genotypes  identified through molecular genotyping. The sera were diluted with phosphate-buffered saline (PBS) to 10-fold serial dilutions ranging from 10The authors declare that they have no competing interests.HJL and HSJ performed much of the ICA and the MEIA, and drafted the manuscripts. BKC, MJJ, and JHK carried out development of the ICA. ANL and KRL tested anti-HAV IgM from patients with acute viral hepatitis, collected positive sera with anti-HAV IgM. DSC critically revised the manuscript and carried out the experiment design. All of the authors read and approved the final version of the manuscript.


Intrauterine devices are often accompanied by various complications, of which the uterine perforation constitutes the most dangerous one.We present a case of a 41-year-old woman complaining of right upper quadrant pain. She had an intrauterine device inserted 12 years earlier without regular follow-up. Abdominal plain X-ray revealed the intrauterine device trans-located into the right subdiaphragmal area. Abdominal ultrasound showed gallbladder stones without any other sonographic pathologic finding. Patient underwent simultaneous laparoscopic cholecystectomy and removal of the intrauterine device from the right subdiaphragmal area.Laparoscopy is an appropriate method for removal of intrauterine device translocated to the right subdiaphragmatic region. The use of laparoscopy enables close inspection of inner organs. Among the advantages of the laparoscopy are its use in trauma setting, blunt and/or penetrating trauma, acute abdomen and peritonitis ,2. Use oA 41-year-old Kosovan Albanian woman presented with right upper quadrant (RUQ) pain for six months. The past medical history revealed the insertion of an intra uterine device (IUD) 12 years ago without regular follow-up. A chest X-ray showed a radio opaque foreign body resembling IUD in the right subdiafragmal area  and 2. TSince the first reported laparoscopy in 1901 - “ventroscopy” and until now the indications for this method of examination has been constantly increasing ,5. DisloAs it has been already stated the dislodgment can bring the IUD in the surrounding organs or the omentum. There were reported cases of the dislodgment in small intestines, rectosigmoid colon, peritoneum, gallbladder, appendix, annexes, iliac vein as well as the omentum -20. By cDue to a potential inflammatory response and consequent obstruction and/or perforation, the majority of authors recommend the removal of the dislodged IUD laparoscopically. Laparotomy remains an option in cases were laparoscopy may not be successful ,20. DrivIn conclusion, we have found the laparoscopy as a method of choice for the removal of the dislodged IUD because of the patients’ comfort and minimal hospital stay.


Gene identification using linkage, association, or genome-wide expression is often underpowered. We propose that formal combination of information from multiple gene-identification approaches may lead to the identification of novel loci that are missed when only one form of information is available.Z-transform test (Stouffer's method).Firstly, we analyze the Genetic Analysis Workshop 16 Framingham Heart Study Problem 2 genome-wide association data for HDL-cholesterol using a "gene-centric" approach. Then we formally combine the association test results with genome-wide transcriptional profiling data for high-density lipoprotein cholesterol (HDL-C), from the San Antonio Family Heart Study, using a We identified 39 genes by the joint test at a conservative 1% false-discovery rate, including 9 from the significant gene-based association test and 23 whose expression was significantly correlated with HDL-C. Seven genes identified as significant in the joint test were not independently identified by either the association or expression tests.This combined approach has increased power and leads to the direct nomination of novel candidate genes likely to be involved in the determination of HDL-C levels. Such information can then be used as justification for a more exhaustive search for functional sequence variation within the nominated genes. We anticipate that this type of analysis will improve our speed of identification of regulatory genes causally involved in disease risk. The ultimate goal of genetic studies of complex diseases is the identification of the genes that are causally involved in risk. Genome-wide association studies (GWAS)  attempt to identify genomic regions that are likely to harbor functional sequence variants influencing disease risk. For linkage studies, the size of the putative target region is on the order of 10 Mb while GWAS generally identify much smaller genomic regions of 500 kb to 1 Mb. Once such a region is found, the critical goal must then be to identify the causal gene(s) involved and their functional variants. In this paper, we propose an approach that leads to the direct nomination of empirically chosen positional candidate genes using independent transcriptional and genetic information. Once nominated, such candidate genes should be examined exhaustively to determine their causal status.In a "successful" genome-wide association study, the result is localization of a genomic region; actual identification of causally involved genes requires substantially more information. Therefore, joint utilization of multiple sources of independent information  is ultimately required to enhance inference about causal relationships. Because genes (or other contiguous genomics regions) remain the primary functional units of the human genome, we focus on gene-based tests of genetic, transcriptional, or proteomic data to determine whether a given gene is likely to be involved in the determination of a complex disease-related phenotype. This gene-centric approach allows replication studies to be focused on genes rather than variants. This analysis approach for GWAS data has been suggested as best practice , howeverGene expression measurements reflect quantitative variation in transcript-specific mRNA levels and thus represent phenotypes lying very close to the direct action of genes. By globally searching for gene transcripts having levels that correlate with more classical measures of disease related phenotypes, it should be possible to nominate or prioritize novel candidate genes for more extensive genetic analysis. Combining such transcriptional information with the results of GWAS should provide a powerful approach for the selection of disease-related genes.As an example of our gene-centric approach, we focus on the dissection of genetic determinants influencing high-density lipoprotein cholesterol (HDL-C) levels, an important endophenotype inversely related to risk of cardiovascular disease. Age, sex, and their interactions as well as smoking status were included as covariates in all analyses.n = 6852) plus the minimal set of untyped individuals required to maintain familial relationships. The trimmed pedigree consists of 12,789 subjects in 1059 extended families.For the genetic component of our tests, we utilized the genome-wide association information available in the Genetic Analysis Workshop 16 Problem 2 single-nucleotide polymorphism (SNP) data obtained from the long-running Framingham Heart Study (FHS)  to incluGenotyping was performed by Affymetrix for approximately 550,000 SNPs. The genotypes were cleaned for mendelian errors using an automated procedure in which a mistyping analysis is conducted with the computer program SimWalk2 ,5 and thFor each cohort fasting HDL-C data were taken from the first visit for which they were available. HDL-C measures were blanked for individuals using cholesterol-lowering drugs.p-value for the best marginal SNP. The measured-genotype analysis [p-values against the effective number of SNPs using corrected = 1 -effective, where corrected is the corrected p-value, nominal is the uncorrected p-value, and effective is the effective number of SNPs. Our approach explicitly allows for non-independence among family members and the effects of other potential covariates.To quantify the evidence for a given gene influencing HDL-C levels we employed an omnibus gene-based test of association. We defined the physical location of each gene (extended by 25 kb in either direction) and then selected the Problem 2 SNPs within each region. We calculated the effective number of SNPs within a gene region using the method of Li and Ji  as impleanalysis  was condA given SNP may fall into the focal bin of more than one gene/transcript. This has the potential of inducing some positive correlation among test statistics for nearby genes. However, our reliance on the false-discovery rate (FDR) approach, with its known robustness in the presence of such positive non-independence, ameliorates this potential problem .2 'tail' test was to assess whether there was a significant excess of samples with transcript-specific expression values above the 95th percentile of the null distribution based on manufacturer-provided negative control samples. This allowed the detection of even those RNA molecules that are clearly present above baseline levels in some individuals. We identified a total of 22,413 transcripts with significantly detectable expression levels [The expression analysis was conducted as part of the San Antonio Family Heart Study, initiated in 1992 to investigate the genetics of cardiovascular disease and its risk factors in Mexican Americans . The expn levels . Using aZ-transform test also known as 'Stouffer's method" [p-values to Z-scores using an inverse normal transformation. The Z-scores are summed and then scaled by the square root of the number of combined tests. The resulting test statistic is distributed as a standard normal variate that is then transformed back to a combined overall p-value. This omnibus test is not dependent upon the distribution of the data but depends only upon the expected uniform distribution of p-values under the null hypothesis.We then simultaneously utilized our transcriptional and genetic information by the application of a joint gene-based test that takes into account the evidence for a phenotypic relationship between HDL-C levels and a gene's expression and the strength of the association between SNPs in (or near) the gene with HDL-C levels. We combine these two tests using a  method" . The metFor each cohort, the first HDL-C measure was included in the phenotype file, along with age at exam and smoking status. HDL-C measures were blanked for individuals using cholesterol-lowering drugs. In total there were 6334 individuals with both HDL-C measures and genotype data, with HDL-C measures ranging from 16 to 206 (mean 53.6 ± 0.2). Within our analysis dataset, age at exam ranged from 5 to 72 (mean 38.3 ± 0.1). There were 6301 individuals with data on both HDL-C and age and 6152 individuals with both HDL-C and smoking status.The genes considered in this investigation were those corresponding to the 22,413 transcripts identified in the expression profiling. Of these, there were 17,350 gene regions with a least one effective SNP located within a 25-kb extension of either side of the physical gene location (NCBI build 36.3). SNP counts ranged from 1 to 597, with an average of 21 ± 1 SNPs per gene region. The 25-kb extension of the boundaries was selected to maximize the number of SNPs that may influence the target gene while minimizing the number of overlapping; this parameter is investigator-driven and can be adjusted as required.p-value for the effective number of SNPs within the region, at a 1% FDR. These results are shown in Table Of the 17,350 gene regions tested, 14 were significantly associated with HDL-C from the measured genotype analysis, following correction of the ABCG1, C12orf62, C6orf64, GPBAR1, LOC283551, LYRM1, and PRPF38A). The results of the joint test are shown in Additional File In the joint test there were a total of 39 genes significant at a highly conservative 1% FDR, including 9 from the significant measured genotype set and 23 with expression that was significantly correlated with HDL-C. Seven genes identified as significant in the joint test were not identified by either the association or expression tests independently (CETP (cholesteryl ester transfer protein), a well known cholesterol binding gene. In total, there are seven well known lipid metabolism genes prioritized by the joint test . Interestingly, the list also prioritizes a number of genes of little-known function, such as NLRC5 (NLR family CARD domain containing 5), TCTN1 (tectonic family member 1), and TPPP3 (tubulin polymerization-promoting protein family member 3), which would not be selected by any form of candidate gene approach.The genes shown in Table 2 are prime candidates for resequencing and variant typing, empirically selected based on evidence both from transcriptional profiling and genome-wide association. One of the most significant genes is IL1R2 (Table 2). Similarly, there are cases (SMURF1) where the association information drives the combined tests. We have retained all genes that exhibit combined significance. An individual reader may choose to further focus on only those genes that exhibit at least nominal significance on each dimension.It can also be seen that there are situations in which genes show a highly significant correlation between their expression and HDL-C, but no evidence of association at the physical location of the gene, such as While this approach shows great potential for speeding gene identification, it also has several limitations. One potential weakness is the focus on regulatory variation. While there is a growing belief that much of quantitative phenotypic variation may stem from regulatory variation, other types of mechanisms  can also be involved. Similarly, genes whose expression is not detected in the target tissue may be missed. Thus, as with all discovery-based approaches, only positive findings admit interpretation. A gene cannot be ruled out using these methods.This paper combines information from two different population studies. Both samples, however, are ascertained without regard to phenotype. It is possible that the relationship between expression levels and disease-related phenotypes may vary across populations. However, we would expect this to diminish signal rather than yield false positives. Optimally, expression and association results would come from the same data set.p-values from two separate tests, both of which are tests of a gene-centric hypothesis. Of course, this approach can be made substantially more powerful when both forms of information are available in a single study and a formal, true joint test is specified. In the current application, our results empirically nominate genes that are likely to be directly involved in quantitative HDL-C variation. Many of these genes would not have been identified using a classical pathways-based combinatorial approach because their functions have yet to be identified. Many would also not have been identified by using each approach in isolation. A logical next step would be either replication or, given the magnitude of current evidence, a direct move to resequencing to identify functional variants.Our results suggest that the formal combination of information from orthogonal sources may lead to the identification of novel loci that are missed when only one form of information is available. For the current study, we have combined existing information on the correlation of gene expression levels with HDL-C and the association between SNPs near these genes with HDL-C levels. Our simple measure of evidence is effectively a measure of significance resulting from the combination of FDR: False-discovery rate; FHS: Framingham Heart Study; GAW16: Genetic Analysis Workshop 16; GWAS: Genome-wide association study; HDL-C: High-density lipoprotein cholesterol; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.JCC and JB wrote the paper. JCC, JMP, ED, HHHG, and TDD processed and analyzed the data. JB conceived the methodology and fundamental structure of the project. JCC, HHHG, and LA supported the conception and design of the project.The 39 significant joint-test results for HDL-C significant at a 1% FDR.Click here for file


Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm  Flatworms (phylum Platyhelminthes) have long been considered the most basal bilaterians, and they have served as models for the bilaterian ancestor in a variety of phylogenetic hypotheses. Generally, morphological data place the Acoela within the Platyhelminthes based on a combination of weak characters: an acoelomate body structure, a densely multiciliated monolayered epidermis leading to a common habitus, a frontal organ, neoblasts, hermaphroditic reproduction with similar reproductive-organ morphology, biflagellate sperms with inverted axonemes (in acoels and rhabditophorans except macrostomorphans), and lack of hindgut and anus We have succeeded in finding two strong synapomorphies between acoel and rhabditophoran flatworms. The stem cell system and the particular mode of replacing epidermal cells represent unique features shared by both acoel and rhabditophoran flatworms, but not by any other bilaterian lineage. At the same time, our phylogenomic data support a separation of acoels from rhabditophoran flatworms.Aurelia aurita and Nematostella vectensis, the sponge Ephydatia fluviatilis, the acoel Isodiametra pulchra, the flatworm Macrostomum lignano, and the annelid Platynereis dumerilii. Applying a phylogenomic approach on the basis of 10,218 amino acid positions of the acoel Isodiametra pulchra, we first identified a set of open reading frames homologous to sequences we generated from major animal taxa or which were represented in public databases. To avoid the use of paralogs, we limited our selection of genes to e-values ≤10−50 in blast searches. We subjected the resulting multi-locus datasets to phylogenetic analyses using Maximum likelihood and Bayesian inference and analyzed two datasets consisting of 23 species represented by 43 loci (Macrostomum lignano), representing diverse animal phyla and including partial sequence data  and gene-expression patterns of the stem-cell marker em cells . HoweverInterestingly, two previous multi-gene phylogenies show some support for the grouping of acoels with other flatworms: “the standard WAG model groups together the two long branches of Platyhelminthes and the acoel” A number of phylogenetic studies based on single molecules have suggested a more basal position of Acoela and Nemertodermatida On the other hand, totipotency of mesodermally located neoblasts in adult animals could be a character uniting all Platyhelminthes, but so far has only been experimentally tested in the Rhabditophora If the similarity of the complex stem-cell system shared between acoels and rhabditophorans was to be explained as a plesiomorphy derived from a hypothetical urbilaterian , and theDiploblasts such as cnidarians lack a mesodermal layer, making a direct comparison with the mesodermally located stem cells in the triploblastic acoel and rhabditophoran flatworms difficult. Moreover, recent phylogenetic studies suggest a placozoan-like ancestor of bilaterians instead of a cnidarian (planula)-like one HydractiniaEpidermal cells are proliferating at least in some cnidarians, and interstitial cells (i-cells) are found and are proliferating in the epidermis between epidermal cells. This is in stark contrast to the neoblasts of acoel and rhabditophoran flatworms, which are entirely lacking in the epidermis. Still, considering the presence of totipotent i-cells in the cnidarian Regardless of whether the neoblast stem-cell system is a plesiomorphy or an apomorphy for acoels, the assumption of an independent development of a very similar stem-cell system in rhabditophorans indicates a similar need in these two taxa for such a peculiar stem-cell system.Only for the Neodermata , the lack of stem cells within the epidermis can be seen as a prerequisite for avoiding the host defense mechanisms by shedding the ciliated epidermis. During postembryonic development, these parasitic rhabditophoran flatworms completely replace their primary epidermis with a newly formed syncytial epidermal layer derived from mesodermally located stem cells piwi-like gene also in somatic, not only in gonadal stem cells. The alternative would be that the highly similar stem-cell system evolved in parallel in Acoela and Rhabditophora, or is a plesiomorphic feature that was retained.Considering the obvious conflict between molecular phylogenies and morphological data, the monophyly of the flatworms remains undecided. Although molecular phylogenies show a position of the Acoela separate from the remaining flatworms, the stem-cell system provides two strong synapomorphies for the Rhabditophora and the Acoela: 1) epidermal replacement exclusively through mesodermally located stem cells, and 2) expression of a Buddenbrockia has been identified as a member of the Cnidaria by molecular means despite striking morphological dissimilarity Buddenbrockia, no such accounting can explain the suite of morphological characters shared among Platyhelminthes Recently, the myxozoan worm Pseudostylochus intermedius) and protease incubation were used for different species, and treatment with 0.1 M HCl after protease incubation was omitted.BrdU labeling was performed according to M. lignano was carried out as described previously I. pulchra, the same protocol was used, except for Proteinase K treatment, which was applied for 7 min only. Sense and antisense riboprobes were generated using the DIG RNA labeling KIT SP6/T7 (Roche), following the manufacturer's protocol. During hybridization, riboprobes were used at a final concentration of 0.05 ng/µl. The following primer couples were used for generating in situ riboprobe templates: For Macpiwi, 5′-TGCTCAAGCTGGTGTTGCTGGTC-3′ and 5′-GTCTTGTTGTTGTGCCGCGTGAG-3′. For Ipiwi15′-CATGCTGGAGATGGGCAAGATCAC-3′ and 5′-GGTGCCGGAGATTTCATTGCTCTC-3. Partial sequences of piwi-like genes were obtained from the Macrostomum lignano EST database (Angu7606)  and unpublished Isodiametra pulchra ESTs (Contig 447) . Both gene sequences were submitted to genebank . Detailed information of both genes will be published separately.Whole mount in situ hybridization (ISH) on We used available EST (expressed sequence tags) and whole genome databases in order to obtain multi-locus data matrices for phylogenetic inferences see . The appIsodiametra pulchra cDNA-sequencing project . These sequences were used as query for tblastx homology searches against all other 23 databases , while the concatenated dataset without Macrostomum lignano (43 loci) had 10,218 amino acid positions. 4,903 positions of the alignment contained gaps in at least one of the taxa. Since the resulting missing data represent less than half of the combined sequence, we included these taxa in the phylogenetic analyses.Maximum likelihood and Bayesian analyses were performed with both datasets and with single loci as well as multi-gene alignments including all sequence fragments. The total length of the concatenated dataset including rotTest with all single-gene and multi-gene alignment files. According to the results obtained, we performed maximum-likelihood analysis and 100 maximum-likelihood bootstrap replicates with PHYML applying the WAG+Γ model (gamma shape parameter α = 0.77) of sequence evolution for the concatenated file including 32 loci, and the WAG+I+Γ model  for the multi-gene dataset containing 43 gene fragments.Prior to phylogenetic analyses, model selections with different model selection strategies  were performed with Pwww.lirmm.fr/mab, We also applied a mixed-model approach to both datasets using CAT, a previously developed model accounting for site-specific amino acid replacement patterns rotTest. The percentage fraction of single-gene trees containing a particular node is depicted on the branches in In order to test the phylogenetic signal and the contribution of each of the single loci to the general topology, we calculated the number of single-gene trees supporting a given partition of the general topology. To this end, maximum-likelihood trees were constructed from each single-gene alignment with PHYML, applying the model of sequence evolution and the respective parameters according to the model selection with PIsodiametra pulchra as most ancestral taxon, sister group to all remaining bilaterians (p>0.01) compared to trees in which the monophyly of the Platyhelminthes was enforced (Alternative topologies constraining monophyly of the Platyhelminthes were compared applying the approximately unbiased (AU) test as implemented in the CONSEL package, using the sidewise likelihood values estimated by PAML. For both datasets, the AU test revealed that the maximum likelihood phylogenies placing aterians , Fig S1,enforced .Figure S1I. pulchra appears as a sister group of the rest of the bilaterians, and not as a member of the platyhelminthes. The macrostomorphan M. lignano lies basal to other rhabditophoran flatworms . Numbers above nodes refer to the maximum likelihood boostraps. Values below nodes represent bootstrap support under CAT. Circled numbers indicate the percentage of individual-loci trees that supported the respective node in the maximum-likelihood analyses of each data-set separately.Tree of phylogenetic analysis of 24 species. Phylogenetic analysis of 24 species using partial sequences of 32 genes. The acoel (0.82 MB TIF)Click here for additional data file.Table S1Species used for the phylogenetic tree reconstruction.(0.05 MB DOC)Click here for additional data file.Table S2GenBank accession numbers. GenBank accession numbers of the sequences used for the phylogenetic analyses.(0.19 MB DOC)Click here for additional data file.Table S3Length of the sequences used. Length of the sequences used for the phylogenetic analyses.(0.03 MB DOC)Click here for additional data file.Table S4Testing the position of the acoel in the tree. Testing the position of the acoel Isodiametra pulchra in the phylogenetic tree.(0.04 MB DOC)Click here for additional data file.


The concentrations of retinol and beta carotene were measured in serum samples taken from 113 women with cervical cancer, 32 with invasive and 81 with pre-invasive disease, and compared with those from 226 age-matched control women. There was little difference in serum retinol levels between women with cancer of the cervix, at any stage, and the control women, after adjusting for potential confounding factors. Serum beta carotene concentrations were likewise similar in women with invasive disease and the controls. However mean beta carotene levels were significantly reduced in women with pre-invasive disease compared to the controls . This reduction was more evident amongst women with a diagnosis of carcinoma-in-situ (mean 213.1 micrograms l-1 than amongst those with severe dysplasia (mean 228.7 micrograms l-1. There is a negative trend between beta carotene and risk of pre-invasive disease which is of borderline significance. These data have also been used to investigate the effects of smoking and oral contraceptive usage on the serum levels of retinol and beta carotene. Both habits tend to increase retinol and decrease beta carotene concentrations.


Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins.Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA). We also identify a novel family of esterases  that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern.We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1) the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58), which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides.This article was reviewed by Gaspar Jekely and Frank Eisenhaber Eukaryotes display a rich complement of secreted and membrane-anchored cell-surface proteins, whose amino acid side-chains are subject to numerous post-translational modifications. These modifications include addition of extensive polysaccharide moieties to asparagine or serine/threonine side chains (N and O linked glycosylation respectively), sulfatation, hydroxylation and cross-linking. These modified surface proteins, together with other biopolymers such as polysaccharides, which might also be highly modified and cross-linked, constitute diverse organic matrices of eukaryotes . The matGiven the profound importance of such cell surface modifying enzymes to host-pathogen interactions, evasion of host-surveillance and developmental processes, we were interested in identifying novel families of such enzymes and computationally predicting their function to facilitate their future exploration by experimental means. In this study we used sensitive sequence and structure analysis methods to investigate eukaryotic proteins with a potential role in cell surface modifications and identified novel enzymes that contain different catalytic domains such as: 1) an esterase with an α/β fold similar to that of the GDSL/SGNH superfamily of esterases, albeit with somewhat distinct active sites; 2) membrane-associated acyltransferases; 3) ATP-grasp enzymes with potential peptide ligase activity. Based on the available genetic data, catalytic site configuration and contextual information we predict that these proteins catalyze previously uncharacterized modifications of surface proteins in various biological roles.Cryptococcus neoformans, which is required for the synthesis of O-acetylated glucuronoxylomannans (GXM), the primary capsular constituent of this fungus [Cryptococcus in animal hosts. Interestingly, homologs of this protein were also found in animals and plants suggesting that such modifications might have a more general function in cell-surface adhesion across eukaryotes. Evidence from Cryptococcus indicates that the multi-TM protein Cas1p is the acyltransferase that adds the acetyl groups to the O-6 position of the mannose residues of the backbone of the GXM [As part of an effort to uncover previously uncharacterized eukaryotic enzymes modifying secreted and cell-surface proteins we investigated the Cas1p protein of the pathogenic fungus s fungus . This ac the GXM . Hence,  the GXM . AdditioCryptococcus neoformans protein Cas1p . The search initially recovers known homologs of the Cas1p family from other eukaryotes. In the 2nd iteration it detects other multi-TM O-acyltransferases, like O-acyltransferase 3 of Clostridium cellulolyticum (gi: 220930016). A reciprocal search with the Clostridium protein retrieves the Cas1p transmembrane region in the 2nd iteration , along with numerous other O-acyltransferases of the classical acyltransferase superfamily that is found in both bacteria and eukaryotes. This superfamily includes proteins such as: 1) Staphylococcus OatA that carries out the O-6 acetylation of N-acetylmuramic acid in peptidoglycan to make it resistant to lysozyme [E.coli and Rhodobacter [Rhizobium-host interactions [Xanthomonas [rd helix; 4) a conserved basic residue (K or R) in 5th helix; 5) a conserved glycine in the 6th helix; 6) a second conserved histidine in the and 9th helix. Together, these are predicted to constitute a previously obscure intra-membrane active site that potentially acylates the sugar moieties in course of their delivery to the cell-surface. Thus, it is likely that the multi-TM domain of Cas1p and its homologs indeed catalyzes the acetylation of the mannosyl moieties of GXM through a mechanism comparable to the classical membrane-linked acyltransferases such as OatA, GumG or GumF.To identify the affinities of the multi-TM domain in Cas1p, we initiated PSI-BLAST searches of the non-redundant database (expect (e)-value for inclusion in threshold = .01, iterated to convergence) with this region from the thomonas ,11. A muAn investigation of the phyletic patterns and affinities of these enzymes suggests that in addition to the Cas1p family there are 3 other distinct 10TM acyltransferase groups in eukaryotes, which have all emerged through independent lateral transfers from bacteria where this superfamily shows an enormous radiation. Some of these are restricted to lineages such as nematodes or fungi while others like the Cas1p and Beltless families are more widespread in eukaryotes , the Arabidopsis thaliana pathogen resistance protein PMR5  and a group of plant freezing resistance/cold-acclimatization proteins typified by Arabidopsis thaliana ESKIMO1, the Caenorhabditis elegans protein D2030.8  and the animal FAM55D proteins  within 5 iterations. Reciprocal PSI-BLAST searches seeded with the newly recovered proteins, in addition to recovering all their orthologs, also recovered several proteins of the former group with significant e-values. A multiple sequence alignment of the predicted domain was prepared with all the members collected in the above searches using the KALIGN program. A search with the PSI-BLAST program of the non-redundant database using a PSSM derived from this alignment recovered the so called FAM113 proteins from animals. Inclusion of the FAM113 proteins in the profiles recovers members of a previously characterized large esterase superfamily known as the SGNH/GDSL superfamily (e~10-3) [1yzf) with highly significant p-values (p = 1.4 × 10-9). A combined secondary structure prediction with the Jpred program by using residue frequencies in columns, a HMM and a PSSM derived from the alignment of the segments homologous to the N-terminal region of Cas1p showed a conserved core of 5 strand-helix units -value for inclusion in threshold = .01) with this region from the human ortholog of Cas1p . In addition to recovering Cas1p orthologs, the search also detected numerous other eukaryotic proteins with significant expect (e)-values in course of successive iterations. For example, the search recovered the (e~10-3) . To furtPC-esterase family (after the Pmr5 and Cas1p proteins and its predicted esterase function) to highlight its differences from the canonical versions GDSL/SGNH superfamily and the fact that most of its members are not recognized as such by the profiles/HMMs of widely used protein alignment databases (e.g. PFAM/Interpro).The GDSL/SGNH superfamily contains numerous well-known esterases from bacteria and eukaryotes such as acyl-coA thioesterase, aryl esterase, platelet activating factor acetylesterase and acetylxylan esterase . The namNaegleria gruberi). At least five distinct subfamilies can be recognized within the PC-esterase family , including some of the early-branching forms such as chlorophyte algae. Given the evolutionary relationship to the larger GDSL/SGNH superfamily it is likely the PC-esterases branched off from them relatively early in eukaryotic evolution  and a few metazoan proteins (e.g. LOC580673 from Strongylocentrotus purpuratus). A profile-profile comparison with the HHpred program significantly recovered the cadherin domain and a comparison of the predicted structure of this domain with the crystal structure of the cadherin showed a congruent seven stranded secondary structure. This suggests that the group of domains prototyped by the second globular N-terminal domain of C7orf58 defines a new family of beta-sandwich domains with a cadherin-like fold. The domain is widespread in bacteria and seen in the firmicutes, actinobacteria, certain proteobacteria, bacteroides and chlamydiae with a notable expansion in Clostridium. In contrast, it is limited in eukaryotes thus far to metazoa and chlorophyte algae, suggesting that it was derived through lateral transfer from bacteria. In prokaryotes, this cadherin-like domain is widely fused to other domains such as FNIII (Fibronectin Type III), TIG, SLH (S-layer homology), discoidin, cell-wall-binding repeat domain and alpha-amylase-like glycohydrolases  recovered several bacterial proteins  Agency for Science, Technology and Research, SingaporeAlthough the theory of sequence homology is general, our sequence-statistical criteria have only been proven for globular domains. Essentially, the conservation of an apparently random hydrophobic pattern that is necessary for composing the hydrophobic core of the globular structure serves as a strong criterion for fold conservation and this is the reason for a high score. For various non-globular segments that have amino acid compositional bias or simple repetitive patterns, sequence similarity does not necessarily mean homology. It is not rare that PSI-BLAST collects groups of proteins that just have transmembrane regions in common, especially if the inclusion cutoff is quite generous as in this work. This is relevant for the 10TM sugar acyltransferase family collection. Are there other arguments for homology than just the conservation of a few functional residues that are hypothesized to form an intramembrane active site? Are there observations of finding irrelevant proteins in the hit lists?1. polar rather than just hydrophobic residues . 3) For at least 4-5 iterations these searches remained "clean" in recovering only known acyltransferases and their homologs. These observations, together the functional congruence between the TM domains of the Cas1p family and the classical acyltransferases, favor homology as the explanation for the detected similarity.Response: It is true that compositional bias can cause recovery of similarly biased non-homologous proteins in profile searches, including in the case of TM proteins. Searches with TM proteins should be examined on a case-by-case basis to rule out spurious relationships. In this case there are number of factors that favor the reported relationship to TM acyltransferases: 1) the similarity consistently extends across 10 TM domains rather than just a few TM segments as is usually seen in the case of spurious hits between membrane proteins. 2) The intra-membrane conservation patterns include a unique set of You write that most PC-esterases have at least a pair of conserved cysteines; "most" being a not very scientific determination. Can this conclusion be supported with quantification?2. Response: Four of the five subfamilies of PC-esterase domains contain these pairs of the cysteines.Gaspar Jekely, European Molecular Biology Laboratory, Heidelberg, GermanyI approve publication of the manuscript. I don't have any major comments, it is a nice work.Material and methods, comprehensive alignments of the 10TM Acytransferase and Cadherin-like domains and a complete list of families are provided.Click here for file


Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol.p-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues.Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence, the present findings should strengthen the notion that modulation of brain phosphatidylinositide signaling probably contributes to the molecular mechanism of diverse antidepressant medications. Neither the pathophysiology of depression nor the mechanism of action of various antidepressant agents is fully understood. Accumulating evidence implicates brain phospholipid metabolism in the actions of diverse antidepressant drugs -3. For e2 serotonin receptors inhibited the effects of antidepressant drugs on inositol phosphate accumulation, but the drug effects on CDP-diacylglycerol production or phosphoinositide synthesis were not substantially reduced by 5HT2 antagonist treatment. These observations suggest that the drug effects on CDP-diacylglycerol and phosphatidylinositide synthesis involve a mechanism that may not depend on increased synaptic serotonin action. Clarifying such a mechanism would increase understanding of depression pathology, and could lead to the development of new and better treatment strategies.CDP-diacylglycerol is a crucial intermediate in the synthesis of phosphatidylinositol and related signaling mediators. Enhanced production of CDP-diacylglycerol can be expected to lead to increased synthesis of phosphatidylinositides. Hence, blood platelets incubated with different antidepressants show higher levels of phosphatidylinositides compared to control platelets, and this newly synthesized pool of phosphatidylinositides could be further available for receptor-coupled cell signaling ,5. AnothThe present study was directed at testing the hypothesis that the nucleolipid effects of antidepressants on CDP-diacylglycerol and its derived inositol phospholipids are substantially independent of the known effects of the drugs to enhance synaptic serotonin levels. We evaluated the neurolipid effects of a selection of drugs in tissues depleted of serotonin, and in neuron-like PC12 cells that are deficient in receptors and transporters for serotonin or norepinephrine . Upon obMale inbred C57BL/6 mice weighing 25-30 g were purchased from Harlan  and housed 5 per cage. Male Sprague-Dawley rats weighing 225-250 g were purchased from Zivic Laboratories  and housed three per cage. Animals were kept for at least three days before use and housing was on a 12-hour light/dark cycle in climate-controlled facilities. Food and water were freely accessible to each cage of animals. Protocols for the care and use of the animals were approved by the Institutional Animal Care and Use Committee and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Buffer reagents and all drugs used were purchased from Sigma-Aldrich . For the neurolipid (CDP-diacylglycerol and phosphatidylinositide) assays and the enzyme activity experiments, test drugs were dissolved in HEPES bicarbonate (HB) buffer, while for the cell culture experiments the drugs were dissolved in phosphate buffered saline. Drugs were prepared fresh before use.To deplete rat brain tissues of serotonin content, the animals were administered the tryptophan hydroxylase inhibitor, p-chlorophenylalanine (PCPA) at the dosage of 150 mg/kg daily for three days, followed by a 12-h washout period prior to testing. Control animals received injections of saline. This regimen of PCPA is known to result in depletion of 85-95% of forebrain serotonin content without affecting the catecholamines -9.3H]cytidine , aliquots of slices from each pretreatment group were incubated with various concentrations of selected antidepressant agents for 90 min, and accumulated [3H]CDP-diacylglycerol measured as previously described cytidine  for 30 min to generate a pool of radiolabeled cytidine triphosphate (CTP). After addition of 5 mM LiCl, solutions of test drugs were added to the cells at indicated concentrations and incubation continued for 3 h. To terminate the reaction, 1.5 ml of chloroform-methanol-1 M HCl (100:200:1) was added with mixing. The lipids were extracted by partitioning to the chloroform layer as described [3H]CDP-diacylglycerol as characterized in previous studies inositol . Subsequent drug treatments were maintained for an incubation period of 3 h, followed by extraction and quantitation of the inositol phospholipids as described CTP  in a 50-μl reaction mixture that contained 0.1 M Tris-HCl pH 7.5, 0.2 M KCl, 1 mg/ml bovine serum albumin, 0.1% Triton X-100, 0.25 mM dithiothreitol, and 200 μM 1,2-dioleoyl-sn-glycero-3-phosphate. The reaction was started by the addition of 10 mM MgCl2 and stopped after 20 min by the addition of chloroform: methanol: 10 M HCl . After incubating at room temperature for 30 min to extract formed [3H]CDP-diacylglycerol, the amount of the incorporated radioactivity was determined by liquid scintillation counting. Assayed enzyme activity was linear with time up to at least 30 min and with protein concentration up to at least 50 μg. One unit of CDP-diacylglycerol synthase activity was defined as the amount of the enzyme that catalyzed the formation of one nanomole of CDP-diacylglycerol per minute.CDP-diacylglycerol synthase activity was assayed in mouse brain membrane preparations according to previously described methods with minor modifications -16. MiceNeurolipid data were calculated as dpm/mg protein. Each set of data was normalized by taking percentages relative to control incubations that excluded drug treatments. Data from two or three separate runs of the experiments were then pooled for statistical analysis and graphical representation as shown. Similarly, enzyme activity data were normalized against each day and tissue controls, and the data pooled for analysis. Statistical analyses were by One-Way ANOVA followed with posthoc Tukey tests to compare all possible pairs of group means or the Dunnett test to compare each treatment mean to its respective control group as indicated in the figure legends. Statistical comparisons were considered significant at p < 0.05 or better.3H]cytidine and then incubated with maximally effective concentrations of the antidepressant drugs fluoxetine , imipramine , or maprotiline ; these dose selections were based on previously published studies with brain slice preparations cytidine were incubated with different concentrations of antidepressant drugs ranging from 0.1 μM to 300 μM and the accumulation of CDP-diacylglycerol assayed after 3 h. The results are shown in Fig. posthoc Dunnett tests, significant effects were obtained at concentrations of 30 μM and higher.PC12 cells prelabeled with [3H]inositol and aliquots tested with the drugs. Maprotiline and imipramine significantly and dose-dependently increased phosphatidylinositide levels in PC12 cells  or 30 μM (paroxetine and maprotiline). Thus, drug stimulation of CDS activity may underlie the positive drug effects on CDP-diacylglycerol synthesis.The enzyme activity of CDP-diacylglycerol synthase (CDS) was determined by measuring the rate of incorporation of tritiated CTP into CDP-diacylglycerol, and the results are shown in Fig. The classic action of antidepressants is to inhibit the reuptake of monoamines which leads to increased synaptic levels of the transmitters. The subsequent postsynaptic actions of the monoamines on their cognate signaling systems may then lead on to produce the clinical antidepressant effect -19. HowePCPA is often used to deplete tissues of serotonin content either in vivo or in vitro -9. ProtoPC12 cells are widely used as a model of neuronal cell metabolism ,24. The It was noted that the antidepressant drugs significantly enhanced phosphatidylinositide levels at concentrations that were clearly lower than the concentrations necessary to observe significant accumulations of CDP-diacylglycerol. Given that CDP-diacylglycerol synthase activity is considered to be rate-limiting, it is likely that any CDP-diacylglycerol formed is quickly converted to phosphatidylinositide. As such, accumulation of CDP-diacylglycerol may not become evident until the mechanism for synthesizing phosphatidylinositides begins to saturate at the higher drug concentrations. Additionally, phosphatidylinositol is able to accumulate because, as explained above, there is no mechanism to stimulate the activity of phospholipase C which is required to break down the phospholipid. Based on the tandem signaling model, one may predict that in the in vivo environment the direct CDP-diacylglycerol effect and the indirect monoamine reuptake effects of the antidepressant drugs would work in concert to enhance both the generation of substrate and the receptor-mediated release of intracellular second messengers.Following the foregoing results, it still remained to be investigated how the antidepressants increase the levels of the neurophospholipids in a direct manner that may not be dependant on monoamine reuptake inhibition. First, we considered if this action was related to the lipidosis that has been associated with a number of amphiphilic compounds, a group of chemical structures that includes the tricyclics and several other antidepressants. Amphiphiles are thought to induce lipidosis  through inhibition of phospholipases that then progresses to the inhibition of phospholipid degradation -27. Othe2 receptors resulted in enhanced accumulations of inositol phosphates over and above the effects due to agonist alone, implying that phosholipase C may not have been inhibited (or at least that enzyme stimulation by a coupled receptor was not prevented) [It is still unclear if imipramine or any of the other agents interact directly with the phospholipids to prevent their degradation or inhibit phospholipases . If the evented) . Taken tBased on the observation that chemically diverse antidepressants increased the synthesis of the phospholipids, we hypothesized that the drugs enhance the activity of the phospholipid synthesizing enzymes. This prompted the need to investigate the effects of the drugs on CDP-diacylglycerol synthase activity. CDS is reported to have high activity in the brain based on distribution analysis in rodent tissues . FurtherOne concern about these observations was the potential toxicity of the antidepressants given that the effective concentrations were in the micromolar range (3-300 μM). A previous report showed that antidepressants such as fluoxetine (selective serotonin reuptake inhibitor) and amitriptyline (non selective reuptake inhibitor) did not adversely affect cell survival of PC12 cells up to 100 μM for 24 or 48 h incubation periods . These dIn conclusion, the results of this study raise the possibility that the neurolipid effects of antidepressant agents, particularly on CDP-diacylglycerol and phosphatidylinositide synthesis, may be independent of monoamine transmitter action. Given that resting cellular levels of CDP-diacylglycerol are thought to be rather low, and that CDS activity is the rate - limiting step in CDP-diacylglycerol synthesis, enhancing the activity of CDS appears to be a logical or plausible explanation for the effect of antidepressants on CDP-diacylglycerol and phosphatidylinositide synthesis.MAA participated in experimental design, conducted the experiments, and drafted the manuscript. ASU designed the study, and participated in data analysis, writing of the manuscript, and securing funds for the study. Both authors read and approved the final manuscript.This research was supported by grant #R01DA017614 from the US National Institutes of Health.


To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe multisystem disease characterized by profuse bleeding with a case-fatality rate as high as 30%. The infection is endemic to the Balkans  M antibodies against CCHFV were detected in the serum sample.The second case-patient was a 34-year-old man who had removed ticks from cows from the same herd as the index case-patient. Symptoms developed on March 23 and he was hospitalized on March 26 with fever, diarrhea, and bloody sputum. Laboratory findings showed moderate leukopenia and thrombocytopenia. His condition improved within 1 week. IgM antibodies against CCHFV were detected in a serum sample collected during the acute phase of the disease.7 copies/mL.The third confirmed case-patient was a 52-year-old woman (nurse) who cared for the index case-patient after his hospital admission. Although she reported wearing gloves, she was extensively exposed to the patient’s blood and vomit and received immunoprophylaxis (specific hyperimmune gamma globulins). On March 28, a mild disease characterized by fever, headache, weakness, and maculopapular rash with petechiae developed; she was hospitalized on April 2. She had leukopenia, thrombocytopenia, and normal levels of liver enzymes. The serum sample collected during the acute phase of the disease was IgM positive, and a 4-fold increase was present in the IgG titer in a sample collected during the convalescent phase (from 160 to 640). Blood and serum samples taken during the acute phase of the disease were positive for CCHFV by real-time PCR  collected from the area and tested by reverse transcription–nested PCR were negative for CCHFV.Two additional suspected CCHF cases occurred in the same area, on March 30 and April 9 (This cluster of CCHF cases has several important highlights. First, it occurred in a region that was considered to have low CCHF endemicity; however, the area is only a few kilometers from Greece, where a human fatal case was observed in June 2008 (In conclusion, CCHF emerged in southwestern Bulgaria near the border with Greece. Person-to-person transmission emphasizes the need for rapid diagnosis of CCHF, especially in cases with atypical clinical manifestations.


The survival to discharge rate after unwitnessed, non-cardiac out-of-hospital cardiac arrest (OHCA) is dismal. We report the successful use of therapeutic hypothermia in a 26-year old woman with OHCA due to intentional poisoning with heroin, amphetamine and insulin.The cardiac arrest was not witnessed, no bystander CPR was initiated, the time interval from the call to ambulance arrival was 9 minutes and the initial cardiac rhythm was asystole. Eight minutes of advanced cardiac life support resulted in ROSC.Upon hospital admission, the patient's pupils were dilated. Her arterial lactate was 17 mmol/l, base excess -20, pH 6.9 and serum glucose 0.2 mmol/l. During the first 24 hours in the ICU, the patient developed maximally dilated pupils not reacting to light and became increasingly haemodynamically unstable, requiring both inotropic support and massive fluid resuscitation. After 1 week in the ICU, however, she made an uneventful recovery with a Cerebral Performance Category of 1 at hospital discharge and at a follow up examination at 6 months.According to most prognostic factors, the patient had a statistical chance for survival of less than 1%, not taking into account her severe state of hypoglyaemia. We suggest that this case exemplifies the need for more studies on the use of TH in non-coronary causes of OHCA. Most primary survivors of out-of-hospital cardiac arrest (OHCA) will succumb to anoxic-ischemic brain injury during their hospital stay .Among the factors known to predict a dismal prognosis are a non-cardiac cause of the OHCA, non-witnessed arrest, asystole as the initial ECG-rhythm, lack of bystander cardiopulmonary resuscitation (CPR) and time interval between distress call and arrival of the ambulance of more than 6 minutes . HypoglyA 26-year old female sustained an OHCA after intentional poisoning. The cardiac arrest was unwitnessed, no bystander CPR was initiated, the interval from the call for help to the arrival of the ambulance and emergency physician was 9 minutes, the initial cardiac rhythm was asystole and the cause of the arrest was non-cardiac. After 8 minutes of standard advanced cardiac life support . After the ROSC, the patient was haemodynamically stable and TH initiated with ice-packs. Her pupils were equal, dilated and not reactive to light. During transport to the hospital, the patient was sedated with 10 mg of diazepam due to irregular spontaneous respiratory efforts and received 500 ml of crystalloid intravenously.ICU for standard post-resuscitation treatment with sedation, controlled ventilation, close metabolic control and TH. The body temperature was maintained in the TH target range (32-34°C) for an additional 26 hours before controlled rewarming was commenced.Upon hospital admission, the patient's tympanic temperature was 31°C, and her ECG showed a sinus rhythm and nonspesific alterations of the ST-segment. Cerebral computer-tomography was normal. The chest x-ray showed opacification of the lower right pulmonary lobe; pO2, pCO2 and O2 saturation were within normal limits. Laboratory findings are depicted in Table During glucose level control in the ICU a severe hypoglycaemia (0.1 mmol/l) was diagnosed and treated with 40 ml of 50% glucose initially. This was followed by continuous glucose infusion for 20 hours to titrate glucose levels ranging from ranged from 4.1-8.2 mmol/l. The cumulative amount of glucose infused was 102 g. When admission documentation was rechecked, it became apparent that a severe hypoglycaemia (0.2 mmol/l) had already been present at admission . Shortly after admittance to the ICU, the patient developed maximally dilated pupils and became increasingly haemodynamically unstable, requiring inotropic and vasopressor therapy as well as substantial fluid resuscitation. Echocardiography verified a significant post-cardiac arrest myocardial dysfunction with an ejection fraction of 30%. The patient required 3 days of inotropic support before weaning was possible. After 8 hours of TH, the maximally dilated pupils decreased in size and became reactive to light. On day 2, pupillary size and reaction to light were normal.During the course in the ICU, the patient required increasing doses of sedation, displayed spontaneous movements in all extremities and responded to endotracheal suctioning and positional change. After discontinuation of midazolam and fentanyl at day 7, she became restless with spontaneous eye opening. Shortly thereafter, she displayed purposeful motion to stimuli. Weaning from mechanical ventilation was delayed by aspiration pneumonia and bilateral pleural effusions, but the patient was extubated on day 7.After transferral to the ward, her cerebral performance progressed continuously. The patient was graded as cerebral performance category (CPC) 1  at hospiThe case is currently under investigation as an attempted homicide with the so-called "Judas-dose", a street term for the drug combination used in our patient .The good outcome in our patient was very surprising. Her statistical prognosis for survival was dismal ,10. Boydhypoglycaemia-, and amphetamine- induced brain damage have also been recognized [The neuroprotective mechanisms of TH have been mostly studied in anoxic-ischemic brain injury, but temperature-dependent neurotoxic mechanisms of cognized ,4,12-14.cognized .pre-cardiac arrest hypothermia in asphyxial CA has been established in the rat model [Whether the low body temperature at admission indicates possible hypothermia before the OHCA is unclear because no on-scene temperature reading was available. The protective effect of at model . In our Post-cardiac arrest myocardial dysfunction is a common but usually transient finding in OHCA survivors, and it cannot be used as a prognostic parameter . In apprAlthough our case does not prove that TH is neuroprotective in non-cardiac OHCA, we suggest that it supports the notion that TH might have an extended role in brain injury due to other aetiologies than cardiac caused, ischemic-anoxic OHCA. Our work also demonstrates that proposed prognostic factors from the pre-TH era may need to be re-evaluated as we gain more experience with the use of TH.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MB carried out the initial resuscitation, clinical follow-up of the patient and conceived the idea of possible publication of the case. MB and ES both participated equally in the literature research and the process of writing the manuscript. Both authors read and approved the manuscript.


The identification of novel drug targets by assessing gene functions is most conveniently achieved by high-throughput loss-of-function RNA interference screening. There is a growing need to employ primary cells in such screenings, since they reflect the physiological situation more closely than transformed cell lines do. Highly miniaturized and parallelized approaches as exemplified by reverse transfection or transduction arrays meet these requirements, hence we verified the applicability of an adenoviral microarray for the elucidation of gene functions in primary cells.Here, we present microarrays of infectious adenoviruses encoding short hairpin RNA (shRNA) as a new tool for gene function analysis. As an example to demonstrate its application, we chose shRNAs directed against seven selected human protein kinases, and we have performed quantitative analysis of phenotypical responses in primary human umbilical vein cells (HUVEC). These microarrays enabled us to infect the target cells in a parallelized and miniaturized procedure without significant cross-contamination: Viruses were reversibly immobilized in spots in such a way that the seeded cells were confined to the area of the viral spots, thus simplifying the subsequent addressing of genetically modified cells for analysis. Computer-assisted image analysis of fluorescence images was applied to analyze the cellular response after shRNA expression. Both the expression level of knock-down target proteins as well as the functional output as measured by caspase 3 activity and DNA fractionation (TUNEL) were quantified.We have developed an adenoviral microarray technique suitable for miniaturized and parallelized analysis of gene function. The practicability of this technique was demonstrated by the analysis of several kinases involved in the activation of programmed cell death, both in tumor cells and in primary cells. In the years following the completion of the human genome sequencing projects, a variety of genome-wide screening procedures for functional analysis were developed. For instance, plasmid vectors were applied to transfer cDNA for over-expression, or, alternatively, shRNA knock-down cassettes for gene silencing . To thisWe have generated a microarray of infectious adenovirus to convey genetic information into the target cells (reverse infection). By choosing an adequate blocking procedure, we managed to strictly confine the cells to the viral spots, preventing adherence to areas without genetic information. By co-expressing fluorescent markers, we could differentiate infected cells from non-infected cells, thus facilitating phenotypic analysis.We have employed the adenoviral array technology to analyze the contribution of seven protein kinases to cell signalling in HUVEC after viral transfer of validated shRNA expression cassettes. This group of enzymes was selected to exemplify the application of miniaturized adenoviral arrays.vice versa   in additHence, our results show that shRNA-mediated knock-down is feasible in cells exposed to adenoviral arrays.A panel of seven adenoviral vectors expressing shRNAs directed against human protein kinases was applied to analyze the phenotypic response of tumor cells after gene silencing. The protein kinases selected are known to be involved in the regulation of apoptosis (BTK (Bruton's tyrosine kinase) -8, p38alThe induction of apoptosis was determined either by terminal-deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling (TUNEL), or by immunodetection of cleaved caspase 3. A representative set of micrographs is given in Figure Fluorescence intensities of both activated (cleaved) caspase 3 and TUNEL in comparison to cells infected with the control adenovirus (shCTR) were quantified as outlined above. The induction factor observed using activated caspase 3 or TUNEL staining as a determinant was up to 4-fold Fig . The kno® 3/7 Assay, Promega), which is adapted to high-throughput cellular assay systems. In this kind of assay, the generated luminescence is proportional to the amount of caspase activity present. For comparison, shRNA sequences against two anti-apoptotic kinases, p38alpha and PKN1, as well as an shRNA sequence against CDK2, the knock-down of which did not induce apoptosis, were chosen, and the induction of caspase 3 activity was depicted. Induction of apoptosis after shRNA-mediated knock-down of p38alpha and PKN1 exceeded the signals of the well-based assay which indicated that the adenoviral chip is a suitable alternative to the well-based assays. No induction of apoptosis was observed with either assay after expression of the vector specific for the knock-down of CDK2.In Figure The signal-to-noise ratios (S/N) in Fig. The advantage of adenoviral infection lies in the fact that adenoviruses allow the genetic manipulation of cells not amenable to classical transfection protocols, such as many primary cells. To make use of this advantage, we investigated both the efficiency of the knock-down and the employability of a functional assay using primary human umbilical vein endothelial cells (HUVEC). The knock-down efficiency was analyzed by seeding HUVEC onto a microspot array of immobilized adenovirus encoding RFP as well as shRNA directed against lamin A/C (shLamin). As a control, adenoviruses encoding RFP as well as a control shRNA (shCTR) were likewise printed. A comparative quantitative analysis was conducted as described above, the result of which is presented in a percentile diagram Fig.  as well Next, we asked whether an array of kinase-specific shRNA expressing adenoviruses would result in detectable induction of apoptosis in HUVEC. As shown above for U-2 OS cells, HUVEC were exposed to arrayed adenoviral shRNA vectors and activated caspase 3 was detected by immunofluorescence analysis. Figure Whereas the knock-down of NEK2 and PBK induced apoptosis in both HUVEC and U-2 OS cells, the cells differed in their response to the knock-down viruses against BTK, CDK2, p38alpha1, and PKN1: The knock-down of the former resulted in apoptosis in HUVEC, and the knock-down of the latter evoked apoptosis in U-2 OS.In conclusion, we show that adenoviral arrays are suitable to perform functional assays even in primary cells. Hence, the adenoviral microarray allows a comparative analysis of kinase functions in different cell types under miniaturized and parallelized conditions.Multiple RNAi platforms are now available for performing large-scale loss of function screens on mammalian cells. Viral-based screens have been introduced for their promise in expanding RNAi screens to models incompatible with conventional siRNA-based approaches.We present here a microarray suitable for miniaturized and parallelized genetic manipulation and screening of primary human cells. As a vector for gene transfer we have employed adenoviruses. The viral vector system applied needs to include stable viral particles to enable long-term storage and transport. Additionally, high-throughput applications for genome-wide screenings require the possibility to produce virus in a small scale, miniaturized format, properties which are adequately met by adenoviral vectors.In addition to the adenoviral microarray presented here, a similar system was also reported for lentiviral  and retrThe decision as to which technology of viral delivery to employ will be situational as each of these three approaches comes along with distinct advantages and drawbacks. The advantage of the adenoviral microarray system presented in this study is an application of an appropriate blocking solution after printing the adenovirus. This implementation serves to confine cell adherence to the distinct spots containing the genetic information. Such confinement resulted in an array of cells, significantly simplifying identification in subsequent analysis. Additionally, it results in a reduced number of cells required for screening, which is especially relevant for the screening of primary cells, as the amount of cells available is frequently restricted. Whether the blocking step is applicable to the lentiviral microarray or any other viral system remains to be shown. As the viral vector system applied is characterised by highly stable viral particles, storage and transport of ready-to-use arrays is also feasible.Genetic manipulations carried out in a microwell format allow for the well-by-well analysis using biochemical assays, the sensitivity of which needed to be achieved in a microarray configuration by fluorescence imaging. We have hence compared a sensitive well-based assay measuring the induction of apoptosis via enzymatic activity of caspase 3 in the lysate with immunodetection of activated caspase 3 on the microarray. As judged by the signal-to-noise ratios, the microarray-based assay provided signals at least in the range of the in-well assay. For optimal sensitivity, it should be noted that any assay carried out on a microarray requires discrimination between cells which have taken up shRNA constructs and untransduced cells. Here we introduced adenoviral vectors which enable the required discriminatory power, provided by a suitable marker incorporated in the vector backbone.In order to test the feasibility of the adenovirus array technology for unravelling signalling pathways in a model primary cell, we studied the influence of shRNA-mediated knock-down of a set of seven protein kinases on the apoptotic process. The applicability of the device for functional assays on primary cells was exemplified using HUVEC. The comparison of signals obtained infecting HUVEC or U-2 OS cells revealed cell-type-specific responses to the knock-down constructs directed against individual kinases. The technology of the adenoviral siRNA microarray allowed to quantitatively measure phenotypical differences between these two distinct cell lines and therefore was qualified as a convenient and suitable tool for the cell-based analysis. Nevertheless, for more fundamental conclusions of the biological impact of these kinases, a more detailed investigation is needed, e.g. by testing several independent shRNA sequences directed against the targets identified to rule out off-target effects.The kinases analyzed have been chosen from two categories: On one hand, we have selected rather general interplayers, such as PBK and NEK2, which have been shown to be important for cell cycle progression, DNA damage, and mitotic regulation ,17,19,20A more specific response was observed for p38alpha and PKN1 in U-2 OS cells. These experiments suggest that osteosarcoma cells exhibit a higher sensitivity towards inhibition of kinases involved in the MAPK pathway than HUVEC. On the contrary, the knock-down of the kinases BTK and CDK2 elicited apoptosis in HUVEC but not in U-2 OS cells. As it was reported by Cai et al., solely depleting CDK2 was not sufficient to induce apoptosis in the osteosarcoma cell line U-2 OS .The depletion of TrkA  showed no effect on the induction of apoptosis in either cell type. This kinase depends on a particular surface membrane receptor for signal transmission, which might be absent or inactive in the cells examined .In general, the results on apoptosis induction upon knock-down of particular targets using the adenoviral chip correlated with published data ,27,28,30HEK293 cells  and U-2 OS cells  were cultivated in DMEM (Gibco), complemented with 200 mM Glutamin and 10% FCS (HyClone). HUVEC (PromoCell) were cultivated in EBM-2 Bulletkit medium (Cambrex).[[, and the Dharmacon siDESIGN® Center . The shRNA sequences are available upon request.The shRNA sequences directed against human kinases were based on siRNA sequences selected using public servers such as the Whitehead siRNA selection program , the Sfo[, the GenFor the generation of shRNA-expressing vectors, oligonucleotide pairs were synthesized encompassing the siRNA sequences (19 nt or 21 nt) and their reversely complementary sequences, connected by the spacer 5'-CGAA-3'. Immediately downstream of the complementary sequences, there is a stretch of five thymidine residues, which serves as a stop signal for RNA polymerase III. Hence, a transcript is generated that can form a hairpin structure, in which the complementary sequences form the stem and the spacer forms the loop ,34.The oligonucleotide pairs were annealed and ligated into pENTR™/U6 according to the manufacturer's protocol . In these vectors, the expression cassette – consisting of the human U6 promoter and the shRNA sequences – is flanked by the attachment sites attL1 and attL2, which allow the transfer of the expression cassette into the adenoviral destination vectors by site-specific LR recombination. The vector pAdEasy1_EGFP_shRNA is used to raise adenoviral stocks for printing as described below. A similar construct encoding RFP was likewise generated . Depending on the presence of suitable restriction sites upstream and downstream of the kinase open reading frames, modified versions of psiCHECK™-2 were generated with extended multiple cloning sites. If no suitable restriction sites were present, the cDNAs were amplified by PCR using primers containing the desired restriction enzyme recognition sites.For each kinase, the cDNA was cloned – sometimes only partially – between the open reading frame of the humanized For the validation of the efficiency of the knock-down, HEK293 cells were co-transfected with a psiCHECK™-2 derivative containing the target cDNA sequence and the corresponding pENTR™/U6 plasmid encoding the shRNA. The co-transfection was carried out in a 96-well format, with 0.2 μg DNA/well using Lipofectamine™ 2000 Transfection Reagent  according to the manufacturer's protocol. Approx. 48 hours after transfection, the cells were lysed and the luciferase activity was determined according to the manufacturer's instructions . Only those shRNA sequences which reduced luciferase activity by > 50% were transferred into a destination vector to generate the corresponding adenoviral shRNA expression vector.The U-2 OS cells were transfected with the indicated constructs and 48 hours later the cells were lysed using Laemmli Buffer, containing 2% SDS, 10% glycerol, 5% 2-mercapto ethanol, 0,002% bromphenol blue and 0,075 M Tris HCl. Correlative to the transfection efficiency, an appropriate amounts of the protein lysate were loaded on 10% SDS-PAGE gel. After transfer and blocking procedures, the nitrocellulose membrane was incubated with mouse anti-Lamin A/C antibody  following immunostaining with HRP-conjugated rabbit anti-mouse secondary antibody. The final blots were treated using ECL™ Plus Western Blot Detection Reagent  for the detection of the signal.attR1-CmR-ccdB-attR2 cassette upstream of the cassette for the expression of the marker genes enhanced green fluorescent protein (EGFP) or red fluorescent protein (RFP), respectively. These adenoviral vectors are based on pAdEasy-1 (Stratagene), into which the above mentioned cassettes were cloned by homologous recombination using Escherichia coli BJ5183[The adenoviral destination vectors used were pAdEasy1_EGFP-DEST and pAdEasy1_RFP-DEST, which contains the li BJ5183. FurtherattR1-CmR-ccdB-attR2 cassette was generated by BP recombination between pDONR221 (Invitrogen) and a suitable cassette containing attB1 and attB2, using the Gateway® BP Clonase™ II enzyme mix  according to the manufacturer's instructions.The U55762) and contains the immediate early promoter of CMV, the EGFP open reading frame as well as the SV40 polyadenylation signal. The RFP expression cassette originated from pDsRed-Express-N1 (Clontech) and contains the immediate early promoter of CMV, the RFP open reading frame as well as the SV40 polyadenylation signal.The EGFP expression cassette originated from pEGFP-N1 .The shRNA expression cassettes in pENTR™/U6 were transferred into the adenoviral destination vectors by site specific recombination R) and the bacterial ccdB gene is replaced by the shRNA expression cassette. Concomitantly, the CmR-ccdB cassette is transferred to the pENTR vector , from which the early genes E1 and E3 have been deleted  ,36. Aden9 infectious units/ml. The adenoviral stocks could be stored at -70°C over months without loosing their infectious capacity by -70 C. The procedure of virus generation is non-laborious and suitable for high-throughput applications.The titres obtained after the third amplification were in the range of 1 × 10Adenoviral stocks, resuspended in PBS, were printed onto glass slides covered with nitrocellulose, using a manual 8-pin arrayer . Nitrocellulose covering was achieved by dipping the microscopic slides in a 25 mg/ml solution of nitrocellulose  in methanol and subsequent evaporation of the solvent under sterile conditions using a laminar air flow bench.The printed adenoviral chips were shown to be not impaired in their infectious capacity till day 17 by storing at 4°C. Longer periods of storage are not recommended since the viruses gradually loose their infectious capacity.®  [After printing, the surface outside the viral spots was blocked by the application of 1 ml of StabilGuardMN, USA) ,38, pass5 trypsinized cells were distributed over the surface of the chip, which was placed into a Petri dish. The cells were left for 8–10 hours at 37°C in 5% CO2. Then, fresh medium was added to the cells so that the chip was completely covered with medium. Three (in the case of cleaved caspase 3 measurement) or four (in the case of the TUNEL assays) days later, the chips were washed twice with PBS and fixed in 4% formaldehyde solution for 15 minutes. After a subsequent wash with PBS, the cells on the chips were permeabilized using 0.25% Triton X-100 in PBS for 15 min. To block unspecific binding of the antibodies, the cells were blocked with 1% BSA  in PBS/Tween-20 for 30 minutes. Afterwards, the chips were treated with either anti-lamin A/C monoclonal mouse antibody  or with anti-cleaved-caspase-3 (Asp175) antibody , diluted in 1% BSA-PBS solution, for at least one hour at 37°C. After decanting the first antibody solution, the chips were washed three times with PBS and incubated for 1 hour with secondary Cy2-coupled goat anti-mouse antibody for lamin staining , or with Cy3-coupled goat anti-mouse antibody for caspase-3 staining . Prior to mounting with DacoCytomation fluorescent mounting medium , the chips were counterstained for 1 minute with DAPI followed by fluorescent microscopy analysis.4–5 × 10The TUNEL assay was performed using the In Situ Cell Death Detection Kit, TMR Red  exactly following the manufacturer's instructions. Shortly, 4 days after infection, the chips were fixed using the Fixation Solution provided with the kit for 1 h at 25 C, washed with PBS and permeabilised with the Permeabilisation Solution from the kit for 2 minutes on ice. After washing with PBS, the chips were overlaid with 100 μl of TUNEL reaction mixture, prepared according to the provider, and kept for 1 hour at 37 C in the cell incubator. After final washing with PBS, the chips were overlaid with cover slips and analysed using a fluorescence microscope.The adenoviral spots were photographed using a microscope (Zeiss Axiovert 200 M) supported by the AxioVision LE Rel 4.5 software. The images were analyzed and evaluated using the ImageJ (Version 1.38x) software. Spot images from immunostaining with antibodies against anti-cleaved caspase 3 or after TUNEL assay were converted into 8-bit pictures and compared with shCTR images using the Image Correlator plug-in.For the evaluation of the lamin A/C knock-down, the fluorescent signal of the markers (EGFP or RFP) was used to identify infected cells, which in turn were used to define the regions of interest (ROI). The ROI were transferred onto the images showing lamin A/C staining. This approach allows to only consider infected cells in the statistical analysis, which was performed using the StatView (SAS Institute Inc.) software.The assay for caspase 3 activity was performed in a 96-well format using the Caspase-Glo 3/7™ assay  according to the manufacturer's protocol.A patent has been filed at the EPO under number EP 1 599 727 B1.AO performed the experimental design and data analysis. AK validated the constructs and assisted in data analysis. MT performed functional assays and drafted the manuscript. FW contributed to data acquisition and drafted the manuscript. UH performed basic viral experiments. RB, DW, and AL contributed to data acquisition, analysis, and drafting the manuscript. FW, TOJ, and HV initially designed the adenoviral microarrays. AK, MHGK, and HV suggested the study and helped interpreting the data and drafting the manuscript. HV coordinated the study and contributed most materials and resources. All authors read and approved the final manuscript.Storage test for the infectivity of the viruses immobilized on the chip. A. Green fluorescence is indicative of successful adenoviral infection. For quantitative evaluation, the number of infected cells was counted within a 2500 μm2 area of each spot. B. Representative images are given from microarrays stored for 1, 10 and 17 days. Scale bar, 10 μm.Click here for file