Recurrent spontaneous abortion (RSA) is defined as the loss of three or more consecutive pregnancies during the first trimester of embryonic intrauterine development. This kind of human infertility is frequent among the general population since it affects 1 to 5% of women. In half of the cases the etiology remains unelucidated. In the present study, we used interspecific recombinant congenic mouse strains (IRCS) in the aim to identify genes responsible for embryonic lethality. Applying a cartographic approach using a genotype/phenotype association, we identified a minimal QTL region, of about 6 Mb on chromosome 1, responsible for a high rate of embryonic death (∼30%). Genetic analysis suggests that the observed phenotype is linked to uterine dysfunction. Transcriptomic analysis of the uterine tissue revealed a preferential deregulation of genes of this region compared to the rest of the genome. Some genes from the QTL region are associated with VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathways. This work may contribute to elucidate the molecular basis of a multifactorial and complex human disorder as RSA. Embryonic development in mammals begins from the female and male interaction which leads to the oocyte fertilization. After 5 to 6 cell divisions inside the zona pellucida, the blastocyst undergoes its development conducing to the implantation in the uterine tissue. The external cells of the blastocyst develop into the placenta, a pivotal organ which allows immune tolerance, bidirectional foeto-maternal exchanges and crucial synthesis of gestational hormones Amnionless gene (AMN) in patients affected by RSA but no causal mutations could be identified et al. described a statistical association between the p.Val617Phe mutation of the Janus kinase 2 protein and RSA At present, although hundreds of mutant mouse models with reproductive phenotypes have been generated Mus spretus SEG/Pas genome fixed at homozygous state on Mus Musculus C57Bl6/J (B6) genomic background. Using IRCS animals we have previously shown that 3 QTL of embryonic lethality mapped on a unique spretus fragment in 3 strains, 66H-MMU13, 66H-MMU1 and 135E. The first, Led1 in 66H-MMU13 strain on the MMU13 (∼2.6 Mb) comprised between the rs120693734 and D13Mit47 polymorphic genetic markers. The second, Led2 in 66H-MMU1 was analyzed in the present study and the third, Led3 located on MMU19 in 135E strain encompassing a unique Spretus fragment of 8 Mb located between D19Mit49 and D19Mit137 markers. The 66H-MMU1 strain, which encompasses a unique spretus chromosomal fragment located on MMU1 is affected by high levels of embryonic death (24.6%). This strain encompasses a QTL of embryonic lethality (named Led2) spreading on 32 Mb and containing 215 genes (143 annotated and 72 predicted) In recent years, in order to overcome these constraints we created an original mouse model of interspecific recombinant congenic strains (IRCS) which permit to localize chromosomal regions associated with complex phenotypes (Quantitative Trait Loci or QTL) Led2. For this purpose, we created 15 substrains from 66H-MMU1 animals, which encompass distinct overlapping spretus fragments. Using in vivo high frequency ultrasonography to follow the embryonic development, we used an approach of type “phenotype/genotype association" to refine this QTL of embryonic death. We identified, into the Led2 QTL, one region of approximately 6 Mb, Led2minA, which has a main effect on the rate of embryonic death. In addition, we pointed out a second region, Led2minB, which could also have a small effect on the phenotype, although statistically not demonstrated.Here, we present a thorough genetic dissection of Procedures for handling and experimentation were conducted in accordance with the policies of the Paris Descartes University, the Cochin Institute and the Guidelines for Biomedical Research Involving Animals. The experiments were approved by the departmental veterinary services of Paris .Mus musculus (C57BL6/J) and Mus spretus SEG/Pas . The design of these crosses was reported in a previous work The 66H-MMU1 strain was created at the Pasteur Institute (Paris) by successive crosses of the two parental species spretus segment present in the 66H-MMU1 genome. Primer microsatellites were retrieved from the Mouse Genome Informatic website (MGI) website of the Jackson Laboratory (www.informatics.jax.org). PCRs were performed using Taq DNA Polymerase (New England Biolabs). PCR products were loaded in a 2% Nusieve, 2% agarose gel .DNA was extracted from mouse tail fragments by a standard procedure. Eight new microsatellites located on MMU1  were genotyped in order to precise the boundaries of the The gestation was obtained by crossing each IRCS female with a C57/BL6 male. Each female was used one time to collect phenotypic data from the primo-gestation. For each group one female per gestation and the number of animals studied is always >4. C57/BL6 females from the control group were crossed with C57/BL6 males. Substrain’s phenotyping was carried out at the small animal imaging facility of the Cochin Institute using high frequency ultrasonography . Eight to 12 weeks IRCS females were mated with C57BL/6J males, during a period of 2.5 days. Then, female mice were anesthetized with 1.5% of isoflurane in order to achieve ultrasound examination . Briefly, a chemical hair remover was used to eliminate abdominal hair. Ultrasonographic contact gel was used to ensure contact between the skin surface and the transducer. Body temperature, electrocardiographic and respiratory profiles were monitored using ultrasound device’s integrated heating pad and monitoring device . Examinations were performed using 2 different high frequency probes depending on the size of the embryos: a 60 MHz transducer for early stages of development (RMV708) and a 40 MHz (RMV704) transducer for late developmental stages.in vivo, three ultrasonographic examinations were performed at three time points (between E7 and E14). During each examination, we assessed the number of implanted embryos in each uterine horn as well as their status  was assessed. For each gestation, the embryonic lethality rate was calculated as the number of resorbed embryos in both horns reported to the total number of implanted embryos.In order to follow the gestation Females from IRCS and B6 strains were crossed with B6 males. Each pregnant female was subjected to ultrasonographic examinations in order to precisely determine the embryonic developmental stage. Female mice were euthanized by cervical dislocation and tissues were taken at E12.5. Total RNA of uterine tissue from six mice of the IRC substrain of interest was extracted using TRIzol Reagent  in accordance with the manufacturer's instructions. Similarly, six B6 were used to extract total RNA. In order to duplicate microarray hybridizations of samples from uterine tissues, two pools of three RNA extractions were created for both IRCS and B6 animals. One microgram of RNA from IRC substrains of interest and the B6 controls was used for hybridization on a NimbleGen expression array. cDNA synthesis, DNA end-labeling, hybridization, scanning, and data normalization were performed at the genomic/transcriptomic platform of the Cochin institute. The average fluorescence values for each transcript were collected chromosome per chromosome for each analyzed strain. To evaluate gene expression modifications in IRC strains, these fluorescence levels  were divided gene per gene by the corresponding ones from B6 which were taken as a reference. The results of the gene expression were deposited at GEO (NCBI) with the accession reference GSE32460.After RNA preparation, the total RNA was treated with DNase I (Invitrogen Life Technologies) for 10 min at room temperature followed by inactivation with EDTA (Sigma-Aldrich). Total uterus RNA was reverse transcribed to obtain cDNA using M-MLV Reverse Transcriptase  following manufacturer’s protocols.Cyclophiline A expression. Primer sequences are listed in Quantitative PCR was carried out using fast SYBR Green Master Mix (Applied Biosystems) and a real time PCR system  according to standard PCR conditions. To validate the primers used in qRT-PCR, four pairs of primers were tested for each gene and four housekeeping genes were also tested to choose the reference gene  samples was evaluated by t-test using the Bonferroni-corrected levels. As we used 7 substrains, a p value <0.007 (0.05/7) was considered as significant. Statistically significant results are labeled as follows in all figures: *: p<0.05; **: p<0.01; ***: p<0.001.Led2 QTL spretus fragment carried by the 66H-MMU1 substrain. However, a uncertainty of ∼6.4 Mb existed at the proximal boundary of this QTL since the interval comprised between D1Mit134 (80.6 Mb) and D1Mit50 (87.0 Mb) markers corresponds to this distance and the breakpoint is located somewhere between these two markers. Indeed, D1Mit134 and D1Mit50 allele markers are of B6 and spretus natures respectively (http://www.pasteur.fr/recherche/unites/Gfons/ircs/ircshome.htm). In an attempt to precise the position of the breakpoint, we genotyped 8 novel markers located on this region that permitted to reduce the recombination region to a 2.5 Mb interval comprised between 84.5 Mb and 87 Mb  caused by the 1 strain . Among 1in vivo ultrasonography as previously described spretus origin (B6/SEG), while the uterus was homozygous SEG/SEG for the same spretus fragment. The control group was obtained by crossing males and females of the B6 strain. A total of 97 gestations (31 and 66 of B6 and IRCS types respectively) were analyzed. For each gestation, we counted the number of implanted and resorbed embryos during three ultrasonographic examinations. There was no correlation between embryonic death and the position inside the womb, which suggests that the death of one embryo did not have deleterious repercussions on the contiguously implanted structures  shared a large spretus region (>84.5 Mb to 90.5 Mb) with the R6 strain (which does not display the embryonic resorption phenotype) and the rest (until <100.3 Mb) is also shared with the other strains  which are not affected. This configuration suggests that two spretus regions, shared by R3 and R5 strains and not present together in the other strains, seem to be necessary to explain the apparition of the phenotype in R3 and R5. We defined a first spretus subfragment called Led2minA which encompasses D1Mit50 to D1Mit305 region (>84.5 Mb to <90.5 Mb) and a second region called Led2minB located at the rs3692309 marker (>92.5 Mb to <100.3 Mb) (see gray boxes in spretus regions (Led2min) are separated as in R6 (that contains Led2minA only) or in R4, R10, R13 or R14 (Led2minB only) the phenotype of embryonic death is absent. The presence of the two spretus regions seems indispensable to permit the manifestation of the phenotype, it’s the case for R3 and R5  bearing these two spretus regions. The results of statistical t-tests are shown in Led2minA QTL. By contrast, the comparison between R6 and R3 (or R5), did not statistically indicate the presence of Led2minB QTL. However the embryonic death rates of R3 and R5 both have a tendency to be higher than that of R6  and R3 (or R5) was 17%–19% which is comparable to 15%–17% difference between B6 (with no spretus regions) and R3 (or R5) also suggesting a nil or very small effect of Led2minB. In consequence this result did not support the presence of an epistatic interaction between Led2minA and Led2minB regions, but a Led2minB additive effect could be revealed by increasing sample size of this “QTL" representative strains. For this raison, the genes present in these two regions, Led2minA and Led2minB are listed in In order to refine at of R6  suggestispretus fragment). Conversely, we performed reverse crosses (♀B6 × ♂IRCS), giving a heterozygous placenta for the genes of the fragment, but a B6 homozygous uterus. In this situation, the would-be disorders ought to find their origin exclusively from a placental-fetal/embryonic defect, caused by the spretus state of the MMU1 fragment, but not from a B6 womb defect. In this optic we realized the cross ♀B6 with ♂R3 (IRCS group2). We observed that the mean of embryonic resorption rate (±SEM) was 0.07±0.04 and not significantly different from the control  whereas the inverse crossing, leading also to a heterozygous foeto-placental complex implanted in homozygous spretus uterus (R3), produced a significantly higher embryonic resorption rate   and homozygous uterine alleles (SEG/SEG) within the same genomic region (= 0.001) . From thspretus fragment or the two Led2min regions were considered  . These results were highly reproducible since they showed strong correlations between experimental duplicates (r = 0.967 for B6 and 0.983 for R3). Thus, for subsequent analysis, we took the average of both values for each transcript. We first focused on transcripts with fluorescence levels higher than 100 AUF, we assumed that values under this threshold were very close to background signals and, with this threshold, we selected 18,085 transcripts . We considered a gene as differentially expressed if a two-fold difference of expression (up or down) was observed. Consequently, 3,436 (19% of the expressed uterine genes) transcripts were modified in R3 uterus when compared to those expressed in B6 . A similnsidered .Led2minA QTL region by quantitative RT-PCR. As shown in In order to validate the differential gene expression obtained by the microarray analysis, we checked 7 genes of the Mus musculus and Mus spretus species, we listed the genes of the Led2min QTL corresponding to these criteria and thus potentially involved in embryonic resorption. Finally we considered the transcripts with an expression level >500 AUF and either exhibiting a deregulation  and the presence of non synonymous polymorphisms between Mus musculus and Mus spretus (provided by SANGER database: http://www.sanger.ac.uk/) (Considering that the uterine dysfunction can take its origin from a deregulation of the gene expression or/and non-synonymous coding polymorphisms accumulated during independent evolutive processes of .ac.uk/) .Then, we searched whether deregulated transcripts could be grouped into functional clusters using DAVID database In the reproductive processes, as in others, hundreds of genes interact into subtle regulatory networks, and this complexity does not permit to easily identify the molecular factors of dysfunctions leading to infertility cases. Moreover, when our interest is turned towards the human clinic, the study of factors involved in reproductive defects is particularly challenging due to obvious ethical constraints, which rends obligatory the use of animal models. However although hundreds of mutant mouse models with infertility/hypofertility phenotypes have been generated Mus spretus genome fixed on a Mus musculus B6 genetic background spretus segments. Mus musculus and Mus spretus diverged ∼2 million years ago meaning that the association of their two genomes has the potential to lead to genetic incompatibilities spretus origin located on MMU1. This QTL was called Led2 and has been mapped to an interval of 32 Mb which contains 215 genes In the present work we used a mouse model including interspecific recombinant congenic strains (IRCS). The originality of this whole model is based on the presence of a small homozygous fragments of Led2 QTL. Each recombinant substrain females were crossed with B6 males, resulting in a fetus/placenta complex with heterozygous B6/SEG genes (at the Led2 locus) and uterine homozygous spretus genes (at the Led2 locus). During each gestation, the substrains were phenotyped in vivo by ultrasonography. This non-invasive technology, based upon a high frequency ultrasound device in vivo real time high resolution observations of embryonic development Led2minA and Led2minB) of approximately 6 Mb each, present together in spretus version only in R3 and R5 strains. In the other recombinant substrains which have not the phenotype, the one or the other of the region is present but not the two regions together. So we defined the first reduced spretus region called Led2minA which encompasses D1Mit50 to D1Mit305 region (>84.5 Mb to 90.5 Mb) and the second called Led2minB located at the rs3692309 marker (>92.5 Mb to <100.3 Mb). Our statistical analysis succeeded in proving the presence of Led2minA QTL responsible for a main effect on embryonic death but it failed it for Led2minB. However, notable differences between the embryonic death rates of certain strains (R6 compared to R3 or R5) led to suppose that this latter region could also have a small effect in the phenotype. Taken together, these data did not support the presence of an epistatic interaction between Led2minA and Led2minB.To accomplish the fine mapping of this QTL, we generated recombinant substrains from 66H-MMU1 by backcrosses, each of them presenting a unique sub-fragment of the spretus fragment were preferentially modified (∼40%). This concentration of deregulated genes located on the spretus fragment has already been reported in a previous study of our group performed on testis transcriptome Mus musculus and Mus spretus are frequent since they appear, in average, every 100 bp. When located on the promoter regions of spretus origin, these nucleotide substitutions could modify the transactivation/transrepression properties of transcription factors of C57BL/6J nature, thus modifying the spretus gene expressions. Additionally, dysfunctions leading to embryonic death could result from non-synonymous coding polymorphisms, accumulated during evolution in the spretus genome. These phenomena should be originated from evolution of separated genomic regions that produces transcription factors/DNA (“transcriptomic shock") and/or protein-protein (“proteomic shock") incompatibilities Reverse crosses using IRCS Group 2 males and B6 females revealed that the genes expressed at heterozygous state in the placental tissues are not deleterious for the gestation. Therefore, we deduced that the high rate of embryonic death occurring during the gestation resulted from dysfunction of genes expressed in the uterine tissue. This is in accordance with the normal embryonic development observed in group 2 IRCS females. Then, we carried out a microarray analysis searching to identify uterine deregulated genes in IRCS animals from Group 2. Although we observed deregulated genes located in all chromosomes (19%) we noticed that those situated on the Led2minA QTL and applying filters from bioinformatics databases, bibliography and our own results, we propose a selection of 7 genes  as putative actors of the embryonic death. These genes play a role in VEGF signaling, mTOR signaling and ubiquitine/proteasome-protein degradation pathway. Their effects could be reinforced by a small participation of genes situated on Led2minB region and which could act in the same signaling pathways .Focusing on genes of the Trip12, Psmd1, Cops7b and Usp40 from Led2minA and Asb1 from Led2minB are involved in protein degradation process through the ubiquitin-proteasome pathway. Trip12 exerts a ligase activity related to ubiquitination Usp40 functions as a deubiquitination enzyme in the same degradation pathway Asb1 is a member of the ankyrin repeat and SOCS box (ASB) family. These family proteins interact with Cul5-Rbx2 to form E3 ubiquitin ligase Psmd1 is a component of the 26S proteasome. Cops7b is a subunit of the eight-subunit heteromeric Cop9 signalosome complex. Genetic invalidations of some Cop9 subunits have been associated with developmental defects of post-implantation embryos Usp40 functions as a deubiquitination enzyme in the same degradation pathway Asb1 is a member of the ankyrin repeat and SOCS box (ASB) family. These family proteins interact with Cul5-Rbx2 to form E3 ubiquitin ligase Led2minA Ncl gene and Led2minB Ramp1 and Col6a3 genes are involved in angiogenesis. Ncl encodes nucleolin and treatment of endothelial cells with anti-nucleolin antibody induces apoptosis of these cells Ramp1 gene, RAMP1 (receptor activity modifying protein) forms a functional receptor for CALCA  which is a proangiogenic growth factor in the human placental development and plays a critical role in embryonic development and fetal growth Collagen typeVI a3 gene, COL IV is a main endometrial extracellular matrix component, and an abnormal increased deposition of collagen might impair uterine function, possibly by interfering with vascularization or retarding remodeling events at implantation In the same manner, Cab39  and Eif4e2 from Led2minA and Traf3ip1 from Led2minB, participate in the mTOR signaling pathway, a regulatory step of protein synthesis and growth. Cab39 effect has been described upstream of mTOR activation while Eif4e2 is a downstream signaling target involved in translation initiation. Interestingly, Mtor genetic disruption in mice leads to early embryonic death Traf3ip1  mutant mice are not viable. Traf3ip1 mutant mouse line was generated and the enlarged mutant cell size in culture was associated with elevated basal mTOR pathway activity Finally, in vivo approach of the embryonic development on a mouse IRCS model to refine a chromosome 1 region (Led2) responsible for embryonic death. The present study succeeded in fine-mapping Led2minA QTL which has a main effect on the embryonic death (about 30%) and pointed out a second region Led2minB which could have a minor effect on the same phenotype. Collecting and analyzing experimental, bioinformatics and literature data on the expression and function of genes present in the two regions (Led2minA and Led2minB), we propose 7 genes from Led2minA that could be related with the phenotype. It appears that the vascularization could be the common denominator at these categories of genes involving angiogenesis and the fluidity of the extracellular matrix. The actual identification of the gene(s) involved in this phenotype will necessarily pass through further molecular approaches. An important outcome of this study is the possibility to evaluate novel promising candidates of RSA in humans We used an Table S1Sequences of used real time RT-PCR primers.(DOC)Click here for additional data file.


We recently reproduced in the SIV 8 PFU of ALVAC-SIV at weeks, 0 (V0), 4 (V2), and with both ALVAC SIV and the SIVgp120 envelope proteins, adjuvanted with ALUM at week 12 (V3) and 24 (V4) and analyzed the profile of gene expression at 24 hours after each vaccination. In parallel, we studied the phenotypical and functional changes in the NK cell subsets after vaccination by multi parametric flow cytometry.In here, we immunized, by the intramuscular route, 6 RM with 10Microarray analysis in the PBMC after V1 and V2 showed the up regulation of anti viral  and IFN responsive genes and a down regulation of pro-inflammatory genes. Significant alteration in the gene expression profile was also observed after the gp120/Alum boost (V3). Interestingly, NK cells associated genes were up regulated after V3 vaccination. These finding paralleled our results observed by FACS analysis that demonstrated an increased frequency of NK22 cells at mucosal sites. These NK22 cells expressed CCR6 (a gut homing marker) and are thought to play a role in mucosal immunity. Similarly, vaccination also increased the frequency of NKG2A+ cells that were either cytotoxic (CD107a+) or cytokine producing (IFNg+).Thus, the ALVAC SIV/gp120 vaccine regimen induces a significant activation of NK cells and other innate immune responses. Given that this vaccine platform has conferred some degree of protection from HIV infection in humans, understanding the role of innate immunity in protection from SIVmac251 may guide our effort in the development of novel vaccine strategies against HIV.


The human immunodeficiency virus type 1 (HIV-1) infection occurs by binding to CD4+ receptor and chemokine receptor 5 (CCR5) or the CXC chemokine receptor (CXCR4). A mutation in the CCR5 gene, 32 base pair deletion consequences a truncated protein that is not expressed on the cell surface. The deletion confers resistance to HIV-1 infection and slows the progression of AIDS in HIV infected individuals. The worldwide distribution of CCR5delta 32 polymorphism was congregated by retrieving the data from literature and genotyping new population samples. A comprehensive resource of frequency data for CCR5delta 32 polymorphism in different population samples was created.The data for different populations was obtained from literature. In order to investigate the genetic variation in CCR5 gene in new population, we analyzed 257 healthy control individuals. We examined the CCR5 32 base pair deletion (CCR5-Δ32) by conventional polymerase chain reaction (PCR).The genotype frequency distribution of CCR5 in new population was found to be  in healthy control.The allele frequency of CCR5-Δ32 observed for new population is 1% which is compared with the other populations. The polymorphism CCR5-Δ32 is primarily found in European population. Compared to our data, frequency of delta 32 deletion is observed at high frequency in European populations. Our data of delta 32 deletion is significantly different from Caucasians (p<0.00000001), Africans (p<0.01458) and Europeans (p< 0.00000001).


For a therapeutic HIV-1 vaccine, it should be considered that the immune system has been confronted with a certain viral sequence and mounted CD8 T cell responses specifically towards the infecting virus. One question is whether the immune system of HIV-infected individuals can create a new response towards a variant epitope in the case of vaccination. To study this in a comparable setting, we addressed the question how frequently a new CD8 T cell response can be generated after the occurrence of a viral escape mutation in its recognized epitope in a population not selected for a certain HLA allele.19 HIV-infected untreated subjects were sampled longitudinally  of these. Peptide titration assays revealed 12 (48%) viral escape mutations with 2 de-novo responses (17%). Here the de-novo response showed less effector functions than the original CD8 T cell response. In addition we identified 5 (20%) shifts in immunodominance. None of the subjects with adaptation to the changing virus carried the HLA alleles B27, B57 or B*5801.Our results show that CD8 T cell responses can adapt to the mutations of HIV. However it was limited to only 28% (7 out of 25) of cases in a cohort not expressing protective HLA alleles.


We have in an epidemiological study identified a group of individuals with multiple asthma symptoms (MSA) that reflect a more severe and uncontrolled disease. An altered innate immunity and adaptive immunity, and effects of microorganisms may play a role in the development of both CRS and asthma. The innate immune system use Toll-like receptors (TLRs) to recognize microbes and activate defense mechanisms within minutes of microbial invasion which is followed by an antigen-specific response by the adaptive immune system. Thus, innate and adaptive immune cells are sequentially activated and reciprocally regulate one another. Glycogen synthase kinase-3β (GSK3β) has been demonstrated as a regulator of both innate and adaptive immunity. GSK3β was shown to modulate inflammatory responses through TLR-mediated production of pro-inflammatory cytokines and inactivation of GSK3β (phosphorylated GSK3β) lead to a reduced IL-12 production, which was suggested to skew the balance towards a Th2 response.To determine the degree of expression of TLRs 2, 4, 7 and 9 on monocytes as well phGSK3β using flow cytometry. We hypothesize that the co-existence of CRS in patients with asthma and especially patients with MSA has an increased TLR expression involved in the innate immune system.Participants were selected from an epidemiological cohort, the West Sweden Asthma Study. Clinical parameters as well as fresh peripheral blood cells were obtained from one group of non-asthmatic subjects with CRS (CRS) and four different groups of asthmatics: 7 subjects with MSA (MSA), 7 subjects with other asthma (OA), 7 subjects with MSA and CRS (MSA/CRS) and 9 subjects with OA and CRS (OA/CRS). These five groups were compared to a control group consisting of 10 healthy subjects without asthma or CRS.+ monocytes compared to controls as well as the expression of phGSK3β and CD14. Individuals in the combined MSA/CRS group showed increased expression of CD14 and TLR2, but increased expression of TLR4 was only found in the CRS group.Individuals with MSA or CRS only, showed significantly increased TLR2, TLR7 and TLR9 expression (rMFI) on CD14The upregulated expression of TLRs in the MSA group compared to control group suggest a higher susceptibility towards an altered immune response that might reflect the degree of severity. Furthermore, the increased phosphorylation of GSK3β may indicate a switch in the immune response from a pro-inflammatory to a dysregulated Th2 response.


As microcystin structure has a significant impact on transporter selectivity, it is potentially possible to develop analogs with even more pronounced OATP1B3 selectivity and thus enable their development as anticancer drugs.Microcystins are potent phosphatase inhibitors and cellular toxins. They require active transport by OATP1B1 and OATP1B3 transporters for uptake into human cells, and the high expression of these transporters in the liver accounts for their selective hepatic toxicity. Several human tumors have been shown to have high levels of expression of OATP1B3 but not OATP1B1, the main transporter in liver cells. We hypothesized that microcystin variants could be isolated that are transported preferentially by OATP1B3 relative to OATP1B1 to advance as anticancer agents with clinically tolerable hepatic toxicity. Microcystin variants have been isolated and tested for cytotoxicity in cancer cells stably transfected with OATP1B1 and OATP1B3 transporters. Microcystin variants with cytotoxic OATP1B1/OATP1B3 IC Microcystis, Oscillatoria, Planktothrix, Nostoc, and Anabaena. They can be considered to be among the best studied cyanobacterial secondary metabolites l-amino acids situated at positions 2 and 4 of the MC backbone. Related compounds are the nodularins, found in Nodularia sp., which also contain Adda but are cyclic pentapeptides instead of heptapeptides (Microcystins (MCs) are cyclic heptapeptides produced by several cyanobacterial genera such as peptides  [57]–+ were isolated in the ion trap, fragmented by collision induced dissociation (CID) using argon as collision gas  to 45.0 kHz), and separated in the TOF analyzer. MS/MS scans were averaged and converted to the mzXML format using the vendor’s software and evaluated using the software mMass  1H-NMR spectra of all isolated compounds can be found in The structures of the isolated MCs have been determined by high-resolution tandem mass spectrometry 3H]-BQ123, a substrate for both transporters. Further, the OATP1B3 clones demonstrated increased uptake of [3H]-CCK-8, a specific substrate for OATP1B3 but not OATP1B1, while the OATP1B1 clones did not show uptake of [3H]-CCK-8. In addition, RKO and HeLa cells stably transfected with an empty expression vector were used as controls. For cytotoxicity assays, the cells were plated in triplicate in 96-well microtiter plates in a medium containing 5% fetal bovine serum at densities of 1000 cells/well. After 24 hours, medium containing MC structural variants was added to the cells. After another 3 days, cell survival was determined with a sulforhodamine-based assay as we have previously described 50 was calculated from the dose response curve as the concentration of drug that produced a 50% decrease in the mean absorbance compared to the untreated wells and reported as the average of at least three independent determinations performed in triplicate using Prism software.Primary hepatocytes are not suitable for studying microcystin toxicity and transport, as OATP transporters are downregulated within hours upon placing cells in culture File S1Details on the Isolation of the Microcystin Congeners.(PDF)Click here for additional data file.File S21H-NMR spectra of all isolated compounds. Raw NMR and MS data of these compounds are available free of charge via the Internet at http://dx.doi.org/10.6084/m9.figshare.880755.Annotated tandem HRMS and (PDF)Click here for additional data file.


Braun et al. discovered an error in the SAS code of their paper “Variability of Urinary Phthalate Metabolite and Bisphenol A Concentrations before and during Pregnancy” [Environ Health Perspect 120:739–745 (2012)]. The error resulted in the reported absolute concentrations of phthalate metabolites and BPA being approximately 50% higher. The authors mistakenly used the median value from the EARTH study men [specific gravity (SG) = 1.025] rather than women (SG = 1.015) in their SG calculations. Thus, the reported concentrations in Table 2 (columns 2 and 3), Table 4 (columns 2 and 3), and Figure 1, as well as in the Supplemental Material, were higher than if the median SG for this group of women had been used. The authors note that this error did not alter the statistical analyses or their interpretations or the conclusions of their paper.The authors regret the errors.


Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine domains . AggregaInducible control and SCA7 models expressing full-length ATXN7 with 10 (FLQ10) or 65 glutamines (FLQ65) were used. The autophagic activity was analyzed by measuring the p62 level and by monitoring the LC3 levels in the presence or absence of the lysosomal inhibitor bafilomycin A. Protein aggregation was analyzed by the filter trap method and protein-protein interactions by co-immunoprecipitation and western blot. Cellular toxicity was determined by the WST-1 assay.We found that mutant ATXN7 causes autophagic dysfunction by altering the cytoplasmic activity of p53 resulting in an increased p53-FIP200 interaction, co-aggregation of p53-FIP200 with mutant ATXN7 and ultimately disruption of the key autophagy regulating FIP200-ULK1 complex. Furthermore, we show that treatment with a p53 inhibitor, or a blocker of ATXN7 aggregation, can restore the soluble levels of FIP200, as well as increase the autophagic activity and reduce mutant ATXN7 toxicity.We have identified a novel p53-mediated mechanism by which aggregating polyQ disease proteins can disrupts autophagic activity and showed that this inhibition contributes to polyQ toxicity. An increased understanding of the molecular mechanism by which autophagy inhibition occurs in neurodegenerative disease is of importance if safe and efficient therapeutic approaches based on autophagy stimulation should be developed.


The aim of our study was to determine the correlation between activated clotting time (ACT) and APTT values in patients receiving unfractionated heparin (UFH) for renal replacement therapy (RRT).A retrospective analysis was made of case notes and laboratory data of 39 critically ill patients who were on UFH for RRT over a 1-year period. There were 183 paired APTT and ACT measurements done at the same time (29 patients). APTT was done at the laboratory and ACT was done at the bedside using an ACTALYKE monitor . Target APTT and ACT ranges for UFH during RRT were 45 to 55 seconds (control 27 to 32 seconds) and 250 to 270 seconds (control 180 to 220 seconds). Datasets were divided into three groups and the correlation coefficient (Pearson's) was calculated using SPSS software.Mean APTT was 129.5 ± 68.29 (range 25.6 to 360) seconds and mean ACT was 234.6 ± 47.02 (range 125 to 387) seconds. APTT and ACT values were divided into three datasets in a 3 × 3 table. There was no correlation between APTT and ACT values (kappa score being 0.12). There were more above-range APTT values (140/183) against above-range ACT values (36/183). See Table Our data demonstrate that monitoring of anticoagulation with UFH using ACT cannot be recommended.


Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In  Saccharomyces cerevisiae to respond to nutrient availability and stress The Target Of Rapamycin (TOR) and Ras/cAMP/Protein Kinase A (PKA) signaling pathways enable The activity of PKA is controlled by intracellular cAMP HXT7 encodes a high affinity hexose transporter and its transcription is induced by low levels of glucose or a non-fermentable carbon source and Hxt7 localizes to the plasma membrane. However, in response to glucose abundance, nitrogen starvation or rapamycin treatment HXT7 transcription is repressed and Hxt7 is degraded in the vacuole HXT3 encodes a low affinity hexose transporter that is actively expressed in glucose abundance, but repressed Hexose transporters are regulated at the transcriptional and post-translational levels to allow yeast to adapt to varying nutrient concentrations in the environment. If conditions become unfavorable for the expression of a specific transporter gene, the cell must repress its transcription and degrade the remaining transporter. This degradation occurs via endocytosis and proteolysis in the vacuole. For example, vid/gid mutants are sensitive to the presence of rapamycin in the media VID/GID genes increases in the presence of non-fermentable carbon sources The Vid/Gid proteins play an important role in the yeast’s adaptation to different nutrient conditions. These proteins assemble into a multi-component complex termed the Vid30 complex (Vid30c) Here we further investigate the link between the Vid30c and the regulatory mechanisms that govern hexose transporter (Hxt) turnover. We expand the known function of the Vid30c in the nitrogen starvation and rapamycin-induced turnover of Hxt7 HXT3 to GFPMET25pro) for the chromosomal fusion of the methionine-repressible MET25 promoter (MET25pro) to the 5′ end of HXT3CUP1pro-GFP) for the chromosomal fusion of the copper-inducible CUP1pro-GFP cassette to the 5′ end of HXT7VID30 with hphMX6PGK1pro) for the replacement of the native VID28 promoter with the constitutively active PGK1 promoter. Following transformation, the correct integration events were verified by PCR. It is important to note that HXT7 is a duplicated gene in the yeast genome with HXT6 being its counterpart. The PCR confirmation of HXT7 tagged strains therefore involved the use of an upstream primer in the upstream region of HXT7 that is unique to HXT7. BYtor1-1 was generated as previously described All the yeast strains used in this study are isogenic to BY4742 and listed in RAS2 and YCp50-VAL19RAS2VAL19RAS allele on Hxt turnover. Yeast strains used to monitor Hxt3-GFP localization and degradation were pre-cultured in synthetic complete media  to generate biomass. Cells were washed and transferred to synthetic media (0.17% YNB without amino acids and ammonium sulfate) containing 2.5% glucose and 0.5% ammonium sulfate. Amino acids were added to complement auxotrophic requirements. Following a three hour incubation to stimulate HXT3 expression, cells were imaged or harvested for protein extraction (time zero). The remaining culture was harvested, washed and transferred to synthetic media (0.17% YNB without amino acids and ammonium sulfate) containing 2% ethanol and 0.5% ammonium sulfate. The 2% ethanol media was used to provide conditions in which glucose repression was alleviated; we will here after refer to its effect as “glucose starvation.” Amino acids were added to complement auxotrophic requirements and/or suppress expression from the MET25 promoter. Samples were subsequently collected at the indicated times and used for fluorescence microscopy or protein extraction.YCp50, YCp50-et al. (2008) with the following exceptions: (1) the four hour pre-shift incubation was performed in raffinose with ammonium media containing 100 µM CuSO4 to stimulate GFP-HXT7 expression from the CUP1 promoter; (2) Cell samples were collected (time zero), and the remaining cells were washed twice with sterile water and resuspended in fresh raffinose with ammonium media devoid of CuSO4 followed by rapamycin treatment.Yeast strains used to monitor GFP-Hxt7 localization and degradation were cultured as described in Snowdon The monitoring of the subcellular localizations of the Hxt-GFP fusion proteins were performed by preparing slides directly from the indicated cell cultures followed by immediate analysis using the 100× objective lens of a Nikon Eclipse E600 microscope. Images were recorded using a Coolsnapfx monochrome CCD digital camera (Roper Scientific) and processed using Metamorph .Harvested cells were resuspended in lysis buffer , added to 0.3 g glass beads and vortexed for two minutes. Lysates were centrifuged at 5,000 rpm for 3 minutes to remove cell debris. Supernatants were collected and the protein concentrations determined using the DC Protein Assay (Biorad) according to the manufacturer’s recommendations. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Mouse anti-GFP (Roche) and rabbit anti-Aldehyde Dehydrogenase (ADH)  were used as primary antibodies. ADH was used as the internal control to confirm equal amounts of protein in each lane as previously described HXT3HXT3-GFP were grown in glucose media to activate the expression of HXT3, and Hxt3-GFP was monitored upon a switch to ethanol as the sole carbon source. In glucose (time zero) HXT3 transcription was activated and Hxt3-GFP was visible on the plasma membrane in all the strains tested  renders PKA constitutively active Val19 would impact the turnover of Hxt3-GFP in a shift to ethanol. Using the MET25pro-HXT3-GFP strain, our fluorescence microscopy and western analysis showed that the native RAS2 allele supported the internalization of Hxt3-GFP at 3 hours and almost complete degradation in the vacuole 6 hours after an ethanol shift, while Ras2Val19 stabilized Hxt3 in the plasma membrane even after 6 hours in ethanol media with little, if any, internalization and degradation visible throughout the entire time course , the internalization of Hxt3-GFP in rim15Δ cells was markedly delayed when the cells were shifted to ethanol; the protein was still visible in the plasma membrane and abundantly present after 6 hours in ethanol  as it functions as an E3 ubiquitin ligase responsible for the ubiquitylation and subsequent degradation of FBPase and Mdh2 when these enzymes are no longer needed ID genes  and 2. Ctor1-1 allele prevented the endocytosis and degradation of Hxt7 in response to rapamycin treatment and the activation of the Ras/cAMP/PKA pathway is known to suppress Tor deficiencies Val19 or lacking BCY1 supports this hypothesis. Similarly, the rapamycin-induced internalization of Hxt7 is largely prevented in cells expressing Ras2Val19. Furthermore, in rich nutrient conditions in the absence of stress, both TORC1 and PKA prevent cell cycle arrest by inactivating Rim15, the kinase that promotes entry into G0Several signaling pathways enable the molecular response to the presence or absence of nutrient abundance in the environment. The Ras/cAMP/PKA pathway facilitates the cellular response and proliferation when glucose is abundant rsp5-1ts mutant, confirming Rsp5 as an essential E3 ubiquitin ligase in Hxt3 turnover. We could not use the tsrsp5-1 allele in the turnover of Hxt7 as the strain could not tolerate rapamycin treatment during the turnover experiment (data not shown). We therefore used the less dominant rsp5-3ts allele to study Hxt7 turnover. Hxt7 was clearly stabilized in the plasma membrane, but some internalization was observed. These observations implicate two E3 ubiquitin ligases, Rsp5 and the Vid30c, in the nutrient-mediated endocytosis and degradation of Hxt3 and Hxt7. Nutrient transporters have been shown to be direct targets for Rsp5-mediated ubiquitylation, suggesting a more indirect function for the Vid30c where it does not directly ubiquitylate the target Hxt. It is therefore of great importance to identify the precise role the Vid30c in Hxt turnover. Investigation into the signaling surrounding Hxt turnover has implicated the Ras/cAMP/PKA pathway and Rim15 in both unique turnover events. The strikingly similar retention of Hxt3 and Hxt7 in the plasma membranes of the vid28Δvid30Δ double mutant and cells expressing Ras2Val19 could serve as the foundation to investigate a potential functional link between the Vid30c and the Ras/cAMP/PKA.Rsp5 is an essential E3 ubiquitin ligase responsible for the ubiquitylation and subsequent endocytosis of nutrient transporters Figure S1Deletion of components of the Vid30c causes a slight increase in HXT3 transcription. BY4742 (WT), vid30Δ, gid2Δ, vid28Δ, gid8Δ, gid9Δ (A) and vid28Δvid30Δ (B) expressing HXT3-GFP were cultured in glucose media as described in the methods (time 0). Following a switch to ethanol media, samples were collected at the indicated times and analyzed by northern blot analysis. Membranes were probed for GFP and ACT1 as a loading control.(TIF)Click here for additional data file.Figure S2The turnover of Hxt7 is dependent on Rsp5. BY4742 (WT) and rsp5-3 (A) and BY4742 (WT), art3Δ, art4Δ, art6Δ and art8Δ (B) expressing HXT7-GFP were cultured in raffinose media as described in the methods, time 0. After treated with rapamycin, samples were collected at the indicated times and analyzed by fluorescence microscopy (Top) and Western analysis with anti-GFP antibodies (Bottom). Identical blots were also probed with anti-ADH antibody as a loading control. (C) BY4742 (WT), art3Δ, art4Δ, art6Δ and art8Δ expressing HXT3-GFP were cultured in glucose media as described in the methods, time 0. Following a switch to ethanol media, samples were collected at the indicated times and analyzed by fluorescence microscopy.(TIF)Click here for additional data file.Table S1Yeast strains used in this study.(DOCX)Click here for additional data file.


Ischemic preconditioning is a neuroprotective mechanism whereby a sublethal ischemic exposure is protective against a subsequent lethal ischemic attack. We previously demonstrated that SIRT1, a nuclear localized stress-activated deacetylase, is vital for ischemic preconditioning neuroprotection. However, a recent study demonstrated that SIRT1 can also localize to the mitochondria. Mitochondrial localized SIRT1 may allow for a direct protection of mitochondria following ischemic preconditioning. The objective of this study was to determine whether ischemic preconditioning increases brain mitochondrial SIRT1 protein levels and to determine the role of PKCɛ and HSP90 in targeting SIRT1 to the mitochondria. Here we report that preconditioning rats, with 2 min of global cerebral ischemia, induces a delayed increase in non-synaptic mitochondrial SIRT1 protein levels which was not observed in synaptic mitochondria. This increase in mitochondrial SIRT1 protein was found to occur only in neuronal cells and was mediated by PKCε activation. Inhibition of HSP90, a protein chaperone involved in mitochondrial protein import, prevented preconditioning induced increases in mitochondrial SIRT1 and PKCε protein. Our work provides new insights into a possible direct role of SIRT1 in modulating mitochondrial function under both normal and stress conditions, and to a possible role of mitochondrial SIRT1 in activating preconditioning induced ischemic tolerance. Ischemic preconditioning (IPC) is an innate neuroprotective mechanism in which a mild ischemic stress protects against a subsequent lethal ischemic exposure. IPC activates both early 0-3h) and delayed 24-72 h) windows of protection -3. The eh and del-72 h win+ dependent deacetylases which is implicated as a metabolic sensor of the cell . In. In39].  peptide  8], and, andp = In this study, we demonstrate a novel regulatory mechanism in targeting SIRT1 to the mitochondria which we believe represents an expansion of the mechanisms and targets by which SIRT1 mediates neuroprotection. We also demonstrate that changes in mitochondrial SIRT1 protein levels were dependent upon PKCε activation, suggesting a novel interaction between the PKCε and SIRT1 signaling pathways.Mitochondrial increases in SIRT1 protein levels following IPC were only observed during the late phase or delayed window of neuroprotection. The increase in mitochondrial SIRT1 protein is also correlated with our previously reported findings of a late phase but not early increase in nuclear SIRT1 activity ,20. ActiAlthough a basal level of SIRT1 protein was observed in all of the mitochondrial preparations investigated, we found that IPC only increased mitochondrial SIRT1 protein levels in non-synaptic mitochondria and in mitochondria from neuronal-only cultures. These results suggest that IPC alters SIRT1 levels primarily in mitochondria localized to neuronal cell bodies. This specific targeting of SIRT1 to a subpopulation of neuronal mitochondria may be explained by our findings that HSP90 is required for mitochondrial import of SIRT1. In the adult rat brain, HSP90 is reported to primarily localize to the neuronal cell body . Gass etWe also demonstrate that the total cellular SIRT1 protein levels were increased following IPC exposure in neuronal but not glial only-cultures which correlates with the observed increases in neuronal but not glial mitochondrial SIRT1 protein levels. We also observed a reduction in nuclear SIRT1 protein levels 48 hrs following IPC exposure. These findings suggest that the increase in mitochondrial SIRT1 protein levels may be the result of both increased SIRT1 expression, as well as, nuclear to mitochondrial shuttling of SIRT1. Our data also demonstrate that SIRT1 is targeted to mitochondria by mechanisms mediated by PKCε. This is based upon our findings that activation of PKCε, in the absence of IPC, increases mitochondrial SIRT1 protein levels and deacetylase activity, whereas inhibition of PKCε prevents increases in mitochondrial SIRT1 protein levels and deacetylase activity following IPC exposure. The subcellular localization of SIRT1 is known to be regulated by posttranslational phosphorylation and sumoylation ,46,47. IIn summary, it has been demonstrated in both the heart and brain that SIRT1 is an important mediator of IPC-induced ischemic tolerance. Based upon the data presented here, part of SIRT1 protection may stem from the ability of IPC to regulate SIRT1 mitochondrial protein levels, which may allow for a direct regulation of mitochondrial function by SIRT1. Our discovery that SIRT1 is targeted to mitochondria by PKCε following IPC may offer new therapeutic directions in the targeting of metabolic dysfunction, which is associated with the pathophysiology of numerous diseases such as ischemia/reperfusion.


TIMP3 and p16 was analyzed with MS-PCR. Of the 105 investigated tumors 59.1% (62/105) were WHO grade I, 33.3% (35/105) were WHO grade II and 7.7% (8/105) were anaplastic meningiomas (grade III), respectively. The histopathological data correlates with the recurrence rate of the investigated meningiomas. Hypermethylation of TIMP3 was detected in 13.3% of all meningiomas: 10.9% in WHO grade I meningiomas, 25.0% in grade II and 14.3% in grade III meningiomas, respectively. No correlation of TIMP3 hypermethylation with tumor recurrence or WHO grade (p = 0.2) was observed. Interestingly, deletion of 1p36 emerged as a significant predictor of shorter overall survival , whereas TIMP3 promoter methylation had no significant effect on overall survival . The results of the current study support the finding that the deletion of chromosome 1p is an independent marker of meningioma recurrence and progression (p = 0.0097). Therefore the measurement of genetic aberrations in meningiomas allows in a combined histological approach a more precise assessment of the prognosis of meningiomas than histopathology alone.Meningiomas are tumors that arise from the coverings of the brain or spinal cord. 5% of the cases turn into malignant forms with aggressive clinical behavior and increased risk of tumor recurrence. One hundred and five patients with meningiomas were operated by open surgery. To investigate predictors of meningioma recurrence in total 124 samples of 105 patients were investigated by iFISH. Dual-probe hybridization was performed to access chromosomal alterations of chromosomes 1p-, 9p- and 22q. Additionally, methylation of  Meningioma is a tumor composed of neoplastic meningothelial cells and accounts for 13 to 26 percent of intracranial tumors. In its sporadic form, it is typically benign and slow growing, appearing mostly in the later decades of life. Histological features allows for the division of meningiomas into three grades: benign meningiomas (WHO grade I), atypical meningiomas (WHO grade II) which represent 4.7 to 7.2 percent of all meningiomaş and anaplastic meningiomas (WHO grade III) representing 1.0 to 2.8 percent of all meningiomas. However, more than eight percent of all meningiomas are characterized by aggressive clinical behavior with increased risk of tumor recurrence NF-2 gene NF2 gene on the long arm of chromosome 22 (22q12.2), often present with multiple meningiomas. However this hereditary alteration has little or no influence on progression TIMP3), which appears to be involved in meningioma progression and a high-grade meningioma phenotype TIMP3 hypermethylation seems to be associated with the allelic loss of 22q12. Moreover, hypermethylation of the TIMP3 promoter has been identified as a common reason of decreased TIMP3 expression levels in numerous tumors like secondary glioblastomas, kidney cancer, or pancreatic adenocarcinomas TIMP3p16Conventional cytogenetic analysis of meningiomas often reveals the frequent entire or partial losses of chromosome 22. Extensive studies by loss of heterozygosity (LOH) also have shown frequent allelic losses on the long arm of this chromosome, pinpointing the tumor suppressor locus in the vicinity of the The p16 pathway, which has been found to be altered in more than 80% of human cancers In contrast to other solid tumors, progression of meningiomas is correlated with increasing hypodiploidy, showing characteristic clonal evolutions that mostly include chromosomes 14,18, and 19 and, more rarely, 6 and 10. Structural aberrations are rare, except for the loss of the short arm of one chromosome 1, which appears to be the decisive step for anaplastic growth In 2001, Zang reported a genetic model of tumor progression in meningiomas Since the publication of Ishino and coworkers in 1998 the fluorescence in situ hybridization (FISH) technique has provided new insights into interphase cytogenetics and is applicable now to clinical practice. Therefore the time-consuming cell culture for conventional chromosome analysis is not further necessary. Instead of paraffin embedded tumor samples, also fresh tumor samples that are speckled on microscope slides can be used.TIMP3 and p16 in meningiomas in order to evaluate the impact of these markers for the biological behaviour of meningiomas.In the current study we examined 124 meningioma samples out of 105 prospective meningioma patients for the partial loss on chromosomes 1p, 9p and 22q, using double-target iFISH. The first aim of this study was to analyze if the iFISH results predict malignant behavior and recurrence of meningiomas as well as conventional cytogenetic analysis. The second aim of this study was to examine the promoter hypermethylation of We performed a prospective study on 124 tumor samples from 105 meningioma patients [76 women and 29 men] operated at the Department of Neurosurgery, Saarland University, between January 1997 and December 2010. The average age of the overall patient population at the date of first surgery was 57.2 years [SD  = 13.3 years], for female patients 58.1 years [SD  = 12.0 years], and for male patients 54.8 years [SD  = 16.3 years]. Written informed consent was obtained from each patient participating in the study.Patients were examined in the neurosurgical outpatient department of the Saarland University, either within routine follow-up or upon appearance of neurological symptoms. A recurrence was defined as new evidence of tumor in CT or NMR after previous complete extirpation (Simpson grades 1 and 2) Complete surgical extirpation of the tumor was defined as Simpson grades I and II. This corresponds to a macroscopically complete tumor resection with bipolar coagulation of the dura insertion.Meningioma grade was assessed by a combined histologic All tumors were classified according to the WHO classification of tumors of the nervous system of 2007 In total 124 different probes were investigated from the 105 patients. In 91 tumors (86.6%), only one surgical procedure was performed, in 10 patients (9.5%) 2 surgery procedures, in 3 patients (2.9%) 3 operations due to recurrence, and in 1 patient 4 surgical approaches was necessary.TIMP3.Tissue specimens from tumors were obtained freshly after surgery and dapped on clean microscope slides previously silanized. After Delauney fixation for 30 sec, slides were stored at −20°C until further FISH analysis. The slides were treated with RNAse for thirty minutes at 37°C and placed three times in 2xSSC for five minutes at RT. Cells were digested in 100 ml 0,01 M HCL with 10 mg pepsin  for 1 min and 45 sec at 37°C. Slides were dipped in 1xPBS for 5 min, 4%PFA/1xPBS for 10 min for fixation and 1xPBS for 5 min. They were then dehydrated in 70%, 80%, 95% ethanol and air dried. Dual-probe hybridization was performed using locus-specific probes for 1p36, 22q11 and 9p21 . Target and probe were separately denatured for 5 minutes at 73°C. Target slides were dehydrated in 70%, 80%, 95% ethanol. Then probes were pipette on slides and incubated overnight at 37°C in a humidified chamber. Stringency wash was performed in 0.4xSSC/0.3%SDS for 2 minutes at 73°C and 2xSSC/0.1% SDS at RT. Finally, slides were counterstained with DAPI antifade .At least 200 non-overlapping nuclei per sample were counted for evaluation according to Hopman-criteria Cut-offs for alterations was determined by comparison with normal human lymphocytes control samples at 10% for deletions of 1p36, 22q11 and 9p21.Extraction of genomic DNA from snap frozen tumor tissue was performed with standard methods with samples more than 0.3 g and QIA DNA Mini Kit for samples with less than 0.3 g . DNA-Isolation from peripheral blood leukocytes was performed according to standard protocols Sodium bisulfite treatment of 500 ng DNA was performed with the EZ Methylation Gold™ Kit (Zymo Research Corporation) according to the manufacturer's protocol. PCR analyses were performed with 70 to 80 ng of the bisulfite-modified DNA as template using 0.2 μl (5 U/μl) of HotStar Taq DNA Polymerase  in 1× Qiagen PCR-buffer with 0.25 mmol/l of each dNTP and 0.1 μmol/l of each primer. The primers used in this study are described in the literature by Herman et al. p16 and TIMP3 by sequencing the regions of CpG-Islands as described in previous studies Also, we analysed methylation status of Comparison of survival times between groups defined by methylation status was performed by Kaplan-Meier curves and with two-sided log rank tests. Univariate and multivariate Cox regression analysis was performed to identify significant predictors for survival and tumor recurrence. Stepwise backward selection based on the AIC (Akaike Information Criterion) was used for variable selection for the multivariate Cox model. Effects in all models were quantified by hazard ratio estimates with corresponding 95% confidence intervals.All p-values were calculated with two-sided tests. Median progression free survival rates were calculated using the Kaplan-Meier method.We obtained ethical approvel from local ethics committee for “The role of high risk of local recurrence in meningioma as indicated by the genetic progression score”  involving both, Department of Human Genetics, Saarland University and Neurosurgical Clinic, Saarland, University Medical School.Written informed consent was obtained from each patient participating in the study.We performed a prospective study on 124 tumor samples from 105 meningioma patients. The sex ratio was 2.6:1 in favor of females (76 women and 29 men). The average age of all patients at the date of first surgery was 57.2 years, and females were slightly but non-significant older on average (58.1 years vs. 54.8 years), see In 91 tumors (86.6%), only one surgical procedure was performed, in 10 patients (9.5%) 2, in 3 patients (2.9%) 3, and one patient had 4 times a tumor resection.The histological results showed in 62 cases (59.05%) a WHO grade I tumor, in 35 cases (33.33%) a WHO grade II and in 8 cases (7.62%) a WHO grade III. The WHO grade was significantly correlated with tumor recurrence , loss of the chromosomal region 1p36 in 35.2% (37/105) and loss of the chromosomal region 9p21 in 6.67% (7/105).Breaking down the FISH results to the respective WHO grading: monosomy 22 was detected in 45.2% (28/62) of WHO grade I, 48.6% (17/35) in grade II and 75% (6/8) in grade III meningiomas. Univariate as well as multivariate Cox regression analysis revealed no correlation with tumor recurrence , in grade II in 27.1% (13/35) and in anaplastic meningiomas in 87.5% (7/8) of cases. Only 1.6% (1/62) of WHO I meningiomas and 5.7% (2/35) of WHO II meningiomas had a deletion of 9p21. On the other hand 50% (4/8) of anaplastic meningiomas showed this deletion see also .TIMP3 was found in 13.3% (14/105) of all cases. Breaking down the methylation results by the WHO grade, 6 cases (9.68%) belonged to WHO grade I, 7 cases (20%) to WHO grade II and 1 case (12.5%) to the anaplastic meningioma grade III (TIMP3 hypermethylation from grade I (9.68%) to grade II (20%). However due to the small number of cases of anaplastic meningiomas these results turned out to be not significant  on 22q12.3 has been described as a tumorsupressor gene in different tumor entities The gene for the tissue inhibitor of metalloproteinase 3 (TIMP3 gene resulting in silencing the tumorsupressor gene was more frequently detected in meningiomas with loss of 1p. Bello and coworkers found a correlations with loss of the genes THSB1 (47%), TIMP3 (24%), p73 (33%), p14ARF (23%) and p16 (23%). Also meningiomas with loss of 22q demonstrated a higher methylation status in this study p16 in 62% of meningiomas with LOH of NF2 (located on 22q12.2) in contrast to the normal karyotype In previous studies by Bello TIMP3 5′-CpG island region in about 24% of meningiomas, Liu TIMP3 hypermethylation with MS-PCR and direct sodium bisulfite sequencing and revealed TIMP3 methylation in 67% of anaplastic meningiomas but less in atypical (22%) or benign meningiomas (17%).While Bello and colleagues TIMP3 methylation increases from 9.68% in grade I to 20% in grade II meningiomas. However, due to the low rate of the TIMP3 methylation in grade III meningiomas (1/8 caseş12.5%) in our study, overall TIMP3 methylation was not significantly correlated with tumor grade .In our present study we found that the rate of patients with TIMP3 gene is significantly associated with a shorter time to recurrence  and was not concordant with the hypermethylation status of p16. Furthermore our results did not confirm the association of hypermethylation of p16 in meningiomas with LOH of the NF2 gene from the previous study by van Tilborg The hypermethylation status of In The significant association of the deletion of 22q with the deletion of one short arm of one chromosome 1 is in line with our previously published results describing the clonal cytogenetic evolution of meningiomas TIMP3 status was associated with a micro deletion on the long arm of chromosome 22, we could not confirm these results. However, it must be given consideration that the monosomy 22 was ascertained by classic cytogenetic analyses in our study, whereas Barski and coworkers used microsatellite analyses using primers who span the TIMP3 region on chromosome 22 Although Barski et al. found in their study that in 20 of 39 investigated meningiomas the methylation of the After chromosome 22 anomalies, aberrations of the short arm of one chromosome 1 are the most frequent alterations detected by cytogenetic analysis of meningiomas In the present study we confirmed these previous results using the high resolution FISH-technology (iFISH) In Our results are in agreement with former cytogenetic investigations which indicated that the deletion of the distal part of the short arm of a chromosome 1 [1p-] is associated with progression in meningiomas In conclusion, in this study we used iFISH that is easily applicable to clinical practice because it is not very time-consuming and can be applied to various clinical materials, including paraffin embedded tumor samples or dapped slides. Consequently, due to the common feasibility of the iFISH method a multimodal approach to meningioma grading, which is in our opinion the most promising for identifying meningiomas with an increased tendency to recur, is now available for almost all patients.TIMP3 inactivation by methylation seems to be also involved in meningioma progression, at least it is associated with a shorter time to recurrence using the multivariate Cox-regression analysis (p = 0.0475).The Based on the results of this study, the deletion of the short arm of one chromosome 1 is an independent prognostic factor which correlates significantly with increased risk of recurrence. After initial speculation on the role of 1p deletion for tumor recurrence


Chlamydomonasreinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX  to form magnesium-protoporphyrin IX , the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that5A7 is missing theCHLI1 gene and at least eight additional functionally uncharacterized genes.5A7 has an intactCHLI2 gene. Complementation with a functional copy of theCHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in5A7. We have identified the firstchli1 (chli1-1) mutant ofChlamydomonas reinhardtii and in green algae. Our results show that in the wild typeChlamydomonas CHLI2 protein amount is lower than that of CHLI1 and thechli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses theCHLI2 gene. Ourchli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis inChlamydomonas,which has never been studied before.The green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants, can grow photo-autotrophically and heterotrophically (it can metabolize exogenous acetate as a carbon source) and possesses a completely sequenced genome. These attributes make it an elegant model organism to study oxygenic photosynthesis and chloroplast biogenesis. In photosynthetic organisms, tetrapyrroles like Chl and heme are essential for energy metabolism (i.e. photosynthesis and respiration). Biosynthesis of Chl and heme occur via a common branched pathway that involves both nuclear- and chloroplast-encoded enzymes in most photosynthetic organisms. In photosynthetic eukaryotes, 5-aminolevulinic acid (ALA) is synthesized from glutamine through glutamyl-tRNA. Conversion of ALA through several steps yields protoporphyrin IX (PPIX), the last common precursor for both heme and Chl biosynthesis. Ferrochelatase inserts iron in the center of PPIX thus committing it to the heme branch of the pathway. Insertion of Mg2+ in PPIX by MgChel leads to Mgproto, the first biosynthetic intermediate in the Chl branch. Magnesium chelatase has three subunits, which are CHLD, CHLH and CHLI. The ATP-dependent catalytic mechanism of the heterotrimeric MgChel complex includes at least two steps: an activation step, followed by the Mg2+ insertion. Activation of MgChel with ATP involves CHLD and CHLI while CHLH is required for the chelation step. CHLI belongs to the AAA+ family of ATPases. Plants have two isozymes of CHLI1 (CHLI1 and CHLI2) which are 70%–81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. Most of the data on CHLI comes from studies onArabidopsis thaliana chli mutants and the functional significance of CHLI1 and CHLI2 has not been studied in green algae. InArabidopsis CHLI2 plays a limited role in Chl biosynthesis because of its lower expression level compared to that of CHLI1. InArabidopsis the CHLI2 protein amount is lower than that of CHLI1. When overexpressed, CHLI2 can fully rescue anArabidopsis chli1chli2 double mutant.The green micro-algachli1-1) mutant ofChlamydomonas reinhardtii (5A7) which possesses an intactCHLI2 gene. Transformation of5A7 with a functional copy of theCHLI1 gene restored Chl biosynthesis. Western analyses show that the CHLI2 protein level is lower than that of CHLI1 in the wild type strain and CHLI2 protein is barely detectable in the mutant strain. In this study, we present our molecular data on the identification of the mutation locus in5A7 and its complementation.We have isolated the first ,5A7/chli1-1 (generated by our laboratory) andchli1-1 rescued transformants (generated by our laboratory) were grown either in Tris-Acetate Phosphate (TAP) heterotrophic media or in Sueoka’s High Salt (HS) photo-autotrophic media. TAP and HS liquid media and agar plates were prepared in the lab using reagents from Fisher Scientific  according to the protocol given in Gorman and Levine (1965) and Sueoka (1960), respectively. The 4A+ strain andchli1-1 rescued transformants were maintained on TAP agar plates and TAP+zeocin  plates, respectively under dim light intensities (10–15 µmol photons m-2s-1) at 25°C. The final zeocin concentration was 15 µg/ml). Thechli1-1 mutant (5A7) was maintained in the dark on TAP 1.5% agar plates containing 10 µg/ml of paromomycin . Liquid algal cultures used for RNA and genomic DNA extractions and protein analyses were grown in 100 ml flasks on the New Brunswick Scientific Excella E5 platform shaker  at 150 rpm in the dark or in the dim light.Escherichia coli clone harboring the pBC1 plasmid  was used for random DNA insertional mutagenesis. This plasmid contains two antibiotic resistance genes:APHVIII andRAmp  and was used as a selection marker for screening ofChlamydomonas transformants.RAmp was used as a selection marker for screening ofE. coli clones harboring the pBC1 plasmid.E. coli was grown in 1 l of Luria Bertani (LB) broth containing 1% tryptone, 0.5% of yeast extract, 1% NaCl and ampicillin [final concentration of ampicillin:100 µg/ml]. LB reagent was prepared in the laboratory using reagents purchased from Fisher . Ampicillin was purchased from Fisher . The culture was incubated at 37°C overnight. Plasmid purification fromE. coli cells was facilitated by Qiagen plasmid mega kit according to the protocol given in the technical manual . Once purified fromE. coli, the circular pBC1 vector was linearized with the restriction enzymeKpn1  according to the protocol given in the technical manual. The linearized DNA was purified using a QIAEX II gel extraction kit  according to the protocol given in the technical manual. All agarose DNA gel electrophoresis was visualized by BioRad Molecular Imager Gel Doc XR+ . Transformation of parental strain 4A+ by the linearized pBC1 vector was performed utilizing the glass bead transformation technique described by Kindleet al. (1989) and Dentet al. (2005). Transformants were plated onto fresh TAP agar plates containing 10 µg/ml paromomycin (TAP+P) in the dark. Single colonies of mutants were picked and transferred onto fresh TAP+P plates using a numbered grid layout. Screening of photosynthetic and pigment deficient mutants was done by visual inspection and monitoring of growth under different light intensities in heterotrophic, mixotrophic and photo-autotrophic conditions.The purified pBC1 plasmid from the DH5αAmpR .APHVIIIchli1-1 rescued transformants complements and5A7/chli1-1 were grown in TAP liquid media in the dark to a cell density of about 5 × 106 cells/ml of the culture. Genomic DNA was purified using a phenol-chloroform extraction method. RNA extraction was facilitated by TRIzol reagent from Invitrogen  following the protocol in the technical manual. DNA and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer from Thermo Fisher Scientific . DNase treatment was performed using Ambion’s TURBO DNA-free kit from Invitrogen  following the protocol in the technical manual to remove genomic DNA from the RNA preparation. Generation of cDNA was performed using Life Technologies Superscript III First-Strand Synthesis System from Invitrogen  following the protocol in the technical manual.4A+,et al. (2005). This protocol was implemented with one modification of utilizing a non-degenerate primer (AD2) derived from the original degenerate primer (AD) for TAIL PCR as this non-degenerate primer was giving us optimum yield without generating excess nonspecific products. HotStar Taq Plus DNA polymerase kit reagents  were used for PCR. The PCR reaction mixture consisted of 1× PCR buffer, 200 µM of each dNTP, 1× Q-solution, 2.5 units of HotStar Taq Plus DNA polymerase, 60 pmoles of the non-degenerate primer AD2 and 5 pmol of theAPHVIII specific primer. Primers were ordered from IDT . TAIL1 PCR product was diluted 10 and 25-fold and 2 μl of the diluted TAIL1 PCR product was used for TAIL2 PCR reactions. The TAIL2 PCR product was gel purified using QIAEX II gel extraction kit  according to the protocol given in the technical manual. The purified TAIL2 PCR product was sequenced at the UC, Berkeley DNA Sequencing Facility .TAIL  PCR was implemented, following the protocol of DentChlamydomonas genome database inPhytozome. Amplifications of genomic DNA and cDNA were executed using MJ Research PTC-200 Peltier Thermal Cycler . HotStar Taq Plus DNA polymerase kit  was used for PCR following the cycling conditions given in the Qiagen protocol booklet. Annealing temperature was between 55 and 60°C depending on the Tm of the primers. Extension time was varied according to the size of the PCR product amplified. Final extension was set at 72°C for ten minutes. All genomic and reverse transcription PCR products were amplified for a total of thirty-five cycles. 50–150 ng of genomic DNA or cDNA templates were used for PCR reactions. For semi-quantitative RT-PCR usingCHLI1 andCHLI2 gene specific primers, 3 μg of total RNA was converted into cDNA and then 150 ng of cDNA templates were used for RT-PCR. Sequences of primers used for genomic and RT-PCR are shown inPrimers were designed based on genomic DNA sequences available in theEcoR1 andNde1  according to the protocol given in the technical manual. TheCHLI1 cDNA template was amplified using primers given inNdeI andEcoR1 digested)CHLI1 cDNA and theNdeI/EcoRI double-digested pDBle vector was done using the T4 ligase and 1 mM ATP . Chemically competent (CaCl2 treated)E. coli cells were used for transformation. After transformation,E. coli cells were plated on LB+ampicillin (100 µg/ml) plates and incubated at 37°C overnight. Single colonies were picked the next day and plasmids were isolated from these clones. Isolated plasmids were double-digested withEcoR1 andNde1 to verify the cloning of theCHLI1 cDNA. TheCHLI1-pDBle construct from the selected clone was sequenced by the UC, Berkeley DNA Sequencing Facility . Chromas Lite (http://technelysium.com.au/) andBLAST were used to analyze DNA sequences.The pDBle vector  was double-digested with restriction enzymeschli1-1 was performed utilizing the glass bead transformation technique described by Kindleet al. 1989. 2 µg of the linearizedCHLI1-pDBle was used to complementchli1-1. Transformed cells were plated onto fresh TAP plates containing 15 µg/ml zeocin (Z) and placed in the dark at 25°C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potentialchli1-1 rescued transformants. Screening ofchli1-1 rescued transformants was done by visual inspection of green coloration and monitoring growth of light adapted complement strain cells either on TAP in the dark or in the dim light or HS plates under medium light (300 μmol photons m-2s-1).Complementation of theChlamydomonas cells from different strains grown in TAP in the dark were harvested, washed twice with fresh medium and resuspended in TEN buffer . Protein concentrations of samples were determined by the method of Lowryet al. (1951) with bovine serum albumin as standard. Gel lanes were either loaded with an equal amount of Chl (4 μg Chl) or with 40 μg of protein. Resuspended cell suspension was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer  and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smithet al. (1990) using reagents from Fisher . After incubation, the solubilized protein samples were vortexed and spun at a maximum speed of 20,000g in a microcentrifuge for five minutes at 4°C. The soluble fraction was loaded on a "any kD™ Mini-PROTEAN® TGX™ Precast Gel"  and SDS-PAGE analysis was performed according to Laemmli (1970) using a Page Ruler prestained or unstained protein ladder  at a constant current of 80 V for 2 hours. Gels were stained with colloidal Coomassie Gel code blue stain reagent  for protein visualization.Arabidopsis thaliana CHLI1 mature protein that lacks the predicted transit peptide. This antibody is a gift from Dr. Robert Larkin (Michigan State University). CHLI1 primary antibodies were diluted to a ratio of 1:2,000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase  and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit .Electrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P–PVDF membrane  was carried out for 2 hours at a constant current of 400 mA in the transfer buffer . The CHLI1 polyclonal antibody was raised in rabbit against the full lengthg for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer . Chla andb concentrations were determined by Arnon (1949) equations, with corrections as described by Meliset al. (1987).Cell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer . Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifugation at 10,0005A7 was generated by random insertional mutagenesis of theChlamydomonas reinhardtii wild type strain 4A+ (137c genetic background).5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media  PCR method was used.APHVIII gene. The PCR results confirmed that the 850 bp DNA product contains the 3´UTR of theAPHVIII gene  is located on chromosome 6.The linearized plasmid pBC1 was used to generate5A7 . To findII gene . Sequenc asUP6 .UP6 (CrUP6 locus with its eight neighboring genesUP4 (Cre06.g306100),UP3 (Cre06.g306150),UP1 (Cre06.g306250),UP2 (Cre06.g306200)CHLI1 (Cre06.g306300),FDX3 (Cre06.g306350),AMT (g7098) andUP5 (Cre06.g306450). It is to be noted that we have named all of these genes arbitrarily for our study except for theCHLI1 andFDX3 genes, which were annotated in theChlamydomonas genome database. Readers are requested to identify these unknown genes by the gene locus number (Cre or g number) in thePhytozome database. PCR analyses with the genomic DNA of 4A+ and5A7 were performed using primers specific to four neighboring genes upstream of theCHLI1 (includingUP6) and four neighboring genes downstream of theCHLI1 locus  are not known. We do not yet know the exact location of the pUC origin (pUC ori) end of the plasmid ;chli1-1 and 4A+.chli1-1 rescued transformants are able to synthesize Chl, are not light sensitive and are capable of photosynthesis (chli1-1 rescued transformants harbor theBle gene (from the pDBle vector) andAPHVIII gene , they can grow both on zeocin and paromomycin media plates unlikechli1-1 and 4A+  that can be loaded in a mini protein gel, was used .chli1-1 rescued transformants with a CHLI1 antibody show that the CHLI1 protein is absent in thechli1-1 mutant but present in thechli1-1 rescued transformants (Arabidopsis CHLI1 antibody detects both the CHLI1 (40 kDa) and CHLI2 (42 kDa) protein inChlamydomonas as theChlamydomonas CHLI2 has about 62% sequence identity to theArabidopsis CHLI1  analyses of steady state tetrapyrrole intermediates will confirm this hypothesis. mutant . Over-acchli1-1 mutant has a deletion of at least nine genes (including theCHLI1 gene). Currently we are investigating the exact insertion point of the pUC ori end of the plasmid in thechli1-1 genome  Is the low abundance of the CHLI2 protein a general effect or is it a specific effect of thechli1-1 due to the specific absence of CHLI1 or due to the absence of both CHLI1 and the near absence of CHLI2 protein?2) Is the total absence of Chl in strainchli1-1 with antibodies raised against any non-photosynthetic and/or photosynthetic protein. If the low abundance of the CHLI2 protein is due to a general effect of the mutation, there will be an overall reduction of different cellular proteins. The second question can be addressed by overexpressing CHLI2 inchli1-1 to see if Chl biosynthesis can occur in the absence of CHLI1 or by silencing CHLI2 in the wild type strain using RNA interference or micro RNA based techniques.The first question can be addressed by performing Western analyses of. InArabidopsis MgPPIX is hypothesized to be a retrograde signal from the chloroplast to the nucleus on the basis of data obtained with mutants that are defective in the NF, induced down-regulation of the transcription of the light harvesting complex protein B(LHCB) expression [gun (genomes uncoupled) phenotype]. InArabidopsis, there are controversies regarding whetherchli1 mutants aregun mutants. To date inArabidopsis, MgPPIX mediated regulation of genes encoding only photosynthetic or chloroplastic proteins, have been documented. InChlamydomonas, hemin and MgPPIX has been shown to induce global changes in nuclear gene expression inChlamydomonas, unlike that inArabidopsis. InChlamydomonas, the above mentioned tetrapyrroles altered expressions of genes encoding TCA cycle enzymes, heme binding proteins and stress response proteins as well as proteins involved in protein folding and degradation (eg. heat shock proteins). Hence the roles of tetrapyrroles in retrograde signaling appears to be distinct in green algae and in higher plants. In summary, in the future ourchli1-1 mutant can be used to clarify the functional role of CHLI1 and CHLI2 in Chl biosynthesis inC. reinhardtii.Norflurazon (NF) causes photo-oxidative damage to the chloroplast by inhibiting carotenoid biosynthesis. I no longer have any comment nor suggestion. The authors have addressed all my queries and have incorporated my suggested recommendations.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this version of the manuscript, the authors have made all of the suggested revisions that I requested. I have no further requests from the authors.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. In this manuscript, the authors identified a Chl deficient mutant ofChlamydomonas reinhardtii by insertional mutagenesis. I have reviewed the manuscript by GrovensteinArabidopsis possesses two CHLI isoforms, CHLI1 and CHLI2, likeChlamydomonas. However, it is not clear whether these isoforms are paralogous or not among these two photosynthetic organisms. The authors should perform phylogenetic analysis to show whether these isoforms are truly phylogenetically related or not.As described in the manuscript,chli1-1 mutant is Chl deficient, it is reasonable to assume that CHLI2 is not functional inChlamydomonas. In my understanding, the expression of CHLI2 is not affected by the mutation ofCHLI1 gene inArabidopsis. The upper band of Fig.11B could be a non-specific band of CHLI1 protein that hides the original band of CHLI2 in Western blot analysis. In this sense, the expression analysis of CHLI2, as well as functional complementation withCHLI2 in the chli1-1 mutant, are apparently necessary for further understanding.Considering theSpecific comments: CHLI1 andCHLI2. As the authors discussed quantities of these transcripts in the Discussion section (p.10), it is necessary to show the levels of housekeeping genes such as ACTIN8 orUbiquitin10 as loading control.In Fig. 7, the authors performed semi-quantitative RT-PCR analysis ofReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors did incorporate the suggestions from version 1 into version 2 of the article.  On my end,  version 2 is already approved with no more revisions required.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis. Using molecular biology techniques they have provided sufficient evidence that the novel mutant is missing a CHLI1 gene and having an intact CHLI2 gene. Their results also provided evidence that CHLI1 inC. reinhardtii is important in chlorophyll biosynthesis and that the presence of its homolog CHLI2 is not sufficient to make up for the function of CHLI1 in chlorophyll biosynthesis. The growth phenotype analysis of the rescued transformants has shown clearly the role of CHLI1 in chlorophyll biosynthesis. It would be interesting to know what percentage of rescued transformants was in their complementation analysis.   Overall this is an interesting article. More importantly, this work reports the firstchli1 mutant to be identified inC. reinhardtii, as well as in green algae. Thechli1 mutant will open up new avenues to further explore the functional roles of CHLI1 and CHLI2 in chlorophyll biosynthesis in particular inC. reinhardtii.The title is appropriate for the content of the article. In addition, the abstract represents a suitable summary of this interesting work. The authors were able to successfully isolate a novelSuggested revisions:5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media .” *Figure 1 should be Figure 2. On page 6, sentence 3 “It is incapable of photosynthesis and is sensitive to light intensities higher than 20 μmol photons m-2s-1 ,” ---*can be more clearly referred to as Figure 2A instead of Figure 2 only.   On page 8, Figures 5A, 5B and 5C mentioned in text actually refers to Figures 6A, 6B and 6C.On page 6, sentence 2 “Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas mutant deficient in chlorophyll biosynthesis using insertional mutagenesis. Through molecular genetic analyses, the authors demonstrated that the deficiency of theCHLI1 gene, which encodes a subunit of Mg-chelatase, caused the chlorophyll-deficient phenotype. AlthoughChlamydomonas has another homologousCHLI2 gene, this mutant failed to accumulate chlorophylls, suggesting the limited function of CHLI2 on chlorophyll biosynthesis. To clarify whether the reduced expression ofCHLI2 caused chlorophyll deficiency, overexpression ofCHLI2 in the mutant should give further insights into the functionality of CHLI2. In addition, it is desirable to determine the levels of chlorophyll intermediates that are correlated to the photosensitivity of the mutant. In this paper, the authors have successfully identified aSuggested revisions:Chlamydomonas andArabidopsis, I am not sure whether this mutant is useful for analysis of the retrograde signaling. Actually, a number of previous papers suggested distinct roles of tetrapyrroles on nuclear gene expression inChlamydomonas . In this sense, the last paragraph of the Discussion section should be largely revised including already known mechanisms inChlamydomonas cells and not be so focused on Arabidopsis.Considering the distinct regulation of chlorophyll biosynthesis and nuclear gene expression betweenReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Chlamydomonas reinhardtii deficient in chlorophyll biosynthesis. In this paper, the authors describe the isolation and characterization of a mutant ofCHLI1 gene encodes a subunit of the enzyme responsible for inserting Mg2+ into protoporphyrin IX. Interestingly, the chli1 mutant also showed decreased expression of a homolog of CHLI1 and CHLI2. The authors also rescued the chli1 mutant phenotype using a wildtype CHLI1 cDNA transgene. The authors screened a population of Chalamydomonas that was transformed with an insertional mutagenesis vector. They found a chlorophyll deficient mutant that only grew under heterotrophic conditions. The authors provided convincing evidence that this mutant lacked the CHLI1 gene as well as several flanking genes. Thechli1 mutant from a green alga. This mutant provides a starting point for additional genetic and biochemical analyses of chlorophyll biosynthesis inChlamydomonas.  This work is important because it is the first report of aSuggested revisions:chli1-1, since it is the first mutant allele isolated. This sets a precedent for naming new alleles as they are discovered. The authors should change their mutant name toInstead of calling the rescued transformants that contain the wildtype CHLI1 cDNA "complements", the authors should consider calling them "rescued transformants". Some geneticists adhere to a more strict use of the term "complementation" and its derivatives where complementation is done by crossing two mutants, and not by genetic transformation of a mutant using a wildtype gene.Reviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.