Saccharomyces cerevisiae genome for fragments conferring a growth-impairment phenotype identified 714 fragments in about 84,000 clones tested.A screen of the  Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.We have screened the genome of  Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Schizosaccharomyces pombe, have revealed a large number of novel genes of unknown functions. In S. cerevisiae, for example, around 1,800 genes  encode proteins that so far remain functionally uncharacterized (compilation from Saccharomyces Genome Database (SGD) [S. cerevisiae has been extensively studied, serving as a test case for novel and important developments in functional genomics. Such developments include transposon-mediated gene inactivation and tagging [in situ gene tagging [The complete genome sequences of various eukaryotic model organisms such as se (SGD) , the gen tagging , the ana tagging -6, two-h tagging -9, prote tagging ,11, two- tagging , proteom tagging ) and pro tagging . Even in tagging ,16 . The complete list of inserts is described in Additional file 1 and 2, and results are summarized in Table S. cerevisiae . In our screening, 32 ORFs were found twice, two ORFs were found three times and one ORF (YHR056c in the CUP1 region) was found four times, the cloned fragments being either overlapping (22 ORFs) or non-overlapping (13 ORFs). Mean size of the coding region of inserts is 659 bp. The chosen cloning strategy favors recovery of central-or carboxy-terminal coding parts of the natural yeast genes, whereas the amino-terminal coding regions are rare [The 493 inserts corresponding to in-frame ORF fragments represent 454 distinct annotated ORFs , which are randomly distributed throughout the 16 chromosomes of are rare . In our To find possible common characteristics, we have compared between themselves all the peptides encoded by in-frame ORF fragments. BLASTP analysis was combined with detection of characterized conserved domains, of COG patterns (clusters of predicted orthologous groups of proteins ), and ofAs well as comparing inserts to each other, we also analyzed the totality of the conserved domains present in all peptides encoded by the 493 toxic inserts . Characterized domains are found, at least partially, in a total of 281 inserts . Of a total of 183 distinct domains, 46 are represented more than once. We have compared the frequency of these 46 domains among the toxic inserts versus their frequency among the 5,803 ORF-encoded proteins of the entire genome (Table These 37 domains correspond predominantly to various transporter domains (11 cases), such as amino-acid permeases and mitochondrial carrier protein domains. The toxicity of these domains is probably due to the presence of transmembrane spans. Indeed, 132 out of the 493 toxic peptides contain at least two transmembrane spans, including cases where one span is putative . Among these, 63 contain three or more predicted spans and 26 have five spans or more. Putative spans were also recognized in 84 other ORF fragments (seven with at least three spans, 15 with two spans, and 62 with one span) .RNA-and DNA-binding domains (nine cases) involved in replication, transcription or translation functions, such as PUF, KH and rrm, are also much more represented than expected Table . The PUFOther important domains for interactions with polypeptides, phospholipids or small molecules (nine cases) are also over-represented. The WD40 motif, a propeller-like platform for stable or reversible binding of proteins in eukaryotes, has been found in inserts of 12 distinct ORFs . The 12 ORFs code for proteins having interactions with other proteins in complexes related to RNA processing or transcription , and ninThe serine/threonine protein kinase domain (S_TKc) is significantly under-represented in our screen. Among the 10 toxic inserts whose cognate genes code for protein kinases (PK), only four contain this domain . In these four cases, the S_TKc domain is either truncated , or flanked by a coiled-coil region and/or a low-complexity segment. Two other inserts contain the PBD (and PH) domains, and the four remaining inserts contain no characterized domain to date. As it is known that overexpression of some protein kinases is deleterious for cells (see  and refeThe remaining 137 domains (out of 183) were found only once each. Many correspond to functional categories described above, such as transport, metabolism, and interactions with nucleotides, other proteins or other ligands. Seven domains associated with ubiquitination functions were also found . Several of the domains encountered have also been isolated as mammalian genetic suppressor elements (GSEs), which are cDNA fragments that inhibit cell growth . A number of these peptides are highly charged, either negatively or positively . Such charged peptides might interact in an artifactual way with other charged domains of proteins or nucleic acids or with small molecules. Interestingly, the prion-like (Q+N)-rich domain was found in eight of the natural peptides having low-complexity regions.We have seen above that 493/714 toxic inserts are in-frame fragments of protein-coding genes. The complete list of the 454 genes corresponding to these toxic inserts is given in Additional dat file 1 and 2. Their sizes range between 282 bp and 14,733 bp. The mean size of this distribution is 2,401 bp (standard deviation (SD) 1,671 bp), to be compared with a mean size of 1,444 bp (SD 1,094 bp) for the entire set of 5,803 ORFs of the yeast genome. The bias towards longer ORFs is expected from our cloning strategy (see above). Note that the 35 ORFs that we found more than once are nearly randomly distributed in various size classes.S. cerevisiae.We examined the distribution of these genes according to different criteria, such as function, subcellular localization, viability and phylogeny Table  and compAmong the 454 ORFs identified, 91 are unclassified, and function is not yet clear for six others . The remaining ORFs represent a variety of functional classes  among the selected ORFs. This is a significantly higher percentage than in the whole genome, where 939/5,803 genes (16.2%) are essential  are ascomycete-specific and 10 (2.2%) are orphan genes. This distribution is significantly different from the distribution among the 5,803 genes of S. cerevisiae, where 64% of protein-coding genes are conserved , we fineas 106 2% are ascNOP4 gene whose disruption is lethal. Six of these genes are singletons; three others have a paralog already known as toxic upon overexpression. Six out of the 13 still have no known function to date , indicating that the toxic effect is not the result of mislocalization of the overexpressed fragment. The gene MSC1 had already been screened [Schizosaccharomyces pombe and has a role in meiotic recombination. The TRM3 gene contains a carboxy-terminal domain responsible for tRNA methyltransferase activity [To compare the phenotypes conferred by overexpression of the entire gene and of the gene fragment, we have cloned the cognate entire genes of 13 in-frame toxic inserts into the vector pCMha191 . One criterion for the choice of the genes was the absence of a mutant phenotype of the corresponding gene disruption at the time this work was started, except for the screened  as a toxactivity , which iactivity .Additional data file 2 analyzes the 221 other toxic inserts which do not correspond to in-frame fragments of annotated ORFs. Sixty-eight inserts correspond to natural ORF fragments cloned in an antiparallel orientation, most of them being entirely included within the ORF sequence (47 cases), the others overlapping the intergenic upstream region of the natural ORF (17 cases) and sometimes the next gene as well (four cases). Their toxicity can result either from the overexpression of an antisense RNA or from the overexpression of a toxic artificial peptide encoded by a fortuitous ORF. Several arguments favor the second hypothesis. First, short ORFs longer than 24 codons (maximum observed 250 codons), and in-frame with the start codon of the cloning vector, are observed in 53 cases . A number of those artificial ORFs are due to the 'mirror' effect produced by codon-biased natural ORFs ,41. But Fifty-three additional inserts correspond to natural ORF fragments cloned out-of-frame with respect to the plasmid-borne ATG codon, of which only 12 code for artificial ORFs longer than 24 codons  reveals no significant similarity. None of these artificial ORFs corresponds to the 137 new annotated yeast genes of Kumar et al. [et al. [et al. [In total, short artificial peptides may be encoded by 92 out of the 162 inserts described above. Comparison of the 92 peptides between themselves reveals several low-complexity sequences , mostly encoded by antiparallel ORF fragments whose direct amino-acid sequence is itself of low complexity. Comparison with the proteins of r et al. , to the  [et al.  or to th [et al. . Even thAmong the 59 remaining inserts, 17 belong to Ty elements, 10 of which are in-frame ORF fragments corresponding to TyB only ), whereas all antisense fragments (three inserts) correspond to TyA. Y' elements, which are present in 20 copies in the genome, are represented by nine inserts, all coding for highly basic or acidic peptides  which contain repeats of amino acids or motifs, and confer a strongly toxic effect . Considering that these inserts are toxic, their observed number is not different from that expected from the size and number of Y' in the genome.REP1, REP2 and FLP1. The seven other inserts are out-of-frame or antisense fragments of FLP1, or fragments of intergenic regions, all (except two) coding for artificial ORFs. Finally, mtDNA is represented by 12 fragments, mostly corresponding to intergenic regions on the minus strand of the chromosome. Artificial peptides highly enriched in the amino acids tyrosine (Y), isoleucine (I), and lysine (K) are encoded by 10 out of the 12 mitochondrial inserts.Four inserts from yeast chromosome XII are fragments of genes coding for 18S or 25S RNA, two inserts being cloned in the sense orientation. The 2 mm plasmid is represented by 17 fragments, 10 of which are in-frame fragments of ORFs coding for The general fitness of living organisms largely depends on a harmonious equilibrium between the various cellular components and on their capacity to maintain homeostasis. The intricate circuitries that regulate gene expression form the basis of these properties, and massive deregulation of single components may result in flagrant phenotypic defects leading to serious growth impairment or even cell death. Our large-scale screening of the yeast genome using random genomic fragments resulted in a collection of several hundreds of inserts showing toxic effects on cell survival or growth when overexpressed. These toxic effects are expected to result from several distinct molecular situations that have been encountered at various frequencies in our experiments. Of the total of 714 toxic inserts studied, a majority (69%) correspond to the overexpression of fragments originating from natural protein-coding genes . But, interestingly, a large minority (23%) correspond to noncoding DNA fragments. The remaining cases  correspond to fragments of Ty or Y' elements, of the 2 μm plasmid or of mtDNA which, after analysis, can be attributed to one of the two previous categories. Toxic fragments of natural gene products are interesting to consider with respect to the functions of the corresponding genes. But the second category may be even more promising in that it offers us a description of DNA sequences that cannot be overexpressed in a cell without a deleterious effect.TOR1 and TOR2 genes, toxicity is specific to a central domain of the proteins distinct from their carboxy-terminal protein kinase domain; overexpression of the entire gene has no effect, and can even cure the negative effect of the overexpressed domain [et al. [TOR1, TOR2, TEL1 and TRA1 - were selected in this work, all represented by inserts of the central region of these proteins , the ZnF C3H1 domain of CTH1 [GCN4 [The toxicity of coding fragments may result from the imbalance between products of tightly controlled genes, or from the titration of active complexes by the presence of truncated proteins and/or isolated domains. In addition, nonspecific effects might also exist, for example, as a result of an abnormal intracellular localization of an artificially overabundant peptide or protein. We did not attempt to distinguish experimentally between these possibilities for all the coding inserts isolated in this work. Taking into account only specific effects, in the limited number of cases in which the entire gene corresponding to a toxic insert was cloned in the overexpression vector (see Results), we verified that toxicity was due, in most cases, to the disruption of the precise dosage of an essential cellular component  and, in some cases, to the titration effect exerted by the incomplete fragment of the natural protein (the entire protein is not toxic when overexpressed). A few examples where the domain responsible for toxicity upon overexpression is known can be found in the literature. In the case of d domain . Alarcon [et al.  have proe effect . In othe of CTH1  and the H1 [GCN4 . All theEven in the absence of precise mapping of the toxic domain present in our clones, we were able to explore the nature of the domains found in each insert. Our experiment has shown a bias towards domains corresponding to transport functions and to various interactions Table . As mentAs our results also showed a bias towards a number of interaction domains, we have examined the known interactions of the proteins encoded by the 454 genes found in this screen . Genetic interactions were also considered, excluding the coexpression results obtained in microarray experiments. It appears that 88.3% of our genes (401/454, of which 70 are of unknown function) code for proteins which have known genetic or physical interactions, or are members of complexes . Moreover, for 60% of these (242/401), at least one of their known partners is also found in our screen . Among the 53 genes having no known interactions, 24 correspond to transporter or membrane proteins .S. cerevisiae, which had previously identified a total 231 genes or gene fragments that were toxic when overexpressed [et al. [et al. experiment. Common biases are, however, observed in favor of transcription, cell-cycle and cellular transport genes. Overall, only 33 of our 454 ORFs were previously identified by the previous authors . Twenty-five other genes from the previous screenings not found here are members of paralogous gene families represented in our work . The limited overlap may result from partial genome coverage. However, by screening 84,086 clones , we must have encountered a total of 4,677 ORFs, each being represented 1.6 times as an ORF fragment . We have thus screened for toxicity around 80% of the natural yeast ORFs. But the limited overlap of results may also be explained by the experimental bias introduced by each technique. The previous experiments were mostly based on cDNA cloning, which favors short and highly expressed genes, whereas our genomic library favors large ORFs (mean size 800 ± 557 codons per ORF) and has no expression bias. In addition, the largest previous experiment [The biases we have observed show little overlap with previous screenings of xpressed ,28-30,49S. cerevisiae. A collection of toxic polypeptides, acting as genetic suppressor elements and interfering with major cellular functions, is of interest not only in antifungal research but also as a means of identifying new domains with major physiological roles.The finding of a large minority of toxic inserts corresponding to noncoding DNA is puzzling. Indeed, some of the toxic inserts originate from annotated but questionable ORFs, and some originate from antisense or intergenic fragments which can artificially be translated into small ORFs. None of these peptides has recognizable characterized domains, but many of them are charged, mostly positively  and some have amino-acid sequences of low complexity. It could be proposed that all these small ORFs represent a reservoir of potentially new gene sequences in the genome. In addition, 100 of the in-frame toxic inserts had no characterized domains and sometimes no predicted secondary structure. These inserts do not contain conserved domains, COGs or TMS, and are not biased in amino-acid composition . They may correspond to domains that have not yet been described, or to domains whose structure has diverged, but another possibility would be that some protein domains are perhaps not structured in a permanent way before evolving towards a structurally functional domain. Interestingly, a significant proportion of the expressed peptides we selected are specific to ascomycetes, or are even true orphan genes that have no known homolog in any other species than Finally, given the large number of inserts encoding very short ORFs , we cannot exclude the possibility that some transcripts are toxic through hypothetical mechanisms that may include, for example, nonspecific interactions with other cellular or nuclear complexes or through overloading of some component(s) involved in RNA metabolism.In a large-scale phenotypic screening of overexpressed random DNA fragments, we selected around 470 genes (including Ty, Y' and the 2 μm plasmid) whose domains inhibit or impair growth when overexpressed. Many functional categories are represented, transporter proteins being especially over-represented, and genes of unknown function represent one-fifth of our selection. Our approach gave access to genes controlling intracellular and membrane structures, as well as to genes whose deficiency is compensated for by genetic redundancy. Comparable approaches, using efficient phenotyping technology  and apprWe have carried out an analysis of toxic protein domains, pointing out the importance of binding domains and of protein-protein interactions correlated to regulation of cell growth and cell division. This provides a large body of data for targeting more specific studies on the modular construction of proteins and the role of interaction domains in multicomponent assembly of physiological complexes. Finally, in some cases, the deleterious effects in our system of inserts that encode very short ORFs may suggest that overexpression of some transcripts is also toxic for cell growth.Mata/α, ura3-52/ura3-52, trp1-Δ63/+, leu2-Δ1/+, his3-Δ200/+) [Matα, ura3-Δ851, trp1-Δ63, leu2-Δ1) [Matα, ura3-Δ851, trp1-Δ63, leu2-Δ1, his3-Δ200, ade2-661) (A. Thierry, unpublished work) were used for transformations and growth defect screening. All strains are isogenic derivatives of S288C.Total yeast DNA from strain FY1679 (-Δ200/+)  was a geEscherichia coli DH10B cells (Electromax DH10B, Gibco-BRL).The yeast genomic library was constructed using Yeast cells transformed by pCMha190 recombinants were grown at 30°C on glucose synthetic complete medium lacking uracil (SC - URA) always supplemented with 10 μg/ml doxycycline (Sigma) (uninduced conditions). Phenotypic tests were done on solid medium (12 cm × 12 cm plates) containing 70 ml of SC - URA + 10 μg/ml doxycycline (uninduced conditions) or SC - URA without doxycycline (overexpression conditions). Yeast cells transformed by pCMha191 recombinants were grown at 30°C on SC - tryptophan medium, with or without addition of doxycycline. Plasmid loss was carried out on SC plates containing uracil (50 mg/l) and 0.1% of 5-fluoroorotic acid (5-FOA).URA3 (pCM189, pCM190) or TRP1 (pCM184) as selection markers [BamHI-NotI fragment was replaced by a synthetic linker with ends compatible with these sites and introducing an ATG codon followed by an in-frame HA-tag, a BamHI cloning site, and stop codons in the three frames  by replacement of the centromere and replication origin with a 2 μm plasmid replication origin. This was PCR-amplified from pCMha190 using primers M1 and M2  using Pfu polymerase (Stratagene), and ligated to the 5,953 bp EcoRI-BglII fragment of pCMha184.Plasmids pCMha184, pCMha189, and pCMha190 were derived from the centromeric (pCM184, pCM189) or episomal (pCM190) overexpression vectors, containing a tetracycline-regulatable promoter system and  markers . In the s Figure . The epiMCM1 and AUAI (861 and 285 bp respectively), which are toxic when overexpressed under the control of a GAL1 promoter [MCM1, cloned in the high-copy pCMha190 vector, had a clear and constant toxic effect on yeast growth when overexpressed. We thus decided to build the library into pCMha190 and to choose the MCM1 gene as a control for toxic phenotype in overexpression conditions.The overexpression system was checked by cloning two short genes, promoter . Both geMCM1 control gene were cloned into the BamHI digested plasmid pCMha191 (TRP1 marker). Genes were PCR-amplified from genomic DNA . For each gene, two independent plasmids were transformed into yeast strain FYBL2-5D. In parallel, the same strain was transformed with the plasmids bearing the corresponding toxic inserts.Thirteen complete genes corresponding to 13 selected toxic inserts (see Results) and the YGL039w and YAL062w/GDH3 ORFs, were modified by PCR synthesis , then recloned in vivo into pCMha190 using homologous recombination [Two toxic inserts, 156C1 and 57B6, which are antiparallel fragments of bination  in yeastThe adaptor-based strategy ,54 was uSonicated total yeast DNA fragments from FY1679 ranging in size from 200 to 1,200 bp were treated with mung-bean nuclease, T4 DNA polymerase and Klenow enzyme following the manufacturers' protocols. Blunt ends of DNA fragments were ligated to the following adaptor:5'-pATCCCGGACGAAGGCC-3'3'-GGCCTGCTTCCGG-5'.BamHI and filled in with dGTP by the Vent (exo-) polymerase (New England Biolabs) was ligated to the purified adaptor-DNA inserts . The ligation result is drawn in Figure Excess of unligated adaptors and small adaptor-DNA fragments were eliminated by two consecutive purifications using Chroma spin+TE-400 columns (Clontech). Vector predigested with E. coli DH10B cells were performed with 1.8 μl of ligation mix and plated onto 2YT medium (16.1 g/l Bacto tryptone, 10.1 g/l Bacto yeast extract, 5 g/l NaCl, 15 g/l Bacto agar) containing 100 μg/ml ampicillin (four 12 × 12 cm plates per transformation) giving 25,000 to 45,000 clones per transformation.Electroporations of 40 μl of A total of 51 independent transformations were made. This corresponds to 1,888,000 clones. We tested 150 clones for the presence of an insert and observed that more than 85% contained one (average size 700 bp, minimum 220 bp, maximum 1,620 bp). Colonies from each transformation were pooled and distinct Qiagen Tip 500 DNA preparations were made and stored separately for yeast transformation. Final concentration of DNA was 300 to 1,300 ng/μl. The detailed protocol of library construction is available on request.Another library had previously been constructed with the same vector ligated to a distinct DNA-adaptor preparation and was partially used, giving rise to 160,000 primary clones. Characteristics of the transformants were the same as described above. Eight pools of plasmid DNA were prepared from this first library.We carried out a total of 28 independent transformations of yeast by the LiAc method : five wiMCM1, respectively) were also inoculated into each microplate. Cultures were grown overnight at 30°C and stored at 4°C before dilutions for phenotypic examination. Screening of the toxic phenotypes after overexpression was done in a two-round selection, using the 'drop test', which allowed us to see even slightly impaired growth effects. Ten-fold serial dilutions in water were made from each 96-well culture microplate with a Beckman Biomek 2000 robot, then manually replicated with the 96-pin Beckman replicator onto SC - URA + doxycycline and SC - URA plates in parallel  or with the Q-Pix robot (54,071 clones) for overnight growth. Non-toxic and toxic control clones  and RNA were examined manually for their position relative to the coding sequences.S. cerevisiae annotated ORFs and to the 308,738 sequences of our internal database (see below).Sequences of inserts other than in-frame ORF fragments were systematically translated into amino-acid sequences from the junction with the adaptor up to the first stop codon encountered in the insert. Sequences coding for more than 24 amino acids were internally compared using BLASTP, then compared to the et al. [S. cerevisiae was filtered to eliminate all spurious ORFs or unlikely real genes, as well as Ty, Y' and mitochondrial ORFs, yielding a final list of 5,803 ORFs [Genetic entities corresponding to the toxic inserts were identified by comparison with the DNA sequences of the 16 chromosomes (available in the Comprehensive Yeast Genome Database (CYGD) at MIPS ); with oet al. ; with thet al. ; and witet al. . The set803 ORFs . For all803 ORFs  containi-3 were examined individually before validation.Comparisons among the peptides encoded by in-frame ORF fragments were done using BLASTP . Alignme-4 for most domains, and 10-3 for short domains (60 amino acids or fewer). Domains were considered as present even when represented only partially. In describing genes  were done using TopPredII  implemenS. cerevisiae [Descriptions of selected genes and their products were retrieved from the Yeast Proteome Database   and in protein complexes using three different sources: YPD  files foet al. ; data coet al. , MIPS [3et al.  and unpuPx of encountering any gene x times is described by a Poisson distribution:We consider that our library contains DNA fragments randomly distributed throughout the genome. Out of 84,086 clones tested, 11% (9,530) contain a DNA fragment cloned in-frame with the frame of the natural ORF , the others containing noncoding, out-of-frame or antisense DNA fragments. If we use the simplifying assumption that all genes are equally represented among the 9,530 clones (not taking into account the size diversity of genes), each of the 5,803 ORFs will be represented 1.64 times (9,530/5,803). The probability m, the mean of the distribution, is 1.64. This is used to estimate the fraction of genes not encountered: for x = 0 and probability p = 0.19, the number of non-encountered genes = 1,126. Thus, by screening a total of 84,086 clones, we have encountered a maximum of 4,677 ORFs (5,803 - 1,126).where The following additional data are available with the online version of this article. Additional data file Lists and coordinates of the 493 in-frame fragments of annotated ORFs giving toxic phenotypes when overexpressed, and short description of their cognate genesClick here for additional data fileA list and description of the 221 DNA toxic inserts other than in-frame ORF fragmentsClick here for additional data fileA description of the peptides encoded by the 493 toxic ORF fragments, and of the cognate proteinsClick here for additional data fileThe content of the 57 groups of peptide inserts sharing similaritiesClick here for additional data fileA list and description of protein domains found only once among the toxic insertsClick here for additional data fileThe genes selected in this work whose products are members of complexesClick here for additional data fileGenes selected in this work whose products are known as interacting with each otherClick here for additional data fileThe sequences of the oligonucleotides used in this workClick here for additional data fileA figure showing the phenotypes induced by overexpression of antiparallel ORF fragments before and after introduction of a stop codon upstream of the artificial ORFsClick here for additional data file


Health-related quality of life instruments are expected to be of particular value in routine care of people with multiple sclerosis (MS), where they may facilitate the detection of disease aspects that would otherwise go unrecognised, help clinicians appreciate patient priorities particularly in terms of treatment goals, facilitate physician-patient communication, and promote shared decision-making. However, it appears that these instruments are little used routine clinical approaches to people with MS. To address this issue, I performed a bibliographic search of studies that evaluated the efficacy of generic or disease-specific health-related quality of life (HRQOL) instruments in MS clinical practice from clinicians' or patients' perspectives. I found only one cross-sectional study, which compared preferences for three instruments, and assessed acceptability in people with MS.Reasons for lack of transfer of HRQOL measurements to clinical practice may be cultural, methodological, or practical. With regard to MS, the proliferation of instruments seems to constitute a barrier, with no particular instrument having gained wide popularity or consensus. Other barriers are lack of resources for the administration, collection and storage of the data, and inability of clinicians to score, interpret, and use HRQOL instrument to guide clinical care. It is therefore important to refine existing tools, extending clinical validation to wider contexts and cultures. More studies assessing acceptability and clinicians' and patients' preferences for different instruments are also required. Multiple sclerosis (MS) is a demyelinating disease of the central nervous system of unknown etiology and poorly understood pathogenesis. There is a north-south gradient of MS prevalence in the northern hemisphere, with highest levels (over 100 per 100,000) in northern regions . It is aInterest in measuring outcomes in MS has increased markedly over the past 20 years. Standardised instruments have been developed, the most-used being the Expanded Disability Status Scale (EDSS)  which isMore recently, the importance of MS outcome assessment from the perspective of the person with the disease has been recognised . After 1HRQOL studies in MS have drawn attention to the multiplicity of domains that may be compromised by the disease, and the effects of this compromise on ability to cope. As expected, people with MS, especially those with a progressive course, report reduced physical functioning compared to the general population ,11,29-31The ultimate aim of measuring HRQOL is to provide a comprehensive assessment of patients' health status, to serve as a baseline from which to tailor interventions, pharmacological or otherwise, and assess their effectiveness, both in the clinical trial setting and in routine care. HRQOL instruments are expected to be of particular value in routine care, where they may improve the detection of disease aspects that would otherwise go unrecognised, help clinicians appreciate patient priorities particularly in terms of treatment goals, facilitate physician-patient communication, and promote shared decision making. In addition HRQOL data from clinical trials can provide information that clinicians can usefully discuss with their patients . UnfortuIt appears that HRQOL instruments are little used in routine clinical approaches to people with MS. To address this issue, I searched MEDLINE (1966–2004), the Cochrane Library (Issue 1, 2005) and the Cochrane MS Group trials register (2004) for studies that evaluated the efficacy of generic or MS-specific HRQOL instruments in clinical practice from the clinicians' or MS patients' perspective, also checking study references. Studies considering patient-reported outcomes other than HRQOL, and domain-specific measures were excluded.I found only one study, a cross-sectional postal survey conducted in Canada, published in 2004 . This stThe reasons for lack of transfer of HRQOL assessment into clinical practice may be cultural, practical, or methodological -43. WithPractical considerations be particularly important in clinical settings, where data must be provided promptly and in an understandable manner to be of use. Instruments must be administered, processed, scored, stored and retrieved – all of which have logistic and financial implications . Most HRAnother factor limiting the dissemination of HRQOL tools in MS clinical practice is likely to be that too many instruments are available, and unlike EDSS, none has emerged as clearly superior to any other.Existing HRQOL tools for people with MS should be refined and their clinical validation pursued in the widest possible cultural context. More studies assessing instrument acceptability and preferences of clinicians and people with MS are also needed. It would be useful for example to implement computer-based technology (touch-screens and adaptive administration to reduce respondent burden by selecting pertinent items and omitting inappropriate ones) and other alternatives to traditional paper-and-pencil or interview methods, which should of course be evaluated for acceptability and reliability . The obj


Drosophila serve as a wonderful model for studying aspects of innate immunity, i.e. the physical, cellular, and molecular features that provide the first lines of defense against infections in flies and man Drosophila melanogaster (http://flybase.bio.indiana.edu/). Components in these pathways perform the same biochemical functions and act in the same order in both fruitfly and mammalian cells.Organisms of vastly differing morphologies, ecologies, and behaviors—such as fruit flies and humans—are now known to share a multitude of molecular, cellular, and developmental processes. Not only is there extensive similarity in the sequences of fly and human genes, but in addition, almost all of the proteins and major signal transduction pathways that control cell division and differentiation in mammals are also found in the fruitfly Evolutionary conservation is of considerable practical and theoretical importance to biologists. First, it provides a valuable source of data for the reconstruction of phylogeny . Evoluti“Innate” immunity refers to the variety of physical, cellular, and molecular features that provide the first lines of defense against infections. The relatively quick innate immune responses operate along with slower but more targeted adaptive immune responses that generate antigen-specific mechanisms that eventually lead to the destruction and elimination of the pathogen.In mammals, the skin and the epithelial lining of the mucosal tissues act as the primary nonspecific barriers, impeding infectious agents from entering the body. The mucous membrane barrier traps microorganisms, and the cilia present on the epithelial cells assist in sweeping the microbes towards the external openings of the respiratory and gastrointestinal tracts. If infectious agents gain entry into the body, internal innate immune responses become activated and rapidly eliminate the infection. Internal innate immune agents and responses include (amongst others) low pH of the stomach and vagina, proteolytic enzymes and bile in the small intestine, and phagocytosis.Phagocytosis is a fundamental innate immune mechanism carried out by a number of different cell types, including macrophages. Specific macrophage subpopulations are associated with different tissues . Their main function is to consume microorganisms, other foreign substances, and old, dying cells.Innate immunity is present from birth, and the information for innate immune responses is inherited. Cells in the mammalian innate immune system (e.g., macrophages) detect “microbial nonself” by recognizing pathogen-associated molecular patterns PAMPs; . PAMPs aAdaptive immunity is characterized by greater specificity than innate immunity, as the adaptive immune response can not only distinguish foreign cells from self, but can also distinguish one foreign antigen from another. Another hallmark of adaptive immunity is memory, which enables the body to remember specific adaptive responses in response to specific antigens. Immunological memory allows the body to make a greater and more rapid second response when the body is reinfected by the same pathogen. Immunological memory underlies both immunization and resistance to reinfection, conferring a tremendous evolutionary advantage to vertebrates. The adaptive immune response has nearly infinite flexibility: the T and B lymphocytes of the acquired immune system can rearrange the elements of their immunoglobulin and T-cell receptor genes to create billions of clones with distinct antigen receptors. In organisms where both innate and acquired immune systems are present, there is a clear interdependence between the two systems. For a fully functional immune system, these components must act in synergy.Drosophila melanogaster serves as a wonderful model for studying aspects of the innate immune system that might otherwise be obscured by the actions of the adaptive immune response. Insects defend themselves against parasites and pathogens by invoking a multitude of innate immune responses .Because it lacks an adaptive immune response, esponses . Like huPLoS Biology, Drosophila. The mammalian ortholog of Collier (Early B-cell Factor) is involved in B-cell differentiation in mice.Once within the body cavity, microbes may be consumed by the phagocytic blood cells called plasmatocytes . Larger Drosophila hemolymph also coagulates and participates in host defense and wound healing  make the fly susceptible to fungal or gram-positive bacterial infections. However, Toll does not act as a pattern recognition receptor in the fly; instead its activation depends on the presence of the processed (active) form of the growth-factor-like polypeptide Spätzle. Processing of Spätzle depends on a serpin-controlled proteolytic cascade (The eceptors . Mutatio cascade .Drosophila Toll pathway were identified in earlier genetic screens for developmental mutants, those in the Imd pathway have been the focus of more recent studies, mainly in the context of Drosophila immunity (While components of the immunity . The effimmunity . Using aDrosophila is now guiding scientists to explore the immune system of other insects such as the mosquito, Anopheles gambiae, that spreads human malaria. Immune responses in this mosquito are linked to the elimination of the malarial parasites (Anopheles and Drosophila reveals the presence of the Toll signaling pathway in the mosquito genome, even though there are some differences in genes encoding pathogen recognition and signal transduction molecules (The impressive progress in our understanding of innate immunity in arasites . A compaolecules . A detaiThe homologs of many genes involved in innate immune responses in flies and humans have also been found in mice, sharks, nematodes, and plants e.g., . In spec


The discovery of experience-dependent brain reactivation during both slow-wave (SW) and rapid eye-movement (REM) sleep led to the notion that the consolidation of recently acquired memory traces requires neural replay during sleep. To date, however, several observations continue to undermine this hypothesis. To address some of these objections, we investigated the effects of a transient novel experience on the long-term evolution of ongoing neuronal activity in the rat forebrain. We observed that spatiotemporal patterns of neuronal ensemble activity originally produced by the tactile exploration of novel objects recurred for up to 48 h in the cerebral cortex, hippocampus, putamen, and thalamus. This novelty-induced recurrence was characterized by low but significant correlations values. Nearly identical results were found for neuronal activity sampled when animals were moving between objects without touching them. In contrast, negligible recurrence was observed for neuronal patterns obtained when animals explored a familiar environment. While the reverberation of past patterns of neuronal activity was strongest during SW sleep, waking was correlated with a decrease of neuronal reverberation. REM sleep showed more variable results across animals. In contrast with data from hippocampal place cells, we found no evidence of time compression or expansion of neuronal reverberation in any of the sampled forebrain areas. Our results indicate that persistent experience-dependent neuronal reverberation is a general property of multiple forebrain structures. It does not consist of an exact replay of previous activity, but instead it defines a mild and consistent bias towards salient neural ensemble firing patterns. These results are compatible with a slow and progressive process of memory consolidation, reflecting novelty-related neuronal ensemble relationships that seem to be context- rather than stimulus-specific. Based on our current and previous results, we propose that the two major phases of sleep play distinct and complementary roles in memory consolidation: pretranscriptional recall during SW sleep and transcriptional storage during REM sleep. Rats exposed to novel objects during periods of wakefulness generate neural activity that is correlated with patterns observed in subsequent sleep episodes Sleep is important for the consolidation of newly acquired memories . The disstrictu sensu neuronal reactivation during sleep in mammals has only been investigated in the hippocampocortical loop  representing larger-scale neural rhythms were simultaneously recorded from four different brain regions: HP, primary somatosensory “barrel field” CX, ventral posteromedial thalamic nucleus (TH), and putamen (PU)  and S7. In our studies, neural signals were continuously recorded across the natural sleep–wake cycle for 48–96 h, with a single 1-h exposure to four complex objects placed in the four corners of the recording box C. All obn= five templates per animal). Control templates were selected from epochs of alert WK 24 h or 48 h before novel stimulation , during which familiar tactile stimulation was produced by the contact of whiskers with the smooth walls of the recording box, to which animals had been habituated. Templates were matched against the entire record of neuronal activity using the neuronal ensemble correlation method . This indicates that the neuronal firing patterns concomitant with novel stimulation persisted significantly more during the ensuing time than patterns sampled 24 h or 48 h before novel stimulation, when animals were in the same behavioral state , but without novel objects to explore. The effect was independently observed, to a variable degree, in all the five animals studied (Bonferroni comparison, p < 0.01). First, we tested whether the neuronal ensemble correlation method could detect any trace of neuronal reverberation lasting at least more than 1 h after exposure to novel stimulation. For this, we examined correlation profiles obtained for all recorded neurons (three to four brain areas pooled together) in each animal. As shown in p values generated by Bonferroni comparisons between post- and prenovelty correlation distributions . This indicates that the neuronal changes associated with exposure to novel stimuli were highly distributed through the neuronal populations sampled. Furthermore, judging by the maximum neuronal ensemble correlations observed . Indeed, experience-dependent changes were not statistically different across different forebrain areas (ANOVA, F = 0.24, d.f. 3, p = 0.86). The temporal evolution of p values (Bonferroni comparison) associated with single-area correlation profiles shows that significant reverberation was present in 16 of 18 recording sites for several hours after exposure to novel stimulation , with SW ratios being significantly higher than those of both WK (Bonferroni comparison, p < 0.05) and REM (Bonferroni comparison, p < 0.003). Indeed, significant state-specific differences in post-/prenovelty correlation ratios were individually detected in four of five animals .Single-area results indicate that neuronal ensemble correlations often peak during discrete epochs that last a few hours. We also noticed marked oscillations of the correlation trace in several recorded sites (e.g., rat 5, CX). These observations suggest that some underlying biological process, with slow evolution but with sharp phase transitions, governs the long-term reverberation of neuronal firing patterns. To test whether transitions in the wake–sleep cycle could amount for these effects, we investigated how experience-dependent changes in neuronal correlations varied across the three major rat behavioral states: WK, SW sleep, and REM sleep. A comparison across states of post-/prenovelty correlation ratios calculated from averages of entire recordings indicated a significant state-specific effect . Next, we tested the possibility reported in hippocampal place cells  that expp values can be observed for speed factors 10 times and 20 times faster than normal WK rates, while speed factors near the physiological range (2×–0.5×) show stronger and similar effects. This was the case even in the HP, in contrast with previous findings in hippocampal place cells recorded in highly trained animals performing a spatial navigation task  are substantially less detectable over time.Our results fend off three major objections to the notion that neuronal reverberation during sleep may underlie memory consolidation. First, significant experience-depen-dent changes in neuronal ensemble correlations can be tracked as late as 48 h after the reference novel experience, being therefore compatible with memory impairment effects of sleep deprivation applied hours or days after training . Second,An important result of the present study is the inverse correlation between neuronal correlations and concurrent firing rates. Although all neuronal activity templates were taken from epochs of high arousal WK when firing rates were generally high, their neuronal reverberation during subsequent WK was not very prominent , agreeing with values previously reported for pairwise  or many-A third important point regards the observation that neural activity sampled when animals were aroused, but not touching the objects, yielded neuronal reverberation that was nearly identical to that obtained when animals made sensory contact with the objects. This indicates that the kind of experience-dependent neuronal reverberation detected by the neuronal ensemble correlation method  does notIt has been suggested that the neuronal reverberation of newly acquired synaptic changes during SW sleep may lead to the recall and storage of new memories by way of “calcium-mediated intracellular cascades” capable of opening the “molecular gates to plasticity” . This hyThe fact that neuronal reverberation is sustained for long epochs during SW sleep suggests that unconsolidated synaptic changes may not only be recalled, but also amplified over time during SW sleep. Indeed, a progressive increase of neuronal correlations across single SW sleep episodes was often observed  so as to reach steady-state behavioral activity and baseline wake–sleep cycles. Behaviors were continuously recorded by way of two infrared-sensitive CCD video-cameras; infrared illumination was used to monitor behavior when visible lights were off. Behavioral states were coded as WK, SW sleep, or REM sleep based on a spectral analysis of LFPs and visual inspection of videotaped behaviors, according to previously described criteria  Figure .TM (MathWorks, Natick, Massachusetts, United States) code running in a computer cluster comprising 32 CPU (EvolocityTM, LNXI, Sandy, Utah, United States). Neuronal ensemble correlations were calculated following the method in CxN matrices corresponding to simultaneously recorded spike trains of C neurons binned into N intervals of a certain length. Our results were obtained using 9 s-long templates binned with 250 ms-long bins with as many neurons as were available for the brain areas in question. Hence, each template pattern yielded a Cx36 matrix. Target data comprising 24 h or 48 h were sparsely sampled (every 30 s) and binned into matrices of the same dimensions. For a C×N template data matrix x and a target data matrix y sampled at time t, the ensemble correlation index Ct is obtained by:Data were processed and analyzed by custom-made MATLABwherec, y¯, and σy).(the same applies to YTM (SAS, Cary, North Carolina, United States) and MATLABTM software were used for descriptive statistics and hypothesis testing.Templates were selected by careful scrutiny of behavior recorded on videotapes and of the corresponding spectral characteristics of hippocampal LFPs, so as to assure sampling during alert WK . ParticuFigure S1Four different objects were used to produce CSS.(9.7 MB PPT).Click here for additional data file.Figure S2All animals were highly habituated to the recording box, so that exposure to novel complex objects caused a general increase in the animals' arousal. Four of five animals showed an increase in time spent in WK with respect to SW and REM sleep during CSS (A), as compared to adjacent pre- and postnovelty periods of equal length (60 min). The only exception was rat 1, which showed nevertheless a marked exploratory drive, spending nearly 20% of the exposure period in direct whisker-contact with the objects (B). Individual object preferences were moderately varied, as indicated in (C).(746 KB PPT).Click here for additional data file.Figure S3Teflon-coated tungsten wires  were assembled in multielectrode arrays shaped to fit different neuroanatomical targets.(282 KB PPT).Click here for additional data file.Figure S4Frontal brain sections stained for cresyl-violet were used to determine the sites of electrode placement. Electrode tracks, tissue scars, and reference electrolytic lesions performed a few days before sacrifice were used to delimit the implant sites, indicated in red in the figure below. Numbers on the right represent standard AP coordinates  in milli(6.3 MB PPT).Click here for additional data file.Figure S5To record neuronal activity, differentiated neural signal was preamplified (2,000×–32,000×) and digitized at 40 KHz. Up to four neuronal action potentials per recording channel were sorted online (SortClient 2002, Plexon Inc.) and validated by offline analysis (Offline Sorter 2.3, Plexon Inc.) according to the following cumulative criteria: voltage thresholds greater than two standard deviations of amplitude distributions; signal-to-noise ratio greater than 2.5 (as verified on the oscilloscope screen); less than 1% of interspike intervals smaller than 1.2 ms; and stereotypy of waveform shapes, as determined by a waveform template algorithm and principal component analysis. In order to continuously record individual neurons for up to 96 h, we used an adaptive algorithm (available on SortClient 2002, Plexon Inc.) that adjusts waveform templates based on the recent accumulated mean shapes (1% of midline every 20 min). This allows for the same neuron to be tracked across consecutive days, as verified by the superimposition of waveforms acquired thoughout the experiment (Wavetracker software, Plexon Inc.).(3 MB PPT).Click here for additional data file.Figure S6Up to 159 neurons were recorded from three to four different brain areas.(1.4 MB PPT).Click here for additional data file.Figure S7LFPs were recorded in parallel with spikes from the same electrodes. Neural signals were split, preamplified (1,000×), and filtered (0.5–400 Hz) by way of a Plexon LFP board. Signals were then fed to the MAP acquisition principal component through a NIDAQ card and digitized at 500 Hz. Behaviors were constantly recorded in videotape by two diametrically opposed infrared-sensitive CCD cameras (model WV-BP332, Panasonic, Laguna, Philippines). A millisecond-precision timer (model VTG-55, For-A Company, Tokyo, Japan) was used to synchronize the acquisition of spikes, LFPs, and videotape records.Behavioral states were identified by the combined inspection of videotapes and the spectral content (1–20 Hz) of LFPs. Behaviors were classified according to the following criteria: (1) alert WK: active exploration with whisking, plus strong hippocampal θ rhythm; (2) quiet WK: stillness or grooming, with eyes open and low-power hippocampal θ rhythm; (3) SW sleep: stillness with eyes closed, plus large-amplitude hippocampal δ rhythm; (4) REM sleep: overall stillness with intermittent whisking, eyes closed, strong hippocampal θ rhythm.The inspection of videotape records readily separates alert and quiet WK from sleep states, but the separation of SW and REM sleep relies strongly on LFP analysis. Hippocampal LFP is particularly useful to disambiguate SW and REM sleep: SW sleep has a strong δ band (2–4 Hz), while REM sleep shows increased θ band (5–8 Hz). The distinction between alert and quiet WK was used only for template selection . For all other purposes, alert and quiet WK data were combined into a single WK category. The graphs depict the θ/δ hippocampal spectral ratios (mean ± SEM) of the three major behavioral states for rat 5 (entire recording).(2.1 MB PPT).Click here for additional data file.Figure S8The figure shows the effect of using different bin sizes to calculate neuronal ensemble correlations. We observed quantitative differences (the larger the bin size, the larger the correlations), but qualitatively the correlations profiles are equivalent, i.e., have very similar shapes.(404 KB PPT).Click here for additional data file.


As the secretory source of vitamins, peptides and hormones for neurons, the choroid plexus (CP) epithelium critically provides substances for brain homeostasis. This distributive process of cerebrospinal fluid (CSF) volume transmission reaches many cellular targets in the CNS. In ageing and ageing-related dementias, the CP-CSF system is less able to regulate brain interstitial fluid. CP primarily generates CSF bulk flow, and so its malfunctioning exacerbates Alzheimers disease (AD). Considerable attention has been devoted to the blood-brain barrier in AD, but more insight is needed on regulatory systems at the human blood-CSF barrier in order to improve epithelial function in severe disease. Using autopsied CP specimens from AD patients, we immunocytochemically examined expression of heat shock proteins (HSP90 and GRP94), fibroblast growth factor receptors (FGFr) and a fluid-regulatory protein (NaK2Cl cotransporter isoform 1 or NKCC1). CP upregulated HSP90, FGFr and NKCC1, even in end-stage AD. These CP adjustments involve growth factors and neuropeptides that help to buffer perturbations in CNS water balance and metabolism. They shed light on CP-CSF system responses to ventriculomegaly and the altered intracranial pressure that occurs in AD and normal pressure hydrocephalus. The ability of injured CP to express key regulatory proteins even at Braak stage V/VI, points to plasticity and function that may be boosted by drug treatment to expedite CSF dynamics. The enhanced expression of human CP 'homeostatic proteins' in AD dementia is discussed in relation to brain deficits and pharmacology. Accumulating evidence supports the idea that continually decreasing choroid plexus (CP) function in advanced ageing exacerbates Alzheimer's disease (AD). As part of a new paradigm to explain brain interstitium deterioration in age-related dementias, increasingly more attention is being paid to the role of compromised blood-CSF  and blooEfficient CSF turnover is essential for a healthy brain. It depends upon an exquisite balance between CSF formation and reabsorption . CompromIn serving the brain's metabolic needs by supplying 'biochemical goods', the CP uses CSF as a conduit for convecting substances . NumerouReduced formation of CSF and stagnated flow in ageing and AD  limits tStructure intimately relates to function in various epithelia. Therefore it is useful to analyze histopathological damage to CP at various stages of AD in order to clarify the onset of functional losses at the blood-CSF barrier. Ageing and AD cause similar degeneration in CP. Structural changes in AD though are usually greater than in non-demented control counterparts . Serot aSeveral features characterizing the CP in AD are highlighted in Table Due to the functional nexus of the CSF with brain interstitial fluid, the neuronal microenvironment in AD is impacted by markedly altered transport and permeability in CP. When neurodegenerative diseases, ischemia ,28 and e+] and immune molecule content of the CNS extracellular fluid [The CP is multifunctional, performing a wide range of homeostatic functions for the CNS . CSF homar fluid . AccordiMoreover to provide steady neuroprotection by regulating the extracellular milieu, the CP must protect itself against various stressor agents that build up during ageing and disease. There have been few investigations of the homeostatic systems within CP that stabilize choroidal functions in the face of ageing and AD. We have explored some candidate systems for compensatory responses by CP. Cytoplasmic heat shock proteins chaperone and perform housekeeping to maintain a healthy steady-state intraepithelial milieu ,32. GrowIt is difficult to functionally assess the CP in vivo ,33, partInformation about functional proteins in human CP is scarce. However protein expression was recently analyzed in autopsied lateral ventricle CP from normal adult brains and in those with confirmed AD ,37. The Ageing and AD dementia tax the CP and other CNS transport interfaces. In late life the deteriorating brain presents many potentially-destructive metabolites to the CSF for multi-site excretion into blood. The greater burden of macromolecule disposal in AD occurs when CP and arachnoid membrane, due to ageing debilities, are less able to transfer solutes. Nevertheless the CP seemingly attempts to maintain its 'epithelial soundness' when challenged to perform additional cleansing acts for the brain extracellular fluid (CSF).In healthly, young adults the CP epithelium sensitively acclimates to chemical and physical distortions in blood, CSF and parenchyma. Following cortical stabbing in rats, the CP upregulates TGFβ presumably to provide this CSF-borne growth factor for repairing the injury . A similAccordingly to assess pathophysiologic consequences of AD we investigated human CP's ability to upregulate certain functional proteins  in advanced states of AD dementia. With advancing knowledge about intricate cell physiology (coordinated interactions among organelles, cytoplasm and membrane-bound proteins) it is relevant to evaluate components of cellular homeostasis. The term 'homeostatic proteins'refers to chaperones, receptors and transporters that stabilize the internal environment of the cell. CSF stability depends in large part upon CP epithelial homeostasis. Our group has analyzed several 'homeostatic proteins' involved in CP intracellular milieu stabilization:A wide spectrum of protection against neurodegeneration is provided by HSPs. The list of protective effects bestowed by HSP molecules is diverse and includes: accelerated degradation of misfolded proteins, maintenance of membrane lipid integrity, prevention of deleterious protein aggregation, and preclusion of damage to the translational apparatus ,42. HSPsTo evaluate human CP expression, we selected HSPs overexpressed in AD brains: GRP94 and HSP90. In contrast to previously found upregulation in brain, there was downregulated GRP94 in lateral ventricle CP. In aged controls there was abundant staining in the epithelial cytoplasm and stroma  ontogeny, ii) normal adult maintenance, and iii) neurodegeneration.FGF and FGFr are also fundamentally important in fostering neuron generation from stem cells in the subventricular zone (SVZ). This requires coordination between the CP-CSF and periventricular regions ,48. Pharnd but not 3rd gestational week [Receptor plasticity in aging and AD is better seen in light of information on FGF/FGFr expression dynamics in early life. In the fetus the formation of CP, neuronal stem cells and brain is promoted by CP growth factor secretion and CSF distribution ,49. InteFGF2 derived from CP is also conveyed by CSF bulk flow to the fetal germinal matrix where it acts on stem cell FGFr to promote neuronal maturation . By thisThe FGF/FGFr system also has a key role in adult CNS fluid homeostasis. CP helps the brain adapt to alterations in blood composition and flow. In otherwise healthy young adults, the imposition of dehydration or sudden ischemia upon the CNS elicits striking adaptive changes in CP epithelium. To endure insults by chemical or physical stressors , it is cDehydration seriously threatens CNS functions. Adjustments to dehydration in the healthy adult brain feature ion and water redistribution among fluid compartments. These compensatory responses to plasma hyperosmolality stabilize neuronal and interstitial volumes. Brain 'barriers' or transport interfaces are sites for the fluid homeostatic mechanisms. A working model for the restoration of fluid balance is offered: Dehydration or hyperosmolality upregulates the FGF2 and AVP peptides in CP ,52. FGF2Ischemia is another disorder with neuropathological consequences that are mitigated by growth factor upregulation or administration . BilaterFGF2 titers and FGFr receptor densities in degenerating CNS compartments provide insight on adaptive responses. FGF2 concentration is augmented in the AD brain . Moreove1-43 [FGF2 has an interesting relationship with amyloid. A worthwhile goal is to probe mechanisms of FGF2 interaction with amyloid in neuronal networks, extracellular matrix and CP-CSF. FGF2 co-localizes with several chemical forms of amyloid. Neuronal coexistence of FGF2 and amyloid precursor protein (APP) ,65 intim1-43  and atte1-43 . In the 1-43 ,70. ThisFGF2-mediated protective regulation of CP transport phenomena potentially affects the course of neurodegeneration. Considerable evidence points to CP's ability to remove Aβ from CSF -73. ThisThe diminished ability of CP to form fluid in advanced ageing and AD begs the question of how epithelial ion transport proteins are altered by distorted neurochemistry in senescence. In very old laboratory mammals the Na-K-ATPase activity of CP and the CSF generated by it are cut in half ,76. AnotThe cation-Cl superfamily of cotransporters includes NKCC1 and consists of 7 isoforms that actively transport Na and/or K electroneutrally with Cl . NKCC1 iThe NKCC1 helps cells and organs adjust to disrupted fluid balance. One thus expects homeostatic upregulation of this CP cotransporter in AD with its altered CSF dynamics. For delineating NKCC1 expression we used the T4 antibody to immunostain CP at various stages of AD dementia. Robust staining of the lateral ventricle plexus (even at Braak stage V/VI) occurred in the apical membrane and cytoplasm , then it might be feasible to prevent the early manifestations of AD (Braak stages I/II) from intensifying into the debilitating pathology of V/VI.One class of agents with considerable potential for AD therapeutics is the growth factor group. A useful paradigm for CSF growth factors and neuroprotection has evolved from experiments on transient forebrain ischemia in rats ,60. The Several other growth factors synthesized by CP and secreted into CSF should be explored in translational research dealing with interacting ischemia, hydrocephalus and AD ,103,104.In searching for agents to modify CP epithelial protein expression, the route of delivery of the active drug is of primary importance. Unlike the brain with its impermeable microvessels, the CP readily takes up water-soluble drugs from the plasma due to the highly-permeable choroidal capillaries. Consequently water-soluble agents freely diffuse to receptors or binding sites at the basolateral surface of the epithelium. Access of blood-borne hydrophilic compounds to the CSF-side of CP however is problematic because the tight junctions and basolateral membrane impede diffusing molecules as small as mannitol . MoleculTo circumvent the blood-CSF barrier, therapeutic agents are delivered into CSF by lateral ventricle catheters in experimental animals  or hydroA compelling aspect of CSF translational research is to identify agents that effectively regulate fluid formation by CP. Whereas the difficulty in congenital hydrocephalus is to downregulate CSF formation, the challenge in AD is to enhance CSF turnover perhaps by accelerating fluid production as well as outflow. Augmented flow of CSF enhances 'sink action' ,109. ThiAnother CP pathology meriting pharmacological attention is the massive fibrosis that progressively envelops the interstitium in old age. This interstitial fibrosis is even more extensive in AD . FibrosiThe CP has the main responsibility for CSF homeostasis. Therefore the functional status of the blood-CSF barrier is of great consequence to the CNS. Maintaining the CSF at a stable, specialized composition is of the utmost importance to neurons. CSF is prominent in regulating brain interstitial fluid with which it exchanges nutrients and waste products. Diseases markedly affect these molecular exchanges. Maintaining healthy bidirectional transport across the CP epithelium (CSF-blood) and ependyma (CSF-brain) is thus integral to a sound brain fluid environment.CSF macrocirculation through the ventriculo-subarachnoid system together with CSF microcirculation in the perivascular Virchow-Robin spaces ,111 perfAs the main generator of CSF, the CP has a pivotal role in helping the brain cope with the twin stressors of ageing and disease. More attention should be focused on the blood-CSF interface for pharmacologic opportunities to stave off CP dysfunction. Our findings on human CP expression of HSP90, FGFr and NKCC1 demonstrate that this epithelium in AD reacts to metabolic insults by upregulating certain proteins. This suggests that even diseased CP could respond to therapeutic agents, thus opening new vistas for treating CSF dysfunction in age-related dementias.Investigation of CP in AD is an area that is opening up. Translational research can now intensely focus on molecular factors that disable the CP to the point of reducing its ability to preserve brain integrity. Systematic CSF analyses using mass spectrometry and other cutting-edge biotechnology should generate neurochemical data specific for disease stages. New imaging approaches are essential to provide much needed functional data for CP, CSF and periventricular regions in AD patients. This should expedite the modeling of CP-CSF malfunctions and their resolution. Deeper insight into the pathophysiology of the blood-CSF transport interface will help to realize the development of novel therapeutic regimens for the AD family of diseases.AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, beta amyloid; AVP, arginine vasopressin; BBB, blood-brain barrier; CP, choroid plexus; FGF2, basic fibroblast growth factor 2; FGFr, receptor for fibroblast growth factor; GRP94, glucose regulatory protein 94; HSP90, heat shock protein 90; NGF, nerve growth factor; NKCC1, Na-K-2Cl cotransporter secretory isoform 1; NPH, normal pressure hydrocephalus; TGFβ, transforming growth factor beta; SVZ, subventricular zone; TFI, transient forebrain ischemia; VEGF, vascular endothelial growth factor; IGF-II, insulin-like growth factor II; HGF, hepatocyte growth factor;The author(s) declare that they have no competing interests.CJ had the primary responsibility of organizing and writing the review, and had NIH support (NS 27601) to do the NaK2Cl cotransporter experiments. PM carried out the NKCC1 immunostaining runs with the T4 antibody and provided interpretation of the regional stainings. RT conducted the experimental analyses of the heat shock proteins and generated figures. AS did the image processing analyses of the cotransporter expression and assisted with the literature analysis. JD contributed ideas for the altered CP-CSF dynamics in hydrocephalus and ventriculomegaly. GS has developed the model that CP-CSF malfunction exacerbates AD progression, and helped to revise the manuscript. ES was responsible for the FGFr experiments and interpreted the human CP data. All authors read and approved the final manuscript.


There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses. Individual doctor attitudes and interest have been demonstrated to influence patient outcomes in some instances. Given the trend towards larger practices and part-time working, continuity of care is likely to fall and thus practice-based rather than individual general practitioner attributes and attitudes are likely to become increasingly important. The aim in this paper was to examine the extent to which individual general practitioner (G.P.) attitudes to the care of people with epilepsy cluster within practices and predict patient-rated quality of care.The sample consisted of 1255 people with active epilepsy (a recent seizure or on anti-convulsant medication for epilepsy) and 199 GPs from 82 general practices. Measures of GP attitudes (a 17-item GP attitudes questionnaire) and patient-rated quality of epilepsy care were obtained. 1210 individuals completed initial questionnaires and 975 patients filled in final questionnaires one year later. Responses were achieved from 64 practices  and 115 GPs .2 main factors were found to underlie GP attitudes to the care of people with epilepsy and these demonstrated clustering within practices "epilepsy viewed as a primary care responsibility"  0.40), and "medication skills". GP-rated scores on "epilepsy care being a primary care responsibility" were a significant predictor of patient-rated quality of GP care (p = 0.031). Other contributory factors were seizure frequency (p = 0.044), and patient-rated "shared decision making" (p = 0.022).Specific general practitioner attitudes to the care of people with epilepsy cluster within practices and are significantly associated with patient-rated quality of epilepsy care. It is important to take these findings into consideration when planning primary care interventions to ensure people with epilepsy receive the benefits of available medical and surgical expertise. There is wide variation in the quality of care provided by primary care practices to individuals with chronic illnesses . IndividThere is a marked trend to larger partnerships in primary care practices and more flexible working practices. It is likely, therefore, that continuity of care will continue to fall, and that patient experience of care of a particular condition will be based on contact with more than one general practitioner . Thus prIn the next few years there is likely to be considerable reorganisation of the way in which epilepsy care within primary care is delivered, with GPs taking on a more active role in providing care. Information on how individual general practitioners view and value their role in providing epilepsy care is considered as important . HoweverIn this paper, data from a completed community-based study on people with epilepsy are used to examine the following questions:1. To what extent do individual general practitioner attitudes to the care of people with epilepsy cluster within practices?2. Do general practitioner attitudes predict how people with epilepsy rate the quality of the general practitioner care of their epilepsy?General practitioners and adults with epilepsy taking part in an intervention study in Greater Manchester provided information for this study. The results of the intervention study  have been reported . No grouEthical approval was obtained in the 4 areas of Greater Manchester from where patients were approached. The patients who consented to participate in the study had their medical records examined to extract data on recording of clinical information about epilepsy and other markers of quality of care. Patients were also sent questionnaires for self-completion. These included both a generic quality of life measure- the EUROQOL 5D and a diGeneral practitioners completed a 17-item GP epilepsy attitudes questionnaire at the end of the study. Responses to items such as "I feel comfortable changing the type of anti epileptic drugs in my patients" were scored using a Likert scale. The attitudes scale was developed and validated for a previous study and results reported in an earlier paper .Mean practice scores for each item on the GP attitudes questionnaire were computed and significant factors underlying the grouping identified . Clustering of responses on the attitudes questionnaire were examined using the intra-class correlation coefficient both for individual items as well as for a mean score of responses to items loading on each of the main factors. Finally linear regression analysis with the patient-rated quality of care provided by the practice as the dependent variable and GP attitudes and other patient derived measures (such as seizure frequency, age, gender, other long term illness) as independent variables was carried out (using aggregated GP attitude and patient scores). Intervention group was adjusted for. P values of 0.05 and 95% confidence intervals were used to assess significance.1255 patients consented to participate in the study and 975 patients filled in final questionnaires. 199 GPs from 82 practices consented to participate in the study. Responses were obtained from 115 GPs  from 64 practices . 29 practices had a single respondent. These practices were excluded from the analysis of attitude clustering. In this study 54% of individuals were seizure-free in the previous year ("controlled" seizures) and 46% had reported a seizure in the previous year ("uncontrolled" seizures).Factor analysis with varimax rotation was undertaken on aggregate GP responses. Four factors had an eigenvalue of above 1.2. Three of these factors were selected after the scree test. Both the Kaiser-Meyer-Olkin measure of Sampling adequacy test (0.778) and Bartletts test for sphericity (Chi-square 413.7, Df 120, p < 0.0001) suggested that factor analysis was appropriate for this data set. Using guidelines for identifying significant factor loadings based on sample size from Hair et al.[Responses to the 11 questions that were included in the first three factors were further examined. The aim was to detect if significant clustering of responses to these items occurred within practices. The average cluster size was 2.74.The results of the factor analysis and clustering of attitudes for the two main factors are listed in Table patient seizure frequency and patient-rated "shared decision making" and GP-rated score on "epilepsy care being a primary care responsibility" (Factor 1). Recording of clinical information about epilepsy was not a significant predictor of patient-rated quality of GP care.Data from 60 practices where both patient and general practitioner data were available were used in the linear regression analysis. As the data were obtained at the end of an intervention study, the intervention group was also included as an independent variable. The results of this analysis are given in Table Some further bivariate analyses were also undertaken. Recording of clinical information about epilepsy by GPs was not significantly associated with the GP-rated score on epilepsy care being a primary care responsibility but was associated with seizure frequency.In this study two main factors ("epilepsy viewed as a primary care responsibility" and "medication skills") were found to underlie GP attitudes to the care of people with epilepsy. Responses to questions constituting these factors demonstrated a high and significant level of clustering within practices. The main factor that accounted for the largest proportion of variance, general practitioner-rated "epilepsy viewed as a primary care responsibility", significantly predicted patient-rated quality of care. Patient-rated shared decision-making and seizure frequency were other significant predictors of patient-rated quality of GP epilepsy care. Recording of clinical information by GPs about epilepsy was not associated with GP attitudes to epilepsy care but was related to patient seizure frequency.In this study general practitioner attitudes to the care of people with epilepsy were found to cluster within practices to a considerable extent. This has not previously been shown in the U.K. A recent Dutch study  showed tThere is relatively little information of the relationship of GP attitudes to patient ratings of the quality of GP epilepsy care. Existing evidence suggests that GPs with a special interest in a particular condition improves outcomes . The resThe results of a multilevel analysis examining patient and doctor predictors of patient satisfaction from the Netherlands  suggestePatient ratings of the quality of care do vary according to whether individuals have controlled or uncontrolled seizures. Individuals with controlled seizures rate the quality of care provided higher than individuals with uncontrolled seizures. However why the ratings of care provided are higher is not clear as individuals with controlled and uncontrolled epilepsy differ from each other in other characteristics that may influence quality ratings apart from seizure frequency .At practice level GP attitudes are not related to mean practice patient seizure frequency. Although it is likely that individual GP attitudes will be influenced by whether an individual patient has controlled or uncontrolled seizures it is not possible to empirically demonstrate this relationship, as nearly all general practitioners will see a mix of individuals with "controlled" and "uncontrolled" seizures in a given year. Their attitudes to the care of people with epilepsy will be influenced by this spectrum of epilepsy severity .In terms of limitations of the results, some practices did not consent to take part in this intervention study and not all GPs who participated completed questionnaires. However there were no significant differences between practices that participated and did not participate in terms of size, average deprivation or training status . MoreoveSpecific general practitioner attitudes to the care of people with epilepsy are significantly associated with patient-rated quality of epilepsy care and cluster within practices. It is important to take these findings into consideration when planning interventions and services. General practitioners need to have good knowledge and skills in the management of epilepsy and should be aware of and utilise current guidelines for good clinical epilepsy care -19 to fuThe author(s) declare that they have no competing interests.AT designed and ran the study, undertook the analysis and wrote the manuscript. MR was involved in the design and running of the study and edited the paper.The pre-publication history for this paper can be accessed here:


Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation.Rad60, Esc2 and mouse NIP45 . Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants.We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs . This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity. Among ubiquitin-related proteins, containing at least one domain with a ubiquitin-like fold, one can distinguish ubiquitin-like modifiers (UBLs) and ubiquitin domain proteins (UDPs) .UBLs can be covalently attached to target proteins analogously to ubiquitin. Unlike ubiquitin, UBLs mostly do not directly target proteins for degradation , althougIn contrast to UBLs, UDPs are not conjugated to other proteins and lack the C-terminal double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. They are a heterogeneous class of usually multi-domain proteins, which are unrelated outside of their ubiquitin-like domain . In seveThe biological relevance of non-conjugatable multi-domain proteins having a domain with clear relationship to UBLs like SUMO, rather than ubiquitin, is yet unknown. Here, we present a detailed sequence analysis of a family of SUMO-like domain proteins (SDPs) containing one or two SUMO-like domains. Members of the proposed RENi family act as factors in transcriptional regulation, chromatin silencing and genomic stability.During the study of the predicted nuclear subset of the Drosophila proteome, we encountered the unknown 424 amino acids long protein CG4449 NP_651134). Initial analysis of its sequence complexity shows that the disordered N-terminal half of the protein is followed by a likely globular segment (predicted using Pdisorder by Softberry, Inc). Indeed, a compositionally biased, polar low-complexity region (LCR) spans almost the entire N-terminal 220 amino acids (AA) as reported by CAST (region 47–165, lysine-rich) [1134. IniThe C-terminal half of CG4449 turns out to contain an internally repeated segment identifiable with RADAR  region .In conclusion, we found that the Drosophila protein CG4449 NP_651134) has a tripartite architecture: with a N-terminal LCR followed by two globular domains with a SUMO-like fold (termed SD1 and SD2). Whereas SD2's similarity to single domain SUMO-like sequences can be easily detected with BLAST tools, the identification of SD1 is non-trivial (Table 134 has aC. briggsae (CAE71155.1, E = 0.001 round 2), H. sapiens (NP_116204.2, E = 0.003 round 2), M. musculus (NP_035030, E = 1e-39 round 3) and C. elegans (NP_497960, E = 2e-12 round 3). All these proteins contain a LCR at the N-terminus followed by two SUMO-like domains, the first of which has mostly diverged away beyond recognition thresholds using traditional sequence-profile searches  including the two SUMO-like domains, collects a family of animal proteins with the same tripartite organization in D. melanogaster CG4449 (220–424) and the corresponding sequences derived from A. gambie, X. laevis, C. elegans, C. briggsae, M. musculus and H. sapiens  A. thaliana (At1g68185.1, NP_564924.1, E = 0.00076), O. sativa (NP_917594.1, E = 0.0019), S. pombe (NP_595995.1, E = 0.00073), S. cerevisiae (NP_010650.1, E = 0.11), Y. lipolytica (CAG82446, E = 0.00087), C. glabrata (CAG57776, E = 0.52) and in other recently published fungi proteomes [S. cerevisiae and S. pombe homologs correspond to the studied Rad60 and Esc2 proteins, respectively [A multiple sequence alignment of the globular C-terminal half of s Figure  was usedroteomes . The S. ectively ,15; the These HMM-search results suggest the most likely plant and lower eukaryote orthologues to the animal NIP45-like proteins. For establishing the orthology relationship, these initial results need to be confirmed by reciprocal searches independently performed for fungal and plant proteins. Further below, we present this evidence for the homology between the C-terminal part of the proteins found in the various taxonomic groups.C. glabrata representative (CAG57776) and retrieving the best and significant hits in the proteomes of S. cerevisiae, S. pombe, K. lactis, C. albicans, Y. lipolytica, D. hansenii, A. nidulans [M. musculus (NP_035030, E = 0.005), A. thaliana (NP_564924, E = 0.016), C. elegans (NP_497960, E = 0.0012).The set of fungal RENi proteins can be autonomously collected using a BLASTP search started with the nidulans . The domnidulans ). A HMM A. thaliana (At1g68185.1, NP_564924.1) against the TIGR Gene indices of barley, maize, rice, potato and soybean [A. thaliana in Y. lipolytica (At vs Yl 1e-07, Yl vs At 6e-04) and H. sapiens proteomes (At vs Hs 3e-05, Hs vs At 8e-05) [Potential plant RENi homologs, derived in a full-length TBLASTN search with  soybean , show a  soybean ) and a ct 8e-05) .We propose to name the collected group of protein sequences the RENi-family after its most studied members Rad60, Esc2 and NIP45. All representatives have a similar sequence architecture involving a N-terminal low complexity region with many polar and (positively) charged residues and a C-terminal globular part with one (plant proteins) or two  SUMO-like domains.The use of a model representing the complete globular region of RENi proteins was essential for the successful collection of the family. A global HMM spanning the SD1 and SD2 domains tests for homology in the whole globular part and, correspondingly, directly collects the RENi family. In contrast, when using the C-terminal half of various RENi family members as query sequence in PSI-BLAST , the seaWhile RENi proteins of the fungal, metazoan and mycetozoan taxa contain two C-terminal SUMO-like domains SD1 and SD2), only the second one can be clearly defined in plant representatives   and rad663E  and A graph representation of the pair-wise similarity relationship for SD1 and SD2 sequences to other known ubiquitin-like domains  is involved in chromatin silencing via the recruitment or stabilization of the Sir (silent information regulators) complex [ complex ,28. It i complex  and, thu complex  and act  complex  as well  complex ,32. With complex  but also complex .NIP45, the one studied RENi in metazoa, has been implied in gene regulation, where it needs its DNA-binding partner NFATp for this activity . StrikinThere is little experimental data on the importance of the predicted SUMO-like domains in RENi proteins. Nevertheless, all listed functions of RENi proteins conform with the known role of SUMO in transcriptional regulation and the control of genome integrity . In the In this report, we use sequence-analytical methods to infer the homology relationships between RENi family members and determine their tripartite (bipartite for plant homologs) domain architecture. A N-terminal polar low-complexity segment and two consecutive SUMO-like domains in the C-terminal half characterize the functionally described fungal and metazoan RENi proteins. While the more C-terminal SD2 is easily detectable, it is the particularly divergent SD1 that was shown in fungi to be essential for the assayed molecular functions. Due to the likely limited sequential-  requirements, this SUMO-like domain is difficult to detect and has been missed in previous analyses of individual family members. The identification of the more N-terminal SUMO-like domain SD1 helps rationalizing experimental findings for mutant fungal RENi family members.RPS-BLAST  and FFASThe sequence analytic work was executed by MN. All authors (MN, AB, BE, FE) contributed to evaluating the results and making the discoveries reported here. MN prepared all the figures and, together with FE, the manuscript text. All authors read and approved the final manuscript.


Nova-1 and Nova-2 genes. Anti-Ri antibodies are typically detected in the serum and cerebrospinal fluids of patients with neurological disorders such as opsoclonus/myoclonus and cerebellar ataxia and in association with gynecologic and breast malignancies.The IgG autoantibody ANNA-2 (anti-Ri) is a type 2 antineuronal antibody that has been found to bind to highly conserved and widely distributed adult brain proteins encoded by the This report describes an unusual example of a 33-year-old female patient who developed short-term memory deficits over a 3-month period. An extensive neurological work-up, including a panel of paraneoplastic markers was negative with the exception of a high titer serum Anti-Ri (1:15,3600). A large left ovarian mass was palpated, surgically resected and eventually diagnosed as a mature cystic teratoma. Post-operatively, memory deficits had disappeared within 1 month and serum Anti-Ri titers had decreased significantly to 1:256. An extensive diagnostic work-up for other malignancies was negative.Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody. Paraneoplastic syndromes (PNS) are signs or symptoms attributable to tissue damage at sites that are remote from a primary malignancy or their metastases. PNS involving virtually every level of the neuro-muscular system have been described -3. TypicA wide variety of anti-neuronal antibodies have been associated with the many PNS-associated neurologic disorders . These aA 33-year-old nulligravid female with no significant past medical history presented to her physician with complaints of a short-term memory loss of approximately three months' duration. This included an inability to remember details about 24-hour old events. There had been no major socio-economic or personal changes in her life over this period. A detailed neurologic examination was notable only for her presenting complaint. Routine laboratory work-up, including a lumbar puncture were all within normal limits. A physical examination revealed a large right adnexal mass, which upon ultrasonographic assessment showed internal features suggestive of a malignancy. A panel of serum paraneoplastic autoantibodies was then requested, including anti-Hu, anti-Yo, anti-Ri, anti-Tr and anti-Ma1/2. All were normal with the exception of IgG anti-Ri, measured at 1:15,360 by an indirect immunofluorescence method. An extensive diagnostic work-up failed to reveal any malignancies. The patient subsequently underwent a right salpingo-oophorectomy, and her adnexal mass was diagnosed as a benign mature cystic teratoma of the ovary. Almost immediately following her surgery, the patient expressed a subjective improvement in her symptoms. Within a month, the serum anti-Ri had decreased to 1:256, and a detailed neurologic examination revealed resolution of her symptoms. She has not experienced any relapse in her symptoms in the 1 year since her surgery.Nova-1 and Nova-2 genes and which may play a role in neuronal maturation and homeostasis [In 1988 and 1991, Budde-Steffen at al  and Luqueostasis -9. As theostasis . Indeed,eostasis . The speeostasis . What theostasis , the vasIs it possible that the presence of the autoantibody and the ovarian tumor are completely fortuitous?. Given the temporal relationship between the resolution of her symptoms and the sharp decrease in her anti-Ri titer following her surgery, we believe this is unlikely. However, the precise mechanistic basis for this association as well as potential influence of outside factors remains to be elucidated. It should also be noted that high titers of Anti-Ri have been identified in patients with a history of ovarian cancer but without any evidence of a PNS, suggesting caution in assessing the specificity of this auto-antibody for PNS.In summary, we describe in this report an association of a common anti-neuronal antibody (anti-Ri) in a patient with a benign neoplasm, mature cystic teratoma of the ovary, and whose neurologic symptoms, short-term memory deficits, was apparently associated with her tumor. Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody.The authors declare that they have no competing interests.Dr Fadare wrote the initial version of the manuscript.Dr Hart managed the patient, provided clinical information, and revised the manuscript.