The expression of both sense and antisense transcripts of 957 NAT pairs was confirmed, and analysis of MPSS data suggested that for most pairs one of the two transcripts is predominantly expressed in a tissue-specific manner.A new computational method for predicting  cis- and trans-NATs may participate in a broad range of gene regulatory events. Genome-wide identification of cis-NATs in human, mouse and rice has revealed their widespread occurrence in eukaryotes. However, little is known about cis-NATs in the model plant Arabidopsis thaliana.Natural antisense transcripts (NAT) are a class of endogenous coding or non-protein-coding RNAs with sequence complementarity to other transcripts. Several lines of evidence have shown that cis-encoded NATs in Arabidopsis and found 1,340 potential NAT pairs. The expression of both sense and antisense transcripts of 957 NAT pairs was confirmed using Arabidopsis full-length cDNAs and public massively parallel signature sequencing (MPSS) data. Three known or putative Arabidopsis imprinted genes have cis-antisense transcripts. Sequences and the genomic arrangement of two Arabidopsis NAT pairs are conserved in rice.We developed a new computational method to predict and identify Arabidopsis genome annotation in our NAT prediction work and reported cis-NAT pairs that could not otherwise be identified by using one of the two datasets only. Analysis of MPSS data suggested that for most Arabidopsis cis-NAT pairs, there is predominant expression of one of the two transcripts in a tissue-specific manner.We combined information from full-length cDNAs and  Cis-NATs are transcribed from the same genomic loci as their sense transcripts but on the opposite DNA strand. By contrast, trans-NATs are expressed from genomic regions distinct from those encoding their sense transcripts [Cis-NATs and their sense RNAs are usually related in a one-to-one fashion, whereas a single trans-NAT may target several sense transcripts; for example, one type of micro RNA (miRNA) could regulate the expression of several distinct target mRNAs [In the past few years, several families of regulatory RNA molecules have been shown to be widely expressed in eukaryotes ,2. Naturnscripts -5. Cis-Net mRNAs .Studies performed in various organisms have suggested that NATs can participate in a broad range of regulatory events, such as transcription occlusion resulting in the reciprocal expression of sense-antisense RNAs ,8 and RNcis-NATs in eukaryotes. Lehner et al. first reported 372 NATs in human by searching for overlapping mRNA sequences in public databases [et al. predicted 2,667 NATs in human and concluded that around 1,600 NAT pairs were transcribed from both strands after experimental validation [Oryza sativa) full-length cDNAs [A. thaliana genome. However, a detailed list of these Arabidopsis antisense RNAs and their complete analysis is not yet available [cis-NATs only.Recent large-scale NAT identifications in several model organisms have revealed the widespread existence of atabases . Using aatabases . In a lalidation . The RIKlidation . A similth cDNAs . Antisenvailable . We notecis-NATs in Arabidopsis. Combining sequence information of Arabidopsis full-length cDNAs from the public databases and Arabidopsis annotated genes from the Arabidopsis genome release, we have identified 1,340 potential cis-NAT pairs. Expression evidence for transcripts derived from both strands of 957 cis-NAT pairs was obtained from the Arabidopsis full-length cDNA and the public Arabidopsis massively parallel signature sequencing (MPSS) database.Here, we present results of a genome-wide computational search to predict and identify cis-encoded Arabidopsis natural antisense transcripts, we aligned all Arabidopsis full-length cDNA sequences collected in the UniGene and RIKEN datasets with the Arabidopsis genome sequences. Pairs of transcripts that satisfied the following criteria were selected as cis-encoded natural sense-antisense transcript pairs (referred to as NAT pairs hereafter): first, cDNAs of both transcripts can be uniquely mapped to the Arabidopsis genome with at least 96% sequence identity; second, the two transcripts are derived from opposite strands of the genome; third, both transcripts are encoded by overlapping genomic loci, and the overlap length is longer than 50 nucleotides; fourth, the sense and antisense transcripts have distinct splicing patterns. Applying all of the above criteria, we identified 332 sense-antisense pairs from Arabidopsis full-length cDNAs. These NAT pairs are referred to as cDNA-NATs.To search for Arabidopsis full-length cDNA datasets, could be identified using the Arabidopsis genome annotation.The 332 pairs of cDNA-NATs can be grouped into two categories. The first category contained 145 NAT pairs in which both the sense and antisense transcripts had nearly perfect annotated gene matches. The second category contained 187 NAT pairs in which at least one transcript had no corresponding annotated gene. This observation led us to hypothesize that additional NAT pairs, whose corresponding cDNAs were not included in the UniGene and RIKEN Arabidopsis annotated genes to search for gene pairs that overlap in an antiparallel manner. Using the criteria described in Materials and methods, 952 putative NAT pairs were identified from the Arabidopsis genome and were named genomic-NATs. Among the 952 genomic-NATs, 145 pairs had corresponding full-length cDNA for both the sense and antisense genes, and therefore were also included in the cDNA-NAT set. The remaining 807 new NAT pairs were predicted using the Arabidopsis genome annotation only and are referred as the unique genomic-NAT set in the following analysis . The 1,291 genomic-cDNA-NAT pairs included the 332 cDNA-NAT pairs and 758 unique genomic-NAT pairs. Therefore, 201 unique NAT pairs were predicted by the cDNA-genome comparison approach and are referred to as unique genomic-cDNA-NAT pairs hereafter  and another 201 genomic-cDNA pairs by combining genome annotation with full-length cDNA sequence information.In total, we have found 1,340 potential NAT pairs from three categories: 332 pairs with cDNA evidence for both sense and antisense transcripts; 807 pairs based on the Arabidopsis NAT pairs (72.1%) overlapped at their 3' end. For almost all NAT pairs (99%), the overlapping region included exon sequences, with a few exceptions in which one transcript was transcribed entirely from the intronic sequences of the other. Figure We classified the 1,340 unique NAT pairs according to the exon-intron structures of each transcript and their overlapping patterns . Among the 455 NAT pairs with unambiguous MPSS data for both transcripts, expression of both transcripts of 78 pairs was only found in distinct libraries, indicating these NAT pairs might have an exclusive transcription relationship. For the other 377 NAT pairs, expression of the sense and antisense transcripts was mainly observed in different libraries or one transcript was dominantly expressed when both transcripts could be detected in the same library  + 332 + 222 + 51 = 957).Arabidopsis small RNA database to investigate the possibility that cis-NAT pairs may generate siRNAs. Similar to the MPSS alignment process, only siRNAs with unique loci on the Arabidopsis genome were used in the comparison to ensure unambiguous assignment. We found 11 pairs of NATs had siRNA sequences mapped uniquely to their overlapping region (Table We compared short interfering RNA (siRNA) sequences collected in the on Table . SiRNAs Arabidopsis NAT pairs with the protein sequences of the 687 predicted rice NAT pairs [Arabidopsis NAT pairs were also encoded by antiparallel genes originated from the same locus in rice .Although NATs are often seen in prokaryotes, their prevalence in eukaryotes was not detected until the past few years -27,34. IArabidopsis NAT pairs were identified from three sources. First, by aligning full-length cDNA sequences to the Arabidopsis genome, we identified 332 cDNA-NAT pairs. However, comparison of these 332 cDNA-NAT pairs with Arabidopsis annotated genes showed that more than half of these NAT pairs had one partner that was not included in the current Arabidopsis genome annotation. Because traditional genome annotation mainly aims at the identification of protein coding genes within a genome, there is the possibility that non-coding antisense transcripts may be overlooked by currently trained gene finders. A recent report using a genome tiling array to examine the transcription activity of the entire Arabidopsis genome also supports this notion [The 1,340 s notion .Arabidopsis cDNA library, we compared the genomic coordinates of all annotated genes with each other and with those of full-length cDNAs. This approach uncovered another 807 overlapping genomic-NAT pairs based on the annotation of their corresponding genes, and 201 genomic-cDNA-NAT pairs, each including a transcript derived from an annotated gene on one strand and a transcript represented in the full-length cDNA database on the other strand. The full-length cDNAs included in genomic-cDNA-NAT pairs either had no annotated gene match or their corresponding transcripts cannot form cis-NAT pairs with transcripts of other genes based on their annotation. These results indicate that although the Arabidopsis genome is currently one of the best annotated eukaryotic genomes, a lot of information is still missing. The identification in eukaryotes of several classes of regulatory RNA genes, such as those encoding natural antisense transcripts, which are the focus here, will not only further our understanding of genome structure and gene regulation, but will also open a new window for improved genome annotation.To search for potential NAT pairs not included in the current full-length Arabidopsis genes in our NAT prediction in order to provide a more complete picture of antisense transcripts in Arabidopsis. The reliability of our approach is supported by the following lines of evidence: first, the expression of both sense and antisense transcripts of 293 pairs of genomic-NATs  was observed in the public MPSS data, and another 222 genomic-NAT pairs  have full-length cDNA evidence for one transcript and associated MPSS data for the other transcript; second, the two NAT pairs which were conserved in rice were also identified in our Arabidopsis genomic-NAT dataset; third, it is known that imprinted genes are likely subject to antisense regulation; three of the six reported Arabidopsis imprinted genes [FIE, FIS2 and MSI1, are included in our genomic-NAT sets. However, it remains possible that some genomic-NAT pairs are false positives if the lengths of their untranslated regions (UTRs) were annotated inaccurately.Most antisense prediction work reported to date has focused on identifying NATs from expressed cDNAs and ESTs -27. In ted genes -39, FIE,Arabidopsis NAT pairs include at least one transcript without coding potential. Non-protein-coding transcripts are more prevalent in cDNA-and genomic-cDNA-NAT pairs in that 170 cDNA NAT pairs and 156 genomic-cDNA-NAT pairs include one non-protein-coding transcript. We used Genescan to evaluate the coding potential of each transcript by screening their corresponding genomic DNA sequence for valid gene structures. Using annotated genes as controls, we estimated the false-negative rate of our definition of coding potential to be 2.3%. Unlike CDS-containing antisense transcripts that may be translated into proteins under certain conditions, transcripts without any protein-coding potential could possess solely regulatory functions.In rice, both transcripts of 86% of the NAT pairs have coding sequence (CDS) regions whereas 28% of the predicted cis-antisense RNAs, which are transcribed from the same genomic loci as their sense RNAs, but on the opposite genome strand. To ensure the cis-antisense relationship of NATs reported here, only cDNAs with unique genomic loci were included in this study. We note that certain number of trans-antisense transcripts also exist in cells. Examples include miRNAs and siRNAs which are widely studied in most model organisms [trans-antisense transcripts in Arabidopsis is being attempted.In our work described here, and in all other genome-wide antisense transcript identification papers published so far -27, the rganisms . Genome-The non-gel-based properties of MPSS technology render it an ideal resource for evaluating the expression profile of NAT pairs for the following reasons: first, because the MPSS technology captures almost all polyadenylated transcripts within cells, this technology is theoretically capable of identifying new, rarely expressed transcripts without prior knowledge of their corresponding genes; second, the digital result of MPSS reflects the expression pattern of a sequenced RNA molecule, and therefore provides a quantitative relationship between the sense and antisense transcript of a NAT pair in different tissues. This information was not available in any of the previous NAT prediction work ,33.Arabidopsis MPSS data, we were able to obtain expression evidence for both transcripts of 957 NAT pairs. The digital nature of MPSS data enabled us to evaluate the expression relationship of the sense and antisense transcripts directly. Our results showed that the sense and antisense transcripts of a NAT pair tend to be expressed in different tissues or under different conditions. In addition, in cases where the sense and antisense transcripts of a NAT pair were expressed in the same library, one type of transcript was usually more abundant than the other. On average, transcripts of NAT pairs were found to be coexpressed in only two libraries, whereas dominant expression (the expression level of one transcript was at least three times higher than that of the other transcript) or absolute expression (only one transcript of a NAT pair was expressed) was observed in nine libraries. The tissue-specific expression of sense and antisense transcripts observed in this study is consistent with the Arabidopsis genome transcription study using a whole genome-tiling array, in which about 7,600 genes were found to have tissue-specific sense and antisense expression [trans-antisense transcripts. This could explain why we predicted fewer NAT pairs than the previous work, as our work only focuses on cis-antisense transcripts.Using the full-length cDNA and public pression . AlthougArabidopsis genome and all annotated mRNAs to remove signatures with multiple genomic loci. Therefore, unless an MPSS signature sequence was derived from the joint-exon region of some transcripts that are not included in the current genome annotation, it should originate from its corresponding transcript.To ensure the MPSS sequences were indeed generated by their matching transcripts, all MPSS data were first aligned with the Arabidopsis siRNA database, which only contains 1,822 unique siRNA sequences, is small and does not cover all siRNAs associated with sequences of the NAT pairs reported here. Second, all NATs identified in this work are cis-antisense transcripts. siRNAs are used to downregulate expression levels of their target mRNA to achieve a low protein concentration. Cis-antisense transcripts can accomplish the same goal by interfering with the transcription of their sense transcripts, and this might be a more energy-efficient mechanism to achieve local gene regulation. This hypothesis predicts that we would expect to find more siRNAs associated with trans-antisense transcripts.One possible function of NATs is to trigger the degradation of their sense transcripts via the RNAi pathway. However, in our study, we found only 11 NAT pairs with known siRNA matches. There are two possible explanations for this observation. First, the current public Arabidopsis genome, indicating that cis-antisense regulation might be a global mechanism for all gene families. Further experiments are needed to investigate the validity of these hypotheses.For most NAT pairs with associated MPSS data for both transcripts, the expression of sense and antisense transcripts tends to occur in different tissues. In these cases, we could speculate that transcription of genes encoding these NAT transcript pairs may be regulated by similar factors but that the production of antisense transcripts might interfere with the transcription of their sense transcripts, resulting in reciprocal expression patterns. Another possibility is that the two genes of a NAT pair are subject to different transcriptional regulation and consequently they are never expressed in the same tissue at the same time. Functional analysis of all NAT pairs using gene ontology reveals no over-representation of any functional category compared to the Xist gene in mouse and human [MEA and PHE genes of Arabidopsis are imprinted [FIS2, FIE, MSI1 and FWA may also be imprinted, although the evidence for these four other genes is not unequivocal [FIS2, FIE and FWA, suggesting that transcription of these three genes might be regulated by antisense transcripts, or their antisense transcripts might be involved in silencing their expression. Genomic imprinting usually involves a chromosomal locus and, in certain cases, may even extend overa chromosomal region. Given the close proximity of the sense-antisense gene transcripts if one member of the pair is imprinted, it is likely that the other would be subject to the same regulation. Unfortunately, because of the absence of data on imprinted genes in rice, we were unable to examine whether imprinted genes were also subject to antisense regulation in rice.Antiparallel transcription and antisense transcripts are known to be involved in genomic imprinting of nd human . There imprinted , and in quivocal -39. NoneArabidopsis NAT pairs are conserved in rice. These conserved NAT pairs could be used to study the antisense regulatory mechanism and the origin of NATs in plants. Given over 150 million years of evolutionary distance between Arabidopsis and rice, the gene order on the two genomes has diverged quite significantly. Therefore, the conservation of these two NAT pairs might have some functional relevance. A closer comparison of the Arabidopsis and rice NAT pairs and the identification of additional conserved NAT pairs could help address this issue.We found that two Arabidopsis. These results will facilitate functional studies of NATs in this model plant, as well as in other plant species, and help to unravel complex gene regulatory networks in eukaryotes.Taken together, our results provide the first genome-wide identification and prediction of NATs in Arabidopsis UniGene (Build 45) dataset  was downloaded from the National Center for Biotechnology Information (NCBI) UniGene Resources [Arabidopsis full-length cDNA dataset, which contains 13,181 sequences, was downloaded from the RIKEN BioResource Center (BRC) [Arabidopsis genome sequences from The Institute for Genomic Research (TIGR) (release version 5) [Arabidopsis genome were used in the following analysis. For pairs of cDNAs encoded by opposite strands of the Arabidopsis genome and sharing overlapping genomic loci, if both their corresponding sense and antisense transcripts had no other genomic locations and exhibited different splicing patterns, they were selected as encoding sense-antisense transcript pairs and are referred to as cDNA-NAT pairs in the text.The esources ,41. A toer (BRC) ,43. The rsion 5)  by BLAT.rsion 5) ,44,45. cA. thaliana genome annotations from TIGR (release version 5) in this study [Arabidopsis genes. If a pair of overlapping genes were located on opposite strands of the Arabidopsis genome and at least one gene had no annotated UTR at the overlap end, their encoded transcripts were selected as a putative NAT pair regardless of the overlap length of the encoded transcripts. Otherwise, if a pair of antiparallel overlapping genes both have annotated UTR regions at the overlap end, the overlap length of their encoded transcripts must be longer than 50 nucleotides to qualify as NAT pairs. NAT pairs from the above two categories are both referred to as genomic-NAT pairs in the text.We used the is study ,45. PutaArabidopsis genome were used in this step. Using the same criteria for genomic NATs, if an annotated gene had a overlap cDNA match on the opposite strand and the transcript of the annotated gene and that derived from the antisense cDNA had different splicing patterns, the gene and its matching cDNA were selected as a genomic-cDNA-NAT pair.Genomic-cDNA-NAT pairs were identified by comparing the genomic loci of full-length cDNAs with those of annotated genes. UniGene and RIKEN full-length cDNAs with unique genomic locations and at least 96% sequence identity to the Arabidopsis genome using the sim4 program [Arabidopsis annotated genes were extracted from the TIGR Arabidopsis genome annotation (release version 5) [Splicing patterns of transcripts encoded by full-length cDNAs were obtained by aligning the cDNA sequences to the  program . Splicinrsion 5) . To evalrsion 5) .Arabidopsis MPSS data at the University of Delaware [Arabidopsis Columbia-0 (Col-0) ecotype were downloaded from [We used the public Delaware  to evaluded from . Each MPArabidopsis genome or to cDNAs had ambiguous origins and were not considered further. For a NAT pair, if both the sense and antisense transcripts had associated MPSS data and their expression values were both significant in one or more libraries, transcripts in this NAT pair were considered as coexpressed in the same tissue. On the other hand, if both transcripts had MPSS data but had no significant coexpression in any of the examined libraries, then the transcripts were considered as expressed, but in different libraries.The public MPSS database contained 87,705 trusted signatures that satisfied the above expression criteria. These signatures were aligned to the sequences of the 1,340 putative NAT pairs to identify MPSS sequences derived from them. Signatures with multiple perfect matches to the Arabidopsis cDNAs were mapped to their corresponding genomes by BLAT [A. thaliana and O. sativa genomes were downloaded from TIGR [Arabidopsis NATs using blastp [E-value less than 10-30 and alignment coverage greater than 50% of query sequence were considered as homologous sequences.Full-length cDNA sequences of the 687 rice NAT pairs were downloaded from the website described in . To faci by BLAT . Both throm TIGR . The corrom TIGR . The prog blastp . High siThe following additional data are available with the online version of this paper. Additional data file Classes of overlap patterns: 1. tail to tail (3' end overlap); 2. head to head (5' end overlap); 3. one transcript is contained entirely within the other transcript; 4.two transcripts overlap only within introns. Coding potential of a transcript: '+' with coding potential; '- ' without coding potentialClick here for fileClasses of overlap patterns: 1. tail to tail (3' end overlap); 2. head to head (5' end overlap); 3. one transcript is contained entirely within the other transcript; 4.two transcripts overlap only within introns. Coding potential of a transcript: '+' with coding potential; '- ' without coding potentialClick here for fileClasses of overlap patterns: 1. tail to tail (3' end overlap); 2. head to head (5' end overlap); 3. one transcript is contained entirely within the other transcript; 4.two transcripts overlap only within introns. Coding potential of a transcript: '+' with coding potential; '- ' without coding potentialClick here for file


Chronic inflammation and autoimmunity likely contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). The aim of this study was to investigate the role of autoimmunity in the etiology of AAAs using a genetic association study approach with HLA polymorphisms.HLA-DQA1, -DQB1, -DRB1 and -DRB3-5 alleles were determined in 387 AAA cases (180 Belgian and 207 Canadian) and 426 controls (269 Belgian and 157 Canadian) by a PCR and single-strand oligonucleotide probe hybridization assay.We observed a potential association with the HLA-DQA1 locus among Belgian males . Specifically, there was a significant difference in the HLA-DQA1*0102 allele frequencies between AAA cases  and controls  in Belgian males . In haplotype analyses, marginally significant association was found between AAA and haplotype HLA-DQA1-DRB1 .This study showed potential evidence that the HLA-DQA1 locus harbors a genetic risk factor for AAAs suggesting that autoimmunity plays a role in the pathogenesis of AAAs. Several distinct processes contribute to the pathologic changes observed in abdominal aortic aneurysms (AAAs). The most apparent of these are chronic inflammation, destructive remodeling of the extracellular matrix, and depletion of vascular smooth muscle cells . Local iThe major histocompatibility complex (MHC) is located at chromosome 6p21.31 and is the most gene-dense and polymorphic region of the human genome identified so far . HistoriThe HLA class II region contains five isotypes, HLA-DM, -DO, -DP, -DQ and -DR, all of which are heterodimers composed of α and β chains . There aSeveral association studies between HLA polymorphisms and AAAs have been performed -19. The A definition of AAA by Johnston et al   and included spouses of AAA cases , or individuals admitted to the same hospitals for reasons other than AAA .The study was approved by the Institutional Review Boards of Wayne State University School of Medicine and each patient recruiting center. All subjects gave informed written consent to participate into the study.Genomic DNA was isolated from peripheral blood using a Puregene kit . Before performing genotyping PCR, a whole-genome amplification using primer extension preamplification (PEP) was carried out to increase the amount of template DNA available for genotyping and to ensure that limited resources are used cost-effectively . The perHLA-DQA1, -DQB1, -DRB1 and -DRB3-5 alleles were determined by PCR and single-strand oligonucleotide probe hybridization assay. HLA-DQA1 alleles were determined according to Kimura et al . Quality2 test for association. Because of the large number of alleles at each locus, we also obtained empirical p-values for the test of association for each allele using a permutation test for the entire table  as implemented in the computer program CLUMP .-19.11-19The HLA-DQA1*0102 allele, which showed a potential association with AAA in our study, has been found to be associated previously with other diseases. The HLA-DQA1*0102 allele is known to be a protective allele against type 1 diabetes mellitus (DM) and systemic lupus erythematosus, which are classical autoimmune diseases ,42. InteAlthough there is growing evidence of the association between the HLA system and autoimmune diseases, including AAA, the precise disease-causing mechanism is yet to be defined. It is also possible that an association between HLA and disease is only a marker for an undiscovered polymorphism in a linked gene. There are at least 120 additional genes in the MHC region. Most of these additional genes in the HLA class II region are involved in immunological functions that relate to the HLA class I and II genes ,43. BecaLimitations of the study were: 1) the statistical power was relatively low , and after correction for multiple testing, none of the results remained significant. Nevertheless, the findings were consistent in the sense that the HLA-DQA1*0102 allele frequency was always higher in the case groups than in the control groups ; 2) although the overall sample size was not small, the number of alleles was large leading to relatively small counts for each allele; 3) we did not test the interaction with other loci and environmental factors, such as age, or smoking; and 4) our sampling scheme including spousal controls could lead to false positive results because of the potential effect of the HLA loci on mate selection. The HLA region has been suggested to influence mate selection based on odor differences coded by the MHC genes, with a preference for dissimilar partners, in both mice and humans , althougWe found evidence for genetic association between the HLA-DQA1 locus and AAA suggesting that this genomic region harbors a genetic risk factor for AAAs in the Belgian male population. The same DQA1*0102 allele was present at higher frequency among the Belgian female, Canadian male and Canadian female cases supporting the hypothesis that autoimmunity contributes to the pathogenesis of AAAs. In future studies, the findings need to be assessed in another, larger sample.The author(s) declare that they have no competing interests.TO carried out the molecular assays, scored results and prepared the manuscript. LG designed the study and scored results. KG designed and carried out statistical analyses as well as prepared the manuscript. MS carried out the molecular assays. GT designed the study and analyzed data. WL designed the study and analyzed data. AP carried out statistical analyses. QL carried out statistical analyses. HS designed the study and carried out some of the molecular assays. NS recruited Belgian patients and controls into the study. RL recruited Belgian patients and controls into the study. GM recruited Canadian patients and controls into the study. CA recruited Canadian patients and controls into the study. TS provided clinical expertise. HK designed the study, analyzed data and prepared the manuscript. All authors gave final approval to the manuscript.The pre-publication history for this paper can be accessed here:


Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals. DNA viruses and some RNA viruses must access the nucleus to replicate. This is also the case for lentiviruses and several excellent reviews have been recently published on the subject -5. The iXenopus Laevis oocytes nuclear pores were obtained using energy-filtering transmission electron microscopy and tomographic 3-D reconstruction [Nuclear pores have a maximum diameter of 120 nm, a depth of 180 nm and an overall mass of approximately 125 MDa in vertebrates -12. Recetruction   [Second problem, regardless of how and where the uncoating step takes place, viral nucleic acids must be imported into the nucleus against a steep gradient, since nucleic acids are compacted to a very high density within the nucleus itself (nearly 0.1 g/cmhocytes) . Some bahocytes) . HoweverThird problem, large nucleic acids molecules are charged and hydrophilic. As mentioned earlier, passage through the central channel of the nuclear pore complex depends on hydrophobic interactions with the highly mobile phenylalanine-glycine (FG)-rich domain of nucleoporins . Thus, sHaving broadly defined the general context and problems relative to nuclear import of viruses' genome in mammalian cells, let us now focus on retroviruses.The ability of lentiviruses to infect terminally differentiated, non-dividing cells has been taken as proof that the genome of such viruses is imported into the nucleus. The inability of simple retroviruses to infect non-dividing cells on the other hand has been taken as proof that the genome of such viruses cannot be imported into the nucleus. The picture is not that clear-cut because different retroviruses infect non-cycling cells with different efficiency.There is overwhelming evidence that lentiviruses infect non-dividing cells with high efficiency. Caprine arthritis-encephalitis virus (CAEV) has increased tropism for differentiated macrophages both in vitro and in vivo -39 and cOn the other hand there is strong evidence that gammaretroviruses can efficiently infect only cycling cells, with one notable exception -61. The So, why can lentiviruses infect non-dividing cells and gammaretroviruses cannot? The basis for this difference may reside in the uncoating process ,74,75. HThe viral capsid of both HIV-1 (broad end) and MLV has a diameter of 60 nm or greater and cannot go across nuclear pore complexes even by an active process. Thus early uncoating may be a necessary, albeit probably not sufficient, step for nuclear import. Two experimental lines of evidence seem to support this possibility. Chimeric viruses in which HIV-1 p24 CA protein has been swapped with MLV p30 CA are unable to infect cells arrested in the cell cycle, hence MLV CA appears to be a dominant-negative factor for nuclear import . HIV-1 pgag protein synthesize normal levels of full length viral DNA but fail to form 2LTR circles, perhaps because they cannot associate properly with nuclear structures [Fv1 gene might directly or indirectly prevent such interaction between nuclear pore components and MLV PICs and hence prevent PIC nuclear retention, viral DNA circularization and integration.Although this is an appealing model to explain the phenotypic difference between HIV and MLV, there are some aspects that merit attention. It is likely that MLV is also targeted to the nucleus. For example, MLV mutants in the p12ructures . Moreoveructures . This isructures . It is tructures . This woin vivo. Nevertheless one can still reconcile this finding with the uncoating model by postulating that NPC permeability in a selected population of macrophages may be greater than usual and allow active passage of macromolecules with a diameter of 60 nm. It will be interesting to test experimentally if this is indeed the case. As mentioned earlier, NPC permeability changes depending on the metabolic state of the cell, its differentiation and its ability to divide. Alternatively, MLV PICs may persist longer in macrophages until some degree of uncoating takes place allowing nuclear import.A recent observation that apparently does not quite fit into the uncoating model for nuclear import suggests that MLV can infect differentiated, post-mitotic macrophages almost as efficiently as HIV-1 based vectors . MLV infgag MA and mutation of two lysines at position 26 and 27 was shown to block HIV-1 replication in terminally differentiated primary macrophages but not in proliferating cells [131) was reported to induce MA incorporation into PICs (via binding to integrase) and to be essential for HIV-1 infection of non-dividing cells [131 did not appear to have an effect on virus replication [Uncoating of the viral capsid is likely to be a pre-requisite for nuclear import but specific signals and import factors are also likely to direct intracellular trafficking of the RTC/PIC. Several such nuclear localisation signals (NLS) have been identified in the HIV-1 PIC but no unequivocal picture has emerged yet. The first NLS within the HIV-1 PIC was reported in the N-terminal region of p17ng cells . These ong cells ,99. Moreng cells ,101. A mlication ,103.Subsequently, however, three studies could not confirm the presence of an NLS in MA -107. Thegag polyprotein [More recently, MA has been reported to have a CRM1-dependent nuclear export signal (NES). Mutations in this NES at position 18 and 22 of MA were shown to cause nuclear localisation/retention of viral RNA and severely impair HIV-1 infectivity. Furthermore, the NES has been proposed to override a presumably masked NLS in the context of the p55yprotein . It is syprotein , yet difyprotein ,109, altyprotein . In summyprotein ,109,111,The viral protein R (Vpr) has been shown to play a role in HIV-1 nuclear import (for a recent review on this subject please refer to ). The gegag [Importantly, Vpr is incorporated into virions at high levels via its interaction with the p6 domain of p55gag . The abigag ,123. Vprgag ,83,124. gag ,126. Indgag ,118,127.gag . Moreovegag , showed gag . Similargag . Taken tIntegrase remains stably associated with the RTC and later the PIC and is the necessary protein for integration of viral DNA into host chromosomes. Because of its tight association with the PIC even after nuclear entry, integrase would be a good candidate to mediate HIV-1 nuclear import. HIV-1 integrase (IN) is karyophilic but there is some disagreement on the mechanisms regulating IN trafficking into the nucleus. Most investigators have reported that IN is imported into the nucleus by an active, saturable and energy -dependent mechanism. Putative NLS have been mapped to several regions of the C-terminus of IN ,132-134 Lentiviral INs specifically bind to lens epithelium-derived growth factor (LEDGF/p75), a hepatoma-derived growth factor that interacts with DNA and this association has been reported to be important for IN nuclear localisation ,143. IN Is it possible to reconcile these findings with the observation that, at least in vitro, IN can be imported into the nucleus by an active mechanism in the absence of LEDGF/p75? It is perhaps interesting that the integrase-binding domain in LEDGF/p75 is structurally closely related to the HEAT repeat found, amongst other proteins, in importin β and other importins . It is tAlternatively, lentiviral INs per se might not have a transferable NLS as shown by two reports ,150 and in vitro by mutagenesis of IN into a clear phenotype of reduced HIV-1 nuclear import. Remarkably, a recent study has analysed the phenotype of HIV-1 chimeric viruses bearing MLV IN in place of HIV-1 IN and shown that such mutants are attenuated but still able to infect non-dividing cells. Only a small increase was observed in the ratio of cytoplasmic to nuclear viral DNA in cells infected with the mutant virus compared with wild-type HIV-1 [Finally, a few words of caution should be spent on the role of IN in HIV-1 nuclear import. There is limited information on the precise conformation of IN within the RTC/PIC and several domains, which are exposed in the recombinant protein, might not be available once the protein is part of the RTC/PIC and bound to nucleic acids ,151. Morpe HIV-1 . Thus, ipe HIV-1 .pol, it is present in all lentiviruses and results in a short (approximately 100 nt) stretch of triple stranded DNA upon completion of reverse transcription [The central polypurine tract (cPPT) was also shown to influence HIV-1 nuclear import. The cPPT is a second origin of DNA plus strand synthesis located in cription ,159. Abscription ,161. Thicription -165. Indcription -165. Thigag ELISA or by enzymatic RT assays and both assays have higher variability than GFP detection by FACS in single cycle assays. Thus, if the variability of the detection assay is greater than the experimental difference to be observed, the results cannot be accurately measured. Alternatively, a difference in the rate rather than absolute amount of HIV-1 DNA nuclear import may not be detectable in spreading assays due to the highly asynchronous infection process, as opposed to a more synchronous infection with viral vectors. Elements in the HIV-1 genome, not included in viral vectors, might also partially compensate for the lack of the cPPT. Recently, a more severe replication defect, consistent with the one found with HIV-1 based vectors, has been reported for different HIV-1 strains lacking the cPPT element in single cycle assays and spreading assays [The picture becomes more complicated when wild type viruses with a normal or mutated cPPT are tested in spreading assays. In this case a modest attenuation of cPPT- viruses is seen only in some cell types and even in single cycle assays the difference between cPPT- and cPPT+ viruses' infectivity has been reported to be approximately two fold ,154. A sg assays . Even ifin vitro assay to screen for potential cellular factors with HIV-1 nuclear import ability. The field of nuclear import has advanced at an impressive pace since the introduction in the early '90s of the so-called "nuclear import assay" [Mutations of all known viral karyophilic elements does not block HIV-1 replication , suggestt assay"   stimulates nuclear import of HIV-1 RTCs and that siRNA-mediated depletion of imp7 inhibits HIV-1 infection, though only by a few fold . Anotherin vitro, such tRNAs promoted HIV-1 RTC nuclear import. Moreover, tRNAs with RTC nuclear import activity were incorporated into and recovered from virus particles. We found that the anticodon loop mediated binding to the viral complex whilst the T-arm may interact with cellular components involved in nuclear import. These tRNAs species were transported into the nucleus on their own in a energy- and temperature-dependent way. We also observed that HIV-1 mutant containing MLV gag [More recently we seek to identify additional cellular factors involved in nuclear import of HIV-1 RTCs. Using chromatographic procedures and the nuclear import assay we have isolated a near-homogeneous fraction from cytoplasmic extracts. This fraction contained tRNAs, most of them with defective 3' CCA ends. When synthesized  MLV gag  did not  MLV gag . Thus, b MLV gag ,176. FutElucidating the mechanisms of HIV-1 nuclear import is clearly a challenging area of research, both from a technical and a conceptual point of view. It is also a promising area of research, likely to reveal new and fundamental cellular pathways. To gain more insight and perhaps a little inspiration, it may be wise to look at similar processes occurring in cells, like mRNA export. Export of mRNA nucleoproteins (mRNPs) involves several factors. One of the best characterized is TAP or nuclear export factor 1 (NXF1) . At leasIn conclusion, I would now like to propose a model on HIV-1 nuclear import. Many parts of this model are still hypothetical; nonetheless I shall be bold enough to put it to the attention and critical mind of the reader. After entry, HIV-1 starts reverse transcription and shortly thereafter sheds, partially or completely, its capsid. Such shedding is sufficient to expose the nucleoprotein complex or RTC, composed of the viral genome (presumably still in part RNA) and some viral and cellular proteins. The RTC then engages with the nuclear import machinery at several levels. There might be adaptors, both viral (for example Vpr) and cellular (for example tRNAs), that promote RTC docking and binding to the nuclear pores. Once at the nuclear pore, additional signals/factors may facilitate the charged and hydrophilic nucleic acids to cross the pore's central channel (for example imp7), and other elements may recruit putative motors at the pore to overcome the steep DNA concentration gradient in the nucleus (for example the cPPT element?). RTCs are likely to undergo substantial conformational changes at different stages (RTCs convert from RNA into double stranded DNA) and viral and cellular factors are also likely to associate and dissociate dynamically from the RTC. The multiplicity of signals ensures that the rate of nuclear transport is fast and individual signals may predominate in specific cell types. Like a good orchestra, the loss of one element will reduce the quality of the performance but, depending on the element lost, it may be noticeable only to the educated ear. It will be important to identify possible bottlenecks in this process to develop effective anti-viral strategies. MLV, on the other hand, may not be able to shed enough capsid to make its RTC fully visible to the nuclear import machinery and/or may lack sufficiently strong NLS . MLV mayThere is of course a fundamental question worth considering: why mammalian cells have an evolutionary conserved mechanism to import DNA into their nuclei? Perhaps the study of nuclear import of viral genomes will shed some light on this problem too.HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus; MLV murine leukaemia virus; RTC, reverse transcription complex; IN, integrase; NPC, nuclear pore complex; cPPT, central polypurine tract; LEDGF, lens ephitelial-derived growth factor; MA, matrix protein; CA, capsid protein; mRNP, messenger RNA ribonucleoprotein.The author(s) declare that they have no competing interests.


Recombination during meiosis is one of the most important biological processes, and the level of recombination rates for a given individual is under genetic control. In this study, we conducted genome-wide association studies to identify chromosomal regions associated with recombination rates. We analyzed genotype data collected on the pedigrees in the Collaborative Study on the Genetics on Alcoholism data provided by Genetic Analysis Workshop 14. A total of 315 microsatellites and 10,081 single-nucleotide polymorphisms from Affymetrix on 22 autosomal chromosomes were used in our association analysis. Genome-wide gender-specific recombination counts for family founders were inferred first and association analysis was performed using multiple linear regressions. We used the positive false discovery rate (pFDR) to account for multiple comparisons in the two genome-wide scans. Eight regions showed some evidence of association with recombination counts based on the single-nucleotide polymorphism analysis after adjusting for multiple comparisons. However, no region was found to be significant using microsatellites. In this study, seeking to identify regions potentially affecting recombination rates, we conducted genome-wide association studies based on microsatellites and single-nucleotide polymorphisms (SNPs) of the Collabroative Study on the Genetics of Alcoholism (COGA) data provided by Genetic Analysis Workshop 14 (GAW14). A total of 315 microsatellites and 10,081 SNPs from Affymetrix on 22 autosomal chromosomes were analyzed. We found eight regions/thirteen SNPs that showed some evidence of association with recombination counts. No region was found to be significant using microsatellites after adjusting for multiple comparisons based on the positive false discovery rate (pFDR) criterion.Recombination between two homologous chromosomes during meiosis generates novel gene combinations and creates genetic diversity among chromosomes. Furthermore, recombination is critical for proper segregation of homologous chromosomes, and is a major factor shaping linkage disequilibrium (LD) patterns in the genome . Much re-6 at these SNPs' genetic map positions to make them distinguishable. To estimate the number of both maternal and paternal recombination events for each female or male meiosis, we used the Best option in the haplotyping analysis in MERLIN [The COGA data consist of 143 pedigrees with 1,614 individuals, including 1,109 male and female meioses. Genetic maps for microsatellites and SNPs were both provided by GAW14. Some of the distinct SNPs have the same genetic map position, which made inferring recombination events between these SNPs impossible. Therefore, we added 1.0 × 10p ≤ 0.025 were excluded [Because genotyping error may lead to double recombinations within a short distance, it can significantly affect the overall recombination counts. To minimize this impact, the error-checking algorithm implemented in MERLIN, which identifies unlikely genotypes based on double recombination events, was applied and the erroneous genotypes were excluded before applying haplotyping analysis. We used the default parameter in MERLIN, where the erroneous genotypes with a likelihood ratio excluded . The samq-values [q-value is a measure of significance in terms of the pFDR, and it is defined to be the minimum pFDR at which the statistic can be called significant. A pFDR of 5% means that among all of the features that are called significant, 5% of them may correspond to the true null hypotheses on average. To get the q-value for each marker, we used the software QVALUE [p-values obtained from the multiple regressions.We used multiple linear regressions to evaluate the relation between recombination counts and markers across 22 autosomal chromosomes with adjustments for age and gender for both SNPs and microsatellites. Analysis was carried out based on Whites only to reduce potential confounding factors related to ethnic differences. To account for the multiple comparison problem in the two whole-genome scans, we used pFDR through q-values , where ae QVALUE  on the pThe genome-wide gender-specific recombination counts of the founders were obtained through averaging recombination counts in all meioses leading to his/her offspring. For SNPs, we inferred the founders' genome-wide recombination counts from the gametes of 1,334 offspring from 130 nuclear families. For microsatellites, we inferred the founders' genome-wide recombination counts from the gametes of 1,409 offspring from 111 nuclear families. This resulted in 189 founders  who were Whites with information for SNPs and 199 founders  who were Whites with information for microsatellies. Among these founders, 14 of them missed all microsatellite genotype information and 23 of them missed all SNP genotype information. They had no contribution in the multiple regression analysis. Therefore, the distributions of the founders' sex-specific recombination counts plotted in Figure We noted that our inferred female and male genome-wide recombination counts were slightly lower than that from previous studies . One reap-values for each marker, which were not adjusted for multiple comparisons. The corresponding q-values based on the pFDR were calculated using the software QVALUE. We applied the 0.05 q-value cut-off, which gave us 8 regions/13 SNPs that showed some evidence of association with recombination counts. The positions of those regions together with the raw p-values and q-values were summarized in Table q-value cut-off suggested that 1 out of these 13 SNPs may not be associated with recombination counts. For microsatellites, no region was found to be significant after adjusting for multiple comparisons using pFDR.Multiple linear regressions with adjustments for age and gender generated In summary, we have identified several candidate SNPs likely associated with recombination events, and further studies on these genes may help us gain valuable knowledge on recombination, better understand LD patterns, and lead to more efficient methods to map disease genes.COGA: Collaborative Study on the Genetics on AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumpFDR: Positive false discovery rateSNP: Single-nucleotide polymorphismSH participated in the design of the study, performed the analysis, and drafted the manuscript. SW helped to obtain recombination counts and preparation of the manuscript. SW, NL, LC, and CO participated in the design and the discussion of the study. HZ conceived the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Neutrophils infiltrate the endometrium pre-menstrually and after long-term progestin only-contraceptive (LTPOC) treatment. Trafficking of neutrophils involves endothelial cell-expressed intercellular adhesion molecule (ICAM-1). Previous studies observed that ICAM-1 was immunolocalized to the endothelium of endometrial specimens across the menstrual cycle, but disagreed as to whether extra-endothelial cell types express ICAM-1 and whether ICAM-1 expression varies across the menstrual cycle.Endometrial biopsies were obtained from women across the menstrual cycle and from those on LTPOC treatment (either Mirena or Norplant). The biopsies were formalin-fixed and paraffin-embedded with subsequent immunohistochemical staining for ICAM-1.The current study found prominent ICAM-1 staining in the endometrial endothelium that was of equivalent intensity in different blood vessel types irrespective of the steroidal or inflammatory endometrial milieu across the menstrual cycle and during LTPOC therapy. Unlike the endothelial cells, the glands were negative and the stromal cells were weakly positive for ICAM immunostaining.The results of the current study suggest that altered expression of ICAM-1 by endothelial cells does not account for the influx of neutrophils into the premenstrual and LTPOC-derived endometrium. Such neutrophil infiltration may depend on altered expression of neutrophil chemoattractants. The premenstrual human endometrium displays increased prostaglandin-generating capacity, elevated levels of inflammatory cytokines ,2 and a Endothelial cell-expressed cellular adhesion molecules mediate leukocyte trafficking . In thisPrior to menstruation and during progestin-only contraception , secretion of MMPs by endometrial leukocytes as well as cytokines that can act as autocrine/paracrine modulators of MMP expression , are thoAfter receiving written informed consent and approval from the Institutional Research Board (IRB) of New York University Medical Center and Bellevue Hospital, specimens of endometrium were obtained across the menstrual cycle  from hysterectomies for benign conditions , and histologically dated by the criteria of Noyes et al . For stuThe subjects had regular menstrual cycles and had not used hormonal or intrauterine contraception in the six months prior to insertion of Norplant or Mirena. Patients did not exhibit symptoms characteristic of endometriosis such as pelvic pain, dysmennorhea, dysparunia, or infertility. The only way to confirm a diagnosis of endometriosis is through exploratory surgery. Such surgery would be prompted by symptoms that would have ruled out the use of those patients for our study. For the cycling endometrium patients were pre-menopausal between 32 and 43 years of age who were not receiving any hormonal treatments. For the LTPOC endometrium patients were premenopausal, between 28 and 45 years of age, had regular menstrual cycles and had not used any hormonal or intrauterine contraception in the six months prior to receiving the LTPOC treatment.Prior to insertion of Norplant biopsies were collected from four women  by Pipelle suction curette . Only patients who experienced bleeding while on the Norplant treatment were used. Biopsies were collected using an operative hysteroscope connected to a video camera to facilitate separate sampling of bleeding and non-bleeding sites as previously described . These sEndometrial biopsies were also obtained from four women  prior to and at 1, 3, 6, and 12 months after intrauterine insertion of the LNG-intrauterine system by Pipelle suction biopsy.Specimens of endometrium obtained across the menstrual cycle as well as from control, and levonorgestrel treated  subjects were fixed in 4% paraformaldehyde and embedded in paraffin. Four μm sections (4 μm) were deparaffinized, rehydrated and washed in Tris-buffered saline , which was used for all washes and for dilution of the antibody. Antigen retrieval was carried-out by incubating sections in sodium citrate buffer  in a microwave oven at 750 Watts for 5 minutes. The sections were then rinsed in 3% hydrogen peroxide to block endogenous peroxidase and incubated for 1 hour at room temperature with either of the following primary antibodies: a goat polycolonal ICAM-1 (CD54) antibody from R&D Systems  or a monoclonal antibody against the Platelet Adhesion Molecule (PECAM) (CD31) from Dako . Staining was visualized using the avidin-biotin peroxidase complex  and the 3,3'-diaminobenzidine tetrahydrochloride  chromogen substrate. Light hematoxylin stain was used for nuclear counterstaining. Negative controls for each tissue section consisted of substituting the corresponding pre-immune serum for the primary antibody.Intensity of ICAM-1 staining was evaluated using a semi-quantitative 4-point rating method with the following scoring system: 0, absence of staining; 1, light staining; 2 moderate staining; and 3, strong staining. Each of these possible scores was established in advance of rating the fields via reference to external stained specimens unrelated to this study. In order to determine inter-rater reliability of this scale, two independent judges scored a series of 35 separate fields on slides from 4 separate patient samples. The degree of concordance was then assessed by use of Cohen's kappa statistic, which yielded a value of 0.67, indicating a high degree of agreement between the judges.Non-parametric statistical analysis was performed by the Mann-Whitney Rank Sum Test with p < 0.05 considered significant.Figure Figure The current study found that human endometrial endothelial cells displayed prominent IHC staining for ICAM-1 in specimens obtained from the follicular, luteal and menstrual phases, and after administration of the LTPOCs, Norplant  and Mirena (intrauterine LNG) and that this staining was of equivalent intensity in all vessels examined. By contrast, the glands exhibited virtually no immunostaining and the stromal cells only weak immunoreactivity. Although IHC staining for ICAM-1 was previously demonstrated in the endometrial endothelium of specimens obtained across the menstrual cycle -19, two 2-dominated follicular phase, the progesterone-exposed luteal phase, and the steroid-withdrawal-initiated menstrual phase suggests that ICAM-1 expression is not under direct ovarian steroid regulation. This conclusion was supported by the ICAM-1 immunostaining results obtained in endometrial biopsies during use of subdermal and intrauterine LNG (Norplant and Mirena respectively) contraception. That both LTPOCs produce a hyperprogestational endometrial milieu is suggested by the observation of significantly high endometrial levels of the progesterone receptor (PR) isoforms PRA and PRB after administration of Norplant [A appears to mediate the long-term effects of LNG in the endometrium during intrauterine LNG contraception [The demonstration in the current study that ICAM-1 levels are equivalent in the endometrial endothelium of specimens from the ENorplant  as well Norplant , whereasaception . Endometaception . HoweverEvidence presented in the current study also argues against a role for the local inflammatory milieu in regulating endothelial cell expressed ICAM-1. Thus, equivalent immunostaining intensity was observed when follicular and luteal phase endometria were compared with menstrual, Norplant, and Mirena-derived endometria, which undergo a marked leukocyte infiltration -5,8,9 anICAM-1 is both constitutively expressed and transcriptionally regulated on the surface of several cell types . ConsistIn vivo studies have generally relied on IHC to localize and assess ICAM-1 levels. Table Regulation of neutrophil migration into inflammatory sites reflects interactions between the IL-8 chemokine and the ICAM-1 adhesion molecule. The former establishes a chemotactic gradient that promotes neutrophil trafficking from the circulation towards the endothelium . This enIn the context of our current observations, constitutive endothelial ICAM-1 expression alone cannot account for the marked neutrophil infiltration that characterizes both premenstrual human endometrium as well as the endometrium resulting from LTPOC therapy.FS designed the study and drafted the manuscript. GK conducted immunohistochemical procedures. RNB assessed immunohistochemical staining. HC performed clinical assessment and collection of specimens related to LTPOC treatment. EK performed the statistical analyses and critically reviewed the manuscript. CJL participated in the conception of the study and critically reviewed the manuscript. All authors read and approved the final manuscript.


The actions and reactions integral to mate recognition and reproduction are examples of multifaceted behaviors for which we are only beginning to comprehend the underlying genetic and molecular complexity. I hypothesized that social interactions, such as those involved in reproductive behaviors, would lead to immediate and assayable changes in gene expression. Such changes may have important effects on individual reproductive success and fitness through alterations in physiology or via short-term or long-term changes in nervous system function.Drosophila Genome arrays to identify genes whose expression profiles would change rapidly due to the social interactions occurring during Drosophila melanogaster courtship. I identified 43 loci with significant expression profile changes during a 5-min exposure period. These results indicate that social interactions can lead to extremely rapid changes in mRNA abundance.I used Affymetrix Odorant-binding protein 99b (Obp99b) and female-specific independent of transformer (fit), is controlled by the Drosophila sex-determination gene hierarchy, which regulates male and female mating behaviors and somatic differentiation. Therefore, additional identified loci may represent other long-elusive targets of Drosophila sex-determination genes.The known functions of the up-regulated genes identified in this study include nervous system signaling and spermatogenesis, while the majority of down-regulated loci are implicated in immune signaling. Expression of two of the up-regulated genes,  Social interactions are known to alter individual physiology, but the extent to which transcriptional or post-transcriptional mechanisms function in inducing these changes is unclear. Immediate responses are likely modulated by environmentally induced changes in synaptic signaling, which may result in alterations in gene expression with long-term consequences. Identifying the genetic basis for physiologic responses is of significant interest because it will clarify how regulated gene expression and the resulting intracellular signaling events modulate complex behaviors.Drosophila geneticists have successfully used mutant analysis to identify loci integral to behaviors such as learning and memory, circadian rhythmicity, foraging and reproduction , and in library . These RDrosophila are genetically programmed and require multi-modal inputs and outputs. Male flies use sight, smell, taste, and touch to identify appropriate mates [Reviewed in [Courtship behaviors in iewed in  and 13]Drosophiliewed in . FemalesD. melanogaster courtship and reproduction have been identified . TherefCG4757 was the only down-regulated gene with a statistically significant difference in expression levels between the two treatments (See Methods). Given the general trend for immunity gene expression to be less abundant in courting males  and spermatogenesis (lectin-24Db and lectin-28C). The three lectin genes are unique at the nucleotide level, so cross hybridization is unlikely to account for the fact that these genes were identified in my analysis. Lectin-24Db and Lectin-28C are possible paralogs of the accessory gland protein Acp29Ab  and areproteins ,53, but proteins -25. fru behavior  and direbehavior ,29,54. Fbehavior .Sxl, tra and tra-2 regulate expression of Obp99b so that it is present at high levels only in males [Obp99b have not been reported, but females over expressing Obp99b do not mate at wild-type frequency [Obp99b has a behavioral function.in males . Loss-ofrequency , possiblfit, is enriched in female heads in the fat body surrounding the brain and is regulated by the sex-determination gene Sxl but does not require tra or tra-2 function [fit expression in males by the sex-determination hierarchy has not been examined. fit expression varies depending upon the genetic background; it is expressed at low levels in males of the w1118 and tra-2/CyO genotypes but is undetectable in Oregon-R [fit are elevated 2 hrs post mating . However, two hours after a courtship exposure, males that do not mate have fit levels similar to those of unexposed animals .The second sex-determination target identified in this experiment, function ; potentiOregon-R . Levels fit, Obp99b, and a third sex-determination target gene, takeout, are expressed in the fat body cells surrounding the adult brain [Obp99b and takeout are expressed in the chemosensory systems of both sexes [takeout has a known role in reproductive behavior; feminization of male takeout-expressing cells results in reduced courtship [Obp99b in females affects their latency to copulation [takeout-related gene, CG5867 [Obp99b and fit also function in male reproduction and support previous work indicating that gene expression in the fat body of the head may have important functions in sex-specific reproductive behavior and physiology [Interestingly, lt brain ,22, and th sexes ,55. takeourtship , and ove, CG5867 , is alsoysiology ,22. The CG4757, showed a statistically significant decrease in expression in courtship-exposed males (See Methods). While the trend indicates a general decrease in the transcript levels of immune response genes as a consequence of courtship interactions, the genes involved appear to be widely variable. Some down-regulated immunity genes are located at adjacent chromosome regions, possibly indicating that they are co-regulated. There are groups of immune genes at chromosome location 36C9 , 51C1 , 55C4  and 99E2 . The 55C region contains a head-specific cluster of co-regulated gene products [AttA and AttB with AttC), so partial cross-hybridization to probe sets may account for correlations among hybridization signals.I also report that 27 of 33 down-regulated genes are implicated in the innate immune response of flies. However, I was able to verify only a small number of the changes using independent samples  or other tissues in only a small number of cells. Future experiments will be designed to identify additional potential candidates in the CNS as well as to differentiate between genes that are specific to reproductive behavior and those involved in more general social interactions among flies.Canton-S males and females and aged them at 25° in groups of 20 or fewer flies. Males were transferred individually to new vials on day 3. On day 4 a single female was aspirated into a vial containing a single male, and the pairs were observed for 5 min at 22°. Males for the unexposed treatment were mock aspirated.I collected virgin, wild-type Thirty-five percent of the males displayed robust courtship without mating , and 36% mated within the 5 min observation period. The remaining males (29%) generally performed at least early courtship behaviors but did not attempt copulation. Males who demonstrated robust courtship toward the females  were collected, quick-frozen and stored at -80° for later RNA extraction. Only males who did not copulate during this 5-min window were collected. The time from completion of courtship to freezing was less than one minute for all samples.Males were randomly pooled in groups of approximately 12 individuals, and total RNA was extracted in Trizol reagent (Invitrogen) following standard protocols. Control samples consisted of males that were treated identically except that they were not exposed to a courtship object. All collections and assays were performed at the same time each day to avoid circadian effects on gene expression.Drosophila Genome Arrays  for a total of six arrays. RNA labeling and hybridization using standard Affymetrix protocols were performed at the University of Kentucky MicroArray Core Facility or at the Texas A&M University Department of Biology Core Facility.Three RNA samples from males that courted females and three RNA samples from unexposed males were used to probe Drosophila reference sequence, while MM (mismatch) refers to a single nucleotide difference between the probe and reference sequence that should affect RNA hybridization to the probe.The statistical analysis of the six arrays  roughly followed that of Lawniczak and Begun . ExpressEach of these three sets of expression values was then analyzed separately with the Bayesian t-test implemented in Cyber-T . In thisst Strand Synthesis Kit (Invitrogen) was used to prepare cDNA from independently obtained RNA samples  as well as from two sets of samples used for microarray analyses. I did not have sufficient RNA remaining from the third array sample to use for cDNA preparation.To validate the microarray results, the Superscript 1cDNA preparations were diluted 1:15, and 1.5 ul was used in each reaction for Real-time PCR using the SYBR Green PCR Mastermix (Applied Biosystems) essentially as described previously . ReactioCG1732, fit, Obp99b, and lectin-28C). Since most of the 33 down-regulated candidates are implicated in immune signaling, I chose a smaller number of candidates for PCR validation. I selected 9 genes that are not closely linked and are either uncharacterized or are known targets of each of the two major immune signaling gene cascades. Since Real-time PCR validated few of the down-regulated immune candidates, I did not continue to test additional genes from this group.I chose five up-regulated candidates for validation; however, only four of the primer pairs generated gene-specific amplification products  between the unexposed and exposed treatments, and all 3 genes are up regulated by courtship exposure. While the levels of CG1732 and lectin-28C are consistently higher in courtship-exposed males compared to mock-exposed males, the two treatments are not statistically different from one another. I attribute this finding to the small sample size and the variation in the absolute levels of transcripts among the different samples for a particular treatment. The levels of one down-regulated gene, CG4757, are significantly different (p < 0.05) between the exposed and unexposed males.I calculated the fold change for the Genechip hybridization samples independently from the three samples that were used only for Real-time PCR confirmation. All of the up-regulated genes tested had the expected directional increase. I grouped the 5 sets of samples together for statistical analysis in order to increase the sample size for the comparisons and performed a two-tailed t-test. Levels of GEC conceived and executed the experiments, participated in the data analysis and wrote the manuscript.


Outcomes of surgical intervention for lumbar synovial cysts have been evaluated in the short and intermediate term. Concerns regarding cyst recurrence, the development of late instability at the involved level, and instability/stenosis at adjacent levels (when concomitant) fusion is performed suggest that long term follow-up is needed. This study aims to fill that void.Forty-six patients operated by a single surgeon not involved in the study were followed up long term at an average of 9.7 years (range 5 to 22 years) post-operatively. All patients underwent decompression (+/- concomitant arthrodesis in the presence of associated degenerative spondylolisthesis) using the operative microscope for magnification/illumination. Outcomes were assessed using a customized questionnaire evaluating: relief of pain/claudicant symptoms, numbness/parasthesias, and weakness; as well as late onset low back pain, new radicular symptoms, need for additional surgery, and patient satisfaction. Outcomes in patients with or without fusion were compared as well.87% of patients noted resolution of their pre-operative pain, numbness, and weakness. 28% of patients developed late onset low back pain. 17% developed late onset radicular symptoms in a new nerve root distribution. 15% required subsequent additional surgery. 89% of patients were satisfied with the surgical outcome. No differences were found for any outcome measure between patients undergoing concomitant fusion and those undergoing decompression alone using the two-sample t-test.This study provides outcome data at an average of nearly ten years post-operative. This information should allow surgeons to provide realistic expectations for their patients regarding outcomes and should enhance the informed consent and surgical decision-making process. Although originally recognized in peripheral joints by Baker in 1877, synoviaWhile multiple non-operative therapies have been implemented -16, few The purpose of this study was to independently evaluate the long-term clinical outcomes in patients who underwent microdecompression with or without concomitant arthrodesis for symptomatic lumbar synovial cysts unresponsive to non-operative measures. The average follow-up of 9.7 years (range five to 22 years) represents the longest follow-up to date; the minimum follow-up for inclusion of five years being greater than the previously reported maximum follow-up of 3.25 years.Patients were placed under general endotracheal anesthesia and placed in a kneeling position on a standard frame. The involved level(s) was marked preoperatively using c-arm imaging. A midline skin incision was made and the dorsolumbar fascia incised just lateral to the midline ipsilateral to the synovial cyst. Unilateral laminae were exposed using the Cobb elevator to the mid-portion of the facet joint. An intraoperative radiograph was used to confirm the level. A laminotomy on the undersurface of the cephalad lamina was undertaken to mirror the cephalad extent of the cyst as determined by pre-operative MRI. A similar caudal laminotomy was performed, again to mirror the extent of the cyst. Ligmantum flavum was then excised and the subarticular and foraminal zones decompressed via complete excision of soft-tissue/bony stenosing lesions to include extirpation of the synovial cyst. If the cyst was adherent to the dura (a common finding), it was carefully teased free so that no cyst pseudocapsule remained. The facet joint was opened and residual synovial tissue excised. If the patient had neurogenic claudication, a contralateral microdecompression as previously described was undeForty-six patients operated between 1984 and 2001 were available for follow-up. All surgeries were performed by a single surgeon who was not involved in the study. Age at surgery ranged from 25 to 96 years with a mean of 73 years. Twenty-nine were females and seventeen males. Twenty-eight cysts were at the L4-L5 level, eight at L5-S1, six at L3-L4, and one each at L1-L2 and L2-L3. Clinical syndromes included unilateral monoradiculopathy in eighteen patients and neurogenic claudication in twenty-eight. Radiographically, twenty-three had an associated degenerative spondylolisthesis and underwent concomitant arthrodesis. This was the only indication for fusion in the study population. This follow-up study was approved by the institutional review board and oral consent was obtained from all participants.Clinical outcomes and patient satisfaction were assessed by two independent spine surgeons using the questionnaire in Table Follow-up averaged 9.7 years with a range of five to 22 years.Forty of the forty-six patients (88%) reported relief of their preoperative pain/symptoms. Six (12%) had persisting complaints ranked in severity at an average of 5.5 on the visual analog scale (VAS: range 2–10) versus an average of 9 on VAS preoperatively. All patients had pain or claudication preoperatively.Twenty-three patients (50%) reported preoperative numbness. Of these, at follow-up, twenty (87%) reported complete or near-complete resolution, two (9%) were the same, and one (4%) was worse.Nineteen (41%) had complained of weakness prior to surgery. Of these, at follow-up, sixteen (84%) reported complete resolution, two (11%) reported no significant change in strength, and one (5%) was worse than preoperatively.After initially doing well, thirteen patients (28%) reported the eventual development of new back pain ranked on average at 7.5 on the VAS.Eight patients (17%) reported the eventual onset of new radicular leg pain (different root involved) with a mean VAS severity of 7.4.Seven patients (15%) reported the need for additional lumbar spine surgery. Three patients who had not undergone fusion at the initial surgery required eventual revision decompression and fusion to include the operated levels due to instablility. Four patients who had undergone concomitant arthrodesis at the primary surgery due to presence of a degenerative spondylolisthesis eventually developed juxtafusional stenosis/instability requiring secondary decompression and fusion at involved adjacent levels.Forty-one patients (89%) reported overall satisfaction with the outcome of their initial procedure and would recommend it to a friend with the same problem.any of the outcome measures above between patients presenting without a degenerative spondylolisthesis  and those presenting with one (decompression with concomitant fusion) using the two-sample t-test.There were no statistically significant differences for This study demonstrates that at an average of nearly ten years following decompressive surgery for symptomatic lumbar synovial cysts (with associated fusion if a degenerative spondylolisthesis is present), patients can anticipate: (a) about an 85% likelihood that their preoperative pain/claudication, numbness, and weakness will be resolved, (b) about a 25% likelihood of developing later onset back pain, (c) about 15% likelihood of developing later onset radicular symptoms in a new nerve root distribution, (d) that they have a 15% likelihood of needing additional lumbar surgery, and that (e) about nine out of ten patients are happy with the results of surgery.These findings are generally commensurate with those of other studies having evaluated outcomes at much shorter intervals. Howington achievedThe value of the current study, given that its follow-up is dramatically longer than any previously published, is that it demonstrates, generally, that the beneficial effects of surgical intervention seen at shorter and intermediate time frames appear to persist, however some patients will develop late-onset low back pain, radicular pain, and may need additional surgery long term. This additional information should allow surgeons to provide realistic expectations for their patients regarding outcomes and should enhance the informed consent and surgical decision-making process.The potential weaknesses of long term studies such as this are two-fold. First, over a period of ten to twenty years patients may go on to further degeneration or develop new medical comorbidities such that their overall health status (SF-36) or disease specific status (ODI) may actually appear worse than their pre-operative status – despite the fact that their specific reasons for surgery  may well have been relieved by the intervention. Long term studies are one of the rare cases where very specific outcome measures are indicated to ferret out this information, hence the choice of custom questionnaire used. Second, over that ten to twenty year period of time, the standards of care and the evidence base may have changed such that the information provided by the study is no longer relevant. This is a common problem in the total hip/knee replacement literature where long term outcomes are provided for prostheses no longer manufactured and surgical approaches no longer used. Just as the first potential weakness was avoided by intention, this second potential weakness was avoided by good fortune. Twenty-two years down the road, the standard of care, commensurate with the current evidence base, remains decompression of involved neurological tissue by complete excision of the cyst , excision of ligamentum flavum and other soft tissue and bony compressive pathology, and concomitant arthodesis in the presence of a degenerative spondylolisthesis.


Scrapie-free farms in scrapie-free areas (n = 9); 2. Scrapie-free farms in scrapie-afflicted areas (n = 17); 3. Scrapie-prone farms  (n = 12); 4. Scrapie-afflicted farms (n = 9). Farms in categories 1 and 2 are collectively referred to as scrapie-free farms. The mean iron concentration in forage samples from scrapie-afflicted farms was significantly higher than in forage samples from farms in the other scrapie categories (P = 0.001). The mean Fe/Mn ratio in forage from scrapie-afflicted farms was significantly higher than in forage from scrapie-free and scrapie-prone farms (P < 0.001). The results indicated relative dominance of iron over manganese in forage from scrapie-afflicted farms as compared to farms in the other categories. Thus thorough knowledge of iron, along with manganese, in soil and vegetation on sheep farms could be a pivot in studies on sporadic scrapie.This study was undertaken in order to examine whether any connection existed between the amounts of iron in forage and the sporadic occurrence of scrapie observed in certain parts of Iceland. As iron and manganese are considered antagonistic in plants, calculation of the Fe/Mn ratios was also included by using results from Mn determination earlier performed in the same samples. Forage samples (n = 170) from the summer harvests of 2001–2003, were collected from 47 farms for iron and manganese analysis. The farms were divided into four categories: 1.  TSEs are always lethal. As the name implies, a distinguishing feature of TSEs is their transmissibility between individuals of the same species, or even between individuals of different species . In this context it is of interest that the high iron levels found in forage from scrapie-afflicted farms border on the toxic levels of the metal for plants [The amounts of soluble iron in Icelandic soil are known to be high . The iror plants .The mean iron concentration in forage samples from scrapie-afflicted farms was significantly higher than in forage samples from farms in the other scrapie categories Fig. . The forWe have previously found that the mean manganese concentration was significantly lower in forage samples from scrapie-afflicted farms than in forage samples from scrapie-prone farms and scrapie-free farms (categories 1 and 2 combined) . How thiAdriano [In this context, it is of interest that in plants, manganese and iron act as biochemical antagonists and may compete with each other with regard to absorption from soil and enzyme binding ,13. AccoAdriano , the Fe/The results presented here would indicate that high amounts of iron in the forage of sheep might somehow premise the occurrence of clinical scrapie through lower, albeit not deficient, levels of manganese. Whether or not high amounts of iron in the forage might per se, or by other mechanisms, have any such effect awaits further research.In conclusion, the forage samples from scrapie-afflicted farms contained significantly more iron and significantly less manganese than the forage samples from scrapie-free and scrapie-prone farms, indicating a relative dominance of iron over manganese in forage from scrapie-afflicted farms as compared to farms in the other categories. A more detailed study of iron, along with manganese, in the soil and vegetation on sheep farms might be the key to understand why scrapie is recurrently diagnosed on some farms and yet never occurs on certain neighbouring farms.


Isolated splenic metastases from colorectal cancer are very rare and there are only 13 cases reported in the English literature so far. Most cases are asymptomatic and the diagnosis is usually made by imaging studies during the evaluation of rising CEA level postoperatively.th postoperative month necessitated further imaging studies including computed tomography of the abdomen which revealed a mass in the spleen that was subsequently confirmed by 18FDG- PET scanning to be an isolated metastasis. The patient underwent splenectomy 17 months after his previous cancer surgery. Histological diagnosis confirmed a metastatic adenocarcinoma with no capsule invasion. After an uneventful postoperative period, the patient has been symptom-free during the one-year of follow-up with normal blood CEA levels, although he did not accept to receive any further adjuvant therapy. To the best of our knowledge, this 14th case of isolated splenic metastasis from colorectal carcinoma is also the first reported case of splenic metastasis demonstrated preoperatively by 18FDG PET-CT fusion scanning which revealed its solitary nature as well.A 76-year-old man underwent an extended left hemicolectomy for synchronous colon cancers located at the left flexure and the sigmoid colon. The tumors were staged as IIIC (T3N2M0) clinically and the patient received adjuvant chemotherapy. During the first year follow-up period, the patient remained asymptomatic with normal levels of laboratory tests including CEA measurement. However, a gradually rising CEA level after the 14Isolated splenic metastasis is a rare finding in the follow-up of colorectal cancer patients and long-term survival can be achieved with splenectomy. From the first case report of isolated metastasis to the spleen in the setting of colorectal carcinoma by Dunbar et al.  in 1969,et al. -13. Twelet al.  reportedet al. . Most ca®, Roche) 5 × 500 mg capsule for 14 days with 7-day interval for a total of 8 courses. Blood CEA level decreased to the 3.54 ng/mL in the early postoperative period and remained normal up to the 14th month of follow-up. A rising CEA level  showed that the tumors located at the left flexure and the sigmoid colon and the endoscopic biopsies revealed the diagnosis of adenocarcinoma for both of the tumors. There was neither liver nor other remote site metastasis in the abdomen. The chest X-ray was normal. However, CEA level was higher  in the preoperative period , whereas it was in cecum and ascending colon in the remaining two. As previously stated by Avesani et al. , this ob [et al. ; a possiDiagnosis of splenic metastasis as well as other remote organ metastasis from colorectal cancer can be made by help of many current imaging studies such as US, CT or MRI. However, FDG-PET scanning provides a better detection of metastatic deposits, especially when they are too small to be shown by other radiological studies in the setting of postoperatively rising CEA level. In addition, PET-CT fusion may specifically suggest that the mass seen by the CT is tumoral in nature. On the other hand, solitary status of metastasis can be demonstrated and predicted preoperatively by PET-CT fusion scanning as in the present case. Therefore, the indication of splenectomy can be justified in such cases since the presence of multiple remote site metastases usually exterminates the chance of surgical intervention.Long-term survival rate following splenectomy in patients with solitary splenic metastasis from colorectal cancer is still unknown. However, the limited data extracted from the case reports in the literature indicate that these patients may survive up to 7 years ,12,13. TIsolated splenic metastasis is a rare finding in the follow-up of colorectal cancer patients and long-term survival can be achieved with splenectomy.CEA: carcinoembryonic antigen18FDG-PET: 18Fluorodeoxyglucose – positron emission tomographyPET-CT: positron emission tomography – computed tomographyUS: ultrasoundCT: computed tomographyMRI: magnetic resonance imagingThe author(s) declare that they have no competing interests.RG performed the operations and prepared the manuscript. FA and GK performed the histopathologic evaluations. NT participated in the redaction of the article. All authors read and approved the final manuscript.


Patients increasingly use the Internet to solicit "tele-advice" via email from physicians. In two different studies we evaluated the quality of the advice given and determined the attitude of physicians and the ethical codes towards giving such tele-advice.In both studies, we posed as a patient describing a medical problem (herpes zoster under immunosuppresion) in lay terms and asked physicians for their advice via email. In the first study, we contacted 17 professional cyberdocs who explicitly offered to give tele-advice. In the second study, we sent an "unsolicited" patient email to 58 physicians who did not offer tele-advice to evaluate their reaction. The second study was also complemented by a questionnaire asking them for their attitude towards answering medical questions via email. Finally, we conducted a review of the ethical, legal and professional codes in the US, Germany, Switzerland to investigate differences in the ethical assessment of giving advice via telecommunication in the absence of a pre-existing patient-physician relationship.From the 17 professional "cyberdocs", only 7 replied giving detailed advice, 2 of the responses were medically wrong. In the second study with the "unsolicited email", 29 (50%) responded to the fictitious patient request; 9 respondents (31%) refused to give advice without having seen the lesion, 27 (93%) recommended that the patient see a physician, and 17 (59%) explicitly mentioned the correct "diagnosis" in their reply. In response to the questionnaire, 8 (28%) of the 29 respondents said that they tended not to answer any patient e-mail, 7 (24%) said they usually reply with a standard e-mail message, and 7 (24%) said they answer each request individually. Slight differences in the professional codes between the US, Germany and Switzerland exist: While the US AMA ethical guidelines only warn to make a diagnosis and considers drug prescriptions unethical, the German code disallows any advice via mail or email in the absence of a physician-patient relationship.These pioneering studies give important hints on the possible economics, problems and ethics of patient-physician communication via email. Responses of physicians and Web masters to e-mail requests for medical advice vary as do approaches to handling unsolicited e-mail. International standards for physician response to unsolicited patient e-mail are needed.


The skeletal muscle is an unusual site for metastasis from renal cell carcinoma (RCC). Metastatic RCC must be differentiated from benign primary soft-tissue tumors because aggressive surgical resection is necessary.We present the case of a 65-year-old man with metastatic RCC in the gluteus maximus muscle (3.8 cm in diameter) found on enhanced computed tomography (CT) 6 years after nephrectomy. Retrospectively, the small mass (1 cm in diameter) was overlooked 5 years earlier on enhanced CT. Because the growth of the lesion was slow, benign tumor was a differential diagnosis. However, magnetic resonance imaging (MRI) showed that the mass had high-signal intensity on T1- and T2-weighted images (WIs) compared to that of skeletal muscle, with mild enhancement by Gadolinium. The MRI features were unusual for most soft-tissue tumors having low-signal intensity on T1-WI and high-signal intensity on T2-WI. Therefore, under a diagnosis of metastatic RCC, the lesion was resected together with the surrounding skeletal muscle. The histology was confirmed to be metastatic RCC.MRI features of metastatic RCC may be beneficial in differentiating it from primary soft-tissue tumor. Renal cell carcinoma (RCC) has widespread and unpredictable metastatic potential , even afMaking a diagnosis of metastatic RCC to the skeletal muscle is challenging, because the site is unpredictable, in addition to it being rare. Furthermore, cases of metastasis arising long after nephrectomy have been reported ,5. The dGenerally, either open or needle biopsy is necessary in order to make a diagnosis, in cases of soft-tissue tumor, because the surgical procedure is different depending upon the histological diagnosis. In the current paper, we present a case of RCC with metastasis to the gluteus muscle. The lesion was treated without biopsy, because the MRI features suggested metastatic RCC. We wish to emphasize the MRI features of metastatic RCC to the skeletal muscle, which could be beneficial in differentiating metastatic RCC from primary soft-tissue tumor.A 59-year-old man underwent right radical nephrectomy for RCC of 3.8 centimeters in diameter stage pT1a N0 M0). One year after the nephrectomy (when he was 60 years old), recurrence was seen in the adrenal gland. Consequently, resection of the adrenal gland was undertaken. Six years after the nephrectomy (when he was 65 years old), a routine checkup using computed tomography (CT) of the chest and abdomen detected a small mass in the gluteus maximus muscle. The tumor was located in the gluteus maximus muscle adjacent to the fascia, and this was enhanced by contrast medium  showed that the lesion adjacent to the fascia had higher signal intensity than that of skeletal muscle on T1- and T2-weighted, and T2-STIR images. The lesion had a rather regular border to the surrounding gluteus maximus muscle, and was capsulated by a thin area with low-signal intensity on both T1- and T2-weighted images Figure . Edema oPrimary soft-tissue tumor was a differential diagnosis. Because the growth of the lesion was slow over 5 years, benign soft-tissue tumor was suspected. The fact that the lesion was capsulated by a thin area as seen on MRI also supported the diagnosis. However, the MRI features were unusual for most soft-tissue tumors having low-signal intensity on T1-weighted imaging and high-signal intensity on T2-weighted imaging. Therefore a diagnosis of metastatic RCC was suspected rather than primary soft-tissue tumor. Because metastatic RCC was strongly suspected, wide resection including the surrounding skeletal muscle was undertaken. Biopsy was not performed in order to avoid unnecessary contamination from the procedure. The resected tumor was composed of alveolar proliferation of carcinoma cells with clear cytoplasm and small round-shaped nucleoli (Fuhrman grade 2), resembling the previously resected RCC, suggesting metastatic RCC Figure .Surgical resection of metastatic RCC reportedly improves the outcome of patients with metastatic RCC, and five-year survival rates are between 35% and 50% after surgical therapy for solitary metastasis . In the Another factor which makes a correct diagnosis difficult is that metastasis sometimes occurs long after primary surgical treatment. It has been reported that 11% of metastatic RCC cases occurred more than 10 years after initial diagnosis  and metaMetastatic RCC to the skeletal muscle must be differentiated from primary soft-tissue tumors. Primary soft-tissue tumors are more common than metastatic tumors to the skeletal muscle . GeneralIn the current case, the characteristic features of MRI for metastatic RCC were beneficial in differentiating it from primary soft-tissue tumor, because most cases of primary soft-tissue tumor have low- to iso-signal intensity relative to the skeletal muscle on T1-weighted images and high-signal intensity on T2-weighted images. Although lipoma, hemangioma , clear cThe high intensity of hemangioma seems to be related with the fat component, as is the case with lipoma. Higher signal intensity on T1-weighted images of clear cell sarcoma  is reported to be correlated with melanocytic differentiation . We haveIt has been reported that angiography shows that RCC metastatic to the skeletal muscle usually appears as hypervascular lesions ,8. The GIn conclusion, we present the case of a 65-year-old man with metastatic RCC to the gluteus maximus muscle. The nodule was slowly progressive over 5 years. MRI of metastatic RCC is characterized by high-signal intensity on T1- and T2-weighted images which may be beneficial in the differential diagnosis from primary soft-tissue tumors. Maintaining a high degree of suspicion of metastatic RCC is required for patients with a history of RCC.The author(s) declare that they have no competing interests.AS drafted the manuscript. TY, KT and ShM participated in the design of the study and performed. SuM, YO and YH and conceived of the study. AY and YI participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All authors read and approved the final manuscript.