Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for ~50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.Nematostella vectensis and the amphioxus Branchiostoma floridae to demonstrate that they are laforin orthologs. We demonstrate that the laforin gene has a unique evolutionary lineage; it is conserved in all vertebrates, a subclass of protists that metabolize insoluble glucans resembling LBs, and two invertebrates. We analyzed the intron-exon boundaries of the laforin genes in each organism and determine, based on recently published reports describing rates of molecular evolution in Branchiostoma and Nematostella, that the conservation of laforin is linked to the molecular rate of evolution and the glucan metabolism of an organism.Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate genomes sequenced to date. We biochemically characterized recombinant proteins from the sea anemone Our results alter the existing view of glucan phosphorylation/dephosphorylation and strongly suggest that glucan phosphorylation is a multi-Kingdom regulatory mechanism, encompassing at least some invertebrates. These results establish boundaries concerning which organisms contain laforin. Laforin is conserved in all vertebrates, it has been lost in the vast majority of lower organisms, and yet it is an ancient gene that is conserved in a subset of protists and invertebrates that have undergone slower rates of molecular evolution and/or metabolize a carbohydrate similar to LBs. Thus, the laforin gene holds a unique place in evolutionary biology and has yielded insights into glucan metabolism and the molecular etiology of Lafora disease. Lafora disease LD; OMIM 254780) is an autosomal recessive neurodegenerative disorder. It is one of five major progressive myoclonus epilepsies (PMEs) . LD comm54780 is While animals normally store glucans as soluble glycogen, LBs are accumulations of poorly branched, hyperphosphorylated, insoluble glucans and are not glycogen. Forty years ago, Sakai and co-workers biochemically characterized LBs and found that they more closely resemble plant starch than glycogen -8. AlthoEPM2A (epilepsy of progressive myoclonus type 2) is mutated in ~50% LD cases [EPM2A encodes a protein named laforin that contains a carbohydrate binding module (CBM) followed by the canonical dual specificity phosphatase (DSP) active site motif, HCXXGXXRS/T (Cx5R) [in vitro and in vivo, and it possesses phosphatase activity in vitro [LD cases ,10. EPM2T (Cx5R) ,11  [SEX4 encodes a protein with similar domains as laforin but in the opposite orientation , a cellular phenotype reminiscent of LD patients [sex4-deficient plants that it rescued the sex4 mutant phenotype. Demonstrating that laforin and SEX4 are functional equivalents and that a laforin-like activity is required to regulate the metabolism of insoluble glucans in multiple Kingdoms [To test this hypothesis, we utilized a recently characterized gene in 4 (SEX4) ,20. SEX4ion Fig. . Disruptpatients . We founKingdoms .We also demonstrated the nature of this activity; we showed that laforin and SEX4 dephosphorylate the glucan itself ,21,22. TNeospora caninum. In addition, we extend our previous results by uncovering putative laforin orthologs in two invertebrates, Nematostella vectensis and Branchiostoma floridae. We cloned the respective genes and biochemically verified that they are laforin orthologs. Furthermore, we present evidence and hypothesize why laforin is conserved in these two invertebrates and is absent in all others sequenced to date. Cumulatively, these results demonstrate that the glucan phosphatase laforin is conserved in a subset of eukaryotic organisms from an array of evolutionary niches, vertebrates, invertebrates, and protists, and highlights the fundamental importance of glucan phosphorylation/dephosphorylation.Herein, we establish definitive boundaries concerning the evolutionary conservation of laforin by probing more than 210 eukaryotic genomes. We utilized criteria that we previously defined to correctly predict a laforin ortholog in the genome of the protozoan Carbohydrate binding modules (CBMs) are domains typically found in glucosylhydrolases and glucotransferases in bacterial, fungal, or plant genomes -27. The Toxoplasma gondii, Eimeria tenella, Tetrahymena thermophila, Paramecium tetraurelia, and Cyanidioschyzon merolae [SEX4 is conserved in green algae and land plants (collectively known as Archaeplastida/Kingdom Plantae) and showed that while laforin and SEX4 are not orthologous proteins that they are functional equivalents [Plasmodium species lack laforin because they do not metabolize insoluble glucans, Cryptosporidium species lack laforin because they lack true mitochondria, and Chlamydomonas lack laforin because they are not of red-algal descent (and instead have SEX4) [Galdieria sulphuraria, Guillardia theta, Neospora caninum, and Sarcocystis neurona [Previously we reported that out of 170 eukaryotic genomes (including 94 protozoan) and 670 bacterial genomes that the laforin gene is only conserved in vertebrate genomes and in five protozoan genomes,  merolae   . Some of neurona .C. merolae (Cm-) laforin, as C. merolae is likely the most evolutionarily ancient organism with laforin and Cm-laforin was the least identical (25%) to Hs-laforin of all the protozoan laforin orthologs previously identified [In light of the relatedness between CBM20 and CBM21 CBMs, we probed 122 protozoan genomes (28 more than previously) searching for a protein containing a CBM20 or CBM21 domain followed by a phosphatase domain. In order to enhance our likelihood of uncovering a laforin ortholog, we performed BLASTp and tBLASTn searches of multiple databases  was only16% and 23% similar to Cm- and Hs-laforin, respectively -laforin and found that these proteins are 82% identical , and Porifera (sponges) [Metazoans are defined as all living animals that contain tissues and are descended from the last common ancestor of Bilateria, Cnidaria , Ctenophora (comb jellies), Placozoa (sponges) . Bilatersponges) ,40.Branchiostoma floridae , Ciona intestinalis (sea squirt belonging to urochordates), Monosiga brevicollis (choanoflagellate and closest known unicellular relative to metazoans), Nematostella vectensis (sea anemone belonging to the ancient metazoan Phylum Cnidaria), and Trichoplax adhaerens  [The gene encoding laforin is an evolutionarily ancient gene, originating in a primitive red alga, or its ancestor, long before the emergence of metazoans . While llacozoa) -45. WhilNematostella vectensis and the small worm-like urochordate Branchiostoma floridae  is 44% identical to Hs-laforin and contains all but one of the known residues conferring glucan binding and phosphatase activity  orthologs are 29–41% identical to Hs-laforin and share the majority of residues important for glucan binding and phosphatase activity  [Characteristic of all dual specificity phosphatases, human DSP VH1-related (VHR) and Hs-laforin exhibit phosphatase activity against the artificial substrate para-nitrophenylphosphate  . MutatioSince laforin is the only phosphatase in any Kingdom Animalia genome with a CBM, it is predicted to be the only phosphatase that binds carbohydrates. Bf- and Nv-laforin bound amylopectin to the same extent as Hs-laforin Fig. . ConversSome DSPs dephosphorylate non-proteinaceous substrates, such as phosphatase and tensin homologue (PTEN), the myotubularin family, and Sac domain phosphatases that all dephosphorylate the inositol head group of phospholipids -56. We rin vitro biochemical properties as Hs-laforin: both utilize p-NPP as an artificial substrate, bind amylopectin, and liberate phosphate from amylopectin. Additionally, the invertebrate laforin orthologs contain the critical signature primary amino acids of both a CBM20 and DSP, and they possess the same predicted secondary structure as Hs-laforin. Thus, our bioinformatics searches for a protein containing a CBM and DSP correctly predicted the proteins biochemical properties and we identified novel laforin orthologs. Our finding of laforin orthologs in two invertebrate genomes reinforces the global function of this protein and yields insights into the evolution of this gene family.Bf- and Nv-laforin possess the same three SEX4 is conserved in diverse members of Archaeplastida/Kingdom Plantae and is necessary for proper starch metabolism in Arabidopsis [As discussed above, we previously reported that laforin is not confined to vertebrate genomes, but it is also conserved in five protozoan genomes . The genbidopsis ,19,20. TWe previously postulated that throughout evolution organisms maintained or lost laforin depending on their "need" to manage insoluble glucans . We suggB. floridae, T. thermophila, and P. tetraurelia. The laforin paralogs have an array of intron-exon numbers and boundaries, from a single exon to similar numbers (4 exons) and boundaries as Hs-laforin to as many as eight exons , two invertebrate genomes, and six protozoan genomes Fig. . Additioons Fig. . The Apions Fig.  all contPhyscomitrella patens), and a single-cell green alga  [Nematostella and Branchiostoma also have laforin in order to combat LBs.Alternatively, d birds) -64. It iNematostella and Branchiostoma may have laforin because their genomes have not evolved as rapidly as other invertebrates, as has been recently demonstrated, and thus still have/need laforin [Lastly,  laforin ,44. If tRegardless of what has driven the evolutionary lineage of laforin, these results define the boundaries of glucan phosphatase conservation. These findings coupled with our previous work establish the conservation of laforin and SEX4 in evolutionary niches from protozoans to plants to algae to vertebrates and now invertebrates. Notably, laforin and SEX4 are absent in bacteria and archaea. While these groups do contain each domain that is found in glucan phosphatases (i.e. a phosphatase domain and carbohydrate binding domain), no protein in their genomes contains both. Given the completeness of bacterial genomes, it is unlikely that a glucan phosphatase exists in bacteria. Therefore, we suspect that we have identified the complete evolutionary lineage of the glucan phosphatases laforin and SEX4. The conservation of laforin across evolutionary niches, coupled with what appears to be complete conservation of SEX4 throughout all Archaeplastida/Kingdom Plantae demonstrate that phosphorylation/dephosphorylation of glucans is pervasive throughout nature.Branchiostoma floridea Gene Collection [Nematostella DNA provided by Dr. Mark Q. Martindale. Bf- and Nv-laforin were cloned into pET-GSTX and pET21a, respectively [2+-agarose  and/or glutathione-agarose affinity chromatography steps as described previously [Wild type and C/S Hs-laforin in pET21a  for use in bacterial expression were described previously ,65. Recollection . The comectively . Mutatioeviously .para-nitrophenylphosphate (p-NPP) was performed in 50 μl reactions containing 1X phosphate buffer , 50 mM pNPP, and 100–500 ng of enzyme at 37°C for 5–30 minutes. The reaction was terminated by the addition of 200 μl of 0.25 M NaOH and absorbance was measured at 410 nm. We tested the specific activity of each enzyme at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. Malachite green assays were performed as described [N-ethylmaleimide and 80 μl of malachite green reagent. Absorbance was measured after 30 minutes at 620 nm. We tested the specific activity of each enzyme at the same pH units as above.Hydrolysis of escribed  with theCarbohydrate binding assays were done similarly as described previously . BrieflyCyanidioschyzon merolae genome project, Department of Energy Joint Genome Institute Resource, The Institute for Genomic Research (TIGR), ToxoDB, GeneDB, Genoscope, UCSC Genome Browser, Tetrahymena Genome Database, and multiple organism specific databases. Accession numbers for small subunit (SSU) ribosomal RNA (rRNA), SEX4 orthologs, and laforin orthologs are listed in Additional File The sequences of laforin and SEX4 orthologs were obtained by performing tBLASTn searches using the GenBank "dbEST" database or BLASTp and PSI-BLAST  searchesin vitro biochemical assays, assisted with interpretation of results, and assisted with writing that paper and generating figures. Both authors read and approved the final manuscript.MSG initiated the study, cloned the various laforin orthologs, designed the experiments, performed experiments, performed all evolutionary analyses, compiled and analyzed data, generated figures, directed the project, and wrote the manuscript. RMP subcloned laforin orthologs into vectors, performed Table of Non-NCBI databases used in this study. The non-NCBI databases used to search for laforin orthologs are listed in blue and the organisms' genome in each database is listed in black. Many of the databases were found by performing a Google search of the organisms genus name and "genome."Click here for fileSummary of metazoan phylogeny. This is a depiction of an unrooted phylogeny showing the major classifications of metazoans . Highlighted in yellow are organisms or groups of organisms that contain laforin. Monosiga brevicollis is not a metazoan, but is arguably the closest unicellular relative to metazoans. For a broad frame of reference visit .Click here for filePurification of recombinant GST-Bf-laforin and GST-Nv-laforin. A, GST-Nv-laforin-HIS6 was purified from soluble E. coli lysate via Ni2+-agarose affinity chromatography. B, GST-Bf-laforin-HIS6 was purified from soluble E. coli lysate via Ni2+-agarose affinity chromatography. U, uninduced; I, induced; PL, post-lysis; P, pellet (insoluble) fraction; S, soluble fraction; NB, not bound to beads; Bds, bound to HIS beads; HIS E, HIS eluate; GST E, GST eluate.Click here for fileIntron-exon numbers and boundaries of laforin genes. Predicted intron-exon boundaries for the genes encoding Hs-, Tg-, Nc-, Et-, Cm-, Tt- and Pt-laforin. The coding region that encodes the CBM is highlighted in red, the DSP in blue, and the rest is in gray. Accession numbers are listed in Additional File Click here for fileTable of small subunit (SSU) ribosomal RNA (rRNA) accession numbers. Listed on the left are the organisms from the phylogeny in Figure Click here for fileTable of accession numbers for SEX4 orthologs.Click here for fileTable of genomes investigated for the presence of laforin. The genome of each organism was searched for laforin and/or SEX4 using the appropriate database (Additional File Click here for fileTable of accession numbers for laforin orthologs.Click here for file


As a proportion of AIDS-defining illnesses, Kaposi's sarcoma (KS) decreased from 1987-89 to 1993-94 in homosexual and bisexual men in all European regions and in the United States. Albeit underestimated, AIDS KS rates in the general male population at ages 25-49 are higher than those of the majority of cancer sites in the same age group.


Involuting lesions as a result of treatment related regression are also presented.This review provides a comprehensive overview of the broad clinicopathologic spectrum of cutaneous Kaposi sarcoma (KS) lesions. Variants discussed include: usual KS lesions associated with disease progression ( Th. Th49]. The only description of intravascular KS is limited to a report of six cases, including four patients with classic KS and two with AIDS-associated KS . HistoloTherapy may result in KS regression, and less likely exacerbation  . The hisThe introduction of highly active antiretroviral therapy (HAART) for patients with human immunodeficiency virus infection (HIV) may lead to complete regression of established AIDS-associated KS lesions ,54. ClinKS clearly has the ability to develop into lesions of varying morphologic appearance. It is important to be able recognize these variants in order to avoid potential misdiagnosis and improper management of afflicted patients. KS has been shown to be of lymphatic origin . This maThe authors declare that they have no competing interests.LP and WG contributed equally to the literature review, photography of cases, and writing of this manuscript. Both authors read and approved the final manuscript.


The strongest causal evidence that customary spanking increases antisocial behavior is based on prospective studies that control statistically for initial antisocial differences. None of those studies have investigated alternative disciplinary tactics that parents could use instead of spanking, however. Further, the small effects in those studies could be artifactual due to residual confounding, reflecting child effects on the frequency of all disciplinary tactics. This study re-analyzes the strongest causal evidence against customary spanking and uses these same methods to determine whether alternative disciplinary tactics are more effective in reducing antisocial behavior.This study re-analyzed a study by Straus et al. on spankThe apparently adverse effect of spanking on antisocial behavior was replicated using the original trichotomous covariate for initial antisocial behavior. A similar pattern of adverse effects was shown for grounding and psychotherapy and partially for the other two disciplinary tactics. All of these effects became non-significant after controlling for latent comprehensive measures of externalizing behavior problems.These results are consistent with residual confounding, a statistical artifact that makes all corrective actions by parents and psychologists appear to increase children's antisocial behavior due to child effects on parents. Improved research methods are needed to discriminate between effective vs. counterproductive implementations of disciplinary tactics. How and when disciplinary tactics are used may be more important than which type of tactic is used. Although physical punishment is used by as many as 94% of parents in some countries, a growinIn the only scientific consensus conference on corporal punishment, co-sponsored by the American Academy of Pediatrics, both criSince the literature review published for that conference, there haGershoff's meta-anaprimary disciplinary method or was too severe (such as beating up a child or striking the face or head). The outcomes of customary spanking were neither better nor worse than for any alternative tactic, except for one study in which spanking reduced drug abuse more than nonphysical punishment compared to a mean of d = .37 [r = .18])[The general failure of spanking to show more adverse effects than grounding and psychotherapy in our closest re-analyses in Table r = .18]). Second,These results are all consistent with the conclusion that the apparent effects of all of these corrective actions are due to residual confounding from the tendency of more oppositional children to be selected more often for disciplinary corrective actions and for professional corrective actions. Statistically controlling for pre-existing differences reduces this selection bias confound, but fallible covariate measures do not eliminate it. When initial differences in levels of externalizing behavior problems were measured more comprehensively and reliably with latent variables, no corrective action predicted significantly higher antisocial behavior two years later. This result and the similar pattern of results for all corrective actions by parents and professionals are what would be expected if these results were due to residual confounding with a selection bias. When we changed the direction of the bias by predicting latent change scores, then the apparent effects of all of these disciplinary tactics were not only non-significant, but reversed the signs of their coefficients. This is because the usual analyses of residualized gain scores in net-effects regression is biased against corrective actions, but analyses of simple gain scores are biased in favor of them,51,64.under-adjustment bias[Overall, this is the same pattern of evidence found in a major early evaluation of Head Start, which coment bias,65. Simiment bias-68.Other research has shown that most of the corrective actions in this study can be used skillfully to reduce behavior problems in children. Beneficial effects have been demonstrated from more causally conclusive designs for some psychotherapies,,28, timeIn addition to residual confounding, three other methodological aspects of this type of longitudinal analysis may suppress the detection of effective corrective actions. The first aspect that might suppress evidence of effectiveness is that overly frequent use of any disciplinary tactic reflects less effective ways of implementing it. The more effectively any disciplinary tactic is used, the less the child will misbehave and the less often a parent will need to resort to that disciplinary tactic again. Therefore more effective use of any disciplinary tactic will be associated with a lower frequency of using it, other things being equal. This would be particularly true of last-resort tactics, such as spanking or psychotherapy. Frequent use of a last-resort tactic is a symptom of dysfunction in the entire disciplinary system as well as a symptom of the challenge to that system by the child's oppositional behavior.A second factor that might suppress evidence of effectiveness is that two years is too long an interval to detect a causal effect of the frequency of any disciplinary tactic during one week. Many events may occur in the course of two years that influence antisocial behavior in the life of a child, including genetic effects, peer effects, and other parenting effects. These other causal influences may account for almost all of the development of antisocial behavior over the next two years, leaving little more to be explained by how often disciplinary punishments were used during a single week two years earlier.rs from .25 to .27), although mothers' and fathers' reports correlate more highly with each other (r = .59)[A third suppressor of effectiveness might be exclusive reliance on maternal report, which has limited reliability and some likely biases. It is well known that parental reports of child behavior problems have low positive correlations with reports from teachers, children, and observers . By aski(r = .59). However(r = .59).Finally, the near-zero effects may represent the average of effective and ineffective use of these disciplinary tactics in reducing antisocial behavior. The failure to find between-tactic differences in effectiveness raises the possibility that within-tactic differences in how and when a disciplinary tactic is used may be more important than which tactic is used. From this perspective, different ways that parents use these forms of punishment may counterbalance each other, yielding the overall non-significant coefficient. This view is consistent with anecdotes from behavioral parent trainers, who train parents how to use time out consistently, even though many referred parents say they have tried time out previously without success.Consistent with this last possibility, the meta-analysis by Larzelere and Kuhn found thFuture research needs to discriminate between effective and counterproductive ways of implementing all disciplinary tactics, so that advice to parents can recommend the mildest effective disciplinary tactics for each situation. Statistically controlled studies of the outcomes of frequency of usage fail to provide those discriminations because frequency measures include no information about how disciplinary tactics were implemented or the disciplinary situations for which they were used.Some limitations of this study should be noted. First, all the data were based on maternal report, identical to the original study by Straus et al. and all A third limitation is that we have not duplicated the original study exactly, although we re-analyzed it as closely as possible. In contrast to the original study, we used a log transformation for the antisocial behavior to reduce the influence of extreme outliers in its skewed distribution. We also dropped 22 cases (2.7%) because they had missing data on one or more of the nonphysical consequences in order to ensure that the comparisons among disciplinary tactics were based on exactly the same sample and types of analyses.Notwithstanding these limitations, the present study has shown that the strongest causal evidence against customary spanking seems to be due to residual confounding because behaviorally difficult children cause parents to use all disciplinary corrective actions more frequently. When the measure of initial child differences is improved, the evidence against customary spanking becomes non-significant, as does similar evidence against grounding and psychotherapy. The apparently adverse effects become nonsignificant more readily for the two milder disciplinary tactics, most likely because of a differential selection bias, because they tend to be selected more often for milder types of misbehavior than the other corrective actions.What are the implications for pediatric advice about disciplinary guidance for parents of young children? The disciplinary goal of parents should be to rely as much as possible on the mildest disciplinary response that will be effective for maintaining age-appropriate levels of cooperation. Verbal correction and reasoning can be effective for children as young as two years of age, especially if it is used emphatically and backed up with nonphysical consequences when necessary,74. MildEven though our results do not show spanking to be causally linked to subsequent antisocial behavior, they should not be understood as an unqualified endorsement of spanking. In the larger debate on whether or not governments should prohibit a parent's right to retain spanking as one disciplinary option, other factors outside of the scope of the present study should be considered, such as relevant moral values and when they are sufficiently compelling to impose non-spanking values on all parents. Parents should choose from the mildest disciplinary tactics that will be effective in any disciplinary situation, but defiant children need stronger tactics to enforce milder tactics to achieve that goal.Although non-empirical considerations may support a ban on spanking, the present study suggests that the strongest causal evidence against non-abusive spanking relies on methods that are inadequate for supporting a general prohibition against spanking or for identifying alternative disciplinary tactics that parents should use instead. Future studies need to use improved research methods to discriminate between effective and counterproductive disciplinary enforcements of all types to provide a stronger scientific basis for disciplinary recommendations, whether parents choose to include spanking as one of their disciplinary options or not.NLSY: National Longitudinal Survey of Youth; HOME: Home Observation for Measurement of the Environment; SES: Socio-Economic Status; SEM: Structural Equation Modeling.The authors declare that they have no competing interests.REL planned, designed, and implemented the study. RBC had a major role in working with REL in revising and finalizing the manuscript. GLS did most of the initial analyses under the supervision of REL. The final manuscript was read and approved by all authors.REL is Associate Professor in the Dept. of Human Development and Family Science (HDFS) at Oklahoma State University. He was a member of the recent Task Force on Physical Punishment of the Child Maltreatment section of Division 37 of the American Psychological Association. RBC is an Assistant Professor in the Dept. of HDFS at Oklahoma State University. GLS is a Senior Research Analyst at Girls and Boys Town, Boys Town, NE, USA.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/10/10/prepub


Cocaine (COC) inhibits the re-uptake of dopamine. However, the dopamine response to COC also depends on dopamine inside storage vesicles. The aim of this study was to investigate whether rats that differentially respond to COC differ in their dopaminergic storage capacity of the nucleus accumbens. Total and vesicular levels of accumbal dopamine as well as accumbal vesicular monoamine transporter-2 levels were established in high (HR) and low responders (LR) to novelty rats. Moreover, the effects of reserpine (RES) on the COC-induced increase of extracellular accumbal dopamine were investigated. HR displayed higher accumbal levels of total and vesicular dopamine than LR. Moreover, HR displayed more accumbal vesicular monoamine transporters-2 than LR. COC increased extracellular accumbal dopamine more strongly in HR than in LR. A low dose of RES prevented the COC-induced increase of accumbal dopamine in LR, but not in HR. A higher dose of RES was required to inhibit the COC-induced increase of accumbal dopamine in HR. These data demonstrate that HR were marked by a larger accumbal dopaminergic storage pool than LR. It is hypothesized that HR are more sensitive to COC than LR, because COC can release more dopamine from accumbal storage vesicles in HR than in LR.J. Neurochem. (2008) 105, 2122–2133. Individual differences in the susceptibility to psychostimulants have extensively been reported, both in humans . PreviouCocaine inhibits the re-uptake of monoamines by blocking plasmalemmal monoamine transporters . HoweverThe drug reserpine (RES) inhibits the VMAT-mediated uptake of cytoplasmatic monoamines into storage vesicles . The preSee The open-field selection is described by 3 = 7.0 mm3). The remaining tissue was fixated in p-formaldehyde solution in order to allow histological verification of the exact position of the punch needle. Purified accumbal vesicles were prepared by ultracentrifugation  were analyzed for VMAT-2 expression. The punches were homogenized in phosphate-buffered saline containing urea, sodium dodecyl sulfate, β-mercaptoethanol, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor . Band inFor quantification of the total levels of accumbal dopamine, the samples that were used to asses the amount of VMAT were diluted and immediately injected into the high performance liquid chromatography–electrochemical detection system. VMAT and total dopamine levels were normalized for variation in protein loading using the levels of tubulin  or the factors type of rat, treatment, and time (experiment 3). In case HR and LR were differentially sensitive to COC, the effects of RES on the effects of COC were statistically analyzed per type of rat. The relationship between the mean COC-induced increase of accumbal extracellular dopamine and the response to novelty on the open-field (traveled distance and habituation time) were evaluated by mean of Pearson's two-tailed correlation analysis. All data were expressed as mean ± SEM. A probability level of p < 0.05 was taken as significant in every test.Data were statistically analyzed using an The open-field selection procedure revealed 24% LR and 30% HR. The average distance traveled in 30 min was 3493 ± 191 and 8643 ± 373 cm in LR and HR, respectively. The average habituation time was 324 ± 28 s in LR and 1340 ± 66 s in HR. Rats that did not fulfill the criteria (46%) were not included in this study.Histological verification revealed that three LR and three HR had to be excluded because incorrect placement of the punch needle. n = 9 and HR: n = 9). The nucleus accumbens of LR was marked by smaller levels of dopamine inside storage vesicles than the nucleus accumbens of HR . The accumbal levels of general protein were equal in LR and HR [anova: type effect: n.s.], demonstrating that similar pieces of tissue were punched from the nucleus accumbens of both types of rat.The vesicular levels of accumbal dopamine are depicted in n = 10 and HR: n = 10). The anti-VMAT-2 antibody labeled both a relatively small protein of ∼70 kDa and a relatively large protein of ∼110 kDa  = 6.490, p = 0.020; F = 6.822, p = 0.018]. The antibody raised against the loading control tubulin selectively labeled a protein of ∼50 kDa (anova: type effect: n.s.), demonstrating that similar pieces of tissue were punched from the nucleus accumbens of both types of rat (n = 10 and HR: n = 10). The total levels of dopamine were significant less in the nucleus accumbens of LR than in the nucleus accumbens of HR .The accumbal VMAT-2 immunoreactivity levels are depicted in ∼110 kDa . A one-wThe dialysis probes of the present study were located in the same region of the nucleus accumbens as the dialysis probes of a previous study see . Histolon = 8 + 8 = 16) and 0.84 ± 0.15 pg/10 μL in HR (mean ± SEM of rats belonging to the pooled groups of solvent-treated HR: n = 9 + 8 = 17). Extracellular dopamine levels after RES were 0.58 ± 0.07 pg/10 μL in LR (1 mg/kg: n = 8) and 0.31 ± 0.05 pg/10 μL in HR (1 and 2 mg/kg pooled: n = 8 + 9 = 17). The RES-induced decrease of the basal levels of dopamine in LR [100%– (0.58/0.68 pg) = 15%] and HR [100%– (0.31/0.84 pg] = 63%) are very similar to the previously reported RES-induced decrease of the basal levels of dopamine in these rats . The accumbal dopamine response to novelty was larger in saline-treated HR than in saline-treated LR . One sample t-test revealed that the extracellular levels of dopamine significantly increased from baseline at 16 out of the 18 time points in control HR, whereas the extracellular levels of dopamine did not differ from baseline at any time point in control LR  on the extracellular amount of accumbal dopamine in novelty-challenged LR and HR are depicted in ntrol LR .n = 8 and HR COC: n = 8]. COC increased the extracellular dopamine levels in both HR  and LR . However, the COC-induced increase of dopamine was stronger in HR than in LR . Accumbal dopamine levels increased during the first 70 min in COC-treated HR (t-test), whereas accumbal dopamine levels increased only during the first 25 min in COC-treated LR (t-test). Pearson's analysis revealed that both traveled distance and habituation time on the open-field positively correlated with the mean COC-induced increase of accumbal extracellular dopamine .The effects of 15 mg/kg of COC on the extracellular amount of accumbal dopamine in novelty-challenged rats are depicted in n = 8 and HR: RES 1 mg/kg + COC: n = 8). The dose of 1 mg/kg of RES strongly reduced the COC-induced increase of extracellular dopamine in LR . In fact, one sample t-test revealed that accumbal dopamine did not anymore increase from baseline at any time point in these rats . A two-way anova revealed that the effects of RES were dose-dependent . The dose of 2 mg/kg of RES strongly reduced the COC-induced increase of accumbal dopamine in HR . One sample t-test revealed that dopamine significantly increased from baseline only during the first 35 min [The effects of 2 mg/kg of RES on the extracellular amount of accumbal dopamine in COC-treated HR are also depicted in t 35 min .Low responders to novelty displayed smaller amounts of dopamine inside the storage vesicles of the nucleus accumbens than HR see . These rCocaine increased the extracellular accumbal dopamine levels more strongly in HR than in LR see . These rNoradrenaline and serotonin are both known to control the release of dopamine . BecauseOne could argue that RES reduced the dopamine response to COC for the reason that RES diminished the basal levels of dopamine. Under the condition that COC blocks the re-uptake of neurotransmitters, low basal levels of extracellular dopamine result in a reduced COC-induced increase of dopamine. This explanation, however, is not supported by the present data. In HR, in which 1 mg/kg of RES strongly reduced the basal levels of dopamine (reduction 63%), the dopamine increase following COC was not inhibited at all . MoreoveThe present data were in agreement with previously reported data demonstrating that the COC-induced release of dopamine is caused by exocytosis . These d1 and D2 receptors promotes the refill of vesicles after dopamine has been released by transporter blockers like COC  might become the drugs of choice for the treatment of COC abuse. Interestingly, recent clinical screening trials on the effects of RES in COC-addicted subjects have already revealed promising results in this respect . These rIn addition to the effects of COC, the effects of methylphenidate  are also known to depend on RES-sensitive storage pools . Given tFinally, vesicular uptake is suggested to protect a neuron against the toxic effects of high levels of cytoplasmatic dopamine . In this


Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) has been associated with several neoplasias, including childhood endemic Kaposi's sarcoma (KS). It is possible that strain genotypes could contribute to the differences in regional presentation (mainly sub-Saharan Africa), childhood infection, lack of male sex bias, distinct disseminated forms and rapid fatality observed for childhood endemic KS. Early studies, at the advent of the HIV/AIDS epidemic, identified only the K1-A5 genotype in childhood KS biopsies as well as blood of a few HIV positive and negative febrile infants in Zambia, a highly endemic region. This current enlarged study analyses blood infections of 200 hospitalized infants (6–34 months age) with symptoms of fever as well as upper respiratory tract infection, diarrhoea, rash or rhinitis. KSHV and HIV viraemia and were prevalent in this group, 22% and 39%, respectively. Multiple markers at both variable ends of the genome  were examined, showing diverse previously adult-linked genotypes  detected in both HIV positive and negative infants, demonstrating little restriction on KSHV genotypes for infant/childhood transmission in a childhood endemic KS endemic region. This supports the interpretation that the acquisition of childhood KSHV infections and subsequent development of KS are due to additional co-factors. J. Med. Virol. 79:1555–1561, 2007. © 2007 Wiley-Liss, Inc. World–wide seroepidemiological analyses of Kaposi's sarcoma associated herpesvirus (KSHV) have associated this virus with KS. Analyses of tissue biopsies by polymerase chain reaction (PCR) have identified KSHV in KS: including HIV/AIDS associated, classic (Mediterranean) and endemic (African). However, little is known about childhood endemic KS, childhood endemic KS (Sub–Saharan African regions). Childhood endemic KS can be rapid and aggressive, often presenting as lymphadenopathy, having disseminated forms. Childhood endemic KS has expanded since the HIV/ AIDS epidemic and extends to half of childhood cancers in some African countries, while practically absent in Europe or USA . Is thisThe highest childhood KSHV seroprevalence are in several African countries including Nigeria (20%), Uganda (21%), Zambia (47%), Egypt (44%), Tanzania (58%), and Cameroon (32%); increasing with age, for example, in adults the seroprevalence is higher in Tanzania (89%) and Cameroon (62%), as reviewed . In contPrevious analyses of KSHV genetic diversity by PCR on DNA extracted from adult KS biopsies followed by nucleotide sequencing, show hypervariable genes at either ends of the genome. These include K1 at the left hand end (genotypes A—F) and, K12 and K14.1/K15  at the right . In adulThese diversity studies were on adult KS biopsies which have relatively high copy numbers, thus amenable to analyses. Less is known about virus genotypes infecting general populations. In adults, studies on PBMC DNA have detected K1–B and A5 genotypes in Gambia and Uganda, with similar results for adults from isolated Botswana tribes, where further variation was demonstrated . In infaPeripheral blood samples were sequentially collected from 200 children, 6 months to 3 years, with non–specific febrile illness during 2003, admitted to Paediatrics Outpatients, University Teaching Hospital, Lusaka, Zambia. Fever (>38°C) was an inclusion criteria, a consistent symptom from the few studies on primary KSHV childhood infection  childhoo2O . PCR was carried in 20 µl reactions using 1 µl DNA with PCR master mix  and pfu polymerase with plus primers for human prolactin gene, KSHV K12 , K14.1/K15 , HIV (BUP/UP/nested BU3) or HCMV  as described [DNA was extracted from 200 µl whole blood  and resuspended in 50 µl nuclease–free Hescribed ; plus K1The amplified PCR products were purified (Qiagen MinElute), followed by nucleotide sequencing using BigDye v3.1 (ABI) and ABI 3730 analyzer as described . SequencDNA was extracted from peripheral whole blood samples from the cohort of 200 febrile infants, hospitalized in Lusaka, Zambia. These were then analyzed by PCR and sequencing using primers specific for KSHV as well as human DNA control (Prolactin). All KSHV sequences collected had unique signatures and there was no evidence for PCR contamination. Moreover, many sequences subsequently derived had not been previously identified in the laboratory. One hundred and forty–one samples were included in the analyses which showed consistent results, and a positive human prolactin gene control.PCR detection of HIV and KSHV was also compared with human cytomegalovirus (HCMV), another herpesvirus related to morbidity and mortality in these HIV positive children. The results showed HIV, KSHV, and HCMV detected in 39%, 22%, and 11% infants, respectively . KSHV anThe PCR reaction (1/50 of 0.2 ml blood with sensitivity of 10–100 copies) detected at least 2,500 copies/ml blood, consistent with KSHV viremia. Given the ages of the patients (6–34 months), these are most likely primary infections, with possible maternal or sibling transmission through non–sexual routes as suggested previously for this population and neighboring regions . Fever wMany samples were at the limit of detection by PCR, which restricted subsequent multiple analyses by nucleotide sequencing. The PCR positive K1 loci was first examined by sequencing. The results showed the K1 A5 genotype, previously identified in infected children and in childhood KS in this region, but also unexpected diversity. The K1 A5 genotype was common 23% (5/22), now linked here with K12 B1 or B2 and K14.1 P genotypes, consistent with previous results for a distinct strain in this region , and preAt the loci from the right hand of the genome K12 and K14.1/K15 genotypes were identified, using primers to amplify regions as characterized previously . PreviouOne K1 sequence overlapped all of the genotypes, ABCD (K111), either a local progenitor virus or effects of recombination  and 2. TIn earlier studies, of a childhood endemic KS region, Zambia, analyses of blood infection in five infants showed only K1–A5 genotypes and this correlated with the genotype identified in different infant and childhood KS samples . Here thDespite the diversity of genotypes identified in the infant blood KSHV infections, is there any evidence for distinct strains for childhood transmission? The K1A5 genotype is prevalent in KS biopsy samples from patients in regions of Africa where there is childhood endemic KS . FurtherSince the data shown here indicates there does not appear to be any strain restriction for infant infection in a region endemic for childhood KS, this implicates other cofactors for childhood KSHV transmission and KS development. Seroepidemiological studies on KSHV transmission, identify factors including genetic predisposition, low socioeconomic status, immunosuppresion from HIV/AIDS or other influences, plus surface water exposure, possibly linking to hygiene . In this


The genetically distinct biotype 3 has penetrated Israeli freshwaters and is causing severe illness in persons who handle tilapia or carp. Vibrio vulnificus biotype 3, which caused severe soft tissue infection after fishbone injury, emerged in Israel. We conducted a follow-up study from 1998 through 2005 to assess changing trends, outcomes, and molecular relatedness of the implicated strains. A total of 132 cases (71% confirmed and 29% suspected) of V. vulnificus biotype 3 infection were found. Most infections (95%) were related to percutaneous fish exposure, mainly tilapia (83%) or common carp (13%). Bacteremia, altered immune status, and history of ischemic heart disease were identified as independent risk factors for death, which reached a prevalence of 7.6%. Pulsed-field gel electrophoresis patterns of strains from 1998 through 2005 and from 1996 through 1997 showed a high degree of homogeneity and were distinct from those of V. vulnificus biotype 1. Infections caused by V. vulnificus biotype 3 continue affect the public’s health in Israel.During 1996–1997, a new  Vibrio vulnificus, a gram-negative bacterium of the family Vibrionaceae, is a worldwide inhabitant of salt water  grown in fresh water, has been associated with V. vulnificus infection. Second, infection is caused by direct injury from the fish backbone while purchasing, cleaning, or handling the live fish, as opposed to contamination of a prior injury by immersion in seawater or ingestion of contaminated seafood. Bacteriologically, the new V. vulnificus biotype differs from other V. vulnificus strains by its biochemical features . These biochemical differences initially prevented correct identification of the strain by routine laboratory methods  and a median age of 56 years. Although no deaths were reported, 41 persons (66%) had conditions that required surgical debridement, 1 had total limb amputation, and 7 had finger amputations (V. vulnificus biotype 3 during a 7-year period (1998–2005) following the initial 1996–1997 cluster. Our study assesses the effects of infection, risk factors for death, possible spread to other fish species, and molecular relatedness of the V. vulnificus biotype 3 strain.We studied the epidemiology and the trends in illnesses associated with the new V. vulnificus infections; a standardized questionnaire was used. Investigation was initiated after passive reporting from primary physicians who treated patients with suspected V. vulnificus infection or when clinical isolates were positively identified as V. vulnificus biotype 3 by the Vibrio Reference Laboratory in the Government Central Laboratories, Israel Ministry of Health, Jerusalem. The data collected were patient’s age, sex, and place of residence; underlying diseases; circumstances of exposure and type of fish involved; time lapse from exposure to seeking treatment in the emergency department; site of infection; length of hospitalization; clinical symptoms; source of isolation; antimicrobial drug treatment; and outcome.Clinical data were obtained from the records of the Infectious Diseases Department, Epidemiology Unit, Israel Ministry of Health, Jerusalem. Subdistrict health offices reported data collected from persons with V. vulnificus or V. vulnificus biotype 3 isolated from blood or soft tissue or as suspected when a patient had suggestive history without positive cultures. Suggestive history was considered to be the development of soft tissue infection or septicemia after recent (within 7 days) fish exposure or immersion in a water pool.Cases were classified as laboratory-confirmed when a patient with suggestive history had V. vulnificus was performed in the microbiology laboratories of hospitals where infected patients had been admitted, using the API 20E strip . The laboratories submitted the isolates for confirmation and further identification to the Vibrio Reference Laboratory, Government Central Laboratories in Jerusalem. Identification of V. vulnificus biotype 3 was performed solely in this laboratory by using biochemical tests .Initial identification of V. vulnificus biotype 3 and biotypes 1 and 2 by demonstrating the unique restriction fragment length polymorphism (RFLP) patterns of biotype 3 (V. vulnificus cytotoxin-hemolysin gene (vvhA) is amplified from crude bacteria lysate (boiling a loop full of bacteria suspended in 100 μL Tris-EDTA buffer for 10 min) using primers with the sequences RRCTH 5′-CAGCTCCAGCCGTTAACCGAACCACCCGC-3′ and LCTH 5′-TTCCAACTTCAAACCGAACTATGAC-3′. This step is followed by RFLP analysis. For the enzymatic restriction reaction, 2 μL of the PCR-amplified DNA was added to a reaction mixture to give a final volume of 20 μL, according to the manufacturer’s instructions , in 2 separate reactions. The restricted DNA was separated by electrophoresis in a 2% gel that was stained with ethidium bromide and visualized for the specific biotype 3 restriction patterns. Restriction sites for 2 enzymes, KpnI and PstI, existed in the sequence of vvhA of V.vulnificus biotype 3 but not in the corresponding vvhA gene of biotypes 1 and 2  plus 4 retrospective isolates from 1997 were sent to Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA, for molecular subtyping. The isolates were recovered from the blood or wound sites of patients with a history of exposure to various fish. An additional isolate was recovered from the remnants of a tilapia fish found in the refrigerator of 1 of the infected patients (the fish remnants were diced and inoculated onto Vibrio-selective agar with and without enrichment). These 25 isolates were available for further molecular studies. Eight submitted human V. vulnificus biotype 1 isolates stored at CDC were used for comparison.Twenty randomly selected laboratory-confirmed isolates of V. vulnificus isolates were subtyped by pulsed-field gel electrophoresis (PFGE) in accordance with the PulseNet protocol for V. cholerae  was routinely added to the electrophoresis running buffer at a final concentration of 50 μmol/L  placed in every fifth lane on the gels .Proportions were compared by using the Fisher exact test or the χV. vulnificus infection were identified during the 8-year study period from 1998 through 2005  showed the unique KpnI, PstI pattern. Two patients with laboratory-confirmed V. vulnificus biotype 3 infection were excluded from further analyses because clinical data were missing. The median age of the remaining 132 patients was 66 years  (>70 years of age (1.7:1). The sites of V. vulnificus isolation included wounds in 61 patients (65% of 94 patients with laboratory-confirmed infection), blood in 24 (26%), or both in 7 (7%). The source was not recorded in 2 patients.A total of 134 cases of ugh 2005 . Most ca3 years) . OverallV. vulnificus biotype 3 infection reported immersion in a natural spring near Jerusalem with an open wound. Most of the injuries affected the hands; only 9 affected the legs.Clinical characteristics of the patients and outcomes are summarized in Cyprinius carpio) in 14 cases (13%), and both in 2 cases (2%). Two other patients reported exposure to saltwater fish: gilt-head sea brim (Sparus aurata) that was purchased in a fish store (laboratory-confirmed infection) and common gray mullet  that was purchased at a fish stand near a fishpond (suspected infection).Most of the infections (74%) occurred in the warm months of the year between June and November . Most paA total of 124 patients (94%) were hospitalized for a mean duration of 10.9 days . Information regarding treatment with antimicrobial drugs was available for 112 patients and included a variety of regimens. The most frequent regimens among these patients included only a single drug, usually ampicillin-clavulanate , doxycycline , and ceftriaxone . Combinations of drugs were also used and included third-generation cephalosporins (ceftriaxone or ceftazidime) plus doxycycline , third-generation cephalosporins plus ampicillin-clavulanate , or doxycycline plus ampicillin-clavulanate .>70 years of age, 2 were in their 50s, and 1 was in his 60s. All patients who died or underwent amputation had laboratory-confirmed infection.Ten patients (7.5%) died and 9 (6.8%) underwent amputation of fingers or part of the arm (7 patients) or a leg (2). Two of the patients who underwent amputation subsequently died. Seven of the patients who died (70%) were Ninety patients with laboratory-confirmed infections were included in the analyses of risk factors associated with death. Factors that were significantly associated with death by a univariate analysis are outlined in V. vulnificus isolates were susceptible to all antimicrobial agents tested by criteria for Enterobacteriaceae. The MIC ranges for ampicillin were 0.5–1.0 μg/mL; cephalothin, 4–8 μg/mL; chloramphenicol, 0.5–0.75 μg/mL; ciprofloxacin, 0.012–0.023 μg/mL; kanamycin, 4–16 μg/mL; nalidixic acid, 0.25–1 μg/mL; streptomycin, 8–16 μg/mL; tetracycline, 0.5–7.5 μg/mL; and trimethoprim-sulfamethoxazole, 0.064–0.094 μg/mL.>88% pattern similarity; 1–3 fragment differences) PFGE fingerprint patterns regardless of the type of fish exposure (tilapia vs. carp) and year of isolation  with reported exposure to tilapia from wound sites. When compared with biotype 1 strains reported from the United States, the biotypes separated into 2 distinct clusters with ≈70% pattern similarity  showed that the selected isolates represented 18 unique but similar  , including 8 (80%) of the 10 patients who died. Previous studies have stressed a male predominance , wound infection (mostly related to immersion in contaminated water or to injury by seafood preparation), and gastroenteritis  , laboratory confirmation of infection was lacking. These patients tended to delay seeking treatment and to have a milder form of infection, compared with patients who had laboratory-confirmed infection. Some of the patients who lacked laboratory confirmation may not have had V. vulnificus  reported exposure to the common carp, or to both tilapia and common carp . Tilapia and common carp are co-cultivated in several fishponds in northern Israel, which may have resulted in cross-contamination. For those exposed to salt water fish , contamination may have occurred at the place of purchase. V. vulnificus infection after exposure to the common carp caused the death of 1 patient and the amputation of a finger in another. One person infected after exposure to the gilt-head sea brim also died. Notably, orthopedic surgeons from hospitals in northern Israel have also pointed out severe soft tissue infections, including those caused by V. vulnificus, after fishbone injury from the common carp  with more severe illness (We identified independent risk factors for death in our group, including bacteremia, altered immune status, and history of ischemic heart disease. Septic shock was also found to be a strong predictor of death; however, septic shock may also be an outcome variable. Nonetheless, prior reports have also identified septic shock or hypotension as important risk factors for death (V. vulnificus biotype 3 into the fish aquaculture no infections were reported in Israel (V. vulnificus infection acquired through marine activities in the Mediterranean Sea. Almost the entire impact of V. vulnificus infections in Israel is associated with the freshwater fish industry. In response to the new threat, the Israeli Ministry of Health has issued regulations forbidding the selling of live, uncleaned tilapia (,Before the introduction of V. vulnificus illnesses in Israel and support a call for more strict regulations of fresh fish marketing as well as public education. Research efforts should focus on how V. vulnificus has penetrated the freshwater aquaculture in Israel and ways in which this trend can be reversed.Our findings outline the substantial effects of


Because two authors were added to the author byline, the funding statement should also include the following: SF was supported by a studentship from the French Ministry of Research. JYG was supported by Programme Amazonie du CNRS and ANR through Projet MIRETTE.


Between 1985 and 1998, 874 cases of CKS and 634 cases of AKS were diagnosed in the study areas. CKS accounted for 16 and 27% of KS cases below 55 years of age in men and women, respectively, but for 91 and 100% of those above age 55. The IRs for CKS were 1.0/ in men and 0.4/100 000 in women, but they varied between 0.3 in Umbria and 4.7 in Sassari in men, and between 0.1 in Parma and 1.7 in Sassari in women. IRs of CKS in both genders were stable between 1985–1987 and 1993–1998. In Northern and Central CRs the IR (adjusted for age and gender) for CKS was 0.5 in individuals born in the same area, but 1.6 in individuals born in Southern Italy or in the Islands (rate ratio=3.2) suggesting that KS-associated herpesvirus, the cause of KS, is acquired early in life.To evaluate incidence rates (IRs) of classic Kaposi's sarcoma (CKS) in Italy after the spread of AIDS, we distinguished CKS from AIDS-related KS (AKS) using an ‘ Before the AIDS epidemic, Kaposi's sarcoma (KS) occurred sporadically in Europe, most notably in elderly men of Mediterranean or Middle Eastern origin  . Some inSince the 1980s, consequent on the AIDS epidemic, KS incidence has increased in all Western countries . KS-assoOwing to the rise in AKS incidence, the evaluation of CKS incidence trends has become difficult. KS rates in older persons (e.g. ⩾65 years), who have an extremely low prevalence of HIV/AIDS, have been used as a proxy for CKS rates (CKS and AKS were distinguished using the record-linkage procedure of Cancer and AIDS Registries Linkage (CARL) study  was developed to perform the record linkage procedure (An ‘The present study was restricted to people who: (1) were diagnosed with KS between 1985 and 1998; (2) reported legal residence in areas covered by CRs; and (3) had KS in periods deemed complete by both CRs and RAIDS. All ages were included.KS cases were considered AIDS-related when: (a) they occurred in patients with AIDS notified at RAIDS; (b) AIDS was one of the three causes mentioned on the death certificate; (c) HIV-infection was mentioned in any additional medical record available at the CR . A total of 16 unlinked KS were reclassified as AKS according to criteria (b) and (c), otherwise the KS cases were considered as CKS.Nine KS cases were excluded because they arose in the transplant patients, information recorded in only three CRs . One KS case notified only by death certificate and three for whom the type of diagnosis  was unknown were also excluded.Standardised IRs were computed by gender, place of residence, place of birth and overall using the direct method  and the Between 1985 and 1998, 874 CKS and 634 AKS were reported to Italian CRs . The ageWith respect to site of cancer presentation, skin predominated in both CKS (88%) and AKS (86%) but skin of the leg and hip was more frequently involved in CKS than in AKS . In totar=−0.16 in men and 0.38 in women, data not shown).r=0.66) between IRs in men and women emerged. Thus, male-female ratios were generally between 2.0 and 2.5 in most of the areas, despite some probability of random variation.A high positive correlation . The practice of applying saliva to children's bite sites to relieve the itching and scratching may represent an efficient route of KSHV transmission  of CKS reported here were histologically confirmed and, therefore, misclassification with sarcomas other than KS or with skin cancer can be ruled out. Misclassification of some AKS as CKS is of greater concern but should not account for our findings. AIDS registration in Italy is nationwide and has been shown to be almost complete  and a non-negligible fraction (42%) of KS in men between 39 and 64 years. Presently, there is no evidence that the improvements in the standard of living in Italy over the last decades are causing a decline in this disease. Separation of CKS from AKS will be important in studies of the disease, especially now that HIV-positive individuals may live into old age.


Incarceration and strangulation are the most feared complications of inguinal hernia. Till date, incarcerated hernias have traditionally been treated by conventional open repair. Reports are now available for the feasibility of laparoscopic repair of incarcerated inguinal hernia. Here, we described our experience with the transperitoneal approach for incarcerated hernias.Between January 2008 and May 2008, four patients were presented with a history of irreducible hernia, abdominal distention and vomiting. All the patients had right-sided inguinal hernia. Reductions of the hernia contents were not possible in any patient. The patients were treated on emergency basis with laparoscopic transabdominal preperitoneal hernia repair. Retrospective analyses of all the patients were done.Reduction of the bowel was achieved in all but one patient, who required the division of the internal ring on lateral side. Transperitoneal mesh repair was performed. No major complications were encountered. One patient developed seroma formation that was treated conservatively.Laparoscopic transperitoneal approach has the advantage of observation of the hernia content for a longer period of time. The division of the internal ring can be done under direct vision. Other intra-abdominal pathology and opposite side hernia can be diagnosed and treated at the same time.. Gangrene in 1990.6 Till re in 1990.–9Here, we described our experience with emergency surgery for incarcerated hernia and review of the literature.Between January and May 2008, four consecutive patients with a mean age of 42 years (range: 26–59) were admitted to our hospital on emergency basis with complaint of incarcerated hernia. All patients were males. The contents of the hernia could not be returned to the abdominal cavity by manipulation. In all four patients, preoperative abdominal x-ray showed increased gas shadow in the small bowel. In all patients, incarcerated hernias were treated laparoscopically.All patients were operated upon under general anaesthesia. A pneumoperitoneum was created by a Veress needle inserted just below the umbilicus. One 10 mm and two 5 mm trocars were inserted. Working trocars were inserted in the midclavicular line on both sides. Hernias were indirect and on the right side in all four patients . LaparosIncarceration and strangulation are one of the most feared complications of the inguinal hernia. It is very important to release incarcerated hernia as soon as possible to avoid obstruction or strangulation of the bowel. Incarcerated hernias have generally been managed using the conventional technique of hernia repair. It has b10Since then few reports are available demonstrating feasibility and safety of laparoscopy in the management of incarcerated inguinal hernia.–9 Both aWe prefer the TAPP approach for incarcerated hernia. There are several advantages of this approach over the TEP approach.The TAPP approach is better in assessing the vitality of the intestine after the reduction of the hernia contents into the abdominal cavity.After reduction of the bowel, it can be observe for a longer period of time that is not possible in the open and TEP approaches.9 This isIt is not always possible to reduce the content of the incarcerated hernia without division of the internal ring as is seen in two of our cases. The division of the internal ring is often very difficult, as it is covered by oedematous bowel loops. The deep ring should be cut gently with scissors. If difficulty is encounter during division, a Maryland forceps or a grasper is inserted into the deep ring just lateral to the bowel and jaws of the instrument should be open. This will give some space to cut the ring safely. The division of the internal ring is under vision in the TAPP approach, which avoids injury to the bowel.In few cases, the TAPP procedure also allows the discovery of additional intra-abdominal pathologies and their possible simultaneous treatment. MoreoverNo attempt was made to dissect the whole sac from the cord structures. In all the patients, the sac was divided at the deep ring and the distal sac was left behind. Thus, less dissection of cord structures during the laparoscopic approach reduces the chances of cord oedema. If the problem of the recurrence after TAPP is considered, similar low rates can be found for repair of both reducible and incarcerated hernias.Nevertheless, laparoscopic hernia repair offers real advantages. These include a gain in diagnostic safety and the possibility it offers for assessing the vitality of incarcerated organs and for exploring additional intra-abdominal complications. Despite a little longer operating time for incarcerated hernias, data suggest that for incarcerated hernias, the TAPP approach is a safe and feasible option and should be considered for repair.In conclusion, the laparoscopic approach is a feasible option for incarcerated inguinal hernia. The TAPP approach allows for the assessment of the bowel for a longer period of time than the TEP approach. The division of the internal ring can be done under vision without injuring the bowel. Undiagnosed inguinal hernia or any other intra-abdominal pathology can be diagnosed and treated at the same time in TAPP repair.


DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively.in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1.To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function  Three evolutionarily-conserved DNA polymerase enzymes play essential roles at the eukaryotic replication fork . DNA polN) [Each of the three replicative polymerases α, δ and ε is a member of the B family of DNA polymerases  and eachN) ,11 and fN) .Schizosacchromyces pombe is an excellent model for studying Pol δ structure and function [Saccharomyces cerevisiae is a three-subunit enzyme; no Cdm1 orthologue is present in this organism. Pol3 binds directly to the B-subunit Cdc1 [The fission yeast function . In thisfunction ,14. Pol3function -18 wherefunction . Ortholonit Cdc1  via one nit Cdc1 . Cdc1 binit Cdc1  which innit Cdc1  and the nit Cdc1 . Cdc27 hnit Cdc1 ,24N complex [N complex  domain. The PDE domain is located in the centre of the complex with the OB domain on one side and the wHTH on the other  method [3-related transposon Tn4430 of Bacillus thuringiensis. Tn4430 transposes efficiently in E.coli and in doing so duplicates 5 bp of host sequence at the insertion point. The transposon also contains KpnI restriction sites 5 bp from the outer end of its terminal repeats. If, following insertion of Tn4430 into the target gene, the bulk of the transposon is deleted by in vitro KpnI restriction and re-ligation, a 15 bp sequence insertion remains. Since Tn4430 inserts with low sequence specificity, the pentapeptide insertions will vary in composition. The usefulness of this system has been demonstrated through its application for random mutagenesis of, amongst others, TEM-1 β-lactamase [Random mutagenesis of the ) method ,31. Thisactamase , XerD reactamase  and phosactamase .cdc1+ is outlined in Figure E.coli FH1046 containing plasmid pHT385 which carries the Tn4430 transposon [E.coli DS941 and resistant colonies identified and plasmids recovered. Restriction mapping was then used in order to identify clones in Tn4430 had inserted into cdc1+ rather than the plasmid backbone, and the bulk of the transposon sequences then removed by cleavage with KpnI followed by ligation under conditions that promoted self-ligation (see Methods).The strategy for mutagenesis of 4430 is not entirely random, as noted previously [cdc1+ gene, with 26 of the 31 insertions mapping within the first 420 bp of the 1386 bp ORF. Clustering towards the 5' end of the ORF was observed in a similar study of the S.pombe rfc2+ gene cloned into pBR322 [cdc1+ sequence. The remaining allele (Cdc1-E2) contained a 16 nt insertion, presumably the result of an inexact transposition event. This insertion leads to the Cdc1 protein being truncated after amino acid 26. Another insertion also led to production of a truncated protein: Cdc1-J17 was truncated after amino acid 27. Two of the insertions resulted in different insertions at the same amino acid position in the protein .In total, 31 mutant alleles were generated by the PSM method (Table o pBR322 . Thirty cdc1 mutations were constructed by oligonucleotide-directed in vitro mutagenesis (see Methods). Eight of these alleles resulting in the encoding proteins (Cdc1-A1 to -A8) differing from the wild-type Cdc1 by a single amino acid substitution. The remaining two alleles encoded proteins (Cdc1-A9 and Cdc1-A10) that featured substitutions of two adjacent amino acids. All the mutated amino acids were amongst those that are found conserved between the eukaryotic DNA polymerase B-subunits and the archaeal PolD small subunit [In addition to the random pentapeptide insertion mutations, ten additional  subunit ,36. MutaS.pombe, each allele was subcloned to plasmid pREP3XH6BN to permit expressing of the mutant proteins under the control of the thiamine-repressible nmt1 promoter .In order to test for function in Figure cdc1Δ background, each was expressed in wild-type cells and in cells carrying the temperature-sensitive cdc1-P13 allele [cdc1-P13 cells, the proteins were tested for their ability to rescue at the restrictive temperature of 36.5°C in cells grown in the presence and absence of thiamine. The results obtained broadly mirrored those seen with the cdc1Δ strain but with the following exceptions: at low expression levels, several of the mutant proteins that could not rescue cdc1Δ could rescue cdc1-P13 at 36.5°C, namely Cdc1-A6, -J3, -J4, -J5, -J10, -J11, -J13 and -J21, and two of the mutants, Cdc1-A10 and -J7, displayed dominant negative properties at 28°C, described further below.In addition to testing the function of the mutant proteins in a 3 allele . Expresscdc1 mutant allele was therefore cloned into the two-hybrid bait pBTM116 (or the related pBTM116BN plasmid) to allow expression of Cdc1 fused to the DNA binding domain of bacterial LexA protein (see Methods). The resulting plasmids were initially co-transformed into S.cerevisiae CTY10-5d along with the prey plasmid pGAD2F-Cdc27, which expresses Cdc27 fused to the yeast Gal4 transcriptional activation domain. Co-transformants were monitored for β-galactosidase activity by X-gal overlay assay (data not shown) and ONPG liquid assay .Two of the Cdc1 mutant proteins exerted a strong dominant negative phenotype when expressed at high level in r Figure . When trThe two-hybrid analysis described above Figure  suggestscdc1-P13 cells expressing Cdc1-J7 and Cdc1-A10 were co-transformed with plasmid pREP4X-Cdc27 which expresses the full-length Cdc27 protein, also from the nmt1 promoter [cdc1-P13 cells expressing Cdc27 and either Cdc1-J7 and Cdc1-A10 were still unable to grow on plates lacking thiamine at 28°C. We also tested whether overproduction of Pol3 or a nuclear-targeted form of the C-terminal ZnF domain of Pol3 would rescue the toxic effects of Cdc1-J7 and Cdc1-A10, despite their apparent inability to bind Pol3 as gauged by two-hybrid assay  appears to rule out the possibility that Pol3-Cdc1 two-hybrid interaction is impaired by direct modification of Cdc1-Cdc27 interaction interface. Instead, the disruption of OB domain fold by J1, J6 and J16 insertions is likely to affect the Cdc1-Cdc27 interaction in part by destabilizing the PDE domain and in part by preventing the correct positioning of N-terminal residues which are important for the function of Cdc1 [in vivo were more severe than for J6 and J21, since neither J1 nor J16 was able to substitute for loss of Cdc1 in cdc1Δ cells under any conditions, while J6 and J21 were able to rescue when overexpressed , cdc1Δ cells expressing Cdc1-J17 were able to grow , it is not surprising these the mutant protein fail to interact with both Pol3 and Cdc27, as both mutations result in incorporation of a stop codon into the cdc1Δ cells. The first sub-group contains six mutations that display either impaired function in vivo (rescuing cdc1Δ cells only when overexpressed from the derepressed nmt1 promoter) or which cannot rescue cdc1Δ cells under either of the conditions tested. This sub-group comprises the pentapeptide insertions J7 and J10, the single point mutations A3 and A6, and the double point mutations A9 and A10  is replaced by serine [This group contains a total of thirteen mutations that can be further divided into two sub-groups on the basis of the ability to substitute for wild-type Cdc1 in 0 Figure . A9 is a0 Figure . Strikin0 Figure . A mutaty serine ,39. Subsy serine . Taken ty serine . The C-tin vivo, even when expressed at near wild-type levels from the repressed nmt1 promoter , the structure of the Cdc1 protein, and more specifically the putative Pol3 binding region described in the preceding section, may be stabilised, effectively insulating the Cdc1-Cdc27 interface from the effects of disruption of the OB domain and β2α2 linker. Cdc1-Cdc27 interactions are also impaired by the J22 insertion (<10% binding in two-hybrid assay) which maps close to the Cdc1-Cdc27 interface  are identical with the residues at the positions 4 and 5 of the pentapeptide insertion  map to loop regions in the human p50 protein Figures  and 6D ain vivo function and for Pol3 and Cdc27 binding using the two-hybrid system. The results obtained can be rationalised in the context of the recently published crystal structure of human p50 bound to the N-terminal domain of human p66 [Using both site-directed and random mutagenesis strategies, thirty new mutant Cdc1 proteins have been test for uman p66  and, in E.coli FH1046 and DS941, used as the donor and recipient strains for pentapeptide mutagenesis, have been described elsewhere [E.coli JM109 and DH5α were used for routine cloning work. Standard molecular cloning methods [lsewhere  and were methods  were usecdc1–223 (mis1–223) his2 leu1–32 h+, cdc1–64 (mis1–64) ade6–704 leu1–32 h-, cdc1-A24 leu1–32 ura4-D18 h-, cdc1-P13 leu1–32 h+, cdc27-P11 leu1–32 h+, leu1–32 ura4-D18 h- and cdc1+/cdc1::ura4+ leu1–32/leu1–32 ura4-D18/ura4-D18 ade6-M210/ade6-M216 h-/h+ [cdc1/mis1 strains [cdc1-A24 was a generous gift of K.L. Gould . Standard growth conditions [S.cerevisiae CTY10-5d and standard methods were used [The fission yeast strains used in this study were as follows: 16 h-/h+ . The cdc strains  were a gnditions  were useere used .cdc1+ cDNA was sub-cloned from pBTM116-Cdc1 [cdc1+ insert in pBR322-Cdc1 is such that the 3' end of the insert is proximal to the unique HindIII site in pBR322. Mutagenesis was carried out as described previously [E.coli strain FH1046 containing the kanamycin-resistant Tn4430-carrying plasmid pHT385. Transformant (donor cell) colonies obtained after overnight growth at 37°C on LB agar plates containing kanamycin (50 μg/ml) and ampicillin (50 μg/ml) were picked and resuspended in 100 μl of LB broth (no antibiotics) and 2.5 μl spotted onto LB agar plates. Next, 2.5 μl aliquots of recipient cells (from an overnight culture of E.coli DS941 in LB supplemented with 50 μg/ml streptomycin) were applied to the donor spots, after which the plates were air-dried and incubated for 3 hours at 37°C to allow mating. The mating mixes were then resuspended in 300 μl of LB broth, and 100 μl plated onto LB agar plates containing ampicillin, kanamycin and streptomycin. Colonies were then screened either by preparing plasmid DNA and digesting this with BamHI to determine if the Tn4430 had inserted within the vector or the BamHI insert, or by PCR analysis of individual colonies using oligonucleotides flanking the BamHI site in pBR322, in which case those isolates in which a 1.6 kb band corresponding to a Tn4430-less cdc1+ insert were discarded. Each pBR322-Cdc1-Tn plasmid was then digested with KpnI, re-ligated and transformed into E.coli JM109. Plasmids were prepared from ampicillin-resistant, kanamycin-sensitive colonies and checked for absence of the transposon by restriction mapping. The 1.5 kb BamHI-NotI fragments from the transposon-free plasmids were then cloned into plasmid pREP3XH6N (below) to facilitate expression in S.pombe, sequence analysis and subsequent sub-cloning to pBTM116.For pentapeptide mutagenesis, a 1.5 kb BamHI fragment containing a 116-Cdc1  into pBR116-Cdc1 . The orieviously . Brieflyin vitro mutagenesis was carried out using the Mutagene II in vitro mutagenesis system (BioRad). The template used was pTZ19R-Cdc1-cDNA [GCTGGTCCTTATG-3', with mutant alanine codon underlined); A2, AANO2 (5'-GTCACTTTAATGCCTGCTCCTTATGATTAC-3'); A3, AANO3 (5'-CCTGGTCCTTATGCTTACAGTTCAACTATC-3'); A4, AANO4 (5'-GTTCAACTATCCTTGCTCAACAGCCTTTGC-3'); A5, AANO5 (5'-ACAAACAGTTACGGCTCCCACTTGGCTTTC-3'); A6, AANO6 (5'-AAACAGTTACGAATGCCACTTGGCTTTCTC-3'); A7, AANO7 (5'-TGGCTACTAGCGCCCAAAACATTAATGATC-3'); A8, AANO8 (5'-AATCATATCACAGCTACCAGCCCTGATACC-3'); A9, AANO1/2 (5'-GTCACTTTAATGGCTGCTCCTTATGATTAC-3'); A10, AANO5/6 (5'-ACAAACAGTTACGGCTGCCACTTGGCTTTC-3'). The resulting mutants were then subcloned to pREP3XH6BN and either pBTM116 or pBTM116BN, as below.Oligonucleotide-directed dc1-cDNA . OligonuGCGGCCGCATCG-3', with NotI site underlined and BamHI 5' overhang in lower case) and 3XH6Not-2 (5'-CGATCGCCGGCGGATGATG-3'). When cloned into pREP3XH6BN, the Cdc1 protein is expressed as a hexahistidine fusion with the 13 amino acid sequence MRGSHHHHHHGIL N-terminal to the native initiating methionine [S.pombe cdc1+/cdc1::ura4+ leu1–32/leu1–32 ura4-D18/ura4-D18 ade6-M210/ade6-M216 h-/h+ [LEU2 selectable marker. Adenine is required to permit the growth of haploid cells either the ade6-M210 or ade6-M216 alleles. The addition of uracil permits growth of cdc1+ haploids; in the absence of uracil, only cdc1::ura4+ haploids expressing functional Cdc1 proteins can grow. The presence of 5 μM thiamine represses the nmt1 promoter in pREP3X, reducing cdc1 expression by a factor of 80–100 compared to cells grown on EMM without thiamine. Following 4–6 days growth at 32°C, colonies were analysed by microscopy and by replica plating to confirm predicted genotypes.Each pentapeptide insertion allele was subcloned as a BamHI-NotI fragment from pBR322-Cdc1-Tn into pREP3XH6BN, a derivative of pREP3XH6  in whichthionine . The res16 h-/h+  by elect16 h-/h+  and tran16 h-/h+ . Individcdc1 alleles were sub-cloned as a BamHI fragment into pBTM116 [GCGGCCGCTtgca-3', with NotI site underlined and BamHI and PstI overhangs, 5' and 3' respectively, in lower case) and PBTMMOD2 (5'-AGCGGCCGCCGGATCG-3'). The plasmids were then co-transformed into S.cerevisiae CTY10-5d along with either pGAD2F-Cdc27  or pGAD2F-Pol3 and pAA-Cdc27 . β-galactosidase activity was determined by an X-gal agar plate overlay assay and later by ONPG liquid culture assay, as previously described [For two-hybrid analysis, mutant  pBTM116  or as a escribed ,21. LiquN and analysis of the possible effects of Cdc1 mutations to Cdc1-Pol3 and Cdc1-Cdc27 interactions. The project was initiated and overseen by SAM. All authors read and approved the final manuscript.The A and J mutant alleles were constructed and analysed in yeast by JSG who also performed all the two-hybrid analysis. The E mutant alleles were constructed and analysed in part by EVK. The remaining analysis was performed by FCG. AGB and THT contributed to the mapping of mutants to the three-dimensional structure of p50•p66


Ortner's syndrome  is described in literature as occurring secondary to a variety of conditions. Spontaneous aneurysm of ductus arteriosus is a rare cause of this condition. We present a case where an adult patient with an aneurysm of the ductus arteriosus presented for the first time at the age of 62 years with hoarseness of voice secondary to left recurrent laryngeal nerve palsy.  A 62-year-old man presented with hoarseness ofvoice of 3 months' duration. The clinical history was negative for any major illness in the past. There was no history of any cardiovascular ailment during childhood. Indirect laryngoscopy showed left vocal cord palsy, without demonstrating any definite laryngeal mass lesion. A CT scan of the chest showed a luminal out-pouching along the inferior surface of the aortic arch. The characteristic location of the out-pouching in the aortopulmonary window suggested the diagnosis of a ductus arteriosus aneurysm. The saccular aneurysm measured approximately 3 cm in diameter and showed a wide neck, without any intraluminal thrombus. The aneurysm showed a distinct bulge toward the main pulmonary artery and appeared thick-walled . There wThe sections through the neck did not reveal any laryngeal pathology. In view of the patient's symptoms, surgery was advised. The patient refused surgery and is presently on regular follow-up.Ductus arteriosus is a normal anatomic structure that provides communication between the systemic and pulmonary circulations during fetal life and closes soon after birth. An indentation of the aortic wall at the site of insertion of the obliterated ductus arteriosus is seen in approximately 9–26% of adults on angiography studies and is referred to as a ductus diverticulum or bump. Aneurysm of the ductus arteriosus may occur either spontaneously or may follow surgical treatment of a patent ductus arteriosus. SpontaneSADA presenting in adults usually shows an obliterated pulmonary end of the ductus, unlike aneurysms in the pediatric age-group which occur in an open ductus arteriosus. The presence of concomitant hypertension can be a contributory factor. Connective tissue disorders such as Marfan syndrome and Ehlers-Danlos syndrome are known to predispose to ductus arteriosus aneurysms as well.6 Hoarseness of voice, cough, anorexia, and chest pain are common presenting symptoms in adults and may be secondary to involvement of the adjacent organs and nerves. Hoarseness of voice occurs due to compression of the recurrent laryngeal nerve as it courses through the aorto-pulmonary window.Ortner's syndrome was first described by Ortner in 1897,Radiologically, SADA can present as a mass lesion in the aortopulmonary window and can be visualized on chest radiographs, CT scan, and MRI. Contrast-enhanced CT scan is the optimal imaging modality to establish the diagnosis. The axiaSADA should also not be mistaken for a ductus diverticulum (‘ductus bump’). The latter is a small conical bulge along the posteroinferior aspect of the aortic arch and is usually incidental and does not cause any compression of the recurrent laryngeal nerve. The diagnosis of aneurysm of ductus arteriosus should therefore be made with caution. Rupture of the aneurysm is reported to be the commonest complication in adults. Erosion 2Thus, SADA is an uncommon but important cause of left recurrent laryngeal nerve palsy in adults.


Anopheles gambiae is polymorphic for chromosomal inversions on the right arm of chromosome 2 that segregate nonrandomly between assortatively mating populations in West Africa. One such inversion, 2Rj, is associated with the BAMAKO chromosomal form endemic to southern Mali and northern Guinea Conakry near the Niger River. Although it exploits a unique ecology and both molecular and chromosomal data suggest reduced gene flow between BAMAKO and other A. gambiae populations, no molecular markers exist to identify this form.The malaria vector To facilitate study of the BAMAKO form, a PCR assay for molecular karyotyping of 2Rj was developed based on sequences at the breakpoint junctions. The assay was extensively validated using more than 700 field specimens whose karyotypes were determined in parallel by cytogenetic and molecular methods. As inversion 2Rj also occurs in SAVANNA populations outside the geographic range of BAMAKO, samples were tested from Senegal, Cameroon and western Guinea Conakry as well as from Mali.j standard homozygotes.In southern Mali, where 2Rj polymorphism in SAVANNA populations was very low and most of the 2Rj homozygotes were found in BAMAKO karyotypes, the molecular and cytogenetic methods were almost perfectly congruent. Elsewhere agreement between the methods was much poorer, as the molecular assay frequently misclassified 2Rj heterozygotes as 2R+j standard and 2Rj inverted homozygotes. Therefore, the proposed approach overcomes the lack of a rapid tool for identifying the BAMAKO form across developmental stages and sexes, and opens new perspectives for the study of BAMAKO ecology and behaviour. On the other hand, the method should not be applied for molecular karyotyping of j-carriers within the SAVANNA chromosomal form.Molecular karyotyping of 2Rj is robust and accurate on 2R+ Anopheles gambiae s.s. is a widespread and efficient vector of malaria across sub-Saharan Africa. This species also is characterized by abundant chromosomal inversion polymorphisms that are considered important sources of ecological flexibility ..Anophelej) or inverted (2Rj) arrangement by importing sequences corresponding to the breakpoint junctions from the A. gambiae reference genome  or from A. gambiae BKO [j proximal breakpoint  and another pair targeting the 2Rj distal breakpoint  . These primers amplify a 494 bp product spanning regions 3–4 that is unique to the uninverted arrangement, as region 3 is no longer adjacent to region 4 on the inverted chromosome  were found among 553 2R+j standard and 409 2Rj inverted homozygotes. Of the 2Rj inverted homozygotes, 377 (92%) carried BAMAKO karyotypes: jcu with b polymorphic.Collections of half-gravid j primers P3 and P4, and (3) only 2Rj primers D1 and D1IN. This procedure gave insight into whether the initial result was repeatable, and which arrangement might be responsible for the anomalous results. Upon repetition, the molecular results were unchanged; the correct size bands also appeared in reactions containing only 2Rj or 2R+j primers. Although the chromosome preparation was not available for validation, a true inversion heterozygote would be difficult to miss cytologically. Nevertheless, human error could have resulted in mistaken data entry, dissociation of carcass and chromosomes into tubes with different numbers, or DNA contamination leading to disagreement between the two methods. The rate of such error is acceptably low, as correspondence between molecular and cytological determinations of 2Rj standard homozygotes was nearly 100% (305/306).The 2Rj multiplex assay was conducted on DNA extracted from 550 of the 968 karyotyped specimens, chosen from the seven villages such that the representation of 2Rj standard and inverted arrangements was approximately equal. Correspondence between cytological and molecular determination of the 2Rj karyotype was high (≥ 99%) for both homokaryotypes Table . In the Among 241 2Rj inverted homozygotes that were successfully amplified by PCR, 238 (98.8%) yielded the 253 bp band in agreement with traditional karyoype analysis. Two of three exceptions yielded a single band larger than 253 bp, but slightly smaller than the 494 bp band expected of standard chromosomes. In both cases, it was possible to verify the chromosomal banding pattern preserved on slides, which was confirmed as 2Rj inverted. Thus, the apparent discrepancy between molecular and cytogenetic determinations could be due to small insertions on the inverted chromosome. The third exception produced a single band indistinguishable in size from that expected of standard chromosomes. Unfortunately the chromosome preparation was unavailable for validation, so an error in recording or interpretation of the banding pattern cannot be ruled out.j heterozygotes by the multiplex PCR assay.Of only 6 2Rj heterozygotes sampled in Mali, two did not amplify and one was lost. The remaining three were successfully scored as 2Rj/+The geographic distribution of the 2Rj inversion greatly exceeds that of the BAMAKO chromosomal form, where it is associated with the cu and bcu arrangements in populations along Niger River in southern Mali and northern Guinea Conakry . Beyond j standard homozygotes and all except one (98.7%) yielded the expected ~500 bp standard band. The exception, a cytologically 2R+j standard specimen collected in Kedougou, Senegal in 2006, was classified as a heterozygote molecularly. This result was confirmed not only by repetition of the molecular diagnostic with primer pairs applied separately and in combination (as described above), but also by different laboratories (at the University of Rome and the MRC) on DNA extracted independently from different portions of the mosquito. It is therefore unlikely that the discrepancy resulted from contamination of primers and template, but other sources of human error cannot be ruled out. An additional 25 specimens from Senegal and Guinea Conakry were 2Rj inverted homozygotes, all of which (100%) revealed the expected ~250 bp inverted band.A total of 181 karyotyped SAVANNA specimens and one karyotyped BAMAKO specimen from the three countries were successfully amplified for molecular karyotype analysis of 2Rj . The vast majority of discordant cytological and molecular results  were due to a cytological heterozygote being scored molecularly as a 2R+j standard. Repetition of the assay with separated primer pairs demonstrated that this outcome reflected the failure of the D1+D1IN primer pair to amplify the 2Rj inverted arrangement.In contrast to the highly congruent molecular and cytological results for 2Rj standard and inverted homozygotes, only ~60% (49/81) of heterozygotes from Senegal, Guinea Conakry and Cameroon were scored as heterozygotes molecularly. The results of three specimens were conservatively included among the molecular "miscalls" despite the fact that two molecularly distinct bands were seen following gel electrophoresis, because one of the two bands was an unexpected size. When the PCR was conducted using primer pairs for standard and inverted arrangements separately, two of these "miscalls" were evidently due to insertions ≥ 400 bp between the standard primers (P3+P4) while the remaining one seemed to be caused by an insertion of ~400 bp between the inverted primers (D1+D1IN). More definitive resolution of these three exceptions requires sequence determination, a step not taken in this study. An additional two of the molecular "miscalls" from Senegal samples collected in 2006 were placed in this category because parallel analyses run at MRC and the University of Rome were in disagreement and could not be resolved despite independent DNA extraction and repeated trials. Whereas one institution correctly scored a specimen as a 2Rj/+j specimens (52/84) were correctly scored as heterozygous. This result is consistent when considering the Senegal, Guinea Conakry and Cameroon data  of tests in which the specimen analysed was a 2Rj standard or inverted homozygote. By contrast, only 62% of cytologically scored 2Rj/+ta Table ; the sizae ~5%; ,22), and, andj spThe development of a molecular karyotyping assay for 2Rj was motivated by the lack of practical tools for the rapid identification of the BAMAKO chromosomal form. The validation efforts described here demonstrate that this assay will serve that purpose very reliably, at least in southern Mali near the Niger River.In the total sample of nearly 1000 karyotyped specimens from Mali, only 6 2Rj heterozygotes were found. This pattern is not due to a Wahlund effect created by pooling samples across different villages, as it is applies equally well to a sub-sample from the village of Kela. Of 608 karyotyped mosquitoes, only 4 2Rj heterokaryotypes were found and a highly significant departure from Hardy-Weinberg equilibrium in the direction of heterozygote deficit was detected (Table A. gambiae reference genome has greatly facilitated study of the ongoing evolution and diversification of this medically important taxon. A case in point is the molecular characterization of the 2Rj inversion breakpoints and the ensuing development of a PCR-based assay for its rapid karyotyping. The intensive efforts at field validation reported here discourage the application of this assay for molecular karyotyping of j-carriers of the SAVANNA chromosomal form. On the other hand, molecular karyotyping of 2Rj is a robust and reliable method to identify BAMAKO in West African sites where this chromosomal form is prevalent and sympatric with a SAVANNA form having a low frequency of 2Rj-carriers. Moreover, cytogenetic analysis of karyotypes from southern Mali is consistent with previous proposals that BAMAKO mates assortatively, raising the possibility that BAMAKO may be in the early stages of speciation within the S form of A. gambiae. Until now only the ovarian polytene chromosome banding pattern was available for recognition of the BAMAKO form, severely limiting its study – especially at the larval stages. Thus, the special significance of the 2Rj karyotyping assay reported here is that it offers for the first time a method to rapidly detect BAMAKO at all stages and both sexes. Application of this assay offers the opportunity to deepen understanding of the genetic, ecological and behavioral processes driving ongoing adaptation and diversification of A. gambiae.As anticipated by Coluzzi , the A. MBC participated in the design of the study, led the field collections in Mali, participated in molecular and cytological karyotyping, data analysis and drafting the manuscript. MP participated in molecular karyotyping of specimens from Senegal and Guinea Conakry, and participated in molecular and cytological karyotyping of the Cameroon sample. BC participated in field collections of specimens from Senegal, and in their molecular and cytological karyotyping. DN participated in molecular karyotyping of specimens from Senegal. MJ, LK, and ID coordinated and led field collections in Senegal and Guinea Conakry. AF participated in cytological karyotyping of specimens from Mali. MK participated in molecular karyotyping of specimens from Mali. VP participated in the cytological karyotyping of samples from Senegal and Guinea Conakry. FS, DC, AdT, and ST helped design and coordinate the study. In addition, AdT and VP helped interpret data and draft the manuscript. NJB participated in design and coordination of the study, data analysis, and drafting the manuscript. All authors read and approved the final manuscript.


Kaposi's sarcoma (KS), common among persons with acquired immunodeficiency syndrome (AIDS), is caused by KS herpesvirus (KSHV) but whether KSHV causes other malignancies is uncertain. Using linked United States AIDS and cancer registries, we measured the incidence of specific malignancies in persons with AIDS (4–27 months after AIDS onset). We identified associations with KSHV by calculating a relative risk: cancer incidence in persons with KS  divided by incidence in persons without KS. Using Poisson regression, relative risks were adjusted for human immunodeficiency virus risk group, gender, age, race, and calendar year. We included 189 159 subjects (26 972 with KS). Immunoblastic lymphoma was significantly associated with KS . Only one immunoblastic lymphoma had pleura as primary site. None of 37 other specified malignancies  was significantly associated with KS. In summary, the association of immunoblastic lymphoma with KS was specific among examined malignancies and remained significant after statistical adjustment. Our findings, and the previously demonstrated presence of KSHV in the histologically related primary effusion lymphoma, suggest that KSHV is involved in the pathogenesis of some immunoblastic lymphomas. © 2001 Cancer Research Campaign