The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.Ciona intestinalis was not present in the completed C. intestinalis genome. The newly discovered relaxin-3 is likely to be the ancestral relaxin. Multiple relaxin-3-like sequences are present in fugu fish (Takifugu rubripes) and zebrafish (Danio rerio), but these appear to be specific to the fish lineage. Possible relaxin-1 and INSL5 homologs were also identified in fish and frog species, placing their emergence prior to mammalia, earlier than previously believed. Furthermore, estimates of synonymous and nonsynonymous substitution rates (dN/dS) suggest that the emergence of relaxin-1, INSL4 and INSL6 during mammalia was driven by positive Darwinian selection, hence these peptides are likely to have novel and in the case of relaxin-1, which is still under positive selection in humans and the great apes, possibly still evolving functions. In contrast, relaxin-3 is constrained by strong purifying selection, demonstrating it must have a highly conserved function, supporting its hypothesized important neuropeptide role.Sequence similarity searches of genomic and EST data identified homologs of relaxin-like peptides in mammals, and non-mammalian vertebrates such as fish. Phylogenetic analysis was used to resolve the evolution of the family. Searches were unable to identify an invertebrate relaxin-like peptide. The published relaxin cDNA sequence in the tunicate, We present a phylogeny describing the evolutionary history of the relaxin-like peptide family and show that positive selection has driven the evolution of the most recent members of the family. The relaxin-like peptide family includes: relaxin-1, relaxin-2, relaxin-3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. All share high structural similarity with insulin due to the presence of six cysteine residues, which confer two inter-chain and one intra-chain disulfide bonds. Thus, it was postulated that relaxin and insulin had derived from a common ancestral gene and were therefore grouped as the insulin superfamily, which later included insulin-like growth factors I and II (IGF-1 and -2) . The stVarious studies have highlighted the importance of a [Arg-X-X-X-Arg-X-X-Ile] motif in the relaxin B chain for interaction with the relaxin receptor and biological activity, which is dependent upon the presence of this motif . CoupledINSL4) is primate specific and likely to have diverged from a common relaxin ancestor before the duplication that led to the two relaxin genes seen in humans and great apes [INSL5 and INSL6) were identified by searching EST databases with the cysteine motif conserved in all insulin-like peptides [Relaxin has been well characterized in a reproductive context.. It is a product of the ovary and/or placenta in most species studied, and has various roles in pregnancy and parturition, which differ between species . Recent eat apes . Insulinpeptides ,12. BothUntil recently, receptors for all peptides of the relaxin family were unknown. Given the high degree of structural similarity between relaxin and insulin, it had been believed that the relaxin receptor would also be a tyrosine kinase receptor, similar to the insulin receptor. However, it was finally demonstrated in 2002, that relaxin activated two previously orphan leucine rich repeat containing heterotrimeric guanine nucleotide binding protein-coupled receptors (GPCR), LGR7 and LGR8 . FurtherRelaxin and INSL3 have primarily been of interest as hormones of pregnancy and reproduction; therefore it was assumed that the largely uncharacterised INSL4, 5 and 6 would share similar functions. However the discovery of the brain specific relaxin-3, and the widespread expression of INSL5 and its receptor GPCR142 have resulted in a re-evaluation of these assumptions and raised the need for a new approach to investigating these peptides.T. pyriformis) [H. momus) [C. intestinalis) [C. intestinalis [Relaxin evolution has confounded researchers for decades. High sequence variability in relaxins across closely related species is a well-known feature of this peptide, however startling similarities have been observed between very distant species such as pigs and whales . Other siformis) , ascidia. momus)  and tunitinalis) . A cDNA stinalis . Contrasstinalis . Despitestinalis  and a mostinalis .The increasing availability of genomic data has provided an opportunity to clarify the area of relaxin evolution using phylogenetic analysis. The relaxin-like peptide family phylogeny shows relaxin-3 is likely to be the ancestral relaxin, emerging prior to the divergence of fish, presumably with a function in the brain. Multiple relaxin-3 sequences are present in the fish and frogs, while the phylogeny also suggests that a relaxin with reproductive functions is present in these lineages. However, relaxin has been lost in the chicken and its genome instead contains two relaxin-3-like sequences. Evolutionary rate analysis shows positive Darwinian selection to be driving the most recent members of the family, INSL6, INSL4 and relaxin-1.R. esculenta, X. tropicalis, X. laevis, fugu fish, zebrafish, rhesus monkey and rainbow trout. New sequences identified during similarity searches have been highlighted. The source of each newly identified sequence is annotated as a footnote (i.e. genomic, ESTs). It should be noted that more mammalian relaxin and INSL3 sequences have been identified than were included in these analyses. Inclusion of these sequences did not improve the accuracy of the phylogeny, and as the aim of this study was to determine the evolutionary history of the entire family, they were omitted. All available non-mammalian relaxin-like sequences with accompanying nucleotide sequences were included.Table Searches of the completed human, mouse and rat genomes did not identify any novel relaxin-like peptides, however, likely non-functional INSL5 genes were revealed in the rat and dog genomes. A sequence with high similarity to INSL5 was found (Genbank Accession No: NW_047717.1), with a frameshift mutation in the first exon of the gene, which introduces a stop codon, resulting in a protein truncated early in the B chain (data not shown). The recently completed dog genome also contains a sequence highly homologous to the human INSL5 peptide (Genbank Accession No: NW_ AAEX01024390.1). However this sequence does not encode an open reading frame, with two stop codons present in the C peptide sequence and one upstream of a homologous B chain sequence. Furthermore, there was no recognizable signal peptide or Methionine start codon upstream of this B chain sequence and it was missing the critical Cys-29 in the B chain.X. tropicalis genome , Oncorhynchus mykiss (rainbow trout) (OmRLX3) and an INSL4 ortholog in the rhesus monkey  with high similarity to human relaxin-3 (79% identity to the translated EST product) starting from the end of the B chain (45F in the human pro-hormone sequence).C. intestinalis relaxin-like cDNA sequence has previously been reported [C. intestinalis genome.Searches of invertebrate genomic and EST databases failed to identify a relaxin-like gene in any invertebrate or prokaryote. Although a reported , our seaR. esculenta, X. laevis and X. tropicalis were aligned using ClustalW and edited to remove all gaps, which are problematic in phylogenetic analysis  and Tree-Puzzle failed to resolve the position of several sequences. This is primarily because of the short sequences used, as the C peptide can not be used to increase sequence length and thus improve the output from the tree generation methods, an inferred tree was produced instead. The Tree-Puzzle tree was resolved using topologies conserved between the MP and NJ trees and then reconciled against a species tree using GeneTree. The inferred gene trees were then edited to minimize the incongruence (the number of losses and inferred duplications) with the species tree. The inferred cluster A Analysis of the reconciled tree shows a major duplication event occurred early in the vertebrate lineage, giving rise to two subfamilies (clusters A and B respectively). Another duplication in subfamily A, prior to the emergence of fish, resulted in two lineages, which evolved into relaxin-3 and INSL5 in mammals. Interestingly, several non-mammalian relaxin-3-like sequences grouped with INSL5, implicating them as possible INSL5 homologs (GgRLX3b, OmRLX3, DrRLX3b, DrRLX3d, TrRLX3d and TrRLX3e). The reconciled tree also shows two additional fish-specific duplications in subfamily A. In the fugu fish genome a third duplication has occurred, resulting in three putative relaxin-3 (TrRLX3a, b, c) and two INSL5 homologs (TrRLX3d, e). In subfamily B there were four duplications, all were after the divergence of birds and reptiles and likely to have occurred during mammalian evolution. These events resulted in INSL3, INSL6, relaxin-1, relaxin-2 and INSL4.RLN1, RLN2 and INSL6 have high dN/dS rate estimates, with results for RLN1 and INSL6 suggesting positive Darwinian selection. The extremely high estimate for INSL6 (99) is caused by having a dS of 0 (i.e. no synonymous substitutions), resulting in a division by 0 for the rate estimate, which is represented as 99 rather than infinity. All other human and chimpanzee sequences compared were identical and thus produced dN/dS  estimates of 0.Results show the relaxin-like peptides are under varying selection pressures table . PairwisRLN2 and INSL6 show the highest estimates again, although are much lower than comparisons with chimpanzee sequences, the INSL6 estimate suggesting weak purifying selection instead of positive selection. The very low substitution rate observed for RLN3 (0.02) shows this peptide to be under strong purifying selection, at a similar rate to insulin (INS) . This comparison yielded a dN/dS estimate of 0.5, indicating weak purifying selection.Human and mouse orthologs were used to estimate rates for the other members of the family. RLN1, RLN2 and INSL6; human, rhesus monkey comparisons were used for INSL4 and human, mouse comparisons were used for RLN3, INSL3 and INSL5  estimates, more sophisticated codon-based substitution models  were usK = 3 site classes was the best fit to the data with a log likelihood value of -1413.8 (table 2 = 2.00), while half (54%) are under weak purifying selection (ω1 = 0.6) and the other 15% constrained under extreme purifying selection (ω0 = 0.01) (listed in table INSL4. The LRT of M3 with its null model (M0) shows these results to be significant  and in the background branches also fits the data significantly better than its null model, M3 with k = 2 (P = 0.007, d.f. = 2, table INSL4 branch (table 2 = 0.58) and 14% under very strong purifying selection , None of the phylogenetic tree construction programs used was able to completely resolve the evolution of the relaxin-like peptide family. This is likely due to variable divergence across the family and the short sequence length . IncorpoSimilarly to previously published results, searches of available genomic and EST data failed to identify any novel members of the relaxin-like peptide family . Given tT. pyriformis [H. momus [C. intestinalis, where a cDNA sequence almost identical to pig relaxin was found [C. intestinalis) failed to identify any relaxin-like sequences, including the published sequence. Multiple insulin-like peptides have been found in several invertebrates, including: Bombyxi mori (silkworm) [D. melanogaster [C. elegans [Ciona intestinalis) almost identical to pig relaxin (Georges and Schwabe, 1999). Completion of the C. intestinalis and other invertebrate genomes has allowed us to conclude that there is not a relaxin-like sequence in any invertebrate sequenced to date. If an invertebrate relaxin does exist, it does not contain the relaxin-specific motif characterized in vertebrates.The presence of an invertebrate relaxin has been of speculation since 1983 when relaxin-like activity was first detected in the protozoa, riformis . SimilarH. momus  and in Cas found . Howeverilkworm) , D. melanogaster  and C. e elegans . As thesA hallmark of relaxin sequences is their high variability, even amongst closely related species. Relaxin-like peptide sequences isolated from two whales are almost identical to porcine relaxin , howeverThe presence of a functional relaxin in the ruminant lineage has yet to be confirmed . More geThe presence of an avian relaxin has also been of speculation. While relaxin-like activity has been reported in the chicken , an aviaThe phylogeny of the relaxin-like peptide family indicates relaxin-3 is the ancestral relaxin, appearing prior to the divergence of teleosts. The finding of multiple relaxin-3-like genes in the fugu fish and zebrafish suggests multiple lineage-specific duplications of a single relaxin-3-like ancestor have occurred in fish . HoweverX. tropicalis genome were placed in this group, while there are members present in the more ancient fish lineage and the younger avian lineage. It is possible that this gene has either been lost, or remains unidentified, in the X. tropicalis genome. A sequence with similarity only to the B chain of relaxin-3 was also found, but a corresponding A chain match was not, however, there is a gap in the genome assembly upstream which might contain the missing domain. Future assemblies of the Xenopus genome should resolve this issue. These results suggest that INSL5 could have emerged during teleost evolution, far earlier than previously believed. Unlike the mammal-specific relaxin-like genes, which are clustered together (on chromosome 9 in the human and chromosome 19 in the mouse), INSL5 is localized independently (chromosome 1 in the human and chromosome 4 in the mouse). These findings are of particular interest in the analysis of INSL5, which is still functionally uncharacterised.While termed relaxin-3-like based on sequence similarity, phylogenetic analysis indicates that several non-mammalian sequences (OmRLX3, DrRLX3b, DrRLX3d, TrRLX3d, TrRLX3e and GgRLX3b) could be INSL5 homologs. None of the sequences found in the complete All the potential non-mammalian INSL5 homologs retain the relaxin-specific B chain [RxxxRxxI/V] motif, hence would be capable of interacting with the relaxin receptor, LGR7, and thus functionally classified as a relaxin. Recent studies have shown INSL5 is a high affinity ligand for GPCR142 but not GPCR135, LGR7 or LGR8 . As the Raja erinacea) [Squalus acanthias) [Dasyatis sabina) [Odontaspis taurus) [R. esculenta gene expressed in the brain using northern blot analysis [Phylogenetic results from this study suggest the presence of a relaxin homolog in fish and frogs, although not in the chicken. Relaxin sequences have previously been isolated and peptide sequenced from either the ovaries or testes of the edible frog , little rinacea) , spiny danthias) , Atlanti sabina)  and the  taurus) . While h taurus)  and has analysis , we beliRLN3 gene is under very strong purifying selection, highlighting the importance of its highly conserved function, likely to be in the brain [RLN2 is under only weak purifying selection. We suggest that this lack of selective pressure has contributed to the high sequence divergence seen between many relaxins (e.g. human and mouse) and the differences in relaxin's functions observed across mammals.The ancestral he brain . As highRLN1 and INSL6 show these to be under positive selection. Positive selection is often difficult to observe using pairwise comparisons that average over the whole length of a sequence, making these results even more striking. While pairwise comparisons failed to confirm positive selection was acting on INSL4, further statistical tests suggested that positive Darwinian selection acted on several sites in the INSL4 sequence after its emergence. Further analysis will be required to confirm these sites as important in the acquisition of a new receptor and a new function by INSL4, particularly in light of recent studies that question the reliability of ML methods to accurately detect positive selection acting on single sites [Information about the selective constraints placed upon these peptides, can provide valuable insight into the nature of interactions with their receptors. Based on selection pressures we can conclude that the interactions between relaxin-3 and GPCR135, INSL5 and GPCR142 are very specific, while the binding of relaxin to LGR7 is much looser. In this context the cross-reactivity seen between LGR7 and INSL3 or H1 relaxin, which are both similar to relaxin in sequence but especially in structure, is understandable, as is the lack of binding between GPCR135 and GPCR142 with any other relaxin-like peptide. Unexpectedly, synonymous and nonsynonymous substitution rate estimates for le sites -50. We aINSL6 emerged during mammalian development, INSL4 and RLN1 during primate evolution, they remain functionally uncharacterized and INSL4 and INSL6 are without known receptors. The low selection pressure on the B domain and the strong constraints placed on the A domain of INSL4 and INSL6 suggests that, unlike the B chain mediated interaction of relaxin and INSL3 with their receptors, the interaction of these peptides with their receptors could be dependant on the A chain instead. The low dN/dS rate observed for INSL5 indicates this peptide to be evolutionary stable and of functional importance. In particular the constraints placed on both A and B chains of INSL5 suggest a well-defined receptor interaction system, while the total absence of these constraints on either chain within relaxin-1 suggests the opposite, that perhaps this peptide is still evolving its function.When the B and A domains of each gene were analyzed separately, further differences in selection pressures became apparent. The interaction between relaxin and its receptor has been thought to be primarily mediated through the B chain of the peptide , so the We present here a phylogeny for the relaxin-like peptide family. Relaxin has long been used as an example of a gene that conflicts with the Darwinian theory of evolution ,32-34. HWe have demonstrated that positive selection has been a driving force in the recent expansion of the relaxin-like peptide family during mammalian evolution. While strong purifying selection has maintained the structural core of these peptides by constraining the insulin superfamily cysteine motif, outside these residues, positive selection has acted after at least three gene duplication events (which generated INSL6, INSL4 and relaxin-1) to allow these new genes to acquire a new receptor and novel functions. Given the known roles of relaxin and INSL3 in reproduction (and the likely similar roles of INSL4 and INSL6 given the specificity of their expression in reproductive tissues) these findings correlate with a general trend towards rapid evolution in several reproduction associated genes -54. We aHomo sapiens) H1 relaxin, H2 relaxin, H3 relaxin, INSL3, INSL4, INSL5, INSL6; mouse (Mus musculus) relaxin, relaxin-3, INSL3, INSL5, INSL6; rat (Rattus norvegicus) relaxin, relaxin-3, INSL3, INSL6; dog (Canis familiaris) relaxin; pig (Sus scrofa) relaxin, relaxin-3, INSL3; edible frog (Rana esculenta) relaxin and tammar wallaby (Macropus eugenii) relaxin (see table Amino acid and nucleotide sequences of cloned relaxin-like peptide family members from the following species were obtained from GenBank : human (Pan troglodytes), fugu fish, zebrafish, fruit fly (Drosophila melanogaster), mosquito (Anopheles gambiae), Caenorhabditis elegans, all yeast, all plant and all bacterial genomes at NCBI [X. tropicalis [C. intestinalis [Sequence similarity searches using TBLASTN  were con at NCBI , X. tropopicalis  and C. istinalis , Expressstinalis . While tAmino acid sequences were aligned using ClustalW  with defPhylogenetic trees were constructed using maximum parsimony (MP): implemented in PHYLIP  using PrGene trees of relaxin-like peptides were reconciled with a species tree using GeneTree . ReconciMaca mulatta) and human and mouse orthologs were constructed using ClustalW [dS) and nonsynonymous (dN) substitution rates were estimated using the methods of Yang and Nielsen [Pairwise nucleotide sequence alignments of human and chimpanzee, human and rhesus monkey  relaxin-2 (Z27245), INSL4 (BK005152); gorilla (Gorilla gorilla) relaxin-2 (Z27228, Z27237); rhesus monkey relaxin (A34936), INSL4 (BK005251); bush baby  relaxin (AF317625); baboon (Papio hamadryas) relaxin (Z27246, Z27224); camel (Camelus dromedarius) relaxin (AF254739); cat (Felis catus) relaxin (AF233688); dog (Canis familiaris) relaxin (AF233687); guinea pig (Cavia porcellus) relaxin (S85964); rabbit (Oryctolagus cuniculus) relaxin (S45940); hamster (Mesocricetus auratus) relaxin (S79879) and horse  relaxin (AB000201). The alignment was edited as described previously. A ML tree was constructed from this alignment using TreePuzzle [P(ω>1) >0.90 were listed.The following relaxin-1 and INSL4 nucleotide sequences were aligned using ClustalW: human H2 relaxin (X00948), INSL4 (L34838), chimpanzee, ML (TreePuzzle) and NJ (Neighbour in PHYLIP) methods and edited in Treeview to minimize species tree incongruence. Human insulin was used as an outgroup. Where possible, confidence values are shown at branches: * >50%, ** >75%, all other branches are inferred. Hsa = Homo sapiens, Pt = Pan troglodytes, Mm = Mus musculus, Rn = Rattus norvegicus, Cf = Canis familiaris, Ss = Sus scrofa, Xt = Xenopus tropicalis, Dr = Danio rerio, Tr = Takifugu rubripes, Gg = Gallus gallus, Om = Oncorhynchus mykiss.Click here for filePhylogeny of cluster B- relaxin-1, 2, INSL3, INSL4 and INSL6. Consensus phylogeny of Cluster B constructed from a ClustalW alignment of the B and A domain amino acid sequences from relaxin 1, 2, INSL3, INSL4, INSL6 peptides. Consensus tree generated from MP (Protpars in PHYLIP), ML (TreePuzzle) and NJ (Neighbour in PHYLIP) methods and edited in Treeview to minimize species tree incongruence. Human insulin was used as an outgroup. Where possible, confidence values are shown at branches: * >50%, ** >75%, all other branches are inferred. Hsa = Homo sapiens, Pt = Pan troglodytes, Mmul = Maca mulatta, Mm = Mus musculus, Rn = Rattus norvegicus, Cf = Canis familiaris, Ss = Sus scrofa, Re = Rana esculenta, Me = Macropus eugenii, Xl = Xenopus laevis, Xt = Xenopus tropicalis, Dr = Danio rerio, Tr = Takifugu rubripes.Click here for file


This is a report on a 37-patient continuation study of the open ended, Omega-3 Fatty Acid (O-3FA) add-on study. Subjects consisted of the original 19 patients, along with 18 new patients recruited and followed in the same fashion as the first nineteen. Subjects carried a DSM-IV-TR diagnosis of Bipolar Disorder and were visiting a Mood Disorder Clinic regularly through the length of the study. At each visit, patients' clinical status was monitored using the Clinical Monitoring Form. Subjects reported on the frequency and severity of irritability experienced during the preceding ten days; frequency was measured by way of percentage of days in which subjects experienced irritability, while severity of that irritability was rated on a Likert scale of 1 – 4 (if present). The irritability component of Young Mania Rating Scale (YMRS) was also recorded quarterly on 13 of the 39 patients consistently. Patients had persistent irritability despite their ongoing pharmacologic and psychotherapy.Omega-3 Fatty Acid intake helped with the irritability component of patients suffering from bipolar disorder with a significant presenting sign of irritability. Low dose (1 to 2 grams per day), add-on O-3FA may also help with the irritability component of different clinical conditions, such as schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability. According to the United States National Institute of Mental Health (NIMH), Bipolar Disorder (BPD), also known as manic-depressive illness, is a serious medical illness that causes shifts in a person's mood, energy, and ability to function. Different from the normal ups and downs that everyone goes through, the symptoms of bipolar disorder are severe. Bipolar disorder is a complex, chronic condition associated with considerable morbidity and mortality, including a high rate of suicide. Bipolar disorder causes dramatic mood swings from overly "high" and/or irritable to sad and hopeless, and then back again, often with periods of normal mood in between. Severe changes in energy and behavior go along with these changes in mood. The periods of highs and lows are called episodes of mania and depression. Most people with bipolar disorder can achieve substantial stabilization of their mood swings and related symptoms over time with proper treatment. A strategy that combines medication and psychosocial treatment is optimal for managing the disorder over time.Omega-3 fatty acids (0-3FA) may have a beneficial effect on irritable mood. Low O-3FA levels in red blood cell membranes of depressed patients hint that O-3FA may be helpful in treating mood disorders . A recenO-3FA may also help with the irritability component of different clinical conditions, such as depression, mania, schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability. Numerous other conditions have an irritability component, including Borderline Personality Disorder, Alzheimer's disease, Premenstrual Dysphoric Disorder, to name a few -12. TherA recent, open ended, O-3FA add-on study has shown beneficial effect of O-3FA on irritability in 19 patients with mood disorders . These pThis is a report on a 37-patient continuation phase of the open ended, O-3FA add-on study. Subjects consisted of the original 19 patients, in addition to the 18 new patients recruited and followed in the same fashion as the first nineteen . SubjectStarting dose and last maintenance dose were available for 37 subjects Table . SubjectThe initial mean was 63.51 (SD 34.17), indicating that on average, subjects were irritable for about six of the previous ten days. The mean for the last recorded percentage was less than half of the initial score: 30.27 (SD 34.03). The decrease was found to be statistically significant using a paired sample t-test (t = 4.36, 36 df, p < .001). The difference between the distributions was examined using the non-parametric sign test. The number of negative differences (25) significantly exceeded positive differences (7); there were five ties, and the pre/post distributions were significantly different (p < .003).Thirty four subjects had initial and last recorded YMRS irritability sub-scores. As with the above means there was a sizable decrease. The initial mean score was 3.18 (SD 1.09). The mean for the last recorded percentage was 1.68 (SD 1.89). The decrease was found to be statistically significant using a paired sample t-test (t = 4.21, 33 df, p < .001).Starting and last recorded YMRS scores were available for 34 subjects. The mean starting score 10.71 (SD 6.77), and the mean for the last recorded score was 4.85 (SD 5.63). The decrease found to be statistically significant using a paired sample t-test (t = 4.14, 33 df, p < .001).Thirty six subjects had initial and last recorded severity scores on the ADE. Again, a decrease was found. The initial mean score was 2.14 (SD 1.22). The mean for the last recorded score was 0.94 (SD 0.92). This decrease was found to be statistically significant using a paired sample t-test (t = 5.23, 35 df, p < .001).As an exploratory measure, a composite score was created by multiplying the ADE severity score, which has a maximum of 4 points, by the percentage of the ten days prior to measurement which the patient was rated as irritable. The initial mean on this composite was 159.72. As with other measures, there was wide variation: SD = 122.92. The mean for this measure on the last recorded scores was percentage was about one-fourth of the initial score: 43.89 (SD 64.38). The decrease was found to be statistically significant using a paired sample t-test (t = 5.00, 35 df, p < .001).Because of apparent wide variation on these two measures and a concern that outliers may have affected some results, the last recorded irritability scores were plotted against the maintenance dose. This revealed a rather bimodal pattern, in which relatively lower irritability measures (≤ 50%) clustered in the quadrant with lower dosage levels (≤ 4,000 mg).In response to a similar observation regarding wide variation in the last recorded values (84 days to 5.5 years) the values were also plotted. A clearly bimodal pattern appeared in which 11 subjects (about one-third of study participants) clustered in the quadrant representing short duration (<500 days) and higher YMRS totals (>7). The remaining two-thirds of subjects clustered in the quadrant representing short duration and lower YMRS totals (<6).The mean start weight was 176.97 lbs (SD 43.13), and the mean for the last weight recorded was slightly higher at 178.59 lbs (SD 43.24). The increase was not statistically significant.Follow-up information, recorded after the collection of the "last" scores for most of the above variables, was available for 13 of the 37 subjects. Final YMRS total or scale scores were not available for this sub-group.The final date recorded for the duration of O3 was derived based from an O3 start date and a "final" date recorded for O3. The time period ranged 84 days to 1995 days (5.46 years). The mean duration of O3 for this group was 439.62 days (SD = 487.46).For these subjects, the mean starting dose was 1807.69 mg (SD 990.34), and the mean for the last maintenance dose was higher at 2615.38 mg (SD 1894.66). The increase was not significant.The initial mean was 82.31 (SD 20.88). The mean for the last recorded percentage was dramatically lower: 25.38 (SD 32.04). The decrease was found to be statistically significant using a paired sample t-test (t = 6.52 12 df, p < .001). The difference between the distributions was examined using a sign test. The number of negative differences (12) significantly exceeded positive differences (0); there was one tie, and the pre/post distributions were significantly different (p < .001).The initial mean score for the 13 subjects with final scores was 2.69 (SD 0.95). The mean for the final score was 0.77 (SD 0.83). This decrease was found to be statistically significant using a paired sample t-test (t = 6.22, 12 df, p < .001).An exploratory composite score, described above, was also created for the subjects with final scores. For these subjects, the initial mean was higher than that of the total group, 223.08. Again, there was wide variation: SD = 104.19. The mean for this measure on the last recorded scores was percentage was much lower that the initial score: 33.08 (SD 39.87). The decrease was found to be statistically significant using a paired sample t-test (t = 6.70, 12 df, p < .001).For these 13 subjects, the mean start weight was 166.23 lbs (SD 35.68), and the mean for the final weight recorded was also slightly higher at 168.23 lbs (33.62). As with the previous finding regarding weight, the increase was not statistically significant.Omega-3 Fatty Acids added onto the existing treatment helped with the irritability component of a significant percentage of patients suffering from bipolar disorder with a persistent sign of irritability.As seen from the standard deviations of several of the variables discussed here, measures ranged widely. This creates difficulty in using descriptive data, such as means, to adequately portray subject attributes and performance. Using data reduction techniques or grouping subjects according to high and low scores on various attributes may be one way to increase the descriptiveness, which would be possible and more reasonable with a larger pool of subjects.A potential limitation or interpretive consideration merits discussion. For many of the variables discussed above, noticeable differences in measures were observed between the "starting" versus "last recorded" group (n = 37) and the "starting" versus "final" measures group (n = 13). Given these differences and the smaller number of subjects in the second set of comparisons, "starting" versus "final" comparisons should be interpreted with caution until differences inherent in this "final" subgroup (n = 13) are more clearly understood. This is clearly seen in the results of sign tests, in which the apparent magnitude of the "final" effects is pronounced.Statistically significant within-subjects differences were found in several independent variables. This is especially notable given the small number of subjects. The preliminary findings suggest that a rigorously designed study tailored especially to the examination of the effects of O-3FA is warranted.The majority of data were collected within an ongoing "best-practice, outpatient bipolar disorder study" that involved medications and talk therapy which we have not reported or discussed herein. Results must, therefore, be interpreted with caution.There are several mechanisms through which O-3FA are theorized to help with mood, irritability, aggression etc. Suggested theories of mechanism converge on the theory of nerve cell membrane stabilization. A recent study has come closest to showing physical proof of effectiveness of O-3FA through indirect demonstration of greater membrane fluidity, as detected by reductions in Tesla-2 (T2) values in MRI scans . The oveWe would like to discuss briefly the issue of daily dosing of O-3FA for nutrition and medicinal purpose: Recent studies give caution that at a 6 gram per day average daily dose, as a single agent, omega 3 fatty acids may not be as effective as an antidepressant . HoweverThe United States (US) accounts for more than 51% of the 430.3 billion dollar expended on pharmaceutical products worldwide each year . World hWe conclude that it is beneficial in many ways to establish a regular intake of 1–2 g per day EPA acid plus DHA, similar to daily intake of vitamins with minerals. Dietary interventions to remedy omega-3 deficiency is necessary . It is t


Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells.To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR  through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway.These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination. The sense of smell originates in the olfactory epithelium when olfactory receptors (ORs), members of the seven transmembrane domain G-protein coupled receptor (GPCR) superfamily, bind odorant ligands ,2. DespiRecently, we elucidated three specific cellular mechanisms responsible for inefficient OR trafficking to the plasma membrane: ORs are retained within the endoplasmic reticulum (ER) due to inefficient folding and poor coupling to ER export machinery, degraded via the ubiquitin-proteasome system, and sequestered in ER aggregates that are degraded by autophagy . Thus, wTo develop rationale means to improve the functional expression of ORs, an approach was devised to quantitate activation of the mouse mOREG OR , which recognizes the odorant eugenol (spicy, cinnamon-like odor) [olf, adenylate cyclase III, and an olfactory CNG, leading ultimately to the sensation of smell [s, a functional homologue of Golf [s and elicits increases in the second messenger cAMP in HEK cells [A cell-based approach was developed to measure functional expression of mOREG in heterologous cells. This system was designed to mimic the signal transduction events involved in OR activation in the olfactory epithelium and utilizes CNG as a cAMP biosensor ,11. In oof smell . Accordi of Golf ,14, and  of Golf . The Rho of Golf ,16,17, pEK cells ,18,19, l50 for eugenol (20.8 +/- 3.4 uM) closely approximated published values (35 uM and 46 uM), thereby validating the utility of our approach [Responses to eugenol, a ligand for mOREG, were detected in cells co-expressing Rho-mOREG and CNG Fig.  and 1B bapproach . Rho-mORapproach ,20. Colls-adenylate cyclase) [Previously, we demonstrated that ORs are inefficiently expressed at the plasma membrane of heterologous cells due, in part, to degradation of ER-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy . Specificyclase) , followi50 = 17.1 +/- 3.0 uM; maximum number of responding cells at 300 uM eugenol = 214 +/- 10) was more robust than untagged mOREG , likely due to an established role of the Rho tag in facilitating chemosensory GPCR functional expression [To determine if increased functional expression of Rho-mOREG following inhibition of ER degradation was attributable to use of an OR fusion protein, we examined the effect of autophagy and proteasome inhibitors on mOREG lacking the Rho tag. Although eugenol activation of Rho-mOREG . These results suggested that either a small pool of properly folded Rho-mOREG was expressed at the plasma membrane in quantities below the threshold of the cell biological techniques employed to visualize the receptor, or that a pool of intracellular Rho-mOREG comprised the functionally responsive population in calcium imaging experiments. Notably, numerous studies have documented the functional expression of GPCRs and requisite signal transduction machinery in intracellular compartments, including ER membranes -29.To differentiate between these two possibilities, we adopted a pharmacological approach to selectively and independently block trafficking from compartments in the early secretory pathway, specifically the ER and Golgi apparatus. If inhibition of ER degradation does not increase Rho-mOREG activity under conditions that block export from ER and Golgi compartments, the functionally responsive Rho-mOREG population is likely derived from an internal pool that is required to traffic to the plasma membrane to function. Conversely, if inhibition of ER degradation increases Rho-mOREG activity under conditions that block export from ER and Golgi compartments, the functionally responsive Rho-mOREG population likely resides in an intracellular compartment.To inhibit protein trafficking from the ER, brefeldin A (BFA), which blocks ER export of cargo proteins by inducing collapse of the Golgi stacks into the ER, was used . As showTo inhibit protein trafficking from the Golgi apparatus, monensin, an ionophore that disrupts Golgi structure and inhibits Golgi trafficking events, was used . MonensiInhibition of Rho-mOREG function by trafficking disrupting agents, specifically BFA, could be due to activation of the unfolded protein response  and inhiCollectively, these data support a model whereby inhibition of ER degradation promotes a small fraction of Rho-mOREG to achieve an ER export competent conformation, thereby satisfying ER quality control processes, and traffic through the secretory pathway to the plasma membrane. Thus, similar to chemical and pharmacological chaperones that promote folding and restore ER export of misfolded GPCR cargo -37, agenWe have developed an expression system for ORs that utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons. Using CNG as a cAMP biosensor to gauge mOREG function, we demonstrate that inhibition of ER degradation, by both autophagy and the ubiquitin-proteasome system, promotes functional expression of Rho-mOREG as well as untagged mOREG. Thus, proteolysis limits mOREG function in heterologous cells. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination.Rho20-mOREG expression vector was generated in pRK5 as previously described . To geneBFA, isoproterenol, monensin, and 3-MA were from Sigma (St. Louis, MO); MG-132 was from Calbiochem (San Diego, CA); eugenol, heptanal and octanal were from Aldrich (Milwaukee, WI).HEK293 cells were maintained and transfected as previously described . For theFunctional expression of mOREG was investigated using calcium imaging methodology as previously described . Cells s50 for isoproterenol activation of the β-AR (3.7 +/- 1.0 nM), measured by counting responding cells, closely matched published values (1.7–3.3 nM), measured either by fluorescent intensity measurements or a cAMP accumulation assay [50 for eugenol activation of Rho-mOREG (20.8 +/- 3.4 uM), measured by counting responding cells, closely approximated the published value (35 uM and 46 uM), determined by monitoring fluorescent intensity of responding cells [Changes in intracellular calcium were monitored by fluorescence microscopy using an Axiovert S100 TV inverted microscope with a 10× long working distance Plan Fluor objective  and a cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ). Images were acquired using a Lambda DG-4 automated wavelength controller (Sutter Instrument Co., Novato, CA) at 480 nm excitation and 535 nm emission and analyzed using Imaging Workbench 4.0 (Axon Instruments, Union City, CA). Counting the number of cells responding to ligands 60 sec following stimulus addition, when cells had achieved a maximal response, quantitated receptor activity. This established methodology has been used to functionally characterize the human T1R1/T1R3 umami receptor, the human T1R2/T1R3 sweet receptor, and the Drosophila Gr5a trehalose receptor ,45. To ing cells . Finallyng cells .We estimate ~40–50% of transfected CNG cells express functional cell surface Rho-mOREG based on the following points. First, ~20–25% of total cells in a microscopic field respond to maximal doses of eugenol. Second, ~50% of cells are transfected, measured by either co-transfection with red fluorescent protein or by immunolabelling permeabilized cells expressing Rho-mOREG with an anti-Rho antibody. Thus, ~40–50% of transfected cells express sufficient Rho-mOREG at the plasma membrane to elicit a functional response.50 values were determined using GraphPad Prism v3.02 software.Data represent the mean +/- SEM. Statistical significance was determined using an unpaired, two-tailed Student's t-test. Dose-response curves were plotted and ECML generated the mOREG expression vectors and carried out most of the calcium imaging experiments. LS generated the cells stably transfected with CNG. FE assisted with the calcium imaging experiments. HX generated the CNG expression vectors. BDM coordinated the study and wrote the paper.


While the basic ethical issues regarding consent may be universal to all countries, the consent procedures required by international review boards which include detailed scientific and legal information, may not be optimal when administered within certain populations. The time and the technicalities of the process itself intimidate individuals in societies where literacy and awareness about medical and legal rights is low.In this study, we examined pregnant women's understanding of group education and counseling (GEC) about HIV/AIDS provided within an antenatal clinic in Maharashtra, India. We then enhanced the GEC process with the use of culturally appropriate visual aids and assessed the subsequent changes in women's understanding of informed consent issues.We found the use of visual aids during group counseling sessions increased women's overall understanding of key issues regarding informed consent from 38% to 72%. Moreover, if these same visuals were reinforced during individual counseling, improvements in women's overall comprehension rose to 96%.This study demonstrates that complex constructs such as informed consent can be conveyed in populations with little education and within busy government hospital settings, and that the standard model may not be sufficient to ensure true informed consent. It is estimated that nearly 7.2 million people in Asia and the Pacific region are now living with HIV/AIDS, one million of whom acquired the virus in 2002 . Of thesIn most industrialized countries, voluntary HIV counseling and testing services are a major component of HIV and AIDS control programs targeted toward pregnant women, although such programs are only now being advocated in developing countries ,5. VolunThe basic ethical issues regarding consent may be universal to all countries. One of the potential limitations with the current approach is that consent has been reduced to a technical issue and is often removed from wider ethical considerations. The consent procedures required by international review boards include having to provide detailed scientific and legal information to participants. Although the intent is to increase knowledge, these messages may lose their meaning when administered within certain populations. The challenge then becomes one of how to balance the requirements of providing complete information while also obtaining true informed consent.To begin establishing voluntary counseling and testing for pregnant women in India, the Indian National AIDS Control Organization (NACO) in 2001 started voluntary HIV counseling and testing for women attending government-run antenatal clinics in 11 sites throughout the country. This study was conducted in one of these sites, Pune, Maharashtra, in order to develop tools to enhance, standardize and improve the communication of messages during the group education and counseling (GEC) sessions, and to enhance the informed consent process.This study was conducted in the antenatal clinic of an urban government hospital in Pune. Eligible patients were newly registered pregnant women between 18 and 44 years of age who were less than 36 weeks gestation and had no physical or mental disabilities (as determined by a physician). After each eligible woman registered in the antenatal clinic and met with the attending physician, they were offered the opportunity to attend the HIV/AIDS GEC sessions.The eight major topics covered in the education component of the GEC included general transmission modes of HIV, sexual transmission, mother-to-infant HIV transmission, precautions to avoid HIV transmission, ways through which HIV does not transmit, identification of HIV in an individual, detection of HIV in the body, and symptoms of AIDS. In addition, nine major topics related to HIV testing and informed consent were covered, including procedural risks of HIV screening, social risks of HIV screening, availability of the report, the right to say no, repercussions of refusing to take the test, confidentiality of the testing procedures, the right to consult others, the benefits of HIV screening, what one's signature means, specific procedures of the HIV ELISA test and, if applicable, specific procedures of the rapid HIV test.We conducted structured observations of 30 GEC sessions over the period from December 2000 to January 2001 to assess how well the information was being covered. The GEC sessions were chosen so that every clinic day (Monday-Saturday) and session time would be equally represented (there were, on average, four GEC sessions during the clinic hours of 9:30 am to 1:00 pm).The structured observation tool included a comprehensive checklist of each of the specific points for each main topic covered during the GEC. A topic would be considered adequately covered if the counselor conveyed all the specific points for the topic. Overall session adequacy was assigned if at least 80% of all topics were presented adequately during the session.To assess women's understanding of the key issues covered we interviewed 136 consecutively consenting women immediately after they attended one of the 30 sessions . Informed consent was obtained from each woman prior to the interview. After the interview, the women met briefly with an individual counselor, where they chose whether to have HIV testing and, if so, signed the appropriate informed consent form.The women's understanding of the GEC sessions was determined through structured interviews containing 17 questions covering each of the main topics discussed in the GEC. Their answers were scored as either adequate or inadequate based on predetermined criteria.Based in part on these observational and interview data we developed and enhanced the content of the GEC sessions. We also developed a comprehension test to evaluate women's understanding of key issues related to informed consent. The key enhancements to the GEC sessions were as follows:• Areas with greater privacy for both group counseling and individual counseling were established.• Posters illustrating the main topics were created and placed in the GEC rooms.• Similar visuals were made into a flipchart and used by the counselor during individual counseling to reinforce the messages.• All visuals developed included substantial input from the counselors.• The visuals were simple, using bold colors and conveying only one message each.• The posters were created to provide informational cues to the counselor to promote and maintain regularity and standardization in presentation.• The counselors completed further training in the use of the visuals.Current standards for ethical treatment of human participants in a study do not require that participants demonstrate comprehension of the study prior to giving informed consent. However, given the volume and complexity of information conveyed during the GEC sessions, we felt it necessary to develop a concise yet complete comprehension test that could be used in HIV screening programs and adapted for future clinical trial enrollments.The questionnaire was developed by choosing eight of the 17 questions from the previous questionnaire used during the baseline evaluation. The eight questions were identified by the field team of counselors, physicians, and behavioral scientists as key issues related to informed consent. The questionnaires were administered by the hospital counselor. Adequacy of women's understanding for any topic was determined if the woman correctly answered the question based on pre-determined criteria. If a woman scored adequately on at least 80%, or six out of eight, questions she became eligible to sign the informed consent form and proceed with the HIV screening. Prior to signing the form, the counselor had the opportunity to clarify any previously misunderstood issues with the woman.The eight questions used to assess comprehension of informed consent issues were as follows:1. What are the modes by which HIV germs are transmitted?2. Can you say "No" to taking the HIV test?3. What happens if you decide not to take the HIV test?4. What do we mean by "the result of the test will be kept confidential"?5. Do you have the right to consult your husband or other family members before taking the test?6. What do you think are the benefits of finding out your HIV status?7. What problems can a woman face on finding out her HIV-positive status?8. What does your signature on the consent form mean?After the enhancements to the GEC were put into place and the comprehension test developed, we conducted structured observations of 40 GEC sessions from May-June 2001. We performed the comprehension test on 224 women who attended one of these 40 sessions . These same women were re-interviewed after the completion of individual counseling. The time between the interview after group counseling and the re-interview after individual counseling was, on average, one-two hours.N = 136) and after (N = 224). In addition, we examined the added improvements in knowledge when the second group of women (N = 224) were tested after their individual counseling. Analysis of the improvements in topic coverage and women's understanding of topics covered was determined by a chi-square test of proportions or Z-scores (for changes in knowledge in the same population) using SPSS version 10 [We compared the adequacy of GEC topic coverage prior to (baseline 30 GEC sessions) and after (40 GEC sessions) the inclusion of the enhancements. Also, improvements in the women's comprehension were assessed through a comparison of their responses to the eight key informed consent questions before GEC enhancements  at Johns Hopkins University, Baltimore, USA, the local IRBs in Pune, the local medical institutions involved in the research and the National Institutes of Health, USA, which funded this study.Topics related to informed consent and HIV testing were not well covered during the GEC sessions. Only three topics–availability of a report, the right to say no, and the benefits of screening–were covered in 90% or more of the sessions. The procedural and social risks of HIV screening, the right to consult others, and confidentiality were discussed in less than 10% of the sessions. The impact of refusing to be screened and what a signature means were discussed in 40% and 70% of the sessions, respectively.P < 0.01). Coverage of "social risk" during sessions showed a statistically significant increase from 7% to 23% (P < 0.05), yet the overall coverage was still very low. Similarly, the coverage of "the right to consult others" showed a statistically significant increase (P < 0.001), however, it was still only covered in 35% of the sessions. Non-significant improvements occurred in the area of "consequences of refusal", increasing from 40% to 55%. Overall, the use of visuals resulted in a statistically significant improvement in adequacy of coverage of the counselling sessions from 30% to 68% of all GEC sessions.Focusing only on the eight key issues related to understanding informed consent (see previously listed questions), we compared topic coverage before (Group A) and after (Group B) the improvements were made to the GEC sessions Table . With thP < 0.01) with enhanced group counseling and enhanced individual counseling, respectively. It should be noted that this topic was well covered in all sessions with or without the addition of visual aids. On the other hand, the discussion of "social risk if found to be HIV-positive" or "the right to consult others" was rarely covered in any of the counseling sessions. In spite of this, a considerable majority of women understood both these issues. Women's understanding of "consequences of refusal" showed marked improvements with use of visuals, rising from 19% at baseline to 75% with enhanced group counselling (P < 0.01) and further to 96% (P < 0.01) with enhanced individual counselling.Table P < 0.01). With the addition of individual counselling with visuals, women's overall understanding showed a statistically significant increase in all eight aspects of informed consent, and an overall increase from 72% to 96% (P < 0.01).Overall, based on adequately answering six out of eight questions, women's knowledge of informed consent topics in enhanced group counselling improved dramatically, from 38% to 72%  by the patient, her husband or an accompanying relative, and is generally considered to serve as legal consent; therefore, it is not interpreted as voluntary. Most often, due to time constraints, very little is explained to the patient about the procedure, risks, and benefits, or what her signature actually means. As found in other regions in India, there is a general perception by clinicians and other healthcare workers that women are "unable" to understand any of the procedures even if explained, because they are illiterate or have no medical background ,18. It iIn creating the modifications to the GEC, we focused on improving communication of those concepts that were the most unfamiliar to these women. For example, for most women in India, there is relatively little sense of autonomy . For manClearly, some of the concepts related to informed consent may already be understood by these women. Women's understanding of "consequences of refusal" showed marked improvements in the second group of women studied. Although some of this improvement may be attributed to the individual counselling and visuals, the fact that only 55% of the GEC sessions actually adequately covered this topic indicates that other factors may have contributed to the women's knowledge of this topic.This process of refining and evaluating the informed consent process can benefit the clinic and research settings in both developed and developing countries. Data from clinics in the USA, Belgium, and France report that even under well suited environments, informed consent for HIV screening was generally only obtained in about 70–85% of cases, and documentation of consent was substantially less ,24. In rWe have modified the standard model of informed consent by adapting it to suit the population that was served, and have documented subsequent improvements in patient understanding of the informed consent process. This study shows that culturally appropriate enhancement of the standard informed consent process moves the process towards its goal of being one that is truly informed and voluntary. Thus it not only fulfills the ethical requirements, but, most importantly, helps to assure that women's rights are preserved. This last point is critical because previous research has pointed out that, although women may be fully informed, they still may not feel their choice is fully voluntary . We sugg. It is hoped that these types of visuals will become an integral part of all voluntary counseling and testing programs throughout India and elsewhere.In an effort to allow all interested organizations involved in HIV counseling and testing to use or modify our visuals for their own programs, we are have created a downloadable version available for public access at our website None declared.JS was involved in developing the research and visuals, implementing the study and preparing the manuscript. HP participated in the design of the visuals and in the analysis and coordination of the study. SS and AJ were involved in data collection and analysis. NK-K participated in the design of the visuals and in the analysis of the study. NS participated in the design of the study and the visuals. KEB, AS and MAP participated in the design and assisted in the implementation of the study. RCB assisted in all aspects of the study design and implementation. AVS participated in the design, conducted the analysis and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Lamivudine inhibits replication of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and is commonly used as part of antiretroviral therapy. The main limitation in the use of lamivudine is resistant mutation selection. Most of these mutations affect the YMDD motif of the HBV DNA polymerase. The resistance occurs through M550V or M550I aminoacid replacements. The M550V variation may be accompanied by L526M mutation, notably in HIV-HBV co-infected patients. The aim of this study was to investigate mutations associated with lamivudine resistance in a hemodialysis patient chronically co-infected with HIV-1 and HBV, who was submitted to several antiretroviral treatments.HBV isolates derived from three blood samples collected at different times of antiretroviral therapies with and without lamivudine, were titred and submitted to nucleotide sequencing.in vitro reduction of virus antigenicity (escape mutant). HBV DNA was detected at high levels (108–109 copies/ml) in the three blood samples.HBV isolate derived from a sample collected in 1999 during an antiretroviral treatment with lamivudine showed the lamivudine resistant double mutation (L526M, M550V). However, no mutation associated with lamivudine resistance was observed in the HBV genome derived from the sample collected during a period of treatment without lamivudine (2001). After reinstitution of lamivudine (2002), the predominant HBV population exhibited a rare triple mutation (V519L, L526M, M550V), which has previously been associated with an Reintroduction of lamivudine as part of antiretroviral treatment in a patient who had developed lamivudine resistant HBV strains favored the predominance of an HBV isolate with reduced antigenicity. The absence of hepatitis acute exacerbation in this patient may be correlated to the absence of significant variations of the viral load, which was independent of the presence of mutations in the HBV DNA polymerase. Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of transmission, mainly sexual, parenteral and vertical. Therefore, the prevalence of HBV serological markers is higher among HIV infected patients than in non-HIV infected individuals ,2. ConsiLamivudine is a nucleoside analogue that inhibits the reverse transcriptase activity of both HIV and HBV  and is cThe consequences of drug resistance mutations for the evolution of HBV induced liver disease are currently under study. Hepatitis B acute exacerbation has been described after withdrawal of lamivudine therapy . Such anThe present study was performed to characterize HBV variants and genetic patterns of lamivudine resistant HBV strains in a patient co-infected with HIV-1 at different periods of an antiretroviral treatment with and without lamivudine.5 copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 105 copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003.A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load , as described previously [4 HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 104 copies/ml by the last positive dilution.Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10eviously . Serial eviously , equivalPre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit  with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed.Nucleotide sequences were aligned using PILEUP . A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows . HBV seqThe patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999  in the blood sample collected in March 1999, during this first period of lamivudine treatment  HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (108copies/ml) after reintroduction of lamivudine  derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A' (genotype A excluding A') and A'  or Ae anTable Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein  or isoleucine (M550I). More rarely, HBV variant presenting M550S replacement may be selected during lamivudine treatment  The M550mivudine , since emivudine . It is bmivudine . Even somivudine . Here, bmivudine ,31, namemivudine . The trint G145R ,31. The 8 copies/ml) in the blood sample collected in 1999, in which the double mutant represents the major viral population. Similar high HBV loads were observed in the sample containing the triple mutant (2002). This result is in agreement with a recent study, showing that V519L mutation also enhances viral replication [There is a general consensus that the lamivudine resistant single mutants in the YMDD motif (M550V/I) replicate substantially more slowly than the wild type. The addition of the mutation at position 526 may act as a compensatory change that partially restores the replication fitness of the virus . Here, ilication .9 copies/ml) was moderately higher during the lamivudine interruption period than that found during lamivudine periods.The HBV population that emerged during the interruption of lamivudine treatment did not show mutations at polymerase positions 519, 526 and 550. However, another substitution, namely G473E, was observed that was associated to G119R substitution in the small S protein. Such a virus might be present as a minor population but not detected during the first lamivudine treatment. Although two out three clones of this population possessed stop codon mutations in S gene, HBV load (108–109 copies/ml) at different periods of antiretroviral treatment with and without lamivudine.HBV chronically infected patients submitted to lamivudine treatment may have acute exacerbation after withdrawal of drug therapy or when lamivudine resistance emerges ,26,35-37A rare HBV triple mutant, belonging to genotype A, subgroup A', appeared predominantly in a patient submitted to lamivudine as part of HIV treatment. This type of mutant, previously found in isolates belonging to genotypes A, D and G ,36, may None declared.EAS and MVS carried out cloning and sequencing of HBV DNA. JA was involved in clinical evaluation of the patient and supervised the antiretroviral treatment. SAG conceived the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


PLoS Biology, Hajime Hirase and his colleagues at Rutgers University have done just that by focusing their microscope on the brains of living rats in order to examine how certain cells called astrocytes function in vivo.Did you ever wish you could peek inside someone's brain and see what was going on in there? In research reported in this issue of Glia is derived from the Latin for “glue”; these cells were thought to “hold it all together.”) One type of glial cell, the astrocyte (named for its starlike shape), is the most populous cell in the brain and forms an intimate association with neurons and their synapses. It was thought that these cells played a supporting role in the brain, ensuring the proper chemical environment for synapses.In the longstanding quest to understand how the brain works, scientists have focused on neurons. Neurons conduct action potentials, electrical signals that transmit information in the nervous system. But the brain also contains several other types of cells called glia. (Recent research, however, has suggested that astrocytes and other glial cells may play a more significant role. When examining astrocytes cultured in the lab, scientists have observed behavior suggesting that astrocytes can communicate with neurons. Though astrocytes cannot propagate electrical signals like neurons do, they can sense the transmission of such signals at the synapse between two neurons. Furthermore, astrocytes are able to propagate a different kind of signal, a chemical signal based on the release of calcium ions. Calcium signaling is a mechanism of chemical signaling that has been observed in many other cell types. The exact properties of neuron–astrocyte communication, however, are not clear because different preparations of these tissues have yielded different results. It has also not been established that this type of communication occurs in the living brain.To explore such questions, Hirase and colleagues have taken the next step by investigating the calcium signaling properties of astrocytes in the brains of living rats. To accomplish this feat, the researchers used a combination of two technologies. They monitored calcium signaling using a fluorescent dye called Fluo-4, which fluoresces in response to calcium ions. Then they used a special type of microscope called a two-photon laser scanning microscope to visualize the dye. Since this type of microscope uses a lower energy laser, it can image the dye in living tissue without causing harm.The researchers applied the dye to the brains of anesthetized rats, washed out the excess dye that had not penetrated into cells, and then imaged the tissue under the microscope. They first confirmed that they indeed were examining astrocytes and noticed that cells displayed a moderate level of baseline calcium signaling activity. They then used a drug called bicuculline to stimulate neurons and observed a significant increase in the calcium signaling activity of the astrocytes. Because bicuculline only affects neurons, this implies that the astrocytes are responding to the activity of the neurons. The researchers also found that neighboring astrocytes often also displayed coordinated calcium signaling activity, suggesting that the communication among astrocytes is facilitated by increased neuronal activity.This research confirms the complexity of astrocyte signaling functions in the living brain and demonstrates that astrocytes play far more than a supporting role in brain function. It also establishes an important experimental system for scientists seeking to understand how these distinct elements of the brain—neurons and astrocytes—work together. Though this research makes it clear that signaling exists both among astrocytes and between neurons and astrocytes, scientists have yet to understand the effect of this signaling. Some possibilities include regulation of synapse formation, modification of synaptic strength, or more complicated roles in information processing resulting from the coordination of neuronal activity. Future research using this and other systems will help reveal these functions.


Critically Appraised Topics (CATs) are a useful tool that helps physicians to make clinical decisions as the healthcare moves towards the practice of Evidence-Based Medicine (EBM). The fast growing World Wide Web has provided a place for physicians to share their appraised topics online, but an increasing amount of time is needed to find a particular topic within such a rich repository.A web-based application, namely the CAT Crawler, was developed by Singapore's Bioinformatics Institute to allow physicians to adequately access available appraised topics on the Internet. A meta-search engine, as the core component of the application, finds relevant topics following keyword input. The primary objective of the work presented here is to evaluate the quantity and quality of search results obtained from the meta-search engine of the CAT Crawler by comparing them with those obtained from two individual CAT search engines. From the CAT libraries at these two sites, all possible keywords were extracted using a keyword extractor. Of those common to both libraries, ten were randomly chosen for evaluation. All ten were submitted to the two search engines individually, and through the meta-search engine of the CAT Crawler. Search results were evaluated for relevance both by medical amateurs and professionals, and the respective recall and precision were calculated.While achieving an identical recall, the meta-search engine showed a precision of 77.26% (±14.45) compared to the individual search engines' 52.65% (±12.0) (p < 0.001).The results demonstrate the validity of the CAT Crawler meta-search engine approach. The improved precision due to inherent filters underlines the practical usefulness of this tool for clinicians. Healthcare has been steadily moving towards Evidence-Based Medicine (EBM) since the term was formally introduced in 1992 by a group led by Gordon Guyatt at McMaster University, Canada . EBM proWith the use of resources on the World Wide Web becoming common practice, several academic and healthcare organizations have built online CAT libraries for knowledge sharing with peer physicians. The repository of CATs has been growing steadily since the setup of the first accessible CATBank developed by the Centre for Evidence Based Medicine, Oxford in 1992 . Among tIt is believed that more CATs will be added into the repositories as more people participate in EBM practice. However, the non-standardized electronic format of CATs has created much difficulty for physicians to access a particular topic. Accordingly, the CAT Crawler was developed at the Bioinformatics Institute, Singapore ,12 to prThe core component of the CAT Crawler is a meta-search engine. Its search is currently based on CAT resources from eight public online libraries . Once thFollowing the user input of a query keyword, a partial search is done through information extracted during an off-line process from six websites that do not hold search engines.The remaining search is carried out by querying the two individual search engines at BestBETs and UMHS. Use of the CAT Crawler is expected to have a quantitative and qualitative improvement of the retrieved results by post-processing obtained raw results from both libraries.The work presented here aims to evaluate the quantity and quality of the obtained results from the CAT Crawler meta-search engine, and thus to evaluate the validity and the usefulness of the application. Recall and precision were estimated to measure the performance of this meta-search engine versus the two individual search engines at BestBETs and UMHS.The workflow of this study is demonstrated in Figure AnalogX Keyword Extractor, which is freely available online [and and day.To find a viable sample of keywords for a test search, the titles of all CATs stored in the two CAT libraries, namely BestBETs and UMHS were submitted to e online . This letechnical relevance of all documents in the dataset must be assessed. In this study, a document is called technically relevant for a given search term if it contains this term in the full-text. Perl scripts were developed to examine all CATs in the two libraries BestBETs and UMHS and the total number of relevant documents as per the above definition in each library was collected for further calculation. This was done for each selected keyword and the process was independent from the search using the three search engines: the CAT Crawler, BestBETs and UMHS.In order to be able to calculate recall as detailed below, the In the next step, those ten keywords were submitted to the search engines at BestBETs and UMHS, and to the CAT Crawler meta-search engine. The retrieved links were evaluated for their relevance by 13 volunteers, who are categorized into three groups. Among them, one physician in Group I represents medical professionals, six persons in Group II represent people who were trained in biology or medicine, and six persons in Group III represent people who do not have any medical background.Recall and precision are two accepted measurements to determine the utility of an information retrieval system or search strategy . They arDespite the relevance evaluation from 13 volunteers, it is necessary to know the total number of the relevant documents in a database for each query keyword in order to estimate the recall. In the present study, a particular CAT in a database was defined as technically relevant if the keyword could be found in its full-text article.The CAT Crawler is designed not to hold permanently any full-text CATs . When a Similarly, the recall and precision of the search engines at BestBETs and UMHS are estimated based on the combined repository of the two individual sites. The revised formula are shown below:The averaged precision and recall over all evaluators are used to evaluate the performance of the CAT Crawler meta-search engine. These values are compared to the estimate based on the search results from the two individual search engines at BestBETs and UMHS.According to the predefined selection criteria, the ten keywords listed in Table To compare the performance of the CAT Crawler meta-search engine to that of the two individual search engines, recall and precision were computed and averaged over the evaluation of all 13 participators. The data recorded are shown in Table The performance evaluation clearly places the CAT Crawler meta-search engine on par with the individual search engines at BestBETs and UMHS as far as recall is concerned, and well above them for precision  lead to numerical results for recall that differ from the absolute true value as determined above. As the same numbers are used throughout, however, the comparison of search results obtained by the individual search engines and the CAT Crawler meta-search engine remains valid.Appendicitis appeared only in the reference section of a document dealing with questions of abdominal pain relief). Due to the relatively small number, no difference could be detected between the various grades of relevance, and results were pooled to relevant/irrelevant and used for calculating recall and precision as described above. If a larger number of volunteers could be recruited, repetition of this evaluation might yield interesting results.Critics have pointed out the over-reliance of researchers on the use of recall and precision in evaluation studies  and the Other approaches have been spawned to evaluating system effectiveness in order to minimize these problems with recall and precision. One example are task-oriented methods that measure how well the user can perform certain tasks -24. ThesIn summary, the data obtained from the analysis of search results obtained from identical queries submitted to the two CAT libraries at BestBETs and UMHS, using either their respective search engines or the CAT Crawler meta-search engine, showed a competitive recall, and superior precision of the meta-search engine compared to the individual search engines.The author(s) declare that they have no competing interests.PD participated in the design of the study, data analysis and drafting of the manuscript. LLW and SN generated raw data for the study. ML was involved in drafting the manuscript. AM designed the study and participated in the drafting of the manuscript.The pre-publication history for this paper can be accessed here:


We assessed whether the Korean version of modified Mini-Mental State Examination (K-mMMSE) has improved performance as a screening test for cognitive impairment or dementia in a general population compared with the Korean Mini-Mental State Examination (K-MMSE).Screening interviews were conducted with people aged 65 and over in Noam-dong, Namwon-city, Jeonbuk province. There were 522 community participants, of whom 235 underwent clinical and neuropsychological examination for diagnosis of dementia and Cognitive Impairment No Dementia (CIND). Sensitivity, specificity and areas under the receiver operating characteristic (ROC) curves for the K-mMMSE and the K-MMSE were the main outcome measures.P < 0.05). For the K-mMMSE, the optimal cut-off score for a diagnosis of CIND was 69/70, which had a sensitivity of 0.86 and a specificity of 0.79, while, for a diagnosis of dementia, the optimal cut-off score of 59/60 had a sensitivity of 0.91 and a specificity of 0.78. The K-mMMSE also had a high test-retest reliability (r = 0.89).Cronbach's alpha for the K-mMMSE was 0.91, compared with 0.84 for the K-MMSE. The areas under the ROC curves in identifying all levels of CIND or dementia were 0.91 for the K-mMMSE and 0.89 for the K-MMSE (Our findings indicate that the K-mMMSE is more reliable and valid than the K-MMSE as a cognitive screen in a population based study of dementia. Considering the test characteristics, the K-MMSE and modified version are expected to be optimally used in clinical and epidemiologic fields. The Mini-Mental State Examination (MMSE) is a brief screening test that quantitatively assesses the cognitive status of elderly people ,2. It isThese limitations led to the development of the Modified Mini-Mental State Examination (3MS) in 1987 , which eth and 10th percentiles [In 2002, a group from the Cache County Study modified the 3MS for use as a cognitive screen in an epidemiologic study of dementia . These mcentiles .In Korea, the MMSE was translated into two versions, the Korean version of the Mini-Mental State Examination (MMSE-K) and the Korean Mini-Mental State Examination (K-MMSE) ,11. Both2 and had an estimated population of 6,883, of whom about 7% were aged 65 or over. All participants gave informed consent, and the study was conducted in accordance with the guidelines in The Declaration of Helsinki and approved by the appropriate research ethics committee.Potential participants for this study were recruited from all inhabitants of Noam-dong, Namwon-city, Jeonbuk-province, South Korea, aged 65 and over in 2003, as recorded in national residents registration lists. The area surveyed covered 8.93 kmP values > 0.1).Among the 522 eligible subjects aged 65 and over identified from registration lists, 235 (45%) completed clinical examinations after the interview and formed the study sample for principal analysis. Of the remaining subjects, we were unable to establish contact with 162 (31%), 75 (14%) refused to participate, 18 (3%) did not complete the survey, 7 (1%) had severe pre-morbid illness including blindness and deafness, 4 (0.8%) had changed address, and 3 (0.6%) had died before the visit. The principal apparent reason for the difficulties in establishing contact was that the person was in a regular daily activity or away from home, visiting family members living elsewhere. The 18 individuals (3%) who did not complete the survey questionnaire or were not examined clinically had a mean age of 74.9 ± 10.3 years; 13 (72%) were females, and 4 (22%) were educated. Of all the eligible subjects, participants and non-participants did not differ in age (73.5 ± 6.8 y vs. 74.6 ± 7.8 y, respectively) or gender  and Korean Instrumental Activities of Daily Living (K-IADL) ,16. The At the second interview, physicians who were blinded to the cognitive scores performed a clinical examination and neuropsychiatric inventory on participants who completed the first survey questionnaire. The clinical examination verified the presence of cognitive impairment. The diagnostic criteria for dementia were based on those of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV)  and wereWe translated the original 3MS-R into Korean according to the guidelines recommended by the modifiers . The iteIn explaining the appropriate questions and answers, we presented a simple example prior to asking the first question, specifically, "the eyes and nose are different, but they are similar in being part of our face." In a pilot study, most elderly subjects could not understand the concept of similarity without this example, and they became intolerant to our interview unless an example was provided. After providing the example, however, most subjects were more cooperative and tried to answer properly. We asked subjects to write a spontaneous sentence and scored whether it was legible and correct, with or without prompting. The total possible score was 100, and a K-MMSE score could be generated from it.For demographic factors, mean or median values and proportions were calculated according to the cognitive impairment strata. Cronbach's alpha was used to quantify internal consistencies of the K-mMMSE and K-MMSE . We asseDescriptive statistics are displayed in Table Severity of cognitive impairment was directly related to mean age and inversely related to number of years of education. Women had higher rates of being cognitively impaired or demented than men. The median scores on the K-mMMSE and K-MMSE decreased with severity of cognitive impairment. Median scores on the informant questionnaires of the S-SDQ and K-IADL increased with poorer cognitive status. The inter-quartile ranges (IQR) also steadily increased, reflecting increasing variability in cognitive and functional status. Four respondents scored perfectly on the K-MMSE, whereas none scored perfectly on the K-mMMSE, suggesting that the K-mMMSE might be less prone to the ceiling effect in this population.The estimated Cronbach's alpha was 0.91 for the K-mMMSE and 0.84 for the K-MMSE. Relative to each cognitive impairment stratum , the alphas were 0.84, 0.81 and 0.81, respectively, on the K-mMMSE and 0.74, 0.72, and 0.63, respectively, on the K-MMSE. Neither age nor gender had any substantial impact on internal consistency. Stratum-specific alphas of the K-mMMSE for the different subgroups ranged from 0.86 to 0.91 in men and 0.89 to 0.90 in women.P = 0.132).The retest of the K-mMMSE was assessed in 30 subjects . The correlation coefficients for the total scores were 0.89 on the K-mMMSE and 0.85 on the K-MMSE. The coefficients of the 15 items of the K-mMMSE were all significant, ranging from 0.37 for similarities to 0.83 for time orientation. The re-tested subjects were representative of the entire study population, in that the sociodemographic characteristics of the 30 retested subjects were similar to the other participants in mean age (74.0 ± 7.4 y vs. 73.5 ± 6.7 y), educational years (3.5 ± 3.2 y vs. 3.4 ± 3.9 y), K-mMMSE scores (63.8 ± 21.0 vs. 63.1 ± 20.4), and proportion of women . The ROC curves plotted in the same graph suggested that the performance of the K-mMMSE was superior to that of the K-MMSE at almost all cut-off points  was significantly superior to that of the K-MMSE . At the cut-off of 59/60, the K-mMMSE had a sensitivity of 0.91 (0.79–0.98), a specificity of 0.78 (0.72–0.84), a positive LR of 4.21 and a negative LR of 0.11. At a cut-off point of 18/19, the K-MMSE had a sensitivity of 0.91 (0.79–0.98), a specificity of 0.76 (0.69–0.82), a positive LR of 3.82 and a negative LR of 0.11.Both the K-mMMSE and the K-MMSE could properly discriminate demented from normal individuals, but there was no significant difference between them  for the 3MS has been reported to be 0.91 in a community study, a value identical to that found here . Anotherr = 0.37), compared with simple items, i.e., temporal orientation (r = 0.81). The discrepancy might be due to a difference of time lag, in that the Stirling County Study retest was performed over a 3 year interval, with individual retests ranging from 0.9 to 4.0 years. Furthermore their retested subjects were not representative of all participants. We observed a correlation coefficient of 0.85 for the K-MMSE over all levels of cognitive status, which is in line with generally acceptable findings [Test-retest reliability results of the K-mMMSE were also comparable to those in previous studies. Correlation coefficients of 0.91 to 0.93 have been reported for small samples of community dwelling residents and dementia patients, which are slightly higher than our value of 0.89 . The Stifindings . Scores findings ,28.The K-mMMSE was superior to the K-MMSE for diagnosis of all levels of CIND or dementia, as well as being slightly superior at almost all cut-off points. Since dementia is usually preceded by CIND or mild cognitive impairment (MCI), the definition of both requires explication ,30. SubjThe two cognitive screening measures did not differ significantly, however, in the detection of dementia. These results are comparable to the findings of McDowell et al. ; howeverEven Cache County modifications to the 3MS showed a good sensitivity and specificity, 3MS-R was also dependent on their cultural and social factors which might limit general use in non-US population . For thiThere are important limitations to our findings. First, the subjects who participated in this study showed very low levels of educational background, perhaps limiting its general usefulness, especially regarding the cut-off points for a diagnosis of CIND or dementia. The low educational attainment, however, has been one of important characteristics of our elder population, because they were largely deprived of education due to Korean War and Japanese colonial dominion over the country . And theWe conclude that the K-mMMSE is a valid, stable, and reliable cognitive screen in an epidemiologic study. The K-mMMSE is more sensitive to all levels of CIND and dementia than the K-MMSE. Future investigations with the K-mMMSE will examine age-, sex-, and education-specific reference values to determine how performance patterns on individual items may discriminate between those with or without cognitive impairment and dementia subtypes.None declared.SKJ performed physical measurements, collected data, and drafted the manuscript. KHC participated in data collection and reviewed the manuscript. JMK conceived of the study and participated in its design, and also performed physical measurements. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: