The circadian rhythm of about 24 hours is a fundamental physiological function observed in almost all organisms from prokaryotes to humans. Identification of clock genes has allowed us to study the molecular bases for circadian behaviors and temporal physiological processes such as hormonal secretion, and has prompted the idea that molecular clocks reside not only in a central pacemaker, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, but also in peripheral tissues, even in immortalized cells. Furthermore, previous molecular dissection revealed that the mechanism of circadian oscillation at a molecular level is based on transcriptional regulation of clock and clock-controlled genes.mBmal1, mNpas2, mRev-erbα, mDbp, mRev-erbβ, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak) showed robust circadian expressions of mRNAs in all tissues except testis, suggesting that these genes are core molecules of the molecular biological clock. The bioinformatics analysis revealed that these genes have one or a combination of 3 transcriptional elements (RORE, DBPE, and E-box), which are conserved among human, mouse, and rat genome sequences, and indicated that these 3 elements may be responsible for the biological timing of expression of canonical clock genes.We systematically analyzed the mRNA expression of clock and clock-controlled genes in mouse peripheral tissues. Eight genes (The observation of oscillatory profiles of canonical clock genes is not only useful for physiological and pathological examination of the circadian clock in various organs but also important for systematic understanding of transcriptional regulation on a genome-wide basis. Our finding of the oscillatory expression of canonical clock genes with a temporal order provides us an interesting hypothesis, that cyclic timing of all clock and clock-controlled genes may be dependent on several transcriptional elements including 3 known elements, E-box, RORE, and DBPE. Circadian rhythms have been known to be generated in pacemaker cells, the suprachiasmatic nuclei (SCN) of hypothalamus in mammals, and entrained by environmental cues, such as light, temperature, noise, feeding or social cues, whereas a recent analysis using mPer2llations . The outllations . Identifllations ,6. Furthllations -10.Drosophila period mutants, originally identified as a circadian mutant brought us the first clock gene, period [hPer2 was recently shown to cause a familial advanced sleep phase syndrome [There is a distinct connection between genes and behaviors in circadian rhythms, which is conserved from fly or other lower organisms to humans ,8. The D, period , while asyndrome . As descsyndrome ,13,14. TIn this study, we systematically examined the mRNA expression of common circadian-regulated genes in several mouse peripheral tissues and made oscillatory profiles of canonical clock genes. Moreover, by bioinformatics, we identified 3 clock elements for circadian transcription (E-box, RORE, DBPE). These 3 elements and their combination would suffice to explain the biological timing of expression of these clock and clock-controlled genes.mBmal1, mNpas2, mRev-erbα, mDbp, mRev-erbβ, mPer3, mPer1 and mPer2; given in the temporal order of the rhythm peak; see also Fig. mBmal1 and mNpas2, in subjective night at CT20-CT0; mRev-erbα, in subjective day at CT4-8; mDbp and mRev-erbβ, at CT8; mPer3, at CT8-12; mPer1, at CT12; and mPer2, at CT12-16  showed weak rhythms of their mRNA expression, while no other genes, including the mPer family, showed any clear oscillation. Surprisingly, the expression of mPer1 transcripts in the testis, which did not show a circadian rhythm, was substantially higher than other tissues, and exceeded RNA expression of other genes studied in the testis. This is consistent with data recently reported [mPer1 in the testis [mPer1 may play an alternative role in the testis, including developmental regulation during spermatogenesis. The rhythm of mCry1 mRNA expression was obviously circadian (peaking at CT16-20, with a trough at CT4-8) except in the testis, but the peak-trough amplitude was relatively smaller than that of the above genes. mCry2 and mClock RNA levels seemed to be rhythmic except in the testis, but the rhythm was rather weak and not clearly circadian. No circadian rhythms were observed in the remaining 3 genes examined, i.e. mCKI-δ, mCKI-ε, and mTim. Despite a central role in Drosophila for timeless (dTim), the mammalian homologue (mTim) was originally found to show weak or no rhythm in the SCN [To examine the circadian expression of mouse clock and clock-related genes in peripheral tissues, we performed the quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) method on mRNAs from 7 different mouse peripheral tissues  proteins, to the E-boxes in the promoter region of Per1 [Pers, as described above, forms another loop [Bmal1 [in silico search identified these 2 ROREs in the promoter region of Npas2  were experimentally studied and confirmed [Using the NCBI database and Celera Database System, we systematically searched for the above 2 elements and another clock element, a DBP-binding element (DBPE), described below. These elements are conserved among human, mouse, and rat genome sequences in the regions 9-kb upstream and 5-kb downstream of the transcription start site  showed a robust circadian rhythm in different peripheral tissues . Further detailed studies of each promoter, combined with systematic analyses using microarrays and real-time RT-PCR, will give us a more detailed comprehension of the intertwined positive and negative regulatory loops of molecular biological clocks.Transcripts of 8 genes , were exposed to 2 weeks of light-dark (LD) cycles and then kept in complete darkness as a continuation of the dark phase of the last LD cycle. mRNA expression was examined in the third dark-dark (DD) cycle. All protocols of experiments using animals in this study were approved by the OBI (Osaka Bioscience Institute) Animal Research Committee.mPer1 FW: CAG GCT AAC CAG GAA TAT TAC CAG C, mPer1 RV: CAC AGC CAC AGA GAA GGT GTC CTG G; mPer2 FW: GGC TTC ACC ATG CCT GTT GT, mPer2 RV: GGA GTT ATT TCG GAG GCA AGT GT; mPer3 FW: CTG CTC CAA CTC AGC TTC CTT T, mPer3 RV: TTA GAC AGC AAG GCT CTG GTT CT; mNpas2 FW: GTA TGC ACA GAG CCA AGT GAT GTT, mNpas2 RV: TGC TCA CTG TGC AGA GAT GTT G; mDbp FW: AAT GAC CTT TGA ACC TGA TCC CGC T, mDbp RV: GCT CCA GTA CTT CTC ATC CTT CTG T; mBmal1 FW: GCA GTG CCA CTG ACT ACC AAG A, mBmal1 RV: TCC TGG ACA TTG CAT TGC AT; mRev-erbα FW: CGT TCG CAT CAA TCG CAA CC, mRev-erbα RV: GAT GTG GAG TAG GTG AGG TC; mRev-erbβ FW: ACG GAT TCC CAG GAA CAT GG, mRev-erbβ RV: CCT CCA GTG TTG CAC AGG TA; G3-PDH FW: ACG GGA AGC TCA CTG GCA TGG CCT T, G3-PDH RV: CAT GAG GTC CAC CAC CCT GTT GCT G; mCry1 FW: CCC AGG CTT TTC AAG GAA TGG AAC A, mCry1 RV: TCT CAT CAT GGT CAT CAG ACA GAG G; mCry2 FW: GGG ACT CTG TCT ATT GGC ATC TG, mCry2 RV: GTC ACT CTA GCC CGC TTG GT; mCKIε FW: GGA TGT GAA GCC CGA CAA CTT, mCKIε RV: TCT CGA CGG CTT TGC TCA AT; mCKIδ FW: CCA GCC TGG AAG ACC TGT TC, mCKIδ RV: TGG CCA GCC CAA AGT CAA; mClock FW: CCT ATC CTA CCT TGG CCA CAC A, mClock RV: TCC CGT GGA GCA ACC TAG AT; mTim FW: ACA TGT GGG CAA TGG CTT, mTim RV: CTG CTC CAC AAA GTG AAA GGT. Specificity of gene amplification was confirmed by measuring the size and purity of the PCR product by gel electrophoresis, and by analyzing the dissociation curve with ABI PRISM 7000 SDS software (Applied Biosystems). For a 25-μl PCR reaction, 50 ng cDNA template was mixed with the forward and reverse primers to a final concentration of 300 nM each and 12.5 μl of 2x SYBR Green PCR Master Mix (Applied Biosystems). The reaction was first incubated at 50°C for 2 min, then at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each gene-specific PCR was performed in triplicate. G3-PDH primers were used as the control.Real-time quantitative RT-PCR was performed by using an ABI PRISM 7000 (Applied Biosystems). The PCR primers were designed with Primer Express software (Applied Biosystems), and the sequences of the forward and reverse primers were as follow: hBmal1 -Luc or mPer2 -Luc, and incubated for 24 hours. The medium was then exchanged for serum-rich medium (DMEM, supplemented with 50 % serum). Two hours later this medium was replaced with normal culture medium. In the presence of 0.1 mM luciferin, light emission was measured and integrated for 1 min at intervals of 15 min, with a photomultiplier tube (Hamamatsu Photonics).NIH3T3 cells were cultured, transfected with The sequences were downloaded from the Celera Database System and the NCBI Gene database. Each gene sequence spanning from 9 kb upstream to 4 kb downstream of the transcription start site was examined in each database. Multiple sequence alignments of these sequences for each gene were obtained by Clustalw version 1.83 with default parameters. The binding elements were then searched from these alignments using a pattern finding tool, fuzznuc, with the following consensus sequences allowing for a 1-base mismatch:DBPE: [GA]T[GT]A[TC]GTAA[TC]E-Box: CACGTGRORE: [AT]A[AT]NT[AG]GGTCABmal1; Human, NT_009237.16, 12054318–12069318, Mouse, NT_081129.1, 107781–122781, Rat, NW_047562.1, 13774073–13789073, Npas2; Human, hCG27614, 95632226–65646226, Mouse, mCG8437, 35980102–35994102, Rat, rCT22431, 39204499–39218499, Rev-erbα; Human, hCG93862, 34926094–34912094, Mouse, mCG15360, 105438925–105424925, Rat, rCG33292, 82492796–82478796, Dbp; Human, NT_011109.15, c21417778–21402778, Mouse, NT_078442.1, 59711–74711, Rat, NW_047558.1, 5120734–5135734, Per3; Human, NT_021937.16, 1962822–1977822, Mouse, NT_039268.2, c4331528–4316528, Rat, NW_047727.1, c8016956–8001956, Per1; Human, NT_010718.14, c6905708–6890708, Moues, NT_039515.2, 65661216–65676216, Rat, rCG34390, 52960430–52974430, Per2; Human, NT_005120.14, c5136562–5121562, Mouse, NT_039173.2, c5833757–5818757, Rat, NW_047817.1, c6827703–6812703.The accession numbers used and the sequence numbers analyzed are as follow: SCN, suprachiasmatic nuclei; RT-PCR, reverse transcription-polymerase chain reaction; CT, circadian time; LD, light-dark; DD, dark-dark; bHLH-PAS, basic helix-loop-helix-Per-Arnt-Sim; RORE, ROR/REV-ERB element; DBPE, DBP-binding element; NCBI, the National Center for Biotechnology Information.TY and YN carried out the molecular biology studies. HS carried out the in silico study. MA carried out the real-time luciferase reporter assay. TM participated in the coordination, and provided financial support. TT conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Possessing two parkin mutations leads to early-onset parkinsonism, while having one mutation may predispose to late-onset disease. This dosage pattern suggests that some parkin families should exhibit intergenerational variation in age at onset resembling anticipation. A subset of familial PD exhibits anticipation, the cause of which is unknown. The aim of this study was to determine if anticipation was due to parkin mutation dosage.parkin analysis included sequencing and dosage analysis of all 12 exons.We studied 19 kindreds that had early-onset parkinsonism in the offspring generation, late-onset parkinsonism in the parent generation, and ≥ 20 years of anticipation. We also studied 28 early-onset parkinsonism cases without anticipation. Patients were diagnosed by neurologists at a movement disorder clinic. parkin mutations, but contrary to our postulate, the affected relative with late-onset parkinsonism did not have a parkin mutation. In effect, none of the anticipation cases could be attributed to parkin. In contrast, 21% of early-onset parkinsonism patients without anticipation had parkin mutations.Only one of 19 cases had compound parkin, and may signify a distinct disease entity.Anticipation is not linked to  Howeverecessive -5, nor iecessive -9. Severse onset -10. Collth decade of life, whereas homozygotes show symptoms at much younger ages, sometimes at birth (OMIM *143890).Several groups have reported what appears to be genetic anticipation in parkinsonism -13. In tparkin mutation dosage? We hypothesized that some parkin mutations are dominant: heterozygotes have incomplete penetrance and may develop late-onset parkinsonism, whereas homozygotes and compound heterozygotes have accelerated disease leading to early-onset parkinsonism. This unifying hypothesis was attractive because if true, it could explain the reported variations in mode of inheritance of parkin (recessive in some families, dominant in others), the range in age at onset from juvenile to late onset, and the significantly earlier onset in some of the children of affected parents. To test this hypothesis, we studied parkin in 19 kindreds with early-onset parkinsonism in the index generation, late-onset parkinsonism in the parent generation, and exhibited ≥ 20 years of anticipation.Is anticipation in parkinsonism related to Families were recruited from a movement disorder clinic. The probands and affected family members had the clinical diagnosis of idiopathic Parkinson's disease (here referred to as parkinsonism due to lack of autopsy confirmation). Diagnosis was made by a neurologist according to the British Parkinson's Disease Brain Bank criteria except that family history was not an exclusion criterion . These fparkin exon amplifications were multiplexed under standard conditions with an 84-bp fragment of a single-copy human β-actin gene (Genbank accession number XM_004814) as an internal control. A standard curve was generated for each parkin exon and for β-actin using 0, 5, 15, 55 and 220 ng of control human genomic DNA. The number of PCR cycles required before the ABI 7700 detects each parkin exon product  was plotted against the corresponding exon standard curve, thus calculating the relative parkin copy number. The copy number for each exon was normalized to the single-copy actin gene within each multiplexed reaction and to a normal control reference individual, allowing an estimate of the number of copies of parkin. Optimal threshold levels for each primer set were maintained between plate analyses. All samples were analyzed in triplicate.DNA was extracted from blood using standard protocol. Genotyping was blind to phenotype. To identify point mutations, we sequenced both DNA strands of all 12 exons. Exons and 50–100 bp of flanking intronic sequences were PCR-amplified ,18. parkparkin analysis based on having parkinsonism in two consecutive generations, late-onset parkinsonism in the parent generation (onset ages 59 to 89 years, mean 71.0 ± 8.5), early-onset parkinsonism in the offspring generation (onset ages 8 to 47 years, mean 37.2 ± 9.8), and ≥ 20 years of anticipation as measured by the difference in mean ages at onset in two generations. In the younger generation, 2 probands had onset before age 20, 1 was in his twenties, and 16 were over 30 years old. It was hypothesized that the affected individuals with early-onset parkinsonism are homozygous or compound heterozygous, and the parents and other relatives with late-onset parkinsonism are heterozygous. For parkin analysis, we began with the family member from the generation with the earlier onset, so as to enrich for parkin mutations. We expected the majority of the index cases to be compound heterozygous. At the minimum, 16–49% of index cases should have had mutations, since this is the range reported for early-onset sporadic and familial parkinsonism [parkin mutations. This patient had compound deletions in exon 3 with onset at age 8. Her sister, onset at age 15, also had the compound mutation. The relative with late-onset was an uncle with onset age of 64. Contrary to our hypothesis, the uncle did not have a parkin mutation. The cause of late-onset parkinsonism in the uncle was different from the nieces. In effect, we did not find any cases where we could attribute the intergenerational difference in age at onset to parkin dosage.Nineteen kindreds were chosen for insonism . Howeverparkin in 28 additional patients with early-onset parkinsonism (onset age 14 to 40 years, mean 32.8 ± 7.0), either with (n = 4) or without (n = 24) family history, but with no evidence of anticipation. In this group, 3 probands had onset at or before age 20, 5 were in their twenties, and 20 were over 30 years old. Nine mutations were found in six individuals. Two subjects were compound heterozygous (onset ages 31 and 37) and four were heterozygous (onset ages 14, 25, 37, 37). The frequency of parkin carriers in early-onset parkinsonism without anticipation was 21% which is in the range 16% – 49% reported in the literature . It was of interest to determine if any of the parents of the three early-onset parkinsonism patients with compound mutations had developed late-onset parkinsonism. All six parents were heterozygous (5 were confirmed by genotyping, one was inferred), and all had remained free of parkinsonism to the ages of 53, 60, 63, 74, 76 and 78 yrs. These individuals may still develop parkinsonism.We also analyzed Familial parkinsonism with anticipation may be more common than classical dominant and recessive subtypes combined. In our clinic population, among 487 patients studied, 145 had a first or second degree relative with PD; that is 30% which is in line with the published figures for other referral clinics. Among the 145 familial cases, 110 had parkinsonism in consecutive generations, which is compatible with autosomal dominant inheritance, and 35 had an affected sibling or cousin, which is suggestive of recessive inheritance. However, among the 110, only 26 were compatible with a classical dominant pattern ; while 63 exhibited 10–68 years of anticipation, and 7 had 10–17 years of reverse anticipation (14/110 had unknown onset ages). Our clinic is a referral center, which explains the relatively high proportions of early-onset and familial cases. The interesting finding was the relative proportions of autosomal dominant, autosomal recessive and anticipation cases within the familial subtype. Despite its relatively high prevalence, at least in our clinic, familial parkinsonism with anticipation has been largely overlooked in genetic research. A few parkinsonism families have been attributed to expansions in known SCA loci, but in the majority of the kindreds, the cause remains unknown. The more obvious possibilities are mitochondrial inheritance, modifier genes, parent-child exposure to environmental triggers, as yet unidentified triplet repeats or dominant genes with dosage effect, and in some cases, artifactual appearance of anticipation due to ascertainment bias. None of these are mutually exclusive; more than one may be true and operative in different families.parkin literature suggest that possessing two parkin mutations is fully penetrant and leads to early-onset parkinsonism, whereas having only one mutation may be incompletely penetrant and lead to later disease onset. While the causative link to early-onset parkinsonism is widely accepted, the association of parkin with late-onset parkinsonism remains controversial [parkin heterozygotes are at risk for late-onset disease, then some parkin families should exhibit intergenerational variation in age at onset resembling anticipation, where heterozygous parents develop late-onset parkinsonism and children who inherit two mutations develop early-onset parkinsonism. We hoped to explain the appearance of anticipation in relation to parkin dosage, but the findings do not support this postulate. While the results rule out a link between parkin and anticipation in these families, they do not negate the association of parkin with late-onset parkinsonism.The current oversial ,10,20,21parkin mutation dosage. The underlying mechanism for anticipation may be genetic or environmental. Identification and a-priori classification of pedigrees that exhibit significant intergenerational age at onset variation, as being distinct from families that display classical dominant pattern, may facilitate gene mapping studies by reducing heterogeneity. Anticipation in parkinsonism merits investigation in its own right, not only because it is a common phenomenon and may account for a large subset of familial parkinsonism, but it may also uncover a novel mechanism in parkinsonism.The phenomenon of anticipation is not due to The author(s) declare that they have no competing interests.PP carried out the molecular genetic studies. LM interviewed the subjects and gathered family histories and medical records. JN performed the neurological examinations. GS participated in study design and supervised molecular genetic studies. JM performed molecular analysis of one large pedigree. HP conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


This study reports on clinical data from an 8-week open-label study of 20 HIV-seropositive individuals, diagnosed with Major Depressive Disorder (DSM-IV), who were treated with SAM-e (S-Adenosylmethionine). SAM-e may be a treatment alternative for the management of depression in a population reluctant to add another "pill" or another set of related side effects to an already complex highly active antiretroviral therapy (HAART) regimen.The Hamilton Rating Scale for Depression (HAM-D) and the Beck Depression Inventory (BDI) were used to assess depressive symptomatology from 1,2,4,6 and 8 weeks after initiation of treatment with SAM-e.Data show a significant acute reduction in depressive symptomatology, as measured by both the HAM-D and the BDI instruments.p < .001), with progressive decreases in depression symptom rating scores throughout the 8 week study.SAM-e has a rapid effect evident as soon as week 1 ( The incidence of depression in people living with HIV (PLWH) has consistently been reported to be higher than the 12–15% rates reported for the general population .In New York City, 27% of all people living with HIV are over 50 years of age and 66% are over 40 years age [Under-treated depression in the HIV population may be the result of multiple factors. People of color, women, substance abusers, and those living in poverty are less likely to have access to or to accept mental health outpatient services -7. Many SAM-e is a compound that has been used for many years in Europe as an antidepressant. It became available in the USA as a non FDA-regulated compound in 1999. SAM-e resembles a naturally occurring compound in the human body, formed by methionine and adenosine triphosphate. SAM-e donates an active methyl group to several acceptor molecules, including catecholamines, fatty acids, phospholipids, proteins and nucleic acids . SAM-e aThe efficacy of SAM-e as an antidepressant has been reported in a number of open-label trials  and in tth International Conference on AIDS. A total of 20 patients were recruited between April 2000 and November 2000.This study is an analysis of the complete dataset from an independently funded, IRB-approved project conducted at ACRIA, a 12-year-old, community-based HIV research and education agency in New York City. A preliminary report, based on a subset of these data, was presented at the 14Recruitment activities included distribution of information brochures to community physicians, announcements at public ACRIA treatment education workshops, and direct calls to persons listed in the ACRIA database. Individuals who responded to recruitment materials were screened via phone by the research coordinator using the PRIME-MD . HIV-posTreatment was initiated at 200 mg of SAM-e twice a day with daily supplementation of 1,000 mcg Vitamin B12 and 800 mcg Folic Acid, which were provided by Pharmavite, LLC. Over the course of the study, the dose of SAM-e was individually adjusted up to 800 mg, twice daily. Patients reporting nausea or insomnia were not increased above 800 mg daily, while patients with no reported side effects could be increased to a total daily dose of 1600 mg. Dosing was adjusted according to the severity of symptoms and clinical treatment response. We defined clinical treatment response as an improvement in depressive symptomatology of greater than 50% reduction on patient scores on the BDI and HAM-D as our treatment endpoint ,26. A stAt each study visit (Baseline, Week 1, Week 2, Week 4, Week 6, and Week 8), patients completed a clinical interview and a series of structured questionnaires on medical/physical symptoms, substance abuse, dementia scales, as well as two instruments designed to measure depressive symptoms, self-administered Beck Depression Inventory (BDI and the clinician-administered HAM-D).The BDI is a self-report measure for depressive symptoms . Total sThe HAM-D is a 17-item, clinician-rated instrument. It was designed to assess the changes in severity of depressive symptomatology over time in patients who had been diagnosed with Major Depressive Disorder . Scores Two separate analyses (e.g., Baseline -v- Week 1 and Baseline -v- Week 8) were identified as the most critical indicators of SAM-e efficacy. The first analysis represented evidence for acute onset of SAM-e, and the second indicated effectiveness of SAM-e over time. BDI and HAM-D scores were centered and the resulting Z-scores were used in a series of t-tests to assess the agreement between patient and investigator ratings of depression. Mean scores from both depression measures (BDI and HAM-D) were compared between time points, using a series of paired t-tests. Finally, an intent-to-treat analysis, using the last score carried forward, was conducted on patient BDI scores for all subjects who initiated treatment.Thirty (30) subjects were screened by telephone and twenty (20) entered into the study. Of these, fifteen 15) were male, five (5) were female and the majority were people of color (50% Black, 30% Hispanic, 20% Caucasian). Table 5 were maM = 33.5, SD = 11.1; Range: 15 – 55) to Week 1  was significant, t(1, 18) = 5.15, p < .001. Mean HAM-D scores declined in a parallel manner, from Baseline  to Week 1 . This change was also statistically significant, t(1, 14) = 3.58, p < .01. Both depression assessment instruments reached the defined clinical treatment response threshold of a greater than 50 percent reduction in depression symptom scores.Table M = 35.1, SD = 12.0; Range: 15 – 55) to Week 8 , t(1, 14) = 8.28, p < .001. Similarly, HAM-D scores were reduced from Baseline  to Week 8 . The reduction in HAM-D scores reached statistical significance, t(1, 13) = 9.92, p < .001.The 15 patients who completed the study also demonstrated clinical treatment response over time. Mean BDI scores dropped significantly from Baseline  were higher than Week 8 . HAM-D scores dropped from Baseline  to Week 8 . The reduction in self-reported (i.e., BDI) depressive symptomatology was significant, t(1, 19) = 8.85, p < .001. The results were equivalent for the psychiatrist-rated HAM-D, t(1, 18) = 7.23, p < .001.The intent-to-treat analysis yielded similar results. The last reported BDI and HAM-D scores for each of the 20 patients who initiated treatment (i.e., received at least one dose of SAM-e) were used in this analysis. Baseline BDI scores (The intent-to-treat remission rate was 79%. Fifteen (15) of the 20 patients achieved a HAM-D of 7 or lower, while one patient did not receive a valid HAM-D rating and four (4) received HAM-D ratings greater than 7 (13, 22, 24, 39, respectively). The intent-to-treat response rate was 74%, with 14 of the 19 patients achieving 50% or greater reduction in HAM-D scores.No patients terminated their participation due to side effects. Two (2) patients reported nausea and one (1) reported diarrhea; all resolved spontaneously. One (1) patient had insomnia and a subjective feeling of 'high energy,' but did not meet criteria for hypomania.Recent data from the HIV Cost and Services Utilization Study (HCSUS) indicate that almost half the nation's adult HIV patients suffered from symptoms of mental disorders. The report indicates that the prevalence of mental disorders range from four to eight times higher than those found in the general populations. Over 60 percent of HIV-positive adults used mental health or substance abuse services during the period studied; 26% visited mental health specialists, 15% had group mental health treatment, 40% discussed emotional problems with medical practitioners and 30% used psychotherapeutic medications .Depression remains under-reported and under-treated in the HIV population. Patient factors include stigma, fear of more medication, and perhaps the assumption that depression is a normal part of HIV disease, or due to HAART. Physicians tend to focus more on physical symptoms, and may be unaware of how to screen for depression in this population. Both physicians and patients are disadvantaged in accessing gay-affirmative, well-trained HIV mental health specialists who can disentangle HIV medical problems from substance abuse and mental illness. Risk factors for depression  are prevalent in those populations who are disproportionately affected by HIV. Many patients are reluctant to add more medication to their complex treatment regimens. The need for effective and safe treatments for depression is clear.Results of this study demonstrate that SAM-e significantly reduces depression in people living with HIV. This finding supports previous research demonstrating the efficacy of SAM-e for use in treating depression. More importantly, the therapeutic effect of SAM-e had an acute onset. There were significant reductions in the severity of depression among study patients within the first week of treatment. Unlike most antidepressants that require several weeks to reach maximum therapeutic efficacy, previous research has shown that serum levels of SAM-e peak within 24 hours of treatment. This may account for the rapid, effects of SAM-e seen on the HAM-D and BDI scores.SAM-e is a safe, effective treatment for depression among people living with HIV. SAMe has few reported side effects, and in our population of PLWH, was extremely well tolerated. SAM-e in this group had rapid onset of clinical efficacy. Both of these results make it an appealing alternative therapy for the management of depression in the HIV population.The results of the current study must be interpreted in the context of the study's limitations. Limitations of this study include the lack of a placebo group, the lack of a double-blind design and small sample size. The criteria for participation and the small sample size may limit the generalizability of our findings to other patient populations. Without a placebo control group, we cannot assess two factors that might have affected our results. Although patients were treatment-experienced with other anti-depressants, we cannot rule out the potential placebo-response that may have contributed to the rapid therapeutic effect of SAM-e. Also, meeting with a study psychiatrist may have contributed to improved depressive symptom ratings. Nonetheless, the study results provide useful and clinically relevant information for treating depression in HIV-positive individuals. Future studies employing a double-blind, placebo-control design are warranted.The absence of depression does not imply wellness. Many people living with HIV/AIDS report feelings of sadness and depression but do not receive treatment for depression. Future studies should also assess health-related quality of life. Such protocols would determine if people with significant, but subclinical depressive symptomatology would benefit from SAM-e.RASrecoded study data, conducted statisticalanalyses andco-wrote the final manuscript. DM provided details of the study design and procedures andwas responsible for extractingclinical data from patient records. IC and KJ served as clinical investigators and provided valuable comments on the final paper. SEKwrote the first draft of the paper and co-wrote the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Faced with new and ongoing threats to public health, researchers are becoming increasingly resourceful in their quest to discover new drugs. Drug researchers have long looked to living organisms for inspiration, either mimicking or extracting chemical formulas from naturally occurring compounds. Bacteria and fungi, for example, produce a wide range of compounds—some of which give them a selective advantage in their own environments—that provide important pharmaceutical activities. One class of these natural compounds are the polyketides, which make up a large portion of the antibiotics (including erythromycin and tetracycline) and antitumor drugs (such as doxorubicin and epothilone) that have been isolated from various microorganisms.Polyketides are synthesized by bacteria and fungi by the appropriately named polyketide synthases (PKSs). PKSs can be thought of as large molecular factories containing a series of enzymes working on an assembly line: each enzyme in the line adds molecules to a primer, or starter, unit—which is usually an acetate molecule—and then hands off the growing chain to the next enzyme. The specific enzymes set all the characteristics of the polyketide, including the chain length, the building blocks used, and the branching pattern of the molecules. Although microorganisms generate polyketides with a variety of characteristics, one goal of drug discovery research is to increase this diversity even further—a larger pool of polyketides promises more drugs with enhanced pharmaceutical applications.Early attempts at creating artificial polyketides focused on altering the functional characteristics of naturally occurring polyketides—the length of the chain, the building blocks, and the patterns of the branches. Chaitan Khosla and colleagues have taken this approach one very large step further. Rather than changing the machinery to modify the growing structure of a polyketide, they engineered bacteria to use an alternative, nonacetate primer molecule. This has important practical implications because some medicinally significant compounds do not use the usual acetate primer unit. By dissecting out the specificities of the “starter” and longer, multiunit “elongation” PKS enzymes and by mixing and matching modules, they have produced novel polyketide analogs (in this case, of anthraquinone) with more effective medically relevant properties. One of the compounds they engineered shows enhanced efficacy in blocking the growth of breast cancer cells that depend on the activity of the estrogen receptor, while a second polyketide inhibits an enzyme linked to adult-onset diabetes, demonstrating just two possible new therapeutic applications for synthesized polyketides. But, as the authors propose, this method promises to reveal new pharmaceutical agents that haven't even been discovered yet.


To examine the relationship between mitochondrial DNA (mtDNA) alterations and colorectal tumorigenesis, we used high-resolution restriction endonucleases and sequencing to assess the mitochondrial genome from three histologic subtypes of colorectal adenomas , colorectal cancer (CRC) tissues = 27, and their matched surrounding normal tissue (MSNT) = 52. The mitochondrial genomes were amplified using 9 pairs of overlapping primers and systematically analyzed by means of high-resolution analysis. DNA fragments showing a shift in banding patterns between the three adenomas, CRC, in comparison to the MSNT were sequenced to identify the mtDNA alterations. A total of thirty-eight germ-line mtDNA variants were observed in this study. Twenty-two of the thirty-eight were identified as mutations and 59% (13 of 22) were silent mutations and one was a 1-bp insertion. Sixteen of thirty-eight were distinct SNPs in flanking regions of the restriction sites and, 6 of the 16 (37%) SNPs were not previously reported. Most of these mutations/SNPs were homoplasmic and distributed in various regions of mitochondrial genes including the 16S and 12S rRNA. Based on our results, mtDNA germline variants increased in prevalence with adenoma CRC progression. To the best of our knowledge, this is the first report to show an increased prevalence of mitochondrial gene variants in CRC tumorigenesis. Colorectal polyps are a frequent occurrence in the general population and adenomatous changes in these polyps are associated with the overwhelming majority of CRC. These adenomas are the precursor lesions of colon cancer . CurrentThis study was approved by the Institutional Review Board of Morehouse School of Medicine and the Research Oversite Committee of the Grady Memorial Hospital, Atlanta, Georgia and the University of Alabama at Birmingham School of Medicine, Birmingham, Alabama. Primary fresh frozen and paraffin embedded tissues from 3 histologic subtypes of adenomatous polyps , 27 CRC tissues, and histologically MSNT, n = 52 were obtained from the Tissue Procurement Network at the University of Alabama at Birmingham and the Department of Surgery of Morehouse School of Medicine/Grady Hospital. Adenomas were defined by histologic type, degree of dysplasia, and presence of infiltrating adenocarcinoma in adenoma and classified as tubular, tubulovillus, and villous. CRC diagnosis was confirmed by histological examinations of biospied specimens for all patients and pathological tumor staging for these was based on American Joint Committee on Cancer [Genomic DNA was extracted from both frozen and paraffin embedded remnant tissues using Tri-Reagent (Molecular, Research Center Inc., Cincinnati, Ohio) and Purgene DNA purification kits (Gentra Systems, Minneapolis, Minnesota) according to the manufacture's protocol. The mtDNA from each tissue sample was amplified by PCR using nine overlapping mtDNA primer pairs as previously described ,11, whicThe analysis of mtDNA samples by restriction digestion and sequencing of the DNA fragments showed non-predicted banding patterns either as homoplasmic single bandshifts or heteroplasmic multiple bands on the gel. The PCR product data point in overlapping regions were counted only once. However overlapping regions were used as internal controls for the identification of the variants. Results of the high-resolution restriction analyses and sequencing of the entire mitochondrial genome yielded 38 sequence variants (including 16 SNPs from the flanking restriction sites) for the precursor adenomatous polyps and cancer tissues. In no instance was a variant detected in the adenomatous tissue found in the histologically matched adjacent surrounding tissue, indicating that these were germ-line origin.Val). However, one was a 9-bp Ins5892C in a non-coding region between MTTY and MTCOI. Some of these mutations are located in ATP synthase genes that are involved in mtDNA genome maintenance and integrity in yeast [Asp), 7335insC , G7256A , T7146A ), G8206A , A16325G (D-loop), T16309G (D-loop), G16294A (D-loop), G16266A (D-loop), G16233A (D-loop), 8269-9bp del (noncoding). Worth mentioning is also the high frequency of 16S rRNA gene variants (> 65%) in the mtDNA among CRC tissue versus the < 25% in the precancerous tissues. Mutations in the 16S rRNA gene were commonly found in different cancers, except those of the thyroid [Based upon our assessment of the mitochondrial DNA from non-cancer, precancerous (adenomatous polyps), and colorectal cancer tissues using a high-resolution restriction analysis, we have identified sequence variants. To our knowledge this is the first assessment of the mitochondrial genome using solely primary tissue rather than cell lines. This is important since tissues in culture can undergo clonal evolution which can distort frequency data. Although, the restriction analyses identified a number of band shifts indicating site gains or losses, these data only indicated that the predicted sequence had changed. They did not identify specific base pair changes. We therefore used the original primers and a series of nested primers to sequence the band shifts in order to identify these specific changes. A total of thirty-eight germ-line mtDNA variants were observed and all mutations/SNPs were considered as germ-line origin since the variants found in the colorectal adenoma polyps or cancerous tissues were also found in the MSNT tissues. Twenty-two of the thirty-eight were identified as mutations and 59% 13 of 22) were mostly silent mutations of T-to-C. or a G-to-A transition, which is consistent with the mutagenic spectra of oxidative, damage ,13. Amon of 22 wein yeast . Seventyin yeast . SixteenWe then sought to determine whether or not any of mtDNA variants observed in flanking regions of these restriction site variants may prove to be informative biomarkers. Although, a total of 38 of the mtDNA variants were found, none of the variants appeared to be a marker for a particular adenoma CRC tumor tissue type. Nevertheless, cumulative frequencies of mtDNA variants in the different tissue types resulted in a high prevalence of mtDNA sequence variants in CRC tissue and the trend was for the number of variants to be lowest in the precancerous Fig. , suggestThese data should be interpreted cautiously as they are based on small number of sample size. Similar studies have looked at precancerous tissue types , howeverMtDNA, mitochondrial DNA; ATP, adenosine triphosphate; CRC, colorectal cancer; MSNT, matched surrounding Normal tissue; SNPs single nucleotide polymorphisms; PCR polymerase chain reaction.FOA performed few assays while MK performed almost all assays. DC provided the statistical support. JO provided some tissue samples and suggestions for finalization of the manuscript. RG provided some technical supervision to MK and suggestions for finalization of the manuscript. All authors read and approved the manuscript.


DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation.We have developed a rapid method for implementing the self-assembly of DNA within a microfluidic system by electrically extracting the DNA from an environment containing an uncharged denaturant. By controlling the parameters of the electrophoretic extraction and subsequent analysis of the DNA we are able to control when the hybridisation occurs as well as the degree of hybridisation. By avoiding off-chip processing or long thermal treatments we are able to perform this hybridisation rapidly and can perform hybridisation, sizing, heteroduplex analysis and single-stranded conformation analysis within a matter of minutes. The rapidity of this analysis allows the sampling of transient effects that may improve the sensitivity of mutation detection.We believe that this method will aid the integration of self-assembly methods upon microfluidic chips. The speed of this analysis also appears to provide information upon the dynamics of the self-assembly process. There has been a rapid growth in the number of applications that are based upon DNA self-assembly, ranging from DNA microarrays (e.g. Affymetrix ) in the Microfluidic devices or 'microchips' are photolithographically-defined networks of microchannels in glass where the microchannels are similar in size to conventional capillaries. These microchips provide compelling advantages in terms of speed, reagent usage and integration over conventional capillary or gel-based methods. The potential of the microfluidic chip has led to the use of terms such as "micro-total analysis systems" and "lab-on-a-chip". These microchips have been demonstrated in conjunction with a range of applications that integrate the polymerase chain reaction (PCR) and capillary electrophoresis (CE) methods with some reaching nanolitre or smaller scale volumes. A powerful advantage of the microchip approach is that it can implement much the same molecular biology protocols and reagents as used with conventional equipment, thereby allowing a wealth of established expertise to be transferred to the microscale.Although the most effective method of mutation detection is sequencing, it is also by far the most expensive . The micMicrofluidic chips may enable extremely high throughputs and high levels of integration. The achievement of this goal has been hindered by the lack of successful integrations of methods of mutation analysis based on single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) – likely due to the difficulties in controlling the degree of hybridisation on chip without time consuming thermal processing. A great advantage would be provided by a method of enabling microchip-based control of a rapid DNA self-assembly process.The term wildtype is used to describe any given genetic sequence that does not contain mutations. Since individuals usually carry two copies of each gene, the genetic sequence of the two copies may be identical (homozygous) or may differ (heterozygous). DNA is normally double stranded, but under some conditions (e.g. high temperature), melts into single strands. Under other conditions, such as a lower temperature, these single strands will self-assemble into the double-stranded form again. The resulting double-stranded DNA is referred to as a homoduplex if the sequences are perfectly complementary, or a heteroduplex if the sequences are nearly complementary (i.e. a mutant sequence paired with a wildtype sequence). The misfit in a heteroduplex creates a "bulge" or "bubble" where the bases do not match and this affects the shape of the assembled molecule, typically lowering its velocity during electrophoretic movement, i.e. the heteroduplexes typically migrate more slowly than the homoduplexes. Any heterozygous sample will generate four different duplexes, two homoduplexes and two heteroduplexes, although the molecules often co-migrate so that fewer than 4 separate electropherogram peaks are resolved.In the HA method, electrophoretic conditions are chosen in order to enhance the velocity differences between the duplexes so that the process of duplex self-assembly can be used to determine the presence of a heterozygous state (hence indicating the presence of a mutation). In SSCP, isolated strands of ssDNA find near-complementary sequences on the same strand, with the result that the strand folds upon itself in a sequence dependent manner forming new conformations. This is a simplistic description since ssDNA without self-similar sequences, and homoduplex dsDNA, may also take complex forms. Techniques such as HA that aim to separate homoduplex fragments from heteroduplex fragments often use some combination of thermally and chemically denaturing conditions to cause the partial melting of the duplex, resulting in a shift in mobility or chromatography column retention time that increases with the degree of mismatch.et al. [et al. achieved 100 % sensitivity by analysing denatured and non-denatured fragments in tandem. Despite being highly effective, the additional sample preparation required by these methods (i.e. separately preparing both single and double stranded DNA and maintaining this strandedness) complicates their implementation on microchips.Many medical diagnostics could be implemented on microchips if an effective implementation of a highly sensitive mutation analysis method could be integrated with PCR/CE. Considerable work has been done in developing SSCP  and HA ,3,12. Anet al.  greatly In this work, we present an electrophoretic method in which DNA is denatured in a microchip (with formamide) and, depending upon the sequence of applied voltages, can be prepared with a widely varying degree of hybridisation . Given the small volumes involved within the microchip, diffusion time plays a small role and the reassembly process can be fast, with dsDNA obtained within minutes. The rapidity of the manipulation possible on this system allows some investigation of the dynamics of the reassembly, indicating that there are well-defined intermediate states where both ssDNA and dsDNA exist in the reassembly process.We have applied our methods to the H63D and S65C mutations from the HFE gene associated with hereditary hemochromatosis (HH). The denaturation technique used enables a combined microchip-based method of HA and SSCP analysis.In our electrophoretic analyses with a double-T chip (described below), the dsDNA arrives at the detection point before the ssDNA (after about 105 s of separation, versus 190 s of separation for the ssDNA). As shown in Fig. As expected, after the addition of formamide the electropherograms showed the presence of ssDNA peaks in addition to the dsDNA peaks Fig. . The dsDIn order to confirm that we are reassembling DNA on chip rather than denaturing to varying degrees we investigated the on-chip production of heteroduplexes from two samples of homoduplexes (i.e. homozygous) samples. Fig. The reassembly of ssDNA can also be shown by denaturating the wildtype and homozygous mutant in separate wells. The two denatured samples were injected simultaneously and their ssDNA mixed in the injection channel of a Y-chip (described below). Subsequent separation and detection showed peak profiles Fig.  similar As will be described in the following section, by varying the electrophoretic parameters we can control the relative amount of dsDNA formed – a significantly larger amount could be obtained.After demonstrating that reassembly occurred within the microchannels after extraction from the formamide-rich sample well, it was of interest to investigate how the sequence and timing of the sample extraction and analysis might affect the degree of rehybridisation. In the work presented thus far we used a 60 s injection  as a means of drawing sample directly from the sample well to the intersection, from whence it could be analysed.After the addition of formamide and an analysis of the resulting sample (60 s injection and 180 s separation), a series of analyses were performed wherein each short injection (10 s) with a lower electric field was followed by a 180 s separation. These short injections sampled DNA that had remained in the microchannel since its extraction during the 60 s injection from the first analysis. The time required for the DNA to travel from the sample well to the intersection with the applied electric field during the 10 s short injections was calculated to be approximately 53 s.  Thus, the two short injections of 10 s each were not enough to bring in fresh samples from the sample well. Fig. Another interesting feature of Fig. et al. [et al. [The integration onto a microchip of an effective means of mutation detection is perhaps one of the most important technological barriers to the implementation of microchip-based medical diagnostics. The best means of attaining sufficiently high sensitivity is by integrating several existing methods of microchip-based mutation detection. The capillary-based analysis procedure developed by Kourkine et al.  is likel [et al.  – througAs demonstrated here, this method also allows on-chip comparisons of one type of DNA with another. A common problem encountered with HA methods is that they cannot distinguish homozygous mutant from homozygous wildtype – the present technique would allow an on-chip comparison of these samples to produce heteroduplexes that will then indicate the mutational status.The on chip denaturation is produced through the addition of formamide. The melting temperature for this sequence of DNA following the addition of formamide was found to be approximately room temperature, as determined bym = 81.5 + 16.6(log M) + 0.41 (% G + C) - 0.72 (% formamide)     (1)Tm is the melting temperature in degrees Celsius, M is the monovalent salt molarity, (% G + C) is the percent of the guanine and cytosine in the DNA strand of interest, and (% formamide) is the percentage of formamide added [where Tde added . The melThe ability to quickly re-hybridise on chip allows for rapid investigation of self-assembly mechanisms. In addition, this re-hybridisation enables the formation of duplexes made from a sample and a set of DNA references – i.e. DNA self-assembly within a microchip could be used to form duplexes that, under electrophoretic analysis, would show the results of comparing the sample DNA with each type of DNA in the reference set. This could avoid the need for DNA sequencing.The rapidity of our method appears to provide additional information upon short-lived conformations. Although we have added a thermal re-annealing step as part of our PCR protocol, that step does not affect the results of analysis after adding formamide – i.e. by re-annealing on-chip the thermal reannealing is not needed. The thermal re-annealing stage was added to allow the direct comparison of heterozygous samples from the PCR with heterozygous samples after on-chip reassembly. After adding formamide, the electropherogram of the first separation analysis following any long injection shows a clearly defined transient peak. For H63D samples the transient peak is a single peak, whereas for S65C the transient peak is clefted. We have found that the transient peaks vary in size significantly depending upon the electrophoretic and PCR protocols used. Initially we had assumed that this transient peak indicated that the reassembly of the DNA was not 'random' but instead hybridised first in a high-melting point region, and only slowly thereafter. In this model, the presence of the split-peak would provide information upon the location of the mutation. This suggests that mutation S65C is within the higher melting point domain, while the H63D is not. However, as determined by the Meltmap program (generously provided by L. Lerman (MIT)), neither the H63D nor S65C mutations were within the high melting point region of the exon (data not shown).et al. [et al. [Several research groups have reported artefacts that arise from ssDNA-primer interactions ,17-19. Ket al. ,18 reporWe therefore suggest that the transient peak is due to the pairing of one product strand with one primer as a result of the renaturation process. The primer-ssDNA complex is primarily ssDNA with a small region of dsDNA at the end(s) of the strand. It is therefore expected to migrate with similar mobility as the ssDNA peaks. The disappearance of the transient peaks with the subsequent short injections may be a result of the complementary single strand binding and displacing the primer. However, the presence of the transient peaks may still provide useful information. The differences in the transient peaks (cleft versus no cleft) between S65C and H63D suggest that their shape may be dependent on the position of the mutation and that the position greatly affects the transport of the transient form of DNA. Thus, the phenomenon of the transient may be a general behaviour that could provide additional mutational information. In corroboration of past work by others ,19, it tWe have developed a method of rapidly disassembling and re-assembling DNA within a microfluidic chip, allowing us control over the relative amount of ss and dsDNA and enabling the performance of rapid hybridisations under electrophoretic control. It has been reported that, when combined, HA and SSCP can provide sensitivities of 100% (e.g. ). In ourThe present work is also an early step towards directing and studying DNA self-assembly within microfluidic systems. The method applied here could be improved significantly by shortening the injection and separation channels and ultimately may even assist in providing the control needed to direct the assembly of DNA-based nanosystems within microfluidic channels.Volunteers who had given informed consent donated lymphocytes from which DNA was extracted and purified by using phenol-chloroform-isoamyl alcohol extractions  or the Q2, 2.5 μL of 10× PCR reaction buffer and 0.5 μL of Platinum Taq DNA Polymerase. All samples were re-annealed following PCR by first heating at 95°C for 3 min, followed by a subsequent ramping down of temperature by 1°C per minute until 65°C. The samples were then stored at -20°C.The two mutations tested were H63D and S65C, from HFE Exon 2. PCR was performed on 25 μL reactions of both mutations. Thermal cycling was performed on all the samples as follows: 94 C for 2 min, 35 cycles of (94°C for 30 s, 55°C for 30 s, 72°C for 30 s), and finally 72°C for 10 min, 4°C thereafter. For H63D and S65C, the PCRs are performed with 5 μL of 30 ng/μL of genomic template DNA, 2 μL of 5 μmol/L each of HEX-HFE-2F primer and H63DR primer (Table PCR reagents (polymerases, buffers and primers) were obtained from Invitrogen (Burlington, ON). GeneScan™ polymer was used for microchip electrophoresis and obtained from PE Applied Biosystems (Foster City, CA). A polymer consisting of 5% GeneScan polymer and 10% glycerol (5GS10G), commonly used for SSCP, was made. Tris borate (Fisher Scientific, Fairland, NJ) with EDTA (Merck KGaA, Darmstadt, Germany) was used as the running buffer in concentrations of 1× and 0.1×. Glycerol (Sigma, Saint Louis, MO) is also added to each in 10% and 1% concentrations respectively (1 × TBE10G and 0.1 × TBE1G). De-ionised formamide (minimum 99.5%) was obtained from Sigma (F9037, Saint Louis, MO). The formamide was aliquotted and kept frozen until required.The microchips were purchased from Micralyne (Edmonton, AB) and unless otherwise mentioned were a 4 port double T design Fig.  consistiThe microchip was loaded with 5GS10G polymer without any pre-treatment. The sample well was loaded with 2.6 μL of 0.1 × TBE1G followed by 0.4 μL of DNA sample and mixed. The remaining wells were loaded with 3 μL of 1 × TBE10G. In the case of the Y-chip, 0.4 μL of wildtype DNA was added to 2.6 μL of 0.1 × TBE1G in the first sample well and mixed. The second sample well was filled with 2.6 μL of 0.1 × TBE1G and 0.4 μL of homozygous mutant. The operation of the μTK (injection and separation) was automated through the use of the LabVIEW interface. LIF detection took place 76 mm downstream from the intersection. We have found that the reproducibility of the peak arrival times is within 2 per cent from one run to the next. As such we have not needed to introduce size standards.The sample DNA was brought from the sample well to the intersection and onto the sample waste well by applying 500 V/cm for 60 s. No initial injection was done with the Y-chip prior to denaturation. During this process the buffer and buffer waste well are left electrically disconnected. In doing so the intersection of the two (three) channels is filled with the sample DNA. This stage is referred to as an injection due to the injection of DNA into the separation channel in the sharply defined volume of the intersection of the channels.Immediately following injection, the DNA caught within the intersection is separated by applying 714 V/cm for 180 s between the buffer and buffer waste wells. During this step, the sample and sample waste wells are left electrically disconnected. The effective separation distance was 76 mm from the intersection.et al. [After the initial run on the 4-port chip, 1.5 μL of the sample mixture was removed and 1.5 μL of formamide was added and mixed. Following Howley et al. , this isYZ performed the experimental work with some assistance from TF. DM performed additional protocol development. CB provided overall direction. All authors contributed to the writing of the manuscript and all made substantial contributions to the work.


A new funding body (the EDCTP) will fund clinical trials in developing countries, particularly in Africa, that help to develop affordable interventions against HIV, TB, and malaria. How is it doing so far? A promising European funding body is stumbling over the details Last year the European Parliament and Council formed the european and Developing Countries Clinical Trials Partnership (EDCTP). The aim of this new funding body, which has a budget of €400 million spread over five years, is a noble one: to fund research in developing countries, particularly in Africa, that contributes to the development of affordable prophylactics and drugs for HIV/AIDS, tuberculosis, and malaria.Unfortunately, the organization has not got off to an auspicious start. Its executive director, Piero Olliaro, was ousted from power at the first EDCTP annual forum at the end of September. There have been rumblings of discontent among grant applicants who say that the first round of grant assessments was administered poorly. And not-for-profit organizations that would like to partner with EDCTP have been left in the dark regarding whom to speak to at the organization.This omission is significant because partnership is one of the key tenets of the EDCTP. European research agencies are slowly beginning to realize that they need to cooperate with each other if they are to be competitive with the United States. The history of many European countries is such that Europe has much stronger ties with Africa than does the United States, so it makes political sense for the European Union to fund research that provides a springboard for European researchers to compete effectively with US scientists.Crucially, the EDCTP was also set up to enable European and African scientists to work together as equal partners. There is increasing recognition that the paternalistic, colonial attitude that pervaded “tropical medicine” in the past just will not do. The EDCTP hoped to change that by having a Partnership Board that contains equal numbers of African and European representatives. However, the EDCTP Assembly, which contains a representative from each of 14 EU member states but none from African countries, has the power to veto the decisions of the Partnership Board, which is supposedly the scientific decision-making authority.Doing clinical trials in Africa is far from easy. There are too few adequately resourced research centers, and those that do consistently perform well are oversubscribed. Therefore, there is a clear need for "capacity building"—development of a research infrastructure, in terms of both equipment and personnel, that is capable of coping with the challenges of clinical trials. The EDCTP hopes to contribute to this essential endeavor by funding clinical trials that are sustainable in the long term. In particular, it believes that the best way to train a new generation of African scientists is by teaching them on the job, that is, involving them fully in the planning and execution of the trial, rather than flying in European experts who leave as soon as the trial is finished.A commitment from European researchers to be engaged for the long term is essential for the success of these projects. In addition, partnerships need to be brokered with national programs in Africa to ensure that the new capacity can be sustained over time. The end goal is to produce centers of excellence that are run by Africans doing internationally recognized research that conforms to Good Clinical Practice guidelines. But this will only happen if African researchers are treated as equal partners and are allowed to be fully engaged in the projects that are taking place in their countries.So can the EDCTP work, or is it doomed to failure? In many ways the organization has a great deal going for it. Although the budget of 3400 million spread over five years is tiny considering the combined burden of HIV/AIDS, tuberculosis, and malaria, it is important to remember that it is the biggest single European project for clinical trials in Africa. In many ways the EDCTP is a demonstration project: if some success can be achieved it is very likely that additional funds will follow. The project is certainly strengthened by the involvement of Pascoal Mocumbi, the former prime minister of Mozambique, as High Representative of the project. Mocumbi is highly respected by the global-health community and carries considerable weight with African politicians. Mobilization of political will within Africa will be essential if research capacity is to be sustained for the long term.On the downside, it seems clear to most insiders that the management structure needs to be radically changed and partnership with other organizations needs to be improved. The EDCTP Assembly met on October 28 and 29 to discuss these issues and to elect a new leader. At the time this editorial went to press, there was still no public announcement of the outcome of this meeting. In addition, the political infighting that pervades European politics at all levels needs to be controlled, or at least managed effectively. This might be a tall order, but it is essential if this worthwhile and high-profile project is to succeed.


The neurophysiological and neuroanatomical foundations of persistent developmental stuttering (PDS) are still a matter of dispute. A main argument is that stutterers show atypical anatomical asymmetries of speech-relevant brain areas, which possibly affect speech fluency. The major aim of this study was to determine whether adults with PDS have anomalous anatomy in cortical speech-language areas.Adults with PDS (n = 10) and controls (n = 10) matched for age, sex, hand preference, and education were studied using high-resolution MRI scans. Using a new variant of the voxel-based morphometry technique (augmented VBM) the brains of stutterers and non-stutterers were compared with respect to white matter (WM) and grey matter (GM) differences.We found increased WM volumes in a right-hemispheric network comprising the superior temporal gyrus , the inferior frontal gyrus (including the pars triangularis), the precentral gyrus in the vicinity of the face and mouth representation, and the anterior middle frontal gyrus. In addition, we detected a leftward WM asymmetry in the auditory cortex in non-stutterers, while stutterers showed symmetric WM volumes.These results provide strong evidence that adults with PDS have anomalous anatomy not only in perisylvian speech and language areas but also in prefrontal and sensorimotor areas. Whether this atypical asymmetry of WM is the cause or the consequence of stuttering is still an unanswered question. Persistent developmental stuttering (PDS) is a relatively severe disturbance characterized by involuntary, audible or silent, repetitions or prolongations of sounds or syllables. These are not readily controllable and often are accompanied by other movements and by negative emotions ,2. DevelSeveral experimental studies have shown that stutterers reveal prolonged manual and vocal reaction times to simple and complex verbal and nonverbal stimuli -10, reduAccording to the present literature four anatomical studies revealed brain abnormalities in stutterers compared to controls. The earliest study examined two left-handed stuttering siblings using CT and revealed an atypical (reduced) anatomical asymmetry of the occipital poles . The firAlthough the aforementioned anatomical studies have focussed on the anatomical foundations of stuttering, several questions are not answered yet. Therefore we re-examined the hypothesis of anatomical differences between stutterers and non-stutterers using voxel-based morphometry (VBM). This approach circumvents the problem of analyzing predetermined regions of interests by analyzing stereotactically normalized brains on a voxel-by-voxels basis with respect to differences in the volume of white matter (WM) or grey matter (GM) ,38. ThisThe sample included adults with PDS (n = 10) and controls (n = 10) matched according to sex, age and education. All subjects were consistent right-handers (CRH) according to the Annett handedness questionnaire (AHQ) [66]. Our sample contained the approximate sex distribution as those reported in population studies of adults who stutter; thus, there were more men (n = 8) than women (n = 2) in this sample. The dysfluent sample was limited to adults with PDS who had been diagnosed with developmental stuttering before the age of 8 years and had undergone treatment at some point, but continued to be dysfluent. None of the subjects was taking centrally acting medications that could have resulted in PDS, and all met the clinical criteria of developmental stuttering – not acquired stuttering. Of the adults who stuttered, 50% had a family history of stuttering; none of the controls had a family history of stuttering. Stuttering severity was determined using the Stuttering Severity Inventory (SSI) [67] with individuals in the sample ranging from mild (2), moderate (7) to severe (1). All participants were native German speakers with no reported history of dyslexia, specific language impairment, attention deficit disorder, traumatic brain injury, substance abuse, or other neuropsychiatric conditions. All participants gave informed consent before participating.) [71]. Preprocessing were guided by the VBM method proposed by Godd et al. [3. The normalized scans were then segmented into grey (GM) and white matter (WM), cerebro spinal fluid (CSF), and other non-brain partitions applying the algorithmus implemented in SPM99 based on the algorithms developed by Ashburner and Friston [We used a Siemens 1.5 T magnet and a 22-min fast-low-angle-shot MR sequence yielding 128 contiguous sagittal slices with 1 × 1 × 1.17 mm image voxel size [68–70]. Data were analyzed on a PC workstation using MATLAB 5.3 (MathWorks, Natick, MA) and SPM 99 .The normalized, smoothed, segmented (and modulated) data were analyzed using statistical parametric mapping (SPM99) employing the framework of the General Linear Model. Regionally specific differences in GM (and WM)  between groups were assessed statistically using a two-tailed contrast. Corrections for the search volume (and implicit multiple comparisons) in terms of the P values were made using Gaussian random field theory, which accommodates spatial correlations inherent in the data and is now established as the conventional approach to inference in smooth spatially extended data. We restricted the search volume to the GM or WM volume enabling us to increase the statistical power of statistical testing. Significance levels for two-sided T statistics were set at T = 5 (corrected for multiple comparisons across the WM or GM volumes) and a spatial extend criterion of k = 50. The spatial extend of k = 50 was introduced because this volume size roughly corresponds to the size of a meaningful anatomical area  including the planum temporale, the precentral gyrus (PrCG), the inferior frontal gyrus (IFG) comprising the pars opercularis (POP), and the middle frontal gyrus (MFD) Table . There w3). The placement pf these ROIs were guided by anatomical landmarks and published probability atlases of the HG and PT [2 = 0.62) and a significant interaction between both factors (F(1, 18) = 31.6, p <= 0.001, ETA2 = 0.64). Subsequent Scheffé contrasts and Figure 2 = 0.34; Group: F(1, 18) = 23.6, p <= 0.001, ETA2 = 0.58) and a significant interaction (F(1, 18) = 23.9, p <= 0.001, ETA2 = 0.57). The strong interaction is qualified by a between-hemisphere difference found for stutterers (with larger WM volumes on the right compared to the left IFG) while there is no between-hemisphere difference in non-stutterers. For the PrCG we found a significant between-group difference (F(1, 18) = 11.1, p = 0.004, ETA2 = 0.38) and a significant interaction (F(1, 18) = 31.0, p <= 0.001, ETA2 = 0.64). The pattern of this interaction resembles the interaction found for the IFG with larger WM volumes on the right hemisphere for stutterers than on the left while non-stutterers show similar values for both hemispheres. For the MFG all two main effects as well as the interaction were strongly significant (Hemisphere: F(1, 18) = 23.5, p <= 0.001, ETA2 = 0.56; Group: (F(1, 18) = 13.2, p = 0.002, ETA2 = 0.42; interaction: (F(1, 18) = 18.4, p <= 0.001, ETA2 = 0.50). The interaction is due to the fact that stutterers revealed larger WM volumes on the right compared to the left hemisphere.In order to understand the differences between stutterers and non-stutterers with respect to the WM volumes in these anatomical areas more precisely, we placed regions of interest (ROI) in these anatomical areas and the homotopic areas on the left hemisphere. For the auditory cortex we used a rectangular ROI including Heschl's gyrus (HG) and the planum temporale (PT) (size of the ROI on both hemispheres: 8.4 cmG and PT ,49. The In addition, we did not find any correlation between the stuttering severity measures (SSI measures) and the anatomical peculiarities neither in the context of the VBM nor the ROI analysis.This study was motivated by the question whether stutterers reveal morphological brain anomalies compared to non-stuttering controls. In fact, we found prominent increases of WM in stutterers within a right-hemispheric network including brain structures relevant for language and speech. These areas comprise the STG (including the auditory areas PT and HG), the IFG (including the pars opercularis which is part of Broca's right-sided homologue), the somatosensory area , and the middle frontal gyrus (MFG). Our findings of regionally increased right-hemispheric WM in stutterers might suggest an increased and possibly atypical intrahemispheric communication within these areas via association fibres ,51 possiThree of the brain areas with different WM composition in stutterers are known to be involved in different speech and language functions. For example, the right IFG (including the pars opercularis) is involved in the perception and generation of phonological or prosodic speech features -55 whileThe auditory cortices in the two hemispheres are relatively specialized in normal subjects ,57. ThusThe reported morphological features complement previous morphological studies comparing stutterers and non-stutterers. Firstly, this study shows again that stutterers reveal atypical anatomical lateralisation in speech-relevant areas. In three areas (PrCG, MFG, and IFG) stutterers reveal more WM volumes on the right than on the left. For the auditory cortex (STG) we found symmetric WM volumes while non-stutterers typically show a leftward asymmetry for this measure. Thus, some kind of hemispheric imbalance seems to be related to persistent developing stuttering. Secondly, using a different method than Foundas et al. , we alsoThese results provide strong evidence that adults with PDS have anomalous anatomy not only in perisylvian speech and language areas but also in prefrontal and sensorimotor areas. These anatomical features might indicate a deficiently working speech system. Whether this atypical asymmetry of WM is the cause or the consequence of stuttering is still an unanswered question.The author(s) declare that they have no competing interests.L.J., J.H. and H.S. conceived the experiment and drafted the manuscript. L.J. and J.H. prepared the exact experimental setup. H.S. supervised data acquisition. J.H. and L.J. performed all data and statistical analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Alu elements are short (~300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30–50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation.Alu elements from the Ye subfamily were extracted from the draft sequence of the human genome. Analysis of these elements resulted in the discovery of two new Alu subfamilies, Ye4 and Ye6, complementing the previously described Ye5 subfamily. DNA sequence analysis of each of the Alu Ye subfamilies yielded average age estimates of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively. In addition, 120 Alu Ye4, Ye5 and Ye6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity.A total of 153 Alu Ye lineage appears to have started amplifying relatively early in primate evolution and continued propagating at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Detailed sequence analysis of several Alu pre-integration sites indicated that multiple types of events had occurred, including gene conversions, near-parallel independent insertions of different Alu elements and Alu-mediated genomic deletions. A potential hotspot for Alu insertion in the Fer1L3 gene on chromosome 10 was also identified.The  Alu elements has had a significant impact on the architecture of primate genomes [Alu elements have achieved this copy number by duplicating via an RNA intermediate in a process termed retroposition [Alu elements are thought to borrow the factors that are required for their amplification from the LINE (long interspersed element) elements [Alu repeats have been a significant source of new mutations as a result of insertion and post-integration recombination between elements [The proliferation of  genomes . They co genomes . Alu eleposition . During position -6. Whileelements , which eelements ,11. Becaelements ,13.Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that appears to have occurred 30–50 million years ago [Alu elements within the human genome, only a small number of these elements are capable of movement [Alu elements, a series of discrete subfamilies of Alu elements that share common diagnostic mutations have been identified in the human genome [Alu repeats are so recent in origin that they are present in the human genome and absent from the genomes of non-human primates, with some of the elements being polymorphic with respect to insertion presence/absence in diverse human genomes [Alu subfamilies which arose around the radiation of Subtribe Hominina (gorillas, chimpanzees, and humans) four to six million years ago [Alu elements as phylogenetic markers [The majority of ears ago ,14-16. Emovement . As a ren genome -21. A sm genomes -25. Indi genomes -34. For ears ago  were useears ago . Relatio markers ,37-40Alu elements found in the human genome that fall in six distinct lineages, termed Ya, Yb and Yc, Yd, Yg and Yi based upon their diagnostic mutations [Alu subfamilies that are members of the Alu Ye lineage [We have previously characterized a large number of recently integrated utations -52. Here lineage  and are Alu Ye elements were identified in the draft sequence of the human genome using BLAST [Alu Ye specific oligonucleotide . Seventeen of the 25 Alu Ye4 elements were unique . There were also 76 unique Ye5 Alu elements and 23 unique Ye6 Alu subfamily members. Multiple alignments of the Alu elements from each subfamily were constructed and the number of mutations from the consensus sequence for each Alu subfamily was determined. In each case the mutations were divided into those that occur at CpG dinucleotides and those that occur at non-CpG positions without including small insertions or deletions as described previously [To estimate the copy number of the Ye4, Ye5 and Ye6 eviously -49. The eviously  as a reseviously .Alu Ye subfamily. For 17 elements from the Alu Ye4 subfamily, the non-CpG and CpG mutation densities were 2.1% (83/3944) and 12.5 % (106/850). Using a neutral rate of evolution of 0.15% per million years for non-CpG positions [Alu Ye5 subfamily 76 elements were analyzed that contained a total of 17632 non-CpG nucleotides and 3800 CpG nucleotides that contained 351 non-CpG and 431 CpG mutations. The mutation densities of the Ye5 subfamily were 1.99% and 11.34% for the non-CpG and CpG nucleotides yielding age estimates based on the average mutation density of 13.27 and 12.60 million years old. For the Alu Ye6 subfamily 23 elements were analyzed that contained a total of 5336 non-CpG nucleotides and 1150 CpG nucleotides that contained 86 non-CpG and 92 CpG mutations. The mutation densities of the Ye6 subfamily were 1.61% and 8% for the non-CpG and CpG nucleotides yielding age estimates based on the average mutation density of 10.75 and 8.89 million years old.Mutation densities were calculated for each ositions  and 0.9%ositions  along wiAlu Ye4, Ye5 and Ye6 subfamily member, we performed a series of PCR reactions using human and non-human primate DNA samples as templates. Unfortunately, not all of the loci identified in the draft sequence were amenable to PCR analysis, as some of them had inserted into other repetitive regions of the genome making the design of flanking unique sequence PCR primers difficult.In order to determine the approximate time of insertion for each Alu repeats beginning with some elements that are recent in origin and unique to the human genome (e.g. Ye5AH110) and ending with elements that are found within all ape genomes (e.g. Ye5AH148). The distribution of all the Ye elements in various primate genomes is summarized in Additional File 2.For the Ye subfamilies, 120 of the 153 elements identified in the draft human genomic sequence were amplified by PCR. Examination of the orthologous regions of the various species genomes displayed a series of different PCR patterns indicative of the time of retroposition of each of the elements into the primate genomes. Results from a series of these experiments showed a gradient of Ye Alu elements and in other regions of the human genome exerts a significant influence on the accumulation of single nucleotide diversity within the human genome [Alu Ye subfamily members, we compared the sequences of the elements found in the human genome to the consensus sequences of other Alu subfamilies. Using this approach, we identified two Alu Ye5 subfamily members that appeared to have been subjected to partial gene conversion at their 3' ends. Alu Ye5AH70 contains three mutations that are diagnostic for the Yb8/9 subfamily. Similarly, Alu Ye5AH173 contains three Alu Sc mutations. Each of the sequence exchanges occurred in a short contiguous sequence suggesting that they were products of gene conversion rather than homoplasic point mutations.Gene conversion between n genome ,50. To eAlu-containing locus that was involved in full gene conversion/ replacement event, (Ye5AH181). In this case, the orthologous Alu elements have similar flanking sequences and direct repeats, although they are not precisely identical due to the random mutations that accumulated over time. DNA sequence analysis of this locus showed that the Alu element of selected new world monkey genomes (spider monkey, woolly monkey and tamarin) belonged to the Alu Sg subfamily. This suggests that a gene conversion of an older, pre-existing Alu Sg may have introduced the Ye5 sequence in the common ancestor of humans, chimpanzees, gorillas and orangutans. Amplification of this locus was unsuccessful in the old world monkey taxa tested.We identified one Alu Ye elements. These deletions were identified at loci Ye5AH24 and Ye5AH27. In the case of Ye5AH24, the deletion was associated with a gene conversion of an Alu Y in both orangutan and siamang to AluYe5 in human, bonobo, common chimpanzee and gorilla and involved the removal of about 500 bp from the 3' flanking region. For Alu Ye5AH27, the deletion was associated with a gene conversion of an Alu Sx element (orangutan and siamang) to AluYe5 (human, bonobo, common chimpanzee and gorilla) and involved the removal of 142 bp from the 3' flanking region. Based on this data, we estimate the frequency of Alu retroposition mediated deletions of approximately 1.67% (2/120).Two deletions of part of the human genome appeared to be associated with newly inserted Alu integration. If a deletion occurred during the integration of an Alu element in the human genome, then the pre-integration product size calculated computationally would be an underestimate of the true size of the locus. To investigate this possibility, we utilized long template PCR reactions of these loci that would facilitate the amplification of larger (up to 25 kb) products. Unfortunately, PCR amplicons were not generated by any of these loci, suggesting that the retrotransposition of these Alu elements in humans may have generated deletions greater than 25 kb in size. Alternately, the orthologous loci in non-human primate genomes may have undergone additional mutations at the oligonucleotide primer sites, preventing PCR amplification.The pre-integration sites for three elements (Ye5AH11, Ye5AH40 and Ye5AH173) did not amplify in any non-human primate species. Previously, the insertion of L1 elements has been shown to be associated with large genomic deletions . Thus, oAlu insertions in human, chimpanzee, gorilla lineage, old world monkey and new world monkey lineages , an Alu Sp has inserted in the 5' flank of the shared Sx element about 58 bp away of the Alu Ye5 present in Hominina. Also, in the woolly and spider monkeys (new world monkeys), there was an independent insertion of an Alu Sx in the 5' flank of the shared Alu Sx. In gibbon, siamang and orangutan, there were no independent Alu insertions at this locus, only the common Alu Sx is present. In orangutan, however, there was an extra 145 bp of genomic sequences inserted inside the old Alu Sx. The pattern discussed suggests that these three independent parallel insertion events occurred sometime after the divergence of these primates from one another. This locus on chromosome 10q23.33 lies in intron 39–40 of the Human Fer1L3 gene, about 50 bp from exon 39. This locus may be considered a hot spot for Alu insertion. An alignment of locus Ye5AH161 is available as .We have also identified one locus (Ye5AH161) that contained multiple paralogous ges Fig. . In the Alu insertion event at human Ye5AH16 locus in all the old world monkey genomes tested (Green monkey, Macaque and Rhesus), within the same locus where an Alu Ye5 element was located in the human, chimpanzee, gorilla and orangutan genomes. Thus, the near-parallel insertion most likely occurred after the divergence of humans and apes from old world monkeys, but before the radiation of the old world monkeys. The element present in the old world monkey genomes is an Alu Y and is 80 bp from the human insertion site.We also identified another near-parallel independent Alu Ye4, Ye5 and Ye6 subfamily members, we performed a series of PCR reactions on a collection of 80 geographically-diverse human genomes. Using this approach, we identified one new Alu insertion polymorphism (Ye5AH167) from the loci analyzed in this report. The allele frequencies, genotypes and heterozygosities for the Alu insertion polymorphism are shown in Table To determine the human genomic diversity associated with each of the ism Ye5AH67 from tAlu Ye5 subfamily resulted in the recovery of two new Alu subfamilies, Ye4 and Ye6. Each of these Alu subfamilies has a relatively small copy number in the human genome. The proportion of polymorphic elements within each of the subfamilies is quite low with only 0.83% of the Alu Ye elements being polymorphic, only one member of Ye subfamilies (Ye5AD167) is polymorphic with respect to insertion presence/absence in the human genome. In contrast, many other young Alu subfamilies have levels of insertion polymorphism in excess of 20% [Alu subfamilies within the human genome has occurred at a very low rate, and may have recently ceased entirely. The estimated average ages of ~14, ~13 and ~9.5 million years old for the Alu Ye4, Ye5 and Ye6 subfamilies, respectively are consistent with their relatively recent origin in primate genomes. It is also consistent with the master gene model of SINE retroposition which suggests that as a master element accumulates mutations over time, the resulting elements will share those mutations [Our detailed analysis of the utations .Alu Ye lineages are dispersed throughout the genomes of all hominoids (humans, greater and lesser apes) suggesting that this subfamily of Alu elements began to amplify about 15–20 million years ago. Therefore, the Ye subfamily appears to have been retroposition competent during hominoid evolution, but must have been relatively inefficient at producing copies. Although the rate of Ye amplification has not been dramatic within the human lineage, it may be quite interesting to recover Alu Ye subfamily members from other ape genomes and to determine the rate of Ye subfamily amplification in these genomes to see if there has been any differential amplification of these elements in non-human primate genomes. The differential amplification of ID SINEs within various members of the rodent lineage has been reported previously suggesting that the amplification of SINEs within various genomes is subject to changes [Members of the  changes ,62.Alu repeats has been reported previously [Alu Ye subfamily members were quite interesting. In one case (Ye5AH181), the Alu-containing locus was involved in full gene conversion event where Alu Sg in new world monkeys is replaced by an Alu Ye5 in Humans, chimpanzees, gorillas and orangutan. In the other two cases (Ye5AH70 and Ye5AH173), only a small portion of the 3' end of the Ye elements were involved in the gene conversion. This is in good agreement with the molecular nature of gene conversion events recently reported for the Ya5 and Yb8/9 Alu subfamilies [Alu Ye elements suggests that gene conversion of these events has been relatively rare, with a rate of 1.96%. However, this rate is comparable to that reported previously for the Alu Ya5 and Yb8 subfamilies within the human genome, as well as that for the Ta subfamily of human LINE elements [Gene conversion between eviously ,63,64. Tfamilies ,48,64,65elements .Alu family members that were involved in the gene conversion were monomorphic for insertion presence within the human genome. In the partial gene conversion events, the Ye Alu repeats were gene converted by Yb8/9 and Sx Alu elements. The Yb8/9 Alu subfamily was one of the first groups of Alu repeats that was ever reported to be involved in gene conversion, and may be more prone to these types of events as a result of a retroposition rate that is slightly higher than other recently integrated Alu subfamilies in the human genome [Alu elements may in part be a function of the length of time that the individual Alu elements have resided in the human genome [Alu elements allows for pairing between regions of sequence identity of different Alu elements initiating the start of gene conversion before cellular control systems can terminate the process resulting in the production of small gene conversion tracts.In all cases, the Ye n genome ,64,65. Tn genome ,50. Basen genome . It is qAlu insertions involving an Alu Ye element (Ye5AH161) in humans, bonobo, common chimpanzee and gorilla lineage, Alu Sp in old world monkeys lineage and Alu Sx in new world monkeys lineage is also interesting. The paralogous insertion of an Alu repeat into the orthologous regions of human and non-human primate genomes is an independent evolutionary event [Alu elements into identical sites within different genomes. The detection of parallel insertions is a function of the rate of retroposition of Alu elements within various primate lineages and the time since the most recent common ancestor [Alu repeats within primate genomes. Future studies on the integration of different SINE elements in syntenic regions of human and rodent genomes may yield new insight into the molecular nature of hotspots for SINE element integration.The identification of multiple paralogous ry event . To dateancestor . HoweverAlu-mediated deletion that resulted in the inactivation of the human CMP-N-acetylneuraminic acid hydroxylase gene [Alu mediated deletions of noncoding genomic sequences have been identified [Alu retroposition-mediated deletions that may have happened by a mechanism similar to that of the LINE element mediated genomic deletions since Alu and L1 elements utilize a common mobilization pathway [Alu Ye5AH24 and Alu Ye5AH27, the deletion appears to have occurred, after the separation of human, chimpanzee and gorillas from orangutan and Siamang, during the process of gene conversion similar to the lineage specific Alu deletion reported previously [Genomic deletions created upon LINE-1 retrotransposition using cell culture assays have been recently identified . The ratase gene  and Alu entified . Here we pathway ,8,72. Ineviously ,71.Alu retroposition associated genomic deletions as approximately 1.67%. The size of the deleted sequences was over 300 bp on average. New Alu integrations have been estimated to occur in vivo at a frequency of one new event in every 10 to 200 births [Alu retroposition events in vivo, the genomic impact of these events could be substantial. This is not a trivial number of deletions when extrapolated to the copy number of Alu elements in the human genome which is over one million [Alu elements may have been involved in retroposition mediated deletion events within primate genomes. If each of these deletion events removes an average of 300 bp of genomic sequence, this would mean that Alu retroposition mediates the deletion of about 5 Mb of the primate genomic sequences. However, if the Alu associated deletions have involved larger sequences similar to those recently reported for LINE elements [Here, we have estimated the frequency of 0 births . If sizaelements , then thThe Alu Ye lineage has had an extended history of expansion in the human lineage. Its expansion appears to have begun soon after the divergence of the hominoids from the remainder of the catarrhine primates and proceeded at a relatively low level since then. Extended periods of relatively low levels of retrotransposition may allow some mobile elements to retain duplication capability for long periods of time. Despite a relatively low level of retrotransposition, the Alu Ye lineage has contributed to the architecture of the human genome through insertion mutations, retrotransposition associated genomic deletions, and gene conversion.Alu Ye elements in the draft sequence of the human genome (August 6, 2001, UCSC GoldenPath assembly), we used Basic Local Alignment Search Tool (BLAST) [Alu elements identified in the searches is located in .To identify  (BLAST)  queries  (BLAST) -49. A coAlu Ye lineage loci analyzed were performed as previously described [Alu Ye lineage members. Diverse human DNA samples were available from previous studies [Pan troglodytes), WES (ATCC CRL1609); gorilla (Gorilla gorilla) lowland gorilla Coriell AG05251B, Ggo-1 (primary gorilla fibroblasts) provided by Dr. Stephen J. O'Brien, National Cancer Institute, Frederick, MD, USA; bonobo (Pan paniscus) Coriell AG05253A; orangutan (Pongo pygmaeus) ATCC CRL6301; green monkey (Chlorocebus aethiops) ATCC CCL70 (old world monkey); and owl monkey (Aotus trivirgatus) OMK (OMKidney) ATCC CRL 1556 (new world monkey). Cell lines were maintained as directed by the source and DNA isolations were performed using Wizard genomic DNA purification (Promega). DNA samples from peripheral lymphocytes or tissue were prepared from the gibbon (Hylobates lar) and siamang (Hylobates syndactylus). Additional non-human primate DNA samples (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Macaca mulatta (old world monkey), Macaca nemestrina (old world monkey), Saquinus labiatus (new world monkey), Lagothrix lagotricha (new world monkey), Ateles geoffroyi (new world monkey) and Lemur catta (prosimian) available as a primate phylogenetic panel (PRP00001) were purchased from the Coriell Institute for Medical Research.Oligonucleotide primers and PCR amplification reactions for each of the escribed -49 using studies -49. The Alu element) has been assigned accession numbers AY849282-AY849301. Sequence alignments of the Ye lineage subfamily members were performed using MegAlign software (DNAStar version 3.1.7 for Windows 3.2). The ages for each of the Alu Ye subfamilies were calculated using mutation densities as previously described [DNA sequencing was performed on a gel purified PCR products that had been cloned using the TOPO TA cloning vector (Invitrogen) using chain termination sequencing  on an Apescribed ,47-49,65escribed .AS performed all experimental work for the project, shared in the analysis and interpretation of the results and wrote the first draft of the manuscript. DAR provided assistance with analysis and interpretation of the data and in preparing the manuscript for submission. DJH wrote the software used to extract Ye elements and the associated flanking sequences from the human genome draft sequence. JJ provided assistance with the analysis and interpretation of the data and input on late drafts of the manuscript. MAB provided the initial input for the project as well as valuable input on each draft of the manuscript.This supplemental file represents a sequence alignment for for locus Ye5AH161 in fasta format.Click here for fileThis supplemental table lists all Alu Ye elements recovered with information on PCR conditions, chromosomal location and phylogenetic origin. It is in Microsoft Word format.Click here for file