Previously, we had shown that persons infected with human T-cell lymphoma leukemia virus 1 or 2 (HTLV-1 or 2) had an increased prevalence of antibodies to a peptide in the Pol protein of the retrovirus HERV-K10, homologous to a peptide in HTLV gp21 envelope protein. The prevalence rate was higher in those with myelopathy vs. non-myelopathy. We have now extended our observations to a cohort restricted to North America in whom the diagnosis of HTLV myelopathy was rigorously confirmed to also test for reactivity to another HERV-K10 peptide homologous to the HTLV p24 Gag protein.Sera from 100 volunteer blood donors (VBD), 53 patients with large granular lymphocytic leukemia (LGLL), 74 subjects with HTLV-1 or 2 infection (58 non-myelopathy and 16 myelopathy) and 83 patients with multiple sclerosis (MS) were evaluated in ELISA assays using the above peptides.The HTLV myelopathy patients had a statistically significant increased prevalence of antibodies to both HERV-K10 peptides (87.5%) vs. the VBD (0%), LGLL patients (0%), MS patients (4.8%), and the HTLV positive non-myelopathy subjects (5.2%).The data suggest that immuno-cross-reactivity to HERV-K10 peptides and/or transactivation of HERV-K10 expression by the HTLV Tax protein may be involved in the pathogenesis of HTLV-associated myelopathy/tropical spastic paraparesis and spastic ataxia. In addition to causing T-cell malignancies, both human T-cell lymphoma/leukemia viruses 1 and 2 (HTLV-1 and HTLV-2) are known to cause myelopathy (HAM) in a minority of infected individuals . The comIn previous studies we had determined that, although only 8% of patients with large granular lymphocytic leukemia (LGLL) were infected with HTLV-2, almost half the patients had antibodies to HTLV p24 Gag and gp21 Env proteins . None ofWe also did molecular studies involving HERV-K113 and HERV-K115, which are members of the HERV-K10 family . They haTable Compared to the VBD (0%), LGLL (6%), HTLVt (28%), HTLVm 94%), HTLVn 10%) and MS (16%) patients all had a significantly higher seroprevalence for anti-HERV-K10 Pol antibodies  HERV-K10 peptides, all of the study populations except the LGLL patients (0%) had a significantly higher seroprevalence than the VBD (0%) Table . HoweverAmong the HTLVm patients, 8 were HTLV-1 positive and 8 were HTLV-2 positive. Six (75%) of the HTLV-2 positives had antibodies to both HERV-K10 peptides, while all of the 8 (100%) HTLV-1 positives had antibodies to the HERV-K10 Gag peptide, and 7 out of 8 (87.5%) had antibodies to HERV-K10 Pol peptide. Obviously, there were no significant differences in these results. Among the 22 HTLV-1n subjects, 3 (13.6%) were reactive to HERV-K10 Gag peptide, 3 (13.6%) were reactive to HERV-K10 Pol peptide and these 3 (13.6%) were reactive to both peptides. Among the 36 HTLV-2n subjects, 1 (2.8%) was reactive to HERV-K10 Gag, and 3 (8.3%) were reactive to HERV-K10 Pol, with 1 (2.8%) being reactive to both peptides. Again, these differences in HERV-K10 seroprevalences between HTLV-1 and −2 infected subjects were not statistically significant.The mean OD minus background for the anti-HERV-K10 Gag results among the HTLV-1 m patients was 0.841, while the same value for the HTLV-2 m patients was 0.261 (p = 0.055). The mean titer for the anti-HERV-K10 Gag results among the HTLV-1 m patients was 925, while the same value for the HTLV-2 m patients was 250 (p = 0.129). The mean OD minus background for the anti-HERV-K10 Pol results among the HTLV-1 m patients was 0.236, while the same value for the HTLV-2 m patients was 0.125 (p = 0.072). The mean titer for the anti-HERV-K10 Pol results among the HTLV-1 m patients was 325, while the same value for the HTLV-2 m patients was 300 (p = 0.202).Table - and HLA-DRB1*0101 alleles, and impaired anti-HTLV cytotoxic T-cell responses [Increased risk for developing HAM is associated with blood-borne viral transmission, increased proviral DNA loads and anti-HTLV antibodies, polymorphisms in the IL-10 promoter and IL-28B gene, HLA-A*02esponses ,15-19. Tesponses .It has been estimated that 8% of the human genome is comprised of endogenous retroviral elements . Most ofHERV-K10 is a member of the HML-2 class of endogenous retroviruses . It has Our data indicate that, compared to VBD and HTLV + non-myelopathy subjects, patients with HTLV myelopathy had higher titers of antibodies to two HERV-K10 peptides. This suggests that cross-reactive or direct immunity to HTLV-transactivated HERV-K expression in the central nervous system could play a role in the pathogenesis of HAM. Obviously, however, this data alone would be insufficient to make such a case. If HAM is associated with higher titers of anti-HTLV antibodies, then it is quite possible that these patients coincidentally would have higher antibody titers against HTLV homologous HERV-K10 peptides. However, it is indeed interesting that MS patients, who were HTLV seronegative, also had a statistically significant higher seroprevalence (7.2% vs. 0%) to both HERV-K10 peptides compared to VBD. Recently, results were published that indicate that broad anti-HERV-K Gag and Env-specific T-cell responses were not detected in HTLV-I infected subjects . This woIn an IRB-approved archival study, serum and peripheral blood mononuclear cells (PBMC) were obtained from 100 VBD, 53 LGLL patients, 74 HTLV-1 or −2 infected individuals (HTLVt), 16 of whom had myelopathy (HTLVm) (8 HTLV-1 and 8 HTLV-2), and 58 who did not (HTLVn) (22 HTLV-1 and 36 HTLV-2), and 83 HTLV negative multiple sclerosis (MS) patients. All LGLL patients had clonal T-cell lymphocytoses, as confirmed by PCR for clonal rearrangements of the T-cell receptor γ- gene and by positivity for CD3, CD8, and CD57 proteins ,38. The 2SO4 to each well and the optical densities of the samples were read at 450 nm. A sample was deemed positive if, at a dilution of 1:200, its optical density was ≥ 2.5 x background plus 0.100 for the Gag peptide or ≥ 2.5 x background for the Pol peptide. Multiple permutations of the above conditions were conducted using the pre- and post-immune rabbit sera and a panel of three VBD sera as putative negative controls before settling on the above conditions. Positive samples were then serially diluted and retested to determine their end point titer.All subjects were screened for anti-HTLV antibodies using a commercial HTLV1/2 EIA assay, as previously described . Seropospol primers SK110/SK111, the HTLV-1 pol probe SK-112, and the HTLV-2 pol probe SK188, as previously described [DNA was extracted from serum or PBMC, and DNA integrity was confirmed by β-globin PCR, as previously described . Only saescribed . Quantifescribed .The presence or absence of HERV-K113 and HERV-K115 was determined with PCR primers spanning the pre-integration sites and sites within the viral sequences. The primers for HERV-K113 were those used by Moyes et al. and those for HERV-K115 were used by Hughes et al. ,43. CondSeroprevalence rates between various subject populations were analyzed using Fisher’s t-test . CompariThe authors declare that they have no competing interests.BJP, RP – conceived and designed the experiments. RP, EG, SS, CW, PB, LA, DL – performed the experiments. JG, TL, WS – contributed the samples. BJP, RP – wrote the paper. WS – edited the manuscript. All authors participated in data analysis. All authors read and approved the final manuscript.


Tax-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1iIn vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of nfected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden.Tax DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared.A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR) and a PvL for p < 0.05), but not between subjects with a low or intermediate PvL (p > 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p < 0.03). No significant difference was found between the groups in relation to age, sex or viral subtypes (p > 0. 05).Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects. Human T-cell leukemia virus type I (HTLV-I) is the retrovirus responsible for adult T-cell leukemia (ATL) and the chronic neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) -4. The vHTLV-1 is classified into seven subtypes : the cosgag, pol and env genes flanked by long terminal repeat (LTR) sequences at both 5' and 3' ends [env and the 3' LTR encodes two key regulatory proteins Tax and Rex, as well as the various nonstructural proteins p12I, p27I, p13II, and p30II [trans-activate HTLV RNA transcription via the viral LTR through its interaction with members of the activating transcription factor/cAMP-responsive element (CRE) binding protein bound to the viral promoter, such as ATF-1, CREB, CREB-2, or cAMP-responsive element modulator [Similar to other retroviruses, HTLV-1 carries a diploid RNA genome comprising 9032 nucleotides that is reverse-transcribed into double-stranded DNA that integrates into host genome as a provirus. This genome contains  3' ends . The LTRnd p30II -22. The nd p30II . Tax modnd p30II ,24,25. Todulator -28. The odulator . These sodulator . Evidencodulator ,32. We hPeripheral blood samples (5 ml) were collected from 256 HTLV-1 positive carriers identified by the HTLV enzyme immunoassays Murex HTLV I + II  and Vironostika HTLVI/II  and confirmed by HTLV BLOT 2.4 . Written informed consent was obtained from each participant. The study was approved by the local review board.Tax region using previously published primers [Taq polymerase . The amplification was performed in the Bio-Rad iCycler iQ system using an initial denaturation step at 95°C for 2 minutes, followed by 50 cycles of 95°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds. The human housekeeping β globin gene primers GH20 and PC04 [2O control) was run with every assay. Standard curves for HTLV-1 Tax were generated from MT-2 cells of log10 dilutions (from 105 to 100 copies). The threshold cycle for each clinical sample was calculated by defining the point at which the fluorescence exceeded a threshold limit. Each sample was assayed in duplicate, and the mean of the two values was considered as the copy number of the sample. The HTLV-1 proviral load was calculated as: the copy number of HTLV-1 (Tax) per 1,000 cells = (copy number of HTLV-1 Tax)/(copy number of β globin/2) × 1000 cells. The method could detect 1 copy per 103 PBMCs cells.DNA was extracted from PBMCs using a commercial kit  following the manufacturer's instructions. The extracted DNA was used as a template to amplify a 158 bp fragment from the HTLV-1  primers . The SYBand PC04  were use<50, between 50 and 100, or > 100 copies/1000 PBMCs were considered to be a low, medium, or high PvL, respectively. An HTLV-1 PvL > 100 copies/1000 PBMCs have previously been shown to be associated with an increased risk of HTLV-1 disease [PvLs of  disease .Taq polymerase on an automated sequencer , according to the protocols recommended by the manufacturer. The complementary sequences for each amplicon were assembled into a contiguous sequence with a minimum overlap of 30 bp with a 97-100% minimal mismatch and edited using the Sequencher program 4.7 . Sequences were then analyzed using BioEdit v.7.0.4.1 [Sequencing of the near complete LTR region of HTLV-1 proviral DNA was performed in 94 clinical isolates. PCR was performed using the primers HFL1 (39) (5' CCCAAGCTTGACAATGACCATGAGC 3') and HFL2 (782) (5'CCCGAATTCCAACTGTGTACTAAATTTC 3') and in conditions as previously described . Complem.7.0.4.1  and Gene.7.0.4.1 . A straiThe HTLV-1 genotypes were determined by comparing the sequence of the LTR region to standard sequences from the GenBank database. HTLV subtyping was performed using the NCBI-Genotyping and REGA-Subtyping websites.p value < 0.05 was considered significant. The data were analyzed with Stata statistical software .For categorical variables, the Fisher's exact test or Chi-square test with Yate's correction was used when appropriate to analyze the association of HTLV-1 PvLs and the LTR mutational frequency. In another analysis, independent of the PvL grouping, the median PvL between subjects infected with mutant viruses to those infected with wild-type viruses was compared using the non-parametric U-Mann-Whitney test. A p = 0.9 for age; p = 0.6 for male to female ratio). A comparison of the stratified PvLs showed no significant differences in any lymphocyte subpopulation percentages (p > 0.05). The transcontinental subgroup A of HTLV-1a was the major subtype; this accounted for 84% of low, 97% of intermediate, and 96% of high PvL groups. There were no differences in the subtype distribution between each group (p = 0.2). Mutational analyses of the non-coding regulatory sequences within the proviral LTR were conducted to determine if a correlation exist between the frequency of mutations and PvLs. These analyses revealed two relevant G232A mutation within the TRE-1 region and an A184G mutation within the TRE-2 region numbered according to the transcription start site. The G232A mutation was detected in 38.7% (12/31) of subjects with a low PvL, 27.5% (8/29) of subjects with an intermediate PvL, and 61.8% (21/34) of subjects with a high PvL (Table p < 0.05 both). Non statistical difference was seen between the low and intermediate PvL groups (p > 0.05). Thirty-six of 41 (87.8%) HTLV-1 G232A mutations were associated with a simultaneous A to G mutation at position 184 , but not between the low and intermediate PvL groups (p > 0.05). To confirm these results, independent of the PvL grouping, a further statistical analysis using a Mann-Whitney test was performed to compare the median PvL between subjects infected with mutant viruses to those infected with wild-type viruses. This analysis revealed that while there was strong evidence for a higher PvL among HTLV-1 positive individuals with the G232A mutation than those without this mutation , there was no similar association among those who were HTLV-1 positive with the A184G mutation  in the low PvL group , 11.3% (n = 29) in the intermediate PvL group , and 17.7% (n = 45) in the high PvL group . Extracted genomic DNA from all subjects with intermediate PvL were subjected to amplification and sequencing of the near full-length HTLV-1 LTR. Due to financial constraints, 31 of the 182 genomic DNA samples from subjects with low PvL and 34 of the 45 genomic DNA samples from subjects with a high PvL were randomly selected and their HTLV-1 LTR was successfully sequenced. The demographic data from the three groups are listed in Table Tax-activated and basal LTR expression. Although the functional importance is unclear, it is possible that the G232A mutation in the TRE1 element may enhance indirect binding to Tax via the CREB family of cellular transcription factors, thereby promoting the proliferation of infected cells and resulting in an increase in patient PvLs. However, the validity of this hypothesis needs to be investigated in an independent series of experiments comparing the expression and activation of the Tax gene in HTLV-1-infected T cells with or without the G232A mutation. Previous reports have shown that mutations that abolish Tax effects are all localized in the CREB of the 21 bp repeats [et al. demonstrated that mutations introduced into domain A or C can severely impair the ability of the Tax protein to transactivate different promoters [In the present cross-sectional, retrospective study, we investigated mutations in the LTR region of HTLV-1 and compared their association with asymptomatic carriers PvL. Our results showed that mutations were relatively rare. However, a solitary, natural mutation at position 232 was significantly associated with HTLV-1 infected subjects' PvL. The G232A mutation is located in domain A of the TRE-1 motif that contains non-consensus CREB response elements and is involved in  repeats . Montagnromoters . Taken tIn our study, the G232A mutation was associated with HTLV-1 PvL. Although the G232A mutation was detected at a higher frequency in asymptomatic HTLV-1 carriers in this study, the frequency of such a mutation is expected to be higher in patients with overt clinical disease, because the PvLs of HTLV-1-infected patients correlates with clinical outcome . It is iAlthough the G232A mutation was detected in some subjects with a low PvL in this study, further study is needed to determine if this mutation is predictive of an increase in PvL in patients who possess the mutation but do not have an elevated PvL yet.As shown in Figure Our analysis was limited to HTLV-1 subgroup A because other subtypes are rare in our patients. Whether similar results will be obtained for other subgroups, is of interest.In conclusion, the results described here suggest the G232A mutation in domain A of the TRE-1 motif may increase HTLV-1 replication in the majority of infected subjects.HTLV-1: Human T Cell Lymphotropic Virus Type 1; TRE: Tax-responsive element; PvL: Proviral load; LTR: Long terminal repeat; ATL: Adult T-cell leukemia; HAM/TSP: HTLV-I-associated myelopathy/tropical spastic paraparesis; CREB: Transcriptional activity of cAMP binding protein; NFkB: Nuclear factor kappa B; SRF: Serum Response Factor; AP-1: Activator protein; CRE: CAMP-responsive element; ATF-1: Activating transcription factor 1.The authors declare that they have no competing interests.WKN and ACC conceived and designed the study, did the data analysis of the sequences. ACSO and VPM conducted the characterization of the LTR sequences and data analysis of the sequences. YN was responsible of the clinical management of patients, acquisition of data; ECS was responsible of the scientific revision, discussion and interpretation of data. SSS wrote the manuscript and directed the study. All authors read and approved the final manuscript.


Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.The alternative oxidase (AOX) of  Although the proper formation of mitochondria requires the expression of genes found in both the nuclear and mitochondrial genomes, the vast majority of mitochondrial proteins are encoded in the nucleus. Thus, communication of mitochondrial status to the nucleus must occur to insure that the organelles meet the requirements for growth and development of the organism and to insure that they can properly respond to changing conditions and stress. Communication from the mitochondria to the nucleus that influences the expression of nuclear genes has been termed retrograde regulation .The alternative oxidase (AOX) is a di-iron carboxylate protein that transfers electrons from ubiquinol to molecular oxygen and exists in the mitochondrial inner membrane . It is fCandida albicans there is evidence for the involvement of a histidine kinase in AOX regulation  family  was used as a control for experiments with the knockout strains.The RIPtom20 sheltered heterokaryon, the methods used to manipulate the heterokaryon, and the control (strain HIV) for experiments with the heterokaryon have been described in detail previously . The other nucleus contains a nonfunctional tom20 allele that was destroyed by repeat-induced point mutation (RIP) mutagenesis. The latter nucleus also carries a mutant allele of the mtr gene that imparts resistance to fpa, as well as a mutation in the his-3 gene. The level of the Tom20 protein can be greatly reduced in the heterokaryon by growth in the presence of fpa plus histidine.The N. crassa cells were grown on solid or in liquid medium according to previously described methods . Proteins were transferred to nitrocellulose and exposed to X-ray film.The procedures used to measure the import of mitochondrial precursor proteins have been described previously , which we had previously identified as a transcription factor necessary for the expression of AOX . Because many of the knocked-out genes are represented in duplicate as both N. crassa . Thus, there are eight new strains that have been identified and assigned to Class 1. The 20 strains in Class 2 exhibited a moderate reduction in the amount of AOX at 24 hr, 48 hr, or both time points. An example is strain 6A1 . There is no obvious relationship among the predicted functions of the eight new class 1 genes (stk-33 (S. cerevisiae homolog (YPL109c) has not yet been assigned a function. However, the proteins from both species are related to the S. cerevisiae YGL119W protein, also known as Coq8  and six kinases or phosphatases . If the previously identified  1 genes  and in mvad-5 and ada-7, respectively, in a study of Neurospora transcription factor knockouts , whereas another two encode predicted transcription factors . The latter two genes were previously identified as N. crassa mitochondria by two-dimensional electrophoresis and mass spectrometry, as well as those predicted to be mitochondrial by various in silico analyses has recently been published  to determine whether the changes in AOX levels were gradual or sudden. As shown in tom70 was somewhat unexpected (strain 96C10). Tom70 is a protein of the outer mitochondrial membrane TOM  holo-complex .Among the genes identified as affecting AOX production, -complex . Tom70 itom70 knockout on AOX, we investigated the phenotype of the strain in more detail. Although we saw no growth of the strain on antimycin A containing medium after 48 hr (RIPtom20 sheltered heterokaryon (tom20 gene resulting in a reduction of the Tom20 protein in the mitochondria of the culture (See Methods). We isolated mitochondria from cultures of the tom70 knockout strain (96C10), a Tom20-depleted strain, and appropriate controls. All strains were grown both in the presence and absence of Cm. Mitochondrial proteins were subjected to SDS-PAGE and analyzed on Western blots for the presence of various mitochondrial proteins. Growth of the RIPtom20 sheltered heterokaryon in the presence of fpa resulted in mitochondria virtually devoid of Tom20, while Tom70 levels were unaffected (RIPtom20 lanes). Levels of AOX in these mitochondria after growth in the presence of fpa and Cm were reduced compared with AOX in RIPtom20 cultures grown without fpa. However, the levels of AOX in the RIPtom20 cultures are virtually identical to the control cells grown under the corresponding conditions (RIPtom20).To validate the effects of the er 48 hr , slow grer 48 hr . We thentom70 knockout strain have levels of Tom20 similar to the control strain, but have much reduced AOX levels compared with the relevant control , a protein that contains a cleavable N-terminal targeting signal, occurred at a slightly higher level in the mitochondria lacking Tom70. For two quantified experiments, the percentage of import into the mitochondria lacking Tom70 was 100% and 123% of the control for F1β; 39% and 47% for AAC; and 35% and 48% for AOX. The simplest interpretation of these data is that Tom70 is the major receptor involved in the import of the AOX precursor. However, we cannot formally eliminate the possibility that another unknown factor that is decreased in mitochondria lacking Tom70 may play a role in AOX import.We also directly measured import of radiolabeled precursor proteins into isolated mitochondria with reduced levels of Tom70 . AOX impN. crassa. Absence of many of the genes has only minor effects on AOX (class 3), whereas loss of others has a more pronounced effect (classes 1 and 2). Many of the knockout strains identified in our screen exhibit reduced growth rates and/or reduced ability to form conidia. Thus, such genes are not likely to be specific for an AOX regulatory pathway. Loss of these genes may affect several functions that include AOX expression. More information on AOX expression at the transcriptional and posttranscriptional levels, as well as the assembly of the enzyme into the mitochondria inner membrane, will be required to understand the effects in each case.This study has identified many genes that are required to achieve full expression of AOX under conditions that induce the enzyme in Although the AOX precursor protein contains a cleavable N-terminal targeting signal, its efficient import appears to be dependent on Tom70. Tom70 was previously shown to enhance the import of a few precursors containing N-terminal cleavable targeting signals  fits nicP. anserina that the orthologs of AOD2 and AOD5  are involved in transcription of AOX but also play a role in transcription of genes involved in gluconeogenesis  are involved in gluconeogenesis and AOX regulation  and/or AcuK (AOD5) to activate the heterodimeric transcription factor  are thought to play a role in mitochondrial biogenesis and in eliciting stress responses in many systems . Increasalbicans , Magnopoic study . One posS. cerevisae proteome demonstrated that NCU09803.5 is most highly related to the monothiol glutaredoxins, Grx3 and Grx4 . These yeast proteins, as well as NCU09803.5, contain a thioredoxin-like domain at their N-terminus, but also the CGFS motif that defines monothiol glutaredoxins . BLAST searches against the N. crassa database identified a GRX5 protein that represents a mitochondrial monothiol glutaredoxin and revealed that NCU09803.5 is the only non-mitochondrial form of the protein in the organism (see Table S1). This is similar to Schizosaccharomyces pombe, which contains only a single Grx3/Grx4 ortholog, called Grx4  was found to exhibit a complex phenotype that included alterations in redox regulation . The CSN is a highly conserved complex in eukaryotes consisting of eight subunits in higher organisms, with some variation in the number of subunits in lower eukaryotes. The CSN is best known for its role in the regulation of protein degradation, but other activities, including possible direct roles in gene expression and regulation of transcription factors, have been associated with the complex or its subunits (reviewed in oduction . At the aod (AOX deficient). Because our previously named aod-2 and aod-5 genes were thought to be AOX-specific transcription factors but are now known to be involved in gluconeogenesis as well (described previously), it may be difficult to assign aod to any gene in the absence of further data regarding the exact function of the gene products. The requirement for many different genes to achieve the full level of AOX in mitochondria suggests that complex pathways of expression exist for the enzyme. Closer examination of the mutants identified here will help to elucidate the nature of these mechanisms.Our study has revealed several new genes that affect AOX production. It is valid to ask whether we have found any genes that should be named


WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 . NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.In contrast to miR-140, the expression of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of  MMP-13) transcription . Together, these results indicate that the TGF-β-induced miR-140 down-regulation is the result of SMAD3 activation and that NFAT3 regulates miR-140 directly, likely at the rsmiR-140 level. NFAT5 could indirectly contribute to the down-regulation of miR-140 by up-regulating the expression of TGF-β, which in turn inhibits miR-140 expression.P ≤0.0001) as well as the ionomycin-induced (P ≤0.0001) expression, indicating the involvement of this site in the positive regulation of miR-140 by NFAT3. The -195 bp CAGA mutation  and TGF-β-induced (P ≤0.005) expression, suggesting its involvement in the negative regulation of miR-140 by TGF-β through the inhibitory action of SMAD3. Mutating the -120 bp TTGGTGTTGG and -209 bp CAGA sites did not affect either basal or TGF-β-induced expression (data not shown). It was also observed that TGF-β treatment of the -195 bp mutated construct resulted in a slightly decreased expression. TGF-β has a number of roles and acts through many intermediates in addition to SMAD3; it is thus possible that TGF-β could act indirectly on rsmiR-140 through other (unknown) binding sites.rsmiR-140 has consensus binding sites for NFAT (GGAAA at position -234 bp) and SMAD3  . Similar experiments done with primer pairs located 800 bp upstream of the binding sites and on the unrelated negative control gene MAP1A revealed no significant binding , and TGF-β triggered that of SMAD3  in OA chondrocytes. Treatment with TGF-β alone had no significant effect on NFAT3 translocation and treatment with ionomycin alone had no effect on SMAD3 translocation. However, when TGF-β was added for the last 30 minutes of the 90-minute ionomycin treatment, there was a significant decrease in the number of NFAT3-positive nuclei (P ≤0.05) when compared to ionomycin alone  in miR-140 compared to ionomycin treatment alone, while the addition of ionomycin to the TGF-β treatment did not significantly affect the miR-140 expression level. All together, these results indicate that the presence of TGF-β interferes with the ionomycin-induced nuclear translocation of NFAT3 and ultimately miR-140 expression.Further experiments were done to verify whether such interference affected miR-140 expression Figure C, D. TheOur previous study in human chondrocytes  and thosWWP2 and TGF-β. In turn, the effects of these factors on miR-140 were translated into the cells and impacted a known direct miR-140 target, IGFBP5. This study also shows the importance of TGF-β as a factor implicated in the decreased miR-140 expression in human OA chondrocytes, thus contributing to the progression of this disease.We report, for the first time, a differential expression between miR-140 and its host gene. We also identify a regulatory sequence located directly upstream of the pre-miR-140 and demonstrate the direct involvement of NFAT3 and SMAD3 on the miR-140 regulatory sequence sites as well as the indirect effect of NFAT5, possibly acting through WWP2 and miR-140 in OA chondrocytes [WWP2 promoter. Other evidence of differential regulation was shown in zebrafish [WWP2 were suggested to play distinct roles in cartilage development, as the separate knockdown of WWP2 and miR-140 caused different effects.It was first believed that the regulation of intronic miRNAs followed that of their host genes as they are often co-expressed. However, recent reports showed that some intronic miRNAs have their own promoter and that their expression/regulation differs from that of their host gene -28. The drocytes ,6 led usebrafish  in whichin silico analysis of the 2 kb sequence located upstream of the mature miR-151 did not reveal any NFAT3 or SMAD3 consensus binding sites, as was the case with miR-140. Although the expression levels of both miR-140 and miR-151 are decreased in OA, their regulation is likely the result of different factors. Thus, the role of miR-151 in OA, direct or indirect, is yet to be determined.Differential regulation between a miRNA and its host gene may not be a rare event as we have also noted that miR-151 expression, like that of miR-140, is decreased in OA independently of its host gene. However, unlike miR-140, miR-151 has not been identified in miRNA profiling of OA cartilage ,31, but et al. [WWP2 gene, upstream of the ATG start of the WWP2-C variant as hypothesized by Soond et al. [et al. [WWP2-C transcription as miR-140 was reported to be co-expressed with WWP2-C[et al. article [WWP2-C is differentially regulated from that of miR-140. The second TSS is located at position 976 bp upstream of the pre-miR-140, thus within the cloned rsmiR-140 sequence, which we have shown to be capable of promoting transcription independently of WWP2, unlike the region identified by Yang et al. [We have identified rsmiR-140 as a regulatory sequence for miR-140 expression independently of its host gene. This sequence is located in a region different from the regulatory sequence identified by Yang et al.  and the et al. , which rd et al.  and desc [et al. . It is pth WWP2-C. In the  article , howeverg et al. .et al. [et al. are not surprising, as the events occurring in mouse limb buds do not necessarily represent what is occurring in human adult articular cartilage.Another difference from the results of Yang et al.  is that Mutagenesis has identified functional NFAT3 (GGAAA) and SMAD3 (CAGA) binding sites. Of interest is the opposite direct regulation of miR-140 by NFAT3 (activator) and SMAD3 (inhibitor). Such competition/cooperation between these two factors has previously been reported in different systems, an example being the human c-Myc transcription, activated by NFATs and repressed by TGF-β .WWP2/miR-140 under hypertonic conditions could occur via the transcription factor Sox9, as WWP2 and miR-140 were reported to be co-expressed and activated by Sox9 [This study also identifies NFAT5 as an indirect regulator of miR-140 expression. The differential activity of NFAT5 and NFAT3, although both belong to the same family of transcription factors, is not surprising as they are activated by different factors, have different binding partners and control the transcription of different genes -40. The  by Sox9  and Sox9 by Sox9 . NFAT5 i by Sox9 . It was  by Sox9  and was  by Sox9 . Here, wMMP-13 expression [IGFBP-5[SMAD3[IGFBP-5 expression in human OA chondrocytes and Baugé et al. [SMAD3 expression, but a longer period resulted in a decreased expression. Thus, TGF-β affects many genes and its regulation through miRNAs is part of this complex network.The importance of TGF-β in OA has been recognized and reviewed ,46 and ipression ,49. Thispression . BesidesP-5[SMAD3. Indeed,é et al.  found thWWP2 and miR-140 expression. As the levels of TGF-β are low in normal cartilage, the end result will be a positive regulation. In OA chondrocytes, the increased expression of TGF-β would activate SMAD3 phosphorylation, thus directly inhibiting miR-140 as well as indirectly down-regulating miR-140 by interfering with the translocation of NFAT3. Therefore, in OA chondrocytes, as the NFAT5 expression is decreased compared to that in normal chondrocytes (data not shown), the NFAT5 contribution in OA will be lower. However, as TGF-β is increased in OA but the expression of NFAT3 is similar in normal and OA (data not shown), the negative regulation of miR-140 levels by TGF-β/SMAD3 would prevail over the positive regulation by NFAT3 and would account for the decreased miRNA in these cells.Data from this study support the hypothesis that miR-140 expression could be regulated at different levels under normal and OA conditions Figure . AlthougIn summary, this study is the first to show the important direct roles of NFAT3 and SMAD3 and the indirect role of NFAT5 in miR-140 expression. Moreover, we highlight a new role for TGF-β in OA chondrocytes as a down-regulator of miR-140 expression, resulting in increased expression of miR-140 target genes, thus contributing to this disease process. These data could open up novel avenues in OA therapeutic strategy.(D)MEM: (Dulbecco’s) modified Eagle’s medium; ACR: American College of Rheumatology; ADAMTS: A disintegrin and metalloproteinase with a thrombospondin type 1 motif; BMP2: Bone morphogenetic protein 2; bp: Base pair; BSA: Bovine serum albumin; ChIP: Chromatin immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole, dihydrochloride; FAK: Focal adhesion kinase; FCS: Fetal calf serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGFBP5: Insulin-like growth factor-binding protein-5; IgG: Immunoglobulin G; MAZ: Myc-associated zinc; miRNA: microRNA; MMP: Matrix metalloproteinase; NFAT: Nuclear factor of activated T-cells; NF-κB: Nuclear factor kappa B; NMP4: Nuclear matrix transcription factor 4; OA: Osteoarthritis; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; PTK2: Protein tyrosine kinase; qPCR: Quantitative PCR; rsmiR-140: miR-140 regulatory sequence; SEM: Standard error of the mean; siRNA: Small interfering RNA; SIS3: Specific inhibitor of SMAD3 phosphorylation; SMAD3: Mothers against decapentaplegic homolog 3; SREBF2: Sterol regulatory element binding factor-2; TGF-β: Transforming growth factor beta; TSS: Transcription start sites; WWP2: WW domain containing E3 ubiquitin protein ligase 2.The authors declare that they have no competing interests.GT, JPP and JMP designed the study. HF, DH, YZ and MK acquired the data. GT, JPP, MK and JMP analysed and interpreted the data. GT, JPP, HF, DH, YZ, MK and JMP participated in manuscript preparation. GT provided statistical analysis. All the authors read and approved the final manuscript.Expression as measured by qPCR of WWP2, miR-140, IGFBP5, and TGF-β in OA control chondrocytes; rsmiR-140 activity in control cells from mutagenesis experiments; basal PCR values of NFAT3 and SMAD3 in OA control chondrocytes in ChIP experiments.Click here for fileSilencing efficiency of siRNAs in human chondrocytes as determined by qPCR and Western blotting.Click here for fileBasal and induced expression of the WWP2 variants 1 and 2 in human chondrocytes.Click here for file


Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent liver diseases around the world, and is closely associated with obesity, diabetes, and insulin resistance. Ursolic acid (UA), an ubiquitous triterpenoid with multifold biological roles, is distributed in various plants. This study was conducted to investigate the therapeutic effect and potential mechanisms of UA against hepatic steatosis in a high-fat diet (HFD)-induced obese non-alcoholic fatty liver disease (NAFLD) rat model.in vitro. HFD-induced adverse changes in the key genes, which participated in hepatic lipid metabolism, were also alleviated by UA treatment. Furthermore, UA significantly ameliorated HFD-induced metabolic disorders, including insulin resistance, inflammation and oxidative stress.Obese NAFLD model was established in Sprague-Dawley rats by 8-week HFD feeding. Therapeutic role of UA was evaluated using 0.125%, 0.25%, 0.5% UA-supplemented diet for another 6 weeks. The results from both morphologic and histological detections indicated that UA significantly reversed HFD-induced hepatic steatosis and liver injury. Besides, hepatic peroxisome proliferator-activated receptor (PPAR)-α was markedly up-regulated at both mRNA and protein levels by UA. Knocking down PPAR-α significantly inhibited the anti-steatosis role of UA These results demonstrated that UA effectively ameliorated HFD-induced hepatic steatosis through a PPAR-α involved pathway, via improving key enzymes in the controlling of lipids metabolism. The metabolic disorders were accordingly improved with the decrease of hepatic steatosis. Thereby, UA could be a promising candidate for the treatment of NAFLD. Non-alcoholic fatty liver disease (NAFLD) encompasses a cluster of liver disorders, ranging from accumulation of fat in liver (steatosis) to nonalcoholic steatohepatitis (necrosis and inflammation), with some people ultimately progressing to fibrosis, cirrhosis and liver failure. Because of its high prevalence in conjunction with obesity, diabetes, and insulin resistance, NAFLD is being increasingly appreciated as a hepatic manifestation of the metabolic syndrome and represents a major cause of liver-related morbidity and mortality The pathogenesis of NAFLD is still unclear, although several hypotheses exist. One generally accepted theory is the “two-hit” hypothesis However, there is currently no Food and Drug Administration (FDA) approved treatment for NAFLD or nonalcoholic steatohepatitis. NAFLD is considered reversible through modifying lifestyle in vitro and in vivo, suggests that UA has a variety of biological roles, including anti-inflammatory, anti-oxidative, anti-mutagenic, anti-carcinogenic, anti-microbial, anti-atherosclerotic, and anti-hyperlipidemic effects Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is widely distributed in various plants, and has become an integral part of human diet All protocols in this study were approved by the Medical Ethics Committee of Harbin Medical University  and were performed in accordance with the National Institutes of Health regulations for the care and use of animals in research.Male Sprague-Dawley rats  were maintained at a 12∶12 h light: dark cycle, and given water ad libitum. Animals were housed in cages individually in an environmentally controlled room at 21±2.0°C, and 50±5% humidity.After one week adaptation, rats (6 weeks of age) were randomly divided into normal-fat diet (NFD) group (n = 13) and HFD group (n = 70) by weight, and fed for 8 weeks, respectively (Diet formula was showed in Obese NAFLD rats (n = 40) were randomly sorted into 4 groups in equation by weight, and fed HFD, 0.125% UA (L-UA), 0.25% UA (M-UA), and 0.5% UA (H-UA) supplemented HFD for 6 weeks, respectively. Rats in control group (n = 10) were maintain with NFD. UA (98% purity) was bought from Zelang Medical Technology Co., LTD .Food intake and body weight were measured daily and weekly, respectively. At week 14, rats were anaesthetised with sodium pentobarbital anaesthesia  and blood was collected from the abdominal aorta. Serum was obtained and stored at −80°C until analysis. Tissues, including liver, skeletal muscle, perirenal and epididymal adipose tissue were removed, weighted and snap-frozen in liquid nitrogen, and stored at −80°C immediately.2 and 95% air. Oleic acid (OA) and fenofibrate were purchased from Sigma.A human normally hepatic immortal cell line HL-7702 obtained from Shanghai Cell Biology Institute, Chinese Academy of Sciences Serum glucose (GLU), TG, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and FFA levels were determined with an automatic analyzer . Serum inflammatory factors and hormones were measured by using various commercially available ELISA kits for rat. Specifically, for tumor necrosis factor (TNF-α), monocyte chemotactic protein-1 (CCL2/MCP-1), leptin, and adiponectin levels measurements, the Enzo Life Sciences ELISA kit was used ; for serum interleukin (IL)-6 levels, the R&D Systems ELISA kit  and for insulin levels the BioVendor ELISA kit . Serum malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured with commercial kits using enzymatic methods .At the time of killing, small pieces of liver were fixed immediately in 10% buffered formalin. After paraffin embedding, 5 µm sections were deparaffinized in xylene and were rehydrated through a series of decreasing concentrations of ethanol. Sections were stained with hematoxylin and eosin (HE). Alternatively, portions of fresh liver were flash-frozen and cryostat sections were cut and prepared for staining with Oil Red O. Photomicrographs were taken on a Nikon Eclipse E600 microscope .Total RNA from rat liver tissue or HL-7702 cells were extracted using TRIzol reagent . Real-time PCR was performed with the SYBR Green PCR Master Mix and a 7500 FAST Real-time PCR System (Applied Biosystems), using the primers shown in RNA interference was performed as described previously β-actin, and carnitine palmitoyltransferase (CPT)-1 , peroxisome proliferator-activated receptor (PPAR)-α, PPAR-γ and sterol regulatory element-binding protein (SREBP)-1c , fatty acid translocase (FAT/CD36) and acyl-CoA: diacylglycerol acyltransferase (DGAT)-1 , histone H3, AMP-activated protein kinase (AMPK) and phosphorylated-AMPK . Experiments were replicated at least three times, and a representative blot is shown.The detailed protocol of whole tissue or HL-7702 cells protein extracting and Western blot in our laboratory was described previously Total lipids  were extracted from tissues (liver and muscle) and measured using the method as described P values less than 0.05 were considered statistically significant.One-way analysis of variance (ANOVA), followed by the post-hoc test, was performed to analyze the data with SPSS software . Data were expressed as means ± S.E.M., and Obese NAFLD rat model was successfully established after 8 weeks HFD feeding. Body and liver weight along with serum and liver TG contents in HFD-fed rats were markedly increased compared to NFD group . After aDynamic change of body weight was measured during 14 weeks feeding period, and body weight gain was obtained by calculating the average change during UA treatment. M- and H-UA supplement significantly decreased HFD-induced increase of body weight at week 14 , and HFDSerum TG, FFA and LDL-C, but not TC and HDL-C were significantly elevated in HFD rats compared to that in NFD rats. All the three supplemental dose of UA significantly decreased HFD-induced increase of serum TG, FFA and LDL-C in a dose-dependent manner . Serum βNAFLD was characterized by the disorder of the expression of a series of lipid metabolic genes, including the increase of DGAT, FAT/CD36, SREBP-1c, ACC, FAS and the decrease of PPAR-α, CPT-1 in vitro. In this study, OA significantly elevated intracellular TG contents in human normally hepatic cells. OA-induced TG deposition was markedly reversed by UA in a dose-dependent manner. Fenofibrate, which was used as a PPAR-α agonist, was shown to reduce OA-induced steatosis , and further fibrosis and cirrhosis Hepatic lipid accumulation and/or it induced inflammation and insulin resistance, act as a powerful oxidative stressors, causing overproduction of reactive oxygen species (ROS) and depleting the concentration of natural cellular antioxidants In summary, we penetratingly reported the curative role of UA on NAFLD with obesity for the first time. Moreover, our results indicated that UA reversed HFD-induced hepatic steatosis. Further, this beneficial effect of UA was associated with a PPAR-α participated pathway. Inflammation, insulin resistance and oxidation stress conditions were accordingly ameliorated with the improvement of lipid metabolism. This is the first study that recommends UA, a safe natural phytochemicals, for the treatment of NAFLD.


Human hearing develops progressively during the last trimester of gestation. Near-term fetuses can discriminate acoustic features, such as frequencies and spectra, and process complex auditory streams. Fetal and neonatal studies show that they can remember frequently recurring sounds. However, existing data can only show retention intervals up to several days after birth.th, 36th, and 37th weeks of gestation. Six weeks later we assessed the cardiac responses of 25 exposed infants and 25 naive control infants, while in quiet sleep, to the descending melody and to an ascending control piano melody. The melodies had precisely inverse contours, but similar spectra, identical duration, tempo and rhythm, thus, almost identical amplitude envelopes. All infants displayed a significant heart rate change. In exposed infants, the descending melody evoked a cardiac deceleration that was twice larger than the decelerations elicited by the ascending melody and by both melodies in control infants.Here we show that auditory memories can last at least six weeks. Experimental fetuses were given precisely controlled exposure to a descending piano melody twice daily during the 35Thus, 3-weeks of prenatal exposure to a specific melodic contour affects infants ‘auditory processing’ or perception, i.e., impacts the autonomic nervous system at least six weeks later, when infants are 1-month old. Our results extend the retention interval over which a prenatally acquired memory of a specific sound stream can be observed from 3–4 days to six weeks. The long-term memory for the descending melody is interpreted in terms of enduring neurophysiological tuning and its significance for the developmental psychobiology of attention and perception, including early speech perception, is discussed. By 35weeks Gestational Age (GA), cochlear biomechanics and frequency selectivity are mature and absolute auditory thresholds are about 10 dB Hearing Level in the premature infant For airborne sounds recorded within the amniotic fluid in the gestating ewe e.g.,  and in win utero after a 6-week retention interval, when they were 1-month old infants. A review of the data supporting the phenomena of prenatal auditory memory in three psycho-biological domains follows.Near term fetuses and newborns can remember simple and complex sounds that frequently occurred earlier in prenatal life. Here, we will refer to fetal auditory memory when fetuses or infants respond to a sound they had previously and frequently experienced before birth differently than do non-exposed fetuses or infants. It has been examined in three developing psycho-biological domains Habituation of fetal gross body movements and heart rate responses to loud airborne noises and tones th–38th weeks GA Fewer studies have examined the effect of repeated exposure to complex auditory streams within the fetal period. Musical stimuli are reported to elicit an increase in movements Most prenatal auditory learning studies in the newborn used various versions of the maternal voice, speech, language and musical stimuli as reinforcers in operant learning tasks. Operant choice procedures with newborns can tell us whether and which characteristics of a sound can affect its power to reinforce behavior , they and a control group were stimulated one time, in quiet sleep, with the descending melody or with an ascending control melody. Both melodies were composed specifically by Claire Gérard  so as to differ mainly in the direction of their contour; they had very similar spectra, almost identical amplitude envelopes but precisely inverse melodic contours. The two melodies had been previously shown to reliably elicit similar HR decelerations in 37–38 weeks GA fetuses during quiet sleep In this study, we assessed prenatal auditory memory for a descending contour melody after a 6-week retention interval. Fetuses were exposed to the melody twice daily between their 352 (1) = 0.35, p = 0.56. Because our interest was in the cardiac orienting or cardiac attention response, not in startles, i.e., somato-cardiac coupling effects, HR accelerations were not analyzed any further. All other infants (n = 32) showed a cardiac deceleration to the melodies. In the Control Group, 60% of the infants (nine boys and six girls) showed a cardiac deceleration; six had the Control Melody and nine the Experimental Melody. In the Experimental Group, 68% of the infants (9 boys and 8 girls) showed a cardiac deceleration; nine had the Control Melody and eight the Experimental Melody. There was no significant difference in the number of cardiac decelerations between groups as a function of the melodies, χ2 (1) = 0.54, p = 0.46. Deceleration responses were examined in detail.All 50 infants tested showed a stimulus elicited HR change. The stimuli evoked small startle movements accompanied by HR acceleration in 18 of them: 10 infants (40%) in the Control Group and 8 infants (32%) in the Experimental Group. The number of accelerations did not differ between groups, χHR during the 15 s Prestimulus Period and the 15 s Stimulation Period were analyzed separately with a mixed ANOVA, having Group  and Melody  as between factors and Time (1–150) as the within factor.The mixed ANOVA was conducted on HR in bpm. The main effects of Group, F  = 1.57, p = .22; Melody, F  = 2.08, p = .16 and the Group x Melody interaction, F  = 0.24, p = .63, were not significant. The main effect of Time was significant, F  = 1.26, p = .018, but all interactions involving Time were not: In fact, all F-values were <1.0 and had p-values between .59 and .91. This analysis indicates that HR did not differ between the 4 SubGroups during the 15 s Prestimulus Period, but small unsystematic moment to moment variations occurred in all groups see .2p = .32; Time x Group, F  = 1.38, p<.0017, η2p = .05 and Time x Melody, F  = 1.79, p<.00001, η2p = .06. Time alone explained a high percentage of variance (32%) because the stimulation always elicited a cardiac deceleration. HRs reached their lowest values between 9 and 10 seconds after stimulus onset  digital HR values during the 15 s Stimulation Period were converted to z-scores based on the subject's mean and standard deviation during the 15 s Prestimulus Period. The normalization controls for individual differences in baseline mean and variability that can affect the amplitude of the stimulus-elicited HR changes us onset . Interpr2p = .37, and SubGroup x Time interaction, F  = 2.48, p.<.00001, η2p = .14. For the contrast with the Control SubGroup tested with the Experimental Melody, the F- statistics were: Time, F  = 8.37, p.<.00001, η2p = .36 and SubGroup x Time interaction, F  = 1.92, p.<.00001, η2p = .11. For the contrast with the Control SubGroup tested with the Control Melody the F- statistics were: Time, F  = 6.22, p.<.0001, η2p = .34 and SubGroup x Time interaction, F  = 1.73, p.<.0001, η2p = .13. When these three SubGroups were assessed together in a mixed ANOVA, only the main effect of Time was statistically significant, F  = 9.40, p.<.0001, η2p = .31. The F values for the effect of SubGroup, F  = .24, and the SubGroup x Time interaction, F  = .44 had P values ≥.79. The results of the non-parametric Mann-Whitney U test were consistent with those of the parametric analyses. The maximum HR deceleration value reached by Experimental infants tested with the Experimental Melody was significantly lower than those of the infants in the three other SubGroups pooled together, U = 52, p = .028, 1-tail test.This interpretation is supported by three separate mixed ANOVAs with SubGroup  and Time (1 – 150) as factors. In each case the main effect of SubGroup was not significant, the F-values for df  and df  ranged between 0.81–1.94 and their p-values ranged between .18–.39. In each case the Time factor was statistically significant, because the stimuli always elicited a cardiac deceleration. Most importantly, all SubGroup x Time interactions were also statistically significant. When the Experimental SubGroup tested with the Experimental Melody was contrasted with the Experimental SubGroup tested with the Control Melody the F- statistics were: Time, F  = 8.76, p.<.00001, ηth, 36th and 37th weeks of gestation affected their cardiac reactions to that melody after a six-week retention interval, when they were one-month old infants.In sum, statistical analyses indicated that there were no differences between the HRs of the four SubGroups during the Prestimulus Period and that onset of the melodies elicited a significant cardiac deceleration in all SubGroups. The deceleration evoked by the Experimental Melody in Experimental infants was 3–5 SD more profound than the decelerations elicited by the Control Melody in the Experimental infants and by both melodies in the Control infants, which did not differ. We concluded that exposing fetuses to a Descending Melody twice each day during the 35th and 38th week GA can last at least six weeks. Fetuses were given precisely controlled exposure to a descending piano melody twice daily during the 35th, 36th, and 37th weeks of gestation. Six weeks later we assessed the cardiac responses of 25 exposed infants and 25 naive control infants, while in quiet sleep, to the descending melody and to an ascending control piano melody. The melodies had precisely inverse contours, but similar spectra, identical duration, tempo and rhythm, thus, almost identical amplitude envelopes. When tested at 1-month of age, 60% of the Control infants and 68% of the Experimental infants displayed a short latency HR deceleration with the two melodies. It peaked below pre-stimulus levels 9–10 s after melody onset and then returned towards baseline, a pattern typical of an orienting response The introductory review showed that memory for sounds repeatedly experienced before birth has been observed up to three-four days after birth. Our results demonstrated that the retention interval of a memory established between the 35Significantly, the HR of the Experimental Group tested with the Experimental Descending Melody decelerated to a level that was twice as low as the HR decelerations of the Experimental Group tested with the Control Ascending Melody and the Control Groups tested with either melody. The present research contributes to the understanding of early auditory development and learning by demonstrating that a mean of 947 presentations of a specific melodic contour, that ended two weeks before birth, is sufficient to impact the autonomic nervous system six weeks later when the infants were one-month old and in quiet sleep. More precisely, the Experimental fetuses showing a HR deceleration to the Experimental Descending Melody had a mean of 39.47, SD = 1.97 (range = 36 – 42) familiarization sessions. Thus, the mean number of presentations of the Experimental Melody was 947 (range = 864 – 1008), among them 631 presentations at 80 dB SPL. The mean retention interval between the end of the exposure period and postnatal testing was 43.47 days, SD = 1.37 (range = 41–46 days).This result and the research cited in the introduction indicate that recurring auditory experience during human prenatal development of the auditory system can impact multiple psychobiological systems. It produces quantitative, not qualitative, differences in the behavior, e.g., magnitude of reinforcer effectiveness in newborns or the magnitude of HR decelerations seen here. These quantitative differences in behavior suggest that a major or common effect of recurring prenatal experience with a sound is an enhanced subsequent perceptual sensitivity to the sound. In this study, it was not the spectra of musical notes, or the tempo, rhythm, duration of the melody or its amplitude envelope that was most salient, but rather, the arrangement of those aspects over time, the descending melodic contour. The high amplitude response to the contour implies that it left some kind of memory trace.One possibility is that the profound cardiac deceleration represents a correlate, mediated by the autonomic nervous system, of tuning of the auditory system. The increased sensitivity to various characteristics of a complex sound repeatedly experienced before birth, and observed in different psychobiological domains see , could bPerceptual tuning of the auditory system can function as a “physiological memory, [which] is an enduring neuronal change sufficiently specific to represent learned information” 5 words spoken by their mother, most of them occurring in normal conversational utterances averaging about 10 words in length in uteroBecause there is no, or little attenuation of the mother's voice, speech and language inside the amniotic fluid see , many ofThe potential capacity of physiological tuning to explain a variety of early auditory perceptual phenomena has an almost exact analog in the domain of prenatal chemosensory functional development, perception and memory in animals More generally, the memory traces of the fetus' auditory environment in which early maturation of the auditory system occurs can result in the infants' differential responding to specific stimuli. Infants' prenatal auditory experiences can result in either a lack of responsiveness to a postnatal auditory stimulus or, on the contrary, an enhancement of orienting and attention to the stimulus. Physiological memories, i.e., sensory tuning, can bridge the temporal and conceptual gaps in our psycho-biological understanding of the early development of attention, perception, discrimination and recognition of complex sounds before and after birth.One hundred and twenty five volunteer pregnant mothers with healthy uncomplicated singleton pregnancies participated in the study. They were recruited during prenatal information sessions at Port-Royal Maternity – CHU Cochin-Saint Vincent de Paul University Hospital, Paris. They were briefed about the research and gave informed written consent to participate.The Experimental Group consisted of 76 infants whose mothers volunteered to listen to a recorded melody each day for three weeks, from the beginning of 35 weeks GA to the beginning of 38 weeks GA, according to instructions. Eighteen infants were not tested because their mothers could not completely follow the prenatal familiarization instructions, or were unable to come to the laboratory at the appointed time. Seventeen infants did not complete testing because they had not entered quiet sleep The Control Group consisted of 49 infants with no prenatal exposure to the melody. Nineteen infants did not complete testing because they had not entered quiet sleep within two-three hours (n = 15) or their HR pattern was sinusoidal during the Prestimulus Period (n = 4). Data from five others were rejected because the HR signal was lost for more than 2 s (n = 2) or experimenter error occurred (n = 3). The final Control Group consisted of 25 infants (15 boys and 10 girls): Ten were tested with the Control Melody (3 girls and 7 boys) and 15 with the Experimental Melody (7 girls and 8 boys).All infants in the final samples were born at term, 39–41 weeks GA, with weights appropriate for gestational age and healthy. At the time of postnatal testing, the Experimental and Control Groups did not differ in GA, respectively, Mean = 44 weeks, SD = 2.2 days vs. Mean = 44 weeks, SD = 1.76 days, t (48) = 0.64, p = .52, or in Postnatal Age, respectively, Mean = 30.44 days, SD = 6.36 vs. Mean = 31.48 days, SD = 6.74), t (48) = 0.56, p = .58.Two melodies were composed for the study. They were played on a piano, synthesized with MIDI software, stored on a PC and normalized (Cool Edit Pro software). The melodies have been previously tested in our lab. They reliably elicited small HR decelerations in 37–38 weeks GA fetuses during quiet sleep at the sound pressure level that was used for the familiarization sessions . Each melody lasted 3.6 s and had nine notes from the same two octave bands, C4 and C5. The Experimental Melody had a descending melodic contour, from G5 (784 Hz) to E4 (330 Hz) and the Control Melody had an ascending melodic contour, from G4 (392 Hz) to B5 (988 Hz). Although the magnitude of pitch change from one note to the next increased from the beginning to the end of each melody in opposite directions, the octave-change ratios of successive notes were identical, viz., 1/8, 1/8, 2/8, 1/8, 1/8, 2/8, 3/8 and 2/8. Constructing inverse melodic contours while keeping their change ratios the same allowed four of the nine notes to be identical but required the other five notes to differ slightly between the melodies. The total “on” time of the melodies was the same and the series of consecutive eighth notes and quarter notes were identical, giving the melodies the same tempo, rhythm, and thus, almost identical amplitude envelopes. In sum, the piano melodies had similar spectra, almost identical amplitude envelopes but precisely inverse melodic contours . A “Stimin utero  of the sequences gradually increased so as not to startle the mother. The first sequence was emitted at 60±2 dB Leq (62 dB peak level), the second sequence at 70±2 dB Leq (72 dB peak level) and the four following sequences at 80±2 dB Leq (82 dB peak level). The maximum SPL of the Experimental Melody, i.e., 80 dB Leq, is conservatively estimated to be 55–60 dB tero see . The ordEach mother was given the same audio equipment to use at home, a Sony tape player and two small loudspeakers with an integrated amplifier (SP 681: 12.5 cm×9 cm×4 cm). The volume controls of the tape player and speakers were calibrated in the laboratory by measuring SPL with a sound level meter (ACLAN-SDH80) placed 5 cm from a mother's abdomen when the loudspeakers were placed at 50 cm from each side of her abdomen. The volume controls were locked and could not be changed by the mothers. The materials were retrieved at the end of the three weeks familiarization period.During postnatal testing, the 15 s Stimulation Sequence of the Experimental and Control melodies was delivered at 60±2 dB Leq, 62 dB peak level, measured with the sound level meter placed where the infants' head would be inside the testing chamber (see procedure). Background noise inside the chamber was 45±2 dB SPL. Sound pressure level of the testing stimulus was checked before each test.th, 36th, and 37th weeks of gestation. Two familiarization sessions occurred each day with a minimum of 5 hours delay between sessions. Mothers were asked to be comfortably seated or lying in a silent room between the two loudspeakers, placed at about 50 cm from their abdomen, to relax and not to make any noise, speak or touch their abdomen. They were allowed to read silently. Each exposure session ran automatically for 12 minutes when the mother started the tape player. If the session was interrupted for any reason the mothers were told to stop the familiarization session and start it again at least 5 minutes later. After each session they filled out a log describing the conditions and time of the session, how they felt physically, how much they felt their fetus move and what they did during the hour before the session.The Experimental subjects were exposed to the Experimental Melody in the mother's home during the 35In principle, over 21 days of exposure a fetus would experience 1008 repetitions of the 3.6 s Experimental Melody (42 sessions×24 occurrences/session). If only the loudest Stimulation Sequences, at 80 dB SPL, are considered, i.e., the ones most likely to be heard by a fetus, there would be 672 repetitions of the Experimental Melody (42 sessions×16 occurrences/session). In fact, the mean number of exposure sessions that occurred in the final Experimental Group was 39.24, SD = 2.05 (range = 35–42 sessions), for a mean of 942 total presentations of the 3.6 s Experimental Melody, among them 628 presentations at 80 dB SPL. The mean retention interval, mean time between the last prenatal familiarization session and postnatal testing, was 44 days (6 weeks and 2 days), SD = 2.62, range = 41–50 days.The mothers entered the laboratory that was quiet and dimly lighted, so as to not wake up sleeping infants and to facilitate the onset of sleep in infants who were awake. They were reminded of the details of the testing procedure and could ask questions. The transducer (7.3 cm in diameter) of a Doppler cardiotocograph (Hewlett-Packard M1351A), connected to a PC computer, was placed on the infant's chest and held in place by a stretched cloth belt. Room-temperature almond oil was used as a contact product for the transducer instead of regular gel whose chilling effect could startle or wake the infants. If the infant was awake, we relied on the mother's judgment about the best way to put her infant to sleep . When asleep, the infant was seated in a semi-reclined position in an infant chair that was placed inside a sound attenuating chamber (62 cm high×57 cm wide×64 cm long) with the infant's head facing the opening in the front (58 cm high×53 cm wide) to allow observation by the experimenters. A piece of cloth that retained the infant's or mother's odor, e.g., a sheet, blanket or a piece of clothing, was placed alongside the infant's head and neck. Inside the chamber two small loudspeakers like the ones used for the prenatal familiarization sessions were located on each side of the infant's head, 20 cm from each ear.Testing started when two experimenters agreed that the infant had been in quiet sleep for 5 consecutive minutes, with a stable low variability HR (SD≤3 bpm), regular slow breathing movements, eyelids closed with no REM, no body movements and no muscle tone Infant HR was recorded continuously during the 5 min before stimulation, during stimulation and the 5 min after stimulation, to see whether behavioral state changed. Two experimenters monitored the continuous analog HR tracing in beats per minute (bpm) that appeared on the PC screen, along with a display of its mean and standard deviation that was updated every 30 s, and on the paper printout of the cardiotocograph (paper speed = 2 cm/m). A proprietary software program (C. Kervella & R. Humbert) controlled stimulus presentations and sampled the analog cardiac signal at a rate of 10 times/second, digitized and stored each value online.After the test session, all mothers answered a questionnaire regarding the infant's health and development and to verify that the daily acoustic characteristics of their home had nothing in particular that could potentially affect the infants' reactions to the test stimuli .: Our criterion for a response was that the subject's HR during the 15 s Stimulation Period increased by at least 3 SD (z≥+3) for a cardiac acceleration, or decreased by at least 3 SD (z≤−3) for a cardiac deceleration, from the Prestimulus Period, and remained at that level for at least two consecutive seconds. The 300 digital HR data were also analyzed with parametric (mixed ANOVAs) and non parametric (Chi-Square and Mann-Whitney U test) statistics with Statistica 8 software.Three experienced observers  examined each cardiac paper tracing to verify that the infant's HR pattern corresponded to a quiet sleep pattern 5 min before, during and 5 min after stimulation. The subject was retained only if the three observers agreed. The raw digital HR values were checked for missing or erroneous values, which were corrected by linear interpolation. Next, we checked whether stimulus-elicited cardiac responses occurred during the 15 s Stimulation Period. For each subject, the 150 (10/s) digital HR values of the 15 s Prestimulus Period (15 s preceding stimulus onset) were converted to z-scores


Infection with the Human T-Lymphotropic Virus 1 (HTLV-1) is associated with bronchiectasis in Indigenous Australians [Retrospective cohort study at Alice Springs Hospital, central Australia. Medical records were reviewed for all Indigenous adults admitted between 2000-2006 with radiologically confirmed bronchiectasis and known HTLV-1 serostatus.One-hundred and twenty patients were admitted during the study period, HTLV-1 serology was performed for 92 (75.8%) patients. Western blots confirmed HTLV-1 infection in 52 (58.4%) cases and were indeterminate for 3 patients. HTLV-1 seropositive patients more often had bilateral bronchiectasis  and ground glass opacities  on HRCT chest, but were less likely to have a pathogen isolated during an infective exacerbation . HTLV-1 seropositive patients were more likely to have cor pulmonale  and to suffer bronchiectasis-related deaths . The mortality rate for the entire cohort during the 7 year period was 34.2%. Median age of death for both groups combined was 42.5 years. Only HTLV-1 seropositive patients were admitted for treatment of infected skin lesions and this was the major predictor of subsequent death from any cause on multivariable analysis .In an Indigenous Australian cohort HTLV-1 infection is associated with bronchiectasis and an increased risk of pulmonary hypertension and death.