Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP  promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into  GAL genes, is tightly coupled to preceding steps in the gene expression pathway by the Sac3-containing TREX-2 complex (Nuclear export of messenger RNA (mRNA) is mediated by transport factors that bind to mature mRNAs and enable them to overcome the barrier function within the nuclear pore complex (NPC) transport channel generated by nuclear pore proteins that have regions rich in phenylalanine-glycine repeats (FG-nucleoporins) . The NXF complex ,16 that  complex ,18. Alth complex ,20. Howe complex . Fluores complex ,22, and  complex , it remaDrosophila  with 25 nM siRNA  was injected into an cells ,8,11,41,an cells ]. The GAan cells A and B. an cells A. For alan cells B as antian cells B. TherefPrevious studies suggested that GANP functions in the recruitment of mRNPs from nuclear processing centres to NPCs . The mRNIn principle, the role of GANP in the nuclear export of specific classes of mRNA could be due either to its function as a scaffold protein for TREX-2 components at nuclear pores or its proposed chaperonin function in the nucleoplasm. At steady state, a large proportion of TREX-2 components ENY2 and PCID2 are stably associated with the nuclear pore basket , consistOur findings indicate that GANP promotes the nuclear export of a specific subset of mammalian mRNAs that use NXF1 as an export factor and also provide biological insight into the function of GANP . RoughlyOur results raise the interesting possibility that GANP may define a ‘fast track’ from the nucleus for export of specific classes of mRNAs. The nuclear mRNA export kinetics of GANP-dependent transcripts were more rapid than those of GANP-independent transcripts. Importantly, although both subsets of transcripts use NXF1 as their primary nuclear export factor, representative transcripts that showed impaired nuclear export following GANP depletion showed faster nuclear mRNA export kinetics, consistent with the hypothesis that the selective participation of GANP may increase the rate of nuclear export of transcripts. This GANP-specific subset of mRNAs is highly enriched for those encoding proteins that are required for RNA processing and splicing, mRNP and ribosome biogenesis and gene expression. The observation that GANP-specific transcripts have short half-lives, are highly expressed, have fewer exons than GANP-independent transcripts and their encoded proteins may facilitate rapid adaptation to changes in gene expression, suggests a functional rationale for why GANP participates in the export of only a subset of NXF1-bound mRNPs. Interestingly, we find that targeting of GANP to nuclear pores requires TPR, a scaffold protein of the NPC that maintains regions of heterochromatin exclusion surrounding NPCs . Based oIn conclusion, we propose that GANP promotes the nuclear export of specific classes of mRNAs that may enable rapid adaptation to changes in gene expression. In this way, export of these transcripts to the cytoplasm is optimized, ensuring that cells can respond quickly to stimuli.The data sets are available in the Gene Expression Omnibus (GEO) database under the accession number GSE54481.Supplementary Data are available at NAR Online.MRC ; MRC Programme grant [G1001522 to A.R.V. supports V.O.W.]; MRC Programme grant [U105178939 to M.S.]; CRUK ; Wellcome Trust . Funding for open access charge: University of Cambridge.Conflict of interest statement. None declared.


West Nile virus (WNV) is transmitted to humans primarily by infected mosquitoes. However, WNV also can be transmitted through infected blood products or solid organs . Since 23  and 46 red blood cells/mm3 . This CSF sample was negative for bacteria by Gram and acid-fast stains, for cryptococcal antigen by latex agglutination, and for cytomegalovirus, BK virus, herpes simplex virus, JC virus, human herpes virus 6, and varicella-zoster virus by polymerase chain reaction (PCR); however, the sample was not tested for WNV. Magnetic resonance imaging showed meningeal and cortical changes consistent with inflammation, and electroencephalogram findings were consistent with diffuse encephalopathy. The patient’s mental status did not improve after discontinuation of sedation; support was withdrawn, and he died on hospital day 47. Postmortem evaluation showed diffuse encephalitis. WNV IgM antibodies were identified in serum collected on hospital day 43. WNV RNA was detected by TaqMan reverse transcription-PCR (RT-PCR) on brain and spinal cord tissues collected at autopsy.In August 2012, a man with non-Hodgkin’s lymphoma was admitted to hospital for chemotherapy and autologous stem cell transplant. His cancer was in partial remission after six cycles of chemotherapy. He had been screened for subclinical infections 14 days before stem cell collection, and all testing, including WNV NAT, was negative. Stem cells were collected 8 days before admission and transplanted on hospital days 8 and 9. The patient developed gastrointestinal symptoms on hospital day 18, followed by fever and hypotension on hospital day 28. On hospital day 29, he developed altered mental status, somnolence, and respiratory failure; sedation and mechanical ventilation precluded a full neurologic assessment. Cerebrospinal fluid (CSF) collected on hospital day 30 showed an elevated glucose  and normal protein  with two white blood cells/mmThe patient was hospitalized continuously for 4 weeks before illness onset without known outdoor exposure; therefore, mosquito-borne WNV transmission was unlikely. A public health investigation was conducted to determine the source of his WNV infection and prevent further transmission by removal of any contaminated blood products. The patient had received allogenic, leukoreduced, irradiated blood products during hospital days 13–30, including 6 units of red blood cells, 2 units of platelets, and 2 units of fresh frozen plasma. He received 2 additional units each of red blood cells and platelets on day 44.The hospital blood bank investigated the blood products received before hospital day 43, when the patient’s first WNV-positive serum was collected. WNV serologic testing was conducted on all 10 associated donors. Nine donors had no evidence of WNV infection by WNV IgM enzyme-linked immunosorbent assay. One apheresis donor was positive for WNV IgM antibodies and for WNV neutralizing antibodies by plaque reduction neutralization testing performed on serum collected 56 days after the implicated donation. This donor reported no history of illness before or after donation and no travel, but did report outdoor exposure with mosquito bites. The apheresis platelet unit was donated on the recipient’s hospital day 9 and transfused on day 14. The implicated donation was initially screened as part of a minipool with five other donations; the MP-NAT was reactive using the Cobas TaqScreen West Nile Virus Test (Roche Molecular Systems). However, all of the units comprising the minipool were nonreactive when tested by ID-NAT using the same assay and were released into the blood supply per FDA guidance at the time of the investigation .In addition to the implicated unit of platelets, fresh frozen plasma was also derived from the original donation but was not transfused. The quarantined unit of fresh frozen plasma was retrieved and tested for WNV. Freshly thawed aliquots from this unit were reactive for WNV on four of five replicate Cobas TaqScreen West Nile Virus Tests, but WNV RNA could not be detected by conventional Taqman RT-PCR. An aliquot from the same plasma unit, tested 58 days after being thawed and stored at 39.2°F (4.0°C) was nonreactive by the Procleix WNV NAT assay (Gen-Probe Incorporated). The unit was positive for WNV IgM antibodies by microsphere-based immunosorbent assay, WNV immunoglobulin G antibodies by enzyme-linked immunosorbent assay, and WNV neutralizing antibodies by plaque reduction neutralization testing.Per FDA guidelines, the donor was deferred from making further donations for 120 days after the implicated donation. The donor had made seven donations of platelets and plasma in the 120 days before the implicated donation, and one donation of platelets and plasma 35 days after the implicated donation. The blood products from those donations had been transfused to nine recipients. Four of these recipients had since died; however, their deaths were thought to be related to causes other than WNV infection because none had discernible signs or symptoms of WNV disease. However, no clinical samples were available for testing. The five surviving recipients were monitored clinically and remained asymptomatic but were not tested for WNV infection.This report describes a fatal case of WNV disease after probable transfusion-associated transmission via platelets collected from an asymptomatic infected blood donor. Although transfusion-transmitted WNV has been reported after initial screening was nonreactive by MP-NAT, it has not been documented after screening was nonreactive by ID-NAT. This case demonstrates the potential for transmission of WNV through transfusion of blood products that screen nonreactive by ID-NAT, despite having detectable WNV-specific IgM and neutralizing antibodies.What is already known on this topic?Blood collection agencies in the United States routinely screen donors for West Nile virus (WNV) RNA using minipool nucleic acid testing (MP-NAT) augmented by individual donation testing (ID-NAT). Since 2003, approximately 3,500 reactive units have been removed from the blood supply, preventing transfusion transmission of WNV. This system is effective; however, 12 transfusion-associated cases have been identified since implementation of WNV NAT screening.What is added by this report?This report describes the first probable case of transfusion-associated WNV infection in which the donation was reactive by MP-NAT but nonreactive by ID-NAT on initial screening. The case occurred in an immunosuppressed patient. Virus-specific immunoglobulin M and neutralizing antibodies could be detected in the implicated donation. These findings are suggestive that transmission occurred from a blood donation with a low concentration of WNV even in the presence of presumptively protective WNV-specific antibodies.What are the implications for public health practice?Clinicians should maintain a high index of suspicion for cases of transfusion-associated WNV disease, especially in immunosuppressed patients with compatible symptoms who have recently received blood products. Further evaluation is required to determine the relative risks and benefits of different strategies to manage MP-NAT–reactive minipools when all constituent donations are nonreactive by subsequent ID-NAT.Persons infected with WNV typically develop a transient viremia that occurs 1–2 weeks after infection and lasts approximately 7 days. Viremia, detectable by NAT, is present before symptom onset and early in the course of illness, but usually resolves as the patient develops WNV antibodies. FDA recommends that blood collection agencies screening for WNV by MP-NAT establish criteria to switch to ID-NAT, which is more sensitive than MP-NAT, during periods of high WNV activity in their collection areas (It is likely that this transfusion-associated transmission occurred because the donor had a waning viremia that was sufficiently low to be inconsistently identified by either MP-NAT or ID-NAT. NATs performed during initial screening and during retrospective testing of the plasma co-component from the implicated donation yielded discrepant results. The Cobas TaqScreen West Nile Virus Test and Procleix WNV NAT assays typically detect lower levels of WNV RNA than conventional RT-PCR. However, at very low levels of viremia, NAT assays performed on minipools or individual units can exhibit test-to-test variability and might require replicate testing to detect viral RNA –8. In adIn published reports of transfusion-associated transmissions of WNV, IgM could not be detected in implicated donations when stored donor segments or co-components were available for testing ,3. HowevSince the implementation of routine screening of blood products for WNV, transfusion-associated WNV infections have been rare. However, clinicians should consider WNV disease in any patient with compatible symptoms who has received a blood transfusion during the 28 days before illness onset. This is particularly important in immunosuppressed patients, such as stem cell recipients, who might be more susceptible to WNV infection at very low viral concentrations in the transfused blood product and, once infected, are at greater risk for developing WNV neurologic disease. Suspected cases should be reported immediately to hospital blood banks and public health authorities to facilitate prompt investigation and quarantine of potentially infectious blood products. Further evaluation is required to determine the relative risks and benefits of different strategies to manage MP-NAT-reactive minipools when all constituent donations are nonreactive by subsequent ID-NAT.


The multifaceted process of aging inevitably leads to disturbances in cellular metabolism and protein homeostasis. To meet this challenge, cells make use of autophagy, which is probably one of the most important pathways preserving cellular protection under stressful conditions. Thus, efficient autophagic flux is required for healthy aging in many if not all eukaryotic organisms. The regulation of autophagy itself is affected by changing metabolic conditions, but the precise metabolic circuitries are poorly understood. Recently, we found that the nucleocytosolic pool of acetyl-coenzyme A (AcCoA) functions as a major and dominant suppressor of cytoprotective autophagy during aging. Here, we propose an epigenetic mechanism for AcCoA-mediated autophagy suppression that causally involves the regulation of histone acetylation and changes in the autophagy-relevant transcriptome. Drosophila AcCoA synthetase enhances autophagic protein clearance and prolongs life span.Besides its role as a fuel for several anabolic pathways, the central energy metabolite AcCoA represents the only known donor of acetyl groups for protein acetylation. This function has lately been recognized as an important regulatory instrument, affecting enzyme activities, and protein complex stability as well as the epigenetic status of chromatin and transcription factor activity. Among others, the autophagic pathway has repeatedly been reported to be subject to regulation by various protein acetylation and deacetylation activities. Therefore, we reasoned that AcCoA availability might directly modulate autophagic activity. Indeed, using the established model of yeast chronological aging, we recently unveiled that the nucleocytosolic pool of AcCoA functions as a metabolic repressor of autophagy in aging cells. Depleting the nucleocytosolic pool of AcCoA is sufficient to induce and maintain high autophagic rates crucial for longevity. This mechanism is phylogenetically conserved, since brain-specific knockdown of nucleocytosolic ACH1 (forming AcCoA from acetate) or the gene for the mitochondrial pyruvate transporter, FMP37/MPC1 (shuttling pyruvate into mitochondria for subsequent conversion to AcCoA) caused a shut-off in autophagic flux upon aging. Both genetic constraints correlate with cytosolic accumulation of the AcCoA precursor acetate. This leads to hyperactivation of the nucleocytosolic AcCoA-synthetase Acs2, culminating in increased acetylation of cellular proteins, particularly histones. Acs2 activity is causally responsible for autophagy limitation since simultaneous knockdown of ACS2 recovers autophagy in ach1 mutant cells—albeit acetate accumulation still occurs.In yeast, AcCoA is synthesized in the mitochondrial and the nucleocytosolic compartments, generating 2 distinct subcellular pools. This enabled us to spatially dissect the role of AcCoA biosynthesis pathways in regulating autophagy. Blocking the mitochondrial route to AcCoA by deletion of either the yeast CoA-transferase-encoding gene ACS2 not only reinstates autophagy but also completely prevents histone hyperacetylation induced by deletion of ACH1. We thus hypothesized that epigenetic changes  guide autophagy in the long-term context of chronological aging, mediating the cellular adaptation to continuous stress. In line with this idea, histone H3 hyperacetylation, resulting from an Acs2-mediated increase in nucleocytosolic AcCoA biosynthesis, is associated with transcriptional downregulation of several autophagy-essential ATG genes . This consequently leads to reduced protein levels (as shown for Atg7), suggesting that limitation of essential ATG transcripts may restrict age-associated autophagic activities. However, complex changes of the autophagy-relevant transcriptome are likely to be present under these conditions and the subset of genes that accounts for AcCoA-mediated repression of autophagy must be elucidated in the future.Importantly, knockdown of H3-K14,18Q/K14,18R allowed normal growth but increased the age-associated autophagic activity above that of wild-type cells  at histone lysyl residues in regulating autophagy during aging, we introduced point mutations at those histone H3 sites that we had found to be hyperacetylated upon increased nucleocytosolic AcCoA production. As the precise stoichiometry of histone acetylation is currently not described for aging yeast cells, we created a panel of (nonacetylable) lysine mutations that likely mimicked differently acetylated states compared with wild-type conditions. Therefore, unique deacetylation-mimicking lysine to arginine (KR) mutations were also combined with lysine to glutamine (KQ) mutations, to simulate various degrees of acetylation “locked” at a certain level. Among the tested mutants, intriguingly, a mixed mutation of pe cells . While ppe cells .Other recent studies reported on the epigenetic modulation of autophagy. Upon modulation of histone H4 acetylation at lysine 16, cells responded by changing their autophagy-relevant transcriptome that determines whether autophagy becomes a vital or lethal process, demonstrating once more that (nuclear) epigenetic events may have been underestimated in their capacity to fine-tune autophagic activities. This may particularly apply to conditions that differ from the most frequently studied case of autophagy induced by acute nutrient withdrawal. Such “tuning” capacities reportedly relate to the general autophagic flux, the frequency of cells with active autophagy, the vital or lethal outcome of autophagy, and the size of autophagosomes. It is tempting to speculate that epigenetic control of the autophagy-relevant transcriptome would also influence the spatiotemporal specificity of cargo selection during macroautophagy.AcCoA integrates vital nutrition pathways and thus translates environmental clues to the epigenome. Therefore, our findings may explain diet-dependent life span and autophagy regulation, and could extend to other pathophysiological conditions known to depend on the status of histone (de)acetylation. Pharmacological interventions targeting the nucleocytosolic pathway of AcCoA generation may represent an attractive strategy to combat autophagy-associated pathological conditions.


Arabidopsis thaliana (Arabidopsis) [total 117 proteins listed in Table Mitochondria and chloroplasts are two distinct organelles essential for plant viability. They evolved from prokaryotic endosymbionts and share a common ancestor with extant Gram-negative bacteria  or Ncytosol-Cintermembrane space (for TA) orientation. β-Barrel proteins are integrated into the membrane via multiple TM-β-strands, whose formation appears to require evolutionarily conserved machinery in the membrane. Marty et al.  machinery, which catalyzes the general pathway to import nuclear-encoded precursor proteins from the cytosol. Among the TOC components are homologous GTPases Toc159 and Toc34, which recognize the precursors and regulate their import, and Toc75, which forms a protein conducting channel. In Arabidopsis, there are four Toc159 isoforms which show substrate selectivity, two catalytically redundant Toc34 isoforms, and one functional Toc75 encoded on chromosome III Table  review tUnder phosphate starvation, phospholipids in the cell membranes, mainly those in extraplastidic compartments, are used as the source of free phosphates and substituted by galactolipids made in the chloroplast outer envelope. Murakawa et al.  have usetoc159-null mutant is seedling-lethal thus has been examined as progenies of heterozygous parents. Tada et al. (toc159 mutants on the sucrose-supplemented media to the point that viable seeds can be obtained. This cost-effective method should be useful to study not only the toc159-null plant but also other recessive lethal mutants of photosynthesis.Hardre et al.  report aa et al.  have estIn summary, the collection highlights various questions about the organelle outer membranes and interdisciplinary approaches employed to address them. The future research should use these and other strategies to answer questions about the proteins of known functions, in particular those involved in protein homeostasis, as well as those of unknown functions Table . The ediThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A substantial proportion of newly diagnosed HIV infections in sub-Saharan Africa occur within serodiscordant cohabiting heterosexual couples. Intimate partner violence is a major concern for couple-oriented HIV preventive approaches. This study aimed at estimating the prevalence and associated factors of intimate partner physical and sexual violence among HIV-infected and -uninfected women in Togo. We also described the severity and consequences of this violence as well as care-seeking behaviors of women exposed to intimate partner violence.WHO Multi-country study on Women’s Health and Life Events questionnaire.A cross-sectional survey was conducted between May and July 2011 within Sylvanus Olympio University Hospital in Lomé. HIV-infected women attending HIV care and uninfected women attending postnatal care and/or children immunization visits were interviewed. Intimate partner physical and sexual violence and controlling behaviors were assessed using an adapted version of the Overall, 150 HIV-uninfected and 304 HIV-infected women accepted to be interviewed. The prevalence rates of lifetime physical and sexual violence among HIV-infected women were significantly higher than among uninfected women . Forty-two percent of the women reported having ever had physical injuries as a consequence of intimate partner violence. Among injured women, only one-third had ever disclosed real causes of injuries to medical staff and none of them had been referred to local organizations to receive appropriate psychological support. Regardless of HIV status and after adjustment on potential confounders, the risk of intimate partner physical and sexual violence was strongly and significantly associated with male partner multi-partnership and early start of sexual life. Among uninfected women, physical violence was significantly associated with gender submissive attitudes.The prevalence rates of both lifetime physical and sexual violence were very high among HIV-uninfected women and even higher among HIV-infected women recruited in health facilities in this West African country. Screening for intimate partner violence should be systematic in health-care settings, and specifically within HIV care services. At a time of increased investments in couple-oriented HIV prevention interventions, further longitudinal research to better understanding of HIV-serodiscordant couple dynamics in terms of intimate partner violence is needed. According to the World Health Organization (WHO), intimate partner violence is defined as the behavior, within an intimate relationship, that causes physical, sexual, or psychological harm or suffering . SeveralBesides its dramatic physical and mental consequences, intimate partner violence has also been identified as an important risk factor for sexually transmitted diseases acquisition and particularly HIV infection . IntimatSub-Saharan Africa is the region reporting the highest epidemiological burden of HIV worldwide, with women accounting for 58% of newly acquired HIV infections . FurtherIn Togo, a West African country with an estimated prevalence rate of HIV infection of 3.4% nationwide and a predominantly heterosexual epidemic , intimatEspoir Vie Togo, one leading organization providing care and psychological support to people living with HIV/AIDS in Togo.This project was an international collaboration between the INSERM  Research Centre U897 based in Bordeaux (France), the Sylvanus Olympio University Hospital in Lomé, and the non-governmental organization A cross-sectional survey was conducted between May and July 2011 within the Lomé University Hospital. The study population was constituted by volunteer participants attending clinical services in the hospital. HIV-infected women attending regular HIV care visits constituted the first group. Women attending postnatal care and/or children immunization visits, and who had been diagnosed HIV-uninfected during their last pregnancy, constituted the second group. For these two study groups, women aged at least 18 years, declaring having a current intimate partner or having ever had one, were eligible to recruitment.The survey was conducted in three stages. The first preparatory phase consisted of a methodological workshop between Togolese and French researchers to discuss the study design, validate the survey instruments, and address relevant ethical issues. A local team of four psychologists was recruited to adapt concepts described in the questionnaires to the local context, train interviewers on the study tool, ethical and privacy terms, and supervise data collection.Second, a pilot study was carried out to test the acceptability, adequacy, and understandability of the recruitment procedures and questionnaire. Survey feasibility data and interviewer’s observations were integrated within the final survey procedures and tools.Finally, every working day during the study period, each eligible woman was systematically asked to participate in the survey and to provide signed informed consent to be sequentially enrolled. Trained female health-care staff administered the questionnaire during a 20-min face-to-face interview in a private room.Espoir vie Togo.All interviews were carried out in French, and all participants were provided information about local support services dedicated to women experiencing intimate partner violence. HIV-infected women were informed about the psychological support available at The study protocol was approved by the National Ethic Committee of the Ministry of Health of Togo (N°0125/2011/MS/CAB/DGS/DPLET/CBRS). Written informed consent was obtained from all participants; and all data collection tools were strictly anonymous.WHO Multi-Country Study on Women’s Health and Life Events questionnaire (version 10)  , 18, 19.All women participating in the survey were asked about their life experience on specific acts of physical and sexual violence induced by their intimate partner. Physical violence was defined as the use of physical force causing bodily harm . Within Additionally, we explored attitudes toward partner controlling behaviors that we summarized in six statements, illustrating different gender submissive situations to which women were asked to agree or not. We developed a scoring system to define a ‘submission index’ summarizing women’s attitudes toward partner controlling behaviors based on the sum of the number of positive answers to six questions detailed in We also documented women’s sociodemographic characteristics , data on the women’s partner  and couple relationships (polygamy and concurrent relationship), women’s employment and financial autonomy , and the modalities of women’s entry in sexual life . We also investigated women’s mental health  and the history of non-partner violence .p-value of <0.25 were included in the multivariable model. To select the final adjusted model presented in this paper, we used a backward elimination method using a p-value of 0.05 . Age of first sexual intercourse was dichotomized into two categories with a cut-off defined by the age at sexual majority in Togo (e.g. 18 years). The prevalence of lifetime physical and sexual violence was estimated separately among HIV-infected and -uninfected women and also compared between the two groups. Chi-square tests were performed to determine statistically significant differences. Sociodemographic and behavioral factors associated with lifetime physical and sexual violence in both groups were then assessed using logistic regression. Both univariate and multivariate analyses were carried out. Variables found to be statistically associated with intimate partner violence with a  of 0.05 . AdjusteOverall, 454 women attending the Sylvanus Olympio University Hospital were informed about the study and screened for eligibility; all of them accepted to be interviewed and were included in the study. One hundred and fifty women were HIV-negative and 304 were HIV-positive.Sociodemographical and behavioral characteristics of women in both groups are described in p<0.01). The proportion of HIV-uninfected women having completed at least primary education was significantly higher than among HIV-positive women . HIV-infected women were significantly more likely than uninfected women to report some degree of financial autonomy . HIV-uninfected women were more likely to be using a contraceptive method at the time of the survey than HIV-infected women .Concerning women’s sociodemographic profile and financial autonomy, HIV-infected women were significantly older than uninfected women . HIV-infected women were significantly more likely to start sexual life through a first forced sexual intercourse than uninfected ones .Concerning modalities of entry to sexual life, the proportion of women having entered sexual life before the age of 18 was higher among HIV-infected than HIV-negative women  and to declare that their partner had concurrent relationships out of the household . Reported rates of partner alcohol consumption  and partner involvement in fights and riots  were more frequent among HIV-infected women than uninfected women.Concerning partner’s profile characteristics, HIV-infected women were more likely to live within polygamous households than uninfected women . The proportion of HIV-infected women reporting loss of interest  and suicidal attempts  during the past 12 months tended to be higher than among uninfected ones but differences were not statistically significant.Concerning women’s mental health, suicidal thoughts during the past 12 months were significantly more frequent among HIV-infected women than among the uninfected ones .Finally, HIV-infected women were slightly more likely to agree to submissive statements than HIV-uninfected women . Similarly, HIV-infected women reported a significantly higher prevalence rate of lifetime sexual violence compared to the uninfected ones . The lifetime prevalence rate of both types of violence combined  was 51.6% (95% CI: 45.3–56.6) among HIV-infected women and significantly higher than among uninfected women  and the frequency of sexual violence during childhood (before 15 years old) was 13.5% among HIV-infected versus 4.0% among uninfected ones , 194 (77.2%) reported physical injuries as a consequence of this violence  reported needing medical care for their injuries. From those needing medical care, 93 (58.1%) received medical care and 14 (7.2%) were even hospitalized. Among injured women in care, 49 (52.6%) disclosed real causes of injuries to medical staff and none of them was referred to local organizations to receive appropriate psychological support .p=0.02, and HIV infected: aOR: 2.2; 95% CI: 1.3–3.6; p<0.001). Otherwise, the profile of HIV-infected and HIV-negative women reporting intimate partner physical violence was different , and education level only for HIV-infected women . Uninfected women not using any contraceptive method at the time of the survey were more likely to be victims of intimate partner physical violence . Among uninfected women, the odds of intimate partner sexual violence were significantly higher among those reporting a first coerced sexual intercourse .Among women’s sociodemographic and sexual characteristics, age was associated with physical violence only for HIV-uninfected women . In the univariate analysis, loss of interest during at least two weeks  and having had suicidal thought at least once over the last 12 months  were also associated with having experienced intimate physical violence among HIV-infected women.In terms of women’s mental health status, having ever attempted suicide was strongly associated with a history of intimate partner physical violence, for HIV-infected women only .As detailed in p=0.02). For HIV-infected women, intimate partner sexual violence was associated with reporting suicidal thoughts , having partners who were involved in fights and/or riots with other men , and reporting a higher submission index .Otherwise, the profile of women reporting intimate partner sexual violence differed according to HIV serological status. HIV-uninfected women reporting having partners maintaining concurrent relationships out of household were more likely to report intimate partner sexual violence  and sexual violence (23–58%) 9% and se, or in eBruises were overall the most frequent injuries reported among African women victims of physical violence , but theAlthough the prevalence of physical and sexual violence varied according to the HIV status among women of our sample, we identified three associated factors that are common to both groups. Reported partner multiple concurrent relationships were associated with higher rates of physical and sexual intimate partner violence regardless of serological status. Other African studies have reported similar findings, whereby women whose partner had several female partners were more likely to report sexual intimate partner violence and women suspecting their partner’s infidelity were at higher risk of any kind of violent act perpetrated by their male partner , 24–26. We observed that starting sexual life before 18 years old was very frequent and appeared to be a risk factor of intimate partner violence for all women, regardless of their serological status. The high proportion of HIV-infected women experiencing a first forced sexual intercourse in our study is consistent with findings in South Africa . Since sFinally, prevailing submissive attitudes among Togolese women, expressed by an overall high acceptance of partner controlling behaviors and high submission index score, were associated with intimate partner violence, principally among HIV-infected women. Many women fearing intimate partner violence, even when aware of an HIV risk, may feel powerless to discuss infidelity, condom use, and HIV testing with their male partner . RenewedSeveral limits to our study need to be acknowledged. First, intimate partner violence may be a very sensitive subject for women and data collection was based on past experiences. We thus should not rule out a potential recall bias. To reduce information bias, however, interviewers were trained before conducting the survey and interviews were carried out in a private office. Further, due to the cross-sectional design of our study, we were not able to demonstrate a causal link between HIV infection and intimate partner violence or to explore the dynamic among these factors; however, our findings may have confirmed some of the factors to target when aiming at preventing intimate partner violence within clinical care services. Moreover, we did not present data on intimate partner psychological violence, as this would have required a thorough psychological assessment that could not be performed at the time of the survey. Finally, our study was conducted among a specific population of women attending a hospital facility in Lomé, and having been tested for HIV at least once in their life; thus, our results are not representative of all women in Togo.WHO Multi-Country Study on Women’s Health and Life Events questionnaire. Our study highlights that intimate partner violence is a true public health issue in Togo, with a high social burden and severe health consequences on women, and especially among HIV-infected women. Our findings argue, in particular, for systematic case detecting of intimate partner violence – as well as any form of violence – within HIV services, to provide adequate medical care to women in need and to advise them about help-seeking strategies. Taking into account the high rate of HIV-discordant stable couples in sub-Saharan Africa, couple-oriented interventions are a priority among primary HIV preventive strategies (One of the main strengths of our study, however, is its contribution to the pool of data available on intimate partner violence, data that can be compared to other settings as it was based on the use of rategies , and becrategies , 13, theFinally, tackling cultural representations and the social construction of masculinity and traditional gender norms in sub-Saharan African contexts is an important challenge to achieve behavior change in terms of sexual health and must be addressed from adolescent ages. Further longitudinal research is needed to understand HIV-serodiscordant couple dynamics with regard to intimate partner violence in African contexts and thus improve the acceptability and efficacy of couple-oriented HIV preventive interventions. Intimate partner violence, being highly prevalent in resource-constrained settings and a major public health concern, global health policies must turn more firmly to this issue.


Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome. The fundamental unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around a histone octamer containing four core histones . Nucleosome positioning in the genome affects the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. Genomic regions associated with regulatory proteins are associated with a pronounced sensitivity to DNase I digestion, and are thus called DNase I hypersensitive sites (DHSs). It is well known that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. However, it is less clear what factors determine genome-wide nucleosome positioning, especially in intergenic regions. We mapped both nucleosome positioning and DHS datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. We confirmed the same association of DHSs with phased nucleosomes in the human genome. We conclude that genomic loci associated with a diverse set of regulatory proteins are major determinants of nucleosome phasing, and this is true in both genic and intergenic regions. In vitro nucleosome assembly studies in yeast (Saccharomyces cerevisiae) and Caenorhabditis elegans have confirmed the DNA sequence preferences in nucleosome formation in vivo is determined by several factors that can override the sequence preferences, including gene transcription, action of nucleosome remodeling complexes, and presence of histone variants and histone modifications in vivo nucleosome positions in S. cerevisiaeThe fundamental unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around a histone octamer containing four core histones  Relationships between nucleosome organization and gene expression have been well demonstrated in several model eukaryotes. Phased nucleosome arrays have been observed on both sides of the promoters of active genes Oryza sativa) has been used as model species for plant genome research. The rice genome is relatively small (∼400 Mb) and is one of the best sequenced genomes in higher eukaryotes Rice  in the rice genome We also examined nucleosome phasing surrounding TSSs in the rice genome independently of DHSs. Clearly-phased nucleosomes were detected downstream of TSSs of expressed genes , but notWe noticed that the average lengths of most DHSs in different genomic regions, except for those located in proximal promoters, were similar in the rice genome, with ∼50% DHSs in the size of 35–150 bp. In contrast, the lengths of DHSs in proximal promoters were more variable, including ∼79% DHSs >150 bp . We suspp<0.001). In addition, 5,197 and 2,898 of the IPA1-binding sites contain the IPA1-binding motif, GTAC, and another over-represented motif, TGGGC[C/T], respectively We wanted to examine if phased nucleosomes are associated with the binding sites of specific rice transcription factors. IDEAL PLANT ARCHITECTURE1 (IPA1), a member of the SPL transcription factor family, is a key regulator in determining plant architecture and enhancing grain yield in rice Mapping of both DNase-seq and MNase-seq datasets revealed peaked MNase-seq reads from both forward and reverse strands on both sides of DHSs . These rThe DHSs in intergenic regions were associated with a unique nucleosomal positioning pattern. The intergenic DHSs lacked the forward MNase-seq peak and the reverse MNase-seq peak, respectively, on the two sides of the DHSs , suggestSince the DHSs in proximal promoters were more variable in lengths , we furtThe sizes of 2,495 DHSs  in proximal promoters were <140 bp, which is shorter than the sequences required to wrap a single nucleosome. These DHSs did not appear to span a nucleosome, but appeared to be enriched in the 3′ portion of the -1 nucleosome  or were p<0.005, Kolmogorov–Smirnov test). We also calculated linker lengths in the human genome using human MNase-seq data We observed a superposition between the forward and reverse MNase-seq reads in genic and promoter regions, which indicates very little or no space between 5' ends of forward and reverse oriented reads . HoweverA weakness of the above method of calculating linker length is that it is influenced by the severity of MNase digestion as MNase can either digest into the nucleosome core DNA or fail to completely digest the linker DNA. Thus, we used an alternative method to estimate the linker lengths in different genomic regions in rice. Since the position of the nucleosome center (dyad), which can be identified as the middle position of each paired-end read, is not affected by different levels of MNase digestion, we can calculate the spacing of between two adjacent nucleosomes using the midway point between paired MNase-seq reads rather than 5' ends. We found that the average spacing between two nucleosomes adjacent to intergenic DHSs was ∼191 bp , which iWe exploited the genomic datasets from the human genome to examine a similar association of DHSs with nucleosome positioning. Human CD4+ T cell line has been extensively used in epigenomics profiling, including histone modifications Since H2A.Z-associated nucleosomes were found in regions that were previously thought to be nucleosome free, we investigated if DHSs in the human genome span H2A.Z-associated nucleosomes. Mapping of H2A.Z ChIP-seq dataset Drosophila melanogasterC. elegansArabidopsis thalianaGenome-wide nucleosome positioning maps have been generated in several eukaryotes, including yeast A barrier model was proposed to explain genome-wide nucleosome positioning S. cerevisiae strains containing large genomic regions from other yeast species Gaffney et al. (2012) recently mapped nucleosomes surrounding the binding sites of 35 different transcription factors in human lymphoblastoid cell lines. Strongly positioned nucleosome arrays were found to flank the binding sites, including those at least 1 kb away from a known TSS Drosophila were quantitatively correlated with DNA accessibility in chromatin that can be measured by the DNase I sensitivity A. thaliana, APETALA1 and SEPALLATA3, were covered by DHSs We demonstrate that DHSs in the rice genome are flanked by phased nucleosome arrays on both sides , which iDrosophila, the binding sites of transcription factors that are flanked with strongly positioned nucleosome arrays were more sensitive to DNase I digestion and have more pronounced DNase I footprints A DHS-based barrier can be permanent, such as the promoters associated with constitutively expressed genes, or be temporarily, such as binding sites of transcription factors associated with tissue- or organ-specific gene expression. Regulatory proteins can bind DNA tightly or loosely , which may result in “hard” barriers or “soft” barriers. Hard barriers will result in well positioned and well phased nucleosome arrays; whereas soft barriers may result in “fuzzy” and less phased nucleosome arrays. In In summary, we demonstrate that DHSs located across the rice genome are flanked by strongly phased nucleosome arrays. We confirmed the same phenomenon in the human genome by analyzing publically available datasets. Our results support the barrier model for nucleosome organization as a general feature of eukaryote genomes. We propose that genome-wide nucleosome positioning in the eukaryotic genomes is orchestrated by genomic regions associated with regulatory proteins.Rice cultivar “Nipponbare” seeds were germinated at room temperature for three days. Germinated seeds were then sowed in soil to continue to grow in the greenhouse. The seedlings continued to grow for two weeks under 12 hrs day/night cycles and 32°C/27°C corresponding to day and night, respectively. The seedlings were then harvested for nuclei isolation, the same growing stage/condition used for developing DNase-seq and RNA-seq datasets previously http://www.r-project.org/).We mapped the MNase-seq reads to the rice genome (TIGR release 5) using MAQ software MNase-seq data has been deposited to NCBI under accession number GSE53027.Figure S1Patterns of nucleosome positioning around DHSs in the rice genome. The nucleosome positioning profiles were shown around the DHSs located in (A) proximal promoters (within 200 bp upstream of a TSS); (B) distal promoters (200–1000 bp upstream of a TSS); (C) within genes; (D) downstream regions of genes (within 200 bp downstream of gene transcription); (E) intergenic region and (F) 10,000 randomly selected genomic regions. Y-axes show normalized reads (read number in per bp genome in per million reads) within 1 kb upstream and downstream around the DHSs. Ellipses indicate the nucleosomes within (grey) and outside (black) of DHSs. Arrows in (a-d) indicate the direction of gene transcription. Paired MNase-seq reads were used in mapping nucleosome positioning.(PDF)Click here for additional data file.Figure S2An illustration of mapping phased nucleosomes with different linker lengths. After MNase digestion, linker DNA was presumably digested and the remaining DNA fragments wrapped on nucleosome core were included in library construction. Longer linkers between adjacent nucleosomes may cause a shift between the sequence reads derived from the forward and reverse strands, respectively.(PDF)Click here for additional data file.


Although paroxysmal atrial fibrillation (AF) is known to be initiated by rapid firing of pulmonary veins (PV) and non-PV triggers, the crucial role of cardiac autonomic nervous system (ANS) in the initiation and maintenance of AF has long been appreciated in both experimental and clinical studies. The cardiac intrinsic ANS is composed of ganglionated plexi (GPs), located close to the left atrium-pulmonary vein junctions and a vast network of interconnecting neurons. Ablation strategies aiming for complete PV isolation (PVI) remain the cornerstone of AF ablation procedures. However, several observational studies and few randomized studies have suggested that GP ablation, as an adjunctive strategy, might achieve better clinical outcomes in patients undergoing radiofrequency-based PVI for both paroxysmal and nonparoxysmal AF. In these patients, vagal reactions (VR) such as vagally mediated bradycardia or asystole are thought to reflect intrinsic cardiac ANS modulation and/or denervation. Vagal reactions occurring during cryoballoon- (CB-) based PVI have been previously reported; however, little is known on resulting ANS modulation and/or prevalence and significance of vagal reactions during PVI with the CB technique. We conducted a review of prevalence, putative mechanisms, and significance of VR during CB-based PVI. Paroxysmal atrial fibrillation (AF) is known to be initiated by rapid firing of pulmonary veins (PVs) and non-PV triggers , 2. In pThe cardiac intrinsic ANS is composed of ganglionated plexi (GPs) and a vast network of interconnecting neurons , 11. TheAblation strategies targeting the PV and aiming for complete PV isolation (PVI) remain the cornerstone of AF ablation procedures . RadiofrSeveral observational studies and few randomized studies have suggested that GP ablation, as an adjunctive strategy, might achieve better clinical outcomes in patients undergoing radiofrequency-based PVI for both paroxysmal and nonparoxysmal AF –22. In tVagal reactions occurring during CB-based PVI have been previously reported; however, little is known on resulting ANS modulation and/or prevalence and significance of VR during PVI with the CB technique. We conducted a review of prevalence, putative mechanisms, and significance of VR during CB-based PVI.p = 0.001 for comparison between groups) [Ganglionated plexi ablation aiming for modulation/denervation of the intrinsic cardiac ANS has been proposed as an adjunctive strategy for radiofrequency catheter ablation of paroxysmal and nonparoxysmal AF –22. Gang groups) .The CB technique targets the PVs and adjacent myocardium with the aim of obtaining a complete PVI –18. It iLittle is known regarding intrinsic cardiac ANS modulation during CB-based PVI. Oswald et al. were the first to investigate the effect on ANS when applying cryoenergy at the PV ostia, although it should be mentioned that the PVIs were performed with the first generation CB  . Similar™, Medtronic) has been redesigned to enhance lesion quality. The goal was to create transmural contiguous lesions in order to achieve durable PVI. The number of refrigerant injection ports has been increased from 4 to 8 with a more distal orientation, resulting in higher refrigerant flow (for Arctic Front Advance 28 mm) that improves balloon cooling uniformity with lower temperature at the CB surface. Compared to the first generation CB, Arctic Front Advance CB has been demonstrated to increase single-shot PVI rate and decrease time to PVI as well as having shorter procedure and radiation exposure times [The second generation of CB . Using a univariate Cox proportional hazard regression analysis, Yorgun et al. found that VR and requirement of atropine administration or cardiac pacing for severe bradycardia decreased the risk of AF recurrence. Using a multivariate Cox proportional hazard regression analysis, the authors demonstrated that the need of atropine administration was an independent predictor of AF recurrence in patients undergoing PVI with the CB technique  while VR was not  [Yorgun et al. specifically studied the impact of VR during PV cryoablation on clinical outcomes in 145 patients with paroxysmal (81%) or persistent drug-refractory AF treated with the second generation CB . After a= 0.225) . Otherwin = 197) or the second generation CB (n = 109 patients). Main conclusion was that the use of the second generation CB resulted in lower late AF recurrence rate. They also demonstrated that both intraprocedural VR and second generation CB use were associated with fewer late AF recurrences. Furthermore, it was found that VR were more frequently observed with the second generation CB compared with the first generation CB . Also, systematic use of the largest 28 mm CB that resulted in more antral CB positioning compared to the 23 mm model might have explained, by itself, the higher prevalence of VR. Multivariate Cox proportional hazard regression analysis related to the late AF recurrence following single cryoablation identified left atrium diameter, early recurrence, and Artic Front Advance CB use as the only predictive factors of late AF recurrence. Vagal reactions tended to be predictive for lower late AF recurrence, but did not reach statistical significance .Aytemir et al. recently tried to define the predictive factors of late AF recurrence with the current CB when compared to the first generation CB . They reIn the three aforementioned studies, several findings are reported. First, the occurrence of VR during CB-based PVI is reported to be frequent, ranging from 36% to 50%, which is comparable to RF studies, and higher with the second generation CB , 26–28. In our practice, we currently use the CB technique for PVI in paroxysmal AF patients for nearly 8 years. In our experience of 980 PV cryoablation procedures performed in our institution  and although we did not specifically investigate the role of VR during PV cryoablation as a predictive factor for AF recurrence, we found a lower prevalence of vagally mediated bradycardia during CB application, about 5 to 10% (unpublished data). Even lower prevalence of VR was observed with the first generation CB compared with the second generation CB. Vagally mediated bradycardia was transient, was self-limited, and required exceptionally temporary pacing or atropine administration. Similar to previous studies, we noticed VR during the CB thawing phase or at time of CB deflation –28. VagaVagal reactions during CB-based PVI are not infrequent. Some authors consider VR during PV cryoablation as a marker of ANS modification. Due to the temporary effect of PV cryoablation on ANS activity with the first generation CB , questions remain regarding the mechanism of improved outcome for patients experiencing VR during CB-based PVI. Larger studies conducted with the second generation CB and achieving long-term follow-up are needed to validate this hypothesis and to confirm the putative mechanisms.


The folK-folP fusion was found across the eukaryotes and a sparse collection of prokaryotes. This suggests an ancient derivation with a number of gene losses in the eukaryotes potentially as a consequence of adaptation to heterotrophic lifestyles. In contrast, the folB-folK-folP gene is specific to a mosaic collection of Amorphea taxa . Next, we investigated the stability of this character. We identified numerous gene losses and a total of nine gene fission events, either by break up of an open reading frame (four events identified) or loss of a component domain (five events identified). This indicates that this three gene fusion is highly labile. These data are consistent with a growing body of data indicating gene fission events occur at high relative rates. Accounting for these sources of homoplasy, our data suggest that the folB-folK-folP gene fusion was present in the last common ancestor of Amoebozoa and Opisthokonta but absent in the Metazoa including the human genome. Comparative genomic data of these genes provides an important resource for designing therapeutic strategies targeting the de novo folate biosynthesis pathway of a variety of eukaryotic pathogens such as Acanthamoeba castellanii.Shared derived genomic characters can be useful for polarizing phylogenetic relationships, for example, gene fusions have been used to identify deep-branching relationships in the eukaryotes. Here, we report the evolutionary analysis of a three-gene fusion of  The resolution of ancient phylogenetic relationships is proving a difficult task . Rare gefolP  are therefore important antimicrobial agents Saccharom pathway  and gene pathway . Here, wAcanthamoeba castellanii sequencing project , we designed a range of overlapping polymerase chain reaction (PCR) primers . Acanthamoeba castellanii Neff strain was grown axenically in a modified M11 defined media  to encourage the transcription of folate biosynthesis pathway genes. Cells were collected and suspended in 1 ml of trizol reagent (Invitrogen) and RNA extracted using the single-step acid guanidinium thiocyanate–phenol–chloroform protocol as described by Chomczynski and Sacchi  to create a 25 µl PCR reaction mix (12.5 µl of Master Mix), 1 µl each primer (10 µM), 9.5 µl of Milli-Q pure water (Millipore), and 1 µl of template cDNA). Acanthamoeba cDNA was diluted to approximately 100 ng/µl using spectrophotometery (NanoDrop ND-1000). Thermocycling followed an initial incubation at 95 °C for 5 min, and cycling conditions details in supplementary table S1, Supplementary Material online followed by a 72 °C–5 min elongation step. See supplementary table S1, Supplementary Material online, for details of PCR primers used. Successfully amplified PCR products were gel-purified  and cloned using TA-cloning . Five clones were selected from each PCR reaction and externally sequenced using the M13/pUC vector primers via Sanger sequencing . The flanking vector sequences were removed; the sequences trimmed to areas of high chromatograph quality and ambiguously defined bases corrected. The overlapping sequences were then assembled into contigs using Sequencher (Gene Codes) version 4.10.1 program (http://www.genecodes.com/) producing a high-confidence consensus sequence for a partial ORF for the folB, folk, and folP gene fusion (GenBank Acc: AFW17812.1). These data demonstrate that the folB, folk, and folP genes are transcribed as a single three-domain gene fusion. It should be noted that subsequently a draft genome and predicted proteome of Acanthamoeba has been released  folB, folk, and folP three gene fusion and Bacillus cereus single domain unfused-genes  as a search query to identify putative homologues using the basic local alignment search tool (tBLASTn) against a set of protistan RNAseq “in-house” data sets. This data set included the unicellular opisthokont Fonticula alba, the amoebozoan Copromyxa protea, and the breviate Pygsuia biforma (PCbi66). From these data, we were able to identify components of the folB, folk, and folP genes from Fonticula and Copromyxa, but not in the breviate P. biforma (PCbi66). Phylogenomic analysis demonstrates that breviate flagellates are related to opisthokonts and the Apusomonadida (We used the monadida .Copromyxa protea (strain CF08-5), the BROAD Institute (Boston) for F. alba (strain ATCC 38817), and Macrogen (South Korea) for the P. biforma (PCbi66). Raw sequence read data were filtered based on quality scores with the fastq_quality_filter program of FASTXTOOLS (http://hannonlab.cshl.edu/fastx_toolkit/), using a cutoff filter . Filtered sequences were then assembled into clusters using the Inchworm assembler of the TRINITY r2011-5-13 package . All unmasked protein alignments are included as supplementary material, Supplementary Material online, on GitHub (DOI: 10.5281/zenodo.11716) as MASE files which includes the alignment mask information  following the protocol supplied by the manufacturer. Construction of cDNA libraries and Illumina RNAseq was performed by the Institut de Recherche en Immunologie et Cancérologie of Université de Montréal (Canada) for  package . The F.  Seaview ).http://genome.jgi-psf.org/), and the Broad Institute (http://www.broadinstitute.org/) genome databases (as of November 2013) using three separate folate biosynthesis domains from B. cereus  and the D. purpureum (XP_003290941) folB, folk, and folP three gene fusion divided into the three-domain regions. Care was taken to survey the major eukaryotic, archaeal, and bacterial groups; to this end additional BLAST searches were conducted using multiple start seeds from diverse taxa to check for alternative sequence hits. The amino acid sequences gathered for each domain were run through the REFGEN tool . Alignments were then manually corrected and masked in SeaView (version 4.2.4)  or multiple highly similar genes from the same genome (sister branches on preliminary phylogenetic trees) were removed from the alignments.Using BLASTp and tBLASTn  we initiGEN tool . The mulGEN tool  was usedn 4.2.4) . Sequencsupplementary figs. S1–S7, Supplementary Material online). Sequences shown to form long branches in the phylogenetic analysis were removed from the alignment to reduce the risk of long-branch attraction artifacts . Each analysis was conducted as two independent runs of four metropolis-coupled Markov chain Monte Carlo [MCMCMC] chains and continued until convergence of these runs as determined using the Tracer (version 1.5) .Phylogenetic analysis was conducted using both Bayesian and maximum-likelihood methodologies with the model of amino acid substitution selected using ProtTest3  . Burn-inion 1.5)  was thenfolB, folk, and folP) that encode sequentially acting enzymes: DHNA, HPPK, and DHPS  folB-folK-folP found in Amoebozoa, the basidiomycete fungi Postia placenta, Coprinopsis cinerea, and Melampsora laricis-populina, and the microsporidian E. hellem,  folK-folP found in a subset of ascomycete fungi, Puccinia graminis, Capsaspora owczarzaki, Sphaeroforma arctica, and a diverse range of Diaphoretickes (At the core of pterin branch of the folate biosynthesis pathway are three genes (and DHPS . In some014143.2—) suggestequences ); 3) folretickes .folB gene using standard BLAST similarity searches (B. cereus folB gene and the folB domain of the D. purpureum folB-folK-folP gene fusion as a search seed against the NCBI GenBank nonredundant (NR) protein database . These analyses failed to identify any additional putative folB encoding genes in the eukaryotic genomes available in the GenBank NR database.In many Diaphoretickes groups, including SAR, Cryptophyta, and the Excavata, we could not identify a searches . To conffolB)  has been suggested to represent a functional replacement of the DHNA enzyme (folB) . To inve sampled . These asupplementary figures S1–S6, Supplementary Material online, with all six trees demonstrating low levels of topology support while many features of the eukaryotic sections of the tree topologies are inconsistent with established multigene phylogenetic trees  and which encompasses ancient and divergent evolutionary groups. These alignment character numbers do not compare favourably to multigene analyses where it has been shown that in excess of 5,000 amino acid alignment characters are required to robustly resolve the Archaeplastida  and weak support in the reduced taxa folK analysis . The comprehensive folB phylogeny also shows the Br. floridae folB-folK gene fusion branching with prokaryote taxa with weak support . Collectively, these trees suggest that the Br. floridae folB-folK branching relationship is consistent with HGT into the Br. floridae genome or, alternatively, contamination of this genome project with a prokaryotic sequence. To explore these possibilities further, we found the genome sequence contig containing the Br. floridae folB-folK gene (GenBank acc: AC150408.2) demonstrating that the prokaryote like Br. floridae folB-folK gene is located in a 180,427 bp contig adjacent to genes that show standard patterns of animal sequence similarity. Analysis of the B. belcheri transcriptome demonstrated that an orthologue of the Br. floridae folB-folK gene is transcribed. Taken together these data suggest that the Br. floridae folB-folK gene is located on native source genome and it is not contamination. Therefore, it is likely to be a prokaryotic-derived HGT into this animal genome. However, it is interesting that an animal lineage could maintain only the first part of a pathway despite lacking the folP gene, whereas many other animal lineages have lost the entire pathway. Further to these data, we detected a putative folB gene in Trichoplax adhaerens and a putative folB-folK fusion gene in Nematostella vectensis. However, these genes were removed from further analyses due to difficulty in alignment of these sequences, as such their provenance and evolutionary ancestry remains questionable as noted on folP step present in Branchiostoma. Furthermore, we see evidence of incomplete pathways in other organisms, for example, the red alga Cyanidioschyzon lacks an identifiable standard folP gene  with weak topology support . Importantly, we note that the only members of the Diaphoretickes and Excavata (formerly the “bikonts”) possessing a putative folB gene are the Archaeplastida and that the folB gene of this eukaryotic group branches separately from the other eukaryotes within a clade of bacterial genes with moderate-to-strong posterior probability/bootstrap support  suggesting a separate evolutionary ancestry of this gene to that of the Opisthokonta and Ameobozoa. Given the taxonomic distribution of the folB gene across the Archaeplastida , this xenologue is most likely to have been derived either by an ancient horizontal gene transfer from a bacterial source into the Archaeplastida lineage or via the cyanobacterial endosymbiosis that gave rise to the plastid organelle, a process that has been suggested to lead to the acquisition of a number of genes of mixed bacterial ancestry  which suggested this gene product was localized to the cytosol or the mitochondria . However, because the Archaeplastida folB is not an orthologue of the Opisthokonta/Amoebozoa version and no additional Diaphoretickes and Excavata folB orthologues are currently available, our folB phylogenetic analysis does not represent a strict test of the monophyly of the folB-folK-folP gene fusion within the eukaryotes.Monophyly of the three-domain gene fusion would signify that the ancestry . Using tfolK and folP genes . This analysis again recovered a tree with low topology support values and taxonomic relationships inconsistent with established eukaryotic phylogenetic relationships . The taxon distribution of this character suggests that the folB tandem exon-duplication represents a novel genetic character that arose in the last common ancestor of the opisthokonts followed by the loss of these genes in Metazoa and some other opisthokont taxa , Naegleria (Excavata), and Thecamonas (Apusomonadida) genomes, suggesting that these protists acquire folate, or precursors of folate , by salvage from external sources. We can therefore infer that these heterotrophic characteristics have resulted in concordant loss of the de novo folate biosynthesis. Likewise the absence, or near absence, of the entire folate biosynthesis pathway in Entamoeba, Trichomonas, and Giardia suggests a dependence on hosts or phagocytosed food for provision of intact folate, as such inhibiting folate synthesis as a therapeutic target is not viable for these parasitic protists, but inhibition of uptake transporters of intact folate may offer an alternative therapeutic strategy.Using a comparative genomic and phylogenetic approach, we have identified the taxonomic distribution of a three protein domain encoding gene fusions in the pterin branch of the folate biosynthesis pathway. In the absence of strong phylogenetic signal demonstrating eukaryote-to-eukaryote HGT our analyses identified multiple gene loss events in different eukaryotic groups , suggesting that the capacity to manufacture folate de novo has been lost on multiple occasions within the eukaryotes. This is consistent with adaptation of these lineages to acquiring folate or folate intermediates from food sources and/or host organisms. Specifically, the comparative genomic data demonstrate that a complete pterin branch is absent from the Metazoa sampled, consistent with the hypothesis that animals acquire folate using “intact folate salvage” from digested food , putativfolK and folP genes were present, but a putative homologue of the folB gene was not identified. These results suggest that this part of the pathway is absent from these taxa or performed by a highly divergent or nonhomologous gene family. A paralogue of folB: folX has been identified in Escherichia coli with 30% identical amino acid residues. This protein was classified as an epimerase and performs the equivalent aldolase type reaction with less than 1% velocity as the DHNA encoded by the Ec. coli folB gene  because other taxa lacked the folB gene and a putative alternative transaldolase-encoding gene; a currently unidentified gene family must encode this enzyme  both olB gene  suggestiomologue  leading s enzyme . Later wsix-PTPS . AnalysiF. alba, Microsporidia, and a range of Amoebozoa . Moreover, two domain variations of these gene fusion forms were identified in a range of eukaryotes  and within the recently completed genome sequence . This work confirmed that Ac. castellanii transcribes a single gene fusion encoding the folB-folK-folP domain architecture and provides evidence of active folate biosynthesis via a complete pterin branch in Ac. castellanii. Acanthamoeba can cause keratitis infection of the cornea linked to use of contaminated contact lenses . Comparisons of the distribution of different folate fusion genes to the established Opisthokonta phylogeny  combinedvolution .folB-folK-folP gene fusion. Our phylogenetic analysis generally proved inconclusive, because we failed to recover tree resolution and specifically because there is no Diaphoretickes and Excavata orthologue of the Amoebozoa and Opisthokonta folB gene. Taken together the phylogenies, therefore, do not constitute an appropriate test of the monophyly of the three-domain gene fusion clade . Furthermore, as the individual folate pathway gene phylogenies were generally unresolved, it is possible that undetected cases of hidden paralogy, multiple folB tandem duplications, and HGT may have occurred in the evolution of this pathway. HGT is especially a concern as some literature suggests that gene clustering increases the possibility that genes become fixed by selection once they have undergone transfer. This is because they lead to the acquisition of functional modules, either as an operon and/or gene fusions , we use the more parsimonious interpretation of vertical inheritance to explain the gene distribution observed.Next, we used phylogenetic analysis to polarize the ancestry of the ns e.g., , 2011. SfolB-folK-folP three-domain gene fusion is monophyletic and arose once and before the diversification of the opisthokonts and amoebozoans. We do acknowledge that alternative hypotheses involving fissions and loss in the Diaphoretickes and Excavata taxa, or convergent gene fusions in the Amoebozoa and Opisthokonta taxa are only slightly less parsimonious given current data. This is an important concern as our data demonstrated that this gene fusion is not a stable character, subject to frequent gene fission and partial and total gene loss. Consequently, perhaps the overriding message of this work is that rare-derived genomic characters, such as gene fusions, can be noisy and therefore these data should not be applied to resolving evolutionary relationships without testing their ancestry and susceptibility to homoplasy.The phylogenies provided no strong support for the paraphyly and convergent evolution of the three-domain gene fusion in the Amoebozoa and Opisthokonta. Therefore, in the absence of strong signal to support an alternative hypothesis and based on current taxonomic distribution of this character, we currently favour the null hypothesis that the Supplementary figures S1–S7 and tables S1–S4 are available at Genome Biology and Evolution online .


The actin cytoskeleton is crucial at many junctures of normal immune function, and consequently there are many immune specific regulators of actin dynamics. A growing number of primary immunodeficiencies are being defined as caused by mutations in the genes encoding these regulators. In addition to immunodeficiency, immune dysregulation and autoinflammation are increasingly recognised to arise from defects within this pathway.To use next generation sequencing, and functional studies to identify the genetic cause for a severe unclassified autoinflammatory disease mimicking Behcet's disease in a consanguineous family.Two affected children in a consanguineous Pakistani kindred suffered from an unclassified autoinflammatory syndrome presenting in the first year of life with: severe oral ulceration resulting in scarring; perianal cutaneous ulceration; severe sterile recurrent fevers; and intermittent episodes of thrombocytopenia associated with intercurrent (presumed) viral infections. Both children were partially responsive to corticosteroids; DMARDS were largely ineffective. TNF-alpha blockade was ineffective; IL-1-beta blockade with Anakinra was partially effective. Despite that the older sibling died at the age of 14.5 years from severe sterile systemic inflammation, thrombocytopaenia, and multi-organ failure. In view of this, the younger (index) sibling underwent allogeneic haematopoietic stem cell transplantation (HSCT) at the age of seven years, and is 100% engrafted 23 months later with complete cure of her illness.DNA from the two patients, two unaffected siblings and their parents were genotyped for homozygosity mapping. One of the affected patients was exome sequenced, and the homozygous regions scrutinised. Variants of interest segregating with disease were confirmed with Sanger sequencing. Patient cells were analysed by flow cytometry and confocal microscopy to visualise polymerised actin levels and phagocytic activity of dendritic cells (DCs). Cellular motility was assessed using a Dunn chamber. These assays were also used to assess THP1 monocytic cell lines modified with shRNA and derived to macrophage-like and DC-like phenotypes. Wild-type and mutant sequence were tagged with mCherry fluorescent protein and overexpressed in HEK393T cells.The index case demonstrated normal T-cell activation to PHA but absent T-cell response to anti-CD3, highly suggestive of a defect in actin-regulated organisation of the immune synapse. A homozygous missense mutation was identified in a gene encoding a regulator of actin stability. CD20+ and CD56+ (but not CD3+) cells showed increased levels of polymerised actin. Dendritic cell and neutrophil motility was disrupted. The mutation identified resulted caused abnormal cellular localisation of the encoded actin-regulating protein.We describe a novel monogenic autoinflammatory disease with sterile inflammation and intermittent thrombocytopenia in association with periodic fever. This resulted from a mutation in a regulator of the actin cytoskeleton, and was resistant to conventional immunosuppression, though ultimately cured by HSCT.


Maladaptive use of the Internet results in Internet addiction (IA), which is associated with various negative consequences. Molecular and functional imaging techniques have been increasingly used for analysis of neurobiological changes and neurochemical correlates of IA. This review summarizes molecular and functional imaging findings on neurobiological mechanisms of IA, focusing on magnetic resonance imaging (MRI) and nuclear imaging modalities including positron emission tomography (PET) and single photon emission computed tomography (SPECT). MRI studies demonstrate that structural changes in frontal cortex are associated with functional abnormalities in Internet addicted subjects. Nuclear imaging findings indicate that IA is associated with dysfunction of the brain dopaminergic systems. Abnormal dopamine regulation of the prefrontal cortex (PFC) could underlie the enhanced motivational value and uncontrolled behavior over Internet overuse in addicted subjects. Further investigations are needed to determine specific changes in the Internet addictive brain, as well as their implications for behavior and cognition. Addiction to substances or activities can profoundly affect people's health and sometimes lead to serious social problems –3. For eWhile IA has drawn growing attention from scientific world, there are currently no standard diagnostic criteria. Several diagnostic criteria have been proposed to quantify IA. The most widely used diagnostic criterion is Young's Diagnostic Questionnaire –10. BaseAs an important subtype of IA, IGD has gained more and more attention from the whole world. IGD has been included in the appendix of the DSM-V, with a goal of encouraging additional studies . The DSMIn the past few years, molecular and functional imaging techniques have been increasingly used to study the neurobiological mechanism underlying IA. Molecular imaging is a rapidly developing field aimed to provide disease-specific molecular information through diagnostic imaging studies . The terMRI is a highly versatile imaging modality which uses magnet and radiofrequency energy to visualize the internal structure and soft tissue morphology of the body . The priUsing MRI, some studies have shown that brain structural changes are associated with IA. Using the Stroop color-word test , which hVoxel-based morphometry is an unbiased technique for characterizing regional cerebral volume and tissue concentration differences in structural magnetic resonance images , 31. VoxDiffusion tensor imaging (DTI) is an approach available to track brain white matter fibers noninvasively. Water molecules' diffusion was found to be much faster along the white matter fibers than perpendicular to them. The difference between these two motions is the basis of DTI , 44. DTIUsing arterial spin-labeling perfusion fMRI, Feng et al. investigated the effects of IGD on resting cerebral blood flow in adolescents . CompareFunctional connectivity impairments are also observed in individuals with IA. A recent study showed that subjects with IGD exhibited increased functional connectivity in the bilateral cerebellum posterior lobe and middle temporal gyrus compared with the control group . The bilIt has been reported that impulsivity is associated with IA . The abiRegional homogeneity is a widely used method in fMRI studies that measures the functional coherence of a given voxel with its nearest neighbors, and it can be used to evaluate resting-state brain activities based on the hypothesis that spatially neighboring voxels should have similar temporal patterns . IGD subSeveral studies investigated brain areas associated with cue-induced gaming urges –65. The An interesting study compared IGD subjects with subjects in remission from IGD and controls in cue-induced craving to play online games . BilaterMRI has also been used to assess therapeutic effects of specific pharmacological treatment on IA. Bupropion is a norepinephrine/dopamine reuptake inhibitor, which has been used in the treatment of patients with substance abuse. A study explored the possible effectiveness of bupropion, assessed brain activity in response to game cues using fMRI . IGD shoNuclear imaging approaches, which include SPECT and PET, have the advantages of high intrinsic sensitivity, unlimited depth penetration, and a broad range of clinically available molecular imaging agents . SPECT a18F-fluoro-deoxyglucose (18F-FDG) PET imaging, a study investigated the differences of cerebral glucose metabolism at resting state between young individuals with IGD and those with normal use [Using rmal use . Imaging11C-labelled raclopride and PET scans to investigate endogenous dopamine release in the human striatum during a video game [11C-raclopride to dopamine D2 receptors is sensitive to levels of endogenous dopamine, which can be detected as changes in binding potential of the radioligand. The authors reported that binding of 11C-raclopride to dopamine receptors in the striatum was significantly reduced during the video game compared with baseline levels of binding, which suggested increased release and binding of dopamine to its receptors. Moreover, they showed that there is a significant correlation between performance level during the task and reduced 11C-raclopride binding potential in the striatum. Similar results have been reported in people with IA [Emerging evidence has shown that the dopaminergic system is involved in drug addiction , 75. A pdeo game . Binding with IA . Individ with IA . They re99mTc-TRODAT-1, our group investigated striatal dopamine transporter density in IA subjects to identify potential presynaptic abnormalities [99mTc-TRODAT-1 uptake ratio of corpus striatum were greatly reduced in individuals with IA compared with controls. Taken together, these results suggest that IA is associated with dysfunction of the brain dopaminergic systems.Dopamine transporter is a plasma membrane protein that actively translocates released dopamine from the extracellular space into the presynaptic neurons . Alteredmalities . We show11C-N-methylspiperone (11C-NMSP) and 18F-FDG, in both states of resting and internet gaming task [11C-NMSP binding was found in the right inferior temporal gyrus in the IGD subjects compared to normal controls  and found that dopamine D2 receptor mediated dysregulation of the OFC could underlie a mechanism for loss of control and compulsive behavior in IGD subjects.Emerging evidence has shown that changes in brain structure and activity related to IA are relevant to brain regions involved in reward, motivation, and memory, as well as cognitive control. Molecular and functional imaging techniques have been increasingly applied to IA research, contributing significantly to our understanding of the neurobiological mechanism. Most of the previous literatures have studied IA individuals only under resting state, verified structural and functional abnormalities in the OFC, dorsolateral PFC, ACC, and PCC. Those regions may play crucial roles in salience attribution, inhibitory control, and decision making. So far, only one PET study with 11C-carfentanil to study the mu-opioid receptor availability and predict treatment outcomes of opioid receptor antagonists and help clinicians determine the most appropriate treatment for individual patients.As IA has become a serious problem worldwide, a need for effective treatment is becoming increasingly urgent. Both psychological and pharmacological treatment approaches have been applied to treat IA. Several drugs have shown to be promising in treating IA, such as antidepressants, antipsychotics, and opioid receptor antagonists . Cogniti


The role of blood flow in regulating signaling pathways and gene expression in the blood vasculature is well known. Recent studies have identified equally important roles of flow-mediated signaling in the lymphatic circulation including control of lymphatic vascular growth, remodeling, regeneration and maintenance of the lymphatic fate. In this review, we summarize these advances focusing on the role of fluid dynamics in control of lymphatic vasculature formation. Blood flow generates shear stress that plays critical role in regulation of vascular growth and remodeling while disturbed shear is central to a number of important vascular disease processes. Thus, physiological shear forces regulate expression of transcription factors such as c-Fos and Egr-1, growth factors  and integrins among other molecules . On the Lymphatic and blood vasculatures are closely related but yet distinctly different vascular systems. The lymphatic vasculature develops from embryonic venous circulation in a series of well-defined steps that are initiated by a fate change in a subset of endothelial cells (ECs) in the cardinal vein that begin expressing lymphatic markers. These newly defined lymphatic endothelial cells (LECs) then migrate out of the cardinal vein forming jugular lymph sacs that then sprout, giving rise to the entire lymphatic vascular network .Unlike the blood vasculature which is a closed system, lymphatic circulation is open with blind-ended lymphatic capillaries serving as an entry point for the interstitial fluid and immune cells that are then transported via progressively enlarging collecting vessels to the thoracic duct that then empties into subclavian vein. Furthermore, while blood flow is propelled by rhythmic contractions of the heart, mammalian lymphatic circulation is driven by a combination of hydrostatic forces and skeletal muscle contraction in the limbs augmented by opening and closing of lymphatic valves. As a result, the lymphatic flow is slower and more irregular than blood flow.Lymphatic vessels serve a variety of roles including draining interstitial fluid and macromolecules extravasated from blood vessels and returning them to blood circulation as well as facilitating immune cell trafficking and absorption of lipids. Therefore, lymphatic circulation is important for the regulation of interstitial fluid balance, tissue homeostasis and immune surveillance. Abnormal development or malfunction of lymphatic vessels is associated with pathological conditions such as lymphedema, late-onset obesity, hypertension and cardiovascular diseases including atherosclerosis -15.Although LECs, similarly to blood endothelial cells (BECs), have the ability to sense flow-mediated signaling, the role played by this signaling in lymphatic vasculature development and function is still poorly understood. Recent studies have identified contributions of lymphatic shear stress signaling to the regulation of lymphatic vascular growth, remodeling and lymphatic fate maintenance that will be the subject of this review.In mammals, the lymphatic vasculature is composed of lymphatic capillaries , pre-collectors and larger collecting lymphatic vessels. Lymphatic capillaries are blind-ended structures consisting of a single layer of overlapping ECs that lack pericyte coverage and have little or no basement membrane . SimilarLECs can also detect changes of shear stress magnitude and adjust their barrier function accordingly . Increas1–8-VEGF121) and LECs which then are pipetted into an interstitial flow culture chamber; interstitial flow enhances the effects of the fibrin-bound VEGF (α2-PI1–8-VEGF121) in promoting lymphatic network formation [1–8-VEGF121) and interstitial flow [1–8-VEGF121) to form a VEGF gradient [Recently, an increasing body of evidence has pointed to an important role of fluid flow in lymphatic vasculature formation. For example, it has been shown that fluid flow can work synergistically with growth factors such as VEGF to promote lymphangiogenesis . In a 3Dormation . Notablyial flow . This regradient .The role of interstitial fluid flow in lymphangiogenesis has been investigated in vivo using a mouse skin regeneration model ,27. In tIn line with these results, reduced interstitial flow during tissue regeneration results in decreased LEC migration and lymphatic vasculature formation. VEGF-C administration fails to induce lymphangiogenesis in this setting . During Interestingly, in the tail skin regeneration model, BEC migration and vascular network organization in CDE are not directional  indicatiInterstitial fluid also generates pressure, which has recently been shown to be involved in lymphangiogenesis in mice. In developing mouse embryos, the volume of fluid in the interstitium is correlated with LEC morphologic changes, proliferation and lymphatic vessel expansion . In respA recent study has identified lymph flow as a factor that regulates lymphatic valve development during lymphatic remodeling . LymphatIn mice, lymph flow in mesenteric lymphatic vessels commences on embryonic day (E) 15.5 and at this time lymphatic valves have not yet formed . As a reUpon exposure to OSS, LECs with high levels of PROX1 and FOXC2 accumulate NFATc1 in the nucleus, which does not occur in LECs expressing low levels of PROX1 and FOXC2. These data suggest that flow-induced shear stress activates distinct signaling pathways in different LEC populations . TogetheFurthermore, it now appears that different types of flow shear stress result in distinct cell morphology and actin cytoskeleton organization changes in LECs . When suSimilar to lymphatic vessels, veins also have luminal valves. Interestingly, both venous and lymphatic valve ECs share common molecular regulators . For insSlp-76 results in the formation of abnormal connections between blood and lymphatic vessels, and thus mixing of blood and lymphatic circulation [Slp-76 null mice lose their lymphatic fate and adapt a venous identity [Fluid flow is also important in the lymphatic fate maintenance. SLP-76 is a hematopoietic intracellular signaling protein, which has been shown to be required for blood and lymphatic vascular separation -43. In mculation -43. MoreRecent research advances have largely improved our knowledge on gene products and signaling pathways involved in lymphatic vascular development and have expanded the list of players that contribute to this process. An increasing amount of evidence has elevated the importance of fluid flow in the formation of lymphatic vasculature. Although our understanding of the molecular mechanism(s) underlying flow-regulated lymphatic vascular development is very limited, the importance of fluid flow in this process will draw increasing research efforts and will help to gain further insight into this fascinating research area.The authors declare that they have no competing interests.YW and MS prepared and wrote the manuscript. All authors read and approved the final manuscript.