Modulated contact frequencies at gene-rich loci support a statistical helix model for mammalian chromatin organization

International audienceABSTRACT: BACKGROUND: Despite its critical role for mammalian gene regulation, the basic structural landscape of chromatin in living cells remains largely unknown within chromosomal territories below the megabase scale. RESULTS: Here, using the 3C-qPCR method, we investigate contact frequencies at high resolution within the interphase chromatin at several mouse loci. We find that, at several gene-rich loci, contact frequencies undergo a periodical modulation (every 90-100 kb) that affects chromatin dynamics over large genomic distances (few hundred kb). Interestingly, this modulation appears to be conserved in human cells and bioinformatic analyses of locus-specific, long-range cis-interactions suggest that it may underlie the dynamics of a significant number of gene-rich domains in mammals, thus contributing to genome evolution. Finally, using an original model derived from polymer physics, we show that this modulation can be understood as a fundamental helix shape that chromatin tends to adopt in gene-rich domains when no significant locus-specific interaction takes place. CONCLUSIONS: Altogether, our work unveils a fundamental aspect of chromatin dynamics in mammals and contributes to a better understanding of genome organization within chromosomal territories

Enhanced normalisation of CD4/CD8 ratio with early antiretroviral therapy in primary HIV infection

Introduction: Despite normalization of total CD4 counts, ongoing immune dysfunction is noted amongst those on antiretroviral therapy (ART). Low CD4/CD8 ratio is associated with a high risk of AIDS and non-AIDS events and may act as a marker of immune senescence [1]. This ratio is improved by ART although normalization is uncommon (7%) [2]. The probability of normalization of CD4 count is improved with immediate ART initiation in primary HIV infection (PHI) [3]. We examined whether CD4/CD8 ratio similarly normalized in immediate vs. deferred ART at PHI. Methods: Using data from the SPARTAC trial and the UK Register of HIV Seroconverters, we examined the effect of ART with time (continuous) from HIV seroconversion (SC) on CD4/CD8 ratio (]1) adjusted for sex, risk group, ethnicity, enrolment from an African site and both CD4 count and age at ART initiation. We also examined that effect by dichotomizing HIV duration at ART initiation (ART started within six months of SC: early ART; ART initiatedsix months after SC: deferred). We also considered time to CD4 count normalization (]900 cells/mm3 ). Results: In total, 353 initiated ART with median (IQR) 97.9 (60.5, 384.5) days from estimated seroconversion; 253/353 early ART, 100 deferred ART. At one year after starting ART, 114/253 (45%) early ART had normalized CD4/8 ratio, compared with 11/99 (11%) in the deferred group, whilst 83/253 (33%) of early ART had normalized CD4 counts, compared with 3/99 (3%) in the deferred group. Individuals initiating within six months of PHI were significantly more likely to reach normal ratio than those initiating later (HR, 95% CI 2.96, 1.755.01, pB0.001). The longer after SC ART was initiated, the reduced likelihood of achieving normalization of CD4/CD8 ratio (HR 0.98, 95% CI 0.960.99 for each 30-day increase). CD4 count at ART initiation was also associated with normalization, as expected (HR 1.002, 95% CI 1.0011.002, pB0.001). There was an association between normal CD4/CD8 ratio and being virally suppressed (B400 copies HIV RNA/ml) pB0.001. CD4 count normalization was also significantly more likely for those initiating early (HR 5.00, 95% CI 1.5216.41, p0.008). Conclusions: The likelihood of achieving normalization of CD4/CD8 ratios was increased if ART was initiated within six months of PHI. Higher CD4/CD8 ratio may reflect a more ‘‘normal’’ immune phenotype conferring enhanced prognosis and predict posttreatment control. Refe

Broadly neutralizing antibody responses in the longitudinal primary HIV-1 infection Short Pulse Anti-Retroviral Therapy at Seroconversion cohort

OBJECTIVE: Development of immunogens that elicit an anti-HIV-1 broadly neutralizing antibody (bnAb) response will be a key step in the development of an effective HIV-1 vaccine. Although HIV-1 bnAb epitopes have been identified and mechanisms of action studied, current HIV-1 envelope-based immunogens do not elicit HIV-1 bnAbs in humans or animal models. A better understanding of how HIV-1 bnAbs arise during infection and the clinical factors associated with bnAb development may be critical for HIV-1 immunogen design efforts. DESIGN AND METHODS: Longitudinal plasma samples from the treatment-naive control arm of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) primary HIV-1 infection cohort were used in an HIV-1 pseudotype neutralization assay to measure the neutralization breadth, potency and specificity of bnAb responses over time. RESULTS: In the SPARTAC cohort, development of plasma neutralization breadth and potency correlates with duration of HIV infection and high viral loads, and typically takes 3-4 years to arise. bnAb activity was mostly directed to one or two bnAb epitopes per donor and more than 60% of donors with the highest plasma neutralization having bnAbs targeted towards glycan-dependent epitopes. CONCLUSION: This study highlights the SPARTAC cohort as an important resource for more in-depth analysis of bnAb developmental pathways

Results of the COVID-19 mental health international for the general population (COMET-G) study.

INTRODUCTION: There are few published empirical data on the effects of COVID-19 on mental health, and until now, there is no large international study. MATERIAL AND METHODS: During the COVID-19 pandemic, an online questionnaire gathered data from 55,589 participants from 40 countries (64.85% females aged 35.80 ± 13.61; 34.05% males aged 34.90±13.29 and 1.10% other aged 31.64±13.15). Distress and probable depression were identified with the use of a previously developed cut-off and algorithm respectively. STATISTICAL ANALYSIS: Descriptive statistics were calculated. Chi-square tests, multiple forward stepwise linear regression analyses and Factorial Analysis of Variance (ANOVA) tested relations among variables. RESULTS: Probable depression was detected in 17.80% and distress in 16.71%. A significant percentage reported a deterioration in mental state, family dynamics and everyday lifestyle. Persons with a history of mental disorders had higher rates of current depression (31.82% vs. 13.07%). At least half of participants were accepting (at least to a moderate degree) a non-bizarre conspiracy. The highest Relative Risk (RR) to develop depression was associated with history of Bipolar disorder and self-harm/attempts (RR = 5.88). Suicidality was not increased in persons without a history of any mental disorder. Based on these results a model was developed. CONCLUSIONS: The final model revealed multiple vulnerabilities and an interplay leading from simple anxiety to probable depression and suicidality through distress. This could be of practical utility since many of these factors are modifiable. Future research and interventions should specifically focus on them

Normal and altered pre-mRNA processing in the DMD gene

International audienceSplicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression controlled by multiple post- and co-transcriptional mechanisms. The large Duchenne muscular dystrophy gene encoding the protein dystrophin provides a striking example of the complexity of human pre-mRNAs. In this review, we summarize the current state of knowledge about canonical and non-canonical splicing in the DMD pre-mRNA, with a focus on mechanisms that take place in the full-length transcript isoform expressed in human skeletal muscle. In particular, we highlight recent work demonstrating that multi-step events are required for long DMD intron removal. The role of temporary intron retention in the occurrence of alternative splicing events is also discussed. Even though the proportion of splicing mutations is lower than reported in other genes, a great diversity of splicing defects linked to point mutations, but also large genomic rearrangements are observed in the DMD gene. We provide an overview of the molecular mechanisms underlying aberrant splicing in patients with Duchenne or Becker muscular dystrophy, and we also detail how alternative splicing can serve as a disease modifier in patients by changing the outcome of the primary defect

Identification of Splicing Factors Involved in DMD Exon Skipping Events Using an In Vitro RNA Binding Assay

International audienceMutation-induced exon skipping in the DMD gene can modulate the severity of the phenotype in patients with Duchenne or Becker Muscular Dystrophy. These alternative splicing events are most likely the result of changes in recruitment of splicing factors at cis-acting elements in the mutated DMD pre-mRNA. The identification of proteins involved can be achieved by an affinity purification procedure. Here, we provide a detailed protocol for the in vitro RNA binding assay that we routinely apply to explore molecular mechanisms underlying splicing defects in the DMD gene. In vitro transcribed RNA probes containing either the wild type or mutated sequence are oxidized and bound to adipic acid dihydrazide-agarose beads. Incubation with a nuclear extract allows the binding of nuclear proteins to the RNA probes. The unbound proteins are washed off and then the specifically RNA-bound proteins are released from the beads by an RNase treatment. After separation by SDS-PAGE, proteins that display differential binding affinities for the wild type and mutant RNA probes are identified by mass spectrometry

A targeted multienzyme mechanism for selective microtubule polyglutamylation.

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level

Polyglutamylation is a posttranslational modification with a broad range of substrates.

Polyglutamylation is a posttranslational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleo-cytoplasmic shuttling proteins, including many chromatin binding proteins. Our work reveals that polyglutamylation is a much more widespread posttranslational modification than initially thought and thus that it might be a regulator of many cellular processes