Caged Dexamethasone to Photo-control the Development of Embryos through Activation of the Glucocorticoid Receptor.

Efficient tools for controlling molecular functions with exquisite spatiotemporal resolution are much in demand to investigate biological processes in living systems. Here we report an easily synthesized caged dexamethasone for photo-activating cytoplasmic proteins fused to the glucocorticoid receptor. In the dark, it is stable in vitro as well as in vivo in both zebrafish (Danio rerio) and Xenopus sp, two significant models of vertebrates. In contrast, it liberates dexamethasone upon UV illumination, which has been harnessed to interfere with developmental steps in embryos of these animals. Interestingly, this new system is biologically orthogonal to the one for photo-activating proteins fused to the estrogen ERT receptor, which brings great prospect for activating two distinct proteins down to the single cell level.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Multiple hPOT1-TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3' toward 5', a feature due to a 3'-end binding preference and to structuring of telomeric DNA.

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5'TTAGGG repeats and ends with a 3' single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1-TPP1 preferentially proceeds from 3' toward 5'. We explain this directionality in terms of two factors: (i) the preference of hPOT1-TPP1 for the binding site situated at the 3' end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1-TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3'-end and proceeding toward 5'.

Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands.

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.

Sex-Specific Response to Caloric Restriction After Reproductive Investment in Microcebus murinus: An Integrative Approach.

In seasonal environments, males and females usually maintain high metabolic activity during the whole summer season, exhausting their energy reserves. In the global warming context, unpredictability of food availability during summer could dramatically challenge the energy budget of individuals. Therefore, one can predict that resilience to environmental stress would be dramatically endangered during summer. Here, we hypothesized that females could have greater capacity to survive harsh conditions than males, considering the temporal shift in their respective reproductive energy investment, which can challenge them differently, as well as enhanced flexibility in females' physiological regulation. We tackled this question on the gray mouse lemur (Microcebus murinus), focusing on the late summer period, after the reproductive effort. We monitored six males and six females before and after a 2-weeks 60% caloric restriction (CR), measuring different physiological and cellular parameters in an integrative and comparative multiscale approach. Before CR, females were heavier than males and mostly characterized by high levels of energy expenditure, a more energetic mitochondrial profile and a downregulation of blood antioxidants. We observed a similar energy balance between sexes due to CR, with a decrease in metabolic activity over time only in males. Oxidative damage to DNA was also reduced by different pathways between sexes, which may reflect variability in their physiological status and life-history traits at the end of summer. Finally, females' mitochondria seemed to exhibit greater flexibility and greater metabolic potential than males in response to CR. Our results showed strong differences between males and females in response to food shortage during late summer, underlining the necessity to consider sex as a factor for population dynamics in climate change models.

AP-TSS: A New Method for the Analysis of RNA Expression from Particular and Challenging Transcription Start Sites.

Alternative promoter usage involved in the regulation of transcription, splicing, and translation contributes to proteome diversity and is involved in a large number of diseases, in particular, cancer. Epigenetic mechanisms and cis regulatory elements are involved in alternative promoter activity. Multiple transcript isoforms can be produced from a gene, due to the initiation of transcription at different transcription start sites (TSS). These transcripts may not have regions that allow discrimination during RT-qPCR, making quantification technically challenging. This study presents a general method for the relative quantification of a transcript synthesized from a particular TSS that we called AP-TSS (analysis of particular TSS). AP-TSS is based on the specific elongation of the cDNA of interest, followed by its quantification by qPCR. As proof of principle, AP-TSS was applied to two non-coding RNA: telomeric repeat-containing RNAs (TERRA) from a particular subtelomeric TSS, and Alu transcripts. The treatment of cells with a DNA methylation inhibitor was associated with a global increase of the total TERRA level, but the TERRA expression from the TSS of interest did not change in HT1080 cells, and only modestly increased in HeLa cells. This result suggests that TERRA upregulation induced by global demethylation of the genome is mainly due to activation from sites other than this particular TSS. For Alu RNA, the signal obtained by AP-TSS is specific for the RNA Polymerase III-dependent Alu transcript. In summary, our method provides a tool to study regulation of gene expression from a given transcription start site, in different conditions that could be applied to many genes. In particular, AP-TSS can be used to investigate the epigenetic regulation of alternative TSS usage that is of importance for the development of epigenetic-targeted therapies.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats - dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

Energy-Dependent Three-Body Loss in 1D Bose Gases.

We study the loss of atoms in quantum Newton's cradles with a range of average energies and transverse confinements. We find that the three-body collision rate in one-dimension is strongly energy dependent, as predicted by a strictly 1D theory. We adapt the theory to atoms in waveguides, then, using detailed momentum measurements to infer all the collisions that occur, we compare the observed loss to the adapted theory and find that they agree well.

Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA).

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

Edwards SAPIEN Transcatheter Pulmonary Valve Implantation: Results From a French Registry.

OBJECTIVES: The aim of this study was to describe and analyze data from patients treated in France with the Edwards SAPIEN transcatheter heart valve (Edwards Lifesciences LLC, Irvine, California) in the pulmonary position. BACKGROUND: The Edwards SAPIEN valve has recently been introduced for percutaneous pulmonary valve implantation (PPVI). METHODS: From April 2011 to May 2017, 71 patients undergoing PPVI were consecutively included. RESULTS: The median age at PPVI was 26.8 years (range 12.8 to 70.1 years). Primary underlying diagnoses were conotruncal malformations (common arterial trunk, tetralogy of Fallot and variants; n = 45), Ross procedure (n = 18), and other diagnoses (n = 8). PPVI indication was pure stenosis in 33.8% of patients, pure regurgitation in 28.1%, and mixed lesions in 38.1%. PPVI was successfully implemented in 68 patients (95.8%). Pre-stenting of the right ventricular outflow tract was performed in 70 patients (98.6%). Early major complications occurred in 4 subjects (5.6%), including 1 death, 1 coronary compression, and 2 pulmonary valve embolizations. Three of the 4 major complications occurred in the first 15 operated patients. No significant regurgitation was recorded after the procedure. Transpulmonary gradient was significantly reduced from 34.5 to 10.5 mm Hg (p < 0.0001). No patient died during a 1-month follow-up period. At 1-year follow-up, the death rate was 2.9%, and 3 patients had undergone surgical reintervention (44%). CONCLUSIONS: Early results with the Edwards SAPIEN valve in the pulmonary position demonstrate an ongoing high rate of procedural success.

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos.

Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca2+ -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+ ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+ ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.

Pou3f transcription factor expression during embryonic development highlights distinct pou3f3 and pou3f4 localization in the Xenopus laevis kidney.

The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis. All vertebrates examined possessed members of the four pou3f subfamilies, excepting the zebrafish, which lacked a pou3f4 gene. Whole mount in situ hybridization and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Xenopus pou3f genes were expressed in the forming neural tube and their expression was maintained in the brain, mostly in the dorsal part, at tailbud stages. The pou3f2, pou3f3, and pou3f4 genes were also expressed in the developing otic vesicle, and pou3f1 in some cells of the epidermis. Besides ectodermal derivatives, pou3f3 and pou3f4 were expressed in the developing kidney. Their expression started at the early tailbud stage in the pronephric anlage and partly overlapped. In the mature pronephric tubule, pou3f3 was restricted to the intermediate tubule, while pou3f4 was also expressed in the distal and connecting tubule. Together, our results highlight a significant conservation of pou3f gene expression in vertebrates and indicate that they may have distinct but also redundant functions during neural and renal development.

Precision of CAPILLARYS 2 for the Detection of Hemoglobin Variants Based on Their Migration Positions.

OBJECTIVES: In this report, we evaluated utility of the capillary electrophoresis (CE) migration position of the CAPILLARYS 2 CE instrument. METHODS: The precision of this x-axis number was determined on a selection of common hemoglobin (Hb) variants (Hb S, Hb C, Hb D-Punjab, Hb E, Hb Hope), and the reproducibility of this number was evaluated by comparing the results obtained by two large reference laboratories on 81 Hb variants. Additionally, the CE migration position is given for a total of 409 Hb variants. RESULTS: The x-axis migration position showed excellent intra- and interassay precision. Comparison of Hb variants seen by both laboratories showed that 83% had a difference in migration position of 1 unit or less. Only three rare Hb variants showed a difference of more than 2 units. CONCLUSION: In summary, the CE migration position is a reproducible value and can be used as an aid in the identification of Hb variants.

The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units.

Replication protein A (RPA) is a single-stranded DNA binding protein involved in replication and in telomere maintenance. During telomere replication, G-quadruplexes (G4) can accumulate on the lagging strand template and need to be resolved. It has been shown that human RPA is able to unfold a single G4. Nevertheless, the G-strand of human telomeres is prone to fold into higher-order structures formed by contiguous G-quadruplexes. To understand how RPA deals with these structures, we studied its interaction with telomeric G-strands folding into an increasing number of contiguous G4s. The aim of this study was to determine whether the efficiency of binding/unfolding of hRPA to telomeric G-strands depends on the number of G4 units. Our data show that the number n of contiguous G4 units (n ≥ 2) does not affect the efficiency of hRPA to coat transiently exposed single-stranded telomeric G-strands. This feature may be essential in preventing instability due to G4 structures during telomere replication.

Short in-Frame Insertions/Deletions in the Coding Sequence of the α-Globin Gene. Consequences of the 3D Structure and Resulting Phenotypes: Hb Choisy as an Example.

A small group of hemoglobin (Hb) variants result from 'in-frame' deletion/insertion (del/ins). We describe a new variant of this group (Hb Choisy), found on the α1 gene, which is the exact counterpart of a previously published deletional variant, Hb J-Biskra [codons 51-58 (or codons 52-59) (-24 bp) (-TCTGCCCAGGTTAAGGGCCACGGC); HBA1: c.157_180del (or HBA2)]. In Hb J-Biskra, the sequence Ser-Ala-Gln-Val-Lys-Gly-His-Gly located from positions α52(E1) to α59(E8) is deleted, while in Hb Choisy the same sequence (Ser-Ala-Gln-Val-Lys-Gly-His-Gly) is inserted at position α52(E1). The variant carrying the insertion appears to be less damaging than the one with the deletion. A possible explanation could be that the additional sequence is located in the C to E interhelical region, and is less disturbing to the general structure of the globin chain. This insertion/deletion (ins/del) is likely favored by the repetition, at an interval of 16 nucleotides, of an eight nucleotide sequence. Comparison of variants of this group, found in the HbVar database, shows that structural modifications resulting from insertions are frequently less damaging than that caused by deletions.

Transcription regulation of CDKN1A (p21/CIP1/WAF1) by TRF2 is epigenetically controlled through the REST repressor complex.

We observed extra-telomeric binding of the telomere repeat binding factor TRF2 within the promoter of the cyclin-dependent kinase CDKNIA (p21/CIP1/WAF1). This result in TRF2 induced transcription repression of p21. Interestingly, p21 repression was through engagement of the REST-coREST-LSD1-repressor complex and altered histone marks at the p21 promoter in a TRF2-dependent fashion. Furthermore, mutational analysis shows p21 repression requires interaction of TRF2 with a p21 promoter G-quadruplex. Physiologically, TRF2-mediated p21 repression attenuated drug-induced activation of cellular DNA damage response by evading G2/M arrest in cancer cells. Together these reveal for the first time role of TRF2 in REST- repressor complex mediated transcription repression.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Infective Endocarditis Risk After Percutaneous Pulmonary Valve Implantation With the Melody and Sapien Valves.

OBJECTIVES: This study compared the risk of infective endocarditis (IE) after percutaneous pulmonary valve implantation (PPVI) with the Sapien and Melody valves. BACKGROUND: The incidence of IE after PPVI is estimated at 3% per year with the Melody valve. The Sapien valve is a more recently marketed valve used for PPVI. METHODS: We retrospectively included consecutive patients who underwent PPVI at a single center between 2008 and 2016. IE was diagnosed using the modified DUKE criteria. RESULTS: PPVI was performed in 79 patients (Melody valve, 40.5%; Sapien valve, 59.5%). Median age was 24.9 years (range 18.1 to 34.6). IE occurred in 8 patients (10.1%) at a median of 1.8 years (minimum: 1.0; maximum: 5.6) after surgery. Causative organisms were methicillin-sensitive Staphylococcus aureus (n = 3), Staphylococcus epidermidis (n = 1), Streptococcus mitis (n = 1), Aerococcus viridans (n = 1), Corynebacterium striatum (n = 1), and Haemophilus influenzae (n = 1). All 8 cases occurred after Melody PPVI (25.0% vs. 0.0%). The incidence of IE was 5.7% (95% confidence interval: 2.9% to 11.4%) per person-year after Melody PPVI. The Kaplan-Meier cumulative incidence of IE with Melody PPVI was 24.0% (95% confidence interval: 12.2% to 43.9%) after 4 years and 30.1% (95% confidence interval: 15.8% to 52.5%) after 6 years, compared with 0.0% with the Sapien PPVI after 4 years (p < 0.04 by log-rank test). There was a trend toward a higher incidence of IE in the first 20 patients with Melody PPVI (who received prophylactic antibiotics during the procedure only) and in patients who had percutaneous interventions, dental care, or noncardiac surgery after PPVI. CONCLUSIONS: IE after PPVI may be less common with the Sapien compared with the Melody valve.

Hb Olivet (HBA1: C.40G > A; p.Ala14Thr), a Novel Silent Hemoglobin Variant in Two Families of Distinct Origin.

We report two families, members of which are carriers of a novel hemoglobin (Hb) variant that was named Hb Olivet [α13(A11)Ala→Thr (α1) (GCC > ACC); HBA1: c.40G > A; p.Ala14Thr]. The analysis of these cases allowed a clear description of this anomaly that behaves as a silent Hb. In the first family, of Portuguese ethnicity living in France, the proband, a 24-year-old male and his 57-year-old mother, both appeared to be carriers. The son presented with borderline mean corpuscular volume (MCV), while the mother was normocytic and normochromic. Hemoglobin separation on capillary electrophoresis (CE) was normal, while a slightly asymmetric peak was observed on high performance liquid chromatography (HPLC). In a second family, originally from Surinam but living in The Netherlands, the proband, a 6-year-old girl, showed a mild microcytosis at low ferritin levels. The abnormal Hb was inherited from the mother who was clearly iron depleted, was not present in the sister and brother of the proband. The microcytic hypochromic anemia was only shown in two out of a total of four carriers. It therefore seems likely that iron depletion is causative as two carriers are completely normal. Characterization and genotype/phenotype correlation are briefly described.