The alkaloid centcyamine increases expression of klotho and lamin B1, slowing the onset of skin ageing in vitro and in vivo.

BACKGROUND: Klotho is a protein known for its beneficial effects on longevity. Centcyamine is an alkaloid present in certain plants whose extracts have an anti-inflammatory effect. Skin fibroblasts are essential to the formation and structure of the dermis. OBJECTIVE: Centcyamine is an indole-based alkaloid composed of coumaric acid, a resveratrol precursor and methoxytryptamine, which can be both a precursor, or a derivative, of melatonin. Given these building blocks and their well-known bioactivities, it was of interest to explore the potential benefits of using this aryl-alkaloid, in cosmetic skincare applications. METHODS: We tested cultured normal human dermal fibroblasts (NHDF) in vitro to observe how supplementation with centcyamine improves properties of the cells to counteract the effect of ageing. The expression of genes and proteins of interest was quantified. The effect on doubling time and cell function was evaluated following treatment of the cells over several replication cycles. Skin firmness, red spot index and skin isotropy were measured with Dynaskin® , Visia® and Primos® equipment, respectively, and compared over two months in a vehicle-controlled clinical trial on 60 persons. RESULTS: Centcyamine activates the expression of the gene KL and the related protein Klotho in dermal fibroblasts. Moreover, centcyamine slows the replicative ageing process of fibroblasts in culture. These cells retain cellular functions identical to those of young cells: the synthesis of lamin B1, a crucial regulatory protein of proliferation, as well as of collagen I and elastin is retained in aged cells. The clinical data are shown to improve skin isotropy in a majority of subjects, to reduce the red spot intensity and to maintain skin firmness in the treated group vs. the vehicle. CONCLUSION: The alkaloid centcyamine induces changes in the metabolism of the ageing process of human dermal fibroblasts. The up-to-now unobserved implication of both Klotho and lamin B1 to maintain homeostasis of the skin opens new venues for the prevention of age-related changes in skin structure. The in vitro and clinical data, while not demonstrated to be causally related, converge towards a common goal of skin repair and slower ageing processes.

CONTEXTE: Klotho est une protéine connue pour ses effets bénéfiques sur la longévité. Le centcyamine est un alcaloïde présent dans certaines plantes et dont les extraits ont un effet anti-inflammatoire. Les fibroblastes cutanés sont essentiels à la formation et à la structure du derme. OBJECTIF: le centcyamine est un alcaloïde à base d'indole, constitué d'acide coumarique, d'un précurseur du resvératrol et de méthoxytryptamine, qui peut être à la fois un précurseur ou un dérivé de la mélatonine. Compte tenu de ces éléments constitutifs et de leurs bioactivités bien connues, il était intéressant d'étudier les bénéfices potentiels associés à l'utilisation de cet alcaloïde arylé dans des applications en soins cosmétiques de la peau. MÉTHODES: nous avons évalué in vitro des fibroblastes dermiques humains normaux (Normal Human Dermal Fibroblasts, NHDF) en culture pour observer comment une supplémentation en centcyamine améliore les propriétés des cellules à contrer l'effet du vieillissement. L'expression des gènes et des protéines d'intérêt a été quantifiée. L'effet sur le temps de doublement et sur la fonction cellulaire a été évalué après le traitement des cellules pendant plusieurs cycles de réplication. La fermeté de la peau, l'indice des taches rouges et l'isotropie cutanée ont été mesurés, respectivement, à l'aide d'appareils Dynaskin®, Visia® et Primos®, et comparés sur une période de deux mois dans un essai clinique contrôlé par un excipient et mené chez 60 personnes. RÉSULTATS: le centcyamine active l'expression du gène KL et de la protéine apparentée Klotho dans les fibroblastes dermiques. De plus, le centcyamine ralentit le processus de vieillissement réplicatif des fibroblastes en culture. Ces cellules conservent des fonctions cellulaires identiques à celles des cellules jeunes : la synthèse de la lamine B1, une protéine essentielle dans la régulation de la prolifération, ainsi que du collagène I et de l'élastine est maintenue dans les cellules âgées. Les données cliniques montrent une amélioration de l'isotropie cutanée chez une majorité de sujets, une réduction de l'intensité des taches rouges et un maintien de la fermeté de la peau dans le bras traité par rapport à l'excipient. CONCLUSION: l'alcaloïde centcyamine induit, dans les fibroblastes dermiques humains, des modifications du métabolisme impliqué dans le processus de vieillissement. Le rôle jamais observé jusqu'à présent des protéines Klotho et lamine B1 dans le maintien de l'homéostasie de la peau offre de nouvelles possibilités en matière de prévention des modifications de la structure cutanée liées à l'âge. Bien qu'elles n'affichent aucun lien de causalité, les données in vitro et cliniques convergent vers un objectif commun de réparation cutanée et de ralentissement du processus de vieillissement.

DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability.

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4.3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer.

Switch Tandem Repeats Influence the Choice of the Alternative End-Joining Pathway in Immunoglobulin Class Switch Recombination.

Immunoglobulin class switch recombination (CSR) plays an important role in humoral imm\une responses by changing the effector functions of antibodies. CSR occurs between highly repetitive switch (S) sequences located upstream of immunoglobulin constant gene exons. Switch sequences differ in size, the nature of their repeats, and the density of the motifs targeted by the activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. CSR involves double-strand breaks (DSBs) at the universal Sµ donor region and one of the acceptor S regions. The DSBs ends are fused by the classical non-homologous end-joining (C-NHEJ) and the alternative-NHEJ (A-NHEJ) pathways. Of the two pathways, the A-NHEJ displays a bias towards longer junctional micro-homologies (MHs). The Sµ region displays features that distinguish it from other S regions, but the molecular basis of Sµ specificity is ill-understood. We used a mouse line in which the downstream Sγ3 region was put under the control of the Eµ enhancer, which regulates Sµ, and analyzed its recombination activity by CSR-HTGTS. Here, we show that provision of Eµ enhancer to Sγ3 is sufficient to confer the recombinational features of Sµ to Sγ3, including efficient AID recruitment, enhanced internal deletions and robust donor function in CSR. Moreover, junctions involving Sγ3 display a bias for longer MH irrespective of sequence homology with switch acceptor sites. The data suggest that the propensity for increased MH usage is an intrinsic property of Sγ3 sequence, and that the tandem repeats of the donor site influence the choice of the A-NHEJ.

Combined deficiency of MSH2 and Sµ region abolishes class switch recombination.

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sµ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sµ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iµ-Cµ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iµ exon that may decrease with transcription elongation across the Sµ region.

Complete cis Exclusion upon Duplication of the Eµ Enhancer at the Immunoglobulin Heavy Chain Locus.

Developing lymphocytes somatically diversify their antigen-receptor loci through V(D)J recombination. The process is associated with allelic exclusion, which results in monoallelic expression of an antigen receptor locus. Various cis-regulatory elements control V(D)J recombination in a developmentally regulated manner, but their role in allelic exclusion is still unclear. At the immunoglobulin heavy chain locus (IgH), the Eµ enhancer plays a critical role in V(D)J recombination. We generated a mouse line with a replacement mutation in the constant region of the locus that duplicates the Eµ enhancer and allows premature expression of the γ3 heavy chain. Strikingly, IgM expression was completely and specifically excluded in cis from the mutant allele. This cis exclusion recapitulated the main features of allelic exclusion, including differential exclusion of variable genes. Notably, sense and antisense transcription within the distal variable domain and distal V(H)-DJ(H) recombination were inhibited. cis exclusion was established and stably maintained despite an active endogenous Eµ enhancer. The data reveal the importance of the dynamic, developmental stage-dependent interplay between IgH locus enhancers and signaling in the induction and maintenance of allelic exclusion.

High-quality animation of 2D steady vector fields.

Simulators for dynamic systems are now widely used in various application areas and raise the need for effective and accurate flow visualization techniques. Animation allows us to depict direction, orientation, and velocity of a vector field accurately. This paper extends a former proposal for a new approach to produce perfectly cyclic and variable-speed animations for 2D steady vector fields (see [1] and [2]). A complete animation of an arbitrary number of frames is encoded in a single image. The animation can be played using the color table animation technique, which is very effective even on low-end workstations. A cyclic set of textures can be produced as well and then encoded in a common animation format or used for texture mapping on 3D objects. As compared to other approaches, the method presented in this paper produces smoother animations and is more effective, both in memory requirements to store the animation, and in computation time.

Tissue-specific inactivation of HAT cofactor TRRAP reveals its essential role in B cells.

The transformation/transcription domain-associated protein (TRRAP) is a common component of many histone acetyltransferase (HAT) complexes. Targeted-deletion of the Trrap gene led to early embryonic lethality and revealed a critical function of TRRAP in cell proliferation. Here, we investigate the function of TRRAP in murine B cells. To this end, we ablated Trrap gene in a B cell-restricted manner and studied its impact on B-cell development and proliferation, a pre-requisite for class switch recombination (CSR), the process that allows IgM-expressing B lymphocytes to switch to the expression of IgG, IgE, or IgA isotypes. We show that TRRAP deficiency impairs B-cell development but does not directly affect CSR. Instead, cells induced to proliferate undergo apoptosis. Our findings demonstrate a central and general role of TRRAP in cell proliferation.