Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration.

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

RNaseH1 regulates TERRA-telomeric DNA hybrids and telomere maintenance in ALT tumour cells.

A fraction of cancer cells maintain telomeres through the telomerase-independent, 'Alternative Lengthening of Telomeres' (ALT) pathway. ALT relies on homologous recombination (HR) between telomeric sequences; yet, what makes ALT telomeres recombinogenic remains unclear. Here we show that the RNA endonuclease RNaseH1 regulates the levels of RNA-DNA hybrids between telomeric DNA and the long noncoding RNA TERRA, and is a key mediator of telomere maintenance in ALT cells. RNaseH1 associated to telomeres specifically in ALT cells and its depletion led to telomeric hybrid accumulation, exposure of single-stranded telomeric DNA, activation of replication protein A at telomeres and abrupt telomere excision. Conversely, overexpression of RNaseH1 weakened the recombinogenic nature of ALT telomeres and led to telomere shortening. Altering cellular RNaseH1 levels did not perturb telomere homoeostasis in telomerase-positive cells. RNaseH1 maintains regulated levels of telomeric RNA-DNA hybrids at ALT telomeres to trigger HR without compromising telomere integrity too severely.

Mitosis inhibits DNA double-strand break repair to guard against telomere fusions.

Mitotic cells inactivate DNA double-strand break (DSB) repair, but the rationale behind this suppression remains unknown. Here, we unravel how mitosis blocks DSB repair and determine the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora B kinase-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. We conclude that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.

Telomerase efficiently elongates highly transcribing telomeres in human cancer cells.

RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription.

Transcription regulates telomere dynamics in human cancer cells.

Telomeres are nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. Although largely heterochromatic, telomeres are transcribed into telomeric repeat-containing RNA (TERRA) molecules by RNA polymerase II. The functions associated with telomere transcription and TERRA remain ill defined. Here we show that the transcriptional activity of human telomeres directly regulates their movement during interphase. We find that chemical inhibition of global transcription dampens telomere motion, while global stimulation promotes it. Likewise, when DNA methyltransferase enzymes are deleted to augment telomere transcription, we observe increased telomere movement. Finally, using a cell line engineered with a unique transcriptionally inducible telomere, we show that transcription of one specific telomere stimulates only its own dynamics without overtly affecting its stability or its length. We reveal a new and unforeseen function for telomere transcription as a regulator of telomere motion, and speculate on the intriguing possibility that transcription-dependent telomere motion sustains the maintenance of functional and dysfunctional telomeres.

TERRA: Long Noncoding RNA at Eukaryotic Telomeres.

Telomeres protect the ends of linear eukaryotic chromosomes from being recognized as DNA double-stranded breaks, thereby maintaining the stability of our genome. The highly heterochromatic nature of telomeres had, for a long time, reinforced the idea that telomeres were transcriptionally silent. Since a few years, however, we know that DNA-dependent RNA polymerase II transcribes telomeric DNA into TElomeric Repeat-containing RNA (TERRA) molecules in a large variety of eukaryotes. In this chapter, we summarize the current knowledge of telomere structure and function and extensively review data accumulated on TERRA biogenesis and regulation. We also discuss putative functions of TERRA in preserving telomere stability and propose future directions for research encompassing this novel and exciting aspect of telomere biology.

[Cost of treatment and follow up of breast cancer: a retrospective assessment in a comprehensive cancer centre]. / Evaluation rétrospective du coût du cancer du sein dans un centre de lutte contre le cancer.

CONTEXT: Breast cancer is one of the major causes of premature death for women. Its cost management is important for both the national health insurance and the individual health care providers. OBJECTIVE: The objective of this study was to assess the global medical cost of breast cancer from diagnosis to follow up in one French medical centre: centre René-Huguenin, Saint-Cloud (92). METHOD: Duration of medical activities and other medical resources utilisations were collected from a retrospective cohort of 120 patients followed from January 1995 to February 2000. Unit costs were obtained from cost accounts of the Centre. RESULTS: The mean medical cost per patient was FF 66,067 [60,318-7,815] (USD $ 10,744 [9,809-11,679]). The mean cost varied from FF 41,875 (UDS $ 6,810) to FF 81,020 (UDS $ 13,175) depending on choice of type of therapy. The initial treatment phase was the most expensive, costing FF 48,397 [46,176-50,617] (USD $ 7,870 [7,509-8,231]) which represented 73.3% of the global cost. CONCLUSION: This study has provided an estimate of the real global cost of managing patients with breast cancer in a single French Comprehensive Cancer Centre (CLCC). The study method used is readily transposable to other treatment contexts and to other types of cancer.