Targeting EDEM protects against ER stress and improves development and survival in C. elegans

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.     

Stereotactic radiotherapy for brain oligometastases

Brain metastases, the most common metastases in adults, will develop in up to 40% of cancer patients, accounting for more than one-half of all intracranial tumors. They are most associated with breast and lung cancer, melanoma and, less frequently, colorectal and kidney carcinoma.Magnetic resonance imaging (MRI) is the gold standard for diagnosis. For the treatment plan, computed tomography (CT ) images are co-registered and fused with a gadolinium-enhanced T1-weighted MRI where tumor volume and organs at risk are contoured. Alternatively, plain and contrast-enhanced CT scans are co-registered. Single-fraction stereotactic radiotherapy (SRT ) is used to treat patients with good performance status and up to 4 lesions with a diameter of 30 mm or less that are distant from crucial brain function areas. Fractionated SRT (2–5 fractions) is used for larger lesions, in eloquent areas or in proximity to crucial or surgically inaccessible areas and to reduce treatment-related neurotoxicity. The single-fraction SRT dose, which depends on tumor diameter, impacts local control. Fractionated SRT may encompass different schedules. No randomized trial data compared the safety and efficacy of single and multiple fractions. Both single-fraction and fractionated SRT provide satisfactory local control rates, tolerance, a low risk of transient acute adverse events and of radiation necrosis the incidence of which correlated with the irradiated brain volume.     

Radiobiology of stereotactic radiotherapy

This paper focuses on the radiobiological mechanisms underlying the effects of stereotactic radiotherapy (SRT ) which, despite SRT expansion, have not yet been fully elucidated. Some authors postulated that radiobiology principles, as applied to conventional fractionations (5R: reoxygenation, repair, repopulation, redistribution, radioresistence), suffice in themselves to account for the excellent clinical results of SRT; others argued that the role of the 5R was limited. Recent preclinical data showed that hypofractionated ablative treatments altered the microenvironment, thus determining cell death either directly or indirectly. Furthermore, dead tumor cells released quantities of antigens, which stimulated antitumor immunity, thus reducing the risk of relapse and metastasis. Better understanding of the radiobiological mechanisms underlying response to high-dose radiation treatment is essential for predicting its short- and long-term effects on the tumor and surrounding healthy tissues and, consequently, for improving its related therapeutic index.     

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C‐to‐T transition at position +6 in exon‐7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C‐to‐T transition in SMN2 creates a putative binding site for the RNA‐binding protein Sam68. RNA pull‐down assays and UV‐crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon‐7 skipping. Moreover, mutations in the Sam68‐binding site of SMN2 or in the RNA‐binding domain of Sam68 completely abrogated its effect on exon‐7 skipping. Retroviral infection of dominant‐negative mutants of Sam68 that interfere with its RNA‐binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon‐7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.     

Phylogeny and biogeography of the Rhinella marina species complex (Amphibia,Bufonidae) revisited: implications for Neotropical diversification hypotheses

Vallinoto, M., Sequeira, F., Sodré, D., Bernardi, J. A. R., Sampaio, I. & Schneider, H. (2009). Phylogeny and biogeography of the Rhinella marina species complex (Amphibia, Bufonidae) revisited: implications for Neotropical diversification hypotheses. —Zoologica Scripta, 39, 128–140. A number of distinct hypotheses have been proposed to account for the origin of the considerable biological diversity found in the Neotropics, which is still a matter of intense debate. Here, we conducted a phylogenetic analysis of the Rhinella marina complex, a group of species widely distributed in Central and South America, combining published data with new sequences of three mtDNA genes (12S, 16S and cyt b) in order to clarify the evolutionary relationships and biogeographical history of the group. We included eight of the ten currently recognized R. marina group species and several outgroups. Maximum parsimony, maximum likelihood, and Bayesian inference analyses produced similar topologies, with two well‐supported main clades, each characterized by a deep subdivision. One of these major clades includes the samples of R. marina from Central America and Ecuador (west of the Andes), whereas the other comprises the remaining species of the group and samples of R. marina from the Amazon basin and other areas east of the Andes. A Bayesian coalescent‐based method (BEAST) dated the divergence between the two major clades, and between the Central American and Ecuadorian clades to the Miocene, matching the timing of other Central‐South American faunal divergences. Taken together, the results highlight the importance of Tertiary events such as the Pebas/marine incursions into the Amazon basin and Andean uplift for the diversification and historical biogeography of R. marina, making such taxa paraphyletic, and provide new perspectives on the debate on its species status.     

Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PT in situ that was accompanied by cytochrome c release and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A(2) inhibitor aristolochic acid. In contrast, tumor necrosis factor alpha caused phospholipid hydrolysis, induction of the PT, cytochrome c release, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A(2) may be a mediator of tumor necrosis factor alpha cytotoxicity in situ through induction of the mitochondrial PT.     

Isochore specificity of AUG initiator context of human genes

The efficiency of AUG start codon recognition in translation initiation is modulated by its sequence context. Here we investigated a non-redundant set of 5914 human genes and show that this context is different in genes located in different isochores. In particular, of the two main consensus start sequences, RCCaugR is five-fold more represented than AARaugR in genes from the GC-rich H3 isochores compared to genes from the GC-poor L isochores. Furthermore, genes located in GC-rich isochores have shorter 5' UTRs and stronger avoidance of upstream AUG than genes located in GC-poor isochores. This suggests that genes requiring highly efficient translation are located in GC-rich isochores and genes requiring fine modulation of expression are located in GC-poor isochores. This is in agreement with independent data from the literature concerning the location of housekeeping and tissue-specific genes, respectively.     

Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.