The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks

Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.     

ROC-guided survival trees and ensembles

Tree-based methods are popular nonparametric tools in studying time-to-event outcomes. In this article, we introduce a novel framework for survival trees and ensembles, where the trees partition the dynamic survivor population and can handle time-dependent covariates. Using the idea of randomized tests, we develop generalized time-dependent receiver operating characteristic (ROC) curves for evaluating the performance of survival trees. The tree-building algorithm is guided by decision-theoretic criteria based on ROC, targeting specifically for prediction accuracy. To address the instability issue of a single tree, we propose a novel ensemble procedure based on averaging martingale estimating equations, which is different from existing methods that average the predicted survival or cumulative hazard functions from individual trees. Extensive simulation studies are conducted to examine the performance of the proposed methods. We apply the methods to a study on AIDS for illustration.     

Osmotic dehydration of Kappaphycus alvarezii

The red alga Kappaphycus alvarezii has been reported to be a potential raw material for functional food due to its high content of soluble dietary fibre, mineral, omega-3 fatty acids as well as a substantial amount of essential amino acids. In order to benefit from these excellent nutritional properties, this project aimed to develop a high-value dehydrated snack from K. alvarezii using osmotic dehydration (OD) treatment prior to hot air-drying. A 3?×?3 factorial design with 50°, 60° and 70°Brix sucrose concentration as well as treatment temperatures of 30, 35 and 40 °C were used. In general, an increase in sucrose concentration and temperature promoted mass transfer. OD treatment using 70°Brix sucrose concentration at 40 °C caused case hardening of the seaweed that reduced the solid gain (p?<?0.05). Firmness of the seaweed increased with sucrose concentration and was not altered by temperature (p?<?0.05). The colour of the seaweed was not affected by OD treatment (p?>?0.05), but dehydrated seaweed became darker at high sugar concentration. Interaction effect between sucrose concentration and temperature was found to affect the water loss and solid gain of the OD treatment (p?<?0.05). The best sensory acceptable dehydrated seaweed was successfully identified. The final product contained high dietary fibre and very low Na/K ratio.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

Late Quaternary (Weichselian) alluvial history and neotectonic control on fluvial landscape development in the southern Körös plain,Hungary

Four drill cores and a clay pit section have been examined in the southern part of the Körös plain to understand the history and controls on alluvial sedimentation for the last ~ 40 ka. Four facies groups were identified, such as channel, channel margin, floodplain and floodbasin with seven distinctive facies. Magnetic susceptibility and mineralogy have further characterized the sedimentary facies indicating shifts in humidity conditions, variations in sediment flux and pedogenesis. Detailed pollen analysis of a 7.5 m thick clayey succession indicated climatic variability within the MIS 3 period. The spatial distribution of the different facies allowed outlining alluvial architecture of the study area. Three depositional units composed of various facies were identified based on OSL and radiocarbon data. These packages correspond to three major phases of channel activity: (F-I) pre-LGM period (> 30 ka to 24 ka), (F-II) post-LGM interstadial (18–16 ka), and (F-III) Late Glacial < 15 ka to ~ 10 ka). The pre-LGM and post-LGM “interstadial” phases are characterized by meandering river patterns, while the Late Glacial fluvial activity is characterized by a braided system in the area. Higher sediment supply feeding this braided river was probably caused by neotectonic uplift of the southern margin of the basin, documented by a significant stratigraphic gap between 25 and 14 ka.     

Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium

We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C. comosa, C. glaucoides, C. goreensis, C. hyssopifolia, C. lathyroides, C. perrottetii, C. podocarpa, and C. retusa) growing in Senegal. Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria. Group I consists of plants solely nodulated by very specific fast-growing strains. Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp. strains already reported to nodulate many tropical legumes. SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia. Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I. The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.     

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.     

An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain

Production of the pathogenic prion isoform PrP^sc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the “protein-only“ hypothesis. In this report, an engineered PrP^sc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentB-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrP^se and will be helpful for further understanding the mechanism of conformational conversion process.     

RADACK, a stochastic simulation of hydroxyl radical attack to DNA

RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes. It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling. The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively. The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed. The calculated relative probabilities of OH* radical attack to all reaction sites are compared to experimental FSB and ARB values. By a fitting procedure, the relative efficiencies of conversion of the C4' and C5'-centred radicals into FSB, epsilon (C4'): epsilon (C5'), and the relative efficiencies of base radicals- to- ARB conversion, epsilon(T) : epsilon(A) : epsilon(C) : epsilon(G), are then deduced for each DNA form. The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems : the lac repressor-lac operator complex and the nuclcosome core.