Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571

Abstract The effect of carbon starvation on growth and poly-β-hydroxybutyrate (PHB) utilization in oxygen-limited chemostat cultures of Rhizobium ORS571 was studied. Under oxygen-limited growth conditions PHB was not degraded. When in a nitrogen-fixing oxygen-limited culture, after stopping the medium supply, the dissolved oxygen concentration was maintained at 10 μM, a slow breakdown of PHB was observed. Addition of ammonia and air to a nitrogen-fixing oxygen-limited culture after the medium supply had been stopped, resulted in the simultaneous utilization of PHB and succinate. The possible use of the energy derived from PHB degradation in Rhizobia bacteria and bacteroids is discussed.     

Using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling in order to predict clinical outcomes of patients with heart failure

Background

Current risk prediction models in heart failure (HF) including clinical characteristics and biomarkers only have moderate predictive value. The aim of this study was to use matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) profiling to determine if a combination of peptides identified with MALDI-MS will better predict clinical outcomes of patients with HF.

Methods

A cohort of 100 patients with HF were recruited in the biomarker discovery phase (50 patients who died or had a HF hospital admission vs. 50 patients who did not have an event). The peptide extraction from plasma samples was performed using reversed phase C18. Then samples were analysed using MALDI-MS. A multiple peptide biomarker model was discovered that was able to predict clinical outcomes for patients with HF. Finally, this model was validated in an independent cohort with 100 patients with HF.

Results

After normalisation and alignment of all the processed spectra, a total of 11,389 peptides (m/z) were detected using MALDI-MS. A multiple biomarker model was developed from 14 plasma peptides that was able to predict clinical outcomes in HF patients with an area under the receiver operating characteristic curve (AUC) of 1.000 (p?=?0.0005). This model was validated in an independent cohort with 100 HF patients that yielded an AUC of 0.817 (p?=?0.0005) in the biomarker validation phase. Addition of this model to the BIOSTAT risk prediction model increased the predictive probability for clinical outcomes of HF from an AUC value of 0.643 to an AUC of 0.823 (p?=?0.0021). Moreover, using the prediction model of fourteen peptides and the composite model of the multiple biomarker of fourteen peptides with the BIOSTAT risk prediction model achieved a better predictive probability of time-to-event in prediction of clinical events in patients with HF (p?=?0.0005).

Conclusions

The results obtained in this study suggest that a cluster of plasma peptides using MALDI-MS can reliably predict clinical outcomes in HF that may help enable precision medicine in HF.

     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Miocene Trace Fossils from Borneo

In the Miocene Belait formation of northern Sarawak, Brunei and Sabah burrows ofOphiomorpha Lundgren were found, described here asOphiomorpha borneensis n. ichnosp. A few associated spiral tunnels of theXenohelix-type were also found. Two species ofHäntzschelinia Vialov occurred on Labuan Island. A third type of structure was found in Brunei and it probably represents castings of an unknown lamellibranch. The burrows and castings were made in a brackish water environment, probably in the littoral zone of an embayment or delta.     

Flyway population delineation in Taiga Bean Geese Anser fabalis fabalis revealed by multi‐element feather stable isotope analysis

Fundamental to effective management of migratory waterbird populations is an understanding of their flyway delineation. Taiga Bean Geese Anser fabalis fabalis wintering in NW Denmark, Scotland and England are considered to originate from northern and central Sweden, southern and central Norway (‘Western flyway’), those wintering in southern Sweden, NE and southern Denmark are considered to originate from northern Fennoscandia and western Russia (‘Central flyway’), and those wintering in eastern Germany and Poland (which show far less favourable conservation status) are thought to come from western Siberia (‘Eastern 1 flyway’), although evidence to demonstrate this has largely been lacking. Evidence for different natal and moult origins of Taiga Bean Geese was investigated using stable isotope analyses of feathers of four elements (δ²H, δ¹³C, δ¹⁵N and δ³⁴S). There were significant differences in isotopic composition of feathers from Swedish (Central) and German (Eastern 1) wintering populations and those moulting in Sweden in late summer (Western), which validated the three proposed major management flyway units above. The strong continental gradient in the stable hydrogen isotope ratios in precipitation (δ²H_p) across the region was used to assign wintering birds geospatially to natal and moulting origin, indicating separate natal and moulting areas for German (n = 37, from western Siberia) and Swedish (n = 20, Fennoscandia and more western Russia) wintering birds. These results confirm the largely discrete nature of these three flyways and contribute significantly to our ability to deliver effective targeted and appropriate research, monitoring and management actions throughout the ranges of these flyways.     

Cell lineage and gene expression in the development of polychaetes

The developmental strategies of embryos within the various taxa of polychaetes are designed to set up the fate of the cell lines. Some of these traits of pattern formation are considered to be ancestral, but we also find a number of derived developmental characteristics, some of which might be useful indicators for phylogenetic relationships. A combination of ooplasmic segregation and anisotropic cleavage rapidly establishes the fate of several larval cell lines in the polychaete embryo. The setting-up of the primary trochoblasts basically concerns the same cell line (la²–ld²) in polychaetes and even in molluscs. Such mechanisms may thus be regarded as ancestral. The determination of the mesoderm precursor occurs very late in both equally cleaving annelids and mollusks, indicating that an equal cleavage pattern may be regarded as an ancestral trait. Since both disproportionate cytoplasmic distribution (either by spindle shift or polar lobe formation) and cell cycle asynchronies appear to speed up the development of the mesoderm-forming cell line, these strategies represent derived traits. An analysis of these derived traits of early development is given and is discussed in the light of the phylogenetic relationships among the polychaetes. These data are extended by an analysis of some of the postlarval structures in polychaetes and the molecular developmenta1 circuitry involved.     

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ~150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.     

Multi-RELIEF: a method to recognize specificity determining residues from multiple sequence alignments using a Machine-Learning approach for feature weighting

MOTIVATION: Identification of residues that account for protein function specificity is crucial, not only for understanding the nature of functional specificity, but also for protein engineering experiments aimed at switching the specificity of an enzyme, regulator or transporter. Available algorithms generally use multiple sequence alignments to identify residue positions conserved within subfamilies but divergent in between. However, many biological examples show a much subtler picture than simple intra-group conservation versus inter-group divergence. RESULTS: We present multi-RELIEF, a novel approach for identifying specificity residues that is based on RELIEF, a state-of-the-art Machine-Learning technique for feature weighting. It estimates the expected 'local' functional specificity of residues from an alignment divided in multiple classes. Optionally, 3D structure information is exploited by increasing the weight of residues that have high-weight neighbors. Using ROC curves over a large body of experimental reference data, we show that (a) multi-RELIEF identifies specificity residues for the seven test sets used, (b) incorporating structural information improves prediction for specificity of interaction with small molecules and (c) comparison of multi-RELIEF with four other state-of-the-art algorithms indicates its robustness and best overall performance. AVAILABILITY: A web-server implementation of multi-RELIEF is available at www.ibi.vu.nl/programs/multirelief. Matlab source code of the algorithm and data sets are available on request for academic use.