G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Measuring the Outcome of Biomedical Research: A Systematic Literature Review

Background

There is an increasing need to evaluate the production and impact of medical research produced by institutions. Many indicators exist, yet we do not have enough information about their relevance. The objective of this systematic review was (1) to identify all the indicators that could be used to measure the output and outcome of medical research carried out in institutions and (2) enlist their methodology, use, positive and negative points.

Methodology

We have searched 3 databases (Pubmed, Scopus, Web of Science) using the following keywords: [Research outcome* OR research output* OR bibliometric* OR scientometric* OR scientific production] AND [indicator* OR index* OR evaluation OR metrics]. We included articles presenting, discussing or evaluating indicators measuring the scientific production of an institution. The search was conducted by two independent authors using a standardised data extraction form. For each indicator we extracted its definition, calculation, its rationale and its positive and negative points. In order to reduce bias, data extraction and analysis was performed by two independent authors.

Findings

We included 76 articles. A total of 57 indicators were identified. We have classified those indicators into 6 categories: 9 indicators of research activity, 24 indicators of scientific production and impact, 5 indicators of collaboration, 7 indicators of industrial production, 4 indicators of dissemination, 8 indicators of health service impact. The most widely discussed and described is the h-index with 31 articles discussing it.

Discussion

The majority of indicators found are bibliometric indicators of scientific production and impact. Several indicators have been developed to improve the h-index. This indicator has also inspired the creation of two indicators to measure industrial production and collaboration. Several articles propose indicators measuring research impact without detailing a methodology for calculating them. Many bibliometric indicators identified have been created but have not been used or further discussed.     

Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).     

Positive interactions of the smooth cordgrass Spartina alterniflora on the mud snail Heleobia australis, in South Western Atlantic salt marshes

The role of positive interactions is often crucial in communities with intense abiotic stress such as intertidal environments. Grasses acting as ecosystem engineers, for example, may ameliorate intertidal harsh physical conditions and modify the community structure. The mud snails Heleobia australis d'Orbigny frequently inhabit the SW Atlantic marshes, mainly associated to intertidal marsh plants (mainly the smooth cordgrass Spartina alterniflora Loisel) probably due to the plant indirect effects. The purpose of this work was to investigate the magnitude of these association and the processes that generate the pattern. Samples of the snail abundance in six SW Atlantic coastal marshes show that H. australis is associated to coastal areas of low energy and low or none freshwater input. This result is important because this species is being used as bioindicator of coastal estuarine systems during the Holocene. Thus the paleontological interpretation based on this species should be revised. Within the studied areas, snails are associated to intertidal marsh plants. However, stable isotope analysis shows that neither plant nor their epiphytes are their main food sources. Field experiments show that snails actively select areas with plants, although tethering experiments show that plants do not provide shelter from predators. However, plants do buffer physical stress factors such as temperature, which generate important mortality outside plants covered areas. These positive interactions have large effects on H. australis distributions in marsh communities; increasing the habitats available for colonization and affecting their local distribution.     

Cutaneous metastasis of occult hepatocellular carcinoma: a case report

BACKGROUND: Skin metastases from hepatocellular carcinoma (HCC) represented only 0.8% of all known cutaneous metastases in a recent large series. The most frequent site appears to be the head, but this fact has received little attention. An accurate cytologic diagnosis is extremely difficult in patients with unknown liver dysfunction. We report the cytologic features of a face metastasis from occult HCC. CASE: A 65-year-old woman presented with a mass in the right preauricular region of 2 months' duration. Her past medical history was noncontributory. Fine needle aspiration cytology was performed. Following the cytologic diagnosis, computed tomography revealed a 6-cm mass in the right lobe of the liver, portal vein thrombosis and involvement of the superior vena cava. The smears were very cellular. The most frequent pattern was trabecular with transmural endothelization. The cells had an epithelial appearance and polyhedral shape, exhibiting distinct borders. The nuclei were centrally placed, with a prominent nucleolus. The cytoplasm was granular. There were numerous atypical bare nuclei. Subsequent staining with antihepatocyte showed positivity in most tumor cells. The final diagnosis was metastatic HCC. CONCLUSION: HCCs should be considered in the differential diagnosis of carcinomas metastatic to the face, even in the absence of liver symptoms.     

Protein disorder,prion propensities,and self-organizing macromolecular collectives

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.     

Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.