Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.     

Computing folding pathways between RNA secondary structures

Given an RNA sequence and two designated secondary structures A, B, we describe a new algorithm that computes a nearly optimal folding pathway from A to B. The algorithm, RNAtabupath, employs a tabu semi-greedy heuristic, known to be an effective search strategy in combinatorial optimization. Folding pathways, sometimes called routes or trajectories, are computed by RNAtabupath in a fraction of the time required by the barriers program of Vienna RNA Package. We benchmark RNAtabupath with other algorithms to compute low energy folding pathways between experimentally known structures of several conformational switches. The RNApathfinder web server, source code for algorithms to compute and analyze pathways and supplementary data are available at http://bioinformatics.bc.edu/clotelab/RNApathfinder.     

Enzymatic cleaning of biofouled thin-film composite reverse osmosis (RO) membrane operated in a biofilm membrane reactor

Application of environmentally friendly enzymes to remove thin-film composite (TFC) reverse osmosis (RO) membrane biofoulants without changing the physico-chemical properties of the RO surface is a challenging and new concept. Eight enzymes from Novozyme A/S were tested using a commercially available biofouling-resistant TFC polyamide RO membrane (BW30, FilmTech Corporation, Dow Chemical Co.) without filtration in a rotating disk reactor system operated for 58 days. At the end of the operation, the accumulated biofoulants on the TFC RO surfaces were treated with the three best enzymes, Subtilisin protease and lipase; dextranase; and polygalacturonase (PG) based enzymes, at neutral pH (~7) and doses of 50, 100, and 150 ppm. Contact times were 18 and 36 h. Live/dead staining, epifluorescence microscopy measurements, and 5 μm thick cryo-sections of enzyme and physically treated biofouled membranes revealed that Subtilisin protease- and lipase-based enzymes at 100 ppm and 18 h contact time were optimal for removing most of the cells and proteins from the RO surface. Culturable cells inside the biofilm declined by more than five logs even at the lower dose (50 ppm) and shorter incubation period (18 h). Subtilisin protease- and lipase-based enzyme cleaning at 100 ppm and for 18 h contact time restored the hydrophobicity of the TFC RO surface to its virgin condition while physical cleaning alone resulted in a 50° increase in hydrophobicity. Moreover, at this optimum working condition, the Subtilisin protease- and lipase-based enzyme treatment of biofouled RO surface also restored the surface roughness measured with atomic force microscopy and the mass percentage of the chemical compositions on the TFC surface estimated with X-ray photoelectron spectroscopy to its virgin condition. This novel study will encourage the further development and application of enzymes to remove biofoulants on the RO surface without changing its surface properties.     

Degradation of pectins with different degrees of esterification by Bacteroides thetaiotaomicron isolated from human gut flora

A complete human fecal flora and cultures of defined species obtained from fecal flora were investigated in vitro to determine their ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli. Macromolecular pectins with different degrees of esterification were used as substrates in microbial degradation studies. The levels of oligogalacturonic acids formed in batch cultures were estimated during a 24- or 48-h incubation period by using high-performance thin-layer chromatography and high-performance anion-exchange chromatography. The spectrum and the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the culture media during incubation with the complete fecal flora. After 24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of a complete fecal flora. Cocultures of B. thetaiotaomicron and E. coli exhibited intermediate levels of degradation activity. In pure cultures of E. coli no pectin-degrading activity was found. Additionally, the rate of pectin degradation was affected by the degree of esterification of the substrate. Saturated oligogalacturonic acids were not found during pectin fermentation. The disappearance of oligogalacturonic acids in the later stages of fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of short-chain fatty acids.     

Recolonisation patterns of benthic invertebrates: a field investigation of restored former sewage channels

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A matrilineal overthrow with destructive aggression inMacaca fascicularis

We report the sequence of a matrilineal overthrow in longtailed macaques (Macaca fascicularis) during which the α-matriline dropped to the third of four ranks. In the initial phase of the overthrow, females from the former second ranking matriline attacked the females of the former α-matriline in a sequence which suggests a strategy. They attacked the highest ranking mother first and then directed their attacks toward her daughter, then toward her grand-daughter, and finally toward her great-grand-daughter. This seems to be the most effective way to disrupt the possible interventions of mothers in favor of their daughters.     

Safety of the Recombinant Cholera Toxin B Subunit, Killed Whole-Cell (rBS-WC) Oral Cholera Vaccine in Pregnancy

Introduction

Mass vaccinations are a main strategy in the deployment of oral cholera vaccines. Campaigns avoid giving vaccine to pregnant women because of the absence of safety data of the killed whole-cell oral cholera (rBS-WC) vaccine. Balancing this concern is the known higher risk of cholera and of complications of pregnancy should cholera occur in these women, as well as the lack of expected adverse events from a killed oral bacterial vaccine.

Methodology/Principal Findings

From January to February 2009, a mass rBS-WC vaccination campaign of persons over two years of age was conducted in an urban and a rural area (population 51,151) in Zanzibar. Pregnant women were advised not to participate in the campaign. More than nine months after the last dose of the vaccine was administered, we visited all women between 15 and 50 years of age living in the study area. The outcome of pregnancies that were inadvertently exposed to at least one oral cholera vaccine dose and those that were not exposed was evaluated. 13,736 (94%) of the target women in the study site were interviewed. 1,151 (79%) of the 1,453 deliveries in 2009 occurred during the period when foetal exposure to the vaccine could have occurred. 955 (83%) out of these 1,151 mothers had not been vaccinated; the remaining 196 (17%) mothers had received at least one dose of the oral cholera vaccine. There were no statistically significant differences in the odds ratios for birth outcomes among the exposed and unexposed pregnancies.

Conclusions/Significance

We found no statistically significant evidence of a harmful effect of gestational exposure to the rBS-WC vaccine. These findings, along with the absence of a rational basis for expecting a risk from this killed oral bacterial vaccine, are reassuring but the study had insufficient power to detect infrequent events.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00709410","term_id":"NCT00709410"}}NCT00709410     

The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42*

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.Macrophages, terminally differentiated cells of the mononuclear phagocytic lineage, are found throughout the body and play important roles in normal tissue development and immune defense. However, in certain circumstances, excessive recruitment of macrophages has been shown to participate in the progression of several diseases, inflammatory (rheumatoid arthritis) or metabolic (atherosclerosis), as well as in tumor progression (1–3). Importantly expression of colony-stimulating factor-1 (CSF-1),⁴ the most pleiotropic macrophage growth factor, has been correlated with the progression of these disease states (for a review, see Ref. 4). Inhibition of undesirable macrophage recruitment to specific sites in response to CSF-1 is therefore an attractive goal for therapies (5).In addition to stimulating survival, proliferation, and differentiation of monocytes and macrophages, CSF-1 is also a potent chemotactic factor inducing the migration of these cell types (for a review, see Ref. 4). CSF-1 stimulation leads to the rapid production of F-actin-rich protrusions and the spreading and migration of macrophages (4). All CSF-1 effects are mediated through its tyrosine kinase receptor (CSF-1R), which upon activation leads to phosphorylation of tyrosine residues in a number of signaling molecules. Downstream molecules essential for macrophage migration in response to CSF-1 include phosphatidylinositol 3-kinase (PI3K) isoforms β and δ (6, 7). PI3K may potentially regulate migration through the activation of guanine nucleotide exchange factor activity to Rac1 and Cdc42, which are required for CSF-1-elicited protrusions (8, 9) and chemotaxis (10). The major means by which Rac and Cdc42 regulate the Arp2/3 complex is through the Wiskott-Aldrich syndrome protein/Wiskott-Aldrich syndrome verprolin-homologous (WASP/WAVE) family of proteins (11). A Rac1-IRSp53-Abi1-WAVE2 complex has been shown to mediate CSF-1-induced macrophage motility (12, 13), and a unique role for WASP in macrophage chemotaxis to CSF-1, formylmethionylleucylphenylalanine, MCP-1, and MIP-1α has been demonstrated (14, 15). WASP is a hematopoietic cell-specific regulator of Arp2/3-dependent actin remodeling. The catalytically active domain of WASP lies in its C terminus, which is conserved among all WASP/WAVE proteins and contains a VCA (verprolin homology, cofilin-like, and acidic region) domain capable of activating the Arp2/3 complex. The other domains found in WASP can regulate, directly or indirectly, the activity of its VCA domain (for a review, see Ref. 16). Both WASP and N-WASP bind activated Cdc42 through their GTPase-binding domain, which is believed to cause a structural transition that results in dissociation of the intramolecular contacts leaving the VCA domain accessible for Arp2/3 binding (17, 18). In addition, biochemical studies have revealed that several signaling molecules, including WASP-interacting SH3 protein, WASP-interacting protein, Grb2, phosphoinositides, and Src family kinases, activate N-WASP (for reviews, see Refs. 16 and 19). Phosphorylation of WASP has also been proposed to activate Arp2/3-mediated actin polymerization in vitro (20–22).Recently different probes have been developed that detect a conformational change in N-WASP and therefore reflect its activation (23–25). Using either a fluorescence resonance energy transfer (FRET)-based biosensor that detects a conformational change in N-WASP (23, 24) or antibodies that can only bind to the open conformation of N-WASP (25), N-WASP has been shown to be activated in response to epidermal growth factor in HEK293 cells and in MTLn3 carcinoma cells. This activity has been temporally localized to subcellular compartments important for carcinoma cell chemotaxis and invasion (24). We have adapted these approaches to explore the signal transduction pathways responsible for the activation of WASP in vivo.     

Energetics of embryonic development: effects of temperature on egg and hatchling composition in a butterfly

Phenotypic plasticity may allow an organism to adjust its phenotype to environmental needs. However, little is known about environmental effects on offspring biochemical composition and turnover rates, including energy budgets and developmental costs. Using the tropical butterfly Bicyclus anynana and employing a full-factorial design with two oviposition and two developmental temperatures, we explore the consequences of temperature variation on egg and hatchling composition, and the associated use and turnover of energy and egg compounds. At the lower temperature, larger but fewer eggs were produced. Larger egg sizes were achieved by provisioning these eggs with larger quantities of all compounds investigated (and thus more energy), whilst relative egg composition was rather similar to that of smaller eggs laid at the higher temperature. Turnover rates during embryonic development differed across developmental temperatures, suggesting an emphasis on hatchling quality (i.e. protein content) at the more stressful lower temperature, but on storage reserves (i.e. lipids) at the higher temperature. These differences may represent adaptive maternal effects. Embryonic development was much more efficient at the lower temperature, providing a possible mechanism underlying the temperature-size rule.