Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.     

Biocompatible coated magnetosome minerals with various organization and cellular interaction properties induce cytotoxicity towards RG-2 and GL-261 glioma cells in the presence of an alternating magnetic field

Background

Biologics magnetics nanoparticles, magnetosomes, attract attention because of their magnetic characteristics and potential applications. The aim of the present study was to develop and characterize novel magnetosomes, which were extracted from magnetotactic bacteria, purified to produce apyrogen magnetosome minerals, and then coated with Chitosan, Neridronate, or Polyethyleneimine. It yielded stable magnetosomes designated as M-Chi, M-Neri, and M-PEI, respectively. Nanoparticle biocompatibility was evaluated on mouse fibroblast cells (3T3), mouse glioblastoma cells (GL-261) and rat glioblastoma cells (RG-2). We also tested these nanoparticles for magnetic hyperthermia treatment of tumor in vitro on two tumor cell lines GL-261 and RG-2 under the application of an alternating magnetic field. Heating, efficacy and internalization properties were then evaluated.

Results

Nanoparticles coated with chitosan, polyethyleneimine and neridronate are apyrogen, biocompatible and stable in aqueous suspension. The presence of a thin coating in M-Chi and M-PEI favors an arrangement in chains of the magnetosomes, similar to that observed in magnetosomes directly extracted from magnetotactic bacteria, while the thick matrix embedding M-Neri leads to structures with an average thickness of 3.5 µm² per magnetosome mineral. In the presence of GL-261 cells and upon the application of an alternating magnetic field, M-PEI and M-Chi lead to the highest specific absorption rates of 120–125 W/g_Fe. Furthermore, while M-Chi lead to rather low rates of cellular internalization, M-PEI strongly associate to cells, a property modulated by the application of an alternating magnetic field.

Conclusions

Coating of purified magnetosome minerals can therefore be chosen to control the interactions of nanoparticles with cells, organization of the minerals, as well as heating and cytotoxicity properties, which are important parameters to be considered in the design of a magnetic hyperthermia treatment of tumor.

     

Calcium-independent cytoskeleton disassembly induced by BAPTA.

In living organisms, Ca2+ signalling is central to cell physiology. The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) has been widely used as a probe to test the role of calcium in a large variety of cell functions. Here we show that in most cell types BAPTA has a potent actin and microtubule depolymerizing activity and that this activity is completely independent of Ca2+ chelation. Thus, the depolymerizing effect of BAPTA is shared by a derivative (D-BAPTA) showing a dramatically reduced calcium chelating activity. Because the extraordinary depolymerizing activity of BAPTA could be due to a general depletion of cell fuel molecules such as ATP, we tested the effects of BAPTA on cellular ATP levels and on mitochondrial function. We find that BAPTA depletes ATP pools and affects mitochondrial respiration in vitro as well as mitochondrial shape and distribution in cells. However, these effects are unrelated to the Ca2+ chelating properties of BAPTA and do not account for the depolymerizing effect of BAPTA on the cell cytoskeleton. We propose that D-BAPTA should be systematically introduced in calcium signalling experiments, as controls for the known and unknown calcium independent effects of BAPTA. Additionally, the concomitant depolymerizing effect of BAPTA on both tubulin and actin assemblies is intriguing and may lead to the identification of a new control mechanism for cytoskeleton assembly.     

Phytoplankton communities of North African wetland lakes: the CASSARINA Project

Seasonal variations in phytoplankton species composition (frequencies) and densities (cell numbers) in nine North African coastal lakes selected in Morocco (Merja Sidi Bou Rhaba, Zerga and Bokka), Tunisia (Chitane, Ichkeul and Korba lakes) and Egypt (Edku, Burullus and Manzala lakes) were investigated during 1998. The main aim was to provide gase-line information about overall phytoplankton diversity and how phytoplanktoncharacteristics differ between these contrasting aquatic systems.Water samples were collected at approximately three monthly intervals and phytoplankton analysis revealed marked seasonal and spatial differences in the quantitative and qualitative composition of the communities at each site. The Egyptian lakes generally had larger crops (Manzala and Burullus had mean crop densities of more than 104 cells ml^–1) but in the western North African sites only Korba and Sidi Bou Rhaba had closely comparable densities. Algae belonging to Bacillariophyceae, Chlorophyceae, Chrysophyceae, Cyanophyceae, Dinophyceae and Euglenophyceae were recorded. Taxa representative of all these algal groups occurred in two lakes (Korba and Manzala) but at the other seven sites only some of the groups were present.The Chlorophyceae was the most dominant group in lakes Burullus, Manzala, Korba and Sidi Bou Rhaba whereas Bacillariophyceae were dominant in lakes Zerga, Bokka and Edku. In Ichkeul and acidic Chitane the Dinophyceae and the Cyanophyceae were the dominant groups, respectively. The maximum percentage of Euglenophyceae occurred in Edku Lake but this group was absent in Sidi Bou Rhaba and Ichkeul. Cyanophyceans were present in significant numbers in all investigated lakes except in Ichkeul. A total of fifty-three genera were recorded, 17 of Chlorophyceae, 18 of Bacillariophyceae, 11 of Cyanophyceae, 3 of Chrysophyceae, 2 of Euglenophyceae and 2 of Dinophyceae. The maximum number of species (34) occurred in Burullus Lake and the minimum (6) in Ichkeul Lake. Only one lake (acidic Chitane) possessed species indicative of oligotrophic conditions. The Nile Delta lakes were the most species diverse sites.The phytoplankton communities of the nine North African lakes were composed entirely of cosmopolitan species but with one new species (Cyclotella choctawatcheeana) was recorded for the region. The data presented provide a contemporary account of the levels of algal diversity present in these sites at the end of the 20th century. The relevance of phytoplankton communities to assessment of lake status and future monitoring studies in the region is emphasised.     

North African wetland lakes: characterization of nine sites included in the CASSARINA Project

Exploitation of land and water resources has increased rapidly in North Africa during the 20th century, paralleling regional population growth. As part of the CASSARINA Project (see Flower, 2001), the environmental status of nine wetland lakes in Morocco, Tunisia, and Egypt was evaluated. All are conservationally important habitats and several are Ramsar Sites (internationally recognized bird reserves) and several support significant fisheries. All are shallow (<2 m in depth) but vary greatly in area.Where available, documentary information on relevant 20th century changes is given. Survey transects for aquatic vegetation were established and used to provide baseline ecological information on the aquatic plant communities during 1997–1999. Unusually, one site (Tunisian Megene Chitane) supported acidophilous vegetation (some taxa being nationally rare). Aquatic macrophytes declined catastrophically at two sites during the 1990s. Merja Bokka was drained in 1998 and, at Garaet El Ichkeul, fringing Phragmites and Scirpus spp. were lost, mainly as a result of salinity changes. Elsewhere, fringing macrophytes remain (extensively so in the Nile Delta lakes) common, despite major land reclamation and water quality problems, or are degraded by grazing (Merja Zerga). Marginal vegetation during 1997/98 changed markedly at Megene Chitane due to water level lowering.Documentary records indicated that throughout the 20th century, reclamation and hydrologic modifications, mainly for agricultural purposes, affected all nine sites. The loss of lake area by reclamation is substantial for the Nile Delta lakes (Edku, Burullus and Manzala). For the western sites, some data indicate increasing salinity in the most recent decade but the Delta lakes have become generally fresher during the 20th century, as supply of Nile water for irrigation increased.Despite intense human disturbance, many of the remaining CASSARINA sites still support regions of high aquatic diversity. Spatial scale monitoring of the larger sites for seasonal and inter-annual changes in open water area and in aquatic plant abundances is a key requirement for integrated environmental change assessment in the 21st century.     

Recent environmental change in North African wetland lakes: a baseline study of organochlorine contaminant residues in sediments from nine sites in the CASSARINA Project

Sediment from 9 lakes and lagoons in 3 North African countries was analysed for a suite of organochlorine insecticides and polychlorinated biphenyls (PCBs). Residues of -HCH and pp-DDE were detected in most cores, and radiometric dating of the sediment enabled time profiles to be constructed which are indicative of recent use of the insecticide lindane and previous use of the insecticide DDT in the region. Absolute concentrations of these pesticides were relatively low compared to reported values for other world-wide locations, but exceeded recommended Canadian and Dutch environmental quality standards at several sites. Maximum fluxes of -HCH and pp-DDE (190 and 95 g m^–2 yr^–1, respectively) were relatively high and comparable to some sediments in North America and the United Kingdom. Other organochlorine pesticides including dieldrin were detected at low levels in some samples. Selected PCB congeners were detected at trace levels at 2 sites only, indicating low levels of industrial contamination at the sampling locations. Tropical and sub-tropical regions of the world have been suggested to be contemporary sources of globally distributed organochlorine contaminants. These data are discussed with respect to this hypothesis.     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.     

Towards a Phylogeny for Coffea (Rubiaceae): identifying well-supported lineages based on nuclear and plastid DNA sequences

BACKGROUND AND AIMS: The phylogenetic relationships between species of Coffea and Psilanthus remain poorly understood, owing to low levels of sequence variation recovered in previous studies, coupled with relatively limited species sampling. In this study, the relationships between Coffea and Psilanthus species are assessed based on substantially increased molecular sequence data and greatly improved species sampling. METHODS: Phylogenetic relationships are assessed using parsimony, with sequence data from four plastid regions [trnL-F intron, trnL-F intergenic spacer (IGS), rpl16 intron and accD-psa1 IGS], and the internal transcribed spacer (ITS) region of nuclear rDNA (ITS 1/5.8S/ITS 2). Supported lineages in Coffea are discussed within the context of geographical correspondence, biogeography, morphology and systematics. KEY RESULTS: Several major lineages with geographical coherence, as identified in previous studies based on smaller data sets, are supported. Other lineages with either geographical or ecological correspondence are recognized for the first time. Coffea subgenus Baracoffea is shown to be monophyletic, but Coffea subgenus Coffea is paraphyletic. Sequence data do not substantiate the monophyly of either Coffea or Psilanthus. Low levels of sequence divergence do not allow detailed resolution of relationships within Coffea, most notably for species of Coffea subgenus Coffea occurring in Madagascar. The origin of C. arabica by recent hybridization between C. canephora and C. eugenioides is supported. Phylogenetic separation resulting from the presence of the Dahomey Gap is inferred based on sequence data from Coffea.     

Age‐dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine‐treated mice

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane‐Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2‐treated mice without change in proteins, while it decreased GluN2B protein in P10‐treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5‐treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5‐treated mice. Microdissection of ketamine‐treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I–IV) and an increase in all subunit expressions in deep layers (V–VI) in P5‐ and P10‐treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A‐enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running‐wheel exercise showed age‐ and sex‐dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short‐term NMDAR cortical developmental profile impairments and long‐term motor activity alterations persisting in adulthood. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 315–333, 2015