Replication Fork Breakage and Restart in Escherichia coli

In all organisms, replication impairments are an important source of genome rearrangements, mainly because of the formation of double-stranded DNA (dsDNA) ends at inactivated replication forks. Three reactions for the formation of dsDNA ends at replication forks were originally described for Escherichia coli and became seminal models for all organisms: the encounter of replication forks with preexisting single-stranded DNA (ssDNA) interruptions, replication fork reversal, and head-to-tail collisions of successive replication rounds. Here, we first review the experimental evidence that now allows us to know when, where, and how these three different reactions occur in E. coli. Next, we recall our recent studies showing that in wild-type E. coli, spontaneous replication fork breakage occurs in 18% of cells at each generation. We propose that it results from the replication of preexisting nicks or gaps, since it does not involve replication fork reversal or head-to-tail fork collisions. In the recB mutant, deficient for double-strand break (DSB) repair, fork breakage triggers DSBs in the chromosome terminus during cell division, a reaction that is heritable for several generations. Finally, we recapitulate several observations suggesting that restart from intact inactivated replication forks and restart from recombination intermediates require different sets of enzymatic activities. The finding that 18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for replication restart, which leads us to speculate that the replicative helicase remains on DNA at intact inactivated replication forks and is reactivated by the replication restart proteins

Recurrent rare copy number variants increase risk for esotropia

Purpose: To determine whether rare copy number variants (CNVs) increase risk for comitant esotropia. Methods: CNVs were identified in 1614 Caucasian individuals with comitant esotropia and 3922 Caucasian controls from Illumina SNP genotyping using two Hidden Markov model (HMM) algorithms, PennCNV and QuantiSNP, which call CNVs based on logR ratio and B allele frequency. Deletions and duplications greater than 10 kb were included. Common CNVs were excluded. Association testing was performed with 1 million permutations in PLINK. Significant CNVs were confirmed with digital droplet polymerase chain reaction (ddPCR). Whole genome sequencing was performed to determine insertion location and breakpoints. Results: Esotropia patients have similar rates and proportions of CNVs compared with controls but greater total length and average size of both deletions and duplications. Three recurrent rare duplications significantly (P = 1 × 10-6) increase the risk of esotropia: chromosome 2p11.2 (hg19, 2:87428677-87965359), spanning one long noncoding RNA (lncRNA) and two microRNAs (OR 14.16; 95% confidence interval [CI] 5.4-38.1); chromosome 4p15.2 (hg19, 4:25554332-25577184), spanning one lncRNA (OR 11.1; 95% CI 4.6-25.2); chromosome 10q11.22 (hg19, 10:47049547-47703870) spanning seven protein-coding genes, one lncRNA, and four pseudogenes (OR 8.96; 95% CI 5.4-14.9). Overall, 114 cases (7%) and only 28 controls (0.7%) had one of the three rare duplications. No case nor control had more than one of these three duplications. Conclusions: Rare CNVs are a source of genetic variation that contribute to the genetic risk for comitant esotropia, which is likely polygenic. Future research into the functional consequences of these recurrent duplications may shed light on the pathophysiology of esotropia

Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres

The viability of lyophilized cultures of Lactobacillus bulgaricus in skim milk, during storage at different temperatures, relative humidities, and atmospheres was investigated. Survival was greatest at 11% relative humidity and at 5°C. Indirect and direct evidence is presented supporting the hypothesis that membrane damage occurs during storage. Experiments on the lipid composition of the cell membrane demonstrate that changes occur with time that are probably the result of oxidation. A study on the lipid composition of the cell membrane by gas chromatography showed that the unsaturated/saturated fatty acid index changes with time during storage

Refining the transit-timing and photometric analysis of TRAPPIST-1: Masses, Radii, densities, dynamics, and ephemerides

We have collected transit times for the TRAPPIST-1 system with the Spitzer Space Telescope over four years. We add to these ground-based, HST and K2 transit time measurements, and revisit an N-body dynamical analysis of the seven-planet system using our complete set of times from which we refine the mass ratios of the planets to the star. We next carry out a photodynamical analysis of the Spitzer light curves to derive the density of the host star and the planet densities. We find that all seven planets' densities may be described with a single rocky mass-radius relation which is depleted in iron relative to Earth, with Fe 21 wt% versus 32 wt% for Earth, and otherwise Earth-like in composition. Alternatively, the planets may have an Earth-like composition, but enhanced in light elements, such as a surface water layer or a core-free structure with oxidized iron in the mantle. We measure planet masses to a precision of 3-5%, equivalent to a radial-velocity (RV) precision of 2.5 cm/sec, or two orders of magnitude more precise than current RV capabilities. We find the eccentricities of the planets are very small; the orbits are extremely coplanar; and the system is stable on 10 Myr timescales. We find evidence of infrequent timing outliers which we cannot explain with an eighth planet; we instead account for the outliers using a robust likelihood function. We forecast JWST timing observations, and speculate on possible implications of the planet densities for the formation, migration and evolution of the planet system

A Screen against Leishmania Intracellular Amastigotes: Comparison to a Promastigote Screen and Identification of a Host Cell-Specific Hit

The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect–infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown. lineage. genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates

Significance of vascular endothelial growth factor in growth and peritoneal dissemination of ovarian cancer

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis which drives endothelial cell survival, proliferation, and migration while increasing vascular permeability. Playing an important role in the physiology of normal ovaries, VEGF has also been implicated in the pathogenesis of ovarian cancer. Essentially by promoting tumor angiogenesis and enhancing vascular permeability, VEGF contributes to the development of peritoneal carcinomatosis associated with malignant ascites formation, the characteristic feature of advanced ovarian cancer at diagnosis. In both experimental and clinical studies, VEGF levels have been inversely correlated with survival. Moreover, VEGF inhibition has been shown to inhibit tumor growth and ascites production and to suppress tumor invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer. In this review, we will focus on VEGF involvement in the pathophysiology of ovarian cancer and its contribution to the disease progression and dissemination