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At least 739 records · Page 41

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon-insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in ΔpriB cells. Taken together, our results highlight roles for several genes in double-strand DNA break homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G > A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. Finally to our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.

59 BASIC BIOLOGICAL SCIENCES↗

Mechanism of efficient double-strand break repair by a long non-coding RNA

The Authors wish to correct an error in Figure 6. In Figure 6B the panel label should read ‘Ku + DNA-PKcs + LINP1, 6 kbp bridge’ instead of ‘Ku + DNA-PKcs + PAXX, 6 kbp bridge’. The published article has been updated. This error does not affect the conclusions of the article.

59 BASIC BIOLOGICAL SCIENCES↗

Repair strategy for fused silica wedged focus lens with high-density filamentary damage

The National Ignition Facility (NIF) is intentionally operated with the final fused silica glass (SiO2) optics exposed to fluences and intensities with the potential to induce damage that will grow with additional laser exposure. Therefore, the NIF operates a recycle loop to refurbish final optics by mitigating any initiated surface damage to arrest its growth. However, the morphology of filamentary damage, caused by local self-focusing in the bulk of a silica optic, adds complexity to optics mitigation and provides a limitation to optic reusability. Here, we evaluate techniques for mitigating isolated and clustered filamentary damage. Optical microscopy before and after installation on NIF was used to determine the efficacy of filamentary mitigation after a series of high fluence and intensity laser exposures. The challenges and success rate of the methods are compared for various filamentary damage mitigation strategies.

National Ignition Facility↗

Exosomes From Induced Pluripotent Stem Cell–Derived Cardiomyocytes Promote Autophagy for Myocardial Repair

Background. Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCMs (iCM-Ex) can be robustly collected in vitro and injected in lieu of live iCMs as a cell-free therapy for myocardial infarction. Methods and Results. iCM-Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM-Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real-time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM-Ex measured ≈140 nm and expressed CD63 and CD9. iCM and iCM-Ex microRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM-Ex demonstrated significant cardiac improvement post–myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM-Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM-Ex groups in vivo and in vitro. Conclusions. iCM-Ex improve post–myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell-free, patient-specific therapy for ischemic cardiomyopathy.

60 APPLIED LIFE SCIENCES↗

Self-repairing properties of OPC clinker/natural zeolite blend in water and alkali carbonate environments at 270°C

The 10 d recoveries of the mechanical properties and crack sealing of an ordinary Portland cement (OPC) clinker/natural zeolite (ferrierite (Fer)) blend modified or unmodified with silica were tested at 270°C in water and alkali carbonate environments. The recoveries of the samples depended on their modification with silica and the curing environment, but were more than 100% after repeated damage under some test conditions. The mechanical properties and phase compositions of recovered samples were evaluated by compressive strength measurements and x-ray diffraction, differential thermogravimetric analyses, Fourier transform infrared analyses and scanning electron microscopy coupled with energy dispersive x-ray spectroscopy. The sealing of 0·25 mm wide and ~2 mm deep cracks was visualised with a three-dimensional optical microscope. Fer decomposed under high-temperature alkaline conditions with the release of hydrolysates that, along with the hydrating clinker, participated in the formation of new phases contributing to strength recoveries. Here, these phases included crystalline magnesium and aluminium-containing silicates, calcium and carbonated calcium silicates and amorphous hydrates. Crack sealing was complete for the silica-modified samples and partial for unmodified ones cured in carbonate environments. The sealing was very poor for samples cured in water. Lastly, the main sealing phases included crystalline and amorphous silica, high-temperature-stable zeolites and talc mineral.

15 GEOTHERMAL ENERGY↗