DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

Energy metabolism disorders in rare and common diseases. Toward bioenergetic modulation therapy and the training of a new generation of European scientists

Energy metabolism alterations are found in a large number of rare and common diseases of genetic or environmental origin. The number of patients that could benefit from bioenergetic modulation therapy (BIOMET) is therefore very important and includes individuals with pathologies as diverse as mitochondrial diseases, acute coronary syndrome, chronic kidney disease, asthma or even cancer. Although, the alteration of energy metabolism is disease specific and sometimes patient specific, the strategies for BIOMET could be common and target a series of bioenergetic regulatory mechanisms discussed in this article. An excellent training of scientists in the field of energy metabolism, related human diseases and drug discovery is also crucial to form a young generation of MDs, PHDs and Pharma or CRO-group leaders who will discover novel personalized bioenergetic medicines, through pharmacology, genetics, nutrition or adapted exercise training. The Mitochondrial European Educational Training (MEET) consortium was created to pursue this goal, and we dedicated here a special issue of Organelle in Focus (OiF) to highlight their objectives. A total of 10 OiFs articles constitute this Directed Issue on Mitochondrial Medicine. As part of this editorial article, we asked timely questions to the PR. Jan W. Smeitink, professor of Mitochondrial Medicine and CEO of Khondrion, a mitochondrial medicine company. He shared with us his objectives and strategies for the study of mitochondrial diseases and the identification of future treatments.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Reference database of the gait cycle for young healthy Tunisian adults

BackgroundQuantified gait analysis is a rising technology used increasingly to assess motor disorders. Normal reference data are required in order to evaluate patients, but there are no reference data available for the Tunisian healthy population.AimTo assess the features of normal Tunisian gait pattern, and examine the intrinsic reliability of spatio-temporal, kinematic and kinetic parameters within a new specific reference database.MethodsEighteen healthy active-young adults (age: 23.30 ± 2.54 years, height: 1.78 ± 0.04 m and, weight: 70.00 ± 4.80 kg) have participated to five trials of step gait where the dominant lower limb were recorded. Two over the five trials were randomly selected to be further analyzed. Twenty-three spatio-temporal, kinematic and kinetic parameters determined from 3-dimensional gait analysis. The intrinsic reliability was examined for each variable and our results were compared with those available in the literature.ResultsTwelve over 23 parameters have an excellent intrinsic reliability (P > 0.05, ICC > 0.9 and SEM < 5% of the grand mean). There are similarities with other studies (P < 0.05) but we noticed the existence of some specificity (the height of hip extension peak and the low cadence of gait) that could characterize the Tunisian population.ConclusionA specific reference database of the gait cycle has been established for healthy Tunisian active-young adults and excellent inter-trial reliability may be observed for different variables.     

Identification of critical regions within the TIR domain of IL-1 receptor type I

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC′ helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.     

Home environment and cord blood levels of lead,arsenic, and zinc on neurodevelopment of 24 months children living in Chitwan Valley,Nepal

In a birth cohort living in Chitwan Valley, lowland Nepal, we have previously reported inverse associations between in utero levels of lead (Pb), arsenic (As) and neurodevelopment at birth measured by the Brazelton Neonatal Behavioral Assessment Scale, third edition (NBAS III). In the present paper, a follow-up of the same cohort was made on 24-month-old infants regarding the neurodevelopmental effects of these metals, taking the postnatal environment into account. In total, the same100 mother-infant pairs as the previous study, whose Pb, As, and Zn concentrations in cord blood were known, were recruited. Postnatal raising environment was evaluated using the Home Observation for Measurement of Environment (HOME) scale. Neurodevelopment of children at 24 months of age (n = 74) was assessed using the Bayley Scale of Infant Development, Second Edition (BSID II). Multivariable regression adjusting for covariates was performed to determine the associations of in utero levels of toxic and essential elements and the home environment with neurodevelopment scores. Unlike the NBAS III conducted for newborns, none of the BSID II cluster scores in 24-month-old infants were associated with cord blood levels of Pb, As, and Zn. The total HOME score was positively associated with the mental development scale (MDI) score (coefficient = 0.67, at 95% CI = 0.03 to 1.31). In this cohort, a detrimental effect of in utero Pb and As on neurodevelopmental indicators observed at birth disappeared at 24 months, while an association between neurodevelopment and home environment continued.     

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1^Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1^Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1^Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1^Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.     

Ostreospis cf. ovata blooms in coastal water: Italian guidelines to assess and manage the risk associated to bathing waters and recreational activities

Ostreopsis cf. ovata is a benthic marine dinoflagellate, which blooms in summer along the coasts of the Mediterranean Sea, including many Italian regions since early 1990s. In tropical areas O. cf. ovata produces mainly palytoxins whereas in the Mediterranean basin it produces ovatoxins, and only trace amounts of a putative palytoxin. The toxicological profile of ovatoxins is still unknown. This paper describes the update of the Italian guidelines for the management of O. cf. ovata blooms related to bathing and recreational activities. The rationale for the guidelines starts from the current scientific knowledge on chemical, toxicological, and epidemiological aspects and on its diffusion, which are briefly summarized in the first part of the paper. On this basis a risk-based approach is developed, with the purpose to protect people from oral, dermal and inhalation exposure (the ones that so far caused major health problems), taking into account also the weather and sea conditions, which are crucial for the onset of blooms. Accordingly, the guidelines provide information on what indicators are needed to set up environmental monitoring activities and actions to take during diverse phases. They also outline a multi-step health surveillance system, and a system of communication to the public. Finally the still important scientific gaps and research needs are highlighted.     

Glycolysis-mediated control of blood-brain barrier development and function

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain.     

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2–protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2–protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.     

Enforced telomere elongation increases the sensitivity of human tumour cells to ionizing radiation

More than 85% of all human cancers possess the ability to maintain chromosome ends, or telomeres, by virtue of telomerase activity. Loss of functional telomeres is incompatible with survival, and telomerase inhibition has been established in several model systems to be a tractable target for cancer therapy. As human tumour cells typically maintain short equilibrium telomere lengths, we wondered if enforced telomere elongation would positively or negatively impact cell survival. We found that telomere elongation beyond a certain length significantly decreased cell clonogenic survival after gamma irradiation. Susceptibility to irradiation was dosage-dependent and increased at telomere lengths exceeding 17 kbp despite the fact that all chromosome ends retained telomeric DNA. These data suggest that an optimal telomere length may promote human cancer cell survival in the presence of genotoxic stress.