ABSTRACT
Methyl p-coumarate (methyl p-hydroxycinnamate) (MH) is a natural compound found in a variety of plants. In the present study, we evaluated the ameliorative effects of MH on airway inflammation in an experimental model of allergic asthma (AA). In this in vitro study, MH was found to exert anti-inflammatory activity on PMA-stimulated A549 airway epithelial cells by suppressing the secretion of IL-6, IL-8, MCP-1, and ICAM-1. In addition, MH exerted an inhibitory effect not only on NF-κB (p-NF-κB and p-IκB) and AP-1 (p-c-Fos and p-c-Jun) activation but also on A549 cell and EOL-1 cell (eosinophil cell lines) adhesion. In LPS-stimulated RAW264.7 macrophages, MH had an inhibitory effect on TNF-α, IL-1ß, IL-6, and MCP-1. The results from in vivo study revealed that the increases in eosinophils/Th2 cytokines/MCP-1 in the bronchoalveolar lavage fluid (BALF) and IgE in the serum of OVA-induced mice with AA were effectively inhibited by MH administration. MH also exerted a reductive effect on the immune cell influx, mucus secretion, and iNOS/COX-2 expression in the lungs of mice with AA. The effects of MH were accompanied by the inactivation of NF-κB. Collectively, the findings of the present study indicated that MH attenuates airway inflammation in mice with AA, suggesting its potential as an adjuvant in asthma therapy.
Subject(s)
Asthma , NF-kappa B , Animals , Mice , Bronchoalveolar Lavage Fluid , Cytokines/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Inflammation/drug therapy , Inflammation/metabolism , Interleukin-6 , Mice, Inbred BALB C , NF-kappa B/metabolism , OvalbuminABSTRACT
Topoisomerase IIIß (Top3ß), the only dual-activity topoisomerase in mammals that can change topology of both DNA and RNA, is known to be associated with neurodevelopment and mental dysfunction in humans. However, there is no report showing clear associations of Top3ß with neuropsychiatric phenotypes in mice. Here, we investigated the effect of Top3ß on neuro-behavior using newly generated Top3ß deficient (Top3ß-/-) mice. We found that Top3ß-/- mice showed decreased anxiety and depression-like behaviors. The lack of Top3ß was also associated with changes in circadian rhythm. In addition, a clear expression of Top3ß was demonstrated in the central nervous system of mice. Positron emission tomography/computed tomography (PET/CT) analysis revealed significantly altered connectivity between many brain regions in Top3ß-/- mice, including the connectivity between the olfactory bulb and the cerebellum, the connectivity between the amygdala and the olfactory bulb, and the connectivity between the globus pallidus and the optic nerve. These connectivity alterations in brain regions are known to be linked to neurodevelopmental as well as psychiatric and behavioral disorders in humans. Therefore, we conclude that Top3ß is essential for normal brain function and behavior in mice and that Top3ß could be an interesting target to study neuropsychiatric disorders in humans.
Subject(s)
Anxiety Disorders/pathology , Behavior, Animal , Circadian Rhythm , Connectome , DNA Topoisomerases, Type I/physiology , Depression/pathology , Animals , Anxiety Disorders/etiology , Depression/etiology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, KnockoutABSTRACT
Importin-11 (Ipo11) is a novel member of the human importin family of transport receptors (karyopherins), which are known to mediate the nucleocytoplasmic transport of protein and RNA cargos. Despite its role in the transport of protein, we found that knockout of Ipo11 nuclear import factor affects normal embryonic development and govern embryo-lethal phenotypes in mice. In this study, we for the first time produced a mouse line containing null mutation in Ipo11 gene utilized by gene trapping. The Ipo11-/- embryos showed an embryonic lethal phenotype. The Ipo11-/- embryos showed a reduced size at embryonic day 10.5 (E10.5) when compared with Ipo11+/+ or Ipo11+/- embryos and died by E11.5. Whereas Ipo11+/- mice were healthy and fertile, and there was no detectable changes in embryonic lethality and phenotype when reviewed. In the X-gal staining with the Ipo11-/- or Ipo11+/- embryos, strong X-gal staining positivity was detected systematically in the whole mount embryos at E10.5, although almost no X-gal positivity was detected at E9.5, indicating that the embryos die soon after the process of Ipo11 expression started. These results indicate that Ipo11 is essential for the normal embryonic development in mice.
Subject(s)
Embryonic Development/genetics , Karyopherins/genetics , Animals , Embryo, Mammalian/metabolism , Female , Gene Expression Regulation, Developmental/genetics , Humans , Karyopherins/antagonists & inhibitors , Mice , Mice, Knockout , PregnancyABSTRACT
E2F3, a member of the E2F family, plays a critical role in cell cycle and proliferation by targeting downstream, retinoblastoma (RB) a tumor suppressor family protein. The purpose of this study, was to investigate the role and function of E2F3 in vivo. We examined phenotypic abnormalities, by deletion of the E2f3 gene in mice. Complete ablation of the E2F3 was fully penetrant, in the pure C57BL/6N background. The E2f3+/ - mouse embryo developed normally without fatal disorder. However, they exhibited reduced body weight, growth retardation, skeletal imperfection, and poor grip strength ability. Findings suggest that E2F3 has a pivotal role in muscle and bone development, and affect normal mouse growth.
Subject(s)
Bone Development/genetics , E2F3 Transcription Factor/genetics , Embryonic Development/genetics , Muscle, Skeletal/growth & development , Animals , Apoptosis/genetics , Body Weight/genetics , Cell Cycle/genetics , Cell Proliferation/genetics , Embryo, Mammalian , Humans , Mice , Mice, Knockout , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , PhenotypeABSTRACT
In this study, we developed a method for the determination of Penicillium griseofulvum-oriented pyripyropene A (PPPA), a selective inhibitor of acyl-coenzyme A:cholesterol acyltransferase 2, in mouse and human plasma and validated it using liquid chromatography-tandem mass spectrometry. Pyripyropene A (PPPA) and an internal standard, carbamazepine, were separated using a Xterra MS C18 column with a mixture of acetonitrile and 0.1% formic acid as the mobile phase. The ion transitions monitored in positive-ion mode [M + H]+ of multiple-reaction monitoring (MRM) were m/z 148.0 from m/z 584.0 for PPPA and m/z 194.0 from m/z 237.0 for the internal standard. The detector response was specific and linear for PPPA at concentrations within the range from 1 to 5,000 ng/mL. The intra-/inter-day precision and accuracy of the method was acceptable by the criteria for assay validation. The matrix effects of PPPA ranged from 97.6 to 104.2% and from 93.3 to 105.3% in post-preparative mouse and human plasma samples, respectively. PPPA was also stable under various processing and/or handling conditions. Finally, PPPA concentrations in the mouse plasma samples could be measured after intravenous, intraperitoneal, or oral administration of PPPA, suggesting that the assay is useful for pharmacokinetic studies on mice and applicable to human studies.
Subject(s)
Chromatography, Liquid/methods , Penicillium/chemistry , Pyridines/blood , Pyridines/pharmacokinetics , Sesquiterpenes/blood , Sesquiterpenes/pharmacokinetics , Tandem Mass Spectrometry/methods , Animals , Drug Stability , Linear Models , Male , Mice , Mice, Inbred ICR , Pyridines/chemistry , Reproducibility of Results , Sensitivity and Specificity , Sesquiterpenes/chemistry , Sterol O-Acyltransferase/antagonists & inhibitors , Sterol O-Acyltransferase 2ABSTRACT
Vibrio parahaemolyticus is one of the leading foodborne pathogens causing seafood contamination. Here, 22 V. parahaemolyticus strains were analyzed for biofilm formation to determine whether there is a correlation between biofilm formation and quorum sensing (QS), swimming motility, or hydrophobicity. The results indicate that the biofilm formation ability of V. parahaemolyticus is positively correlated with cell surface hydrophobicity, autoinducer (AI-2) production, and protease activity. Field emission scanning electron microscopy (FESEM) showed that strong-biofilm-forming strains established thick 3-D structures, whereas poor-biofilm-forming strains produced thin inconsistent biofilms. In addition, the distribution of the genes encoding pandemic clone factors, type VI secretion systems (T6SS), biofilm functions, and the type I pilus in the V. parahaemolyticus seafood isolates were examined. Biofilm-associated genes were present in almost all the strains, irrespective of other phenotypes. These results indicate that biofilm formation on/in seafood may constitute a major factor in the dissemination of V. parahaemolyticus and the ensuing diseases.
Subject(s)
Biofilms/growth & development , Hydrophobic and Hydrophilic Interactions , Quorum Sensing , Vibrio parahaemolyticus , Fimbriae, Bacterial , Food Contamination/prevention & control , Foodborne Diseases/microbiology , Foodborne Diseases/prevention & control , Humans , Seafood/microbiology , Vibrio parahaemolyticus/pathogenicity , Vibrio parahaemolyticus/physiologyABSTRACT
Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Replication/genetics , Translocation, Genetic/genetics , Chromosome Aberrations , Chromosomes/metabolism , DNA Repair , DNA-Binding Proteins , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Recombination, Genetic , Saccharomyces cerevisiaeABSTRACT
During Saccharomyces cerevisiae mating-type switching, an HO endonuclease-induced double-strand break (DSB) at MAT is repaired by recombining with one of two donors, HMLα or HMRa, located at opposite ends of chromosome III. MATa cells preferentially recombine with HMLα; this decision depends on the Recombination Enhancer (RE), located about 17 kb to the right of HML. In MATα cells, HML is rarely used and RE is bound by the MATα2-Mcm1 corepressor, which prevents the binding of other proteins to RE. In contrast, in MATa cells, RE is bound by multiple copies of Fkh1 and a single copy of Swi4/Swi6. We report here that, when RE is replaced with four LexA operators in MATa cells, 95% of cells use HMR for repair, but expression of a LexA-Fkh1 fusion protein strongly increases HML usage. A LexA-Fkh1 truncation, containing only Fkh1's phosphothreonine-binding FHA domain, restores HML usage to 90%. A LexA-FHA-R80A mutant lacking phosphothreonine binding fails to increase HML usage. The LexA-FHA fusion protein associates with chromatin in a 10-kb interval surrounding the HO cleavage site at MAT, but only after DSB induction. This association occurs even in a donorless strain lacking HML. We propose that the FHA domain of Fkh1 regulates donor preference by physically interacting with phosphorylated threonine residues created on proteins bound near the DSB, thus positioning HML close to the DSB at MAT. Donor preference is independent of Mec1/ATR and Tel1/ATM checkpoint protein kinases but partially depends on casein kinase II. RE stimulates the strand invasion step of interchromosomal recombination even for non-MAT sequences. We also find that when RE binds to the region near the DSB at MATa then Mec1 and Tel1 checkpoint kinases are not only able to phosphorylate histone H2A (γ-H2AX) around the DSB but can also promote γ-H2AX spreading around the RE region.
Subject(s)
Cell Cycle Proteins , Enhancer Elements, Genetic , Forkhead Transcription Factors , Genes, Mating Type, Fungal , Recombination, Genetic , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Bacterial Proteins , Cell Cycle Checkpoints , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatin/genetics , DNA Breaks, Double-Stranded , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Fungal , Histones/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phosphorylation , Phosphothreonine/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Serine EndopeptidasesABSTRACT
Chromosome translocations are frequently associated with many types of blood-related cancers and childhood sarcomas. Detection of chromosome translocations assists in diagnosis, treatment and prognosis of these diseases; however, despite their importance to such diseases, the molecular mechanisms leading to chromosome translocations are not well understood. The available evidence indicates a role for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs) in their origin. Here we develop a yeast-based system that induces a reciprocal chromosome translocation by formation and ligation of breaks on two different chromosomes. We show that interchromosomal end joining is efficiently suppressed by the Tel1- and Mre11-Rad50-Xrs2-dependent pathway; this is distinct from the role of Tel1 in telomeric integrity and from Mec1- and Tel1-dependent checkpoint controls. Suppression of DSB-induced chromosome translocations depends on the kinase activity of Tel1 and Dun1, and the damage-induced phosphorylation of Sae2 and histone H2AX proteins. Tel1- and Sae2-dependent tethering and promotion of 5' to 3' degradation of broken chromosome ends discourage error-prone NHEJ and interchromosomal NHEJ, preserving chromosome integrity on DNA damage. Our results indicate that, like human ATM, Tel1 serves as a key regulator for chromosome integrity in the pathway that reduces the risk for DSB-induced chromosome translocations, and are probably pertinent to the oncogenic chromosome translocations in ATM-deficient cells.
Subject(s)
Cell Cycle Proteins , Chromosome Breakage , DNA Repair , DNA-Binding Proteins , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Translocation, Genetic , Tumor Suppressor Proteins , Ataxia Telangiectasia Mutated Proteins , Chromosomes, Fungal/genetics , Chromosomes, Fungal/metabolism , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases , Exodeoxyribonucleases/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Aromadendrin is a phenolic compound with various biological effects such as anti-inflammatory properties. However, its protective effects against acute lung injury (ALI) remain unclear. Therefore, this study aimed to explore the ameliorative effects of aromadendrin in an experimental model of lipopolysaccharide (LPS)-induced ALI. In vitro analysis revealed a notable increase in the levels of cytokine/chemokine formation, nuclear factor kappa B (NF-κB) activation, and myeloid differentiation primary response 88 (MyD88)/toll-like receptor (TLR4) expression in LPS-stimulated BEAS-2B lung epithelial cell lines that was ameliorated by aromadendrin pretreatment. In LPS-induced ALI mice, the remarkable upregulation of immune cells (ICs) and IL-1ß/IL-6/TNF-α levels in the bronchoalveolar lavage fluid (BALF) and inducible nitric oxide synthase (iNOS)/cyclooxygenase-2 (COX-2)/CD68 expression in lung was decreased by the oral administration of aromadendrin. Histological analysis revealed the presence of cells in the lungs of acute lung injury (ALI) mice, which was alleviated by aromadendrin. In addition, aromadendrin ameliorated lung edema. This in vivo effect of aromadendrin was accompanied by its inhibitory effect on LPS-induced NF-κB activation, MyD88/TLR4 expression, and signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, aromadendrin increased the expression of heme oxygenase-1 (HO-1)/ NAD(P)H quinone dehydrogenase 1 (NQO1) in the lungs of ALI mice. In summary, the in vitro and in vivo studies demonstrated that aromadendrin ameliorated endotoxin-induced pulmonary inflammation by suppressing cytokine formation and NF-κB activation, suggesting that aromadendrin could be a useful adjuvant in the treatment of ALI.
ABSTRACT
In this study, the Poly(vinylidene fluoride-trifluoethylene) (PVDF) electrospun fibers were successfully prepared by electrospinning. Processing parameters, such as solvents and solution temperature were varied to study their influence on fiber dimensions. Electrospun PVDF fibers were characterized by scanning electron microscope (SEM), Fourier transform infrared spectrophotometer (FT-IR), wide angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC). The result indicated that the solvent component and temperature have great influence on fiber dimensions. 19% PVDF dissolved in DMF/MEK mixed solvents with the ratio of 8:2 was considered to be most suitable in this study. Furthermore, the increasing of solution temperature can probably induce the formation of beta-phases in electrospun PVDF Fibers.
ABSTRACT
In this work, novel poly(ε-caprolactone) (PCL) fibrous membranes incorporating amphiphilic polyhedral oligosilsesquioxane (POSS) telechelic (PEG-POSS telechelic) were prepared via electrospinning. The unique microstructure, morphology, thermal stability of the resulting PCL/PEG-POSS telechelic electrospun nanowebs were investigated by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis, respectively. The addition of amphiphilic PEG-POSS telechelic strongly influenced the fiber diameters, microstructures of the resultant PCL/PEG-POSS telechelic nanofibers, compared to pure PCL nanofibers. The potential biomedical applications of such PEG-POSS telechelic nanowebs as a scaffolding material were also evaluated in vitro using mouse osteoblast-like MC3T3-E1 cells. The cell adhesion, spreading, and interaction behavior of pure PCL and PCL/PEG-POSS telechelic fibrous membranes were explored. It was found that electrospun PCL fibrous membranes incorporating amphiphilic PEG-POSS telechelic showed higher initial cell attachment than pure PCL due to the higher surface free energy of POSS siloxanes. Moreover, the obtained PCL/PEG-POSS telechelic fibrous scaffolds were found to be nontoxic and to maintain the good adhesion ratio between cells and surface (about ~93 %) after cell culturing for 24 h.
Subject(s)
Organosilicon Compounds/chemistry , Osteoblasts/cytology , Polyesters/chemistry , Polyethylene Glycols/chemistry , Tissue Scaffolds , Animals , Cell Adhesion/drug effects , Cell Culture Techniques/instrumentation , Cell Proliferation/drug effects , Cells, Cultured , Electroplating/methods , Mice , Organosilicon Compounds/pharmacology , Osteoblasts/drug effects , Osteoblasts/physiology , Polyesters/chemical synthesis , Polyethylene Glycols/pharmacology , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , Tissue Scaffolds/chemistry , X-Ray DiffractionABSTRACT
The term 'Blue Economy (BE)' is increasingly popular in modern environmental research. The concept seeks to explore ocean-based development opportunities with environmental stewardship and protection. Yet different scholars and actors adopt this term often in conflicting ways without attempting to explore the relevance and the link between the blue economy and the broader environment viz., total environment. The potential opportunities to resolve the conflicts require a better understanding of the impacts and/or interactions of the BE on the total environment. This paper aims to map the interface between the two for a better understanding of the total environment and implications for the BE. Using a systematic literature review, this study finds that the field of the blue economy in association with the total environment is very new and emerging in the literature, and the link between the BE and the total environment is increasingly being invoked, yet clarity on the link or interactions remain vague. By analysing the co-occurrence of selected keywords and networks, we present six clusters (three for general relationship, and the other three for specific dimensions of total environment). In a general relationship between the BE and the total environment, clusters of environmental sustainability, marine resource, and economic development are identified to link directly to the BE. In specific dimensions of the total environment, clusters of growth and sustainable development, spatial planning and environmental management, and environmental sustainability and the BE are presented. The analysis outcomes show that specific areas from the total environment (growth, spatial planning, environmental management, and environmental sustainability) are directly linked to the BE where a call for a wider range of studies in the future is identified.
Subject(s)
Economic Development , Sustainable DevelopmentABSTRACT
The "Blue Economy (BE)" is an increasingly popular concept as a strategy for safeguarding the world's oceans and water resources. It may emerge when economic activity is in balance with the long term capacity of ocean ecosystems to support the activity in a sustainable manner. Importantly, the concept of BE posits the inherent conflicts between two discourses-growth and development, and protection of ocean resources. The inherent conflicts require solutions to embrace the opportunities associated with the ocean economy while recognizing and addressing its threats. The potential solutions on a global scale are advocated by the United Nations in their Sustainable Development Goals (SDGs). However, we notice that the identification of the scope and boundaries of the BE in line with the UN's SDGs is vague even challenging, and the key stakeholders and their interests and roles in the BE are also vague. This review examines the scientific evidence of the association between the BE and the UN's SDGs, and relevance and alignment of stakeholders on the link between the BE and SDGs. Based on a literature survey between 1998 and 2018, we find that BE is highly associated with SDGs 14-17. Notably, we find that stakeholders prefer SDG 3 Good Health & Well-Being and SDG 8 Decent Work & Economic Growth in the BE context. As stakeholder involvement shows some differences and variations in the relationship between the BE and SDGs, we consider that stakeholders can play some roles directly or indirectly in the BE-SDGs context. In order to set achievable goals and targets in BE-SDGs, we support that key stakeholders should be identified to play several important roles in prosperous economic, societal development and setting tolerable ranges for the ocean biosphere.
Subject(s)
Ecosystem , Sustainable Development , Economic Development , Global Health , United NationsABSTRACT
Because of the nature of their work, physical and occupational therapists are at high risk of burnout, which is associated with decreased job satisfaction, medical errors, and mental wellbeing in healthcare professionals. To well manage and minimize potential impact of burnout, risk factors should be determined. This study examined burnout and job stress in physical and occupational therapists in various Korean hospital settings. Physical and occupational therapists from several rehabilitation facilities in South Korea completed a survey between March-May 2019. A set of questionnaires, including the Maslach Burnout Inventory and Job Content Questionnaire, were distributed to all participants. In total, 325 professionals (131 men and 194 women) were recruited. Burnout and work-related stress differed significantly according to several factors. Hospital size, gender, and age were the main contributory factors affecting at least two dimensions of the questionnaires. The more vulnerable group consisted of female therapists in their 20s at small- or medium-sized hospitals with low scores for quality of life. High levels of job stress and burnout were observed in female therapists in their 20s at small- or medium-sized hospitals. Hospitals and society should create suitable environments and understand the nature of therapists' work to improve healthcare.
Subject(s)
Burnout, Professional/psychology , Burnout, Psychological/psychology , Occupational Stress/psychology , Occupational Therapists/psychology , Quality of Life/psychology , Age Factors , Burnout, Professional/epidemiology , Burnout, Psychological/epidemiology , Child , Cross-Sectional Studies , Female , Health Facility Size , Humans , Job Satisfaction , Male , Occupational Stress/epidemiology , Republic of Korea/epidemiology , Sex Factors , Surveys and QuestionnairesABSTRACT
Salmonella is one of the main foodborne pathogens that affect humans and farm animals. The Salmonella genus comprises a group of food-transmitted pathogens that cause highly prevalent foodborne diseases throughout the world. The aim of this study was to appraise the viability of Salmonella Typhimurium biofilm under water treatment at room temperature on different surfaces, specifically stainless steel (SS), plastic (PLA), rubber (RB), and eggshell (ES). After 35 D, the reduction of biofilm on SS, PLA, RB, and ES was 3.35, 3.57, 3.22, and 2.55 log CFU/coupon without water treatment and 4.31, 4.49, 3.50, and 1.49 log CFU/coupon with water treatment, respectively. The dR value (time required to reduce bacterial biofilm by 99% via Weibull modeling) of S. Typhimurium without and with water treatment was the lowest on PLA (176.86 and 112.17 h, respectively) and the highest on ES (485.37 and 2,436.52 h, respectively). The viability of the S. Typhimurium on ES and the 3 food-contact surfaces was monitored for 5 wk (35 D). The results of this study provide valuable information for the control of S. Typhimurium on different surfaces in the food industry, which could reduce the risk to consumers.
Subject(s)
Biofilms , Egg Shell , Food Microbiology , Microbial Viability , Salmonella typhimurium , Animals , Chickens , Colony Count, Microbial/veterinary , Egg Shell/microbiology , Food Handling , Salmonella typhimurium/physiology , Temperature , WaterABSTRACT
In budding yeast, a single DNA double-strand break (DSB) triggers the activation of Mec1ATR-dependent DNA damage checkpoint. After about 12 h, cells turn off the checkpoint signaling and adapt despite the persistence of the DSB. We report that the adaptation involves the autophosphorylation of Mec1 at site S1964. A non-phosphorylatable mec1-S1964A mutant causes cells to arrest permanently in response to a single DSB without affecting the initial kinase activity of Mec1. Autophosphorylation of S1964 is dependent on Ddc1Rad9 and Dpb11TopBP1, and it correlates with the timing of adaptation. We also report that Mec1's binding partner, Ddc2ATRIP, is an inherently stable protein that is degraded specifically upon DNA damage. Ddc2 is regulated extensively through phosphorylation, which, in turn, regulates the localization of the Mec1-Ddc2 complex to DNA lesions. Taken together, these results suggest that checkpoint response is regulated through the autophosphorylation of Mec1 kinase and through the changes in Ddc2 abundance and phosphorylation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , DNA Damage , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction , Amino Acid Sequence , DNA Mutational Analysis , DNA Repair , Mutation/genetics , Phosphorylation , Phosphoserine/metabolismABSTRACT
Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair/genetics , DNA Repair/genetics , Saccharomyces cerevisiae/genetics , DNA-Binding Proteins/genetics , Endonucleases/genetics , Rad52 DNA Repair and Recombination Protein/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion/geneticsABSTRACT
Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.
Subject(s)
DNA, Fungal/genetics , DNA, Fungal/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Base Sequence , DNA Breaks , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Polymerase beta , DNA Repair , DNA Repair Enzymes , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Genes, Fungal , Models, Genetic , Single-Strand Specific DNA and RNA EndonucleasesABSTRACT
FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl-/- mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl-/- mice died soon after birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl-/- neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl-/- neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.