Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

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Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

Journal of Virology ◽

10.1128/jvi.00705-19 ◽

2019 ◽

Vol 93 (18) ◽

Cited By ~ 22

Author(s):

Artem Baidaliuk ◽

Elliott F. Miot ◽

Sebastian Lequime ◽

Isabelle Moltini-Conclois ◽

Fanny Delaigue ◽

...

Keyword(s):

Aedes Aegypti ◽

Dengue Virus ◽

Cell Line ◽

Content Type ◽

Cells In Culture ◽

Mosquito Cells ◽

Wide Range ◽

Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

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Enhancement of In Vitro Conidia Production in Monilinia vaccinii-corymbosi (Reade) Honey by Cellulose Acetate Membranes

HortScience ◽

10.21273/hortsci.36.7.1290 ◽

2001 ◽

Vol 36 (7) ◽

pp. 1290-1291

Author(s):

A.W. Stretch ◽

M.K. Ehlenfeldt ◽

V. Brewster

Keyword(s):

Cellulose Acetate ◽

Light Environment ◽

Ambient Light ◽

Conidium Production ◽

Cellulose Acetate Membranes ◽

Wide Range ◽

Conidia Production ◽

Higher Temperature ◽

Very High

In vitro conidia production by Monilinia vaccinii-corymbosi (Reade) Honey, the cause of mummy berry disease in blueberry, was significantly enhanced by cellulose acetate membranes placed on the surface of V-8 juice agar for most of the pathogen isolates tested, compared to V-8 juice agar alone. Temperature and light affected conidia production, but the effects were not consistent. Higher temperature (22 vs. 15 °C) yielded better sporulation, but the effects of light environment were variable. When 55 isolates from various sources were rated visually for sporulation on cellulose acetate membranes at 22 °C under ambient light/dark cycles, a wide range of conidium production was observed, and three of 55 isolates (6%) were identified as having very high conidia production.

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40 VARIOUS DNA METHYLATION LEVELS OF IMPRINTED GENES IN CLONED COWS FROM THE SAME DONOR CELLS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab40 ◽

2011 ◽

Vol 23 (1) ◽

pp. 126

Author(s):

M. Kaneda ◽

S. Watanabe ◽

S. Akagi ◽

T. Somfai ◽

S. Haraguchi ◽

...

Keyword(s):

Dna Methylation ◽

Environmental Changes ◽

Monozygotic Twins ◽

Preimplantation Embryos ◽

Imprinted Genes ◽

Imprinted Gene ◽

Donor Cells ◽

Pcr Products ◽

Epigenetic Patterns

Somatic cell nuclear transferred (SCNT) animals are genetically identical to the donors; however, because of epigenetic abnormalities caused by incomplete reprogramming during nuclear transfer, the efficiency of SCNT is still very low. Monozygotic twins are also genetically identical, but it is reported that their epigenetic patterns on the genome, the so-called epigenome, are different. The epigenome is easily influenced by aging, environmental changes and nutrients, therefore these effects can be predicted by comparing epigenetic differences between genetically identical animals. Here we analysed DNA methylation levels of imprinted genes, which express in a parent-of-origin specific manner, in various tissues of cloned cows derived from the same donor cells. Imprinted gene expression is controlled by DNA methylation and other epigenetic modifications and abnormal expression/methylation patterns of imprinted genes have been observed in cloned animals. These alterations also occur during in vitro development of preimplantation embryos, which suggests that imprinted genes are easily influenced by environmental changes. Therefore, we chose H19 and PEG3 imprinted genes for the analysis to determine the epigenetic differences between individual cloned cows derived from the same donor cells. From 5 cloned and 5 non-cloned cows, we isolated DNA from 8 tissues (heart, lung, liver, kidney, spleen, intestine, muscle, and spinal cord) and analysed DNA methylation levels by bisulfite sequencing method. Briefly, genomic DNA was isolated by QIAGEN DNeasy Blood & Tissue Kit and bisulfite converted by QIAGEN EpiTect Bisulfite Kits (Qiagen, Valencia, CA). After amplification, the PCR products were cloned into TA vector and at least 10 clones were sequenced in each gene/sample. In every tissue analysed, the methylation levels largely differ among tissues and individuals. On average, the paternally imprinted gene H19 was 9.4 to 47.9% methylated (average 27.6 ± 10.3%) in clones and 0.5 to 69.8% methylated (average 29.0 ± 16.8%) in non-clones. The maternally imprinted gene PEG3 was 18.8 to 82.2% methylated (average 43.5 ± 15.8%) in clones and 8.0 to 98.7% (average 48.2 ± 18.8%) in non-clones. Even though there were large variations in DNA methylation levels, the variability tends to be low in clones compared to non-clones. More specifically, the variabilities of H19 methylation levels in spleen and intestine were significantly lower in clones than those in non-clones (32.3 ± 5.4% v. 27.0 ± 19.0% and 25.1 ± 4.2% v. 45.1 ± 14.3%, respectively, F-test; P < 0.05). These results suggest for the first time that epigenetic patterns in some tissues of both clones and non-clones are influenced by genetic background; however, mostly they are varied depending on non-genetic factors.

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Flavones, Flavonols, and Glycosylated Derivatives—Impact on Candida albicans Growth and Virulence, Expression of CDR1 and ERG11, Cytotoxicity

Pharmaceuticals ◽

10.3390/ph14010027 ◽

2020 ◽

Vol 14 (1) ◽

pp. 27

Author(s):

Marija Ivanov ◽

Abhilash Kannan ◽

Dejan S. Stojković ◽

Jasmina Glamočlija ◽

Ricardo C. Calhelha ◽

...

Keyword(s):

Candida Albicans ◽

Fungal Infections ◽

Ergosterol Biosynthesis ◽

Fungal Virulence ◽

Wide Range ◽

Genes Encoding ◽

Increasing Demand ◽

Hyphal Formation ◽

Further Development

Due to the high incidence of fungal infections worldwide, there is an increasing demand for the development of novel therapeutic approaches. A wide range of natural products has been extensively studied, with considerable focus on flavonoids. The antifungal capacity of selected flavones (luteolin, apigenin), flavonols (quercetin), and their glycosylated derivatives (quercitrin, isoquercitrin, rutin, and apigetrin) along with their impact on genes encoding efflux pumps (CDR1) and ergosterol biosynthesis enzyme (ERG11) has been the subject of this study. Cytotoxicity of flavonoids towards primary liver cells has also been addressed. Luteolin, quercitrin, isoquercitrin, and rutin inhibited growth of Candida albicans with the minimal inhibitory concentration of 37.5 µg/mL. The application of isoquercitrin has reduced C. albicans biofilm establishing capacities for 76%, and hyphal formation by yeast. In vitro treatment with apigenin, apigetrin, and quercitrin has downregulated CDR1. Contrary to rutin and apigenin, isoquercitrin has upregulated ERG11. Except apigetrin and quercitrin (90 µg/mL and 73 µg/mL, respectively inhibited 50% of the net cell growth), the examined flavonoids did not exhibit cytotoxicity. The reduction of both fungal virulence and expression of antifungal resistance-linked genes was the most pronounced for apigenin and apigetrin; these results indicate flavonoids’ indispensable capacity for further development as part of an anticandidal therapy or prevention strategy.

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Bacterial promoter opening underpins ubiquitous transcriptional regulation by DNA supercoiling

10.1101/2020.10.01.322149 ◽

2020 ◽

Author(s):

Raphaël Forquet ◽

Maïwenn Pineau ◽

William Nasser ◽

Sylvie Reverchon ◽

Sam Meyer

Keyword(s):

Transcription Initiation ◽

Environmental Changes ◽

Double Helix ◽

Transcriptional Regulator ◽

Dna Supercoiling ◽

Differential Response ◽

Transcriptomic Response ◽

Specific Step ◽

Wide Range

AbstractDNA supercoiling acts as a basal transcriptional regulator, which contributes to the quick and global transcriptional response of bacteria to many environmental changes. In spite of this importance, mechanistic models explaining the differential response of promoters to global topological variations of the chromosome remain essentially lacking. Here, we present the first quantitative transcriptional regulatory model by DNA super-coiling, focusing on the specific step of promoter opening during transcription initiation. Based on the known physico-chemical properties of DNA denaturation, it involves only one global adjustable parameter and is yet able to predict the global supercoiling response of promoters in a wide range of bacteria, based on the sequence content of their “discriminator” element. We first show that it quantitatively predicts both in vitro and in vivo data from transcription assays focusing on individual model promoters. We then assess the universality of the mechanism by analyzing transcriptomes of phylogenetically distant bacteria under conditions of supercoiling variation: (1) by gyrase-inhibiting antibiotics, (2) by biologically relevant environmental stresses, (3) naturally acquired and inherited in the longest-running evolution experiment. The model robustly predicts a significant contribution of the entire transcriptomic response to transient or inherited supercoiling variations in various species. This study strongly suggests that the proposed physical mechanism is used as an ubiquitous regulatory mechanism in the whole prokaryotic kingdom, based on the fundamental mechanical properties of the double-helix.ImportanceDNA supercoiling acts as a global yet underestimated transcriptional regulator in bacteria. We propose the first quantitative model of this regulation mode, based on the specific step of promoter opening during transcription initiation, explaining the differential response of promoters to global topological variations of the chromosome. In contrast to classical mechanisms requiring dedicated regulatory molecules to bind target promoters, we show that global deformations of the DNA template itself underpin a selective response of each particular promoter, according to its “discriminator” sequence, by modulating the ability of RNA Polymerase to initiate transcription. This study defines the first systematic rule underpinning the ubiquitous regulatory action of DNA supercoiling on the core transcriptional machinery, in particular in response to quick environmental changes.

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In vitro antifungal potential of surfactin isolated from rhizospheric Bacillus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe

Acta agriculturae Slovenica ◽

10.14720/aas.2021.117.4.2345 ◽

2021 ◽

Vol 117 (4) ◽

pp. 1

Author(s):

MUDDASIR KHAN ◽

Muhammad SALMAN ◽

Syed Hussain SHAH ◽

Muhammad ISRAR

Keyword(s):

Zea Mays ◽

Bacillus Thuringiensis ◽

Fusarium Graminearum ◽

Antimicrobial Agents ◽

Significant Loss ◽

Plant Growth Promoting Bacteria ◽

Maize Crop ◽

Wide Range ◽

Fungal Phytopathogen

Fusarium graminearum fungus cause significant loss in maize (Zea mays L.) and other cereal crops all over the world. The usage of chemical agents cause severe environmental problems. Bacillus species and other plant growth-promoting bacteria (PGPR) play key role in biopesticide development. A wide range of environmentally safe antimicrobial agents are already being manufactured. The current investigation was focused on exploring the antifungal activity of Bacillus thuringiensis lipopeptide surfactin against fungal phytopathogen Fusarium graminearum. B. thuringensis was isolated from the rhizosphere of maize crop and cultivated to produce lipopeptides. Surfactin was identified by high-performance liquid chromatography (HPLC) from the extract at 210 nm, retention time 3-5 minutes and the obtained peaks area was 3.990. The growth of F. graminearum was successfully inhibited by surfactin at different concentrations. Among these, 80 % concentration showed the highest zone of inhibition in comparison to 60 %, 40 % and 20 % concentrations (p < 0.005), respectively. The current study concludes B. thuringensis lipopeptide surfactin has a high potential to inhibit the growth of F. graminearum.

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Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

Nuklearmedizin ◽

10.1055/s-0038-1629552 ◽

1991 ◽

Vol 30 (01) ◽

pp. 35-39 ◽

Cited By ~ 1

Author(s):

H. S. Durak ◽

M. Kitapgi ◽

B. E. Caner ◽

R. Senekowitsch ◽

M. T. Ercan

Keyword(s):

Soft Tissue ◽

Room Temperature ◽

Normal Subjects ◽

Left Testis ◽

Wide Range ◽

Activity Ratios ◽

The Right ◽

Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

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In Vitro Clot Lysis as a Potential Indicator of Thrombus Resistance to Fibrinolysis – Study in Healthy Subjects and Correlation with Blood Fibrinolytic Parameters

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656041 ◽

1997 ◽

Vol 77 (04) ◽

pp. 725-729 ◽

Cited By ~ 11

Author(s):

Mario Colucci ◽

Silvia Scopece ◽

Antonio V Gelato ◽

Donato Dimonte ◽

Nicola Semeraro

Keyword(s):

Plasminogen Activator ◽

Clot Lysis ◽

Individual Response ◽

Plasminogen Activator Inhibitor 1 ◽

Autologous Plasma ◽

Wide Range ◽

Blood Clots ◽

Physiological Variations ◽

Pai 1

SummaryUsing an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. 125I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37° C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-anti-plasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p <0.001) and age (p <0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-ginduced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.

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