scholarly journals Occurrence and Characterization of Dimethachlon Insensitivity in Sclerotinia sclerotiorum in Jiangsu Province of China

Plant Disease ◽  
2009 ◽  
Vol 93 (1) ◽  
pp. 36-42 ◽  
Author(s):  
Hui-Xia Ma ◽  
Xi-Jie Feng ◽  
Yu Chen ◽  
Chang-Jun Chen ◽  
Ming-Guo Zhou
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
In Vitro Selection ◽  
Jiangsu Province ◽  
Resistance Factor ◽  
Cross Resistance ◽  
Sclerotinia Stem Rot ◽  
Wild Type ◽  
Resistance Level ◽  
Significant Difference

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 μg/ml dimethachlon with the resistance factor ≈10 (resistance factor was estimated as ratios between the EC50 values of resistant isolates and the average EC50 values of sensitive ones) compared to the sensitive isolates (EC50 is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum, together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.

scholarly journals Effects of the Novel Fungicide Benzothiostrobin on Sclerotinia sclerotiorum in the Laboratory and on Sclerotinia Stem Rot in Rape Fields

Plant Disease ◽  
2015 ◽  
Vol 99 (7) ◽  
pp. 969-975 ◽  
Author(s):  
Congying Xu ◽  
Xiaoyu Liang ◽  
Yiping Hou ◽  
Mingguo Zhou
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Pathogenic Fungus ◽  
Field Trials ◽  
Stem Rot ◽  
Cross Resistance ◽  
Synergistic Activity ◽  
The Novel ◽  
Sclerotinia Stem Rot ◽  
Sclerotial Germination

We determined the effects and efficacy of benzothiostrobin, a new strobilurin-derived fungicide, against the plant-pathogenic fungus Sclerotinia sclerotiorum (the causal agent of Sclerotinia stem rot). Mycelial growth and sclerotial germination in vitro were strongly inhibited by benzothiostrobin in the presence of salicylhydroxamic acid. On detached rapeseed leaves, benzothiostrobin at 40 μg/ml reduced lesion development by 87%. No cross-resistance was detected between benzothiostrobin and carbendazim, iprodione, fludioxonil, or boscalid. A formulated mixture of benzothiostrobin and fluazinam at 1:1 had synergistic activity against S. sclerotiorum in vitro. In field trials, benzothiostrobin alone or formulated with fluazinam at 1:1 (150 g a.i. ha−1) was significantly (P < 0.05) superior to iprodione in controlling Sclerotinia stem rot of rapeseed. These results suggest that benzothiostrobin has substantial potential for the control of Sclerotinia stem rot.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants

2021 ◽  
Vol 7 (8) ◽  
pp. eabf1738 ◽  
Author(s):  
Kui K. Chan ◽  
Timothy J. C. Tan ◽  
Krishna K. Narayanan ◽  
Erik Procko
Keyword(s):  
In Vitro Selection ◽  
Protein S ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Host Cell Membrane ◽  
Decoy Receptor ◽  
Decoy Receptors ◽  
Binding Surface ◽  
Type Receptor

The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, because of close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. We find that an engineered decoy receptor, sACE22.v2.4, tightly binds S of SARS-associated viruses from humans and bats, despite the ACE2-binding surface being a region of high diversity. Saturation mutagenesis of the receptor-binding domain followed by in vitro selection, with wild-type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild-type receptor. We conclude that resistance to engineered decoys will be rare and that decoys may be active against future outbreaks of SARS-associated betacoronaviruses.

scholarly journals Multiple Novel Inhibitors of the NorA Multidrug Transporter of Staphylococcus aureus

1999 ◽  
Vol 43 (10) ◽  
pp. 2404-2408 ◽  
Author(s):  
Penelope N. Markham ◽  
Eric Westhaus ◽  
Katya Klyachko ◽  
Michael E. Johnson ◽  
Alex A. Neyfakh
Keyword(s):  
Staphylococcus Aureus ◽  
Clinical Setting ◽  
In Vitro Selection ◽  
Antibacterial Activities ◽  
Multidrug Transporter ◽  
Wild Type ◽  
Chemical Library ◽  
Fluoroquinolone Antibiotics

ABSTRACT The multidrug transporter NorA contributes to the resistance ofStaphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

scholarly journals Effect of some fungicides against the growth inhibition of Sclerotinia sclerotiorum mycelial compatibility groups

2015 ◽  
Vol 55 (4) ◽  
pp. 354-361 ◽  
Author(s):  
Alireza Dalili ◽  
Saeed Bakhtiari ◽  
Hossein Barari ◽  
Majid Aldaghi
Keyword(s):  
Growth Inhibition ◽  
Sclerotinia Sclerotiorum ◽  
Integrated Management ◽  
In Vitro Tests ◽  
Sclerotinia Stem Rot ◽  
Mazandaran Province ◽  
Brassica Napus L ◽  
Northern Iran ◽  
Mycelial Compatibility

Abstract Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of Sclerotinia stem rot, is one of the most important pathogens of Brassica napus L. in northern Iran. In this study, 13 mycelial compatibility groups (MCGs) of the fungus were identified among 31 isolates sampled from four regions of Mazandaran province, Iran. Effective fungicides are useful in the integrated management of the disease. So, the effect of tebuconazole, propiconazole, cyproconazole, and Rovral-TS at five doses (0.0001, 0.001, 0.01, 0.1, and 1 ppm) was studied on the growth inhibition of S. sclerotiorum as in vitro tests. Maximum inhibition (100%) of S. sclerotiorum mycelial growth was obtained by the highest dose (1 ppm) of all tested fungicides, as well as by the doses of 0.1 and 0.01 ppm of propiconazole, cyproconazole, and tebuconazole. In this investigation, the reaction of S. sclerotiorum isolates belonging to different MCGs was evaluated against tebuconazole, propiconazole, cyproconazole, and Rovral-TS at their EC50 ranges. The results revealed that there was high variation of S. sclerotiorum MCGs against different fungicides. The inhibition percentage varied between 4.29% and 71.72%.

scholarly journals Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

1996 ◽  
Vol 40 (1) ◽  
pp. 40-46 ◽  
Author(s):  
J L McKimm-Breschkin ◽  
T J Blick ◽  
A Sahasrabudhe ◽  
T Tiong ◽  
D Marshall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Receptor Binding ◽  
Liquid Culture ◽  
Plaque Assay ◽  
Enoic Acid ◽  
Cross Resistance ◽  
Receptor Binding Site ◽  
Significant Difference

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

scholarly journals Peptide Deformylase in Staphylococcus aureus: Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene

2000 ◽  
Vol 44 (7) ◽  
pp. 1825-1831 ◽  
Author(s):  
Peter S. Margolis ◽  
Corinne J. Hackbarth ◽  
Dennis C. Young ◽  
Wen Wang ◽  
Dawn Chen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Library ◽  
Specific Inhibitor ◽  
Peptide Deformylase ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Resistant Mutants

ABSTRACT Peptide deformylase, a bacterial enzyme, represents a novel target for antibiotic discovery. Two deformylase homologs, defA and defB, were identified inStaphylococcus aureus. The defA homolog, located upstream of the transformylase gene, was identified by genomic analysis and was cloned from chromosomal DNA by PCR. A distinct homolog, defB, was cloned from an S. aureus genomic library by complementation of the arabinose-dependent phenotype of a P BAD -def Escherichia coli strain grown under arabinose-limiting conditions. Overexpression in E. coli of defB, but not defA, correlated to increased deformylase activity and decreased susceptibility to actinonin, a deformylase-specific inhibitor. ThedefB gene could not be disrupted in wild-type S. aureus, suggesting that this gene, which encodes a functional deformylase, is essential. In contrast, thedefA gene could be inactivated; the function of this gene is unknown. Actinonin-resistant mutants grew slowly in vitro and did not show cross-resistance to other classes of antibiotics. When compared to the parent, an actinonin-resistant strain produced an attenuated infection in a murine abscess model, indicating that this strain also has a growth disadvantage in vivo. Sequence analysis of the actinonin-resistant mutants revealed that each harbors a loss-of-function mutation in the fmt gene. Susceptibility to actinonin was restored when the wild-type fmt gene was introduced into these mutant strains. An S. aureusΔfmt strain was also resistant to actinonin, suggesting that a functional deformylase activity is not required in a strain that lacks formyltransferase activity. Accordingly, thedefB gene could be disrupted in an fmt mutant.

scholarly journals Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

2000 ◽  
Vol 191 (5) ◽  
pp. 859-870 ◽  
Author(s):  
Melanie Allen ◽  
Linne Svensson ◽  
Marsha Roach ◽  
John Hambor ◽  
John McNeish ◽  
...  
Keyword(s):  
Map Kinase ◽  
Es Cells ◽  
Interleukin 1 ◽  
Embryonic Stem ◽  
Wild Type ◽  
Antiinflammatory Drugs ◽  
Gene Encoding ◽  
Significant Difference ◽  
Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

CEP701 and PKC412 Predictably and Reliably Inhibit FLT3 Phosphorylation in Primary AML Blasts but Their Induction of a Cytotoxic Response Appears to Be Much More Variable.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Steven Knapper ◽  
Alan K. Burnett ◽  
Amanda F. Gilkes ◽  
Kenneth I. Mills ◽  
Val Walsh
Keyword(s):  
Statistical Significance ◽  
Annexin V ◽  
Expression Level ◽  
Wild Type ◽  
Response Curves ◽  
Allele Ratio ◽  
Cytotoxic Response ◽  
Significant Difference ◽  
Flt3 Expression

Abstract Activating mutations of the receptor tyrosine kinase FLT3 are present in approximately one-third of AML cases and are associated with an adverse prognosis. FLT3 is expressed in over 90% of cases of AML and many non-mutants show evidence of FLT3 activation, which may play a significant signalling role in leukaemogenesis, making FLT3 an attractive therapeutic target. CEP701 (Cephalon) and PKC412 (Novartis) are orally-bioavailable indolocarbazole derivatives that potently inhibit FLT3 phosphorylation. We studied the relationship between in vitro inhibition of FLT3 phosphorylation and induction of cytotoxicity in primary AML blasts from 12 patients. 7 of the cases were FLT3 mutants (6 ITDs and 1 D835 point mutant), the amount of mutant RNA varying between 7% and 84% of total FLT3 RNA expressed. The blasts were exposed for 1 hour to a range of concentrations of CEP701 and PKC412, lysed and immunoprecipitated with an anti-FLT3 antibody. After sequential immunoblotting with anti-phosphotyrosine and anti-FLT3 antibodies, inhibition of FLT3 phosphorylation was measured by densitometry. Both drugs inhibited FLT3 phosphorylation in all samples with lower concentrations required in FLT3 mutants. CEP701 inhibited FLT3 phosphorylation with median IC50s of 3.7nM and 11.9nM in mutant and wild type (WT) cases respectively (p=0.0006). IC50s for PKC412 were 7.7nM and 59.8nM in mutant and WT cases (p=0.0268). Induction of cytotoxicity was assessed by MTS assay following 72-hour exposure of blasts to a range of concentrations of CEP701 and PKC412. Cytotoxic responses to both drugs were greater in FLT3 mutants than WT cases at each dose studied and in terms of IC50 dose (median IC50s in mutant and WT cases: 95nM and 231nM with CEP701, 1.24 μM and 1.61μM with PKC412) although these differences did not reach statistical significance. Annexin V binding apoptosis assay produced similar dose response curves. Both agents showed greater inter-case variability in cytotoxic response than in sensitivity to inhibition of FLT3 phosphorylation. A lack of cytotoxic response to FLT3 inhibition with CEP701 was seen in the ITD mutant with the lowest ratio of mutant to WT FLT3 RNA (0.08) and several WT samples displayed resistance to in vitro induction of cytotoxicity despite almost complete inhibition of FLT3. Induction of cytotoxicity with PKC412 in both mutant and WT cases generally required doses well in excess of those required to fully inhibit FLT3 phosphorylation. Cases were further stratified by flow cytometric measurement of surface FLT3 expression, and by immunoblotting to measure STAT5 dephosphorylation in response to both drugs. No significant difference in overall FLT3 expression was seen between mutant and WT cases. Interestingly the highest FLT3 expression level was seen in a wild type case that was highly sensitive to CEP701. Inhibition of STAT5 phosphorylation appeared closely linked to FLT3 inhibition, although in some cases a good cytotoxic response was achieved despite failure to inhibit STAT5, suggesting involvment of other signalling pathways. In summary, although both CEP701 and PKC412 predictably and reliably inhibit FLT3 phosphorylation in primary AML blasts, their induction of cytotoxicity appears to be much more variable. A number of factors may influence this including variations in level of dependency on FLT3 signalling for blast survival, mutant to WT allele ratio and overall FLT3 expression level. Effects on targets other than FLT3 also need to be considered.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

Parasitology ◽  
2017 ◽  
Vol 145 (3) ◽  
pp. 355-370 ◽  
Author(s):  
SIMONE S. C. OLIVEIRA ◽  
INÊS C. GONÇALVES ◽  
VITOR ENNES-VIDAL ◽  
ANGELA H. C. S. LOPES ◽  
RUBEM F. S. MENNA-BARRETO ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Oncopeltus Fasciatus ◽  
Calpain Inhibitor ◽  
Insect Vector ◽  
Peptidase Activity ◽  
Wild Type ◽  
Calcium Dependent ◽  
Calpain Inhibitors ◽  
Selection Of

SUMMARYThe species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µm (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

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