scholarly journals Nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Joanna Katarzyna Strzelczyk ◽  
Anna Slemp-Migiel ◽  
Magdalena Rother ◽  
Karolina Gołąbek ◽  
Andrzej Wiczkowski
Keyword(s):  
Candida Albicans ◽  
Sequence Analysis ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Clinical Isolates ◽  
Primary Target ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Conformation Polymorphism

One of the mechanisms of Candida albicans resistance to azole drugs used in antifungal therapy relies on increased expression and presence of point mutations in the ERG11 gene that encodes sterol 14α demethylase (14DM), an enzyme which is the primary target for the azole class of antifungals. The aim of the study was to analyze nucleotide substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant clinical isolates. The Candida albicans isolates represented a collection of 122 strains selected from 658 strains isolated from different biological materials. Samples were obtained from hospitalized patients. Fluconazole susceptibility was tested in vitro using a microdilution assay. Candida albicans strains used in this study consisted of two groups: 61 of the isolates were susceptible to azoles and the 61 were resistant to azoles. Four overlapping regions of the ERG11 gene of the isolates of Candida albicans strains were amplified and sequenced. The MSSCP (multitemperature single strand conformation polymorphism) method was performed to select Candida albicans samples presenting genetic differences in the ERG11 gene fragments for subsequent sequence analysis. Based on the sequencing results we managed to detect 19 substitutions of nucleotides in the ERG11 gene fragments. Sequencing revealed 4 different alterations: T495A, A530C, G622A and A945C leading to changes in the corresponding amino acid sequence: D116E, K128T, V159I and E266D. The single nucleotide changes in the ERG11 gene did not affect the sensitivity of Candida albicans strains, whereas multiple nucleotide substitutions in the ERG11 gene fragments indicated a possible relation with the increase in resistance to azole drugs.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

1997 ◽  
Vol 41 (3) ◽  
pp. 540-543 ◽  
Author(s):  
A Scorpio ◽  
P Lindholm-Levy ◽  
L Heifets ◽  
R Gilman ◽  
S Siddiqi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Direct Sequencing ◽  
Single Strand Conformation Polymorphism ◽  
Therapy Resistance ◽  
Sscp Analysis ◽  
Clinical Isolates ◽  
Nucleotide Substitutions ◽  
Resistant Strains ◽  
Made In

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

Promoter region single nucleotide polymorphism of siglec-8 gene associates with susceptibility to allergic asthma

2020 ◽  
Vol 17 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Mohammad Sajay-asbaghi ◽  
Mahnaz Sadeghi-shabestrai ◽  
Amir Monfaredan ◽  
Narges Seyfizadeh ◽  
Alireza Razavi ◽  
...  
Keyword(s):  
Allergic Asthma ◽  
Normal Subjects ◽  
Single Strand Conformation Polymorphism ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Potential Therapeutic Target ◽  
Single Nucleotide ◽  
Study Materials ◽  
Protective Allele ◽  
Conformation Polymorphism

Aim: Siglec-8 is exclusively expressed on mast cells, eosinophils and basophils. Possible association of six siglec-8 single nucleotide polymorphisms (SNPs) with susceptibility to allergic asthma in the Azeri population of Iran was investigated in this study. Materials & methods: A total of 194 patients and 190 normal subjects were enrolled. PCR single strand conformation polymorphism (PCR-SSCP) was used to determine the genotypes of the studied SNPs. Results: The rs36498 showed significant association with allergic asthma (odds ratio [OR]: 0.65; p = 0.022) and the T allele was found as a protective allele (OR: 0.61; p = 0.008). Also, eosinophil count in the CC genotype was significantly higher than that in the other genotypes (p = 0.026). Conclusion: The rs36498 is thought to influence the expression level of siglec-8. Siglec-8 could be a potential therapeutic target for allergic asthma.

scholarly journals Antifungal activity of thymol against clinical isolates of fluconazole-sensitive and -resistant Candida albicans

2009 ◽  
Vol 58 (8) ◽  
pp. 1074-1079 ◽  
Author(s):  
Na Guo ◽  
Jingbo Liu ◽  
Xiuping Wu ◽  
Xingming Bi ◽  
Rizeng Meng ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Concentration Index ◽  
Inhibitory Concentration ◽  
Clinical Isolates ◽  
Standard Strain ◽  
Antagonistic Action ◽  
Microdilution Method ◽  
Interaction Intensity

Thymol (THY) was found to have in vitro antifungal activity against 24 fluconazole (FLC)-resistant and 12 FLC-susceptible clinical isolates of Candida albicans, standard strain ATCC 10231 and one experimentally induced FLC-resistant C. albicans S-1. In addition, synergism was observed for clinical isolates of C. albicans with combinations of THY–FLC and THY–amphotericin B (AMB) evaluated by the chequerboard microdilution method. The interaction intensity was determined by spectrophotometry for the chequerboard assay, and the nature of the interactions was assessed using two non-parametric approaches [fractional inhibitory concentration index (FICI) and ΔE models]. The interaction between THY–FLC or THY–AMB in FLC-resistant and -susceptible strains of C. albicans showed a high percentage of synergism by the FICI method and the ΔE method. The ΔE model gave results consistent with FICI, and no antagonistic action was observed in the strains tested.

scholarly journals Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

2002 ◽  
Vol 196 (5) ◽  
pp. 629-639 ◽  
Author(s):  
Carmela Gurrieri ◽  
Peter McGuire ◽  
Hong Zan ◽  
Xiao-Jie Yan ◽  
Andrea Cerutti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Lymphocytic Leukemia ◽  
Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

scholarly journals Structure and Temporal Dynamics of Populations within Wheat Streak Mosaic Virus Isolates

2001 ◽  
Vol 75 (21) ◽  
pp. 10231-10243 ◽  
Author(s):  
Jeffrey S. Hall ◽  
Roy French ◽  
T. Jack Morris ◽  
Drake C. Stenger
Keyword(s):  
Mosaic Virus ◽  
Temporal Dynamics ◽  
Consensus Sequence ◽  
Single Strand Conformation Polymorphism ◽  
Wheat Streak Mosaic Virus ◽  
Relative Fitness ◽  
Nucleotide Sequencing ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Constant Rate

ABSTRACT Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G6016) in NIa that was restored to A6016 in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.

scholarly journals Mutations in the fks1 Gene in Candida albicans, C. tropicalis, and C. krusei Correlate with Elevated Caspofungin MICs Uncovered in AM3 Medium Using the Method of the European Committee on Antibiotic Susceptibility Testing

2008 ◽  
Vol 52 (9) ◽  
pp. 3092-3098 ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Stéphane Bretagne ◽  
Dorothée Raoux ◽  
Damien Hoinard ◽  
Françoise Dromer ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Isolates ◽  
Antibiotic Susceptibility Testing ◽  
Wild Type ◽  
European Committee ◽  
Rpmi Medium

ABSTRACT Mutations in two specific regions of the Fks1 subunit of 1,3-β-d-glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans, 62 C. tropicalis, and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis, and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC90) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis, one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 μg/ml for wild-type isolates and from 1 to 8 μg/ml for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 μg/ml of caspofungin, while caspofungin MICs for all mutant isolates were ≥0.5 μg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis, and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

A963G single nucleotide variant of BMP15 is not common bio-marker for fecundity in goat

2016 ◽  
Author(s):  
Guang-Xin E ◽  
Yong-Fu Huang ◽  
Jian-Ning He ◽  
Wei-Wei Ni ◽  
Yong-Ju Zhao
Keyword(s):  
Polymerase Chain Reaction ◽  
Single Strand Conformation Polymorphism ◽  
Single Nucleotide Variant ◽  
Chain Reaction ◽  
Single Nucleotide ◽  
Exon 2 ◽  
Morphogenetic Protein ◽  
Polymerase Chain ◽  
Bone Morphogenetic Protein 15 ◽  
Conformation Polymorphism

Bone morphogenetic protein 15 (BMP15) is crucial factor for ovulation as well as for increasing litter size. In the present investigation efforts had been carried out to assess the genetic variations in Exon 2 region of BMP15 in goat, using polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) sequencing methods and cooperated frequency distribution to discuss its possibility of related fecundity. Across the 144samples from six breeds were identified in the A963G location of BMP15 using PCR-SSCP and sequences technology. A963A genotype was the most frequent (85.4%) and G963G was the least frequent with a frequency of 4.2% and A963G is 10.4%. It revealed non significant different between high and low fecundity breed. Therefore, this single nucleotide variant is not common Bio-Marker for fecundity in Goat.

scholarly journals Distribution of Fitness and Virulence Effects Caused by Single-Nucleotide Substitutions in Tobacco Etch Virus

2007 ◽  
Vol 81 (23) ◽  
pp. 12979-12984 ◽  
Author(s):  
Purificación Carrasco ◽  
Francisca de la Iglesia ◽  
Santiago F. Elena
Keyword(s):  
Single Case ◽  
Site Directed Mutagenesis ◽  
Relative Fitness ◽  
In Planta ◽  
Nucleotide Substitutions ◽  
Phenotypic Effect ◽  
Single Nucleotide ◽  
Mutant Genotype

ABSTRACT Little is known about the fitness and virulence consequences of single-nucleotide substitutions in RNA viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. Here we used site-directed mutagenesis to create a collection of 66 clones of Tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. Competition experiments between each mutant and the ancestral nonmutated clone were performed in planta to quantitatively assess the relative fitness of each mutant genotype. Among all mutations, 40.9% were lethal, and among the viable ones, 36.4% were significantly deleterious and 22.7% neutral. Not a single case of beneficial effects was observed within the level of resolution of our measures. On average, the fitness of a genotype carrying a deleterious but viable mutation was 49% smaller than that for its unmutated progenitor. Deleterious mutational effects conformed to a beta probability distribution. The virulence of a subset of viable mutants was assessed as the reduction in the number of viable seeds produced by infected plants. Mutational effects on virulence ranged between 17% reductions and 24.4% increases. Interestingly, the only mutations showing a significant effect on virulence were hypervirulent. Competitive fitness and virulence were uncorrelated traits.

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