Adriana Fiorini

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs.

Mapping of intrinsic bent DNA sites in the upstream region of DNA puff BhC4-1 amplified gene

We have identified bent DNA sites in the distal and proximal DNA puff BhC4‐1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B − 9 to + 1. The 1847 bp fragment (− 3697 to − 1850) in relation to the transcription start site shows multiple bending sites, BhC4B − 9 to BhC4B − 4, with periodicity ∼300 bp. The analysis of the other identified bent region, starting at position − 957, reveals that the BhC4B + 1 bent site colocalizes with the putative BhC4‐1 minimal promoter. The sequence analysis of bent site BhC4B − 4 shows a distribution of dA•dT at ∼10 bp intervals between the middle of each tract, but intervals with more than one turn, ∼20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B − 6 and BhC4B − 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4‐1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R‐values) were determined, and a high R‐value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R‐value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and ΔG, were determined. The role of these bent sites in the BhC4‐1 transcription regulation is discussed.

β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent). Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO) activity, and the release of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

Colonization of the oral cavity by yeasts in patients with chronic renal failure undergoing hemodialysis

OBJECTIVES To determine the frequency of yeast in the oral cavity of patients with chronic renal failure, undergoing hemodialysis (PCRFH); identification and antifungal susceptibility profile of yeast and demographic profile of patients. METHODS We performed mouthwash in 146 PCRFH; the rinse fluid was collected and cultured, yeasts grown were identified by phenotypic and molecular methods. The antifungal susceptibility profile was determined against nystatin, amphotericin B, fluconazole, voriconazole, and caspofungin based in Clinical and Laboratory Standards Institute (document M27-A3). RESULTS Positive culture was observed in 39% of patients, of whom 53% were women; the median of dialysis time was 2.9 years. The age of the colonized patients varied between 26 and 84 years, with a median of 52.5 years. PCRFH over 45 years were significantly more colonized (P = 0.0108) as well as denture wearers (84.0%). We isolated 81 yeasts, predominantly Candida albicans (63%) followed by Candida glabrata. In general, yeasts were sensitive to the evaluated antifungal agents, but there was significant variation in the minimum inhibitory concentration, especially among non-C. albicans Candida (NCAC) compared to fluconazole, caspofungin, and amphotericin B. NCAC required significantly higher concentrations of fluconazole (P < 0.01). CONCLUSION The rate of colonization by yeasts in PCRFH was high, and there was variability in species distribution and antifungal susceptibility profile. These results are little known in this group of patients and are important for controlling the risk of developing invasive fungal infections.

MOLECULAR TYPING OF Candida albicans ISOLATES FROM HOSPITALIZED PATIENTS

SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

Structural Changes and Differentially Expressed Genes in Pseudomonas aeruginosa Exposed to Meropenem-Ciprofloxacin Combination

ABSTRACT The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa. The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.

Intrinsically bent DNA sites in the Drosophila melanogaster third chromosome amplified domain

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila ampliconin the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-β. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-β region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes.

Intrinsic Bent DNA Sites in the Developmentally Amplified C3-22 Gene Promoter of Rhynchosciara americana (Diptera: Sciaridae)

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b−3, b−2, and b−1) were localized in the promoter, and one was localized downstream of the gene (b+1). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.

Genotypic variability and antifungal susceptibility of Candida tropicalis isolated from patients with candiduria

BACKGROUND Candida tropicalis is an emerging major human pathogen in nosocomial infections, and it is considered the second or third species of Candida most isolated from urine cultures. AIMS The study aimed at characterizing genotypically C. tropicalis strains from patients with candiduria in a university hospital, and assessed the antifungal susceptibility profile. METHODS The study was conducted with hospitalized patients who developed urinary tract infection from C. tropicalis from June 2010 to June 2011 at the Grande Dourados University Hospital of the Federal University, Dourados, MS, Brazil. Susceptibility to the antifungal agents amphotericin B and fluconazole was determined by broth microdilution. The genotypic variability of isolates of C. tropicalis was analyzed by microsatellite markers and RAPD-PCR. RESULTS Only one isolate was resistant to amphotericin B (MIC→16μg/ml); the others were susceptible to fluconazole and amphotericin B. The genotypic variability by RAPD-PCR resulted in distinct profiles for RAPD markers. A total of 10 alleles were observed for the microsatellite loci, URA3 and CT14, which were grouped differently, and four associations were observed for locus URA3 and eight for locus CT14. CONCLUSIONS C. tropicalis isolates from urine were susceptible to the antifungal agents tested. The genotyping techniques make possible proving the similarity and genetic diversity among isolates of C. tropicalis involved in nosocomial infections. This knowledge is important for the control and prevention of nosocomial infections caused by this yeast species.