The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.     

Primary intramuscular infestation of Echinococcus granulosus misdiagnosed as a soft tissue tumor: a case report

BACKGROUND: The occurrence of a primary intramuscular infestation of Echinococcus granulosus is extremely rare. CASE: A 70-year-old woman with primary skeletal muscle hydatidosis initially presented with a soft tissue mass. Clinical and radiologic examination revealed a huge cystic mass in the right quadriceps muscle without any visceral organ involvement. Since the differential diagnosis included a soft tissue tumor, fine needle aspiration cytology was performed, and a diagnosis of hydatid disease was made. CONCLUSION: This very rare case of primary intramuscular infestation of E granulosus was clinically misdiagnosed as a soft tissue tumor. Hydatid disease, albeit rare, should be considered in the differential diagnosis of a soft tissue mass.     

Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues

Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.     

Role of D-Limonene in Autophagy Induced by Bergamot Essential Oil in SH-SY5Y Neuroblastoma Cells

Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005–0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70^S6K (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125–750 µM) and linalyl acetate (62.5–375 µM), were individually tested at concentrations comparable to those found in 0.005–0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by d-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by d-limonene.     

Axitinib Improves Radiotherapy in Murine Xenograft Lung Tumors

A third of patients with non-small cell lung cancer (NSCLC) present with un-resectable stage III locally advanced disease and are currently treated by chemo-radiotherapy but the median survival is only about 21 months. Using an orthotopic xenograft model of lung carcinoma, we have investigated the combination of radiotherapy with the anti-angiogenic drug axitinib (AG-013736, Pfizer), which is a small molecule receptor tyrosine kinase inhibitor that selectively targets the signal transduction induced by VEGF binding to VEGFR receptors. We have tested the combination of axitinib with radiotherapy in nude mice bearing human NSCLC A549 lung tumors. The therapy effect was quantitatively evaluated in lung tumor nodules. The modulation of radiation-induced pneumonitis, vascular damage and fibrosis by axitinib was assessed in lung tissue. Lung irradiation combined with long-term axitinib treatment was safe resulting in minimal weight loss and no vascular injury in heart, liver and kidney tissues. A significant decrease in the size of lung tumor nodules was observed with either axitinib or radiation, associated with a decrease in Ki-67 staining and a heavy infiltration of inflammatory cells in tumor nodules. The lungs of mice treated with radiation and axitinib showed a complete response with no detectable residual tumor nodules. A decrease in pneumonitis, vascular damage and fibrosis were observed in lung tissues from mice treated with radiation and axitinib. Our studies suggest that axitinib is a potent and safe drug to use in conjunction with radiotherapy for lung cancer that could also act as a radioprotector for lung tissue by reducing pneumonitis and fibrosis.     

Increased N-Glycosylation Efficiency by Generation of an Aromatic Sequon on N135 of Antithrombin

The inefficient glycosylation of consensus sequence on N135 in antithrombin explains the two glycoforms of this key anticoagulant serpin found in plasma: α and β, with four and three N-glycans, respectively. The lack of this N-glycan increases the heparin affinity of the β-glycoform. Recent studies have demonstrated that an aromatic sequon (Phe-Y-Asn-X-Thr) in reverse β-turns enhances N-glycosylation efficiency and stability of different proteins. We evaluated the effect of the aromatic sequon in this defective glycosylation site of antithrombin, despite of being located in a loop between the helix D and the strand 2A. We analyzed the biochemical and functional features of variants generated in a recombinant cell system (HEK-EBNA). Cells transfected with wild-type plasmid (K133-Y-N135-X-S137) generated 50% of α and β-antithrombin. The S137T, as previously reported, K133F, and the double mutant (K133F/S137T) had improved glycosylation efficiency, leading to the secretion of α-antithrombin, as shown by electrophoretic and mass analysis. The presence of the aromatic sequon did not significantly affect the stability of this conformationally sensitive serpin, as revealed by thermal denaturation assay. Moreover, the aromatic sequon hindered the activation induced by heparin, in which is involved the helix D. Accordingly, K133F and particularly K133F/S137T mutants had a reduced anticoagulant activity. Our data support that aromatic sequons in a different structural context from reverse turns might also improve the efficiency of N-glycosylation.     

The impact of a 6-year comprehensive community trial on the awareness, treatment and control rates of hypertension in Iran: experiences from the Isfahan healthy heart program

Background

Acute mental stress may contribute to the cardiovascular disease progression via autonomic nervous system controlled negative effects on the endothelium. The joint effects of stress-induced sympathetic or parasympathetic activity and endothelial function on atherosclerosis development have not been investigated. The present study aims to examine the interactive effect of acute mental stress-induced cardiac reactivity/recovery and endothelial function on the prevalence of carotid atherosclerosis.

Methods

Participants were 81 healthy young adults aged 24-39 years. Preclinical atherosclerosis was assessed by carotid intima-media thickness (IMT) and endothelial function was measured as flow-mediated dilatation (FMD) using ultrasound techniques. We also measured heart rate, respiratory sinus arrhythmia (RSA), and pre-ejection period (PEP) in response to the mental arithmetic and speech tasks.

Results

We found a significant interaction of FMD and cardiac RSA recovery for IMT (p = 0.037), and a significant interaction of FMD and PEP recovery for IMT (p = 0.006). Among participants with low FMD, slower PEP recovery was related to higher IMT. Among individuals with high FMD, slow RSA recovery predicted higher IMT. No significant interactions of FMD and cardiac reactivity for IMT were found.

Conclusions

Cardiac recovery plays a role in atherosclerosis development in persons with high and low FMD. The role of sympathetically mediated cardiac activity seems to be more important in those with impaired FMD, and parasympathetically mediated in those with relatively high FMD. The development of endothelial dysfunction may be one possible mechanism linking slow cardiac recovery and atherosclerosis via autonomic nervous system mediated effect.     

Structure and Dynamics of a Benthic Invertebrate Community in an Intertidal Area of the Tagus Estuary, Western Portugal: A Six Year Data Series

A predictive model, incorporating macroinvertebrate and environmental data, similar to that developed for Australian rivers (AUSRIVAS) and British rivers (RIVPACS) was constructed using a dataset collected from 23 reference (least altered) wetlands on the Swan Coastal Plain, Western Australia, sampled in summer and spring, 1989 and spring, 1990. Four main groups of reference wetlands were identified by UPGMA classification (using the Bray–Curtis dissimilarity measure). Distinguishing environmental variables identified by Stepwise Multiple Discriminant Function Analysis were: calcium, colour (gilvin), latitude, longitude, sodium and organic carbon. Observed to expected ratios of taxa with a >50% chance of occurrence (OE50) derived from the model for a suite of 23 test wetlands sampled in spring, 1997, were significantly correlated with pH and the depth of the sampling sites. Greater discrimination between the test wetlands was provided by the OE50 ratios than either raw richness (number of families) or a biotic index (SWAMPS). Results obtained for a subset of 11 test wetlands sampled with both a rapid bioassessment protocol (incorporating field picking of 200 invertebrates collected in 2 min sweeps from selected habitats) and a semi-quantitative protocol (incorporating laboratory picking of all invertebrates collected in sweeps along 10 m transects at randomly allocated sites) were not significantly different, indicating that the former could be used to reduce the time and costs associated with macroinvertebrate-based wetland monitoring programs. In addition to providing an objective method of assessing wetland condition, predictive modelling provides a list of taxa expected to occur under reference conditions, which can be used as a target in wetland restoration programs. The probable impediment to widespread adoption of predictive modelling for wetland bioassessment is the need to produce models tailored to specific geographic regions and specific climatic conditions. This may incur significant costs in countries, such as Australia, which span a wide range of climatic zones.     

Modulation of calcium signalling by the actin-binding protein cofilin

Cofilin is a small protein that belongs to the family of actin-depolymerizing factors (ADF). The main cellular function of cofilin is to change cytoskeletal dynamics and thus to modulate cell motility and cytokinesis. We have recently demonstrated that the actin cytoskeleton is involved in the modulation of Ca(2+) signalling in starfish oocytes. To extend these observations, we have explored whether cofilin influences Ca(2+) signalling in the oocytes. Here we show that microinjection of the functionally active cofilin alters the Ca(2+) signalling mediated by the three major second messengers, InsP(3), NAADP, and cADPr. Cofilin intensifies the Ca(2+) signals induced by InsP(3) and NAADP, and delays those induced by cADPr. Furthermore, the injection of cofilin increases the Ca(2+) signals during hormone-induced oocyte maturation and fertilization. The results suggest that the dynamic regulation of F-actin by its binding proteins may play an important role in the modulation of intracellular Ca(2+) signalling.