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1.
Environ Sci Technol ; 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32401017

RESUMO

Selenium (Se) is an essential dietary element for humans and animals, and the atmosphere is an important source of Se to soils. However, estimates of global atmospheric Se fluxes are highly uncertain. To constrain these uncertainties, we use a global model of atmospheric Se cycling and a database of more than 600 sites where Se in aerosol has been measured. Applying Bayesian inference techniques, we determine probability distributions of global Se emissions from the four major sources: anthropogenic activities, volcanoes, marine biosphere, and terrestrial biosphere. Between 29 and 36 Gg of Se are emitted to the atmosphere every year, doubling previous estimates of emissions. Using emission parameters optimized by aerosol network measurements, our model shows good agreement with the aerosol Se observations (R2=0.66), as well as with independent aerosol (0.59) and wet deposition measurements (0.57). Both model and measurements show a decline in Se over North America in the last two decades, due to changes in technology and energy policy. Our results highlight the role of the ocean as a net atmospheric Se sink, with around 7 Gg yr-1 of Se transferred from land through the atmosphere. The constrained Se emissions represent a substantial step forward in understanding the global Se cycle.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31338201

RESUMO

Background: In resource-limited settings, such as Rwanda, health care profession (HCP) to neonate ratios are low, and therefore caregivers play a significant role in providing care for their admitted neonates. To provide such Family Integrated Care, caregivers need knowledge, skills, and confidence. The objective of this study was to identify consensus from key stakeholders regarding the priority topics for a "parental neonatal curriculum." Methods: A three-round Delphi-study was conducted. During Round-1, face-to-face interviews were undertaken and responses coded and categorized into themes. In Round-2, participants were presented with Round-1 feedback and asked to provide additional topics in respective themes. In Round-3, respondents were asked to rank the importance of these items using a 9-point Likert scale. Results: Ten, 36 and 40 stakeholders participated in Rounds-1, - 2 and - 3 respectively, including parents, midwives, nurses and physicians. Twenty and 37 education topics were identified in Rounds-1 and -2 respectively. In Round-3 47 of the 57 presented outcomes met pre-defined criteria for inclusion in the "parental neonatal curriculum." Conclusion: We describe a "parental neonatal curriculum," formed using robust consensus methods, describing the core topics required to educate parents of neonates admitted to a newborn care unit. The curriculum has been developed in Rwanda and is relevant to other resource-limited settings.

4.
J Phys Chem Lett ; 10(15): 4484-4489, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295402

RESUMO

The diffusivity of molecules relevant to condensed-phase chemistry within viscous secondary organic aerosol (SOA) remains highly uncertain. Whereas there has been an effort to characterize water diffusivity as well as the diffusivity of larger compounds, data are lacking almost entirely for small molecules, such as carbon dioxide (CO2). Here we use photochemically generated CO2 in single particles of aqueous citric acid as a SOA proxy, levitated in an electrodynamic balance, to deduce CO2 diffusivity in the particle with unprecedented accuracy. For medium viscosities at intermediate relative humidities (∼25-40% RH), we find CO2 diffusivities DCO2 ≈ 10-14 m2 s-1, agreeing with the Stokes-Einstein relationship based on current viscosity data but 10 times lower than that for water. Conversely, under dry high-viscosity conditions, we find that DCO2 ≈ 10-16 m2 s-1, which is 10 times higher than for water. We infer that the chemical degradation of atmospheric SOA particles will likely not be limited by CO2 diffusivity.

5.
J Geophys Res Atmos ; 124(9): 5058-5087, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31245233

RESUMO

The method to derive aerosol size distributions from in situ stratospheric measurements from the University of Wyoming is modified to include an explicit counting efficiency function (CEF) to describe the channel-dependent instrument counting efficiency. This is motivated by Kovilakam and Deshler's (2015, https://doi.org/10.1002/2015JD023303) discovery of an error in the calibration method applied to the optical particle counter (OPC40) developed in the late 1980s and used from 1991 to 2012. The method can be applied to other optical aerosol instruments for which counting efficiencies have been measured. The CEF employed is the integral of the Gaussian distribution representing the instrument response at any one aerosol channel, the aerosol counting efficiency. Results using the CEF are compared to previous derivations of aerosol size distributions (Deshler et al., 2003, https://doi.org/10.1029/2002JD002514) applied to the measurements before and after Kovilakam and Deshler's correction of number concentration for the OPC40 calibration error. The CEF method is found, without any tuning parameter, to reproduce or improve upon the Kovilakam and Deshler's results, thus accounting for the calibration error without any external comparisons other than the laboratory determined counting efficiency at each aerosol channel. Moments of the new aerosol size distributions compare well with aerosol extinctions measured by Stratospheric Aerosol and Gas Experiment II and Halogen Occultation Experiment in the volcanic period 1991-1996, generally within ±40%, the precision of OPC40 moments, and in the nonvolcanic period after 1996, generally within ±20%. Stratospheric Aerosol and Gas Experiment II and Halogen Occultation Experiment estimates of aerosol surface area are generally in agreement with those derived using the new CEF method.

6.
Paediatr Int Child Health ; 39(4): 265-274, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31079590

RESUMO

Background: Monitoring and evaluation is vital in the quest to improve the quality of care and to reduce the morbidity and mortality of neonates in a resource-limited setting. Databases offer several advantages such as data on large cohorts of neonates and from multiple centres. Aim: To establish a minimal dataset neonatal database in Kigali, Rwanda and to assess the quality and timing of the data entry process. Secondary objectives were to describe survival rates and associated risk factors. Methods: A cross-sectional, observational study was undertaken at a tertiary hospital in Kigali, Rwanda. The Rwanda Neonatal Data Collection Form was designed specifically for the database, based on the Vermont-Oxford Network neonatal data-collection tool with locally relevant amendments. All admitted neonates were enrolled during the study period of 2011-2017 with ongoing data-collection. Infants were recruited and data collected prospectively and cross-checked retrospectively with the inclusion of basic data on neonates who were not initially recruited prospectively. Results: 3391 analysable cases were recruited: 1420 prospective and 1971 retrospective cases. Prospective data collection peaked at 90%. Data entry was not always complete with data-points left blank with only 21% having adequate data available (0-25% missing). All-cause mortality during the study period was 16% and annual mortality ranged from 12% to 24%. On multivariate analysis, place of birth (AOR 2.17), small-for-gestational-age (AOR 2.05) and gestational age were all positively associated with survival. Conclusions: An academic setting in a low- or middle-income country can create and maintain a neonatal database without funding and produce a wealth of actionable results. Throughout the process, there were considerable challenges which must be addressed if such a database is to be optimised, maintained and created in other clinical sites. Abbreviations: CHUK: Centre Hospitalier et Universitaire de Kigali (University Teaching Hospital of Kigali); CPAP: continuous positive airway pressure; HCP: Healthcare professional; HRH, Human Resources for Health Programme; LMIC: low- and middle-income countries; MeSH: Medical subject headings; MoH: Ministry of Health; NAR: Newborn admission record; QI: Quality improvement; REDCap: Research electronic data capture; RNDB: Rwanda neonatal database; RNDCF: Rwanda neonatal data collecion form; SGA: Small for gestational age; STROBE: Strengthening the reporting of observational studies in epidemiology; VON: The Vermont-Oxford Network.

7.
J Microbiol Methods ; 161: 63-70, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31022418

RESUMO

The fast and non-destructive detection of bacterial attachment on food contact surfaces is important for the prevention of the unwanted formation of biofilms. Biofilms constitute a protected growth mode that allows bacteria to survive even in hostile environments. Therefore, the fast detection of bacterial attachment may be an effective strategy for biofilm control. In this study cyclic voltammetry (CV) was used to detect Bacillus subtilis ssp. subtilis, Paenibacillus polymyxa, Pseudomonas fragi attachment on interdigitated microelectrodes. The differences in current between the uncolonized sterile microelectrodes and the microelectrodes after bacterial attachment were determined. In addition, the surface coverage of microelectrodes was visualized using microscopy techniques. The results showed that the cyclic voltammetry in combination with interdigitated platinum microelectrodes can be used to detect bacterial biofilms.

8.
Langmuir ; 35(8): 2966-2975, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30767535

RESUMO

Herein, we present an easy-to-use protein and cell patterning method relying solely on pipetting, rinsing steps and illumination with a desktop lamp, which does not require any expensive laboratory equipment, custom-built hardware or delicate chemistry. This method is based on the adhesion promoter poly(allylamine)-grafted perfluorophenyl azide, which allows UV-induced cross-linking with proteins and the antifouling molecule poly(vinylpyrrolidone). Versatility is demonstrated by creating patterns with two different proteins and a polysaccharide directly on plastic well plates and on glass slides, and by subsequently seeding primary neurons and C2C12 myoblasts on the patterns to form islands and mini-networks. Patterning characterization is done via immunohistochemistry, Congo red staining, ellipsometry, and infrared spectroscopy. Using a pragmatic setup, patterning contrasts down to 5 µm and statistically significant long-term stability superior to the gold standard poly(l-lysine)-grafted poly(ethylene glycol) could be obtained. This simple method can be used in any laboratory or even in classrooms and its outstanding stability is especially interesting for long-term cell experiments, e.g., for bottom-up neuroscience, where well-defined microislands and microcircuits of primary neurons are studied over weeks.

9.
J Pathol ; 247(2): 214-227, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30350370

RESUMO

Metaplastic breast carcinoma (MBC) is relatively rare but accounts for a significant proportion of global breast cancer mortality. This group is extremely heterogeneous and by definition exhibits metaplastic change to squamous and/or mesenchymal elements, including spindle, squamous, chondroid, osseous, and rhabdomyoid features. Clinically, patients are more likely to present with large primary tumours (higher stage), distant metastases, and overall, have shorter 5-year survival compared to invasive carcinomas of no special type. The current World Health Organisation (WHO) diagnostic classification for this cancer type is based purely on morphology - the biological basis and clinical relevance of its seven sub-categories are currently unclear. By establishing the Asia-Pacific MBC (AP-MBC) Consortium, we amassed a large series of MBCs (n = 347) and analysed the mutation profile of a subset, expression of 14 breast cancer biomarkers, and clinicopathological correlates, contextualising our findings within the WHO guidelines. The most significant indicators of poor prognosis were large tumour size (T3; p = 0.004), loss of cytokeratin expression (lack of staining with pan-cytokeratin AE1/3 antibody; p = 0.007), EGFR overexpression (p = 0.01), and for 'mixed' MBC, the presence of more than three distinct morphological entities (p = 0.007). Conversely, fewer morphological components and EGFR negativity were favourable indicators. Exome sequencing of 30 cases confirmed enrichment of TP53 and PTEN mutations, and intriguingly, concurrent mutations of TP53, PTEN, and PIK3CA. Mutations in neurofibromatosis-1 (NF1) were also overrepresented [16.7% MBCs compared to ∼5% of breast cancers overall; enrichment p = 0.028; mutation significance p = 0.006 (OncodriveFM)], consistent with published case reports implicating germline NF1 mutations in MBC risk. Taken together, we propose a practically minor but clinically significant modification to the guidelines: all WHO_1 mixed-type tumours should have the number of morphologies present recorded, as a mechanism for refining prognosis, and that EGFR and pan-cytokeratin expression are important prognostic markers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Mutação , Neoplasias Complexas Mistas/genética , Antígenos CD/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Caderinas/análise , Classe I de Fosfatidilinositol 3-Quinases/genética , Estudos Transversais , Transição Epitelial-Mesenquimal , Receptores ErbB/análise , Feminino , Predisposição Genética para Doença , Humanos , Queratinas/análise , Metaplasia , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Complexas Mistas/química , Neoplasias Complexas Mistas/classificação , Neoplasias Complexas Mistas/patologia , Neurofibromina 1/genética , PTEN Fosfo-Hidrolase/genética , Fenótipo , Carga Tumoral , Proteína Supressora de Tumor p53/genética
12.
J Phys Chem A ; 121(48): 9284-9296, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29111734

RESUMO

Mixed organic/inorganic aerosols may undergo liquid-liquid phase separation (LLPS) when the relative humidity drops in the atmosphere. Phase-separated particles adopt different morphologies, which will have different consequences for atmospheric chemistry and climate. Recent laboratory studies on submicron particles led to speculation whether LLPS observed for larger drops might actually be suppressed in smaller droplets. Here, we report on micron-sized droplets of a ternary mixture of ammonium sulfate (AS), carminic acid, and water at different temperatures, which were exposed to typical atmospheric drying rates ranging from 0.34 to 5.0% RH min-1. Our results reveal that increasing the drying rate and lowering the temperature results in different morphologies after LLPS and may suppress the growth and coalescence of the inorganic-rich phase inclusions due to kinetic limitations in a viscous matrix. The coalescence time was used to estimate the viscosity of the organic-rich phase within a factor of 20, and based on the Stokes-Einstein relationship, we estimated AS diffusivity. Furthermore, we evaluated the initial growth of inclusions to quantitatively determine the AS diffusivity in the organic-rich phase, which is about 10-8 cm2 s-1 at room temperature. Extrapolation of diffusivity to lower temperatures using estimations for the diffusion activation energy leads us to conclude that the growth of the inorganic phase is not kinetically impeded for tropospheric submicron particles larger than 100 nm.

14.
Langmuir ; 33(35): 8594-8605, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28792773

RESUMO

Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.


Assuntos
Oxazóis/química , Adsorção , Animais , Células Cultivadas , Humanos , Polietilenoglicóis , Polilisina , Polímeros , Ratos , Reprodutibilidade dos Testes , Propriedades de Superfície
16.
Sci Rep ; 7: 45257, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28349934

RESUMO

Sporadic solar energetic particle (SEP) events affect the Earth's atmosphere and environment, in particular leading to depletion of the protective ozone layer in the Earth's atmosphere, and pose potential technological and even life hazards. The greatest SEP storm known for the last 11 millennia (the Holocene) occurred in 774-775 AD, serving as a likely worst-case scenario being 40-50 times stronger than any directly observed one. Here we present a systematic analysis of the impact such an extreme event can have on the Earth's atmosphere. Using state-of-the-art cosmic ray cascade and chemistry-climate models, we successfully reproduce the observed variability of cosmogenic isotope 10Be, around 775 AD, in four ice cores from Greenland and Antarctica, thereby validating the models in the assessment of this event. We add to prior conclusions that any nitrate deposition signal from SEP events remains too weak to be detected in ice cores by showing that, even for such an extreme solar storm and sub-annual data resolution, the nitrate deposition signal is indistinguishable from the seasonal cycle. We show that such a severe event is able to perturb the polar stratosphere for at least one year, leading to regional changes in the surface temperature during northern hemisphere winters.

18.
Cell Physiol Biochem ; 39(3): 939-49, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27513568

RESUMO

BACKGROUND/AIMS: The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, caspase activation and/or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. METHODS: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+, by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-V-binding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM). CONCLUSIONS: Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase.


Assuntos
Antifúngicos/farmacologia , Cálcio/metabolismo , Equinocandinas/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Lipopeptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Compostos de Anilina , Anexina A5 , Caseína Quinases/antagonistas & inibidores , Caseína Quinases/genética , Caseína Quinases/metabolismo , Caspases/genética , Caspases/metabolismo , Caspofungina , Células Cultivadas , Ceramidas/metabolismo , Eritrócitos/química , Eritrócitos/citologia , Citometria de Fluxo , Fluoresceínas , Corantes Fluorescentes , Expressão Gênica , Hemólise/efeitos dos fármacos , Humanos , Oligopeptídeos/farmacologia , Estresse Oxidativo , Fosfatidilserinas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Xantenos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Cell Physiol Biochem ; 39(2): 584-95, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27394133

RESUMO

BACKGROUND/AIMS: The antifungal drug Micafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. Side effects of Micafungin treatment include microangiopathic hemolytic anemia and thrombocytopenia with microvascular thrombosis. The development of thrombosis may be fostered by stimulation of eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. The present study explored, whether Micafungin induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Micafungin (10 - 25 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Micafungin (25 µg/ml) did not significantly modify Fluo3-fluorescence, DCFDA fluorescence, or ceramide abundance. The effect of Micafungin on annexin-V-binding was not significantly modified by removal of extracellular Ca2+, by PKC inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). CONCLUSIONS: Micafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane.


Assuntos
Cálcio/metabolismo , Equinocandinas/farmacologia , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Lipopeptídeos/farmacologia , Fosfatidilserinas/metabolismo , Antifúngicos/farmacologia , Benzamidas/farmacologia , Caseína Quinase I/antagonistas & inibidores , Caseína Quinase I/metabolismo , Tamanho Celular/efeitos dos fármacos , Ceramidas/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Citometria de Fluxo , Hemólise/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Micafungina , Microscopia Confocal , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estaurosporina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Cell Physiol Biochem ; 38(6): 2272-84, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27197532

RESUMO

BACKGROUND/AIMS: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). CONCLUSIONS: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.


Assuntos
Antifúngicos/efeitos adversos , Equinocandinas/efeitos adversos , Eriptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Anidulafungina , Cálcio/metabolismo , Tamanho Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritrócitos/patologia , Humanos , Espécies Reativas de Oxigênio/metabolismo
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