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Study of the conformational and mechanical behaviors of biomolecular assemblies is vital to the rational design and realization of artificial molecular architectures with biologically relevant functionality. Here, we revealed DNA-modulated and mechanoresponsive excitonic couplings between organic chromophores and verified strong correlations between the excitonic chiroptical responses and the conformational and mechanical states of DNA self-assemblies irrespective of fluorescence background interference. Besides, the excitonic chiroptical effect allowed sensitive monitoring of DNA self-assembled nanostructures due to small molecule bindings or DNA strand displacement reactions. Moreover, we developed a new chiroptical reporter, a DNA-templated dimer of an achiral cyanine5 and an intrinsically chiral BODIPY, that exhibited unique multiple-split spectral line shape of exciton-coupled circular dichroism, largely separated response wavelengths, and enhanced anisotropy dissymmetry factor (g-factor). These results shed light on a promising chiroptical spectroscopic tool for studying biomolecular recognition and binding, conformation dynamics, and soft mechanics in general.
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Nanoestructuras , Nanoestructuras/química , ADN/química , Conformación Molecular , Dicroismo CircularRESUMEN
Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis.
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Cilastatina/metabolismo , Cisplatino/efectos adversos , Riñón/efectos de los fármacos , Riñón/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Imagen Molecular , Animales , Citoprotección/efectos de los fármacos , Femenino , Riñón/diagnóstico por imagen , Peroxidación de Lípido/efectos de los fármacos , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The quest for internal standards useful in MALDI imaging studies goes on to get not only lateral distribution but also reliable relative quantitative information. We developed a method based on application of matrix and dual internal standards to allow intra- and intersample normalization of lipids intensities in kidney sections of control and cisplatin-treated Wistar rats. An inkjet printer was used to deposit a custom-prepared ink with DHB as MALDI matrix, a primary lipids-based internal standard, and a spiked lanthanide as a secondary internal standard. We applied different laser energy and varied the amounts of matrix-internal standards mixture to evaluate the normalization potential of the internal standards. Successful correction of intensity artifacts caused by instrumental drifts was possible, but not those resulting from uneven matrix application. ICP-MS absolute quantification of the lanthanide in the printed layer ensured the reproducibility of the matrix and internal standards application with RSD of 10-15%. Internal standard-normalized data allowed intrasample modification of the MALDI image to make it compatible with the optical image. Normalization to internal standards corrected a 2-fold difference in lipids intensity, which allowed a meaningful comparison of tissue lipids in control and cisplatin-treated kidneys. More importantly, normalization of lipid relative abundances based on the same adduct type (H+, Na+, and K+) for analyte and internal standard corrected for different ionization efficiencies showing a realistic signal level and enabling reliable comparison of different samples on relative quantitative basis.
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Riñón/química , Lípidos/análisis , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Tulio/química , Animales , Femenino , Ratas Wistar , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Summary: Given the type of pathology and age of patients in the inpatient unit of Neurology, Hospital de Cabueñes (Gijon), acute confusional syndrome (ACS) is very common in this service. Objective: To determine the influence of the family, within the limits of the hospital, in patients with neurological disease suffering from SCA. Methodology: A prospective, observational, descriptive study of 53 patients with neurological disease that suffered from SCA was performed. The study variables were: disorientation, agitation, medication, effectiveness, and body grip on each shift with and without family. All those patients suffering from any dementia or psychiatric problem before entering were discarded. Results: The mean age of the patients was 83 years old. The most common pathology was ICTUS. The patients with family were 47.4% versus 52.6% without family in three shifts. 70.3% of the patients became disoriented with family; for patients without family the percentage increased up to 83.8%. As far as nervousness is concerned, 11.4% of the patients with family experienced this behaviour in the three shifts; no-family patients suffering from nervousness were 29.4%. Medication was administered to 33% of the patients with family, and to 44% of no-family patients. The effectiveness was 66.5% for patients with family and 48% for no-family patients. The use of body grip in the three shifts was 7.6% for patients with family and 64.5% for no-family patients. Conclusions: Family has a beneficial effect on neurological patients with ACS, specially by reducing the use of mechanical restraint, helping to preserve the dignity of elderly patients in an adverse environment such as a hospital.
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Confusión/epidemiología , Confusión/etiología , Familia , Enfermedades del Sistema Nervioso/complicaciones , Enfermedad Aguda , Anciano de 80 o más Años , Confusión/psicología , Femenino , Hospitalización , Humanos , Masculino , Enfermedades del Sistema Nervioso/psicología , Estudios Prospectivos , SíndromeRESUMEN
The study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts. In this work, an internal standardization method based on printing an Ir-spiked ink onto the surface of the sample has been developed to evaluate the different distributions and accumulation levels of the aforementioned drugs along the kidney of a rat model. A conventional ink-jet printer was used to print fresh sagittal kidney tissue slices of 4 µm. A reproducible and homogenous deposition of the ink along the tissue was observed. The ink was partially absorbed on top of the tissue. Thus, this approach provides a pseudo-internal standardization, due to the fact that the ablation sample and internal standard take place subsequently and not simultaneously. A satisfactory normalization of LA-ICP-MS bioimages and therefore a reliable comparison of the kidney treated with different Pt-based drugs were achieved even for tissues analyzed on different days. Due to the complete ablation of the sample, the transport of the ablated internal standard and tissue to the inductively coupled plasma-mass spectrometry (ICP-MS) is practically taking place at the same time. Pt accumulation in the kidney was observed in accordance to the dosages administered for each drug. Although the accumulation rate of cisplatin and oxaliplatin is high in both cases, their Pt distributions differ. The strong nephrotoxicity observed for cisplatin and the absence of such side effect in the case of oxaliplatin could explain these distribution differences. The homogeneous distribution of oxaliplatin in the cortical and medullar areas could be related with its higher affinity for cellular transporters such as MATE2-k.
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Carboplatino/toxicidad , Cisplatino/toxicidad , Tinta , Riñón/efectos de los fármacos , Compuestos Organoplatinos/toxicidad , Impresión , Animales , Carboplatino/metabolismo , Cisplatino/metabolismo , Riñón/metabolismo , Compuestos Organoplatinos/metabolismo , Oxaliplatino , Ratas , Estándares de ReferenciaRESUMEN
BACKGROUND: Evidence for the effectiveness of linezolid in neurosurgical infections (NSIs) is growing. The comfortable oral dosage and tolerance of linezolid opens the possibility for sequential antimicrobial treatment (SAT) in stable patients after a period of intravenous treatment. METHODS: To evaluate the efficacy and safety of SAT with oral linezolid in patients with NSI and to analyse the cost implications, an observational, non-comparative, prospective cohort study was conducted on clinically stable consecutive adult patients at the Neurosurgical Service. Following intravenous treatment, patients were discharged with SAT with oral linezolid. RESULTS: A total of 77 patients were included. The most common NSIs were: 41 surgical wound infections, 20 subdural empyemas, 18 epidural abscesses, and 16 brain abscesses. Forty-four percent of patients presented two or more concomitant NSIs. Aetiological agents commonly isolated were: Propionibacterium acnes (36 %), Staphylococcus aureus (23 %), Staphylococcus epidermidis (21 %) and Streptococcus spp. (13 %). The median duration of the SAT was 15 days (range, 3-42). The SAT was interrupted in five cases due to adverse events. The remainder of the patients were cured at the end of the SAT. A total of 1,163 days of hospitalisation were saved. An overall cost reduction of 516,188 was attributed to the SAT. Eight patients with device infections did not require removal of the device, with an additional cost reduction of 190,595. The mean cost saving per patient was 9,179. CONCLUSIONS: SAT with linezolid was safe and effective for the treatment of NSI. SAT reduces hospitalisation times, which means significant savings of health and economic resources.
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Antibacterianos/efectos adversos , Costos y Análisis de Costo , Linezolid/efectos adversos , Procedimientos Neuroquirúrgicos/efectos adversos , Infecciones Estafilocócicas/prevención & control , Infección de la Herida Quirúrgica/prevención & control , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/administración & dosificación , Antibacterianos/economía , Femenino , Humanos , Linezolid/administración & dosificación , Linezolid/economía , Masculino , Persona de Mediana Edad , Procedimientos Neuroquirúrgicos/métodos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/etiología , Infección de la Herida Quirúrgica/tratamiento farmacológicoRESUMEN
Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS.
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A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin-, oxaliplatin-, and carboplatin-protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum-peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLC-MS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum-peptides from cisplatin-HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodology.
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Carboplatino/química , Compuestos Organoplatinos/química , Platino (Metal)/química , Proteínas/química , Espectrometría de Masas en Tándem/métodos , Antineoplásicos/sangre , Antineoplásicos/química , Carboplatino/sangre , Cisplatino/sangre , Cisplatino/química , Humanos , Masculino , Compuestos Organoplatinos/sangre , Oxaliplatino , Péptidos/sangre , Péptidos/química , Platino (Metal)/sangre , Unión ProteicaRESUMEN
A study was performed in order to understand the development of digestive enzymes during initial ontogeny of Cichlasoma trimaculatum, for which the activity of acidic and alkaline proteases, lipases, amylases and phosphatases was determined by means of biochemical and electrophoretic analysis. Our results showed that the activity of alkaline proteases, trypsin and chymotrypsin is present from day 6 after hatching (dah) during exogenous feeding with Artemia nauplii. The activities of carboxypeptidase A and leucine aminopeptidase are present from the first days, increasing at 6 dah and reaching their maximum activity at 9 dah while acid protease activity started at 9 dah. Furthermore, the lipase activity is detected on 6 dah and keeps increasing and decreasing on 17 dah. Amylase activity is detected on 3 dah, presenting fluctuations until 45 dah, where it reaches its maximum activity. Acid and alkaline phosphatases are detected from 3 dah and reach a maximum activity between 13 and 19 dah. The SDS-PAGE electrophoresis revealed six types of bands in the alkaline proteases, with molecular weight between 113.4 and 20.4 kDa. First three bands appear on 6 dah, but it is until 11 dah when all isoforms appear. Based on these results, it is considered that this species completes its digestive enzymatic machinery from day 9 after hatching, therefore is recommended to perform the transition from live feed to inert feed at 15 dah.
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Acuicultura/métodos , Cíclidos/crecimiento & desarrollo , Sistema Digestivo/enzimología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Factores de Edad , Amilasas/metabolismo , Animales , Carboxipeptidasas A/metabolismo , Quimotripsina/metabolismo , Cíclidos/metabolismo , Sistema Digestivo/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Leucil Aminopeptidasa/metabolismo , Lipasa/metabolismo , Péptido Hidrolasas/metabolismo , Tripsina/metabolismoRESUMEN
The elemental distribution of self-organized In-rich In(x)Ga1-xN nanowires grown by plasma-assisted molecular beam epitaxy has been investigated using three different techniques with spatial resolution on the nanoscale. Two-dimensional images and elemental profiles of single nanowires obtained by x-ray fluorescence and energy-dispersive x-ray spectroscopy, respectively, have revealed a radial gradient in the alloy composition of each individual nanowire. The spectral selectivity of resonant Raman scattering has been used to enhance the signal from very small volumes with different elemental composition within single nanowires. The combination of the three techniques has provided sufficient sensitivity and spatial resolution to prove the spontaneous formation of a coreshell nanowire and to quantify the thicknesses and alloy compositions of the core and shell regions. A theoretical model based on continuum elastic theory has been used to estimate the strain fields present in such inhomogeneous nanowires. These results suggest new strategies for achieving high quality nonpolar heterostructures.
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In this work, we aimed to evaluate human intake of triclosan (TCS) associated with real-life use of different brands of Microban™ microwave-safe food packaging. Calculations were based on: TCS migration data (under the worst-case foreseeable conditions), MPs abundance and TCS bioaccessibility from microplastics (MPs), leached from containers under microwave heating. Bioaccessibility studies were performed with in vitro digestion of MPs, followed by liquid-liquid extraction of TCS from digestive fluids and LC-QqQ-MS analysis yielding values of 46 ± 9%. The estimated weekly intake (EWI) of TCS ranged between 11 and 42 µg/kg body weight/week, with migration being the largest contribution (0.6-2.3 mg/week), compared to leaching of MPs (75-300 µg/week). These values represent a significant source of human exposure to TCS, emphasizing the need to harmonize the ban of TCS in food contact materials worldwide and improve compliance testing of food contact articles, particularly those marketed through online sales platforms.
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Embalaje de Alimentos , Polipropilenos , Triclosán , Triclosán/análisis , Triclosán/química , Embalaje de Alimentos/instrumentación , Humanos , Polipropilenos/química , Contaminación de Alimentos/análisis , Exposición Dietética/análisisRESUMEN
Acute kidney injury (AKI) is one of the most serious complications of cisplatin anticancer therapies. Cilastatin is a highly promising nephroprotective agent to eventually enter clinical use, but its biochemical mechanism is still not fully understood. We have employed an untargeted metabolomics approach based on capillary electrophoresis mass spectrometry (CE-MS) analysis of serum and urine from an in vivo rat model, to explore the metabolic pathways involved in cisplatin-induced AKI and cilastatin nephroprotection. A total of 155 and 76 identified metabolites were found to be significantly altered during cisplatin treatment in urine and serum, respectively. Most of these altered metabolites were either partially or totally recovered by cilastatin and cisplatin co-treatment. The main metabolic pathways disturbed by cisplatin during AKI involved diverse amino acids metabolism and biosynthesis, tricarboxylic acids (TCA) cycle, nicotinate and nicotinamide metabolism, among others. Cilastatin was proved to protect diverse cisplatin-altered pathways involving metabolites related to immunomodulation, inflammation, oxidative stress and amino acid metabolism in proximal tubules. However, cisplatin-altered mitochondrial metabolism (especially, the energy-producing TCA cycle) remained largely unprotected by cilastatin, suggesting an unresolved mitochondrial direct damage. Multivariate analysis allowed effective discrimination of cisplatin-induced AKI and cilastatin renoprotection based on metabolic features. A number of potential serum and urine biomarkers could also be foreseen for cisplatin-induced AKI detection and cilastatin nephroprotection.
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Lesión Renal Aguda , Cilastatina , Cisplatino , Metabolómica , Animales , Cisplatino/efectos adversos , Cisplatino/toxicidad , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/prevención & control , Metabolómica/métodos , Masculino , Cilastatina/farmacología , Ratas , Antineoplásicos , Redes y Vías Metabólicas/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: Cerebrospinal fluid leaks to the sinonasal cavities (rhinoliquorrhoea) represent a process with diagnostic and therapeutic difficulties. The endoscopic transnasal approach is the method of choice, but with many possible variants. The purpose of this paper was to make a critical review of our diagnostic and therapeutic protocol used for 11years. MATERIAL AND METHODS: We operated on 31patients. The diagnosis was based on the biochemical analysis of rhinorrhoea, CT and MRI. TREATMENT: endoscopic nasal surgery after preoperative intrathecal injection of 5% fluorescein (2cc). Closure was performed using a free overlay graft from middle turbinate mucosa. RESULTS: Two patients had meningitis as the first sign. All patients were diagnosed by biochemical analysis of rhinorrhoea. CT and MRI gave clear evidence of the leakage location. The skull base defect was always less than 1cm. Fluorescein allowed clear visualisation of the fistulous area without other instruments and produced no side effects. One patient had a postoperative frontal abscess, which evolved favourably. All fistulas were closed and there was only one recurrence at 10years, which debuted as pneumococcal meningitis. DISCUSSION AND CONCLUSIONS: Our surgical protocol, based on the use of intrathecal fluorescein and free grafting of middle turbinate mucosa overlay onto the fistulous area, achieves successful long-term results in the management of rhinoliquorrhoea secondary to small skull base defects.
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Rinorrea de Líquido Cefalorraquídeo/cirugía , Endoscopía , Mucosa Nasal/trasplante , Anciano , Rinorrea de Líquido Cefalorraquídeo/diagnóstico , Rinorrea de Líquido Cefalorraquídeo/etiología , Protocolos Clínicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trasplante/métodos , Adulto JovenRESUMEN
The evaluation of the migration of ionic silver and nanoparticulated silver (AgNPs) from antimicrobial plastic packaging to food is crucial to ensure its safety. Migration assays were performed on reusable silver-containing polypropylene (PP) food containers and a silicone baby bottle, using food simulants, under conventional or microwave heating and repeated use. The PP containers released significant amounts of silver, increasing with temperature, contact time, acidity and lower crystallinity. Silver migration in the silicone bottle was much lower. Risk assessment of released silver was done considering European authorities safety recommendations, with some containers far exceeding these levels. No significant AgNPs release was detected in the simulants by single particle-ICPMS. Silver-containing microplastics and silicone microparticles were detected by SEM in the food simulants after the migration assays. Consumers may be continuously exposed to the harmful effects of ionic silver and microplastics, which can potentially lead to health issues.
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Nanopartículas del Metal , Plásticos , Embalaje de Alimentos , Microplásticos , Plata/análisis , Calefacción , Microondas , Nanopartículas del Metal/análisis , Antibacterianos , Polipropilenos , Medición de Riesgo , Contaminación de Alimentos/análisisRESUMEN
The COVID-19 is a "unique" stressor, which can produce physical and psychological trauma. Coping styles can buffer this psychological impact. Consequently, this paper aims to psychometrically adapt the Fear of COVID-19 scale (FCV-19S) to Spanish and examines the relationships between FCV-19S, stress response, and coping strategies. The sample comprised a convenience sample of 1146 participants (12-83 years), 880 from Spain (76.8%), and 266 from Dominican Republic (23.2%). Overall, the findings support a one-factor structure for FCV-19S, consisting of 7-items, and was invariant across age, sex, occupational status, and cross-national. Therefore, indicating evidences of construct validity. Evidences of reliability were also observed (Cronbach's α = .86, McDonald's ω = .86, Guttmann's λ6 = .86, greatest lower bound = .91, composite reliability = .85, and average variance extracted = .44). Moreover, as regards criterion-related validity, the mediation analysis indicated that the relationship between FCV-19S and acute stress was positive and high, with maladaptive coping styles mediating the relationship, and with a stronger mediation for men. The findings give evidences of the reliability and validity of the Spanish version of FCV-19S among Spanish-speaker participants, which provides the chance of cross-cultural studies.
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A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 µm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 µm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.
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Antineoplásicos/toxicidad , Cisplatino/toxicidad , Riñón/patología , Espectrometría de Masas/métodos , Animales , Cilastatina/farmacología , Cobre/química , Femenino , Corteza Renal/patología , Túbulos Renales Proximales/patología , Espectrometría de Masas/instrumentación , Platino (Metal)/análisis , Ratas , Ratas Wistar , Zinc/químicaRESUMEN
[This corrects the article DOI: 10.1007/s11469-021-00615-x.].
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A major area in cancer therapy is the search for protective strategies against cisplatin-induced nephrotoxicity. We investigated the protective effect of cilastatin on cisplatin-induced injury to renal proximal tubular cells. Cilastatin is a specific inhibitor of renal dehydrodipeptidase I (DHP-I), which prevents hydrolysis of imipenem and its accumulation in the proximal tubule. Primary cultures of proximal cells were treated with cisplatin (1-30 microM) in the presence or absence of cilastatin (200 microg/ml). Apoptosis and mitochondrial injury were assessed by different techniques. Cisplatin uptake and DNA binding were measured by inductively coupled plasma spectrometry. HeLa cells were used to control the effect of cilastatin on the tumoricidal activity of cisplatin. Cisplatin increased cell death, apoptotic-like morphology, caspase activation, and mitochondrial injury in proximal tubular cells in a dose- and time-dependent way. Concomitant treatment with cilastatin reduced cisplatin-induced changes. Cilastatin also reduced the DNA-bound platinum but did not modify cisplatin-dependent up-regulation of death receptors (Fas) or ligands (tumor necrosis factor alpha, Fas ligand). In contrast, cilastatin did not show any effects on cisplatin-treated HeLa cells. Renal DHP-I was virtually absent in HeLa cells. Cilastatin attenuates cisplatin-induced cell death in proximal tubular cells without reducing the cytotoxic activity of cisplatin in tumor cells. Our findings suggest that the affinity of cilastatin for renal dipeptidase makes this effect specific for proximal tubular cells and may be related to a reduction in intracellular drug accumulation. Therefore, cilastatin administration might represent a novel strategy in the prevention of cisplatin-induced acute renal injury.
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Antineoplásicos/toxicidad , Cilastatina/farmacología , Cisplatino/toxicidad , Dipeptidasas/antagonistas & inhibidores , Túbulos Renales Proximales/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cilastatina/metabolismo , ADN/metabolismo , Proteína Ligando Fas/biosíntesis , Proteína Ligando Fas/genética , Células HeLa , Humanos , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/enzimología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , ARN Mensajero/biosíntesis , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Receptor fas/biosíntesis , Receptor fas/genéticaRESUMEN
We performed a retrospective and observational study of 51 patients treated with tigecycline, as the treatment for nosocomial infections due to multidrug-resistant microorganisms, to evaluate the superinfection rate and their etiologies. Superinfections were diagnosed in 12 (23.5%) patients (seven due to Pseudomonas aeruginosa, 13.7%) and one patient had P. aeruginosa colonization. Five patients with superinfection died (41.6%), three due to superinfections and two to underlying diseases. The superinfection rate observed during tigecycline treatment is higher than that previously reported. Pseudomonas aeruginosa is the most frequent agent, being the cause of 58.5% of all superinfections.
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Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Infección Hospitalaria/tratamiento farmacológico , Minociclina/análogos & derivados , Sobreinfección/epidemiología , Adulto , Anciano , Bacterias/clasificación , Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/mortalidad , Infección Hospitalaria/microbiología , Infección Hospitalaria/mortalidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Minociclina/uso terapéutico , Prevalencia , Estudios Retrospectivos , Sobreinfección/microbiología , Sobreinfección/mortalidad , TigeciclinaRESUMEN
The characterization of the interaction of platinum drugs with proteins has been previously performed using bottom-up proteomics approaches (enzymatic digestion followed by MS analysis). Nevertheless, the study of the stability of the Pt-protein bonds along the whole process has been obviated for the moment. Herein the suitability of the treatments implied during enzymatic digestion of Pt-protein adducts has been evaluated, focusing on the stability of the Pt bonds. Insulin-cisplatin adducts were generated in vitro and separated from unreacted cisplatin by HPLC, the separation being checked by HPLC-ICP-MS. The chromatographically isolated Pt-insulin adducts have been proved to resist overnight digestion including treatment with Urea, DTT, IAA and trypsin in a Tris buffer. Direct analysis of the peptides generated by nESI-LIT MS allowed the determination of Pt-binding sites in insulin as: B Chain N-terminus, His5, His10, Cys7, Cys19 and A Chain Cys6, Cys7, Cys20. Results have been compared to a previous top-down approach, indicating that more complete information can be obtained with the bottom-up approach. Reactivity of free cysteines has been proved to prevail to N-donor groups, but when cysteines participate in disulfide bonds, their reactivity is comparable to N-donor sites (N-terminus, His). Preliminary results indicate that the use of High Intensity Focused Ultrasound for accelerating the enzymatic digestions is compatible with preserving Pt-protein bonds, allowing a reduction in the total digestion time to 5 min. Pt-containing peptides were fragmented and sequenced by CID, and results were compared with those obtained by the use of ETD, being CID spectra far more informative.