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BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with cirrhosis being a major risk factor. Traditional blood markers like alpha-fetoprotein (AFP) demonstrate limited efficacy in distinguishing between HCC and cirrhosis, underscoring the need for more effective diagnostic methodologies. In this context, extracellular vesicles (EVs) have emerged as promising candidates; however, their practical diagnostic application is restricted by the current lack of label-free methods to accurately profile their molecular content. To address this gap, our study explores the potential of mid-infrared (mid-IR) spectroscopy, both alone and in combination with plasmonic nanostructures, to detect and characterize circulating EVs. RESULTS: EVs were extracted from HCC and cirrhotic patients. Mid-IR spectroscopy in the Attenuated Total Reflection (ATR) mode was utilized to identify potential signatures for patient classification, highlighting significant changes in the Amide I-II region (1475-1700 cm-1). This signature demonstrated diagnostic performance comparable to AFP and surpassed it when the two markers were combined. Further investigations utilized a plasmonic metasurface suitable for ultrasensitive spectroscopy within this spectral range. This device consists of two sets of parallel rod-shaped gold nanoantennas (NAs); the longer NAs produced an intense near-field amplification in the Amide I-II bands, while the shorter NAs were utilized to provide a sharp reflectivity edge at 1800-2200 cm-1 for EV mass-sensing. A clinically relevant subpopulation of EVs was targeted by conjugating NAs with an antibody specific to Epithelial Cell Adhesion Molecule (EpCAM). This methodology enabled the detection of variations in the quantity of EpCAM-presenting EVs and revealed changes in the Amide I-II lineshape. SIGNIFICANCE: The presented results can positively impact the development of novel laboratory methods for the label-free characterization of EVs, based on the combination between mid-IR spectroscopy and plasmonics. Additionally, data obtained by using HCC and cirrhotic subjects as a model system, suggest that this approach could be adapted for monitoring these conditions.
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Biomarcadores de Tumor , Carcinoma Hepatocelular , Vesículas Extracelulares , Neoplasias Hepáticas , Espectrofotometría Infrarroja , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico , Espectrofotometría Infrarroja/métodos , Oro/química , Molécula de Adhesión Celular Epitelial/metabolismo , Nanopartículas del Metal/químicaRESUMEN
The pressing need for multifunctional materials in medical settings encompasses a wide array of scenarios, necessitating specific tissue functionalities. A critical challenge is the occurrence of biofouling, particularly by contamination in surgical environments, a common cause of scaffolds impairment. Beyond the imperative to avoid infections, it is also essential to integrate scaffolds with living cells to allow for tissue regeneration, mediated by cell attachment. Here, we focus on the development of a versatile material for medical applications, driven by the diverse time-definite events after scaffold implantation. We investigate the potential of incorporating graphene oxide (GO) into polycaprolactone (PCL) and create a composite for 3D printing a scaffold with time-controlled antibacterial and anti-adhesive growth properties. Indeed, the as-produced PCL-GO scaffold displays a local hydrophobic effect, which is translated into a limitation of biological entities-attachment, including a diminished adhesion of bacteriophages and a reduction of E. coli and S. aureus adhesion of â¼81% and â¼69%, respectively. Moreover, the ability to 3D print PCL-GO scaffolds with different heights enables control over cell distribution and attachment, a feature that can be also exploited for cellular confinement, i.e., for microfluidics or wound healing applications. With time, the surface wettability increases, and the scaffold can be populated by cells. Finally, the presence of GO allows for the use of infrared light for the sterilization of scaffolds and the disruption of any bacteria cell that might adhere to the more hydrophilic surface. Overall, our results showcase the potential of PCL-GO as a versatile material for medical applications.
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PURPOSE: Nearly, 40% of the causes of male infertility remain idiopathic. The only suggested treatment in idiopathic oligo- and/or asthenozoospermia in normogonadotropic patients is the FSH. In the current clinical practice, efficacy is exclusively assessable through semen analysis after 3 months of treatment. No molecular markers of treatment efficacy are appliable in clinical practice. The aim of the present work is to evaluate the combination of extracellular signal regulated kinase (ERK) 1 and 2 and prolactin inducible peptide (PIP) as potential markers of idiopathic infertility and FSH treatment efficacy. METHODS: Western blot and confocal microscopy were performed to analyze the modulation of PIP and ERK1/2 in idiopathic infertile patients (IIP) sperm cells. Taking advantage of mass spectrometry analysis, we identified these proteins unequivocally in sperm cells. RESULTS: We demonstrated a significant decrease of both PIP protein and of ERK1/2 levels in spermatozoa obtained from IIP in comparison to healthy fertile patients (HFP). Conversely, we reported a significant increase of these markers comparing infertile patients before and after 3 months of FSH treatment. Importantly, this correlated with an increase in total number of sperm and sperm motility after FSH treatment. Finally, we identified of PIP and ERK2 proteins in sperm samples by proteomic analysis. CONCLUSIONS: The combined evaluation of ERK1/2 and PIP proteins might represent a useful molecular marker to tailor FSH treatment in the management of male normogonadotropic idiopathic infertility.
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Infertilidad Masculina , Prolactina , Masculino , Humanos , Quinasas MAP Reguladas por Señal Extracelular , Proteómica , Semen , Motilidad Espermática , Espermatozoides , Infertilidad Masculina/tratamiento farmacológico , Resultado del Tratamiento , Hormona Folículo Estimulante/uso terapéuticoRESUMEN
2-deoxy-2-fluorine-(18F)fluoro-d-glucose Positron Emission Tomography/Computed Tomography (18F-FDG-PET/CT) is widely used in oncology mainly for diagnosis and staging of various cancer types, including lung cancer, which is the most common cancer worldwide. Since histopathologic subtypes of lung cancer show different degree of 18F-FDG uptake, to date there are some diagnostic limits and uncertainties, hindering an 18F-FDG-PET-driven classification of histologic subtypes of lung cancers. On the other hand, since activated macrophages, neutrophils, fibroblasts and granulation tissues also show an increased 18F-FDG activity, infectious and/or inflammatory processes and post-surgical and post-radiation changes may cause false-positive results, especially for lymph-nodes assessment. Here we propose a model-free, machine-learning based algorithm for the automated classification of adenocarcinoma, the most common type of lung cancer, and other types of tumors. Input for the algorithm are dynamic acquisitions of PET data (dPET), providing for a spatially and temporally resolved characterization of the uptake kinetic. The algorithm consists in a trained Random Forest classifier which, relying contextually on several spatial and temporal features of 18F-FDG uptake, generates as an outcome probability maps allowing to distinguish adenocarcinoma from other lung histotype and to identify metastatic lymph-nodes, ultimately increasing the specificity of the technique. Its performance, evaluated on a dPET dataset of 19 patients affected by primary lung cancer, provides a probability 0.943 ± 0.090 for the detection of adenocarcinoma. The use of this algorithm will guarantee an automatic and more accurate localization and discrimination of tumors, also providing a powerful tool for detecting at which extent tumor has spread beyond a primary tumor into lymphatic system.
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Adenocarcinoma , Neoplasias Pulmonares , Fluorodesoxiglucosa F18 , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Aprendizaje Automático , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Tomografía de Emisión de Positrones/métodos , RadiofármacosRESUMEN
In physiological conditions, red blood cells (RBCs) are capable of dramatic deformations when passing through the microvasculature. This extreme deformability is closely related to the RBC biconcave shape, to the fluidic nature of the haemoglobin and the cell membrane structure, primarily consisting of a phospholipid bilayer with an underlying two-dimensional spectrin network. In many pathological and inflammatory conditions, the shape and the extreme deformability of erythrocytes appear to be significantly altered. These findings have stimulated intense research towards the search and validation of novel erythrocyte-based mechanical biomarkers, useful for disease diagnosis and therapy monitoring. In this study, we investigated with Atomic Force Microscopy (AFM) the mechanical properties of erythrocytes obtained from a 68 years old cirrhotic man diagnosed with spur cell anaemia and cold agglutinated disease, before and after liver transplantation. Mechanical changes are compared with ultrastructural alterations as studied by scanning electron microscopy and discussed according to confocal fluorescence microscopy results, showing possible alterations induced by the cirrhotic environment at the level of the RBCs cytoskeletal organisation and lipidic composition. Taken together, the results here presented show that liver transplantation not only contributes to restoring the proper RBC morphology, but it also induces recovery of the physiological viscous behaviour of cells, further stressing the relevance of viscous and dissipative forces in determining the RBC biomechanical response.
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Membrana Celular/fisiología , Elasticidad/fisiología , Eritrocitos/fisiología , Eritrocitos/ultraestructura , Trasplante de Hígado/métodos , Anciano , Anemia/patología , Membrana Celular/ultraestructura , Humanos , Cirrosis Hepática/patología , Cirrosis Hepática/cirugía , Masculino , Microscopía de Fuerza Atómica/métodos , Microscopía Electrónica de RastreoRESUMEN
The present paper concerns a new description of changing in metabolism during incremental exercises test that permit an individually tailored program of exercises for obese subjects. We analyzed heart rate variability from RR interval time series (tachogram) with an alternative approach, the recurrence quantification analysis, that allows a description of a time series in terms of its dynamic structure and is able to identify the phase transitions. A transition in cardiac signal dynamics was detected and it perfectly reflects the aerobic threshold, as identified by gas exchange during an incremental exercise test, revealing the coupling from the respiratory system toward the heart. Moreover, our analysis shows that, in the recurrence plot of RR interval, it is possible to identify a specific pattern that allows to identify phase transitions between different dynamic regimes. The perfect match of the occurrence of the phase transitions with changes observed in the VO2 consumption, the gold standard approach to estimate thresholds, strongly supports the possibility of using our analysis of RR interval to detect metabolic threshold. In conclusion, we propose a novel nonlinear data analysis method that allows for an easy and personalized detection of thresholds both from professional and even from low-cost wearable devices, without the need of expensive gas analyzers.
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Prueba de Esfuerzo , Frecuencia Cardíaca , Obesidad , Consumo de Oxígeno , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/fisiopatologíaRESUMEN
Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways.
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Enfermedades Autoinmunes/sangre , Crioglobulinemia/sangre , Hepacivirus , Hepatitis C/sangre , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Enfermedades Reumáticas/sangre , Anciano , Enfermedades Autoinmunes/inmunología , Crioglobulinemia/inmunología , Femenino , Hepatitis C/inmunología , Hepatitis C/patología , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Masculino , Persona de Mediana Edad , Enfermedades Reumáticas/inmunologíaRESUMEN
The umbilical cord is a complex structure containing three vessels, one straight vein and two coiled arteries, encased by the Wharton Jelly (WJ) a spongy structure made of collagen and hydrated macromolecules. Fetal blood reaches the placenta through the arteries and flows back to the fetus through the vein. The role of the WJ in maintaining cord circulation proficiency and the ultimate reason for arterial coiling still lack of reasonable mechanistic interpretations. We performed biaxial tension tests and evidenced significant differences in the mechanical properties of the core and peripheral WJ. The core region, located between the arteries and the vein, resulted rather stiffer close to the fetus. Finite element modelling and optimization based inverse method were used to create 2D and 3D models of the cord and to simulate stress distribution in different hemodynamic conditions, compressive loads and arterial coiling. We recorded a facilitated stress transmission from the arteries to the vein through the soft core of periplacental WJ. This condition generates a pressure gradient that boosts the venous backflow circulation towards the fetus. Peripheral WJ allows arteries to act as pressure buffering chambers during the cardiac diastole and helps to dissipate compressive forces away from vessels. Altered WJ biomechanics may represent the structural basis of cord vulnerability in many high-risk clinical conditions.
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Fenómenos Biomecánicos , Fuerza Compresiva , Cordón Umbilical/fisiología , Gelatina de Wharton/fisiología , Adulto , Algoritmos , Anisotropía , Colágeno/fisiología , Elasticidad , Femenino , Análisis de Elementos Finitos , Análisis de Fourier , Hemodinámica , Humanos , Imagenología Tridimensional , Sustancias Macromoleculares , Placenta/fisiología , Embarazo , Presión , Estrés Mecánico , Resistencia a la Tracción , Adulto JovenRESUMEN
Graphene oxide is the hot topic in biomedical and pharmaceutical research of the current decade. However, its complex interactions with human blood components complicate the transition from the promising in vitro results to clinical settings. Even though graphene oxide is made with the same atoms as our organs, tissues and cells, its bi-dimensional nature causes unique interactions with blood proteins and biological membranes and can lead to severe effects like thrombogenicity and immune cell activation. In this review, we will describe the journey of graphene oxide after injection into the bloodstream, from the initial interactions with plasma proteins to the formation of the "biomolecular corona", and biodistribution. We will consider the link between the chemical properties of graphene oxide (and its functionalized/reduced derivatives), protein binding and in vivo response. We will also summarize data on biodistribution and toxicity in view of the current knowledge of the influence of the biomolecular corona on these processes. Our aim is to shed light on the unsolved problems regarding the graphene oxide corona to build the groundwork for the future development of drug delivery technology.
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Proteínas Sanguíneas/metabolismo , Grafito/sangre , Adsorción , Animales , Línea Celular Tumoral , Eritrocitos/efectos de los fármacos , Grafito/química , Grafito/metabolismo , Grafito/farmacocinética , Humanos , Macrófagos/efectos de los fármacos , Nanotubos/química , Unión ProteicaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for serious hospital infections worldwide and represents a global public health problem. Curcumin, the major constituent of turmeric, is effective against MRSA but only at cytotoxic concentrations or in combination with antibiotics. The major issue in curcumin-based therapies is the poor solubility of this hydrophobic compound and the cytotoxicity at high doses. In this paper, we describe the efficacy of a composite nanoparticle made of curcumin (CU) and graphene oxide (GO), hereafter GOCU, in MRSA infection treatment. GO is a nanomaterial with a large surface area and high drug-loading capacity. GO has also antibacterial properties due mainly to a mechanical cutting of the bacterial membranes. For this physical mechanism of action, microorganisms are unlikely to develop resistance against this nanomaterial. In this work, we report the capacity of GO to support and stabilize curcumin molecules in a water environment and we demonstrate the efficacy of GOCU against MRSA at a concentration below 2 µg ml-1. Further, GOCU displays low toxicity on fibroblasts cells and avoids haemolysis of red blood cells. Our results indicate that GOCU is a promising nanomaterial against antibiotic-resistant MRSA.
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BACKGROUND: Mycobacterium tuberculosis (MTB), the aetiological agent of tuberculosis (TB), is capable of interfering with the phagosome maturation pathway, by inhibiting phagosome-lysosome fusion and the autophagic process to ensure survival and replication in macrophages. Thus, it has been proposed that the modulation of autophagy may represent a therapeutic approach to reduce MTB viability by enhancing its clearance. OBJECTIVE: The aim of this study was to investigate whether transglutaminase type 2 (TG2) is involved in the pathogenesis of MTB. RESULTS: We have shown that either genetic or pharmacological inhibition of TG2 leads to a marked reduction in MTB replicative capacity. Infection of TG2 knockout mice demonstrated that TG2 is required for MTB intracellular survival in macrophages and host tissues. The same inhibitory effect can be reproduced in vitro using Z-DON, a specific inhibitor of the transamidating activity of TG2. Massive cell death observed in macrophages that properly express TG2 is hampered by the absence of the enzyme and can be largely reduced by the treatment of wild-type macrophages with the TG2 inhibitor. Our data suggest that reduced MTB replication in cells lacking TG2 is due to the impairment of LC3/autophagy homeostasis. Finally, we have shown that treatment of MTB-infected murine and human primary macrophages with cystamine, a TG2 inhibitor already tested in clinical studies, causes a reduction in intracellular colony-forming units in human macrophages similar to that achieved by the anti-TB drug capreomycin. CONCLUSION: These results suggest that inhibition of TG2 activity is a potential novel approach for the treatment of TB.
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Proteínas de Unión al GTP/metabolismo , Mycobacterium tuberculosis/patogenicidad , Transglutaminasas/metabolismo , Tuberculosis/metabolismo , Animales , Autofagia , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Macrófagos/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica de Transmisión , Proteína Glutamina Gamma Glutamiltransferasa 2 , Tuberculosis/microbiología , Tuberculosis/patologíaRESUMEN
PURPOSE: EPID-based in vivo dosimetry (IVD) has been implemented for stereotactic body radiotherapy treatments of non-small cell lung cancer to check both isocenter dose and the treatment reproducibility comparing EPID portal images. METHODS: 15 patients with lung tumors of small dimensions and treated with volumetric modulated arc therapy were enrolled for this initial experience. IVD tests supplied ratios R between in vivo reconstructed and planned isocenter doses. Moreover a γ-like analysis between daily EPID portal images and a reference one, in terms of percentage of points with γ-value smaller than 1, Pγ<1, and mean γ-values, γmean, using a local 3%-3mm criteria, was adopted to check the treatment reproducibility. Tolerance levels of 5% for R ratio, Pγ<1 higher than 90% and γmean lower than 0.67 were adopted. RESULTS: A total of 160 EPID images, two images for each therapy session, were acquired during the treatment of the 15 patients. The overall mean of the R ratios was equal to 1.005±0.014 (1 SD), with 96.9% of tests within±5%. The 2D image γ-like analysis showed an overall γmean of 0.39±0.12 with 96.1% of tests within the tolerance level, and an average Pγ<1 value equal to 96.4±3.6% with 95.4% of tests with Pγ<1>90%. Paradigmatic discrepancies were observed in three patients: a set-up error and a patient morphological change were identified thanks to CBCT image analysis whereas the third discrepancy was not fully justified. CONCLUSIONS: This procedure can provide improved patient safety as well as a first step to integrate IVD and CBCT dose recalculation.
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Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Tomografía Computarizada de Haz Cónico/métodos , Dosimetría in Vivo/métodos , Neoplasias Pulmonares/radioterapia , Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia de Intensidad Modulada/métodos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Carcinoma de Pulmón de Células no Pequeñas/patología , Tomografía Computarizada Cuatridimensional/métodos , Humanos , Pulmón/diagnóstico por imagen , Pulmón/patología , Pulmón/efectos de la radiación , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Aceleradores de Partículas , Seguridad del Paciente , Traumatismos por Radiación/prevención & control , Dosificación Radioterapéutica , Radioterapia Guiada por Imagen/métodos , Radioterapia de Intensidad Modulada/instrumentación , Respiración , Carga TumoralRESUMEN
Dystroglycan (DG) serves as an adhesion complex linking the actin cytoskeleton to the extracellular matrix. DG is encoded by a single gene as a precursor, which is constitutively cleaved to form the α- and ß-DG subunits. α-DG is a peripheral protein characterized by an extensive glycosylation that is essential to bind laminin and other extracellular matrix proteins, while ß-DG binds the cytoskeleton proteins. The functional properties of DG depend on the correct glycosylation of α-DG and on the cross-talk between the two subunits. A reduction of α-DG glycosylation has been observed in muscular dystrophy and cancer while the inhibition of the interaction between α- and ß-DG is associated to aberrant post-translational processing of the complex. Here we used confocal microscopy based techniques to get insights into the influence of α-DG glycosylation on the functional properties of the ß-DG, and its effects on cell migration. We used epithelial cells transfected with wild-type and with a mutated DG harboring the mutation T190M that has been recently associated to dystroglycanopathy. We found that α-DG hypoglycosylation, together with an increased protein instability, reduces the membrane dynamics of the ß-subunit and its clustering within the actin-rich domains, influencing cell migration and spontaneous cell movement. These results contribute to give novel insights into the involvement of aberrant glycosylation of DG in the developing of muscular dystrophy and tumor metastasis.
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Movimiento Celular , Distroglicanos/metabolismo , Seudópodos/metabolismo , Animales , Línea Celular , Distroglicanos/genética , Glicosilación , Ratones , Microscopía Confocal , Estabilidad Proteica , Seudópodos/genéticaRESUMEN
Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for ß-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and α-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.
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Ácidos Grasos/química , Fluidez de la Membrana , Humanos , Metabolismo de los LípidosRESUMEN
It is now established that the gastric pathogen Helicobacter pylori has the ability to form biofilms in vitro as well as on the human gastric mucosa. The aim of this study is to evaluate the antimicrobial effects of Clarithromycin on H. pylori biofilm and to enhance the effects of this antibiotic by combining it with Alginate Lyase, an enzyme degrading the polysaccharides present in the extracellular polymeric matrix forming the biofilm. We evaluated the Clarithromycin minimum inhibition concentration (MIC) on in vitro preformed biofilm of a H. pylori. Then the synergic effect of Clarithromycin and Alginate Lyase treatment has been quantified by using the Fractional Inhibitory Concentration index, measured by checkerboard microdilution assay. To clarify the mechanisms behind the effectiveness of this antibiofilm therapeutic combination, we used Atomic Force Microscopy to analyze modifications of bacterial morphology, percentage of bacillary or coccoid shaped bacteria cells and to quantify biofilm properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1584-1591, 2016.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Claritromicina/farmacología , Helicobacter pylori/efectos de los fármacos , Polisacárido Liasas/metabolismo , Antibacterianos/química , Claritromicina/química , Relación Dosis-Respuesta a Droga , Helicobacter pylori/metabolismo , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
By mimicking naturally occurring superhydrophobic surfaces, scientists can now realize artificial surfaces on which droplets of a few microliters of water are forced to assume an almost spherical shape and an extremely high contact angle. In recent decades, these surfaces have attracted much attention due to their technological applications for anti-wetting and self-cleaning materials. Very recently, researchers have shifted their interest to investigate whether superhydrophobic surfaces can be exploited to study biological systems. This research effort has stimulated the design and realization of new devices that allow us to actively organize, visualize and manipulate matter at both the microscale and nanoscale levels. Such precise control opens up wide applications in biomedicine, as it allows us to directly manipulate objects at the typical length scale of cells and macromolecules. This progress report focuses on recent biological and medical applications of superhydrophobicity. Particular regard is paid to those applications that involve the detection, manipulation and study of extremely small quantities of molecules, and to those that allow high throughput cell and biomaterial screening.
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Materiales Biocompatibles/química , Materiales Biomiméticos/química , Interacciones Hidrofóbicas e Hidrofílicas , Agua/química , HumectabilidadRESUMEN
In order to pass through the microcirculation, red blood cells (RBCs) need to undergo extensive deformations and to recover the original shape. This extreme deformability is altered by various pathological conditions. On the other hand, an altered RBC deformability can have major effects on blood flow and can lead to pathological implications. The study of the viscoelastic response of red blood cells to mechanical stimuli is crucial to fully understand deformability changes under pathological conditions. However, the typical erythrocyte biconcave shape hints to a complex and intrinsically heterogeneous mechanical response that must be investigated by using probes at the nanoscale level. In this work, the local viscoelastic behaviour of healthy and pathological red blood cells was probed by Atomic Force Microscopy (AFM). Our results clearly show that the RBC stiffness is not spatially homogeneous, suggesting a strong correlation with the erythrocyte biconcave shape. Moreover, our nanoscale mapping highlights the key role played by viscous forces, demonstrating that RBCs do not behave as pure elastic bodies. The fundamental role played by viscous forces is further strengthened by the comparison between healthy and pathological (diabetes mellitus) RBCs. It is well known that pathological RBCs are usually stiffer than the healthy ones. Our measures unveil a more complex scenario according to which the difference between normal and pathological red blood cells does not merely lie in their stiffness but also in a different dynamical response to external stimuli that is governed by viscous forces.
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Viscosidad Sanguínea , Diabetes Mellitus Tipo 2/sangre , Elasticidad , Eritrocitos/metabolismo , Diabetes Mellitus Tipo 2/patología , Eritrocitos/patología , Femenino , Humanos , MasculinoRESUMEN
SW480 and SW620 colon carcinoma cell lines derive from primary tumour and lymph-node metastasis of the same patient, respectively. For this reason, these cells represent an ideal system to analyse phenotypic variations associated with the metastatic process. In this study we analysed SW480 and SW620 cytoskeleton remodelling by measuring the cells' mechanics and morphological properties using different microscopic techniques. We observed that different specialized functions of cells, i.e. the capacity to metastasize of elongated cells inside the primary tumour and the ability to intravasate and resist shear forces of the stream of cells derived from lymph node metastasis, are reflected in their mechanical properties. We demonstrated that, together with stiffness and adhesion between the AFM tip and the cell surface, cell shape, actin organization and surface roughness are strictly related and are finely modulated by colorectal cancer cells to better accomplish their specific tasks in cancer growth and invasion.
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Neoplasias Colorrectales/ultraestructura , Citoesqueleto/ultraestructura , Ganglios Linfáticos/ultraestructura , Invasividad Neoplásica/ultraestructura , Línea Celular Tumoral , Forma de la Célula , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Citoesqueleto/química , Humanos , Ganglios Linfáticos/química , Metástasis Linfática/patología , Metástasis Linfática/ultraestructura , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Invasividad Neoplásica/patología , Propiedades de SuperficieRESUMEN
BACKGROUND: Despite the recognised contribution of the stroma to breast cancer development and progression, the effective targeting of the tumor microenvironment remains a challenge to be addressed. We previously reported that normal fibroblasts (NFs) and, notably, breast cancer-associated fibroblasts (CAFs) induced epithelial-to-mesenchymal transition and increases in cell membrane fluidity and migration in well- (MCF-7) and poorly-differentiated (MDA-MB-231) breast cancer cells. This study was designed to better define the role played, especially by CAFs, in promoting breast tumor cell migration. METHODS: Fibroblast/breast cancer cell co-cultures were set up to investigate the influence of NFs and CAFs on gene and protein expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, as well as on the protein level and activity of its transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), in MCF-7 and MDA-MB-231 cells. To assess the role of SREBP1 in the regulation of SCD1 expression, the desaturase levels were also determined in tumor cells treated with an SREBP1 inhibitor. Migration was evaluated by wound-healing assay in SCD1-inhibited (by small-interfering RNA (siRNA) or pharmacologically) cancer cells and the effect of CAF-conditioned medium was also assessed. To define the role of stroma-derived signals in cancer cell migration speed, cell-tracking analysis was performed in the presence of neutralising antibodies to hepatocyte growth factor, transforming growth factor-ß or basic fibroblast growth factor. RESULTS: A two to three fold increase in SCD1 mRNA and protein expression has been induced, particularly by CAFs, in the two cancer cell lines that appear to be dependent on SREBP1 activity in MCF-7 but not in MDA-MB-231 cells. Both siRNA-mediated and pharmacological inhibition of SCD1 impaired tumor cells migration, also when promoted by CAF-released soluble factors. Fibroblast-triggered increase in cancer cell migration speed was markedly reduced or abolished by neutralising the above growth factors. CONCLUSION: These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Movimiento Celular/genética , Fibroblastos/patología , Comunicación Paracrina/genética , Estearoil-CoA Desaturasa/genética , Neoplasias de la Mama/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Membrana Celular/genética , Técnicas de Cocultivo/métodos , Transición Epitelial-Mesenquimal/genética , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factor de Crecimiento de Hepatocito/genética , Humanos , Células MCF-7 , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The zona pellucida (ZP) is a specialized extracellular matrix surrounding the developing oocyte. This thick matrix consists of various types of glycoprotein that play different roles in the fertilization process. Nowadays, several techniques are available for assessing ZP's mechanical response. The basic assumption behind these methods is that the ZP behaves like an elastic body: hence, dissipative forces are neglected and Young's modulus remains unaffected by probe dynamics. However, dissipative forces are strongly regulated by the slippage of ZP chains past one another while reaction forces related to elastic deformations (driven by the ability of each chain to stretch) depend on the ZP structure (i.e. number of cross-links and distances between knots). Although viscous reaction forces generated by the ZP are one of the main factors regulating sperm transit, their peculiar behaviour along the ZP structure remains poorly understood and rarely investigated. In order to overcome this limitation, a novel visco-hyperelastic model describing the porcine ZP reaction forces generated by nanoindentations at different probe rates is developed and verified in this study. Visco-hyperelastic parameters of porcine ZP membranes are determined by means of a hybrid characterization framework combining atomic force microscopy nanoindentation measurements, nonlinear finite-element analysis and nonlinear optimization. Remarkably, it is possible to separate the contributions of hyperelastic and viscous terms to ZP mechanical response and evaluate the error made in the determination of ZP mechanical properties if viscous effects were not considered.