RESUMEN
Expansion microscopy (EM) is an emerging approach for morphological examination of biological specimens at nanoscale resolution using conventional optical microscopy. To achieve physical separation of cell structures, tissues are embedded in a swellable polymer and expanded several fold in an isotropic manner. This work shows the development and optimization of physical tissue expansion as a new method for spatially resolved large-scale proteomics. Herein we established a novel method to enlarge the tissue section to be compatible with manual microdissection on regions of interest and MS-based proteomic analysis. A major issue in expansion microscopy is the loss of protein information during the mechanical homogenization phase due to the use of proteinase K. For isotropic expansion, different homogenization agents were investigated, both to maximize protein identification and to minimize protein diffusion. Best results were obtained with SDS for homogenization. Using our modified protocol, we were able to enlarge a tissue section more than 3-fold and identified up to 655 proteins from 1 mm in size after expansion, equivalent to 330 µm in their real size corresponding thus to an average of 260 cells. This approach can be performed easily without any expensive sampling instrument. We demonstrated the compatibility of sample preparation for expansion microscopy and proteomic study in a spatial context.
Asunto(s)
Microscopía , Proteómica , Polímeros , Proteínas , Manejo de EspecímenesRESUMEN
Although there are many types of epilepsy, temporal lobe epilepsy (TLE) is probably in humans the most common and most often studied. TLE represents 40% of the total epilepsy form of the disease and is difficult to treat. Despite a wealth of descriptive data obtained from the disease history of patients, the EEG recording, imaging techniques, and histological studies, the epileptogenic process remains poorly understood. However, it is unlikely that a single factor or a single mechanism can cause many changes associated with this neuropathological phenomenon. MALDI mass spectrometry imaging (MSI) coupled to protein identification, because of its ability to study a wide range of molecules, appears to be suitable for the preparation of molecular profiles in TLE. Seven neuropeptides have been have been identified in Dental gyrus regions of the hippocampus in relation with TLE pathology. Shot-gun studies taking into account gender influence have been performed. Tissue microextraction from control (10) toward 10 TLE patients have been analyzed after trypsin digestion followed by separation on nanoLC coupled to LTQ orbitrap. From the shot-gun analyses, results confirmed the presence of specific neuropeptides precursors and receptors in TLE patients as well as proteins involved in axons regeneration including neurotrophins, ECM proteins, cell surface proteins, membrane proteins, G-proteins, cytoskeleton proteins and tumor suppressors. Among the tumor suppressors identified, the Leucine-rich glioma inactivated 1 (LGI1) protein was found. LGI1 gene recently been demonstrated being implicated in heritability of TLE. We have also demonstrate the presence a complete profile of tumor suppressors in TLE patients, 7 have been identified. Refining this analysis taken into account the gender influence in both control and in TLE reflected the presence of specific proteins between male and female and thus mechanisms in pathology development could be completely different.
Asunto(s)
Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , Proteómica/métodos , Adulto , Giro Dentado/metabolismo , Giro Dentado/cirugía , Epilepsia del Lóbulo Temporal/cirugía , Femenino , Hipocampo/cirugía , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Proteínas/metabolismo , Caracteres Sexuales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
Amphotericin B (AmB) is the most widely used antibiotic to treat systemic fungal infections. However, the molecular mechanism of its activity is still not completely understood. In the present work we have used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes using the 2850 cm(-1) band, associated with the methylene symmetric stretching mode. The liposomes were either binary mixtures of the lipid with AmB, cholesterol or ergosterol, or ternary systems of these constituents. The two sterols contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergosterol. In the gel state, AmB does not change the conformational order of DPPC even at high concentration. In the fluid phase, however, the drug clearly structures its lipid environment. Our results show that AmB can initiate a redistribution of the ergosterol in the plane of the membrane, but not of the cholesterol molecules, which might constitute an additional mechanism to explain the activity of the antibiotic.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , Anfotericina B/química , Colesterol/química , Ergosterol/química , Membrana Dobles de Lípidos/química , Anfotericina B/farmacología , Calorimetría , Liposomas/química , Modelos Moleculares , Conformación Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: Alterations in renal function have been described with telaprevir (TVR). We examined the relationship between ribavirin (RBV) trough concentration (C), estimated glomerular filtration rate (eGFR) and severe anaemia, before and after TVR introduction in HIV-HCV-coinfected patients included in ANRS HC26 TelapreVIH study. METHODS: 69 HIV-HCV genotype-1 coinfected patients received 4 weeks of pegylated interferon (PEG-IFN)-α2a/RBV, followed by 12 weeks of TVR/PEG-IFN/RBV, then 32 to 56 weeks of PEG-IFN/RBV. RBV C was determined at week (W)4, W8 and W20/24. eGFR was estimated by the Modification of the Diet in Renal Disease (MDRD) equation. Severe anaemia was defined as haemoglobin <70 g/l, RBV dose reduction, prescription of erythropoietin or blood transfusion. RESULTS: 67 patients were analysed. eGFR remained normal between baseline (97.9 ml/min) and W4 (103.4 ml/min), declined to 86.3 ml/min at W8 (P<0.0001), stabilized until W16 and increased back to baseline level at W20 (98.4 ml/min). RBV C increased from 1.88 mg/l at W4 to 2.88 mg/l at W8 (P<0.0001), then decreased to 2.73 mg/l at week 20/24 (P=0.015). An inverse correlation was observed between W8 eGFR and W8 RBV C (r2=0.429; P=0.0005). RBV C≥3 mg/l was observed in 12% of patients at W4, 45% at W8 (P<0.0001) and 38% at W20/24 (P=0.0005). Severe anaemia was observed in 23.9% of patients at W4 and 45.3% at W8. RBV C≥3 mg/l at W8 (OR 7.7 [95% CI 2.2, 27.4]) and baseline haemoglobin <150 g/l (OR 6.4 [1.7, 23.8]) were independently associated with W8 severe anaemia. CONCLUSIONS: Association of TVR to PEG-IFN/RBV was associated with a decrease in eGFR and increase in RBV C, leading to severe anaemia in 45% of patients.