text
string | predicted_class
string | confidence
float16 |
|---|---|---|
In order to complement the promising in vitro results, the purpose of this study was to test, in vivo in a suitable efficacy model, the performance of the two innovative formulations, i.e., the Na-DOC gel containing 0.05% w/w CP and the nanoparticle suspension in chitosan gel (NP) containing 0.005% w/w CP. A commercial cream containing CP 0.05% w/w was used for comparison purposes. In vivo pharmacodynamic activity was evaluated using the carrageenan-induced hind paw edema test on rats . The effect of formulations on the barrier properties of the stratum corneum was determined using TEWL; histological analysis was performed to evaluate the possible induction of morphological changes.
|
study
| 100.0 |
The clobetasol propionate-loaded nanoparticles were characterized before inclusion in the chitosan gel. The average particle size obtained by dynamic light scattering (DLS) was 248 ± 15 nm with a narrow nearly monodisperse distribution (Polidispersity Index 0.1105), while the ζ potential measured by phase analysis light scattering (PALS) had a positive value, i.e., +34 ± 2 mV, in agreement with previous data . Indeed, the structure of the particles has been investigated previously: chitosan and lecithin interact electrostatically to form essentially a multilayered structure . These particles are able to load lipophilic drugs in the phospholipid bilayers and the loading can be improved by adding pharmaceutical oils such as triglycerides or fatty acid esters. In fact, isopropyl myristate (IPM) affects the structure of the multilayer chitosan/lecithin nanoparticles by intercalating the phospholipid bilayers, allowing the loading of a much higher quantity of corticosteroid .
|
study
| 100.0 |
To further confirm the presence of a rigid shell, a scanning electron microscopy (SEM) was performed. The images obtained (Figure 1) show particles with a diameter of 100–150 nm. Particles were almost perfectly spherical and despite not having being metal-coated, no sign of melting or damage from the electron beam was visible, suggesting a rigid shell surface. The discrepancy between the size results obtained by SEM and DLS could be ascribed to the fact that DLS measures a hydrodynamic diameter, based on the NP diffusion coefficient, and thus it also takes into account the hydration layer surrounding the particles .
|
study
| 100.0 |
The anti-inflammatory effect of CP-loaded NP and Na-DOC Gel after topical administration was evaluated using carrageenan-induced acute edema in a rat paw model . This model is well-established and has been previously used by numerous authors to evaluate the anti-inflammatory and anesthetic properties of topically applied semisolid formulations.
|
study
| 100.0 |
The results obtained are illustrated in Figure 2, which shows the percent increase in paw thickness as a function of time after carrageenan injection. The data obtained with Na-DOC gel and with the nanoparticles have been compared to the ones obtained using a reference commercial cream (Dermovate®, GSK, Uxbridge, UK).
|
study
| 100.0 |
The increase in paw thickness was significantly lower (p < 0.05) than for the control group at all time points for each and every formulation tested. However, while the commercial formulation and Na-DOC gel had similar efficacy, the NP formulation performed significantly (p < 0.05) better at the third and fourth hour. It is worth mentioning that this higher activity is obtained despite a 10-fold lower drug concentration in the nanoparticle-containing formulation, supporting the evidence that the formulation is more relevant than the drug concentration in the process of CP absorption .
|
study
| 100.0 |
The table includes also in vitro data obtained in previous papers using isolated pig skin . These in vitro data, obtained after 6 h of application, were characterized by: A 10-fold higher CP accumulation in the epidermis starting from the Na-DOC gel compared to NP and commercial cream;A comparable CP level in the dermis for the three formulations (see the very high data variability).
|
study
| 100.0 |
It appears that no clear correlation exists between in vitro epidermal drug levels (highest skin retention is observed for Na-DOC gel) and in vivo efficacy (highest edema inhibition is observed for NP). Furthermore, the comparable CP levels found in the dermis for the 3 formulations cannot explain the superior in vivo performance of the NP-containing formulation.
|
study
| 100.0 |
However, considering the distribution of CP between epidermis and dermis, commercial cream and NP-containing gel are characterized by deeper skin accumulation. The percentages accumulated in the dermis, in fact, are 8% for NP and 12% for cream, but less than 2% in the case of NA-DOC gel.
|
study
| 99.25 |
Skin barrier properties and possible skin irritation and skin damage induced by the formulation applied can be assessed by transepidermal water loss (TEWL) measurements and histology. In fact, it has been shown that, in rat skin, TEWL increase is directly correlated to barrier function impairment produced by irritants. In particular, sodium lauryl sulfate exposure increased TEWL approximately 8 times compared to the control value (10.3 g·m−2·h−1) . Typical basal values of TEWL obtained in the present work were in the order 11.1–12.6 g·m−2·h−1, close to the literature data reported before. The TEWL value of rat skin is typically higher than the value of human skin (5–6 g·m−2·h−1), probably due to the high density of hair follicles in rat skin .
|
study
| 100.0 |
Figure 3 shows the variation of TEWL before the application of the formulations and 5 min, 2 and 4 h after its removal (application time was 1 h). It is well known that corticosteroids increase the TEWL value as a signal of the disturbance of skin barrier function . However, for all tested formulations TEWL did increase in a non-significant way when measured 5 min post-application, then decreased gradually to reach the basal value after 4 h. The reason for the increase of TEWL after formulation removal is probably the evaporation of residues of water contained in the formulation. Overall, at least for the application time examined in this study, the commercial cream, NP and Na-DOC gel formulation produced no significant disturbance on the barrier function of stratum corneum after a single application.
|
study
| 100.0 |
Skin morphological changes, such as epidermal liquefaction, edema of collagen fibers and cell infiltration can be used as parameters to evaluate the possible damaging effects of the formulation. However, as already suggested by transepidermal water loss (TEWL) data, histological analysis of the rat skin did not reveal morphological tissue changes, at least after a single application of the formulations in this study. No cell infiltration signs were observed, and the structures of the stratum corneum, epidermis and dermis were preserved (Figure 4).
|
study
| 100.0 |
The use of nanoparticles in topical formulations is a topic of debate, especially concerning their safety and their ability to penetrate skin layers . In this study, the potential of nanoparticles for the improvement of the topical administration of glucocorticoids was demonstrated by the higher efficacy shown by a gel containing clobetasol propionate-loaded chitosan/lecithin nanoparticles in comparison to a conventional formulation and a commercial cream. In the carrageenan-induced hind paw edema test in rats, the nanoparticle formulation outperformed the other preparations despite containing a drug concentration ten times lower. Edema development after carrageenan injection has been described as a biphasic event, where the initial phase is mainly histamine- and serotonin-mediated, while prostaglandin-like substances are responsible for the second phase that is thus more sensitive to anti-inflammatory drugs .
|
study
| 99.94 |
Interestingly, the differences between the nanoparticle-containing formulation and the other formulations were evidenced in this second phase, as shown in Figure 2 where differences between the formulations appear more significant after 2 h. This may suggest a higher and faster availability of CP from the NP formulation at the site of action that, in this case, is represented by the edema where the inflammatory cells are mainly located.
|
study
| 100.0 |
However, this result appears not to be directly correlated to the in vitro accumulation of the drug in skin layers. Data reported in previous studies, in fact, indicate that the sodium deoxycholate gel is the formulation that provides the highest accumulation in the most superficial skin layers (Table 1). Reasonably, the high CP amount found in epidermis using Na-DOC gel is mainly located in the stratum corneum, and thus not available to elicit anti-inflammatory activity on the edema. This is plausible, considering the high lipophilicity of this molecule, characterized by a logP of 3.98 ± 0.61 (ACD/I-Labs, version 15.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2015). At the same time, the comparable CP levels found in the dermis for the three formulations (Table 1) cannot explain the superior in vivo performance evidenced by the formulation containing the chitosan/lecithin nanoparticles. However, these differences could be attributed to the fact that the in vitro situation is probably different from the in vivo situation, when inflammation and edema are present. In this case, in fact, the augmented capillary permeability can cause a higher CP absorption into the systemic circulation, resulting in a faster clearance from the inflammation site. In this context, nanoparticles can have a positive effect since they can accumulate inside the hair follicles, forming a drug reservoir less susceptible of fast drainage and clearance. Indeed, follicular accumulation of topically applied nanoparticles has been reported by many authors . Follicular accumulation of NP can take place in the hind paw model used here, since, contrarily to human palms and plants, which are hairless, Wistar rat foot pads contain hair follicles, although with a much lower density than normal rat skin .
|
study
| 99.94 |
In addition, recently, the boost of tamoxifen intestinal permeation after encapsulation in chitosan/lecithin nanoparticles and in presence of specific enzymes such as lysozyme and lipases has been highlighted . Lysozyme in particular is an antibacterial protein found in many physiological secretions, able to degrade chitosan and hence to biodegrade the chitosan/lecithin nanoparticle structure. Interestingly, lysozyme is among skin-produced antimicrobial peptides, and pilosebaceous follicle cells, hair bulb cells and eccrine sweat glands have been found to be positive for lysozyme . This suggests that nanoparticle accumulation in the skin annexes could contribute to explain the outstanding performance of the nanoparticle formulation containing clobetasol propionate in two ways: By forming a reservoir of the drug;Through a progressive enzyme-controlled delivery of the drug from the nanometric delivery system.
|
study
| 99.94 |
Considering these two aspects, the formulation containing the nanoparticles appears to provide an optimal timing for the delivery of the drug and is probably the reason why the efficacy of this formulation is more than 10 times that of a traditional gel or commercial cream.
|
other
| 99.8 |
Clobetasol-17-propionate (CP) was a kind gift from GlaxoSmithKline (Ankara, Turkey). Lecithin (Lipoid S45) was purchased from Lipoid AG (Ludwigshafen, Germany) and chitosan (Chitoclear FG, specifications: deacetylation degree 95%, viscosity 93 cP for 1% w/v solution in 1% v/v acetic acid) for nanoparticles preparation was from Primex (Haugesund, Norway). Medium molecular weight chitosan (specifications: MW 190-310 kDa, deacetylation degree 75%–85%, viscosity 200–800 cP for 1% w/v solution in 1% v/v acetic acid) for gel preparation as well as λ carrageenan used for hind paw test was obtained from Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol 400 (PEG 400) and mannitol were provided from Merck (Darmstadt, Germany). Sodium deoxycholate (Na-DOC) was purchased from Fluka (Munich, Germany). All other chemicals were of analytical grade.
|
other
| 99.9 |
Briefly, an ethanol solution of lecithin 2.5% (w/v) containing 0.625% w/v of CP and 2% w/v of isopropyl myristate (IPM) was prepared. Nanoparticles were obtained by rapidly injecting 4 mL of the lecithin ethanol solution through a glass pipette (internal diameter 0.75 mm, injection rate 40 mL/min) under mechanic stirring (Ultraturrax TP 18/10-10N, IKA Werke, Staufen, Germany), into 46 mL of a chitosan solution obtained by diluting with distilled water 0.5 mL of 1% (w/v) chitosan solution in 0.275 N HCl. In the colloidal suspension, lecithin/chitosan ratio was 20:1 (w/w) and the final CP content was 0.050% w/v.
|
study
| 100.0 |
In the case of particle size measurements, samples were diluted in ultrapure 0.45 µm filtered water in order to obtain an average count rate below 150 kcps to avoid multiple scattering. Measurements were performed at 25 °C allowing the sample to equilibrate for 1 min and acquiring six measurements of 60 s each for each sample. The average size and PDI were expressed as mean and standard deviation of the measures obtained for three different batches.
|
study
| 99.94 |
For surface charge measurements, samples were measured directly without dilution, allowing the instrument to automatically optimize signal intensity of the sample. Measurements were performed at 25 °C, collecting scattered light at 15° and repeated 10 times for each sample. The instrument software, applying Smoluchowski approximation, calculated the ζ potential of samples. The ζ potential values were expressed as mean and standard deviation of the measures obtained for three different batches.
|
study
| 99.94 |
Particles morphology was evaluated by scanning electron microscopy (SEM). The sample was diluted 1:10 in ultrapure water (Purelab Flex, ELGA-Veolia LabWater, Zoppola, Italy) and 20 µL were deposed on an aluminium stub and left to dry. The sample was then observed without further metallization using an Ultra High Resolution Field Emission SEM (SUPRA 40, CarlZeiss, Oberkochen, Germany) operated at an operating voltage of 1.00 kV.
|
study
| 99.94 |
The composition and method of preparation of the sodium deoxycholate gel was taken from a previous work published by Valenta and collaborators . In particular, 0.5% sodium deoxycholate (Na-DOC) was dissolved in phosphate buffer saline (0.1 M phosphate buffer pH 7.2 containing 0.9% sodium chloride) and 5% of mannitol was added. Finally, 0.05% (w/w) CP dissolved in 10% (w/w) PEG-400 was added to Na-DOC Gel with continuous stirring until uniform distribution.
|
study
| 100.0 |
A chitosan gel in water was prepared by dissolving 2 g of chitosan in 100 mL of acetic acid 1.5% w/v aqueous solution. Then, the colloidal suspension of CP-loaded nanoparticles was incorporated in the chitosan gel in 1:9 (w/w) ratio to obtain a final CP concentration of 0.005% w/w.
|
study
| 99.94 |
Male Albino Wistar rats, weighing 180–220 g, were used. They were housed in standard environmental conditions and fed with standard rodent diet with water ad libitum. The experimental protocol was approved by the Local Animal Ethical Committee of Ege University, Faculty of Pharmacy (Approval No. 2007/12-1).
|
study
| 95.44 |
To determine the anti-inflammatory activity of the formulations prepared, carrageenan-induced hind paw edema test was performed . Twenty rats were divided into four groups, one being the untreated control. Commercial cream, Na-DOC Gel and nanoparticle colloidal suspension incorporated in chitosan gel (NP) were applied to the plantar surface of the rat hind paw at a fixed CP dose of 0.125 mg/cm2 for one hour, before the induction of inflammation. In order to produce inflammation, 50 µL of a 1% (w/v) solution of λ carrageenan in saline was injected into the plantar side of the right hind paw of rats. The contralateral paw received the same volume of sterile saline. The paw thickness (indication of edema) was measured using digital micrometer (Mitutoyo Corp., Kawasaki, Japan) before carrageenan injection and then after 1, 2, 3, 4 and 5 h. The % of increase in the paw thickness was calculated as the difference between the values obtained in right and left paw expressed as percentage. The obtained values were reported as a function of time after carrageenan injection; from the graph, AUC was calculated using Kaleidagraph software (Version 4.01, Synergy Software, Reading, PA, USA). The percentage of inhibition of carrageenan-induced paw edema was then calculated according to the Equation (1) presented above: (1)%Inibition=AUCC−AUCfAUCC×100 where AUCC is the average area under the curve of the control rats (no treatment) and AUCf is the area under the curve of the rats treated with the different formulations.
|
study
| 100.0 |
Before TEWL measurements, rats were anesthetized by intraperitoneal injection of ketamine (30 mg/kg). After recording pre-treatment TEWL values, 0.5 g of formulation (Na-DOC Gel, nanoparticle colloidal suspension incorporated in chitosan Gel or commercial cream) was spread uniformly on 2 × 2 cm area of shaven rat abdomen (4 cm2) and left for 1 h. Transepidermal water loss (TEWL) measurements were obtained a Tewameter TM 300 (Courage + Khazaka electronic GmbH, Köln, Germany) applied on the skin for 1 min. TEWL values were recorded 5 min, 2 and 4 h after the removal of the formulation applied on the skin by means of a dry cotton pad. The laboratory temperature and relative humidity were kept constant in the range of 24–25 °C and 45%–50% RH, respectively.
|
study
| 100.0 |
Twenty-four hours after treatment, animals were humanely euthanized under anesthesia (urethane 1 g/kg and α-chloralose 50 mg/kg). In order to evaluate the histology of the skin at the site of application, the skin was dissected and fixed with buffered 10% formalin solution for 48 h. Tissue samples were embedded in paraffin following a routine protocol. Subsequently, sections 5 µm thick were stained with hematoxylin and eosin (H&E). The slides were examined using a light microscopy (Olympus BH-2, Tokyo, Japan) and the histopathological appearance of tissues in the different groups were compared. Possible structure changes and cell infiltration were evaluated by two analyzers blinded to the treatments.
|
study
| 99.94 |
Each experiment was replicated at least five times and the values reported are expressed as mean ± standard deviation. Statistical differences were determined using ANOVA test. Significance was determined by Bonferroni test as a post hoc test. Data were considered significant at p < 0.05.
|
study
| 99.94 |
In this study, clobetasol propionate-loaded lecithin/chitosan nanoparticles in chitosan gel demonstrated a significantly higher anti-inflammatory activity compared to a sodium deoxycholate gel and commercial cream (Dermovate) containing the same drug. This result was obtained despite the 10-fold lower (0.005% vs. 0.05%) drug concentration in the nanoparticle-enabled formulation. This result can be explained by considering the well-known capability of nanoparticles to accumulate inside the hair follicles, thus forming a reservoir that slowly releases the drug. In this latter case, the biodegradability of the nanoparticles could be the basis of a further capability of these nanocarriers to promote absorption through the skin. In fact, as a consequence of their specific composition, i.e., polysaccharides and lipids, lecithin/chitosan nanoparticles are most likely biodegraded by enzymes present in skin secretions, such as lysozyme.
|
study
| 100.0 |
Concerning the formulation tolerability, according to the results of TEWL all tested formulations did not significantly disturb the skin barrier. Furthermore, histological analysis of rat abdominal skin did not show morphological tissue changes or cell infiltration signs after application of the formulations.
|
study
| 99.94 |
Finally, lecithin/chitosan nanoparticle-in-gel formulation showed high skin tolerability with significantly improved efficacy over a classic gel formulation and marketed product. This, along with the use of a ten times lower dose, may represent remarkable progress to reduce the dose-dependent side effects and to increase the risk-benefit ratio of clobetasol propionate.
|
study
| 99.9 |
Hearing loss is a sensory impairment caused by genetic and environmental factors (Dror and Avraham, 2009; Roth et al., 2011), whose incidence is increasing according to reports of the World Health Organization. Factors such as noise and ototoxic drugs are well-known effectors of this impairment, but more recently several epidemiological studies have shown the association between the nutritional status and hearing loss. Precisely, inadequate levels of folic acid have been correlated with hearing loss in the presence of reduced vitamin B12 concentrations or hyperhomocysteinemia (Houston et al., 1999; Cadoni et al., 2004; Lasisi et al., 2010). Additional reports have explored the putative association of mutations in genes of the folate cycle and the impact of hearing loss, their results being somewhat inconsistent (Durga et al., 2006; Uchida et al., 2011). In contrast, two animal studies have demonstrated the link between homocysteine (Hcy) metabolism and hearing loss (Cohen-Salmon et al., 2007; Martinez-Vega et al., 2015a) and the effects of a folate-deficient (FD) diet in the auditory system (Martinez-Vega et al., 2015a). Thus, remarkable increases in auditory brainstem response (ABR) thresholds were detected in C57BL/6J mice after 2-months on a FD diet (Martinez-Vega et al., 2015a). These changes correlated with the presence of severe histological damage in the cochlea and with important alterations in cochlear metabolism. Folate deficiency induced a decrease in Hcy remethylation and its flux through trans-sulfuration, together with an increase in adenosine elimination, as deduced from the important changes observed in the enzymes involved. Altogether these changes led to cochlear accumulation of Hcy, resulting in enhanced protein homocysteinylation, and in redox stress in FD cochleae (Martinez-Vega et al., 2015a). C57BL/6J is a strain known to develop premature hearing loss, a phenotypic trait that was accelerated in FD mice. Therefore, we set up a new experiment using the CBA/Ca mouse strain, less prone to hearing loss, to further evaluate the impact of folate deficiency in the development of this impairment.
|
study
| 99.94 |
Two month-old CBA/Ca female mice were purchased from Harlan Interfauna Ibérica S.A. and housed under standard conditions. Mice were randomly divided into two experimental groups (n = 10 each) that were fed the A04/A04C/R04 diet (Panlab/SAFE) containing standard folate levels (normal folate 2 mg/kg; NF) or a FD diet (folic acid ≤0.1–0.2 mg/kg, Harlan Tecklad TD.95247) ad libitum for 8 months. Weight gain was measured weekly for both dietary regimes. All experiments were approved by the CSIC Bioethics Committee and carried out in full accordance with the guidelines of the European Community (2010/63/EU) and the Spanish regulations (RD 53/2013).
|
study
| 99.94 |
Blood samples were collected through the external maxillary vein (2-months of age; 2M) or by cardiac puncture after CO2 asphyxiation (10-months of age; 10M), and directly placed in either regular or heparin coated tubes (Laboratorios Farmacéuticos Rovi). Isolation of serum and plasma fractions was performed after centrifugation at 2500× g for 10 min. The hematological analysis was performed with Abacus Junior Vet 5 automatic equipment (Diatron). Fresh blood (2/mice) extensions were prepared for Wright staining (Reagan et al., 2011).
|
study
| 99.94 |
For histological analysis mice were injected a pentobarbital overdose and perfused with PBS/paraformaldehyde as previously described (Camarero et al., 2001; Sanchez-Calderon et al., 2010), before tissue extraction (cochlea and femur; Rodriguez-de la Rosa et al., 2014). Decalcified samples were dehydrated and embedded in paraffin (Panreac Química), as previously described (Aburto et al., 2012). Cochlear and bone marrow cytoarchitecture was evaluated using representative paraffin sections (7 μm thick) and Nissl or hematoxylin-eosin staining (Reagan et al., 2011).
|
study
| 100.0 |
Mice were anesthetized with a mixture of ketamine (100 mg/kg; Imalgene 1000, Merial) and xylazine (10 mg/kg; Rompun 2%, Bayer) for ABR analysis using a Tucker Davis Technologies workstation. The electrical responses to broadband click and 8, 16, 20, 28 and 40 kHz pure tone stimuli, with an intensity range 90–20 dB SPL in 5–10 dB steps, were recorded as previously reported (Cediel et al., 2006; Murillo-Cuesta et al., 2012). The electrical responses were amplified and averaged to determine hearing thresholds for each stimulus. Peak and interpeak latencies were analyzed at 15–20 dB SPL above hearing threshold after click stimulation. Recording of distortion product otoacoustic emissions (DPOAEs) was performed after stimulation with f1 and f2 primary tones, with a ratio f2/f1 = 1.2 using a TDT equipment, as described previously (Martinez-Vega et al., 2015b). Primary tones for 8, 10, 14, 18 and 22 kHz frequencies were tested.
|
study
| 100.0 |
Total Hcy (tHcy) was derivatized using the Reagent kit for HPLC analysis of Hcy in plasma/serum (Chromsystems Instruments and Chemicals GmbH, Munich, Germany). The resulting samples (50 μl) were then injected into the HPLC column and fluorescence measured at 515 nm upon excitation at 385 nm.
|
study
| 99.94 |
Statistical analysis was carried out between NF and FD groups at the specified time points using the Student’s t-test for unpaired samples was performed with SPSS v 19.0 software package (SPSS, Chicago, IL, USA). No statistical evaluation was performed within each dietary group during the whole experiment.
|
study
| 99.94 |
The CBA/Ca mouse strain, known to present with late-onset of hearing loss (18-months onward; Li and Borg, 1991; Zheng et al., 1999), was chosen to evaluate the impact of a FD diet during a long-term study carried out for 8 months. No statistical differences in the daily food intake were found between NF and FD groups during the experiment. However, animals in the FD group gained less weight than those on the NF diet, this difference becoming significant at 9–10 months of age (Figure 1A). Nevertheless, both daily ingestion and weight gain remained above normal values for this strain1.
|
study
| 100.0 |
(A) Weight gain along the study. The size of each group is specified in Table 2. Several mice from the folate-deficient (FD) group were sacrificed due to their condition, and hence the group size was reduced (n = 5) from 7 months of age onward. (B) Serum folate concentrations were measured at 2-months of age (2M) in the normal folate (NF; n = 9) and FD (n = 5) groups. At 10-months (10M) of age serum folate and homocysteine (Hcy) concentrations were determined in the NF (n = 9) and FD groups (n = 5). (C) Hematoxylin-eosin staining of bone marrow (a–d) and Wright staining of peripheral blood (f–i) at 10M (n = 5 for both dietary groups). Megakaryocytes (yellow arrows), scheme of normal erythrocytes and white series (e), anisocytosis and Howell-Jolly bodies (f, arrows). Bar scale 50 μm (a,c); 25 μm (b,d); 10 μm (remaining panels). Statistical analysis was performed between NF and FD groups at each of the specified time points; no evaluation within the same dietary groups was performed. **p < 0.01; ***p < 0.001.
|
study
| 100.0 |
Additional effects of the dietary treatment were analyzed in blood samples obtained at the start and the endpoint of the study (Figure 1B). No differences in serum folate levels between groups were detected at the beginning of the experiment (NF-2M vs. FD-2M), whereas a five-fold decrease was measured in the FD-10M group. This decrease correlated with a seven-fold increase in tHcy levels in FD-10M mice (Figure 1B), as expected for the role of folate in Hcy remethylation. Altogether, these systemic metabolic changes confirmed the efficacy of the dietary treatment.
|
study
| 100.0 |
FD-10M mice also presented decreased hemoglobin levels, packed cell volume and hematocrit values, lower red and white blood cell counts and reduced lymphocyte percentage, together with increased mean corpuscular volume, mean corpuscular hemoglobin and an enhanced degree of anisocytosis (Table 1). Their bone marrow showed a decrease in megakaryocyte number, which also had lower differentiation and volume (Figure 1C). Along with these alterations an increased number of Howell-Jolly bodies, hypersegmented polymorphonuclear neutrophils and strong anisocytosis were observed in peripheral blood extensions of FD-10M mice (Figure 1C). Altogether, these changes indicated signs of megaloblastic anemia that jointly with the presence of signs of stress in the FD-10M mice, led to the interruption of our study at 10M, despite the late-onset of HL described for these mice.
|
study
| 100.0 |
Blood parameters were measured at the end of the study, when the mice were 10 months of age, and had been fed the normal (NF) or folate deficient (FD) diets for 8 months. Statistical analysis was performed between NF and FD groups. 1mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001.
|
study
| 100.0 |
The auditory function was evaluated every month in both dietary groups. No significant changes in ABR threshold, latencies, or amplitudes were evident between NF and FD mice along the whole study (Table 2). Both NF-10M and FD-10M groups showed a decreased amplitude of wave I, which correlated with a slight increase in hearing thresholds. Histological analysis revealed damage in the organ of Corti (OC) at the low basal turn of the cochlea, together with a slight loss in type IV fibrocytes at the spiral ligament (Spl) and accumulation of melanin granules in the stria vascularis (StV; Figure 2A). Altogether, these alterations are indicative of the initial phases of hearing loss in both dietary groups, but seem more severe in cochleae of FD mice in which the absence of outer hair cells is noticed (Figure 2Ag).
|
study
| 100.0 |
Hearing parameters were measured during the whole study in the normal (NF) and folate deficient (FD) dietary groups, although only three key points of the experiment that correspond to the start, 5 and 8 months of diet administration (2, 7 and 10 months of age) are shown. 1mean ± SEM.
|
study
| 99.94 |
Representative histological images of the Nissl-stained basal regions and distortion product otoacoustic emissions (DPOAE) results. (A) General views of the cochlear basal turn (a,f) for NF (n = 3) and FD mice (n = 3) 10-months old. Detailed views of the organ of Corti (OC; b,g), where collapse of the inner and outer pillar cells is observed in both dietary groups (#) and the presence of outer hair cells in the NF (arrows) and their absence in the FD group (***) are indicated. Additional subpanels show detailed views of the stria vascularis (StV; c,h) with melanin granules in both dietary groups (arrows); spiral ligament (Spl; d,i) and cochlear ganglion (CG; e,j). Bar scale: 150 μm (a,f); 25 μm (remaining panels). (B) DPOAE analysis at 2- (2M; NF n = 9 and FD n = 10) and 10-months (10M; NF n = 9 and FD n = 5) of age; DP thresholds (a) and 2f1-f2 amplitudes (b) are shown (mean ± SEM). *p < 0.05.
|
study
| 100.0 |
The presence of the 2f1-f2 component after pure tone stimulation was evident in both groups in DPOAE studies. However, FD-10M mice presented increased DP thresholds at all the frequencies studied as compared to NF-10M mice, the differences being significant at 8–14 kHz (Figure 2B). Concomitantly, decreased 2f1-f2 amplitude was detected in the FD-10M group, which became significant at 10 kHz (Figure 2Bb). These results suggested that the initial signs of hearing loss and the outer hair cell damage were more pronounced in the FD than in the NF group after 8 months of nutritional intervention.
|
study
| 100.0 |
The relationship between nutritional deficiencies in vitamins, including folate deficiency, and hearing loss has been explored in a variety of epidemiological studies (Houston et al., 1999; Cadoni et al., 2004; Lasisi et al., 2010; Attias et al., 2012; Karli et al., 2013). However, the data obtained have not always been consistent. This fact, led several authors to the evaluation of the putative contribution of human mutations to these disparities and, among them, the analysis of modifications in key genes of the one-carbon metabolism. Evaluation of the relationship between the polymorphism C677T in the methylenetetrahydrofolate reductase gene, a key player in folate metabolism, and hearing loss rendered contradictory results (Durga et al., 2006; Uchida et al., 2011; Fusconi et al., 2012). In contrast, animal studies have provided clues to the interplay between folate, Hcy and hearing loss, showing alterations in Hcy metabolism correlating with the onset of this impairment (Cohen-Salmon et al., 2007; Kundu et al., 2012; Martinez-Vega et al., 2015a). In this line of evidence, a previous study using the C57BL/6J mouse strain, known by its early onset of hearing loss, showed that a decrease in folate intake accelerates development of this sensory decline (Martinez-Vega et al., 2015a). A fact that correlated with a systemic decrease of the vitamin levels and concomitant hyperhomocysteinemia, as expected from the critical role of folate in Hcy remethylation (Pajares and Pérez-Sala, 2006). These systemic changes are also detected in the present study using CBA/Ca mice during long-term feeding with a folate deprived diet, following the same trend than in C57BL/6J mice after 2 months of FD diet (Martinez-Vega et al., 2015a). However, hyperhomocysteinemia was more severe in CBA/Ca mice after 8 months on the FD diet than in C57BL/6J mice after 2 months (Martinez-Vega et al., 2015a).
|
study
| 99.7 |
Folate deficiency induced premature signs of hearing loss in both strains, although their appearance is delayed more than 6 months in the CBA/Ca strain as compared to C57BL/6J mice (Martinez-Vega et al., 2015a). Moreover, the extent of the damage is also very different, since most C57BL/6J mice showed profound hearing loss after 2 months on the FD diet (Martinez-Vega et al., 2015a), whereas the changes in hearing parameters in CBA/Ca mice are small, but consistent (increased DP thresholds and 2f1-f2 amplitude) and appear after 8 months on the deficient diet. In fact, histological data corroborated cochlear damage in CBA/Ca mice that was more evident in the FD animals. Folate-induced damage seemed restricted to the low basal turn of the cochlea, thus explaining that stimulation required high frequency sounds (Müller et al., 2005). This higher resistance of the CBA/Ca cochleae against vitamin deficiency may rely in differences in the genetic background of the strains used that remain largely unknown (Willott et al., 1995).
|
study
| 99.94 |
The high resistance against FD-induced hearing loss exhibited by CBA/Ca mice, as compared to the C57BL/6J strain used in our previous work (Martinez-Vega et al., 2015a), suggested putative differences in genes involved in folate metabolism. However, the small size of the mouse cochleae and the histological procedures carried out precluded an expression study on the remaining samples. Nevertheless, a search carried out into the Mouse Genome Project database showed a number of single nucleotide polymorphisms (SNPs) on several genes of folate metabolism in CBA/J mice as compared to the reference C57BL/6J strain (Keane et al., 2011). No differences in Mtr, Bhmt, Dhfr, Tyms, Mthfd1 and Mthfd2 genomic sequences were found between both strains (Table 3). In contrast, several SNPs for Bhmt2, Folr1, Folr2, Mtrr, Mthfr, Shmt1 and Shmt2 were identified. Among them, a missense mutation on Folr1 for which the functional impact remains unknown, and those on Bhmt2, a gene whose hepatic expression levels have been proposed as a dietary-dependent factor conferring protection against drug intoxication (Liu et al., 2010). In this work, Liu et al. (2010) suggested that strains with higher hepatic BHMT2 levels/activity would be adapted for a lower dependence on folate for Hcy recycling by the use of S-methylmethionine as methyl donor. Clarification on whether this postulate is valid for the auditory system would require an extensive work on genomic sequencing and evaluation of several SNPs that is out of the scope of the present article.
|
study
| 99.94 |
The table shows results of a search for SNPs in key genes of folate metabolism of CBA/J mice carried out in The Mouse Genome Project web page (http://www.sanger.ac.uk/science/data/mouse-genomes-project), where the C57BL76J strain is used as reference. *Multiple consequences, only the main one listed. aNumber indicates position. bGene assignment differs between MGP and NCBI databases.
|
study
| 53.72 |
Despite the interest of a prolonged study to get deeper insight the interplay between Nutrition and cochlear aging, the continuity of this long-term nutritional treatment was precluded by the presence of megaloblastic anemia. This fact was described in previous studies analyzing low intakes of folate or vitamin B12 for extended periods of time (Cox et al., 1961; Bills et al., 1992), but was not evident in C57BL/6J mice after 2 months of diet (Martinez-Vega et al., 2015a). Altogether, our data support the role of folate deficiency in premature development of hearing loss, although new experiments aimed to analyze its impact in strains with delayed onset of this sensory impairment should consider beginning nutritional interventions at later stages of animal development. Moreover, studies on the putative value of folate supplementation against hearing loss are needed, as well as the analysis of the incidence of this sensory decline in human populations living in countries with mandatory folate fortification for which only preliminary data are available: http://www.cdc.gov/ncbddd/hearingloss/data.html. In this line, nutrigenomic studies may render clues about the interaction between folate deficiency and the cochlear genome.
|
study
| 99.75 |
The results presented herein reinforce the crucial role of folic acid status in the cochlea. Moreover, our data suggest that differences encountered in animal and human studies analyzing the interplay between nutritional deficiencies and hearing loss, especially in age-related hearing loss, may depend on the combinatory effects of both diet and genetic background.
|
study
| 99.94 |
RM-V carried out animal experimentation, histological evaluation and statistical analyses; RM-V and SM-C performed hearing assessment; TP and GV-M performed metabolite analysis; IV-N, GV-M and MAP conceived, designed and coordinated the study; MAP drafted the manuscript. All authors gave final approval for publication.
|
other
| 99.94 |
Autophagy plays important function in regulating cell homeostasis. Autophagy performs homeostatic ‘quality control’ functions to eliminate unfolded proteins or damaged organelles. Under conditions of high cellular stress, such as during nutrient starvation, bulk autophagy is induced to degrade intracellular components for recycling and building blocks necessary for cell survival.1 Dysfunction of autophagy machinery often results in a variety of human diseases, such as cancer.2, 3, 4, 5
|
review
| 99.75 |
In the context of cancer, the role of autophagy is controversial and resembles the mythical ‘janus face’. Autophagy plays tumor suppressive function by clearing unfolded proteins or damaged organelles, thereby preventing genome instability.6, 7, 8 Loss of autophagy genes perturbs homeostasis and potentially primes cells for tumor development. Many core autophagy genes, including BECN1, UVRAG, SH3GLB1 (Bif-1), ATG2B, ATG5, ATG9B, ATG12 and RAB7A, are frequently mutated in human cancers.9 In various murine models, depletion of essential autophagy genes promotes tumorigenesis. Heterozygous disruption of BECN1 leads to spontaneous tumor formation in mice, indicating BECN1 is a halpoinsufficient tumor suppressor,10, 11, 12 mice lacking ATG5, ATG7 or AMBRA1 show similar effect.13, 14, 15 However, autophagy is also involved in oncogenic process in some circumstances. Autophagy promotes tumor cell survival and growth within tumor microenvironment, such as hypoxia or metabolic stress. Dysfunction in autophagy pathway suppresses tumor proliferation, dissemination and metastasis.15 Inhibition of autophagy increases sensitivity of tumor cells to chemotherapeutic agents as well as radiation therapy.16, 17, 18 Deletion of FIP200, a component of the ULK1–ATG13–FIP200 complex that is required for autophagosome formation, suppresses tumor initiation and progression. These findings strongly support the role of autophagy in oncogenesis in vivo.19
|
review
| 99.8 |
MicroRNAs (miRNAs) contributes to almost all physiological and pathological processes. It is well established that miRNAs control gene expression through a post-transcriptional mechanism that involves mRNA degradation and translation repression.20, 21, 22, 23 miRNAs have crucial function in cancer; more than 50% of miRNA genes are located at chromosomal regions that include fragile sites or regions that exhibit genetic deletions or amplifications.24, 25, 26 miR-17/92 is the best-characterized oncogenic miRNA cluster.27 Human miR-17/92 gene is frequently amplified in solid tumors and several hematopoietic malignancies.28, 29 We examined the expression patterns of miR-20a, a member of the miR-17/92 cluster, from TCGA breast cancer data set. A significant upregulation of miR-20a is observed in breast cancer, especially in triple-negative breast cancer. Gene Set Enrichment Analysis (GESA) suggests that miR-20a negatively correlates with the autophagy/lysosome pathway. Studies herein show that miR-20a inhibits basal and nutrient starvation-induced autophagic flux and lysosomal proteolytic activity, increases cellular reactive oxygen species (ROS) levels and DNA damage response by downregulating several key regulators of autophagy, including BECN1, ATG16L1 and SQSTM1. Low levels of BECN1, ATG16L1 and SQSTM1 are predominantly observed in triple-negative cancers than in other subtypes. A large fraction of copy-number-altered genome and DNA mutations are also detected in breast cancer patients with elevated levels of miR-20a. Collectively, our results suggest that miR-20a-mediated dysfunction of autophagy contributes to mammary tumorigenesis by promoting genomic damage and instability.
|
study
| 99.94 |
Recent studies show that the expression levels of the oncogenic miR-17/92 cluster in triple-negative breast cancer is three times higher than other subtypes.29, 30 Here, we analyzed miR-20a expression from TCGA miRNA-seq data set; miR-20a expression was markedly higher in breast cancer tissues (Figure 1a). We identified miR-20a expression signature predictive of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor 2 (HER2). The TCGA samples were subdivided into clinically the relevant subgroups: ER negative (n=109), ER positive (n=384), PR negative (n=164), PR positive (n=328), HER2 negative (n=411) or HER2 positive (n=71) based on immunohistochemical expression of hormone receptors. A significant upregulation of miR-20a expression was observed in ER-negative or PR-negative tumors (Supplementary Figure S1). Tumors with the triple-negative immunophenotyped account for up to 15% of breast cancer. Comparison of miR-20a levels between triple-negative breast cancers (n=82) and other subtypes (n=391) revealed that miR-20a is upregulated in triple-negative subtype (Figure 1b). These results demonstrate that miR-20a might be a potential diagnostic marker to distinguish triple-negative breast cancer from other subtypes.
|
study
| 99.94 |
Autophagy/lysosome pathway plays important roles in malignant transformation and cancer progression.15 To make the connection between miR-20a and the autophagy/lysosome pathway at a molecular level, we performed Gene Set Enrichment Analysis. The expression level of miR-20a was used as a phenotype label to identify biological processes and signaling pathways associate with it. Enrichment analysis of gene expression profiles revealed that expression of certain components of the autophagy/lysosome pathways correlate with lower expression of miR-20a (Figure 1c).
|
study
| 100.0 |
To investigate the role of miR-20a in autophagy, we transfected miR-20a and GFP-LC3 into MDA-MB-231 triple-negative breast cancer cells and cultured cells in Earle's Balanced Salt Solution (EBSS) medium to induce nutrient starvation. miR-20a significantly reduced GFP-LC3 puncta formation in nutrient-starved cells. Immunofluorescence assay with anti-LC3 confirmed these results (Figure 1d and Supplementary Figure S2a). Next, we used two breast cancer cell lines MDA-MB-231 and MCF7 to examine LC3-I to LC3-II conversion. Quantitative real-time PCR showed MDA-MB-231 cells expressed much higher levels of endogenous miR-20a than MCF7 cells, but much lower amounts of miR-20a after transfection, indicating MCF7 cells have much better transfection efficiency than MDA-MB-231 cells (Supplementary Figures S3a and b). miR-20a attenuated LC3-II protein expression in both cells, especially in MCF7 cells presumably because MCF7 cells have higher levels of miR-20a after transfection (Figure 1e).
|
study
| 100.0 |
We used TargetScan, an miRNA target prediction algorithm, to discover the downstream targets of miR-20a in the autophagy pathway. Several autophagy related genes, including BECN1, ATG16L1 and SQSTM1 were identified as putative targets. To validate, we performed qPCR and immunoblotting analysis. Overexpression of miR-20a markedly reduced the transcript abundance of BECN1, ATG16L1 and SQSTM1 in both MDA-MB-231 and MCF7 cells (Figures 2a and c). Immunoblotting revealed that miR-20a suppressed BECN1, ATG16L1 and SQSTM1 protein expression in both cells (Figures 2b and d and Supplementary Figure S4). We were also interested to know whether knockdown of endogenous miR-20a would affect target genes expression. LNA-modified miR-20a inhibitor (LNA-20a) efficiently suppressed endogenous miR-20 expression (Supplementary Figure S3c). LNA-20a moderately increased the amounts of BECN1, ATG16L1 and SQSTM1 in both MDA-MB-231 and MCF7 cells (Figures 2b and d and Supplementary Figure S4).
|
study
| 100.0 |
Next, we employed luciferase assay to examine whether miR-20a directly regulates target genes expression. We cloned the wild-type (WT) 3′-UTRs of BECN1, ATG16L1 or SQSTM1 gene to a luciferase reporter. In the presence of miR-20a, the relative luciferase activities for BECN1, SQSTM1 or ATG16L1 wild-type constructs were reduced by 47%, 28% and 39%, respectively. We also disrupted the putative miR-20a paring sites in the 3′-UTRs by site-directed mutagenesis, the mutated reporters (Mut) restored miR-20a-induced inhibition of luciferase activity (Figure 2e). Because BECN1, ATG16L1 and SQSTM1 are key autophagy regulators, we propose that miR-20a may negatively regulate the autophagy/lysosome pathway.
|
study
| 100.0 |
To investigate the role of miR-20a in regulating the autophagy/lysosome pathway, we used lysosome inhibitors to evaluate autophagic flux.31, 32 Immunoblotting revealed that starvation-induced LC3-II turnover was inhibited by bafilomycin A1 (Baf A1) or hydroxychloroquine (HCQ), miR-20a inhibits basal level and starvation-induced LC3-II turnover (Figure 3a and Supplementary Figure S5). Because MCF7 cells express higher amounts of LC3-II and much less visible LC3-I, we performed more assays to precisely measure autophagic flux. SQSTM1/p62 is commonly used to monitor autophagic flux. Because we have shown that miR-20a directly targets SQSTM1, we used OPTN (Optic neuropathy-inducing protein, Optineurin), a most recently identified selective autophagy substrate, to determine autophagic flux.33 Nutrient starvation destabilized endogenous OPTN protein, overexpression of miR-20a caused OPTN accumulation, especially in nutrient-starved cells (Figure 3b). Then, we used the tandem mCherry-GFP-LC3 probe to distinguish autolysosomes (mCherry positive/GFP negative; red dots) and autophagosomes (mCherry positive/GFP positive; yellow dots).34 Nutrient starvation increased red and yellow LC3 dots per cell; miR-20a-transfected cells showed decreased number of autophgosomes and autolysosomes in nutrient-starved cells, indicating that miR-20a inhibits starvation-induced autophagic flux (Figures 3c and d).
|
study
| 100.0 |
The final stage in autophagy pathway is the maturation of autolysosomes and degradation by lysosomal hydrolases. To visualize and quantify lysosomal proteolytic activity, we employed a fluorescent probe DQ Red BSA.35 The probe was nearly undetectable in control cells, dequenching of red fluorescence was observed in cells under nutrient starvation, indicating that starvation stimulates lysosomal activity. Compared with the NC-transfected cells, starvation-induced lysosomal proteolytic activity was markedly reduced in miR-20a-transfected MDA-MB-231 and MCF7 cells. Reduction of lysosomal proteolytic activity was more pronounced in MCF7 cells because MCF7 cells had higher amounts of miR-20a after transfection (Figures 3e and f).
|
study
| 100.0 |
To explore the physiological relevance of the above findings, we inhibited endogenous miR-20a expression by LNA-20a. We observed that LNA-20a remarkably increased LC3 puncta formation under nutrient starvation (Figure 4a and Supplementary Figure S2b). Additionally, immunoblotting showed that LNA-20a enhanced the ratios of membrane-bound lipidated LC3-II in both MDA-MB-231 and MCF7 cells (Figure 4b).
|
study
| 100.0 |
Next, we determined whether knockdown of endogenous miR-20a would affect autophagic flux and lysosomal proteolytic activity. In comparison, LNA-20a-transfected cells showed measurable lower levels of OPTN in control and starved cells (Figure 4c). LNA-20a also increased the numbers of autophagosomes and autolysosomes (Figure 4d and Supplementary Figure S6). We used DQ Red BSA to measure proteolytic activity; LNA-20a-transfected cells showed higher levels of fluorescence intensity compared with LNA-NC-transfected cells (Figures 4e and f). These results demonstrate that endogenous miR-20a negatively regulates autophagic flux and lysosomal proteolytic activity.
|
study
| 100.0 |
Previous studies have shown that cancer cells with defective autophagic apparatus accumulate DNA damage and deregulated ROS.36 We exploited γH2AX, a sensitive marker of DNA damage response, for assessing DNA damage in our system. Overexpression of miR-20a caused distinct γH2AX foci formation in MCF7 cells (Figure 5a). In line with this observation, immunoblotting revealed that miR-20a markedly increased the levels of γH2AX under normal and nutrient-starved conditions, whereas knocking down of endogenous miR-20a showed the opposite effect (Figure 5b). Comet assay and quantification of the tail moment (TM) confirmed that DNA damage was much more pronounced in miR-20a-transfected cells under nutrient starvation (Figure 5c). miR-20a also increased intracellular ROS levels in control and starved cells (Figure 5d).
|
study
| 100.0 |
We examined whether knockdown of miR-20a targets phenocopy the effect of miR-20a on DNA damage and ROS. Immunoblotting revealed that depletion of ATG16L1, BECN1 or SQSTM1 moderately reduced LC3-II levels; DQ Red BSA assay showed that both siRNAs significantly reduced EBSS-induced lysosomal proteolytic activity, indicating that these siRNAs can efficiently block autophagy and lysosomal activity (Figure 5g). Knockdown of ATG16L1, BECN1 or SQSTM1 increased expression of γH2AX and intracellular ROS levels in nutrient-starved cells (Figures 5d–f). These results indicate that defective autophagy increases ROS and activates the DNA damage response.7
|
study
| 100.0 |
We also observed whether re-introduction of exogenous BECN1, SQSTM1 or ATG16L1 could provide resistance to miR-20a-mediated autophagy inhibition and prevents DNA damage. Both immunoblotting and DQ Red BSA fluorescence assay showed that overexpression of BECN1, SQSTM1 or ATG16L1 construct was sufficient to rescue miR-20a-induced inhibition of autophagy and lysosomal proteolytic pathway (Figures 5h and j). Additionally, miR-20a-induced γH2AX accumulation was markedly reduced by exogenous BECN1, SQSTM1 or ATG16L1 in nutrient-starved cells (Figures 5h and i).
|
study
| 100.0 |
Evidence from the above-mentioned in vitro experiments indicates that miR-20a suppresses autophagy pathway, induces ROS and DNA damage response. To further define the clinical relevance of our findings, we examined miR-20a expression in normal mammary tissues (n=30) and breast cancer tissues (n=30) by in situ hybridization. Consistent with the TCGA miRNA-seq data set analysis, miR-20a expression was markedly higher in tumor tissues (Figures 6a and b). Immunohistochemical staining of consecutive sections of triple-negative breast cancer specimens revealed that tumor tissues expressed lower levels of BECN1, ATG16L1 and SQSTM1 than normal tissues (Figure 6c). We also performed immunoblotting to explore the expression of BECN1, ATG16L1 and SQSTM1 in tissues from triple-negative subtype. Compared with the normal mammary tissues, breast cancer tissues showed an obvious reduction in BECN1, ATG16L1 and SQSTM1 levels, whereas OPTN expression was higher in cancer tissues (Figure 6d).
|
study
| 100.0 |
We analyzed mRNA expression profiles from TCGA breast cancer cohort (n=500). miR-20a expression correlates negatively with that of BECN1, ATG16L1 or SQSTM1 in breast cancer (Figure 7a). The mRNA expression patterns of BECN1, ATG16L1 or SQSTM1 were also compared across different breast cancer subtypes. In TCGA data set, the gene expression of BECN1, ATG16L1 or SQSTM1 were markedly lower in triple-negative breast cancers (n=82) compared with other subtypes (n=391) (Figure 7b).
|
study
| 100.0 |
Immunohistochemistry showed low levels of miR-20a targets (BECN1, ATG16L1 and SQSTM1) in tumor tissues. However, γH2AX foci formation was dramatically increased in tumor tissues, whereas the staining of γH2AX in normal tissues was nearly undetectable (Figure 6c). In TCGA data set, breast cancer patients with higher levels of miR-20a show higher frequency of copy-number alterations and DNA mutations than cancer patients with lower miR-20a expression (Figure 8a), indicating that miR-20a promotes DNA damage response and increases genomic instability.
|
study
| 100.0 |
To investigate the role of miR-20a in tumorigenesis, we generated MDA-MB-231 cell lines stably expressing NC or miR-20a through the lentivirus expression system. Stable transfection of miR-20a markedly suppressed mRNA and protein expression of ATG16L1, SQSTM1 and BECN1 in MDA-MB-231 cells. miR-20a stable transfection also led to reduced LC3-II level and increased OPTN expression, indicating that miR-20a inhibits autophagic flux (Supplementary Figure S7). Nude mice injected with the miR-20a-expressing cells produced more tumors than the mice that were injected with the control cells (Figure 8b). Tumor volume was also significantly larger in mice that were injected with miR-20a-expressing cells (Figure 8c). These results suggest that miR-20a promotes tumor initiation and progression in vivo.
|
study
| 100.0 |
Several studies report that the oncogenic miR-17/92 cluster is frequently deregulated in human cancers.28, 29 Analysis of miRNA expression profiles revealed a three-fold increase in miR-17/92 expression in triple-negative breast carcinoma. However, comparison of absolute miRNA levels between normal breast tissue and tumor tissues did not show any significant differences.30 Here, we analyzed TCGA miRNA-seq data set, upregulation of miR-20a was observed in breast cancer, especially in triple-negative subtype.37
|
study
| 99.94 |
The oncogene c-MYC directly activates miR-17/92 gene expression. In addition, E2F1 and E2F3 also activate the transcription of miR-17/92 cluster, suggesting that miR-17/92 plays key roles in regulating cell proliferation.38, 39 By contrast, the tumor suppressor TP53 transcriptionally represses miR-17/92 expression, through binding to the TATA box of the miR-17/92 promoter.40 A recent study report that both two members of the miR-17/92 cluster, miR-20a and miR-17, target the TP53-activating kinase DAPK3, which leads to inactivation of TP53 and upregulation of the miR-17/92 cluster.41 Several studies demonstrate that miR-20a promotes proliferation, sustains cell survival and increases angiogenesis by targeting a number of genes, including the E2F family members.28, 42, 43 However, in some circumstances, which depends on the physiological context and the cell type, miR-20a plays tumor suppressive function.44, 45, 46 Our results show that miR-20a inhibits nutrient starvation-induced autophagic flux. miR-20a suppresses autophagy and lysosomal activity through downregulating the expression of ATG16L1, BECN1 and SQSTM1. Autophagy-deficient cells have deregulated ROS and accumulate DNA damage, which possibly contribute to genome instability and tumor-initiating capacity (Figure 8d).
|
study
| 100.0 |
Autophagy constitutes a barrier to prevent malignant transformation.15 Several lines of evidence demonstrate that BECN1 is a haploinsufficient tumor suppressor.10, 11 Monoallelic deletion of BECN1 gene is frequently observed in a high proportion of sporadic breast and ovarian carcinomas.47 Low expression of BECN1 is also found in colorectal, gastric and intracranial tumors.48 A recent study reported that BECN1 expression is reduced in triple-negative breast cancers compared with other subtypes, lower expression of BECN1 is also associated with poor prognosis.49 In line with this report, we demonstrate that miR-20a directly targets BECN1, SQSTM1 and ATG16L1. Upregulation of miR-20a strongly associates with downregulation of BECN1, SQSTM1 and ATG16L1 in human breast cancers, especially in triple-negative subtypes. Therefore, in addition to the monoallelic loss of BECN1 in breast cancer, miR-20a-mediated downregulation of autophagy pathway might be another mechanism that promotes malignant transformation.
|
study
| 100.0 |
Autophagy-deficient cells are impaired in DNA repair by the error-free process of homologous recombination. Cells lacking autophagy machinery largely depend on the non-homologous end joining, the error-prone repair process of DNA double-strand breaks. Sustained reliance on non-homologous end joining results in loss of genetic integrity.50 Interestingly, Gene Set Enrichment Analysis analysis indicate that high expression miR-20a is associated with reduction in the levels of proteins involved in DNA repair pathways (data not shown). Thus, we propose that in cancers with higher miR-20a, there is parallel inactivation of autophagy and impairment of DNA repair; this may impair the ability of the cells to adapt to environmental stress, which may subsequently enable gene amplification, mutation and accumulation of DNA damage, thereby promoting genomic instability and cancer progression. Future work should address these questions.
|
study
| 100.0 |
We downloaded TCGA breast cancer data sets from http://tcga-data.nci.nih.gov/tcga/. miRNA-seq analysis was performed from data of 694 breast cancer tissues and 83 normal tissues. RNA-seq analysis was performed from data of 500 breast cancer cohorts. The fraction of copy-number altered genome and mutation counts data were from 347 breast cancer cohorts.
|
study
| 100.0 |
Gene Set Enrichment Analysis (version 2.0) was used to investigate the biological processes that correlate with miR-20a expression in breast cancer as described in the references.51, 52 The expression levels of miR-20a were used as phenotype label, and ‘Metric for ranking genes’ was set to Pearson Correlation. All other basic and advanced fields were set to default.
|
study
| 100.0 |
MDA-MB-231 and MCF7 cells were purchased from ATCC (Mannassas, VA, USA). MCF7 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA), MDA-MB-231 cells were cultured in ATCC-formulated Leibovitz’s L-15 medium (ATCC), supplemented with 10% fetal bovine serum (PAA Laboratories, Pasching, Austria) and 10 U/ml penicillin/streptomycin (Invitrogen). Cells were cultured in Earle’s Balanced Salt Solution (EBSS; Sigma, St Louis, MO, USA) to induce nutrient starvation.
|
study
| 99.6 |
Lipofectamine 2000 (Invitrogen) was used to transfect miRNAs or siRNAs. miRNAs were purchased from GenePharma Co. Ltd (Shanghai, China). siRNA pools of SQSTM1 (sc-29679) and ATG16L1 (sc-72580) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). LNA-NC (199020-00) and LNA-20a (426943-00) were purchased from Exiqon (Vedbaek, Denmark). BECN1 siRNA is a pool of two target specific siRNA designed by GenePharm. BECN1 siRNAs: 5′-CAGUUUGGCACAAUCAAUATT-3′ and 5′-AAGAUCCUGGACCGUGUCACCTT3′ Scramble control: 5′-UUCUCCGAACGUGUCACGU-3′.
|
other
| 99.5 |
mRNA or miRNA expression was determined by quantitative reverse transcriptase–PCR analysis as previously described.53 mRNA expression was normalized to GAPDH and miRNA was normalized to U6 snoRNA. The following primer are used for PCR analysis: GAPDH forward 5′-AAGGCTGTGGGCAAGG-3′, GAPDH reverse 5′-TGGAGGAGTGGGTGTCG-3′ BECN1 forward 5′-ACCTCAGCCGAAGACTGAAG-3′, BECN1 reverse 5′-AACAGCGTTTGTAGTTCTGACA-3′ ATG16L1 forward 5′-AACGCTGTGCAGTTCAGTCC-3′, Atg16L1 reverse 5′-AGCTGCTAAGAGGTAAGATCCA-3′ SQSTM1 forward 5′-GATGAGGAAGATCGCCTTGGA-3′, SQSTM1 reverse 5′-TTCGGATTCTGGCATCTGTAGG-3′.
|
study
| 99.94 |
miR-20a and LC3 plasmids (GFP-LC3 or mCherry-GFP-LC3) were co-transfected into MDA-MB-231 or MCF7 cells. Nutrient starvation was induced by treating cells with EBSS for 4 h. Fluorescent images of GFP-LC3 or mCherry-GFP-LC3 were acquired by confocal microscope (FV1000; Olympus, Tokyo, Japan). LC3 dots per cell was calculated from 150 cells in each sample.
|
study
| 99.94 |
The 3′ UTRs fragments of human BECN1, ATG16L1 or SQSTM1 genes were cloned into pRL-TK luciferase vector (Promega, Madison, WI, USA), the resulting plasmids were called wild-type reporters (WT). To ablate the miR-20 bindings sites, we used site-directed mutagenesis kit (Takara, Kusatsu, Japan) to generate mutated vectors (Mut). MCF7 cells were transfected with luciferase vectors and miR-20a or NC using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured and normalized by protein concentration.
|
study
| 100.0 |
Cells were lysed in ice-cold whole-cell extract buffer as described previously.53 Antibodies used are LC3 (L7543, Sigma), ATG16L1 (ab106354, Abcam, Cambridge, UK), BECN1 (#3738, Cell Signaling Technology, Danvers, CA, USA), SQSTM1/p62 (610832, BD Biosciences, Franklin Lakes, NJ, USA), OPTN (A301-831A-T, Bethyl Laboratories, Montgomery, TX, USA), γH2AX (05-636, Millipore, Billerica, MA, USA), GAPDH (Proteintech, Wuhan, China), ACTB (SAB1403520, Sigma), HRP-conjugated secondary antibodies (KPL, Milford, MA, USA). Protein bands were visualized by chemiluminescence (Thermo Scientific, Rockford, IL, USA).
|
study
| 99.9 |
Cells were fixed in freshly prepared 4% paraformaldehyde solution for 15 min, washed three times with phosphate-buffered saline (PBS) and then treated with 0.25% Triton X-100/PBS for 5 min. The fixed preparations were blocked in 3% BSA/PBS for 1 h, incubated with primary antibodies for 1 h at room temperature, washed three times with PBS and incubated with Alexa Fluor 555 or Alexa Fluor 488 conjugated secondary antibody for another 1 h (Invitrogen). Fluorescent images were acquired by confocal microscopy.
|
other
| 97.9 |
MCF7 cells transfected with miRNAs, siRNAs or LNAs were treated with 10 μg/ml DQ Red BSA (D-12051, Invitrogen) for 12 h at 37 °C. Samples were cultured in normal medium or EBSS solution to induce nutrient starvation. The fluorescent images of DQ Red BSA were acquired by confocal microscopy and the fluorescence intensity was measured by Image J software (National Institute of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/).
|
study
| 99.94 |
MCF cells were transfected with NC or miR-20a, cultured in normal medium or EBSS for 24 h. Cells suspension was mixed with low melting agarose (AB0015, Life Technologies, Carlsbad, CA, USA) and plated onto comet slide to perform single-cell gel electrophoresis.54 Comet images were captured using a fluorescence microscope (Leica, Wetzlar, Germany); DNA damage was quantified by measuring the Tail Moment (TM) using CASP software (http://casplab.com).
|
study
| 99.94 |
Paraffin-embedded consecutive slides of normal tissues (n=30) and breast cancer tissues (n=30) were obtained from Shanghai Outdo Biotech Co., Ltd (Shanghai, China). For in situ detection of miR-20a, the slides were hybridized with DIG-labeled oligonucleotide (Exiqon) complementary to miR-20a. Hybridization was visualized by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color substrate (Roche, Basel, Switzerland). The staining intensity of miR-20a was determined using a visual grading system.
|
study
| 99.94 |
For immunohistochemistry, slides were deparaffinized and rehydrated, blocked in sheep serum for 30 min, and then incubated with anti-Beclin 1 (ab55878, Abcam), anti-ATG6L1 (GTX129093, GeneTex, Irvine, CA, USA), anti-SQSTM1 (PM045B, MBL, Woburn, MA, USA) or anti-γH2AX (#9718, Cell Signaling Technologies) overnight at 4°C. The slides were mounted and the images were captured and analyzed by a fluorescence microscope.
|
other
| 99.9 |
Four-week-old female BALB/c nude mice were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China) and kept in pathogen-free conditions for 1 week. The mice were randomly divided into two groups (n=12 for each group). miR-20a or NC was transfected into MDA-MB-231 cells by lentivirus infection. Stable cells were trypsinized and suspended in PBS. Each mouse was subcutaneously injected with approximately 5 × 106/0.2 ml cells at the abdominal region. The tumor size was measured using digital calipers daily for 3 weeks.
|
study
| 99.94 |
All data were expressed as mean±s.d. of three independent experiments. Measurement data between two groups were performed using nonparametric Mann–Whitney test. The correlation of miR-20a and target gene expression was examined by Spearman correlation test. Statistical analyses were performed using two-tailed Student’s t-test.
|
study
| 99.94 |
Consistent, safe, efficient, and unified critical care training is needed within graduate medical education (GME). Increasing demands on physicians, resident work hour restrictions, increased patient acuity, and varying clinical experiences within GME have led to inconsistent exposure to topics and procedures crucial to preparing for intensive care unit (ICU) rotations. This apparent inconsistency in education has led residents to report being ill-prepared for ICU rotations .
|
other
| 99.9 |
The American College of Critical Care Medicine has recommended the use of simulation to enhance resident training in critical care . Additionally, the Institute of Medicine report “To Err is Human” has recommended simulation training for physicians to reduce preventable errors . Growing literature supports the use of simulation to educate and improve knowledge, skills, and attitudes in complex communication such as medical error disclosure and death notification . Within our hospital system, residency programs use simulation in variable capacities. Importantly, no overarching simulation curriculum exists for critical care emergencies and complex communication skills; rather, residents have varied exposure through bedside experiences.
|
other
| 99.56 |
To address these challenges, two emergency medicine faculty members trained in simulation debriefing and a behavioral psychologist developed and implemented a longitudinal, multidisciplinary critical care simulation curriculum. This program aimed to enhance and unify critical care education for all interns caring for adult patients within a tertiary care hospital system. The primary goal of the curriculum was to increase confidence in common critical care topics and select procedural skills. Secondary goals included improving confidence in communication skills and obtaining a high level of learner satisfaction.
|
other
| 99.9 |
This novel simulation curriculum provided instruction on common critical care topics. Curricular design was based on a thorough needs assessment considering learner characteristics, institutional and system priorities, and time and space constraints. Curricular support was provided by a team of emergency medicine and critical care experts, ethicists, pastoral care, and experts in risk management.
|
other
| 99.9 |
Our learners were multi-specialty adult learners (internal medicine, general surgery, emergency medicine, family medicine, obstetrics/gynecology, and orthopedics) with busy clinical schedules and demands. As such, our curriculum focused on best practices in andragogy. Curricular design blended asynchronous, online, self-paced educational components with hands-on, reflective, and cooperative learning. Although learners had variable experience with simulation as an educational modality, simulation education was chosen for the curriculum due to its experiential and activating nature.
|
other
| 99.9 |
Topics and procedural skills imperative for all residents caring for ICU patients within our hospital were determined by a survey given to medical and surgical critical care colleagues and incorporated so that the curriculum would be applicable to all specialties. Included in these critical topic areas were important public health and system initiatives including the Surviving Sepsis campaign, target temperature management in postcardiac arrest patients, and expeditious use of thrombolytics in acute ischemic stroke care. Additionally, ethical and spiritual topics, such as medical error disclosure and death notification, were integrated to enhance communication skills perceived as lacking in residency education.
|
other
| 99.8 |
Each intern participated in three mandatory 4-h simulation-based sessions over a 6-month period. The curriculum began in January to avoid hospital orientation activities held in the first half of the educational year, to facilitate scheduling nearly 60 interns each year, and to allow appropriate time for interns to become comfortable with patient care requirements within their new hospital setting. Scheduling of simulation sessions was based on intern clinical duties, rotation call schedules, and resident work-hour restrictions. To avoid unexpected absenteeism, a designated scheduling coordinator reminded interns of their scheduled simulated sessions via e-mail as well as digital page the day before each session.
|
other
| 99.9 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.