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By using AFM as a nanografting tool, we grafted micrometer-sized DNA platforms into inert alkanethiol SAMs. Tuning the grafting conditions (surface density of grafting lines and scan rate) allowed us to tailor the molecular density of the DNA platforms. Following the nanografting process, AFM was operated in the low perturbative Quantitative Imaging (QI) mode. The analysis of QI AFM images showed the coexistence of molecular domains of different heights, and thus different densities, within the grafted areas, which were not previously reported using contact AFM imaging. Thinner domains corresponded to low-density DNA regions characterized by loosely packed, randomly oriented DNA strands, while thicker domains corresponded to regions with more densely grafted DNA. Grafting with densely spaced and slow scans increased the size of the high-density domains, resulting in an overall increase in patch height. The structure of the grafted DNA was compared to self-assembled DNA, which was assessed through nanoshaving experiments. Exposing the DNA patches to the target sequence produced an increase in the patch height, indicating that hybridization was accomplished. The relative height increase of the DNA patches upon hybridization was higher in the case of lower density patches due to hybridization leading to a larger molecular reorganization. Low density DNA patches were therefore the most suitable for targeting oligonucleotide sequences.
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ADN , Oligonucleótidos , Microscopía de Fuerza Atómica , ADN/química , Hibridación de Ácido NucleicoRESUMEN
The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABAA receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABAA receptors, containing the α6 subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell.
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Neuropéptidos , Receptores de GABA-A , Animales , Cerebelo/fisiología , Mamíferos/metabolismo , Neuronas/fisiología , Neuropéptidos/metabolismo , Ratas , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
SARS-CoV-2 is causing an increasing number of deaths worldwide because no effective treatment is currently available. Remdesivir has shown in vitro activity against coronaviruses and is a possible antiviral treatment for SARS-CoV-2 infection. This prospective (compassionate), open-label study of remdesivir, which was conducted at Luigi Sacco Hospital, Milan, Italy, between February 23 and March 20, 2020, involved patients with SARS-CoV-2 pneumonia aged ≥18 years undergoing mechanical ventilation or with an oxygen saturation level of ≤94 % in air or a National Early Warning Score 2 of ≥4. The primary outcome was the change in clinical status based on a 7-category ordinal scale (1 = not hospitalised, resuming normal daily activities; 7 = deceased). The 35 patients enrolled from February 23 to March 20, 2020, included 18 in intensive care unit (ICU), and 17 in our infectious diseases ward (IDW). The 10-day course of remdesivir was completed by 22 patients (63 %) and discontinued by 13, of whom eight (22.8 %) discontinued because of adverse events. The median follow-up was 39 days (IQR 25-44). At day 28, 14 (82.3 %) patients from IDW were discharged, two were still hospitalized and one died (5.9 %), whereas in ICU 6 (33.3 %) were discharged, 8 (44.4 %) patients died, three (16.7 %) were still mechanically ventilated and one (5.6 %) was improved but still hospitalized. Hypertransaminasemia and acute kidney injury were the most frequent severe adverse events observed (42.8 % and 22.8 % of the cases, respectively). Our data suggest that remdesivir can benefit patients with SARS-CoV-2 pneumonia hospitalised outside ICU where clinical outcome was better and adverse events are less frequently observed. Ongoing randomised controlled trials will clarify its real efficacy and safety, who to treat, and when.
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Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Betacoronavirus , Ensayos de Uso Compasivo/estadística & datos numéricos , Infecciones por Coronavirus/tratamiento farmacológico , Hospitalización/estadística & datos numéricos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Neumonía Viral/tratamiento farmacológico , Lesión Renal Aguda/inducido químicamente , Adenosina Monofosfato/efectos adversos , Adenosina Monofosfato/uso terapéutico , Anciano , Alanina/efectos adversos , Alanina/uso terapéutico , Antivirales/efectos adversos , Antivirales/uso terapéutico , COVID-19 , Infecciones por Coronavirus/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/sangre , SARS-CoV-2 , Transaminasas/sangre , Resultado del TratamientoRESUMEN
BACKGROUND: The current effective delivered dose is a quality indicator for continuous renal replacement therapy. Its periodic assessment might enable physicians to deliver personalised treatments. Yet, its quantification as by extracorporeal urea clearance (Cl) is cumbersome and thus often neglected in routine practice. The aim of this in vitro study is to demonstrate the non-inferior effectiveness of assessing the current effective delivered dose using a simpler, cheaper and faster approach based on measurement of fluoride rather than urea extracorporeal Cl. METHODS: We compared urea and fluoride removal in 3 post-dilution continuous veno-venous haemofiltration (CVVH) and 3 continuous veno-venous haemodialysis (CVVHD) in vitro experimental models. Experiments ran for 180 min, using 3 L of human blood, heparin anticoagulation and a machine dose of 30 mL/kg/h. Urea and fluoride were measured in the inflow, outflow and effluent lines to compare sieving coefficients (SC), saturation coefficients (SA) and transmembrane Cls. RESULTS: In CVVH, the median SC values were 1.06 (1.02-1.07) and 1.02 (1.01-1.04) for fluoride and urea, respectively (discrepancy of 4.3%), while transmembrane convective Cls were 31.28 (30.01-31.31) mL/kg/h and 30.30 (29-31.85) mL/kg/h (discrepancy of 3.13%), respectively. In CVVHD, the median SA values were 1.01 (0.96-1.02) and 1 (0.95-1.01) for fluoride and urea, respectively (discrepancy of 1.6%), while transmembrane dialytic Cls were 30.26 (29.52-31.32) mL/kg/h and 31.16 (30-31.75) mL/kg/h (discrepancy of -2.97%), respectively. CONCLUSION: Fluoride transmembrane removal was close to that observed with urea, in terms of SC, SA and transmembrane Cl. Fluoride seems as much accurate as urea in assessing the current effective delivered dose during both CVVH and CVVHD and might therefore be adopted for dose measurement. Besides accuracy, fluoride bedside assessment could present many advantages over urea, particularly in terms of availability, costs, time requirement and rapidity of assessment.
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Terapia de Reemplazo Renal Continuo/normas , Fluoruros/sangre , Indicadores de Calidad de la Atención de Salud , Lesión Renal Aguda/terapia , Nitrógeno de la Urea Sanguínea , Terapia de Reemplazo Renal Continuo/métodos , Hemofiltración/métodos , Hemofiltración/normas , Humanos , Diálisis Renal/métodos , Diálisis Renal/normas , Resultado del Tratamiento , Urea/sangreRESUMEN
The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.
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Antígenos/aislamiento & purificación , Técnicas Biosensibles , Inmunoensayo/métodos , Dispositivos Laboratorio en un Chip , Anticuerpos/química , Antígenos/química , Dimetilpolisiloxanos/química , Humanos , Nanomedicina/tendenciasRESUMEN
BACKGROUND: The novel analysis of high-frequency QRS components (HF/QRS) has been proposed in patients with chest pain (CP) referred for exercise tolerance test (ex-ECG). We sought to evaluate the prognostic role of exercise high-frequency QRS-analysis (ex-HF/QRS) in patients with recent-onset stable CP, in the emergency setting. METHODS: Patients with CP underwent ex-ECG. A decrease greater than or equal to 50% of the signal of HF/QRS intensity was considered as index of ischemia as ST-segment depression greater than or equal to 2 mm or greater than or equal to 1 mm associated with CP. Exclusion criteria were QRS duration greater than or equal to 120 milliseconds and inability to exercise. Baseline characteristics were adjusted with the propensity score matching specifying nearest-neighbor matching in cardiovascular risk factors and risk scores. The primary end point was the composite of coronary stenosis greater than or equal to 70% or acute coronary syndrome, revascularization, and cardiac death on the 6-month follow-up. RESULTS: Of 589 patients, 22 achieved the end point. On the univariate analysis, known cardiovascular disease, GRACE score, and ex-HF/QRS were predictors of the end point. On the multivariate analysis, only ex-HF/QRS was predictor of the end point (odd ratio, 28; 95% confidence interval [CI], 6-120; P < .001). Overall, the ex-HF/QRS when compared to ex-ECG showed higher sensitivity (91% vs 27%; P = .02), lower specificity (74% vs 86%; P = .09), and comparable negative predictive value (99% vs 97%; P = .78). Receiver operating characteristic curve analysis showed the larger area of ex-HF/QRS (0.83; 95% CI, 0.75-0.90) over ex-ECG (0.57; CI, 0.44-0.70) and GRACE score (0.65; CI, 0.54-0.76); P < .03 on C-statistic. Women showed the largest area (0.89; CI, 0.83-0.95; P < .03) vs the other clinical data. CONCLUSIONS: In patients with CP, the novel ex-HF/QRS analysis has a valuable incremental prognostic role over ex-ECG, especially in women.
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Dolor en el Pecho/diagnóstico , Electrocardiografía , Prueba de Esfuerzo , Anciano , Biomarcadores/sangre , Femenino , Humanos , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
We present the first detailed experimental observation and analysis of nanoparticle electrophoresis through a nanochannel obtained with synchronous high-bandwidth electrical and camera recordings. Optically determined particle diffusion coefficients agree with values extracted from fitting electrical transport measurements to distributions from 1D Fokker-Planck diffusion-drift theory. This combined tracking strategy enables optical recognition and electrical characterization of nanoparticles in solution, which can have a broad range of applications in biology and materials science.
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Electroforesis/instrumentación , Dispositivos Laboratorio en un Chip , Nanopartículas/análisis , Difusión , Dimetilpolisiloxanos/química , Diseño de Equipo , Colorantes Fluorescentes/análisis , Nanotecnología/instrumentación , Óptica y Fotónica/instrumentación , Tamaño de la Partícula , Grabación en VideoRESUMEN
BACKGROUND: The relationship between troponin and atrial fibrillation (AF) without acute coronary syndrome is still unclear. We sought to investigate the presence of coronary atherosclerosis and adverse outcomes in patients with AF. METHODS: Consecutive patients with recent-onset AF and without severe comorbidities were enrolled between 2004 and 2013. Patients with a troponin rise or with adverse outcomes were considered for coronary angiography and revascularization when "critical" stenosis (≥70%) was recognized. Propensity score matching was performed to adjust for baseline characteristics; after matching, no differences existed between the groups of patients with or without troponin rise. The primary end point was the composite of acute coronary syndrome, revascularization, and cardiac death at 1- and 12-month follow-ups. RESULTS: Of 3627 patients enrolled, 3541 completed the study; 202 (6%) showed troponin rise; and 91 (3%), an adverse outcome. In the entire cohort, on multivariate analysis, the odds ratio for the occurrence of the primary end point of troponin rise was 14 (95% confidence interval [CI], 10-23; P<.001), and that of known coronary artery disease was 3 (CI, 2-5; P=.001). In the matching cohort, the odds ratio of troponin rise was 10 (CI, 4-22; P<.001), and that of TIMI score greater than 2 was 4 (CI, 2-9; P≤.001). In the entire cohort, patients with or without troponin rise achieved the primary end point in 38 (19%) and 43 (1%) patients, respectively (P<.001). Stroke occurred in 4 (2%) and 20 (1%), respectively (P=.018). Critical stenosis and revascularization account for 23 (12%) and 15 (1%), respectively (P<.001). In the matching cohort, results were confirmed, but incidence of stroke was comparable. CONCLUSIONS: Patients with recent-onset AF and troponin rise showed higher prevalence of coronary atherosclerosis and adverse cardiac events. Stroke per se did not succeed in justifying the high morbidity. Thus, beyond stroke, coronary atherosclerosis might have a pivotal role in poor outcomes.
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Fibrilación Atrial/sangre , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/diagnóstico , Revascularización Miocárdica , Troponina/sangre , Anciano , Biomarcadores/sangre , Enfermedad de la Arteria Coronaria/terapia , Electrocardiografía , Servicio de Urgencia en Hospital/estadística & datos numéricos , Femenino , Estudios de Seguimiento , Humanos , Italia , Masculino , Pronóstico , Puntaje de PropensiónRESUMEN
BACKGROUND: The novel exercise computer-assisted high-frequency QRS analysis (HF/QRS) has demonstrated improved sensitivity and specificity over the conventional ST/electrocardiogram-segment analysis (ST/ECG) in the detection of myocardial ischemia. The aim of the present study was to compare the diagnostic value of the validated exercise echocardiography (ex-Echo) with the novel exercise ECG (ex-ECG) including HF/QRS and ST/ECG analysis. METHODS: A prospective cohort study was conducted in the emergency department of a tertiary care teaching Hospital. Patients with chest pain (CP), normal resting ECGs, troponins, and echocardiography, labeled as "intermediate-risk" for adverse coronary events, underwent the novel ex-ECG and ex-Echo. An ST-segment depression of at least 2 mV or at least 1 mV when associated with CP was considered as an index of ischemia, as well as a decrease of at least 50% in HF/QRS intensity, or new wall motion abnormalities on ex-Echo. Exclusion criteria were QRS duration of at least 120 milliseconds, poor echo-acoustic window, and inability to exercise. Patients were followed up to 3 months. The end point was the composite of coronary stenoses of 50% or greater at angiography or acute coronary syndrome, revascularization, and cardiovascular death on the 3-month follow-up. RESULTS: Of 188 patients enrolled, 18 achieved the end point. The novel ex-ECG and ex-Echo showed comparable negative predictive value (97% vs 96%; P = .930); however, sensitivity was 83% vs 61%, respectively (P = .612), and specificity was 64% vs 92%, respectively,(P = .026). The areas on receiver operating characteristic analysis were comparable (ex-ECG: 0.734 [95% confidence interval, or CI, 0.62-0.85] vs ex-Echo: 0.767 [CI, 0.63-0.91]; C statistic, P = .167). On multivariate analysis, both ex-ECG (hazard ratio, 5; CI, 1-20; P = .017) and ex-Echo (HR, 12; CI, 4-40; P < .001) were predictors of the end point. CONCLUSIONS: In intermediate-risk CP patients, the novel ex-ECG including HF/QRS added to ST/ECG analysis was a valuable diagnostic tool and might be proposed to avoid additional imaging. However, the novel test needs additional study before it can be recommended as a replacement for current techniques.
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Dolor en el Pecho/diagnóstico , Electrocardiografía , Prueba de Esfuerzo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y EspecificidadAsunto(s)
Bilirrubina/genética , Virus de la Hepatitis A/aislamiento & purificación , Hepatitis A/genética , Hiperbilirrubinemia/genética , Adulto , Bilirrubina/sangre , Bilirrubina/metabolismo , Hepatitis A/diagnóstico , Hepatitis A/metabolismo , Humanos , Hiperbilirrubinemia/diagnóstico , Hiperbilirrubinemia/metabolismo , Masculino , Índice de Severidad de la EnfermedadRESUMEN
Glioblastoma (GBM), one of the deadliest brain tumors, accounts for approximately 50% of all primary malignant CNS tumors, therefore novel, highly effective remedies are urgently needed. Boron neutron capture therapy, which has recently repositioned as a promising strategy to treat high-grade gliomas, requires a conspicuous accumulation of boron atoms in the cancer cells. With the aim of selectively deliver sodium borocaptate (BSH, a 12 B atoms-including molecule already employed in the clinics) to GBM cells, we developed novel cell membrane-derived vesicles (CMVs), overcoming the limits of natural extracellular vesicles as drug carriers, while maintaining their inherent homing abilities that make them preferable to fully synthetic nanocarriers. Purified cell membrane fragments, isolated from patient-derived GBM stem-like cell cultures, were used to prepare nanosized CMVs, which retained some membrane proteins specific of the GBM parent cells and were devoid of potentially detrimental genetic material. In vitro tests evidenced the targeting ability of this novel nanosystem and ruled out any cytotoxicity. The CMVs were successfully loaded with BSH, by following two different procedures, i.e. sonication and electroporation, demonstrating their potential applicability in GBM therapy.
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Terapia por Captura de Neutrón de Boro , Neoplasias Encefálicas , Membrana Celular , Glioblastoma , Humanos , Terapia por Captura de Neutrón de Boro/métodos , Glioblastoma/radioterapia , Glioblastoma/patología , Glioblastoma/terapia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patología , Membrana Celular/metabolismo , Borohidruros/química , Línea Celular Tumoral , Portadores de Fármacos/química , Nanopartículas/química , Compuestos de SulfhidriloRESUMEN
We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.
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Envejecimiento , Organoides , Humanos , Organoides/metabolismo , Envejecimiento/metabolismo , Proteínas de la Membrana/metabolismo , Senescencia Celular , Femenino , Andamios del Tejido/química , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/citologíaRESUMEN
HYPOTHESIS: Spinodal dewetting is one of the basic processes inducing a spontaneous withdrawal of a liquid from a substrate surface. In the accepted theory, thickness fluctuations generated by thermally activated capillary waves are amplified by the competing actions of surface tension and disjoining pressure. Ubiquitous sub-nanometric substrate roughness also produces thickness fluctuations and may play a role analogous but even more efficient in seeding the process. MODELLING: Analytic calculations valid at the early linear stage of the process and simulations extending the study to its whole non-linear development have been performed to compare features and the relative relevance of the two seeding mechanisms. FINDINGS: Calculations and simulations have shown that substrate roughness can replace capillary waves in seeding spinodal dewetting. A typically larger amplitude and a steady nature compared to the transitory one of capillary waves allow us to conclude that, contrary to the common view, substrate roughness is the prevailing seed of the spinodal instability. The consequence of our statement is that spinodal dewetting loses most of its stochastic nature and becomes, in principle, a process that can be tuned by engineering substrate roughness.
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Semillas , Tensión SuperficialRESUMEN
The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo.
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Proteínas de Choque Térmico , Proteínas Serina-Treonina Quinasas , Proteínas Adaptadoras Transductoras de Señales/química , Autofagia , Proteínas de Choque Térmico/química , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/química , Humanos , AnimalesRESUMEN
Childbirth education classes represent an antenatal tool for supporting pregnant women and couples in increasing knowledge on pregnancy, delivery, breastfeeding, and newborn care. The aim of this study was to investigate the impact of an additional lesson during the prenatal course regarding the advantage of vaccination to mitigation of maternal anxiety. An observational study was designed that included participants in childbirth education classes and compared courses enhanced by the extra lesson on vaccination during pregnancy versus those who did not receive it. Assessment of the impact of prenatal educational on vaccination was measured by using validated questionnaires (State-Trait Anxiety Inventory, STAI; Perceived Stress Scale, PSS; World Health Organization- Five Well-Being Index, WHO-5). A total of 145 pregnant women participated to the investigation by answering to the online survey. Of them, 33 patients (22.8%) belonged to the course without a lesson on vaccine, while 112 (77.2%) participated to online prenatal education that included an additional meeting on the usefulness of getting vaccinated during pregnancy. No statistical differences were found between study groups in terms of demographics and perinatal outcomes. Participants in the enriched course reported lower basal anxiety levels than those without the vaccine lesson (STAI-State, normal score < 40, 30 vs. 19%, p-value 0.041; STAI-State, mild score 40-50, 78 vs. 67%, p-value 0.037). With reference to the prior two weeks, maternal wellbeing level was improved by the added class (score > 13 as measurement of wellbeing: 62% vs. 80%, p-value < 0.05). Moderate perceived stress assessed by PSS was found in those pregnant women without prenatal education on vaccination (64 vs. 50%, p-value 0.042). The introduction of a lesson regarding vaccination during pregnancy in the program of prenatal education courses improved maternal anxiety levels and wellbeing, in addition to reducing perceived stress.
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The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors' distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the "RuBi-cage" from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the "cage" molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell.
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Neuronas , Ácido gamma-Aminobutírico , Animales , Neuronas/fisiología , Ratas , Receptores de GABA-A/fisiología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Ruthenium-bipyridinetriphenylphosphine-GABA (RuBi-GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is bound to a caged group that blocks the interaction of the neurotransmitter with its receptor site. Following linear-one-photon (1P)-and non-linear-multi-photon-absorption of light, the covalent bond of the caged molecule is broken, and GABA is released. Such a controlled release in time and space allows investigating the interaction with its receptor in four dimensions (X,Y,Z,t). Taking advantage of this strategy, we succeeded in addressing the modulation of GABAA in rat cerebellar neurons by coupling the photoactivation process, by confocal or two-photon excitation microscopy, with the electrophysiological technique of the patch-clamp in the whole-cell configuration. Key parameters have been comprehensively investigated and correlated in a temporally and spatially confined way, namely: photoactivation laser power, time of exposure, and distance of the uncaging point from the cell of interest along the X, Y, Z spatial coordinates. The goal of studying specific biological events as a function of controlled physical parameters has been achieved.
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Neuronas/metabolismo , Neuronas/fisiología , Neurotransmisores/metabolismo , Receptores de GABA-A/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Cerebelo/citología , Luz , Microscopía Confocal , Imagen Molecular/métodos , Técnicas de Placa-Clamp , Ratas Sprague-DawleyRESUMEN
Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.
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Ingeniería de Tejidos , Andamios del Tejido , Diferenciación Celular , Humanos , Microfluídica , PoliésteresRESUMEN
This paper describes a procedure to measure the permeability P, diffusivity D, and rate of adsorption k1, thus determining the solubility S and rate of desorption k2 of He, N2, O2, CH4, and CO2 on a polydimethylsiloxane (PDMS) membrane. The described procedure is able to determine experimentally all the physical quantities that characterize the gas transport process through a thin rubber polymer membrane. The experiments were carried out at room temperature and at a transmembrane pressure of 1 atm. The results are in good agreement with the available data in the literature and offer an evaluation of k1 and k2.
RESUMEN
Nanofluidic structures are often the key element of many lab-on-chips for biomedical and environmental applications. The demand for these devices to be able to perform increasingly complex tasks triggers a request for increasing the performance of the fabrication methods. Soft lithography and poly(dimethylsiloxane) (PDMS) have since long been the basic ingredients for producing low-cost, biocompatible and flexible devices, replicating nanostructured masters. However, when the desired functionalities require the fabrication of shallow channels, the "roof collapse" phenomenon, that can occur when sealing the replica, can impair the device functionalities. In this study, we demonstrate that a "focused drop-casting" of h-PDMS (hard PDMS) on nanostructured regions, provides the necessary stiffness to avoid roof collapse, without increasing the probability of deep cracks formation, a drawback that shows up in the peel-off step, when h-PDMS is used all over the device area. With this new approach, we efficiently fabricate working devices with reproducible sub-100 nm structures. We verify the absence of roof collapse and deep cracks by optical microscopy and, in order to assess the advantages that are introduced by the proposed technique, the acquired images are compared with those of cracked devices, whose top layer, of h-PDMS, and with those of collapsed devices, made of standard PDMS. The geometry of the critical regions is studied by atomic force microscopy of their resin casts. The electrical resistance of the nanochannels is measured and shown to be compatible with the estimates that can be obtained from the geometry. The simplicity of the method and its reliability make it suitable for increasing the fabrication yield and reducing the costs of nanofluidic polymeric lab-on-chips.