Real-Time Autophagic Flux Measurements in Live Cells Using a Novel Fluorescent Marker DAPRed.

Autophagy is a conserved homeostatic mechanism involved in cellular homeostasis and many disease processes. Although it was first described in yeast cells undergoing starvation, we have learned over the years that autophagy gets activated in many stress conditions and during development and aging in mammalian cells. Understanding the fundamental mechanisms underlying autophagy effects can bring us closer to better insights into the pathogenesis of many disease conditions (e.g., cardiac muscle necrosis, Alzheimer's disease, and chronic lung injury). Due to the complex and dynamic nature of the autophagic processes, many different techniques (e.g., western blotting, fluorescent labeling, and genetic modifications of key autophagy proteins) have been developed to delineate autophagy effects. Although these methods are valid, they are not well suited for the assessment of time-dependent autophagy kinetics. Here, we describe a novel approach: the use of DAPRed for autophagic flux measurement via live cell imaging, utilizing A549 cells, that can visualize and quantify autophagic flux in real time in single live cells. This approach is relatively straightforward in comparison to other experimental procedures and should be applicable to any in vitro cell/tissue models. Key features • Allows real-time qualitative imaging of autophagic flux at single-cell level. • Primary cells and cell lines can also be utilized with this technique. • Use of confocal microscopy allows visualization of autophagy without disturbing cellular functions.

Control of tissue homeostasis, tumorigenesis, and degeneration by coupled bidirectional bistable switches.

The Hippo-YAP/TAZ signaling pathway plays a critical role in tissue homeostasis, tumorigenesis, and degeneration disorders. The regulation of YAP/TAZ levels is controlled by a complex regulatory network, where several feedback loops have been identified. However, it remains elusive how these feedback loops contain the YAP/TAZ levels and maintain the system in a healthy physiological state or trap the system in pathological conditions. Here, a mathematical model was developed to represent the YAP/TAZ regulatory network. Through theoretical analyses, three distinct states that designate the one physiological and two pathological outcomes were found. The transition from the physiological state to the two pathological states is mechanistically controlled by coupled bidirectional bistable switches, which are robust to parametric variation and stochastic fluctuations at the molecular level. This work provides a mechanistic understanding of the regulation and dysregulation of YAP/TAZ levels in tissue state transitions.

A plausible accelerating function of intermediate states in cancer metastasis.

Epithelial-to-mesenchymal transition (EMT) is a fundamental cellular process and plays an essential role in development, tissue regeneration, and cancer metastasis. Interestingly, EMT is not a binary process but instead proceeds with multiple partial intermediate states. However, the functions of these intermediate states are not fully understood. Here, we focus on a general question about how the number of partial EMT states affects cell transformation. First, by fitting a hidden Markov model of EMT with experimental data, we propose a statistical mechanism for EMT in which many unobservable microstates may exist within one of the observable macrostates. Furthermore, we find that increasing the number of intermediate states can accelerate the EMT process and that adding parallel paths or transition layers may accelerate the process even further. Last, a stabilized intermediate state traps cells in one partial EMT state. This work advances our understanding of the dynamics and functions of EMT plasticity during cancer metastasis.

An organic electrochemical transistor integrated with a molecularly selective isoporous membrane for amyloid-ß detection.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with severe memory loss and impaired cognitive skills. A common pathological change found in AD-affected brains is the accumulation of a peptide named amyloid-ß (Aß) that can form plaques. Aß aggregates are visible to structural scanning tools; however, these bulky and expensive instruments are accessible to trained personnel in clinical settings only, thus hampering timely diagnosis of the disease, particularly in low-resource settings. In this work, we design an organic electrochemical transistor (OECT) for in vitro detection of Aß aggregates in human serum. The OECT channel is integrated with a nanostructured isoporous membrane which has a strong affinity for Aß aggregates. The detection mechanism relies on the membrane capturing Aß aggregates larger than the size of its pores and thus blocking the penetration of electrolyte ions into the channel underneath. Combining the high transconductance of the OECT with the precise porosity and selectivity of the membrane, the device detects the presence of Aß aggregates in human serum samples with excellent sensitivity. This is the first-time demonstration of a biofunctionalized, nanostructured, and isoporous membrane integrated with a high-performance transistor for biosensing. This robust, low-power, non-invasive, and miniaturized sensor aids in the development of point-of-care tools for early diagnosis of AD.

Isolation of Rat and Mouse Alveolar Type II Epithelial Cells.

The gas exchange surface of the lungs is lined by an epithelium consisting of alveolar type (AT) I and ATII cells. ATII cells function to produce surfactant, play a role in host defense and fluid and ion transport, and serve as progenitors. ATI cells are important for gas exchange and fluid and ion transport. Our understanding of the biology of these cells depends on the investigation of isolated cells. Here, we present methods for the isolation of mouse and rat ATII cells.

Lead isotope ratios in lichen samples evaluated by ICP-ToF-MS to assess possible atmospheric pollution sources in Havana, Cuba.

Epiphytic lichens, collected from 119 sampling sites grown over "Roistonea Royal Palm" trees, were used to assess the spatial distribution pattern of lead (Pb) and identify possible pollution sources in Havana (Cuba). Lead concentrations in lichens and topsoils were determined by flame atomic absorption spectrophotometry and inductively coupled plasma (ICP) atomic emission spectrometry, respectively, while Pb in crude oils and gasoline samples were measured by ICP-time of flight mass spectrometry (ICP-ToF-MS). Lead isotopic ratios measurements for lichens, soils, and crude oils were obtained by ICP-ToF-MS. We found that enrichment factors (EF) reflected a moderate contamination for 71% of the samples (EF > 10). The 206Pb/207Pb ratio values for lichens ranged from 1.17 to 1.20 and were a mixture of natural radiogenic and industrial activities (e.g., crude oils and fire plants). The low concentration of Pb found in gasoline (<7.0 µg L-1) confirms the official statement that leaded gasoline is no longer used in Cuba.

Factores epidemiológicos en la inmunopatogénesis de la alergia a los alimentos / Epidemiological factors in food allergy immunopathogenesis

La alergia a los alimentos (A A) es un problema de salud en los países desarrollados. En estos países, las prevalencias oscilan entre el 6 y el 8% en niños y alrededor del 3% en adultos. Un reciente metaanálisis mostró una marcada heterogeneidad en las cifras de prevalencia, atribuida principalmente a diferencias metodológicas o poblacionales. De otra parte, hay muy pocos estudios que revelen la verdadera dimensión del problema en los países en vías de desarrollo. Los escasos estudios existentes se realizaron con cuestionarios y sin confirmación de los diagnósticos con pruebas objetivas. En general, los síntomas de alergia a los alimentos se presentan más frecuentemente en las primeras etapas de la vida y casi siempre son desencadenados por un número limitado de alimentos, como la leche, el huevo, el maní, etc. Recientemente se han desarrollado varios modelos animales que han facilitado el entendimiento de algunos de los mecanismos inmunopatogenicos involucrados en esta enfermedad. En diversos estudios epidemiológicos y experimentales se ha sugerido que algunos factores genéticos y ambientales (nutricionales, microbianos, farmacológicos y contaminantes) podrían jugar algún papel importante en la epidemiología y la patogénesis de la A A...

Food allergy (FA) is an important health problem in developed countries. In these countries the prevalence ranging between 6 to 8% in children and about 3% in adults. A recent metaanalysis showed a marked heterogeneity in the prevalence of food allergy that could be a result of differences in study design, methodology, or differences between populations. In addition, there are very few studies to reveal the true dimension of the problem in developing countries. In these countries the few existing studies were conducted with questionnaires and without confirmation of the diagnoses with objective test. In general the symptoms of food allergy occurs more frequently in the early stages of life and are almost always triggered by a limited number of food, such as milk, eggs, peanuts, etc. Recently, several animal models have been developed and they have facilitated the understanding of some of the immunopathogenic mechanisms involved in this disease. In various epidemiological and experimental studies have suggested that some genetic and environmental factors (Nutritional, microbial, pharmacological and pollutants) could play an important role in the epidemiology and the pathogenesis of FA...

Characterization of mouse alveolar epithelial cell monolayers.

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epithelial type II (AT2) cells into an alveolar epithelial type I (AT1) cell-like phenotype. Primary mouse AT2 cells plated on laminin 5-coated polycarbonate filters formed monolayers with transepithelial resistance (R(T)) and equivalent short-circuit current (I(EQ)) of 1.8 kOmega.cm(2) and 5.3 microA/cm(2), respectively, after 8 days in culture. Amiloride (10 microM), ouabain (0.1 mM), and pimozide (10 microM) decreased MAECM I(EQ) to 40%, 10%, and 65% of its initial value, respectively. Sequential addition of pimozide and amiloride, in either order, revealed that their inhibitory effects are additive, suggesting that cyclic nucleotide-gated channels contribute to amiloride-insensitive active ion transport across MAECM. Ussing chamber measurements of unidirectional ion fluxes across MAECM under short-circuit conditions indicated that net absorption of Na(+) in the apical-to-basolateral direction is comparable to net ion flux calculated from the observed short-circuit current: 0.38 and 0.33 microeq.cm(-2).h(-1), respectively. Between days 1 and 9 in culture, AEC demonstrated increased expression of aquaporin-5 protein, an AT1 cell marker, and decreased expression of pro-surfactant protein-C protein, an AT2 cell marker, consistent with transition to an AT1 cell-like phenotype. These results demonstrate that AT1 cell-like MAECM grown on laminin 5-coated polycarbonate filters exhibit active and passive transport properties that likely reflect the properties of intact mouse alveolar epithelium. This mouse in vitro model will enhance the study of AEC derived from mutant strains of mice and help define important structure-function correlations.