Travelling-waves consistent with turbulence-driven secondary flow in a square duct

We present numerically determined travelling-wave solutions for pressure-driven flow through a straight duct with a square cross-section. This family of solutions represents typical coherent structures (a staggered array of counter-rotating streamwise vortices and an associated low-speed streak) on each wall. Their streamwise average flow in the cross-sectional plane corresponds to an eight vortex pattern much alike the secondary flow found in the turbulent regime

Indentation modulus, indentation work and creep of metals and alloys at the macro-scale level: Experimental insights into the use of a primary Vickers hardness standard machine

In this work, the experimental method and the calculation model for the determination of indentation moduli, indentation work, and indentation creep of metallic materials, by means of macroscale-level forces provided by a primary hardness standard machine at the National Institute of Metrological Research (INRIM) at the at room temperature were described. Indentation moduli were accurately determined from measurements of indentation load, displacement, contact stiffness and hardness indentation imaging and from the slope of the indentation unloading curve by applying the Doerner-Nix linear model; indentation work, representing the mechanical work spent during the force application of the indentation procedure, was determined by calculating the areas under the loading–unloading indentation curve, through fitting experimental data with a polynomial law. Measurements were performed with a pyramidal indenter (Vickers test). The applied force was provided by a deadweight machine, and the related displacement was measured by a laser interferometric system. Applied forces and the occurring indentation depths were simultaneously measured: The resulting loading–unloading indentation curve was achieved. Illustrative tests were performed on metals and alloy samples. Discussion and comments on the suitability of the proposed method and analysis were reported

A respiração de Cheyne-Stokes em pacientes com insuficiência cardíaca congestiva: causas e conseqüências

Cheyne-Stokes respiration is a form of periodic breathing in which central apneas and hypopneas alternate with periods of hyperventilation, producing a waxing and waning pattern of tidal volume. This review focuses on the causes and consequences of Cheyne-Stokes respiration in patients with congestive heart failure, in whom the prevalence is strikingly high and ranges from 30% to 50%. Several factors have been implicated in the genesis of Cheyne-Stokes respiration, including low cardiac output and recurrent hypoxia. The key pathophysiological mechanism triggering Cheyne-Stokes respiration is hyperventilation and low arterial CO2 (PaCO2) that when below the apneic threshold triggers a central apnea. Hyperventilation is associated with pulmonary congestion, and Cheyne-Stokes respiration is more prone to occur during sleep, when the respiratory system is mainly dependent on chemical control. It is associated with recurrent dips in oxygen saturation and arousals from sleep, with oscillations in blood pressure and heart rate, sympathetic activation and increased risk of ventricular tachycardia. Cheyne-Stokes respiration is an independent marker of poor prognosis and may participate in a vicious cycle, further stressing the failing heart.A respiração de Cheyne-Stokes é uma forma de respiração periódica na qual apnéias e hipopnéias se alternam com períodos de hiperpnéias que apresentam um padrão crescendo e decrescendo de volume corrente. Esta revisão enfoca as causa e conseqüências da respiração de Cheyne- Stokes em pacientes com insuficiência cardíaca congestiva na qual a prevalência é extremamente alta e varia entre 30 a 50%. Vários fatores foram implicados na gênese da respiração de Cheyne-Stokes, incluindo baixo debito cardíaco e hipoxia recorrente. Hiperventilacão e baixos níveis de CO2 arterial (PaCO2), que quando abaixo do limiar de apnéia desencadeiam apnéia central são os mecanismos fisiopatológicos chave na gênese da respiração de Cheyne-Stokes. Hiperventilação está associada com congestão pulmonar, e a respiração de Cheyne-Stokes tem uma tendência maior de ocorrer durante o sono, quando o centro respiratório é dependente principalmente do controle químico. A respiração de Cheyne-Stokes está associada a quedas recorrentes da saturação de oxigênio e ao despertar do sono, com oscilações recorrentes na freqüência cardíaca, pressão arterial, aumento da atividade simpática e risco aumentado de taquicardia ventricular. A respiração de Cheyne-Stokes é um marcador independente de mau prognostico e provavelmente participa de um ciclo vicioso que contribui para a deterioração cardíaca

Hydrogels for 3D neural tissue models: understanding cell-material interactions at a molecular level.

The development of 3D neural tissue analogs is of great interest to a range of biomedical engineering applications including tissue engineering of neural interfaces, treatment of neurodegenerative diseases and in vitro assessment of cell-material interactions. Despite continued efforts to develop synthetic or biosynthetic hydrogels which promote the development of complex neural networks in 3D, successful long-term 3D approaches have been restricted to the use of biologically derived constructs. In this study a poly (vinyl alcohol) biosynthetic hydrogel functionalized with gelatin and sericin (PVA-SG), was used to understand the interplay between cell-cell communication and cell-material interaction. This was used to probe critical short-term interactions that determine the success or failure of neural network growth and ultimately the development of a useful model. Complex primary ventral mesencephalic (VM) neural cells were encapsulated in PVA-SG hydrogels and critical molecular cues that demonstrate mechanosensory interaction were examined. Neuronal presence was constant over the 10 day culture, but the astrocyte population decreased in number. The lack of astrocytic support led to a reduction in neural process outgrowth from 24.0 ± 1.3 μm on Day 7 to 7.0 ± 0.1 μm on Day 10. Subsequently, purified astrocytes were studied in isolation to understand the reasons behind PVA-SG hydrogel inability to support neural network development. It was proposed that the spatially restrictive nature (or tight mesh size) of PVA-SG hydrogels limited the astrocytic actin polymerization together with a cytoplasmic-nuclear translocation of YAP over time, causing an alteration in their cell cycle. This was confirmed by the evaluation of p27/Kip1 gene that was found to be upregulated by a twofold increase in expression at both Days 7 and 10 compared to Day 3, indicating the quiescent stage of the astrocytes in PVA-SG hydrogel. Cell migration was further studied by the quantification of MMP-2 production that was negligible compared to 2D controls, ranging from 2.7 ± 2.3% on Day 3 to 5.3 ± 2.9% on Day 10. This study demonstrates the importance of understanding astrocyte-material interactions at the molecular level, with the need to address spatial constraints in the 3D hydrogel environment. These findings will inform the design of future hydrogel constructs with greater capacity for remodeling by the cell population to create space for cell migration and neural process extension

Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut

Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest kcat/KM value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane. © 2006 Elsevier Ltd. All rights reserved

Structural Characterization of LRRK2 Inhibitors

Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a structure-function analysis of such inhibitors, we mutated the active site of Dictyostelium Roco4 kinase to resemble LRRK2. Here, we show saturation transfer difference (STD) NMR and the first cocrystal structures of two potent in vitro inhibitors, LRRK2-IN-1 and compound 19, with mutated Roco4. Our data demonstrate that this system can serve as an excellent tool for the structural characterization and optimization of LRRK2 inhibitors using X-ray crystallography and NMR spectroscopy

Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy.

Chitosan-based nanoparticles (NPs) deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier) and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB) photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus