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

First thermostable CLIP-tag by rational design applied to an archaeal O6-alkyl-guanine-DNA-alkyl-transferase

Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP-tag, CLIP-tag and Halo-tag). Commercially available SLPs are highly useful in studying molecular aspects of mesophilic organisms, while they fail in characterizing model organisms that thrive in harsh conditions. By applying an integrated computational and structural approach, we designed a engineered variant of the alkylguanine-DNA-alkyl-transferase (OGT) from the hyper-thermophilic archaeon Saccharolobus solfataricus (SsOGT), with no DNA-binding activity, able to covalently react with O6-benzyl-cytosine (BC-) derivatives, obtaining the first thermostable CLIP-tag, named SsOGT-MC8. The presented construct is able to recognize and to covalently bind BC- substrates with a marked specificity, displaying a very low activity on orthogonal benzyl-guanine (BG-) substrate and showing a remarkable thermal stability that broadens the applicability of SLPs. The rational mutagenesis that, starting from SsOGT, led to the production of SsOGT-MC8 was first evaluated by structural predictions to precisely design the chimeric construct, by mutating specific residues involved in protein stability and substrate recognition. The final construct was further validated by biochemical characterization and X-ray crystallography, allowing us to present here the first structural model of a CLIP-tag establishing the molecular determinants of its activity, as well as proposing a general approach for the rational engineering of any O6-alkylguanine-DNA-alkyl-transferase turning it into a SNAP- and a CLIP-tag variant

Microfluidic encapsulation method to produce stable liposomes containing iohexol

Since the discovery of X-rays in the late 1890s, several medical imaging techniques have been developed, such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI) and Ultrasound Imaging, which are used daily to diagnose, monitor, or treat medical conditions. Some of these techniques include the use of contrast agents to enhance the contrast images, therefore, toxic effects must be considered. Among these, Contrast-Induced Nephropathy (CIN) is an acute renal failure resulting from the administration of iodinated contrast media (CM). To date, there is no definitive treatment for CIN and several prevention approaches have been evaluated. Nanoparticles (NPs) represent a promising strategy for treatment and prevention of CIN, due to their ability to deliver CM during diagnosis imaging. In this study, iohexol-containing liposomes were produced using microfluidic technique for first time. Several phosphocholine lipids (e.g. DMPC, DOPC, DPPC and DSPC) with cholesterol (2:1 ratio) were investigated and DLS, FTIR and in vitro release studies at 37 °C were performed, with stability studies conducted on the best formulation. The microfluidic method allowed to obtain a high encapsulation efficiency (over 70%), and release profiles showed an iohexol release around or less than 0.12 mg/ml after 2 h for the majority of the formulations, which is not toxic to the kidney cells

Analysis of residual plastic deformation of blanked sheets out of automotive aluminium alloys through hardness map

Reducing overall vehicle weight is essential to reduce fuel consumption and pollutant emission and to improve noise, vibration, and harshness (NVH) performances. The substitution with lighter alloys can involve the grand majority of vehicle components, depending on the market sector. In several applications, e.g., chassis, pulleys, and viscodampers, metal sheets are formed in several steps, each of whom work-hardens the material reducing the available residual plasticity. Typically, the process is designed via FEM, whose results are affected by the initial conditions, often neglected, and is performed on pre-processed materials from suppliers. In this regard, correctly simulating the first step of the process is critical. However, the related initial conditions, in terms of residual stress and strain induced by former preliminary operations, are often neglected. This work proposes a quick and economical experimental procedure based on a hardness map to estimate initial conditions and to validate FEM results. The procedure allows evaluating the material's residual plasticity, which is necessary to process engineers to design following manufacturing steps. The approach is demonstrated on an industrially relevant case study, i.e., the blanking of an AA 5754, in use for water pump pulleys

Minimization of defects generation in laser welding process of steel alloy for automotive application

Laser welding (LW) thanks to its flexibility, limited energy consumption and simple realization has a prominent role in several industrial sectors. LW process requires careful parameters' tuning to avoid generating internal defects in the microstructure or a poor weld depth, which reduce the joining mechanical strength and result in waste. This work exploits a supervised machine learning algorithm to optimize the process parameters to minimize the generated defects, while catering for design specifications and tolerances to predict defect generation probability. The work outputs a predictive quality control model to reduce non-destructive controls in the LW of aluminum for automotive applications

Overcritical Rotation of a Trapped Bose-Einstein Condensate

The rotational motion of an interacting Bose-Einstein condensate confined by a harmonic trap is investigated by solving the hydrodynamic equations of superfluids, with the irrotationality constraint for the velocity field. We point out the occurrence of an overcritical branch where the system can rotate with angular velocity larger than the oscillator frequencies. We show that in the case of isotropic trapping the system exhibits a bifurcation from an axisymmetric to a triaxial configuration, as a consequence of the interatomic forces. The dynamical stability of the rotational motion with respect to the dipole and quadrupole oscillations is explicitly discussed.Comment: 6 pages, 3 postscript figure

The effect of Process Parameters on Alignment of Tubular Electrospun Nanofibers for Tisue Regeneration Purposes

Electrospinning is known to be an effective and straightforward technique to fabricate polymer non woven matrices made of nano and microfibers. Micro patterned morphology of electrospun matrices results to be outmost advantageous in the biomedical field, since it is able to mimic extracellular matrix (ECM), and favors cell adhesion and proliferation. Controlling electrospun fibers alignment is crucial for the regenerative purposes of certain tissues, such as neuronal and vascular. In this study we investigated the impact of electrospinning process parameters on fiber alignment in tubular nanofibrous matrices made of Poly (L-lactide-co-ε-caprolactone) (PLA-PCL); a Design of Experiments (DoE) approach is here proposed in order to statistically set up the process parameters. The DoE was studied keeping constants the previously set material and environmental parameters; voltage, flow rate and mandrel rotating speed were the process parameters here investigated as variables. Orientation analysis was based on ImageJ and plugin Orientation J analysis of SEM images. The results show that voltage combined with flow rate has significant impact on electrospun fiber orientation, and the greatest orientation is achieved when all the three input parameters (voltage, flow rate and mandrel rotation speed) are at their maximum value

A Gene Expression Panel is Accurate for Diagnosis and Monitoring Treatment of Eosinophilic Esophagitis in Adults

Eosinophilic esophagitis (EoE) can be difficult to diagnose. We aimed to evaluate whether a gene expression score could differentiate adult EoE cases from non-EoE controls and to determine whether scores normalized after treatment for EoE

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes