Additive manufacturing assisted investment casting: a low-cost method to fabricate periodic metallic cellular lattices

Metallic cellular solids are a class of materials known for their high specific mechanical properties, being desirable in applications where a combination of high strength or stiffness and low density are important. These lightweight materials are often stochastic and manufactured by foaming or casting. If regular (periodic) lattice structures are desired, they may be manufactured by metallic additive manufacturing techniques. However, these have characteristic issues, such as un-melted powders, porosity and heterogeneous microstructures. This study reports a novel low-cost route for producing regular lattice structures by an additive manufacturing assisted investment casting technique. Fused filament fabrication is used to produce the lattice structure pattern which is infiltrated with plaster. The pattern is then burnt off and the aluminum is cast in vacuum. In this way we can manufacture non-stochastic metallic lattices having fine struts/ribs (0.6mm cross-section using a 0.4mm nozzle) and relative densities down to 0.036. X-ray micro computed tomography (μCT) showed that as-cast A356 Aluminium alloy frameworks have high dimensional tolerances and fine detail control. Frameworks based on units of six connected struts ranging from intruding (auxetic) to protruding (hexagonal) strut angles are studied. Vertical struts are finer than expected, reducing their moment of area which could impact their compressive strength. This new, low cost, route for producing high precision metallic cellular lattices offers an attractive alternative to other additive manufacturing techniques (e.g. selective laser and electron beam melting).European Research Council through the ERC grant CORREL-CT, number 695638 to enable VHC to visit the Henry Royce Institute to undertake the X-ray CT studies. This work was supported by the Henry Royce Institute for Advanced Materials, funded through EPSRC grants EP/R00661X/1, EP/S019367/1, EP/P025021/1 and EP/P025498/1. Also, this work was supported by Portuguese FCT, under the reference project UIDB/04436/2020. project iRAIL Innovation in Railway Systems and Technologies Doctoral Programme funds and by national funds through FCT - Portuguese Foundation for Science and Technology and was developed on the aim of the Doctoral grant PD/ BD/114096/2015

Phagocytosis and digestion of pH-sensitive fluorescent dye (Eos-FP) transfected E. coli in whole blood assays from patients with severe sepsis and septic shock

The function of phagocytic and antigen presenting cells is of crucial importance to sustain immune competence against infectious agents as well as malignancies. We here describe a reproducible procedure for the quantification of phagocytosis by leukocytes in whole blood. For this, a pH-sensitive green-fluorescent protein- (GFP) like dye (Eos-FP) is transfected into infectious microroganisms. After UV-irradiation, the transfected bacteria emit green (≈5160 nm) and red (≈581 nm) fluorescent light at 490 nm excitation. Since the red fluorescent light is sensitive to acidic pH, the phagocytosed bacteria stop emitting red fluorescent light as soon as the phagosomes fuse with lysosomes. The green fluorescence is maintained in the phagolysosome until pathogen degradation is completed. Fluorescence emission can be followed by flow cytometry with filter settings documenting fluorescence 1 (FL 1, FITC) and fluorescence 2 (FL 2, phycoerythrin, PE). Eos-FP transfected bacteria can also be traced within phagocytes using microscopical techniques. A standardized assay has been developed which is suitable for clinical studies by providing clinicians with syringes pre-filled with fixed and appropriately UV-irradiated Eos-FP E. coli (TruCulture™). After adding blood or body fluids to these containers and starting the incubation at 37°C, phagocytosis by granulocytes proceeds over time. Cultures can be terminated at a given time by lysing red blood cells followed by flow cytometry. A pilot study demonstrated that Eos-FP E. coli phagocytosis and digestion was up-regulated in the majority of patients with either severe sepsis or septic shock as compared to healthy donors (p < 0.0001 after o/n incubation). Following treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in selected patients with sepsis, phagolysosome fusion appeared to be accelerated

Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution

Ligand Migration and Binding in Nonsymbiotic Hemoglobins of Arabidopsis thaliana

We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions

Nerve globins in invertebrates

The expression of nerve hemoglobins in invertebrates is a well-established fact, but this occurrence is uncommon. In the species where nerve globins occur, they probably function as an oxygen store for sustaining activity of the nerves during anoxic conditions. Although invertebrate nerve globins are functionally similar with respect to O-2 a finity, they are by no means uniform in structure and can differ in size, cellular localization and heme-coordination. The best-studied nerve globin is the mini-globin of Cerebratulus lacteus, which belongs to a class of globins containing the polar TyrB10/GlnE7 pair in the distal pocket. The amide and phenol side chains normally cause low rates of O-2 dissociation and ultra-high O-2 affinity by forming strong hydrogen bonds with bound ligands. Cerebratulus hemoglobin, however, has a moderate O-2 affinity, due to the presence of a third polar amino-acid in its active site, ThrE11, which inhibits hydrogen bonding to bound oxygen by the B10 tyrosine side chain

Dimeric variants of the red fluorescent protein eqFP611 generated by site-directed mutagenesis

The red fluorescent protein eqFP611 shows favorable properties for applications as molecular marker. Its usefulness is, however, limited by its tendency to form tetramers at physiological concentrations. To provide a basis for the rational design of monomeric variants, we examined the monomer interfaces in the x-ray structure of eqFP611. The arrangement of the four ß cans is very similar to that of other GFP-like proteins such as DsRed and RTMS5. In eqFP611, the monomers are linked by comparatively weak interactions, as inferred from the dissociation into monomers in the presence of SDS or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. Some structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. Functional dimeric variants could be generated by altering the A/B interface by single point mutations (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface resulted as yet in essentially complete loss of fluorescence. Presumably, the folding of eqFP611 into its functional form relies on A/C interfacial interactions.<br/

Biofunctionalized, ultrathin coatings of cross-linked star-shaped poly(ethylene oxide) allow reversible folding of immobilized proteins.

Dense, ultrathin networks of isocyanate terminated star-shaped poly(ethylene oxide) (PEO) molecules, cross-linked at their chain ends via urea groups, were shown to be extremely resistant to unspecific adsorption of proteins while at the same time suitable for easy biocompatible modification. Application by spin coating offers a simple procedure for the preparation of minimally interacting surfaces that are functionalized by suitable linker groups to immobilize proteins in their native conformations. These coatings form a versatile basis for biofunctional and biomimetic surfaces. We have demonstrated their advantageous properties by using single-molecule fluorescence microscopy to study immobilized proteins under destabilizing conditions. Biotinylated ribonuclease H (RNase H) was labeled with a fluorescence resonance energy transfer (FRET) pair of fluorescent dyes and attached to the surface by a biotin-streptavidin linkage. FRET analysis demonstrated completely reversible denaturation/renaturation behavior upon exposure of the surface-immobilized proteins to 6 M guanidinium chloride (GdmCl) followed by washing in buffer. A comparison with bovine serum albumin (BSA) coated surfaces and linear PEO brush surfaces yielded superior performance in terms of chemical stability, inertness and noninteracting nature of the star-polymer derived films