Advanced 3D Printing of Polyetherketoneketone Hydroxyapatite Composites via Fused Filament Fabrication with Increased Interlayer Connection.

Additively manufactured implants, surgical guides, and medical devices that would have direct contact with the human body require predictable behaviour when stress is applied during their standard operation. Products built with Fused Filament Fabrication (FFF) possess orthotropic characteristics, thus, it is necessary to determine the properties that can be achieved in the XY- and Z-directions of printing. A concentration of 10 wt% of hydroxyapatite (HA) in polyetherketoneketone (PEKK) matrix was selected as the most promising biomaterial supporting cell attachment for medical applications and was characterized with an Ultimate Tensile Strength (UTS) of 78.3 MPa and 43.9 MPa in the XY- and Z-directions of 3D printing, respectively. The effect of the filler on the crystallization kinetics, which is a key parameter for the selection of semicrystalline materials suitable for 3D printing, was explained. This work clearly shows that only in situ crystallization provides the ability to build parts with a more thermodynamically stable primary form of crystallites.

Lap Shear Strength and Fatigue Analysis of Continuous Carbon-Fibre-Reinforced 3D-Printed Thermoplastic Composites by Varying the Load and Fibre Content.

This study focuses on evaluating the fatigue life performance of 3D-printed polymer composites produced through the fused deposition modelling (FDM) technique. Fatigue life assessment is essential in designing components for industries like aerospace, medical, and automotive, as it provides an estimate of the component's safe service life during operation. While there is a lack of detailed research on the fatigue behaviour of 3D-printed polymer composites, this paper aims to fill that gap. Fatigue tests were conducted on the 3D-printed polymer composites under various loading conditions, and static (tensile) tests were performed to determine their ultimate tensile strength. The fatigue testing load ranged from 80% to 98% of the total static load. The results showed that the fatigue life of the pressed samples using a platen press was significantly better than that of the non-pressed samples. Samples subjected to fatigue testing at 80% of the ultimate tensile strength (UTS) did not experience failure even after 1 million cycles, while samples tested at 90% of UTS failed after 50,000 cycles, with the failure being characterized as splitting and clamp area failure. This study also included a lap shear analysis of the 3D-printed samples, comparing those that were bonded using a two-part Araldite glue to those that were fabricated as a single piece using the Markforged Mark Two 3D printer. In summary, this study sheds light on the fatigue life performance of 3D-printed polymer composites fabricated using the FDM technique. The results suggest that the use of post-printing platen press improved the fatigue life of 3D-printed samples, and that single printed samples have better strength of about 265 MPa than adhesively bonded samples in which the strength was 56 MPa.

Identification of Small Molecule Inhibitors of Human Cytomegalovirus pUL89 Endonuclease Using Integrated Computational Approaches.

Replication of Human Cytomegalovirus (HCMV) requires the presence of a metal-dependent endonuclease at the C-terminus of pUL89, in order to properly pack and cleave the viral genome. Therefore, pUL89 is an attractive target to design anti-CMV intervention. Herein, we used integrated structure-based and ligand-based virtual screening approaches in combination with MD simulation for the identification of potential metal binding small molecule antagonist of pUL89. In this regard, the essential chemical features needed for the inhibition of pUL89 endonuclease domain were defined and used as a 3D query to search chemical compounds from ZINC and ChEMBL database. Thereafter, the molecular docking and ligand-based shape screening were used to narrow down the compounds based on previously identified pUL89 antagonists. The selected virtual hits were further subjected to MD simulation to determine the intrinsic and ligand-induced flexibility of pUL89. The predicted binding modes showed that the compounds reside well in the binding site of endonuclease domain by chelating with the metal ions and crucial residues. Taken in concert, the in silico investigation led to the identification of potential pUL89 antagonists. This study provided promising starting point for further in vitro and in vivo studies.

Impact of orientation and flexibility of peptide linkers on T. maritima lipase Tm1350 displayed on Bacillus subtilis spores surface using CotB as fusion partner.

Fusion protein construction often requires peptide linkers for prolonged conformation, extended stability and enzyme activity. In this study a series of fusion between Thermotoga maritima lipase Tm1350 and Bacillus subtillis coat protein CotB, comprising of several peptide linkers, with different length, flexibility and orientations were constructed. Effects of temperature, pH and chemicals were examined, on the activity of displayed enzyme. The fusion protein with longer flexible linkers L9 [(GGGGS)4] and L7 (GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS) possess 1.29 and 1.16-fold higher activity than the original, under optimum temperature and pH respectively. Moreover, spore surface displaying Tm1350 with L3 (EAAAK-GGGGS) and L9 ((GGGGS)4) showed extended thermostably, maintaining 1.40 and 1.35-fold higher activity than the original respectively, at 80 °C after 5 h of incubation. The enzyme activity of linkers with different orientation, including L5, L6 and L7 was determined, where L7 maintained 1.05 and 1.27-fold higher activity than L5 and L6. Effect of 0.1% proteinase K, bromelain, 20% ethanol and 30% methanol was investigated. Linkers with appropriate Glycine residues (flexible) showed higher activity than Alanine residues (rigid). The activity of the displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers, to evaluate high activity and stability in industrial processes.

Progress in Bacillus subtilis Spore Surface Display Technology towards Environment, Vaccine Development, and Biocatalysis.

Spore surface display is the most desirable with enhanced effects, low cost, less time consuming and the most promising technology for environmental, medical, and industrial development. Spores have various applications in industry due to their ability to survive in harsh industrial processes including heat resistance, alkaline tolerance, chemical tolerance, easy recovery, and reusability. Yeast and bacteria, including gram-positive and -negative, are the most frequently used organisms for the display of various proteins (eukaryotic and prokaryotic), but unlike spores, they can rupture easily due to nutritive properties, susceptibility to heat, pH, and chemicals. Hence, spores are the best choice to avoid these problems, and they have various applications over nonspore formers due to amenability for laboratory purposes. Various strains of Clostridium and Bacillus are spore formers, but the most suitable choice for display is Bacillus subtilis because, according to the WHO, it is safe to humans and considered as "GRAS" (generally recognized as safe). This review focuses on the application of spore surface display towards industries, vaccine development, the environment, and peptide library construction, with cell surface display for enhanced protein expression and high enzymatic activity. Different vectors, coat proteins, and statistical analyses can be used for linker selection to obtain greater expression and high activity of the displayed protein.

Influences of Various Peptide Linkers on the Thermotoga maritima MSB8 Nitrilase Displayed on the Spore Surface of Bacillus subtilis.

In the present study, fusion genes composed of Thermotoga maritima MSB8 nitrilase and Bacillus subtilis 168 outer coat protein CotG were constructed with various peptide linkers and displayed on B. subtilis DB 403 spores. The successful display of CotG-nit fusion proteins on the spore surface of B. subtilis was verified by Western blot analysis and activity measurement. It was demonstrated that the fusion with linker GGGGSEAAAKGGGGS presented the highest thermal and pH stability, which is 2.67- and 1.9-fold of the fusion without linker. In addition, fusion with flexible linker (GGGGS)3 demonstrated better thermal and pH stability than fusions with linkers GGGGS and (GGGGS)2. Fusion with rigid linker (EAAAK) demonstrated better thermal stability than fusions with linkers (EAAAK)2 and (EAAAK)3. Fusions with linker (EAAAK)2 demonstrated better pH stability than fusions with linkers (EAAAK) and (EAAAK)3. In the presence of 1 mM dithiothreitol, 1% (v/v) sodium dodecyl sulfate, and 20% (v/v) ethanol, the optimal linkers of the fusions were MGSSSN, GGGGSEAAAKGGGGS, and (GGGGS)3, respectively. In summary, our results showed that optimizing the peptide linkers with different type, length, and amino acid composition of the fusion proteins would be an efficient way to maintain the stability of fusion proteins and thus improve the nitrilase display efficiency, which could provide an effective method for rational design peptide linkers of displayed nitrilase on B. subtilis.