Search Results (35)

The Golgi of goblet cells represents a specialized machine for mucin glycosylation. This process occurs in a specialized form of the secretory pathway, which remains poorly examined. Here, using high-resolution three-dimensional electron microscopy (EM), EM tomography, serial block face scanning EM (SBF-SEM) and immune EM we analyzed the secretory pathway in goblet cells and revealed that COPII-coated buds on the endoplasmic reticulum (ER) are extremely rare. The ERES vesicles with dimensions typical for the COPII-dependent vesicles were not found. The Golgi is formed by a single cisterna organized in a spiral with characteristics of the cycloid surface. This ribbon has a shape of a cup with irregular perforations. The Golgi cup is filled with secretory granules (SGs) containing glycosylated mucins. Their diameter is close to 1 µm. The cup is connected with ER exit sites (ERESs) with temporal bead-like connections, which are observed mostly near the craters observed at the externally located cis surface of the cup. The craters represent conus-like cavities formed by aligned holes of gradually decreasing diameters through the first three Golgi cisternae. These craters are localized directly opposite the ERES. Clusters of the 52 nm vesicles are visible between Golgi cisternae and between SGs. The accumulation of mucin, started in the fourth cisternal layer, induces distensions of the cisternal lumen. The thickness of these distensions gradually increases in size through the next cisternal layers. The spherical distensions are observed at the edges of the Golgi cup, where they fuse with SGs and detach from the cisternae. After the fusion of SGs located just below the apical plasma membrane (APM) with APM, mucus is secreted. The content of this SG becomes less osmiophilic and the excessive surface area of the APM is formed. This membrane is eliminated through the detachment of bubbles filled with another SG and surrounded with a double membrane or by collapse of the empty SG and transformation of the double membrane lacking a visible lumen into multilayered organelles, which move to the cell basis and are secreted into the intercellular space where the processes of dendritic cells are localized. These data are evaluated from the point of view of existing models of intracellular transport. Full article

Fused filament fabrication of thermoplastic composites has grown exponentially owing to its efficiency, thereby meeting numerous engineering demands. However, these materials have limitations owing to their structural vulnerability to elevated temperatures. To address this drawback, this study aims to investigate the tensile behavior of 3D-printed composites in a broad thermal domain from ambient temperature to the crystallization point. For this purpose, a commercial high-temperature-resilient polyamide carbon fiber was selected. To assess the optimal bead configuration and application range, the methodology includes tensile testing of five infill orientations across the four principal thermal domains of the polymers. The results highlight different bead arrangements under constant thermal conditions and demonstrate how temperature effects the tensile performance at similar raster angles, as further correlated with fracture mechanism analysis via scanning electron microscopy. The key findings indicate that raster orientation has a minor influence compared to temperature change. In accordance with the literature, a significantly decreased strength and an abrupt increase in plasticity is observed above the glass transition temperature. Nevertheless, the material retains one-third of its ambient tensile strength at 150 °C, demonstrating its potential for high-temperature applications. Full article

Rapidly growing industrial biotechnology and bio-manufacturing require simple and cost-effective bioseparation tools. A novel strategy of bioseparation based on the streptavidin-decorated polyhydroxyalkanoate (PHA) nano-granules was developed in this study. By fusing to the N-terminus of PHA-associated phasin protein, the streptavidin was one-step immobilized on the surface of PHA nano-granules simultaneously with the accumulation of PHA in recombinant Escherichia coli. About 1.95 g/L of PHA nano-granules (54.51 wt% of cell dry weight) were produced after 48 h bacterial cultivation. The following qualitative and quantitative characterizations demonstrated that the streptavidin accounted for approximately 6.78% of the total weight of the purified PHA nano-granules and confirmed a considerable biotin affinity of 0.1 ng biotin/μg surface protein. As a proof of concept, the nano-granules were further functionalized with biotinylated oligo(dT) for mRNA isolation and about 1.26 μg of mRNA (occupied 2.59%) was purified from 48.45 μg of total RNA, achieving good integrity and high purity with few DNA and rRNA contaminations. Moreover, the nano-granules retained more than 80% of their initial mRNA recovery efficiency after ten cycles of repeated use. The PHA-SAP nano-granules were also functionalized with biotinylated magnetic beads, allowing magnetic recovery of the PHA nano-granules from cell lysates that still needs optimization. Our study provides a novel and expandable platform of PHA nano-granules that can be further functionalized with various biological groups for bioseparation applications. The functional PHA nano-granules have a great potential to serve as bioseparation resin for large-scale purification processes after suitable optimizations for “bench-to-factory” translation, contributing to scalable and sustainable bioprocessing. Full article

Fused Deposition Modeling (FDM) is a well-established manufacturing method for producing both prototype and functional components. This study investigates the mechanical properties of FDM components by material and process-related influencing variables. Tensile tests were conducted on seven different materials in their raw filament form, two of which were fiber-reinforced, to analyze their material-related influence. To cover a wide range from standard to advanced materials relevant for load-carrying components as well as their respective variations, polylactic acid (PLA), 30% wood-fiber-reinforced PLA, acrylonitrile butadiene styrene (ABS), polycarbonate (PC), a blend of ABS and PC, Nylon, and 30% glass-fiber-reinforced Nylon were selected. The process-related influencing variables were studied using the following process parameters: layer thickness, nozzle diameter, build orientation, nozzle temperature, infill density and pattern, and raster angle. The first test series revealed that the addition of wood fibers significantly worsened the mechanical behavior of PLA due to the lack of fiber bonding to the matrix and significant pore formation. The polymer blend of ABS and PC only showed improvements in stiffness. Significant strength and stiffness improvements were found by embedding glass fibers in Nylon, despite partially poor fiber–matrix bonding. The materials with the best properties were selected for the process parameter analysis. When examining the impact of layer thickness on part strength, a clear correlation was evident. Smaller layer thicknesses resulted in higher strength, while stiffness did not appear to be affected. Conversely, larger nozzle diameters and lower nozzle temperatures only positively impacted stiffness, with little effect on strength. The part orientation did alter the fracture behavior of the test specimens. Although an on-edge orientation resulted in higher stiffness, it failed at lower stresses. Higher infill densities and infill patterns aligned with the load direction led to the best mechanical results. The raster angle had a significant impact on the behavior of the printed bodies. An alternating raster angle resulted in lower strengths and stiffness compared to a unidirectional raster angle. However, it also caused significant stretching due to the rotation of the beads. Full article

The potential of bisphenol A (BPA)-binding peptides fused to magnetic beads is demonstrated as novel adsorbents that are reusable and highly selective for BPA removal from aqueous environments, in which various interfering substances coexist. Magnetic beads harboring peptides (peptide beads) showed a higher BPA removal capacity (8.6 mg/g) than that of bare beads without peptides (2.0 mg/g). The BPA adsorption capacity of peptide beads increased with the number of peptides fused onto the beads, where monomeric, dimeric, or trimeric repeats of a BPA-binding peptide were fused to magnetic beads. The BPA-adsorbing beads were regenerated using a methanol–acetic acid mixture, and after six regeneration cycles, the adsorption capacity remained above 87% of its initial capacity. The selective removal of BPA was confirmed in the presence of BPA analogs with high structural similarity (bisphenol F and bisphenol S) or in synthetic wastewater. The present work is a pioneering study that investigates the selective affinity of peptides to remove specific organics with high selectivity from complex environmental matrices. Full article

Endotoxin, a synonym for lipopolysaccharide (LPS), is anchored in the outer membranes of Gram-negative bacteria. Even minute amounts of LPS entering the circulatory system can have a lethal immunoactivating effect. Since LPS is omnipresent in the environment, it poses a great risk of contaminating any surface or solution, including research products and pharmaceuticals. Therefore, monitoring LPS contamination and taking preventive or decontamination measures to ensure human safety is of the utmost importance. Nevertheless, molecules used for endotoxin detection or inhibition often suffer from interferences, low specificity, and low affinity. For this reason, the selection of new binders that are biocompatible, easy to produce, and that can be used for biopharmaceutical applications, such as endotoxin removal, is of high interest. Powerful techniques for selecting LPS-binding molecules in vitro are display technologies. In this study, we established and compared the selection and production of LPS-specific, monoclonal, human single-chain variable fragments (scFvs) through two display methods: yeast and phage display. After selection, scFvs were fused to a human constant fragment crystallizable (Fc). To evaluate the applicability of the constructs, they were conjugated to polystyrene microbeads. Here, we focused on comparing the functionalized beads and their LPS removal capacity to a polyclonal anti-lipid A bead. Summarized, five different scFvs were selected through phage and yeast display, with binding properties comparable to a commercial polyclonal antibody. Two of the conjugated scFv-Fcs outperformed the polyclonal antibody in terms of the removal of LPS in aqueous solution, resulting in 265 times less residual LPS in solution, demonstrating the potential of display methods to generate LPS-specific binding molecules. Full article

This work explored the possibility of producing a compositional functionally graded material (FGM) between mild steel and Inconel 625. An in-house fabricated, dual filler wire tungsten inert gas welding set up was used to deposit weld beads on a mild steel substrate. Filler wire feed rates were controlled independently and the combinations of filler material volumes, i.e., mild steel–Inconel 625 of (100:0), (75:25), (50:50), (25:75), and (0:100) were fed into the arc simultaneously and individual weld beads deposited. Preliminary studies revealed that defect free completely fused new alloys were formed and intermetallic phases rich in Nb observed. Compositional analysis showed that the content of each element changed from one alloy to the other gradually, smoothly indicating the feasibility of a mild steel–Inconel 625 functionally graded material. Full article

This study investigates the effect of annealing on the mechanical properties of fused deposition modeling (FDM) 3D-printed recycled carbon fiber (rCF)-reinforced composites. In this study, filaments for FDM 3D printers are self-fabricated from pure acrylonitrile butadiene styrene (ABS) and ABS reinforced with fiber content of 10 wt% and 20 wt% rCF. This study explores the tensile and flexural properties as a function of the annealing temperature and time for the three different fiber content values. In addition, dimensional measurements of the shape changes are performed to determine the suitability of applying annealing in practical manufacturing processes. The results show that annealing improves the mechanical properties by narrowing the voids between the beads, which occur during the FDM process, and by reducing the gaps between the fibers and polymer. Following annealing, the largest tensile and flexural strength improvements are 12.64% and 42.33%, respectively, for the 20 wt% rCF content samples. Moreover, compared with the pure ABS samples, the annealing effect improves the mechanical properties of the rCF-reinforced samples more effectively, and they have higher dimensional stability, indicating their suitability for annealing. These results are expected to expand the application fields of rCF and greatly increase the potential use of FDM-printed parts. Full article

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry. Full article

The industrial demand for functional filaments made of bio-sourced, biocompatible, biodegradable, and/or recyclable polymers and composites for material extrusion (MEX) 3D printing is continuously growing. Polylactic acid (PLA), the most popular filament, combines such properties, yet its reinforcement with low-cost, inert, and/or recycled fillers remains challenging. Herein, glass in three different micro/nano-forms was the reinforcement agent in PLA. Three different experimental tiers were elaborated by producing composite filaments with glass in powder, beads, and flake forms in various loadings to optimize the concentrations. A thermomechanical process, i.e., melt filament extrusion, was exploited. The composites were evaluated for their thermal degradation stability and composition using thermogravimetric analysis and Raman. MEX 3D printing was used to produce tensile, flexural, impact, and microhardness specimens, to quantitatively evaluate their mechanical response. Field emission scanning electron microscopy evaluation and fractography were carried out to depict fracture patterns of the specimens after their tests. All three glass types induced impressive reinforcement effects (up to 60% in flexural loading), especially in the flake form. The impact of the additional process cost through glass fillers implementation was also assessed, indicating that such composites are cost-effective. Full article

FDM (Fused Deposition Modeling) is one of the most used and industrially applied additive manufacturing processes due to its fast prototyping and manufacturing, simplicity, and low cost of the equipment. However, the mechanical properties of the printed products have a large dependence on orientation and interface strength between layers which is mainly related to the thermal union obtained. This thermal union has a large dependence on the melting and cooling down process. Additionally, the materials used must be extruded in a continuous filament before their use, which limits the materials used. However, a pellet extruder could be used directly in the printing equipment, avoiding filament extrusion. In this work, specimens of PLA (Poly(lactic acid)) with different bead orientations have been produced via filament or pellet extrusion to compare the effect of the different melting processes in the manufacturing methodology. Pellet extruded specimens showed higher infill and mechanical properties. These results were related to better adhesion between layers due to the longer melting and cooling process. The result was confirmed using DSC and XRD techniques, where a higher crystallinity was observed. A bicomponent specimen (50% pellet–50% filament) was prepared and tested, showing higher mechanical results than expected, which was, again, due to the better thermal union obtained in the pellet extruder. Full article

At present, 3D printing is experiencing a great boom. The demand for new materials for 3D printing is also related to its expansion. This paper deals with manufacturing innovative polymer composite filaments suitable for the Fused Filament Fabrication method in 3D printing. As a filler, common and uncostly glass beads were used and mixed with biocompatible and biodegradable poly (ε-caprolactone), as a polymer matrix. This material was characterized via several physical-chemical methods. The Youngs modulus was increasing by about 30% with 20% loading of glass beads, and simultaneously, brittleness and elongations were decreased. The glass beads do not affect the shore hardness of filaments. The rheological measurement confirmed the material stability in a range of temperatures 75–120 °C. The presented work aimed to prepare lightweight biocompatible, cheap material with appropriate mechanical properties, lower printing temperature, and good printing processing. We can assess that the goal was fully met, and these filaments could be used for a wide range of applications. Full article

Friction welding (FW) FW of dissimilar polymer rods is capable of manufacturing green products swiftly and economically. In this study, a green manufacturing technique of joining dissimilar polymer rods was proposed, and the effects of rotational speed on the joint characteristics of friction-welded dissimilar polymer rods fabricated by the fused deposition modeling process were investigated experimentally. The shore surface hardness test, impact test, three-point bending test, and differential scanning calorimetry analysis were carried out on the weld joints. The impact energy for FW of polylactic acid (PLA) and PLA, PLA and acrylonitrile butadiene styrene (ABS), PLA and PLA filled with glass fiber (GF), PLA and PLA filled with carbon fiber (CF), PLA and polycarbonate (PC), and PLA and polyamide (PA) rods can be increased by approximately 1.5, 1.5, 1.3, 1.3, 2.1, and 1.5 times by increasing the rotational speed from 330 rpm to 1350 rpm. The bending strength for FW of PLA and PLA, PLA and ABS, PLA and PLA filled with GF, PLA and PLA filled with CF, PLA and PC, and PLA and PA rods can be increased by approximately 1.3, 1.7, 1.3, 1.2, 1.2, and 1.2 times by increasing the rotational speed from 330 rpm to 1350 rpm. However, the surface hardness of the weld bead is not proportional to the rotational speed. The average surface hardness of the weld bead was increased by approximately 5% compared to the surface hardness of the welding base materials. Full article

The common method for producing casting molds for the fabrication of polydimethylsiloxane (PDMS) chips is standard photolithography. This technique offers high resolution from hundreds of nanometers to a few micrometers. However, this mold fabrication method is costly, time-consuming, and might require clean room facilities. Additionally, there is a need for non-micromechanics experts, who do not have specialized equipment to easily and quickly prototype chips themselves. Simple, so-called, makerspace technologies are increasingly being explored as alternatives that have potential to enable anyone to fabricate microfluidic structures. We therefore tested simple fabrication methods for a PDMS-based microfluidic device. On the one hand, channels were replicated from capillaries and tape. On the other hand, different mold fabrication methods, namely laser cutting, fused layer 3D printing, stereolithographic 3D printing, and computer numerical control (CNC) milling, were validated in terms of machine accuracy and tightness. Most of these methods are already known, but the incorporation and retention of particles with sizes in the micrometer range have been less investigated. We therefore tested two different types of particles, which are actually common carriers for the immobilization of enzymes, so that the resulting reactor could ultimately be used as a microfluidic bioreactor. Furthermore, CNC milling provide the most reliable casting mold fabrication method. After some optimization steps with regard to manufacturing settings and post-processing polishing, the chips were tested for the retention of two different particle types (spherical and non-spherical particles). In this way, we successfully tested the obtained PDMS-based microfluidic chips for their potential applicability as (bio)reactors with enzyme immobilization carrier beads. Full article

It is of great significance to reduce welding hot cracks and improve the corrosion resistance of nuclear power components by controlling the dilution ratio of the cladding weld bead formed by gas tungsten arc welding (GTAW). This paper presents a method to predict the dilution ratio by using the ratio of the thermal power of forming a unit length weld to the cross-section of the fuse, that is, the linear energy of forming a unit volume of deposited metal, which is characterized by the “Heat Equivalent for Melting Welding Wire” (HEMW). It is found that there is a linear positive correlation between the HEMW and the dilution ratio. With the increase in HEMW, the proportion of deposition heat energy in the whole effective heat input energy decreases, the proportion of base metal melting heat energy increases, and the proportion of non-melting heat energy diffused to the base metal remains basically unchanged. The HEMW is used to predict and control the dilution rate under the condition of pulse welding. By increasing the wire feeding speed under the condition of pulse peak current and reducing the welding speed, a high deposition efficiency, low dilution rate and low heat input is realized, which plays an important guiding role in improving the deposition quality. Full article