Biotechnology and Bioprocess Engineering最新文献

The fabrication of scaffolds that mimic the structure of extracellular matrix molecules using electrospinning has great potential for various tissue engineering applications because it is advantageous for cell ingrowth and tissue formation. In particular, in the case of non-adhesive scaffolds, tissue adhesives (e.g., fibrin, glutaraldehyde) that provide strong adhesion to tissue surfaces or cells are sometimes used to efficiently utilize the desired function. Therefore, scaffolds that combine the advantages of adhesive materials while maintaining cell growth and functionality could further expand tissue engineering applications. In the present study, we developed nanofibers with greatly improved adhesive ability by blended cellulose acetate and levan, a bioadhesive polymer. Nanofibers manufactured through electrospinning exhibited stable mechanical properties through citric acid cross-linking and exhibited excellent adhesive performance with an adhesion strength of up to 3.67 MPa. In future research, we aim to expand the utility of bioadhesive nanofibers by loading nanofiber scaffolds with useful substances for diagnostic and therapeutic purposes.

A two-step optimization protocol was attempted to optimize the condition for dextran production from pineapple waste using Leuconostoc mesenteroides NCIM-2198. Response surface methodology, in combination with numerical optimization technique, was explored for this purpose. In the first step, the initial medium pH (6.5–7.5), incubation temperature (25–35 °C), and time (12–60 h) were optimized from 45 full factorial runs. The maximum dextran yield was estimated as 1.46 g·[100 mL]⁻¹ while incubated at 29.2 °C for 57.2 h at an initial pH of 7.15. Further, sucrose concentration (2–10 g·[100 mL]⁻¹) and culture volume (3–7 mL·[100 mL]⁻¹) were optimized from 15 experimental runs. The maximum dextran yield (1.47 g·[100 mL]⁻¹) was obtained at 7.6 g·[100 mL]⁻¹ of sucrose with 3 mL·[100 mL]⁻¹ of culture volume at the previously optimized fermented broth. The response surface models were validated to explain the interaction between factors affecting dextran yield. The structural characteristics of the exopolysaccharide were analyzed. Fourier-transform infrared spectra showed that the exopolysaccharide contains similar spectral peaks as that of standard dextran. Nuclear magnetic resonance spectroscopy confirms the exopolysaccharide was dextran with mainly α-1-6 glycosidic bonds. Scanning electron microscopy explained its porous structure, which would be useful in retaining water and thus giving texturizing and viscosifying properties.

Abstract

Xenograft bone granules are bone graft materials that are widely used to reconstruct bone tissue in clinical practice, and their clinical success requires aggregation with a biocompatible binder. While the catechol mussel bioadhesive has been used as a key universal biomedical glue moiety, the thiols in mussel adhesive proteins have recently been shown to play key roles in mussel wet adhesion. Here, pentaerythritol tris(3-mercaptopropionate)-di-catechol (PETMP-di-catechol), a catechol- and thiol-containing binder molecule that mimics mussel adhesive proteins, was used to aggregate xenograft bone granules and reconstruct bone tissue. The PETMP-di-catechol-aggregated xenograft bone granules were most resistive to compression stress when both the catechol and thiol moieties in PETMP-di-catechol were chemically oxidized with sodium periodate. The xenograft bone granules aggregated with the oxidized PETMP-di-catechol exhibited enhanced osteogenic cellular behavior in vitro and in vivo in a rat calvarial defect model, compared to the control group treated with catechol-conjugated chitosan, a catechol-containing polymer binder devoid of thiol moieties. Hence, the thiol-containing catechol-functionalized binder exhibits improved bone tissue reconstruction properties by mimicking mussel wet adhesion. In addition, our study suggests that PETMP-di-catechol is a potential biocompatible bone binder for use in clinical settings.

Asthma is a chronic inflammatory disease triggered by allergic reactions in the bronchia. These reactions lead to swelling of mucous membranes, hypersecretion of mucus, and bronchoconstriction, resulting in a restricted opening of the lung airway. Allergic pulmonary inflammation and airway hyperresponsiveness are induced when Th2 cytokines, such as interleukin (IL)-4 and IL-13, bind to their cognate receptors on lung epithelial cells. Specifically, IL-13 stimulates inflammation through a multi-subunit receptor, mainly the alpha chain of the IL-4 receptor (IL-4Rα), which also plays a role in IL-4 signaling. In this study, we employed a lung epithelial cell-targeting siRNA carrier composed of a rabies virus glycoprotein-derived small peptide coupled with cationic nona-arginine and trileucine before cysteine peptide (RVG9R3LC). This carrier was complexed with siRNA, enabling targeted delivery of therapeutic siRNA to IL-4Rα (siIL4Rα) expressed in lung epithelial cells within an asthma model in vivo. Our approach demonstrated efficient gene knockdown in cultured lung epithelial cells and in vivo. Furthermore, two administrations of therapeutic siIL4Rα protected the ovalbumin-sensitized and challenged asthma mouse model from airway inflammation and excessive mucus secretion. Our findings suggest that the peptide-siRNA carrier system presents a promising therapeutic approach for respiratory inflammation.

Graphical abstract

Abstract

Adeno-associated virus (AAV)-mediated gene therapy holds significant promises to treat or potentially cure various human diseases. Although AAV holds promise for their significant therapeutic potential, batch-to-batch differences can exist from manufacturing; and therefore, a potency assay is required for clinical development of AAV. Among different serotypes, due to its ability to cross blood–brain barrier and wide-spread transduction capability in vivo upon systemic administration, AAV9 has been widely utilized for the development of treatment of neurological disorders. However, as AAV9 is known to show poor transduction in vitro, establishing a robust in vitro potency assay have been difficult. To this end, we engineered HEK293T and Schwann-like cell lines to express previously identified common AAV receptor, AAVR or endogenous host factor involved in AAV endosomal escapes, GPR108 that can increase infectivity of AAVs in an attempt to increase transduction capability of AAV9. We found that AAVR overexpressed Schwann-like cell line showed significant increase in AAV9 transduction; whereas, GPR108 overexpression showed no effect on AAV9 transduction. On the other hand, GPR108 engineered HEK293T showed increase in AAV9 transduction; whereas, AAVR overexpressed HEK293T cell line showed modest increase in AAV9 transduction. Gene expression analysis showed that AAVR is highly expressed in HEK293T compared to Schwann-like cell line; whereas, GPR108 is highly expressed in Schwann-like cell line when compared to HEK293T. These results indicate that different cell lines may require different gene engineering to increase AAV9 infectivity and analysis of endogenous expression of AAV entry factors for cell line to be engineered can improve efficiency of cell line engineering for AAV transduction.

Synthetic biology aims to establish engineering principles for biological systems by leveraging the design-build-test-learn (DBTL) cycle. Central to the success of the DBTL cycle is the selection of a suitable chassis, which is the environment in which biological designs are tested. Every step of this cycle is strongly influenced by the properties of chassis. A successful chassis must meet various criteria, prompting ongoing research regarding new candidates. Recently, species within the Vibrio genus, notably Vibrio natriegens and related strains, have emerged as promising next-generation chassis due to their rapid growth rates, versatile substrate utilization, and biosafety level 1 classification. These properties make them highly attractive for accelerating the DBTL cycle with the potential for efficient protein and metabolite production. This review emphasizes the foundational requirements for efficient engineering in synthetic biology, including genetic parts, vectors, and genome engineering technologies tailored to Vibrio species. Practical applications, such as metabolic engineering and protein expression, have been discussed, offering a comprehensive summary of recent advances. This paper also outlines the future directions and suggestions for fully unlocking the potential of Vibrio species as next-generation chassis.

Abstract

Being able to perform modular design of artificial transcription factors is useful in bioengineering and synthetic biology, particularly in the development of biosensors and therapeutics. This study aimed to develop a two-fragment transcription factor system by splitting a lactose repressor (LacI). To fragment LacI, we screened potential split positions from transposon-based insertional libraries that we generated to identify those positions that did not disturb the intrinsic activity of LacI. The interaction of protein tags fused with fragments induces the reassembly of LacI and recovers the isopropyl-β-D-thiogalactoside-dependent regulatory function. The split LacI-based biosensor was implemented on an in vitro platform using a cell-free protein expression system to facilitate accurate analytical studies with high reproducibility. This versatile platform holds great potential to realize the rapid and simple detection of protein–protein interactions in cell-free systems; thus, it can be further extended to disease diagnosis, particularly at the point-of-care.

With a better understanding of the health benefits of β-carotene, the precursor of vitamin A, as well as its coloring property, the need for this carotenoid has increased in various sectors. In order to meet the increasing demand efficiently, cheaply, and sustainably, interest in heterologous β-carotene production through metabolic engineering strategies has increased in recent years. In this context, although it is not a native producer of β-carotene, Yarrowia lipolytica yeast stands out due to its metabolic, physiological, and genomic properties. Successful results have been obtained by using a series of engineering strategies, including biosynthesis pathway engineering, morphological engineering, and fermentation engineering strategies, in the production of heterologous β-carotene from Y. lipolytica. However, these strategies have various strengths and weaknesses against each other, and there are also some points open to improvement. In this review, the engineering strategies that have been applied and have the potential to be applied for the production of β-carotene from Y. lipolytica have been examined in depth, including their advantages and disadvantages, and compared with each other. Moreover, a future perspective has been presented to increase the potential of using Y. lipolytica yeast as a cell factory in β-carotene production.

The interdisciplinary nature of synthetic biology merges engineering principles with biology and provides innovative solutions for issues in the biomanufacturing industry. To develop industrially applicable biocatalysts and/or microbial cell factories, a design-build-test-learn cycle-based iterative process is necessary, which is often time-consuming and labor-intensive. The integration of microfluidic technologies into synthetic biology can accelerate these processes, particularly for achieving high-throughput phenotyping and screening. In this review, we examine the potential of microfluidic technologies to revolutionize synthetic biology. Although commercial microfluidics demonstrate superior throughput for single-cell assays, their application can be limited, for example, in cases where products are retained inside the cells. Droplet microfluidics, on the other hand, is a rather flexible platform and shows high diversity in single-cell, cell-to-cell interaction-based, and cell-free reaction-based analyses. By examining previous studies, we have summarized the potential of microfluidic technologies to foster sustainable biomanufacturing and advanced biological engineering.

Nowadays, the fabrication and characterization of plant extract-based gold nanoparticles (AuNPs) as well as their anticancer activities have gained significant attention. Acorus calamus is a typical Chinese medicinal herb that has been utilized since a period of thousand years for treating rheumatism, asthma, tracheitis, etc. In the current study, the reaction parameters are optimized for managing the size of NPs as categorized by the high-resolution transmission electron microscopy. Various characterization methods such as X-ray diffraction, UV-visible spectroscopy and Fourier-transform infrared spectroscopy were carried out to confirm the fabrication of AuNPs mediated by the leaf extract of A. calamus. Further, the neuroprotective potential of fabricated AuNPs was studied by evaluating the cholinesterase inhibitory activity and antioxidant potential. The nanogold aqueous extracts exhibited the highest antioxidant and anti-acetyl cholinesterase activity, confirming that AuNPs can cross the blood-brain barrier and raise the level of acetyl cholinesterase, at the same time reduces the oxidative stress levels. The obtained results concluded their important potential for development of anti-Alzheimer drug having antioxidant and anticholinesterase activity against oxidative stress.