Objective: To construct ferritin nanoparticles modified with near-infrared fluorescent probes, and to study their bio-safety as an in vivo imaging system, biological distribution in mice, and tumor targeting. Methods: By artificially designing cysteine point mutation on the surface of ferritin, the surface of ferritin nanoparticles was functionalized with Dy731 fluorescent probe by sulfhydryl coupling of cysteine sulfhydryl groups and the maleimide group of the Dy731 dye. Ferritin nanoparticles (Dy731-Ft) modified with near-infrared fluorescent probes were mixed with 5% w/v agarose solution and cooled to form a gel. The gel was placed under the skin and abdominal muscle wall of mice to determine the ability of Dy731-Ft to penetrate skin and muscle tissue. Dy731-Ft protein nanoparticles were injected into mice through the tail vein to observe whether acute adverse reactions occurred in mice and to detect the in vivo near-infrared fluorescence intensity and observe the bio-distribution of Dy731-Ft as an in vivo imaging system in mice. U87-MG tumor xenograft models were constructed and the distribution of Dy731-Ft within the tissues and organs of tumor-bearing mice and its targeting to tumor tissues were verified by tail vein injection. Results: The results showed that no acute adverse reactions were observed during and after the injection of ferritin nanoparticles into mice, indicating that ferritin nanoparticles have good biocompatibility. Dy731-Ft accumulates mainly in the liver after in vivo injection. Conclusion: Near-infrared Dy731-Ft protein nanoparticles can target U87-MG tumor tissues, and its accumulation in tumor tissues may be related to the synergistic effect of the transferrin receptor 1 (TfR-1) mediated cellular endocytosis and enhanced permeability and retention effect (EPR) in tumor tissues.
Objective: To identify the anillin-related protein in Yarrowia lipolytica and to study its function in septin organization. Methods: We identified the anillin-related protein in Y. lipolytica by aligning the protein sequence of Saccharomyces cerevisiae Bud4 in the Y. lipolytica genome database. The sequences of these anillin-related proteins were amplified and fused with green fluorescent protein GFP to observe their intracellular localization. In the model organism budding yeast, these anillin-related proteins of Y. lipolytica were overexpressed and their effects on cell morphology and septin organization were determined to investigate their function in this process. Results: Two potential anillin-related proteins, named YlBud4A (protein number YALI0D11880 in the database) and YlBud4B (protein number YALI0B07623), were identified by sequence comparison in the Y. lipolytica genome database, both of which were conserved in their C-terminal containing DUF1709 and PH domain. YlBud4A had no bud neck localization, whereas YlBud4B localized to the bud neck as a double ring in medium and large bud cells, a localization pattern identical to Bud4. Overexpression of YlBud4B in budding yeast caused elongated buds and septin disruption, which clustered into dots. Conclusion: YlBud4B may interact with the septins and it is likely to be the functional homolog of anillin-related protein whereas YlBud4A is not.
Objective: To screen the protease which can be used in washing industry through NCBI database and express it efficiently using Bacillus licheniformis. Methods: The protease gene was integrated into the genome of Bacillus licheniformis by homologous recombination, the fermentation was studied, and the enzymatic characteristics and washing effect of the recombinant protease were investigated. Results: The optimum temperature and pH of the recombinant enzyme were 60℃ and 11.0, respectively. AH-101 has good heat resistance, which can be kept stable in the range of 20 ~ 55℃, and the residual activity is still higher than 30% at 60℃ for 1 h. In addition, the enzyme has good compatibility with surfactants and liquid detergents, and the loss of activity of the recombinant enzyme is less than 30% when incubated for 2 h in surfactants and liquid detergents with final concentrations of 0.1% and 0.5%. Conclusion: A recombinant Bacillus licheniformis strain containing exogenous protease was successfully constructed, and the properties and washing effect of the recombinant protease were investigated. The recombinant protease can be used as an environmentally-friendly enzyme preparation in the washing industry.
Objective: To optimize the fermentation and purification process of human α1- microglobulin (A1M), an index protein of kidney injury, in order to prepare recombinant A1M with high purity and high affinity in gram scale. Methods: The optimal pH of A1M fermentation was optimized by a single factor experiment in a 1 L shake flask culture system, followed by a 5 L large-scale fermentation, and the key feeding points and fermentation time were optimized. According to the characteristics of A1M antigen, the protein purification process was established, and N-glycosylation and purity identification were carried out. Finally, A1M immunoaffinity column was prepared and its loading was evaluated. Results: The optimal initial pH of human recombinant A1M Pichia pastoris was 6.0, the expression level reached 888 mg/L in 1 L shake flask culture system, and the degree of glycosylation was relatively lowest. During the 48~120 h of 5 L scale fermentation, DO≥30% reverse correlation methanol was used to feed 4 ~ 6 mL/(h·L-1), which can effectively control the core area of DO>20%. The 48th, 72nd and 96th hours of fermentation were the key feeding points, and the expression level was 12.5 g/L. The space-time yield of fermentation from 58th to 78th hours was 160 ~ 210 mg/(L·h), and the batch of fermentation in a 5-L tank for 120 h was 49 g. The purification process of A1M was established by ultrafiltration concentration, cation exchange chromatography and desalting chromatography column, and the total yield was 18%. The coupling rate of A1M antigen affinity column was 96%, the coupling density was 2.46 mg/mL, and the loading capacity of A1M sheep polyclonal antibody was 37 mg/mL. Conclusion: The yeast fermentation and purification process of recombinant human A1M antigen in 5 L scale was successfully established, and the high purity and high affinity A1M antigen in gram scale was prepared.
Objective: To establish an ultra-fast nucleic acid test for Orientia tsutsugamushi that can be used in grassroots medical institutions. Methods: Using quantitative real-time PCR (qPCR), we compared and analyzed the whole genome sequences of Orientia tsutsugamushi in the database, identified the target genes, designed qPCR-specific primers and probes, optimized the reaction system and reaction procedure, and established a multiplex qPCR ultra-rapid assay. The sensitivity, reproducibility, and minimum detection limit of the method were evaluated using viral nucleic acids quantified by droplet digital PCR (ddPCR) as standards. Comparative genomic analysis was performed to verify the specificity of Orientia tsutsugamushi with pathogens of the same family or genus that tend to cause similar symptoms. Specificity was evaluated using nucleic acids from 24 (45 cases in total) other pathogens that tend to cause similar symptoms. The results of the multiplex qPCR ultra-rapid assay and the conventional qPCR assay were compared using 116 clinical whole blood samples by four-grid table chi-square test, and the agreement between methods was analyzed using the Kappa consistency test. Results: Orientia tsutsugamushi has low homology to pathogens of the same family or genus that tend to cause similar symptoms. Multiplex qPCR ultra-rapid assay showed good specificity and no cross-reactivity with 24 common pathogens. The minimum detection limit was 156 copies/mL. Repeatability comparison analysis showed a coefficient of variation of ≤ 3.13% for Ct values for each concentration assay. The differences between the multiplex qPCR ultra-rapid assay and the conventional qPCR assay for simultaneous testing of 116 clinical whole blood samples were not statistically significant (P>0.05), the two methods had high agreement (Kappa=0.939 2,P<0.001),with total compliance rate 97.42% (CI: 92.67%~99.12%), and the testing time could be be reduced to 30 min. Conclusion: The multiplex qPCR ultra-rapid assay has good sensitivity, reproducibility and specificity, which can rapidly diagnose Orientia tsutsugamushi nucleic acid in clinical whole blood samples and achieve timely and rapid treatment in grassroots medical institutions.
Small extracellular vesicles (sEVs) are a class of extracellular vesicles with a lipid bilayer membrane structure, which can participate in intercellular communication and be used as nanovesicles for drug delivery. The microRNA (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), and proteins in sEVs are important mechanisms to regulate the development of tumors, and reflect the physiological and functional states of cells. They are present in large quantities in the plasma, urine, and saliva of patients, so the analysis and detection of sEVs may become a novel means of tumor diagnosis. In addition to being a biomarker for tumor diagnosis, sEVs can also regulate the invasion and metastasis of tumor cells, drug resistance, cytokine secretion and expression, and immune response via their contents or as specific drug delivery carriers to exert their anti-tumor effects. This paper summarizes the last five years of research on the use of RNAs (miRNAs, lncRNAs, circRNAs) and proteins in sEVs as biomarkers for breast cancer diagnosis and their therapeutic potential in breast cancer, and describes the advantages and shortcomings of sEVs as a new type of diagnostic and therapeutic method to provide references for the future clinical application of sEVs in the field of breast cancer diagnosis and treatment.
Pulmonary fibrosis is a disease characterized by irreversible structural and functional changes in the lung caused by excessive deposition of extracellular matrix proteins, resulting in fibrotic remodeling and alveolar destruction. As extracellular vesicles, exosomes can mediate intercellular communication by delivering functional nucleic acids and proteins produced by specific cell types. Recent studies have shown that exosomes play an important role in the diagnosis and treatment of pulmonary fibrosis and are considered to be a potential biological treatment method and drug delivery carrier. This review aims to summarize the current research on the role and therapeutic application of exosomes in pulmonary fibrosis, and to provide insights into the future development direction and prospects of exosome research, so as to provide new ideas for the treatment of exosomes in pulmonary fibrosis.
Hydrogels have emerged as a promising treatment for skin wounds due to their ability to maintain a moist wound environment, mimic the extracellular matrix, encapsulate and deliver drugs and nanoparticles, and relieve pain. However, conventional hydrogels may not be effective for wounds with high levels of inflammation or microbial contamination, such as diabetic wounds and severe infections, due to their limited antibacterial and anti-inflammatory properties. Researchers have recently explored the potential of combining hydrogels with MXene nanomaterials to address these challenges. MXene is a two-dimensional layered nanomaterial that is widely employed in skin wound treatment due to its exceptional antibacterial, anti-inflammatory, antioxidant, drug delivery, and conductive properties. Combining MXene with hydrogel has effectively controlled wound infection, removed reactive oxygen species, and accelerated wound tissue regeneration. Furthermore, this combination can enhance the tissue adhesion and stability of MXene while reducing potential side effects associated with systemic administration of MXene. However, a comprehensive review of the MXene hydrogel as a wound dressing is still lacking. This review summarizes the latest research progress on MXene hydrogels for wound healing and discusses their challenges and prospects.
Vitamin B6 (VB6) is a water-soluble vitamin, composed of six transformable pyridine compounds: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), pyridoxal 5'-phosphate (PLP), pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP). PLP is the bioactive form of VB6, and is the cofactor of over 180 enzymes. VB6 is an important dietary chemical with several physiological functions such as anti-inflammatory, antioxidant, neuromodulatory, and anti-tumor. Pyridoxine hydrochloride is currently the most common commercial form of VB6, which is fully synthesized using expensive and toxic chemicals via the oxazole method. Compared with chemical synthesis, the biosynthesis of VB6 has significant advantages in terms of non-toxicity, high purity, and sustainability, and has great potential for application. Although high-level production of VB6 by microbial cell factories is still in its infancy, in-depth research on the metabolic pathways, key enzymes and homeostatic regulation of VB6 biosynthesis will gradually increase the yield of VB6, and it is expected to fundamentally replace chemical synthesis. This article mainly reviews the physiological functions, chemical synthesis, and biosynthesis of VB6, focusing on the metabolic pathways and homeostatic regulation involved in biosynthesis. It proposes relevant strategies for the development of high-yielding VB6 strains, with the aim of exploring the potential of VB6 biosynthesis and providing a reference for the efficient biosynthesis of VB6.
Dunaliella salina (D. salina) has become a promising expression system in genetic engineering due to its low cost, easy cultivation, simple transgenic operation, and controllable transcription and translation characteristics. However, there are technical bottlenecks such as low expression and unstable inheritance in the D. salina expression system, which severely limit its application. With the advantages of high stability, the high target specificity and programmability, the CRISPR/Cas system can be used as an important tool to promote the improvement and application of the D. salina expression system, thereby greatly increasing the production of high value-added products such as recombinant proteins, lipid accumulation and carotene. Due to the dual characteristics of prokaryotic and eukaryotic cells, salt algae have greater difficulties and limitations in the gene editing process. Therefore, it is necessary to continuously improve the accuracy and editing efficiency of gene editing technology in D. salina to ensure that the target genes are accurately modified. On this basis, the advantages of genetic engineering of D. salina expression system were comprehensively reviewed, including the transformation methods, exogenous gene expression and the current research status of gene editing. Meanwhile, the main challenges of CRISPR/Cas editing technology in D. salina are highlighted, the future development direction and improvement strategy are proposed, and the application prospect of this technology is envisioned to accelerate the early maturation and popularization of D. salina expression system.
Phytosterols are natural compounds with a cyclopentane nucleus and are widely present in various foods of plant origin, especially enriched in corn oil. As a novel food additive, phytosterols have been added into many fortified foods, including low-fat beverages, dairy products and bread. Moreover, phytosterols also exhibit versatile biological activities, including serum cholesterol lowering and anti-inflammatory and antioxidant activities. However, the practical application effectiveness of phytosterols has been severely underestimated due to their extremely low bioavailability, high melting point and low solubility in water and in oil. In rencent decades, enzymatic modification of the side chain and core of phytosterols has made great progress. This review focuses on the molecular modification of phytosterols using various biocatalysts to redesign their character. Moreover, the prospects of synthetic biotechnology for phytosterol biotransformation are also discussed in this review.
In the context of the increasingly severe global climate change and the advocacy of “green recovery” after the COVID-19 pandemic, biotechnology-enabled agricultural research (the so-called bio-agriculture) is gradually becoming a key point for the development of global agriculture. Bio-agriculture has immense potential to address societal challenges such as food security. Key areas of research in global bio-agriculture include bio-breeding, agricultural bio-agents, and future foods, which play a critical role in aspects such as food security, environmental protection, and nutritional health. This article uses literature review and quantitative research methods to analyze the policy planning, research trends, and the current status and future development trends of the capital markets in global bio-agriculture from 2013 to 2022. The results indicate a promising momentum in the development of global bio-agriculture, with various countries adopting multifaceted strategies. Currently, bio-breeding is the mainstream field in global bio-agriculture development, while future foods represent an important growth area. Overall, China and the United States are leading the global development of bio-agriculture. We believe that seizing the opportunities presented by the transformation of agricultural production is essential to improving the competitiveness of our country’s agricultural development, ensuring food security, and achieving our country’s dual carbon goals.
