Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

Effects of ultrasonic pretreatment on drying characteristics and water migration characteristics of freeze-dried strawberry.

The effects of ultrasonic pretreatment on the drying characteristics and microstructure of strawberry slices were investigated. The rehydration characteristics of freeze-dried products, which were pre-frozen at -20 °C and - 80 °C were explored, with a focus on water mobility and distribution. The ultrasonic pretreatment significantly increased the water mobility of the strawberry slices, resulting in a reduction in their water content. However, the application of ultrasound significantly decreased the rehydration speed, indicating a lower moisture absorption capacity in the pretreated sample. The micrographs revealed that the structure of the tissue was more uniform after ultrasonic treatment, and water loss was accelerated. In addition, the contact angle measurements showed that the samples were more hydrophobic after ultrasonic treatment, and the eutectic temperature and fold point of the samples increased. Therefore, this study found that ultrasonic-assisted freeze vacuum drying technology effectively reduces hygroscopicity, improves product storage, and represents a potential method for dried production.

A comprehensive review of engineered exosomes from the preparation strategy to therapeutic applications.

Exosomes exhibit high bioavailability, biological stability, targeted specificity, low toxicity, and low immunogenicity in shuttling various bioactive molecules such as proteins, lipids, RNA, and DNA. Natural exosomes, however, have limited production, targeting abilities, and therapeutic efficacy in clinical trials. On the other hand, engineered exosomes have demonstrated long-term circulation, high stability, targeted delivery, and efficient intracellular drug release, garnering significant attention. The engineered exosomes bring new insights into developing next-generation drug delivery systems and show enormous potential in therapeutic applications, such as tumor therapies, diabetes management, cardiovascular disease, and tissue regeneration and repair. In this review, we provide an overview of recent advancements associated with engineered exosomes by focusing on the state-of-the-art strategies for cell engineering and exosome engineering. Exosome isolation methods, including traditional and emerging approaches, are systematically compared along with advancements in characterization methods. Current challenges and future opportunities are further discussed in terms of the preparation and application of engineered exosomes.

The prognostic value of LAYN in HPV-related head and neck squamous cell carcinoma and its influence on immune cell infiltration.

BACKGROUND: HPV-positive head and neck squamous cell carcinoma (HNSCC) exhibits different characteristics from HPV-negative tumors in terms of tumor development, clinical features, treatment response, and prognosis. Layilin (LAYN), which contains homology with C-type lectins, plays a critical role in tumorigenesis and cancer progression. However, the prognostic value of LAYN and the relationship between LAYN and immune infiltration levels in HPV-related HNSCC patients still require a comprehensive understanding. Herein, we aimed to assess the prognostic value of LAYN and to investigate its underlying immunological function in HPV-related HNSCC. METHODS: Through various bioinformatics methods, we analyzed the data from The Cancer Genome Atlas (TCGA), Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) databases to explore the potential underlying oncogenic impression of LAYN, including the relevance of LAYN to survival outcomes, clinicopathological factors, immune cell infiltration, and immune marker sets in HPV-related HNSCC. The expression levels of LAYN and HPV were also verified in HNSCC patient tissues. RESULTS: LAYN was differentially expressed in a variety of tumors. The expression of LAYN in HNSCC was significantly higher than that in adjacent normal tissues (P < 0.0001), and high expression of LAYN was correlated with poor overall survival (OS) in HNSCC patients (Hazard Ratio (HR) = 1.3, P = 0.035). Moreover, LAYN expression level in HPV-positive HNSCC patients was significantly lower than that in HPV-negative patients, with HPV-positive HNSCC patients displaying a trend of favorable prognosis. In addition, the relationship between LAYN expression and immune infiltration levels in HPV-positive HNSCC group was less tightly correlated than that in HPV-negative HNSCC group, and there was a strong relationship between LAYN expression and markers of M2 macrophage (P < 0.001) and exhausted T cells (P < 0.05) in HPV-negative HNSCC. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis suggested that LAYN potentially influenced tumor progression through HPV infection and other cancer-related pathways. CONCLUSIONS: LAYN might contribute to tumorigenesis via its positive correlation with immune checkpoint molecules and tumor-associated macrophages (TAMs). Our study might provide a novel prognostic biomarker and latent therapeutic target for the treatment of HPV-related HNSCC.

LncRNA MCM3AP-AS1 serves as a competing endogenous RNA of miR-218 to upregulate GLUT1 in papillary thyroid carcinoma

ABSTRACT Objective: MCM3AP-AS1 has been characterized as an oncogenic long non-coding RNA (lncRNA) in several cancers including papillary thyroid cancer (PTC), but its role in PTC has not been fully elucidated. Considering the critical role of lncRNAs in cancer biology, further functional analysis of MCM3AP-AS1 in PTC may provide novel insights into PTC management. Subjects and methods: Paired tumor and non-tumor tissues were collected from 63 papillary thyroid carcinoma (PTC) patients. Expression levels of MCM3AP-AS1 , miR-218 and GLUT1 in tissue samples were analyzed by qRT-PCR. Cell transfection was performed to explore the interactions among MCM3AP-AS1 , miR-218 and GLUT1 . Cell proliferation assay was performed to evaluate the effects of MCM3AP-AS1 and miR-218 on cell proliferation. Results: MCM3AP-AS1 accumulated to high levels in PTC tissues and was affected by clinical stage. MCM3AP-AS1 showed a positive correlation with GLUT1 across PTC tissues. RNA interaction prediction showed that MCM3AP-AS1 could bind to miR-218 , which can directly target GLUT1 . MCM3AP-AS1 and miR-218 showed no regulatory role regulating the expression of each other, but overexpression of MCM3AP-AS1 upregulated GLUT1 and enhanced cell proliferation. In contrast, overexpression of miR-218 downregulated GLUT1 and attenuated cell proliferation. In addition, miR-218 suppressed the role of MCM3AP-AS1 in regulating the expression of GLUT1 and cell proliferation. Conclusions: MCM3AP-AS1 may serve as a competing endogenous RNA of miR-218 to upregulate GLUT1 in PTC, thereby promoting cell proliferation. The MCM3AP-AS1/miR-218/GLUT1 pathway characterized in the present study might serve as a potential target to treat PTC.