Application of Aptamer-SERS Nanotags for Unveiling the PD-L1 Immunomarker Progression Correlated to the Cell Metabolic Bioprocess.

In recent years, the expression and progression of programmed cell death ligand 1 (PD-L1) as an immunomarker in the context of a cell metabolic environment has gained significant attention in cancer research. However, intercellular bioprocesses that control the dynamics of PD-L1 have been largely unexplored. This study aimed to explore the cell metabolic states and conditions that govern dynamic variations of PD-L1 within the cell metabolic environment using an aptamer-based surface-enhanced Raman scattering (SERS) approach. The aptamer-SERS technique offers a sensitive, rapid, and powerful analytical tool for targeted and nondestructive detection of an immunomarker with high sensitivity and specificity. By combining aptamer-SERS with cell state profiling, we investigated the modulation in PD-L1 expression under different metabolic states, including glucose deprivation, metabolic coenzyme activity, and altered time/concentration-based cytokine availability. The most intriguing features in our findings include the cell-specific responses, cell differentiation by revealing distinct patterns, and dynamics of PD-L1 in different cell lines. Additionally, the time-dependent variations in PD-L1 expression, coupled with the dose-dependent relationship between glucose concentration and PD-L1 levels, underscore the complex interplay between immune checkpoint regulation and cellular metabolism. Therefore, this work demonstrates the advantages of using highly-sensitive and specific aptamer-SERS nanotags for investigating the immune checkpoint dynamics and related metabolic bioprocess.

Astaxanthin targets IL-6 and alleviates the LPS-induced adverse inflammatory response of macrophages.

Numerous natural compounds are recognized for their anti-inflammatory properties attributed to antioxidant effects and the modulation of key inflammatory factors. Among them, astaxanthin (AST), a potent carotenoid antioxidant, remains relatively underexplored regarding its anti-inflammatory mechanisms and specific molecular targets. In this study, human monocytic leukemia cell-derived macrophages (THP-1) were selected as experimental cells, and lipopolysaccharides (LPS) served as inflammatory stimuli. Upon LPS treatment, the oxidative stress was significantly increased, accompanied by remarkable cellular damage. Moreover, LPSs escalated the expression of inflammation-related molecules. Our results demonstrate that AST intervention could effectively alleviate LPS-induced oxidative stress, facilitate cellular repair, and significantly attenuate inflammation. Further exploration of the anti-inflammatory mechanism revealed AST could substantially inhibit NF-κB translocation and activation, and mitigate inflammatory factor production by hindering NF-κB through the antioxidant mechanism. We further confirmed that AST exhibited protective effects against cell damage and reduced the injury from inflammatory cytokines by activating p53 and inhibiting STAT3. In addition, utilizing network pharmacology and in silico calculations based on molecular docking, molecular dynamics simulation, we identified interleukin-6 (IL-6) as a prominent core target of AST anti-inflammation, which was further validated by the RNA interference experiment. This IL-6 binding capacity actually enabled AST to curb the positive feedback loop of inflammatory factors, averting the onset of possible inflammatory storms. Therefore, this study offers a new possibility for the application and development of astaxanthin as a popular dietary supplement of anti-inflammatory or immunomodulatory function.

Potentialities of Ganoderma lucidum extracts as functional ingredients in food formulation.

Owing to the recognized therapeutic characteristics of G. lucidum, it is one of the most extensively researched mushrooms as a chemopreventive agent and as a functional food. It is a known wood-degrading basidiomycete possessing numerous pharmacological functions and is termed a natural pharmacy store due to its rich number of active compounds which have proved to portray numerous therapeutic properties. This current review highlights studies on the potentialities of G. lucidum extracts as functional ingredients on organoleptic and nutritional properties of food products (e.g., dairy, wine, beverage, bakery, meat, and other products). In addition, the study delved into various aspects of encapsulated G. lucidum extracts, their morphological and rheological characteristics, prebiotic and immunomodulatory importance, the effects on apoptosis, autophagy, cancer therapy, inflammatory responses, oxidative stress, antioxidant activities, and safety concerns. These findings have significant implications for the development of new products in the food and pharmaceutical industries. On the other hand, the various active compounds extracted from G. lucidum exhibited no toxic or adverse effects, and the appeal for it as a dietary food, natural remedy, and health-fortifying food is drastically increasing as well as attracting the interest of both the industrial and scientific communities. Furthermore, the formation of functional foods based on G. lucidum appears to have actual promise and exciting prospects in nutrition, food, and pharmaceutical sciences.

A dynamic fluorescence probed glycolysis suppression process in HeLa cells treated with trichostatin A.

Acetylation can be regulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs), and the imbalance between HDACs and HATs can lead to cancers. Trichostatin A (TSA), as one of the typical HDAC inhibitors (HDACis), may regulate the acetylation level and thus prevent the proliferation of cancer cells, in which the metabolic change of glycolysis is involved. However, the dynamic process of glycolysis has not yet been explored, and the mechanism is still elusive. In this work, we constructed GFP-actin-HeLa cells to observe the dynamic change of glycolysis in situ and found that the GFP fluorescence enhanced significantly after TSA treatment, which was consistent with the change of pH in the cells (pHi) depending on the change of lactate originated from glycolysis. Furthermore, we confirmed that the glycolysis was inhibited after TSA treatment, and this process was associated with HIF-1α and c-Myc regulation. As such, this work not only demonstrates the usefulness of the GFP fluorescent probe for monitoring the metabolic process of glycolysis in situ, but also sheds new light on the mechanism of glycolysis suppression in the cancer cells treated with HDACi.

Study of detoxification of methyl parathion by dielectric barrier discharge (DBD) non-thermal plasma at gas-liquid interface：mechanism and bio-toxicity evaluation.

Methyl parathion (MP) as an organophosphorus pesticide has been used in the control of agricultural pests and diseases. Due to its high toxicity and persistence in the environment, MP may pose threat to human health when it is released into environmental water. For MP treatment, people have found that oxidative degradation of MP may generate some intermediates which are more toxic than MP itself, such as methyl paraoxon. Herein, we proposed a new method of applying dielectric barrier discharge (DBD) non-thermal plasma technology to treat MP in aqueous solution, and investigated the influences of different gases, pH value, discharge voltage/power, and main active species on the MP removal efficiency. In particular, the safety of DBD treatment was concerned with analysis of the biological toxicity of the byproducts from the DBD oxidation, and the DBD-induced degradation together with the involved mechanism was explored therein. The results showed that the production of toxic intermediates could be effectively suppressed or avoided under certain treatment conditions. As such, this work demonstrates that the proper application of DBD plasma technology with necessary caution can detoxify methyl parathion effectively, and also provides a practical guide for low-temperature plasma application in treatment of various organophosphorus pesticides in agricultural wastewater.

Green synthesis of broccoli-derived carbon quantum dots as effective photosensitizers for the PDT effect testified in the model of mutant Caenorhabditis elegans.

The clinical application of photodynamic therapy (PDT) is still limited because of the drawbacks of the traditional photosensitizers, such as low singlet oxygen (1O2) quantum yield and the problem of photobleaching. Herein, carbon quantum dots (CQDs) derived from broccoli natural biomass as a carbon source were fabricated via a simple hydrothermal method and showed outstanding PDT ability as an effective photodynamic agent tested in Caenorhabditis elegans (C. elegans) models. The as-prepared broccoli-derived CQDs (BCQDs) showed excellent water solubility and optical properties and could generate singlet oxygen (1O2) effectively under irradiated light with a wavelength of 660 nm. The in vivo experiment revealed that the PDT efficiency of the BCQDs was dependent on the induction of germline apoptosis through the cep-1/p53 pathway. Further investigation confirmed the DNA damage of the worm by the BCQDs after sufficient light irradiation, which was tested by measuring the egl-1-fold induction in hus-1(op244), and cep-1(w40) mutants that have a loss of function in the genes involved in DNA damage response such as hus-1 (DNA checkpoint gene) and cep-1/p53 (tumor suppressor). The lack of germline apoptosis in the loss of function mutants egl-1(n487), hus-1(op244), and cep-1(w40) exposed to light irradiation compared with the control proved the necessity of these genes in DNA damage-induced germline apoptosis. Therefore, this work has not only provided a new photodynamic agent but also introduced C. elegans as an easy and high-throughput model for the rapid evaluation of the efficiency of PDT.

Regulating the synthesis rate and yield of bio-assembled FeS nanoparticles for efficient cancer therapy.

Biosynthesis has gained growing interest due to its energy efficiency and environmentally benign nature. Recently, biogenic iron sulfide nanoparticles (FeS NPs) have exhibited excellent performance in environmental remediation and energy recovery applications. However, their biosynthesis regulation strategy and application prospects in the biomedical field remain to be explored. Herein, biogenic FeS NPs are controllably synthesized by Shewanella oneidensis MR-1 and applied for cancer therapy. Tuning the synthesis rate and yield of biogenic FeS NPs is realized by altering the initial iron precursor dosage. Notably, increasing the precursor concentration decreases and delays FeS NP biosynthesis. The biogenic FeS NPs (30 nm) are homogeneously anchored on the cell surface of S. oneidensis MR-1. Moreover, the good hydrophilic nature and outstanding Fenton properties of the as-prepared FeS NPs endow them with good cancer therapy performance. The intracellular location of the FeS NPs taken up is visualized with a soft X-ray microscope (SXM). Highly efficient cancer cell killing can be achieved at extremely low concentrations (<12 µg mL-1), lower than those in reported works. Such good performance is attributed to the Fe2+ release, elevated ROS, reduced glutathione (GSH) consumption, and lipid hydroperoxide (LPO) generation. The resulting FeS NPs show excellent in vivo therapeutic performance. This work provides a facile, eco-friendly, and scalable approach to produce nanomedicine, demonstrating the potential of biogenic nanoparticles for use in cancer therapy.

Two FD homologs from London plane (Platanus acerifolia) are associated with floral initiation and flower morphology.

The flowering-time gene FD encodes a bZIP transcription factor that interacts with FLOWERING LOCUS T (FT) to induce flowering in Arabidopsis. Previous research has identified two FT homologs of Platanus acerifolia, PaFT and PaFTL, which each have different expression patterns and are involved in diverse developmental processes. However, it is not known whether such FT/FD complexes participate in the flowering processes in P. acerifolia. Therefore, we isolated two closely related FD homologs, PaFDL1 and PaFDL2, and investigated their functions through the analysis of expression profiles, transgenic phenotypes, their interactions with different FT proteins, and potential cis-regulatory elements in their promoters. The PaFDL genes were found to display their maximal expression levels during the stage of floral transition, and subsequent expression patterns were also seen to be related to inflorescence developmental stage. In addition, both PaFDL1 and PaFDL2 were found to be subject to post-transcriptional alternative splicing, each gene producing two transcript forms. Transgenic tobacco overexpressing each of the four resulting transcript types displayed accelerated floral initiation and produced abnormal flowers. The results suggested that the complete PaFDL proteins may interact with different PaFT/PaFTL proteins in order to fulfill both conservative and diverse functions in floral initiation and floral development.

Development of IL-15/IL-15Rα sushi domain-IgG4 Fc complexes in Pichia pastoris with potent activities and prolonged half-lives.

BACKGROUND: Interleukin-15 (IL-15) is a critical cytokine for the development, proliferation, and function of natural killer (NK) cells, NKT cells, and CD8+ memory T cells and has become one of the most promising protein molecules for the treatment of cancer and viral diseases. However, there are several limitations in applying IL-15 in therapy, such as its low yield in vitro, limited potency, and short half-life in vivo. To date, there are several recombinant IL-15 agonists based on configurational modifications that are being pursued in the treatment of cancer, such as ALT-803, which are mainly produced from mammalian cells. RESULTS: In this study, we designed two different forms of the IL-15 complex, which were formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15Rα)-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc, respectively. The two IL-15 complexes were expressed in Pichia pastoris (P. pastoris), and their activities and half-lives were evaluated and compared. Pharmacokinetic analysis showed that IL-15/SuIL-15Rα-dFc had a half-life of 14.26 h while IL-15/SuIL-15Rα-mFc had a half-life of 9.16 h in mice, which were much longer than the 0.7-h half-life of commercial recombinant human IL-15 (rhIL-15). Treatment of mice with intravenous injection of the two IL-15 complexes resulted in significant increases in NK cells, NKT cells, and memory CD8+ T cells, which were not observed after rhIL-15 treatment. Treatment of human peripheral blood mononuclear cells (PBMCs) from healthy donors with the two IL-15 complexes yielded enhanced NK and CD8+ T cell activation and proliferation, which was comparable to the effect of rhIL-15. CONCLUSIONS: These findings indicate that the IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc produced in P. pastoris exhibit potent activities and prolonged half-lives and may serve as superagonists for immunotherapy in further research and applications.

Ganoderic acid T improves the radiosensitivity of HeLa cells via converting apoptosis to necroptosis.

The use of natural substances derived from traditional Chinese medicine and natural plants as safe radiosensitizing adjuvants is a new trend for cancer radiotherapy. Ganoderma lucidum has been used as a traditional Chinese medicine with a history of more than 2000 years. Ganoderic acid T (GAT) is a typical triterpene of G. lucidum, which has strong cytotoxicity to cancer cells, but whether it has radiation sensitization effect has not been explored. In this work, we treated the HeLa cells with different concentrations of GAT before exposure to gamma-ray radiation and investigated its influence on the radiosensitivity. The cell viability, apoptosis rate, necoptosis rate, intracellular ATP level, cell cycle, the amount of H2AX and 53BP1, reactive oxygen species, and mitochondrial membrane potential were examined. Apoptotic, necroptotic, and autophagic biomarker proteins, including caspase 8, cytochrome c, caspase 3, RIPK, MLKL, P62, and LC3, were analyzed. As a result, we confirmed that with treatment of GAT, the gamma-ray radiation induced both apoptosis and necroptosis in HeLa cells, and with increase of GAT, the percentage ratio of necroptosis was increased. The involved pathways and mechanisms were also explored and discussed.

Low Dose of Trichostatin A Improves Radiation Resistance by Activating Akt/Nrf2-Dependent Antioxidation Pathway in Cancer Cells.

Numerous studies have shown that histone deacetylase inhibitors (HDACis) improve cellular acetylation while also enhancing the radiation sensitivity. In this work, however, we confirmed that low-dose trichostatin A (TSA) as a typical HDACi could reduce rather than increase the radiosensitivity of cancer cells, while the cellular acetylation was also increased with TSA-induced epigenetic modification. The surviving fraction of HeLa/HepG2 cells pretreated with 25 nM TSA for 24 h was higher at 1 Gy/2 Gy of γ-ray radiation than that of the cells with the same radiation dose but without TSA pretreatment. To understand the underlying mechanism, we investigated the effect of low-dose TSA on HO-1, SOD and CAT induction and activating Akt together with its downstream Nrf2 signaling pathway. Our results indicated that TSA activated HO-1, SOD and CAT expression by increasing the phosphorylation level of Nrf2 in an Akt-dependent manner. In addition, we also observed that the 25-nM-TSA-pretreated group showed a significant increase in the antioxidant capacity in terms of SOD and CAT activities. Therefore, our results suggest that low-dose TSA can activate the Akt/Nrf2 pathway and upregulate expression of HO-1, SOD and CAT to stimulate the cellular defense mechanism. This work demonstrates that low-dose TSA treatment may activate the adaptation mechanism against the oxidative stress induced by ionizing radiation, and application of HDACi treatment should be undertaken with caution to avoid its possible radioresistance in radiotherapy.

Highly Sensitive Detection of Elevated Exosomal miR-122 Levels in Radiation Injury and Hepatic Inflammation Using an Aptamer-Functionalized SERS-Sandwich Assay.

Radiation-induced organ injury is one of the major fallouts noticed during radiotherapy treatment of malignancies and other detrimental radiation exposures. MicroRNA (miRNA), which is involved in multiple critical cellular processes, is released from the cells of damaged organs in cellular vesicles, commonly known as exosomes. Specifically, exosomal miR-122 is reported to be actively involved in radiation-actuated rectal and hepatic injuries or inflammation. In this work, we developed a surface-enhanced Raman spectroscopy (SERS) assay for the quantitative and targeted detection of exosomal miR-122 in mice after drug/radiation treatments. In particular, an aptamer-functionalized magnetic capturing element and Au shell nanoparticle (NP)-based SERS tags were utilized, which upon recognition of the target miRNA constituted a "sandwich" formation, with which an 8 fM limit of detection (LOD) could be achieved. Using this SERS assay, we further found that radiation injury led to the elevated expression of exosomal miR-122 in mice at 4 h postirradiation, confirmed by the quantitative real-time PCR method. It was demonstrated that the drug-induced hepatic inflammation could also be assessed via detecting miR-122 using this SERS method. As such, this work has demonstrated the achievement of a highly selective and sensitive probe of exosomal miRNA, which may thus open a gateway for promising usage in drug/radiation-induced inflammation.

Remodeling Chromatin Induces Z-DNA Conformation Detected through Fourier Transform Infrared Spectroscopy.

The SWI/SNF complex is a highly conserved chromatin remodeling complex and can hydrolyze ATP by its catalytic subunit BRG1 or BRM to reconstruct the chromatin. To investigate whether this ATP-dependent chromatin remodeling could affect the DNA conformation, we therefore regulated (knocked down or overexpressed) BRG1/BRM in the cells and applied Fourier transform infrared (FTIR) spectroscopy to probe DNA conformational changes. As a result, we found that BRG1/BRM was indeed associated with the DNA conformational changes, in which knockdown of BRG1/BRM reduced Z-DNA conformation, while overexpression of BRG1/BRM enhanced Z-DNA conformation. This Z-DNA conformational transformation was also verified using the Z-DNA-binding proteins. Therefore, this work has provided a direct analytical tool to probe Z-DNA transformation upon ATP-dependent chromatin remodeling.

Ganoderic acid D induces synergistic autophagic cell death except for apoptosis in ESCC cells.

ETHNOPHAMACOLOGICAL RELEVANCE: Ganoderma lucidum has been used as a medicinal mushroom for more than 2000 years in China. Ganoderic acid D (GAD) as a representative active triterpenoid from Ganoderma lucidum is known to possess anticancer activity. However, the mechanism involved in its anticancer cell process is still largely elusive. AIM OF THE STUDY: Our study aimed to investigate the anticancer effects of GAD on the esophageal squamous cell carcinoma (ESCC) cells and the underlying mechanisms at the cell level. MATERIALS AND METHODS: EC9706 and Eca109 cells were treated with GAD (0, 10, 20, 40 µM) for 24 h. The cell viability, cell cycle, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis rate, caspase-3 activity, autophagic flux, lysosomal function were examined. Cell cycle, apoptotic, autophagy and mTOR signal pathway related proteins such as P53, Cyclin B1, CytoC, PARP, Beclin-1, P62, LC3, PI3K, AKT and mTOR were analyzed by Western blot approach. RESULTS: GAD inhibited cell proliferation and induced both apoptosis and autophagic cell death. In particular, we found that in the early stage of the autophagic process, GAD could initiate and enhance the autophagy signal while in the late stage it on the contrary could block the autophagic flux by impairing the autophagosome-lysosome fusion and inhibited the lysosomal degradation. Besides the autophagic cell death, GAD also induced the apoptosis mediated by caspase-related process in parallel. The mechanism involved for the synergistic apoptotic and autophagic cell death was also explored. We found that GAD down-regulated the expression of PI3K, AKT and mTOR phosphorylated proteins in the mTOR signaling pathway which thus led to the synergistic effect on apoptosis and autophagic cell death in the ESCC cells. CONCLUSIONS: In summary, this study has documented that GAD may inhibit cell proliferation through the mTOR pathway in ESCC cells, and induce synergistic apoptosis and autophagic cell death by disrupting the autophagic flux. This work therefore also suggests that GAD may be used as an efficient anticancer adjuvant for ESCC cancer therapy.

Label-free SERS diagnostics of radiation-induced injury via detecting the biomarker Raman signal in the serum and urine bio-samples based on Au-NPs array substrates.

A sensitive approach based on surface enhanced Raman spectroscopy (SERS) has been developed to evaluate the radiation caused biological injury. To achieve the effective SERS substrate, canonical anodic aluminum oxide (AAO) templates with regular array of nanotips were fabricated, and by plasma sputtering the gold nanoparticles (Au-NPs) were distributed on the nanotips to form the Au-NPs array with plenty of hotspots. The SERS substrates were utilized to examine the serum samples taken from the mice with the treatment of total body irradiation (TBI) of X-ray. The impact of TBI on the mice was analyzed and it was found that the SERS peak intensity at 532 cm-1 increased as a function of duration or dose of TBI. We confirmed that this Raman signature belongs to the myoglobin as a biomarker for the muscle damage due to the radiation caused injury. Furthermore, we also tested several blood and urine specimen of cancer patients who received radiotherapy. The results showed that our approach to some extent could distinguish the bio-samples from normal, X-ray treated and untreated individuals. Therefore, the proposed methodology may have the potential for prompt prognosis of radiation injury at early stage.

Single-repeat R3 MYB transcription factors from Platanus acerifolia negatively regulate trichome formation in Arabidopsis.

MAIN CONCLUSION: Four R3 MYB genes were cloned and identified from Platanus acerifolia and analysed according to endogenous gene expression profiles, protein-protein interaction patterns, phenotypic effects and related gene expression profiles in transgenic Arabidopsis, suggesting that London plane R3 MYB genes inhibit trichome formation in Arabidopsis. The CPC-like MYB transcription factors including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC 1, 2 and 3 (ETC1, ETC2 and ETC3), TRICHOMELESS1 (TCL1) and TRICHOMELESS2(TCL2) play important roles in controlling trichome patterning in Arabidopsis. In this study, four sequences homologous with the Arabidopsis CPC family were identified from London plane and named PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3. Over-expression of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 in Arabidopsis resulted in glabrous phenotypes. In addition, expression of endogenous GL2, GL1, MYB23, TTG2 and a set of R3 MYB-encoding genes was markedly reduced. Furthermore, the protein products of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 were shown to interact with PaGL3 in yeast two-hybrid assays. Together, these results likely suggest that the mechanisms of trichome regulation in London plane have similarities with those in Arabidopsis.

Is level of acetylation directly correlated to radiation sensitivity of cancer cell?

It is known that histone deacetylase inhibitors (HDACis) can modify acetylation of tumor cells and affect radiation sensitivity, or, radiosensitivity. While previous studies showed that trichostatin A (TSA, a typical HDACi) could enhance cell acetylation and so be used as radiation-sensitizer in radiotherapy, we questioned if the radiosensitivity is correlated with cellular acetylation directly. So we inspected radiation response of HeLa cells treated with TSA and investigated the time-dependent effect on radiosensitivity. To our surprise, HeLa cells treated with 200 nM TSA for shorter period (6 h) had relatively higher acetylation level but apparently less radiation damage compared to the irradiated cells with same radiation dose treatment of TSA but for longer time (24 h) with lower acetylation level, gainsaying the direct relationship between acetylation and radiosensitivity. We explained that the anti-oxidation activity for the combined treatments with TSA-radiation gave rise to the enhanced radiation protection of the cells at certain period of time. This work has therefore for the first time presented the experimental evidence showing that there is no direct relationship between acetylation and radiation sensitivity, and our results may also provide the guidance for optimized radiotherapy assisted by HDACis in cancer treatment.

Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate.

Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and ß-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and ß-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.