Structures of human γδ T cell receptor-CD3 complex.

Gamma delta (γδ) T cells, a unique T cell subgroup, are crucial in various immune responses and immunopathology1-3. The γδ T cell receptor (TCR), which is generated by γδ T cells, recognizes a diverse range of antigens independently of the major histocompatibility complex2. The γδ TCR associates with CD3 subunits, initiating T cell activation and holding great potential in immunotherapy4. Here we report the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR-CD3 complexes5,6, revealing two distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR-CD3 complex is monomeric, with considerable conformational flexibility in the TCRγ-TCRδ extracellular domain and connecting peptides. The length of the connecting peptides regulates the ligand association and T cell activation. A cholesterol-like molecule wedges into the transmembrane region, exerting an inhibitory role in TCR signalling. The Vγ5Vδ1 TCR-CD3 complex displays a dimeric architecture, whereby two protomers nestle back to back through the Vγ5 domains of the TCR extracellular domains. Our biochemical and biophysical assays further corroborate the dimeric structure. Importantly, the dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. These findings reveal organizing principles of the γδ TCR-CD3 complex, providing insights into the unique properties of γδ TCR and facilitating immunotherapeutic interventions.

Constructing the Interconnected and Hierarchical Nanoarchitectonics in Coal-Derived Carbon for High-Performance Supercapacitor.

Because of the deep and zigzag microporous structure, porous carbon materials exhibit inferior capacitive performance and sluggish electrochemical kinetics for supercapacitor electrode materials. Herein, a single-step carbonation and activation approach was utilized to synthesize coal-based porous carbon with an adjustable pore structure, using CaO as a hard template, KOH as an activator, and oxidized coal as precursors to carbon. The obtained sample possesses an interconnected and hierarchical porous structure, higher SSA (1060 m2 g-1), suitable mesopore volume (0.25 cm3 g-1), and abundant surface heteroatomic functional groups. Consequently, the synthesized carbon exhibits an exceptionally high specific capacitance of 323 F g-1 at 1 A g-1, along with 80.3% capacitance retention at 50 A g-1. The assembled two-electrode configuration demonstrates a remarkable capacitance retention of up to 95% and achieves Coulombic efficiency of nearly 100% with 10,000 cycles in a 6 M KOH electrolyte. Furthermore, the Zn-ion hybrid capacitor also exhibits a specific capacity of up to 139.1 mA h g-1 under conditions of 0.2 A g-1. This work offers a simple method in preparation of coal-based porous carbon with controllable pore structure.

The Relationship Between Remnant Cholesterol and Visceral Adipose Tissue: A National Cross-Sectional Study.

The aim of our study is to explore the relationship between remnant cholesterol (RC) levels and visceral adipose tissue (VAT) in the US adult population. This cross-sectional study utilized data from 5301 participants aged 20 to 59 years gathered by the National Health and Nutrition Examination Survey (NHANES). RC was determined by deducting both high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) from total cholesterol (TC), and VAT was measured using dual-energy X-ray absorptiometry. Visceral obesity is defined as a VAT area ≥ 100 cm2. With increasing quartiles of RC levels, the prevalence of visceral obesity rises (16.51% vs. 36.11% vs. 55.66% vs. 74.48%, p<0.001). After adjusting for confounders, RC levels positively correlate with visceral obesity risk (OR=1.039, 95% CI 1.031-1.048, p<0.001). Additionally, individuals with low LDL-c/high RC and those with high LDL-c/low RC showed 2.908-fold (95% CI 1.995-4.241) and 1.310-fold (95% CI 1.022-1.680) higher risk of visceral obesity, respectively, compared to those with low LDL-c/low RC. Receiver Operating Characteristic (ROC) and Decision Curve Analysis (DCA) show RC's superior predictive ability over other lipid markers. Subgroup analysis showed that the relationship between RC and visceral obesity was more ronounced in those with cardiovascular disease. Smooth curve fitting indicated a nonlinear relationship between RC levels and VAT area. Our study highlights that elevated levels of RC are associated with adverse accumulation of VAT. However, the causal relationship between RC and visceral obesity requires additional investigation.

The mediating effect of self-efficacy on social support and cancer screening behavior among Chinese women: a cross-sectional study.

BACKGROUND: Breast and cervical cancer are the most common cancers in women, and are associated with high morbidity and mortality rates. Cancer screening can facilitate early diagnosis, reduce mortality, and ease the burden of cancer. Social support and self-efficacy are strongly associated with cancer screening behavior. The present study aimed to explore the mediating effect of self-efficacy on social support and cancer screening behavior. METHODS: In this cross-sectional survey study conducted from June to October 2023, 312 women aged 35-65 years were recruited from the East Coast area of China. A general information questionnaire, cancer screening behavior questionnaire, social support scale and self-efficacy scale were used to collect data. Descriptive statistics were used to analyze the general characteristics of participants; one-way analysis of variance was used to test for differences in the measured variables; and Pearson's correlation analyses were used to describe the relationship among social support, self-efficacy, and cancer screening behavior. A mediation model was constructed and analyzed using the PROCESS macro for SPSS. RESULTS: The mean (standard deviation) screening behavior score for breast cancer and cervical cancer was 3.98 (2.79), representing an intermediate level. Self-efficacy was closely related to social support and cancer screening behavior. Social support showed a significant positive correlation with self-efficacy (r = 0.37, p < 0.01) and cancer screening behavior (r = 0.18, p < 0.01). Self-efficacy was also significantly positively correlated with cancer screening behavior (r = 0.19, p < 0.05). Self-efficacy showed a full mediating effect between social support and cancer screening behavior, with an explanatory power of 32%. CONCLUSIONS: The findings emphasize the need to increase women's level of social support and self-efficacy, which in turn can increase women's participation in breast and cervical cancer screening.

The relationship between non-high-density lipoprotein cholesterol to high-density lipoprotein cholesterol ratio (NHHR) and hyperuricaemia.

PURPOSE: The ratio of non-high-density lipoprotein cholesterol (non-HDL-c) to high-density lipoprotein cholesterol (HDL-c) (NHHR) is a novel comprehensive lipid index. The aim of this study was to investigate the relationship between the NHHR and the prevalence of hyperuricaemia (HUA) in the adult population of the U.S. METHODS: This cross-sectional study collected data from the National Health and Nutrition Examination Survey (NHANES) (2007-2018). HUA was defined as a serum uric acid (SUA) concentration ≥ 7 mg/dL in men and ≥ 6 mg/dL in women. Multivariate logistic regression models and the restricted cubic spline (RCS) method were applied to examine the relationship between the NHHR and the risk of developing HUA. Subgroup analyses and interaction tests were also performed. RESULTS: The prevalence of HUA increased with increasing NHHR values (9.01% vs. 13.38% vs. 17.31% vs. 25.79%, P < 0.001). The NHHR was independently correlated with the risk of developing HUA (OR = 1.10, 95% CI: 1.05-1.16; P < 0.001). Furthermore, the risk of developing HUA was significantly greater among individuals with the highest NHHR quartile than among those with the lowest NHHR quartile (OR = 1.94, 95% CI: 1.62-2.33; P < 0.001). This relationship was consistent across subgroups. According to the RCS analysis, an inverted U-shaped relationship existed between the NHHR and the risk of developing HUA. CONCLUSIONS: The NHHR was closely associated with an increased risk of developing HUA. Further studies on the NHHR could be beneficial for preventing and treating HUA.

Targeting ferroptosis using Chinese herbal compounds to treat respiratory diseases.

BACKGROUND: Respiratory diseases pose a grave threat to human life. Therefore, understanding their pathogenesis and therapeutic strategy is important. Ferroptosis is a novel type of iron-dependent programmed cell death, distinct from apoptosis, necroptosis, and autophagy, characterised by iron, reactive oxygen species, and lipid peroxide accumulation, as well as glutathione (GSH) depletion and GSH peroxidase 4 (GPX4) inactivation. A close association between ferroptosis and the onset and progression of respiratory diseases, including chronic obstructive pulmonary disease, acute lung injury, bronchial asthma, pulmonary fibrosis, and lung cancer, has been reported. Recent studies have shown that traditional Chinese medicine (TCM) compounds exhibit unique advantages in the treatment of respiratory diseases owing to their natural properties and potential efficacy. These compounds can effectively regulate ferroptosis by modulating several key signalling pathways such as system Xc- -GSH-GPX4, NCOA4-mediated ferritinophagy, Nrf2-GPX4, and Nrf2/HO-1, thus playing a positive role in improving respiratory diseases. PURPOSE: This comprehensive review systematically outlines the regulatory role of ferroptosis in the onset and progression of respiratory diseases and provides evidence for treating respiratory diseases by targeting ferroptosis with TCM compounds. These insights aim to offer potential remedies for the clinical prevention and treatment of respiratory diseases. STUDY DESIGN AND METHODS: We searched scientific databases PubMed, Web of Science, Scopus, and CNKI using keywords such as "ferroptosis","respiratory diseases","chronic obstructive pulmonary disease","bronchial asthma","acute lung injury","pulmonary fibrosis","lung cancer","traditional Chinese medicine","traditional Chinese medicine compound","monomer", and "natural product" to retrieve studies on the therapeutic potential of TCM compounds in ameliorating respiratory diseases by targeting ferroptosis. The retrieved data followed PRISMA criteria (preferred reporting items for systematic review). RESULTS: TCM compounds possess unique advantages in treating respiratory diseases, stemming from their natural origins and proven clinical effectiveness. TCM compounds can exert therapeutic effects on respiratory diseases by regulating ferroptosis, which mainly involves modulation of pathways such as system Xc- -GSH-GPX4,NCOA4-mediated ferritinophagy, Nrf2-GPX4, and Nrf2/HO-1. CONCLUSION: TCM compounds have demonstrated promising potential in improving respiratory diseases through the regulation of ferroptosis. The identification of specific TCM-related inducers and inhibitors of ferroptosis holds great significance in developing more effective strategies. However, current research remains confined to animal and cellular studies, emphasizing the imperative for further verifications through high-quality clinical data.

Simultaneous aerobic nitrogen and phosphorus removal by novel halotolerant fungus Mucor circinelloides SNDM1: Function and metabolism pathway.

Fungi capable of simultaneous nitrogen and phosphorus removal from wastewater is rarely found. Here, a novel fungal strain (SNDM1) performing heterotrophic nitrification, aerobic denitrification, and phosphate removal was isolated and identified as Mucor circinelloides. The favorable nutrient removal conditions by the strain using glucose were C/N ratios of 25-30, salinities of 0 %-3 %, and pH of 7.5. Strain SNDM1 achieved ammonium, nitrite, nitrate, and phosphate removal rates of 5.23, 10.08, 4.88, and 0.97 mg/L/h. Nitrogen balance indicated that gaseous (18.60 %-24.55 %) and intracellular nitrogen (43.76 %-70.63 %) were primary fate of initial nitrogen. Enzyme activity revealed that ammonium removal occurred through heterotrophic nitrification and aerobic denitrification. Removed phosphorus was mainly transformed into cell membranes (56 %-64 %) and extracellular polymeric substances (20 %-26 %). Orthophosphate was the major intracellular phosphorus species, while polyphosphate and pyrophosphate existed extracellularly. These findings highlight the potential of this fungal strain for bioremediating polluted wastewater.

Cerebral venous sinus stenting and jugular bulb embolization for pulsatile tinnitus: A case report.

Background: Pulsatile tinnitus (PT) is a rare form of tinnitus that aligns with the heartbeat. It is typically brought on by lesions with significant vascularity, which produce aberrant sound conduction and increase the risk of mental health issues and hearing loss. Venous PT is more prevalent than arterial PT. Open procedures or interventional procedures can be used to treat PT. We present here a case of PT caused by venous luminal stenosis combined with jugular bulb (JB) malformation, which was improved by stenting and JB embolization. Case presentation: A 59-year-old woman presented with long-term tinnitus consistent with heart rhythm and hearing loss, accompanied by anxiety, insomnia, and depression. The results of brain MRV, CT, and DSA showed stenosis of the right sigmoid sinus and high jugular bulb (JB) with dehiscence of the JB wall. The patient saw a significant improvement in PT symptoms following sigmoid sinus stenting and spring coil embolization of the high JB, following the diagnosis of PT. The patient had no PT recurrence for the course of the 31-month follow-up period. Conclusion: In the present PT case, there was a simultaneous onset of the right sigmoid sinus stenosis and the high JB with the JB wall abnormalities. Sigmoid sinus stenting and spring coil embolization of high JB may be a treatment for the PT, but the prevention of post-stenting complications is still an issue that requires great attention and needs further study.

Erdheim­Chester disease of multisystem involvement with delayed diagnosis: A case report and literature review.

Erdheim-Chester disease (ECD) is a rare tumor of histiocytic origin, characterized by foamy or lipid-laden histiocytes mixed or surrounded by fibrosis that infiltrate multiple organs. Misdiagnosis is common due to the diversity of clinical presentations. The present study reported a case of ECD with the involvements of bone, cardiac, aorta and retroperitoneum. The patient had no obvious clinical symptoms and no noteworthy foamy histiocytes or Touton giant cells were found on pathological examination, delaying the diagnosis. The patient was a young male found to have pericardial effusion on physical examination, and computed tomography (CT) revealed soft tissue infiltrates in the retroperitoneum and around the aorta. A mediastinal biopsy revealed fibrous connective tissue with small-vessel hyperplasia and acute-chronic inflammatory cell infiltration. The initial diagnosis was retroperitoneal fibrosis (RPF), and hormonal and tamoxifen treatments were administered. The patient presented with oliguria, eyelid edema and fever four years later. A repeat CT revealed an increase in the extent of tissue infiltration and pericardial effusion compared with the previous CT. Subsequent cardiac magnetic resonance imaging revealed massive thickening in the form of fibrotic tissue infiltrating the heart and surrounding thoracic and abdominal aorta. Single photon emission CT revealed multiple areas of increased bone metabolism, particularly symmetrical involvement of the long bones of both lower extremities. A biopsy of the perirenal tissue revealed fibrous tissue and a small number of lymphocytes and macrophages [typical foamy histiocytes observed via x200 magnification and hematoxylin-eosin (HE) staining, no presence of xanthogranuloma or Touton giant cells]. After a comprehensive evaluation and ruling out other diseases, the diagnosis of ECD was determined. The prognosis of this disease is poor; early diagnosis is critical and requires accurate judgment by clinicians. Biopsies of all involved sites and refinement of genetic tests to guide treatment, if possible, are both necessary.

The role expectations of young women as wives after breast cancer treatment: A qualitative study.

Objectives: Through the reflection of young breast cancer women on their selves and identities, we explored expectations of the wife role that they need to fulfill to return to their families, aimed to provide a reference basis for medical professionals to develop interventions related to cancer family rehabilitation. Methods: Descriptive phenomenological methods and purposive sampling were used. Young breast cancer patients and their spouses were selected for semi-structured face-to-face interviews in the study from March to April 2023 at the department of breast surgery and oncology center of a Class A tertiary hospital in Xuzhou City, China. The interviews were transcribed verbatim and analyzed using Colaizzi's phenomenological approach. Results: Twenty patients and six spouses were interviewed. The mean patient age was (35.95 ± 3.36) years, and the mean spouse was (37.67 ± 5.28) years. Young breast cancer patients were concerned about three main wife expectations during their treatment and rehabilitation: preserving self-love and self-esteem (paying attention to physical health, embracing the disease, and regaining confidence in female characters); adjustment of conjugal relationships (harmonious and effective couple communication, providing support for marriage and love, and creating a beautiful married life together); assisting in family recovery (relieving stress on spouses from caregiving and finances, and management of daily household chores). Conclusions: The wife role expectations of young breast cancer women and their spouses encompass three core aspects: self, couple, and family. Self-esteem and self-love are the most fundamental expectations of the wife role, while adjusting the couple's relationship and assistance in family rehabilitation represent higher expectations. This study can help healthcare professionals and cancer families gain a more comprehensive understanding of the wife role expectations for young cancer women, thereby enabling the development of couple-centered interventions to promote patient recovery and enhance the resilience of marriages and families.

MAGE-A11 is a potential prognostic biomarker and immunotherapeutic target in gastric cancer.

Gastric cancer poses a serious threat to human health and affects the digestive system. The lack of early symptoms and a dearth of effective identification methods make diagnosis difficult, with many patients only receiving a definitive diagnosis at a malignant stage, causing them to miss out on optimal therapeutic interventions. Melanoma-associated antigen-A (MAGE-A) is part of the MAGE family and falls under the cancer/testis antigen (CTA) category. The MAGE-A subfamily plays a significant role in tumorigenesis, proliferation and migration. The expression, prognosis and function of MAGE-A family members in GC, however, remain unclear. Our research and screening have shown that MAGE-A11 was highly expressed in GC tissues and was associated with poor patient prognosis. Additionally, MAGE-A11 functioned as an independent prognostic factor in GC through Cox regression analysis, and its expression showed significant correlation with both tumour immune cell infiltration and responsiveness to immunotherapy. Our data further indicated that MAGE-A11 regulated GC cell proliferation and migration. Subsequently, our findings propose that MAGE-A11 may operate as a prognostic factor, having potential as an immunotherapy target for GC.

Development of a reliable cell-based reporter gene assay to measure the bioactivity of anti-HER2 therapeutic antibodies.

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.

Reduced sensitivity to thyroid hormones is associated with lung function in euthyroid individuals.

Background: The thyroid gland exhibits a subtle interconnection with the lungs. We further investigated the correlation between thyroid hormone sensitivity and lung function in euthyroid individuals. Methods: Data on spirometry and mortality for participants aged 19-79 years were extracted from the NHANES database. Obstructive lung function was defined as a forced expiratory volume in 1 s to forced vital capacity ratio (FEV1/FVC) < 0.70, while restrictive lung function was considered when FEV1/FVC ≥0.70 and baseline FVC <80 % predicted. Central and peripheral sensitivities to thyroid hormones were mainly evaluated by Thyroid Feedback Quantile-based Index (TFQI) and Free Triiodothyronine/Free thyroxine (FT3/FT4) ratio. Logistic regression and subgroup analysis were used to examine potential associations between thyroid hormone sensitivity and lung function. The association between TFQI and all-cause mortality risk was also investigated. Results: A total of 6539 participants were analyzed, 900 with obstructive lung function and 407 with restrictive lung function. The prevalence of impaired lung function, both obstructive and restrictive, increased with higher TFQI levels. Logistic regression analysis showed that increased TFQI and decreased FT3/FT4 levels were independent risk factors for obstructive and restrictive lung function (P < 0.05). After adjusting for the impact of lung function, TFQI (HR = 1.25, 95 % CI 1.00-1.56, P = 0.048) was an independent risk factor for all-cause mortality. Conclusion: Reduced sensitivity to thyroid hormones has been linked to impaired lung function. TFQI and FT3/FT4 are potential epidemiological tools to quantify the role of central and peripheral thyroid resistance in lung function.

An acid-activatable fluorouracil prodrug for colorectal cancer synergistic therapy.

5-Fluorouracil has demonstrated certain efficiency in patients with colorectal cancer. However, significant side effects of use by injection are common. To address this issue defects, a reengineered 5'-deoxy-5-fluorocytidine (DFCR) based drug delivery system (POACa) is developed as a prominent tumor-selective nano-activator. Investigations demonstrate that the constructed nano-activator exhibits good biocompatibility and high therapeutic efficiency in mice with subcutaneous and orthotopic SW-480 colorectal tumors, as its activity is strictly dependent on the tumor-associated acid environment and thymidine phosphorylase. These strategies diminish the off-target toxicity and improve the specificity and sensitivity of human colorectal cancer cells to 5-Fu, obtaining potent efficiency by the combination of H2O2 mediated oxidative stress, calcium overload and 5-Fu-induced chemotherapy (the combination index is 0.11). Overall, the engineered nano-activator exhibits a high therapeutic index in vitro and in vivo. STATEMENT OF SIGNIFICANCE: In this study, we designed and prepared a pH-responsive polymer to synchronously deliver DFCR (5'-deoxy-5-fluorocytidine, a prodrug of 5-Fu), Ca2+ and H2O2. The constructed nano-activator was denoted as POACa. (1) To address the problem of premature leakage of cargo by physical embedding, our research modified the inactive prodrug DFCR through chemical bonding. (2) The activation of the prepared nano-activator was strictly dependent on the tumor-associated acid environment and thymidine phosphorylase, providing the drug delivery system with inherent safety. (3) A distinctly low combination index value (0.11) of CaO2 and DFCR indicated that POACa has a prominent tumor suppression effect by tumor calcium overload sensitized chemotherapy and H2O2 mediated cytotoxicity.

An Assessment of Trends in HIV-1 Prevalence and Incidence and Spatio-Temporal Analyses of HIV-1 Recent Infection Among MSM During the Surveillance Period Between 2018 and 2022 in Sichuan, China.

Background: Men who have sex with men (MSM) is one main type of high-risk activities facilitating HIV-1 transmission in Sichuan province. Previous works on HIV-1 incidence and prevalence among MSM only concentrated before 2018, the situation after that is unknown. In addition, the distribution of hot-spots related to current HIV-1 epidemic is also rarely known among MSM in Sichuan. Objective: To update trends of HIV-1 prevalence and incidence and to visualize hot-spots of ongoing transmission in Sichuan province during surveillance period among MSM between 2018 and 2022. Methods: Limiting Antigen Avidity assay was performed to detect recent infection within new HIV-1 diagnoses founded during surveillance period among MSM. The HIV-1 prevalence and incidence were calculated according to an extrapolation method proposed by publications and guidelines. Trend tests were performed using χ2 tests with linear-by-linear association. The spatial analysis was conducted with ArcGIS 10.7 to figure hot-spots of HIV-1 recent infections among MSM. Results: Between 2018 and 2022, 16,697 individuals participated in HIV-1 MSM sentinel surveillance program, of which 449 samples (98.25%) were tested with LAg-Avidity EIA, and 230 samples were classified as recent infection. Respectively, the overall prevalence and incidence were 2.74% and 3.69% (95% CI: 3.21, 4.16) and both had significant declining trends (p < 0.001). Luzhou city had a highest HIV-1 incidence (10.74%, 95% CI: 8.39, 13.10) over the study period and was recognized as a hot-spot for recent HIV-1 infection among MSM. Conclusion: During the surveillance period, both HIV-1 prevalence and incidence were declining. However, Luzhou city had an unusually high HIV-1 incidence and became an emerging hot-spot of recent HIV-1 infection among MSM. This finding suggested focused attention, cross-regional intervention strategies, and prevention programs are urgently required to curb the spread of ongoing transmission.

Improving product quality and productivity of an antibody-based biotherapeutic using inverted frustoconical shaking bioreactors.

The Chinese hamster ovarian (CHO) cells serve as a common choice in biopharmaceutical production, traditionally cultivated in stirred tank bioreactors (STRs). Nevertheless, the pursuit of improved protein quality and production output for commercial purposes demand exploration into new bioreactor types. In this context, inverted frustoconical shaking bioreactors (IFSB) present unique physical properties distinct from STRs. This study aims to compare the production processes of an antibody-based biotherapeutic in both bioreactor types, to enhance production flexibility. The findings indicate that, when compared to STRs, IFSB demonstrates the capability to produce an antibody-based biotherapeutic with either comparable or enhanced bioprocess performance and product quality. IFSB reduces shear damage to cells, enhances viable cell density (VCD), and improves cell state at a 5-L scale. Consequently, this leads to increased protein expression (3.70 g/L vs 2.56 g/L) and improved protein quality, as evidenced by a reduction in acidic variants from 27.0% to 21.5%. Scaling up the culture utilizing the Froude constant and superficial gas velocity ensures stable operation, effective mixing, and gas transfer. The IFSB maintains a high VCD and cell viability at both 50-L and 500-L scales. Product expression levels range from 3.0 to 3.6 g/L, accompanied by an improved acidic variants attribute of 20.6%-22.7%. The IFSB exhibits superior productivity and product quality, underscoring its potential for incorporation into the manufacturing process for antibody-based biotherapeutics. These results establish the foundation for IFSB to become a viable option in producing antibody-based biotherapeutics for clinical and manufacturing applications.

Highly drug/target-tolerant neutralizing antibody (NAb) assay development through target-based drug depletion and drug-based NAb extraction for an anti-EGFR therapeutic monoclonal antibody.

The reduction of immunogenicity is fundamental for the development of biobetter Erbitux, given that the development of an immune response reduces treatment efficacy and may lead to potential side effects. One of the requirements for the clinical research of a Erbitux biobetter candidate (CMAB009) is to develop a neutralizing antibody (NAb) assay, and sufficient drug and target tolerance for the assay is necessary. Here, we describe the development of a competitive ligand binding (CLB) assay for CMAB009 with high drug and target tolerance through target-based drug depletion and drug-based NAb extraction, the integrated experimental strategy was implemented to simultaneously mitigate drug interference and enhance target tolerance. Following troubleshooting and optimization, the NAb assay was validated for clinical sample analysis with the sensitivity of 92 ng/mL, drug tolerance of 70 µg/mL and target tolerance of 798 ng/mL. The innovative drug depletion and NAb extraction achieved though the combination of drug and target beads would enable the development of reliable NAb assays for many other therapeutics that overcome drug and its target interference for more precise and sensitive NAb assessment.

Rapid Mass Spectrometry-Based Multiattribute Method for Glycation Analysis with Integrated Afucosylation Detection Capability.

The multiattribute method (MAM) has emerged as a powerful tool for simultaneously screening multiple product quality attributes of therapeutic antibodies. One such potential critical quality attribute (CQA) is glycation, a common modification that can impact the heterogeneity, functional activity, and immunogenicity of therapeutic antibodies. However, current methods for monitoring glycation levels in MAM are rare and not sufficiently rapid and accurate. In this study, an improved mass spectrometry (MS)-based MAM was developed to simultaneously monitor glycation and other quality attributes including afucosylation. The method was evaluated using two therapeutic antibodies with different glycosylation site numbers. Treatment with IdeS, Endo F2, and dithiothreitol generated three distinct subunits, and the glycation results obtained were similar to those treated with PNGase F, which is routinely used to release glycans; the sample processing time was greatly reduced while providing additional quality attribute information. The MS-based MAM was also employed to assess the glycation progression following forced glycation in various buffer solutions. A significant increase in oxidation was observed when forced glycation was conducted in an ammonium bicarbonate buffer solution, and a total of 23 potential glycation sites and 4 significantly oxidized sites were identified. Notably, we found that ammonium bicarbonate was found to specifically stimulate oxidation, while glycation had a synergistic effect on oxidation. These findings establish this study as a novel methodology for achieving a technologically advanced platform and concept that enhances the efficacy of product development and quality control, characterized by its broad-spectrum, rapid, and accurate nature.

Biosynthesis of lactones from diols mediated by an artificial flavin.

BACKGROUND: Lactones are important compounds in the field of medicine, material and chemical industry. One of the promising accesses to these flexible scaffolds is NAD(P)+-dependent alcohol dehydrogenases-catalyzed oxidative lactonization of diols, which relies on the construction of an efficient NAD(P)+ regeneration system. RESULTS: In this study, a novel system combining horse liver alcohol dehydrogenase (HLADH) with the synthetic bridged flavin cofactor was established for biosynthesis of lactones. The reaction conditions of this system were optimized and a variety of lactones including chiral lactones were efficiently obtained from various diols. Compared to the previously reported NAD(P)+-regeneration systems, this system showed better regeneration efficiency and product yield. A two-phase system was further applied to solve the problem of product inhibition, and 80% yield was obtained at the condition of 300 mM substrate. CONCLUSIONS: This study provides an efficient method to synthesis of lactones from diols under mild conditions. We believe this system will be a promising alternative to promote the synthesis of other valuable compounds.