Characterization of antigen presentation capability for neoantigen-based products using targeted LC-MS/MS method.

The generation of an immune response in neoantigen-based products relies on antigen presentation, which is closely analyzed by bioassays for T-cell functions such as tetramer or cytokine release. Mass spectrometry (MS) has the potential to directly assess the antigen-presenting capability of antigen-presenting cells (APCs), offering advantages such as speed, multi-target analysis, robustness, and ease of transferability. However, it has not been used for quality control of these products due to challenges in sensitivity, including the number of cells and peptide diversity. In this study, we describe the development and validation of an improved targeted LC-MS/MS method with high sensitivity for characterizing antigen presentation, which could be applied in the quality control of neoantigen-based products. The parameters for the extraction were carefully optimized by different short peptides. Highly sensitive targeted triple quadrupole mass spectrometry combined with ultra-high performance liquid chromatography (UHPLC) was employed using a selective ion monitoring mode (Multiple Reaction Monitoring, MRM). Besides, we successfully implemented robust quality control peptides to ensure the reliability and consistency of this method, which proved invaluable for different APCs. With reference to the guidelines from ICH Q2 (R2), M10, as well as considering the specific attributes of the product itself, we validated the method for selectivity, specificity, sensitivity, limit of detection (LOD), recovery rate, matrix effect, repeatability, and application in dendritic cells (DCs) associated with neoantigen-based products. The validation process yields satisfactory results. Combining this approach with T cell assays will comprehensively assess cell product quality attributes from physicochemical and biological perspectives.

An Adaptive ORB-SLAM3 System for Outdoor Dynamic Environments.

Recent developments in robotics have heightened the need for visual SLAM. Dynamic objects are a major problem in visual SLAM which reduces the accuracy of localization due to the wrong epipolar geometry. This study set out to find a new method to address the low accuracy of visual SLAM in outdoor dynamic environments. We propose an adaptive feature point selection system for outdoor dynamic environments. Initially, we utilize YOLOv5s with the attention mechanism to obtain a priori dynamic objects in the scene. Then, feature points are selected using an adaptive feature point selector based on the number of a priori dynamic objects and the percentage of a priori dynamic objects occupied in the frame. Finally, dynamic regions are determined using a geometric method based on Lucas-Kanade optical flow and the RANSAC algorithm. We evaluate the accuracy of our system using the KITTI dataset, comparing it to various dynamic feature point selection strategies and DynaSLAM. Experiments show that our proposed system demonstrates a reduction in both absolute trajectory error and relative trajectory error, with a maximum reduction of 39% and 30%, respectively, compared to other systems.

GMP-grade microcarrier and automated closed industrial scale cell production platform for culture of MSCs.

Efficient and large-scale expansion of mesenchymal stem/stromal cells (MSCs) has always been a formidable challenge to researchers in cell-based therapies and regenerative medicine. To reconcile major drawbacks of 2D planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on GMP-grade microcarrier for culture of umbilical cord-mesenchymal stem/stromal cells (UCMSCs), in accordance with the criteria of stem cell bank. ACISCP system is a fully closed system, which employs different models of vivaSPIN bioreactors (CytoNiche Biotech, China) for scale-up cell culture and vivaPREP (CytoNiche Biotech, China) for automated cell harvesting and cell dosage preparation. To realize industrial scale expansion of UCMSCs, a three-stage expansion was conducted with 1 L, 5 and 15 L vivaSPIN bioreactors. Using 3D TableTrix® and ACISCP system, we inoculated 1.5 × 107 of UCMSCs into 1 L vivaSPIN bioreactor and finally scaled to two 15 L bioreactor. A final yield of 2.09 × 1010 cells with an overall expansion factor of 1975 within 13 days. The cells were harvested, concentrated, washed and prepared automatically with vivaPREP. The entire process was realized with ACISCP platform and was totally enclosed. Critical quality attributes (CQA) assessments and release tests of MSCs, including sterility, safety, purity, viability, identity, stability and potency were performed accordingly. The quality of cells harvested from 3D culture on the ACISCP and conventional 2D planar culture counterpart has no significant difference. This study provides a bioprocess engineering platform, harnessing GMP-grade 3D TableTrix® microcarriers and ACISCP to achieve industrial-scale manufacturing of clinical-grade hMSCs.

Oxygen vacancy engineering of novel ultrathin Bi12O17Br2 nanosheets for boosting photocatalytic N2 reduction.

The conversion of N2 to NH3 is one of the most promising processes in maintaining natural life and chemical production. Photocatalytic nitrogen reduction reaction (NRR) has the advantage of clean and sustainable, which is considered to be an ideal synthesis technology. In this work, we report the successful synthesis of Bi12O17Br2 ultrathin nanosheets through simple alkali treatment and solvothermal method. The Bi12O17Br2 ultrathin nanosheets can improve the separation of carriers and the transfer of photogenerated electrons to N2 molecules, thus improving the photocatalytic efficiency. Of note, the higher Bi/Br atomic ratio in Bi12O17Br2 is beneficial to broaden the light absorption edge, and the high concentration of O atoms is easy to produce oxygen vacancies on the surface during the synthesis process of Bi12O17Br2. The abundant oxygen vacancies and high specific surface area enable N2 molecules and water to have powerful chemical adsorption and activation. In addition, the photocatalytic reduction of N2 to NH3 in pure water shows excellent and stable performance, and the average generation rate of NH3 reaches up to 620.5 µmol·L-1·h-1. This study discovers that rich oxygen vacancies and ultrathin morphology may have a significant part in the process of the photocatalytic nitrogen reduction reaction.

Requirements for human-induced pluripotent stem cells.

'Requirements for Human-Induced Pluripotent Stem Cells' is the first set of guidelines on human-induced pluripotent stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, and instructions for use, labeling, packaging, storage, transportation, and waste handling for human-induced pluripotent stem cells, which apply to the production and quality control of human-induced pluripotent stem cells. It was released by the Chinese Society for Cell Biology on 9 January 2021 and came into effect on 9 April 2021. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human-induced pluripotent stem cells for applications.

Deconstructing transcriptional variations and their effects on immunomodulatory function among human mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapies are being actively investigated in various inflammatory disorders. However, functional variability among MSCs cultured in vitro will lead to distinct therapeutic efficacies. Until now, the mechanisms behind immunomodulatory functional variability in MSCs are still unclear. METHODS: We systemically investigated transcriptomic variations among MSC samples derived from multiple tissues to reveal their effects on immunomodulatory functions of MSCs. We then analyzed transcriptomic changes of MSCs licensed with INFγ to identify potential molecular mechanisms that result in distinct MSC samples with different immunomodulatory potency. RESULTS: MSCs were clustered into distinct groups showing different functional enrichment according to transcriptomic patterns. Differential expression analysis indicated that different groups of MSCs deploy common regulation networks in response to inflammatory stimulation, while expression variation of genes in the networks could lead to different immunosuppressive capability. These different responsive genes also showed high expression variability among unlicensed MSC samples. Finally, a gene panel was derived from these different responsive genes and was able to regroup unlicensed MSCs with different immunosuppressive potencies. CONCLUSION: This study revealed genes with expression variation that contribute to immunomodulatory functional variability of MSCs and provided us a strategy to identify candidate markers for functional variability assessment of MSCs.

Design and synthesis of sinomenine isoxazole derivatives via 1,3-dipolar cycloaddition reaction.

A novel structure of sinomenine isoxazole derivatives is synthesised from sinomenine hydrochloride and aromatic aldehydes and requires six steps. 19 target compounds have been obtained in good yields. The sinomenine hydrochloride transforms to 4-alkynyl sinomenine, which is a key intermediate product to synthesise the target sinomenine isoxazole compounds, after a neutralisation reaction with ammonia and substitution reaction with 3-chloropropyne. Another key intermediate product is 1,3-dipole, which can be obtained from aromatic aldehyde. After treatment with hydroxylamine hydrochloride and then sodium carbonate solution, aromatic aldehyde is converted to aldehyde oxime, which reacts with N-chlorosuccinimide (NCS) to afford aryl hydroximino chloride. 1,3-Dipole is eventually formed in situ while triethylamine (TEA) in DMF is added dropwise. Then 4-alkynyl sinomenine is added to provide the sinomenine isoxazole derivative via 1,3-dipolar cycloaddition reaction as the key step. All the target compounds are characterised by melting point, 1H NMR, 13C NMR, HRMS and FT-IR spectroscopy.

The DLC-1 tumor suppressor is involved in regulating immunomodulation of human mesenchymal stromal /stem cells through interacting with the Notch1 protein.

BACKGROUND: Immunomodulatory activities of human mesenchymal stromal /stem cells (hMSCs) has been widely recognized as the most critical function of hMSCs for exerting its therapeutic effects. However, the detailed mechanisms responsible for regulating the immunomodulation of hMSCs still remain largely unknown. Previous studies revealed that the Notch1 protein exerted a pro-immunomodulatory function probably through interacting with the protein(s) subjective to proteasome-mediated protein degradation. The DLC-1 protein represents a well characterized tumor suppressor subjective to proteasome-mediated degradation. However, the detailed signaling pathway of Notch1 and the involvement of DLC-1 in regulating the immunomodulation of hMSCs have not been studied before. METHODS: The transfection with cDNA or siRNA into hMSCs assisted by co-culture of hMSCs with peripheral blood mononuclear cells and small molecule inhibitors of signaling proteins, followed by immunoprecipitation, Western blotting, RT-PCR, and flowcytometry, were employed to characterize the Notch1 signaling, to identify DLC-1 as a candidate proteasome-targeted protein, and to characterize DLC-1 signaling pathway and its interaction with the Notch1 signaling, in the regulation of immunomodulation of hMSCs, specifically, the inhibition of pro-inflammatory CD4+-Th1 lymphocytes, and the release of immunomodulatory molecule IDO1. STATISTICAL ANALYSIS: One-way ANOVA was utilized as a statistical tool to analyze the data presented as means ± SEM of at least three separate experiments. RESULTS: The present study revealed that the Notch1-Hey1 axis, but not the Notch1-Hes1 axis, was likely responsible for mediating the pro-immunomodulatory function of the Notch1 signaling. The DLC-1 protein was found subjective to proteasome-mediated protein degradation mediated by the DDB1 and FBXW5 E3 ligases and served as an inhibitor of the immunomodulation of hMSCs through inhibiting Rock1, but not Rock2, downstream the DLC-1 signaling. The Notch1 signaling in the Notch1-Hey1 pathway and the DLC-1 signaling in the DLC-1-Rock1-FBXW5 pathway exhibited a mutual exclusion interaction in the regulation of immunomodulation of hMSCs. CONCLUSIONS: The present study uncovers a novel function of DLC-1 tumor suppressor in regulating the immunomodulation of hMSCs. It also proposes a novel mutual exclusion mechanism between the DLC-1 signaling and the Notch1 signaling that is possibly responsible for fine-tuning the immunomodulation of hMSCs with different clinical implications in hMSCs therapy.

DLC-1 tumor suppressor regulates CD105 expression on human non-small cell lung carcinoma cells through inhibiting TGF-ß1 signaling.

Acquisition of features of mesenchymal cells represents a key step of metastatic progression of cancer cells and searching for mechanisms underlying the acquisition will help design novel clinical strategies for suppressing the metastatic progression. The Deleted in Liver Cancer-1 (DLC-1) gene is a p122/RhoGAP tumor/metastatic suppressor gene. However, the mechanism underlying DLC-1's inhibition of metastasis still remains largely unknown. In this study, we revealed that the DLC-1-deficient, but not the DLC-1-competent, human non-small cell lung carcinoma cells (NSCLCs) could acquire the TGF-ß1-induced expression of CD105, a common surface marker of mesenchymal stem cells, with consequent increase in CD105-associated cell motility. Interestingly, the induced CD105 expression and cell motility were subjected to the inhibition by the DLC-1-RhoA-Rock1 signaling through inhibiting the serine phosphorylation at a linker region, but not at the C-terminus, of the Smad3 protein and Smad3 protein nuclear translocation down the canonical TGF-ß1 signaling. In addition, the evidence suggested that DLC-1 very likely exerted its inhibitory effects on the TGF-ß1 signaling and the associated CD105 acquisition in both the cytoplasm and the nucleus. Consistent to the in vitro findings, a reverse correlation between CD105 and DLC-1 in protein expression was identified in primary NSCLC tissues and their surrounding non-tumor tissues. In summary, this study revealed a novel anti-metastasis mechanism governed by the DLC-1 tumor/metastasis suppressor, thus helping design new diagnostic and therapeutic approaches for NSCLCs.

Dendritic core-shell silica spheres with large pore size for separation of biomolecules.

Monodispersed core-shell silica spheres with fibrous shell structure and tunable pore size were prepared by using a one-pot oil-water biphase method. The pore size could be tuned from 7 nm to 37 nm by using organic solvents with different polarities as oil phase. The spheres synthesized by using benzene as organic solvent had the maximum pore size of 37 nm and possessed a surface area of 61 m2 g-1. The obtained wide pore core-shell silica spheres were applied for rapidly separating small molecules, peptides, small proteins, and large proteins with molecular weight up to 200 kDa. Since the pore size of the core-shell silica spheres was sufficiently large for the free access of all the solutes, sharp and symmetric peaks were obtained. The separation performance was as high as 264,531 plates m-1 for fluorene. The great efficient separation demonstrates that the wide pore core-shell silica spheres have a great potential for rapid analysis of both small and large solutes with high performance liquid chromatography.

The hUC-MSCs cell line CCRC-1 represents a novel, safe and high-yielding HDCs for the production of human viral vaccines.

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.

RIG-I and IL-6 are negative-feedback regulators of STING induced by double-stranded DNA.

The stimulator of interferon genes (STING) protein has emerged as a critical signal transduction molecule in the innate immune response. Sustained activation of the STING signaling induced by cytosolic DNA has been considered to be the cause of a variety of autoimmune diseases characterized by uncontrolled inflammation. Therefore, it is important to understand the molecular basis of the regulation of STING signaling pathway. Here we demonstrate that the STING protein undergoes a proteasome-mediated degradation in human diploid cell (HDC) lines including MRC-5 following the transfection of double-stranded DNA (dsDNA). The degradation of STING is accompanied by the increased expression of both RIG-I and IL-6. Employing the RIG-I siRNA knockdown and an IL-6 neutralizing antibody greatly inhibits the degradation of STING induced by dsDNA. We further demonstrate that both IL-6 and RIG-I are downstream molecules of STING along the DNA sensor pathway. Therefore, STING degradation mediated by RIG-I and IL-6 may serve as a negative feedback mechanism to limit the uncontrolled innate immune response induced by dsDNA. We have further shown that RIG-I and IL-6 promote STING degradation by activating/dephosphorylating UNC-51-like kinase (ULK1). Interestingly, the STING protein is not significantly affected by dsDNA in non-HDC HEK293 cells. Our study thus has identified a novel signaling pathway for regulating STING in HDCs.

EDTA functionalized Fe3O4/graphene oxide for efficient removal of U(VI) from aqueous solutions.

In this study, a regenerable magnetic ligand material (EDTA-mGO) was fabricated by binding chelating groups (EDTA) onto Fe3O4/graphene oxide and applied to remove U(VI) from aqueous solution. The sorption experiments were investigated under different experimental conditions, such as temperature, contact time and pH. EDTA-mGO showed fast removal capacity for U(VI) (<1.5h) with high sorption ability (277.43mg/g). The removal mechanisms mainly attributed to the metal-binding organic ligand (EDTA) and the electrostatic attractions between U(VI) and oxygenic functional groups. In addition, the as-prepared EDTA-mGO dispersed in water could be easily collected after the sorption by the external magnetic field within 10s. The removal of U(VI) by EDTA-mGO followed the pseudo-second-order rate better than the pseudo-first-order. The thermodynamic data indicated the endothermic and the spontaneous nature of U(VI) sorption. Furthermore, the reproducibility studies suggested that EDTA-mGO has a good reusability and a high stability.

Core-shell silica particles with dendritic pore channels impregnated with zeolite imidazolate framework-8 for high performance liquid chromatography separation.

Metal-organic frameworks (MOFs) have been emerged as promising stationary phases for separations. However, the irregular shapes and wide size distribution of MOF particles have led to the high column backpressure and low column efficiency. We described here a kinetic controlling method to deposit zeolitic imidazolate framework (ZIF-8) onto the pore surface using core-shell silica spheres as support. By varying the volume ratio of N,N-dimethylformamide and methanol, the formation speed of ZIF-8 crystals were greatly suppressed, resulting in the growth of very thin layer (∼3.4nm) of ZIF-8 nanocrystals on the pore surface of the spheres instead of freedom growth of micron sized crystals in the solution. Thus prepared hybrid particles combined the merits of high selectivity of MOFs and the high separation performance of core-shell silica spheres. As a result, separation performance as high as 210000 plates/m for a mixture of xylene isomers was achieved.

Rods-on-sphere silica particles for high performance liquid chromatography.

Silica spheres covered with rods perpendicular to the particle surface were prepared by a simple one-pot sol-gel process. Thus prepared rods-on-sphere silica particles possessed core-shell structure. Compared to other core-shell silica particles in which the shell was synthesized by the time-consuming multiple-step layer-by-layer coating technique, the shell of our rods-on-sphere particles was formed by directly grown rods from the silica spheres. The coverage of the rods on the particle surface could be tuned by changing the amount of water in the reaction. The rods on the particle surface increased the surface roughness which may help decreasing the A-term. Therefore, the calcined and modified rods-on-sphere silica particles were packed into stainless steel columns and then assessed for the separation of various samples including small molecules and proteins. In comparison with a commercially available Kromasil column, the pressure of the rods-on-sphere column is much lower under the same separation conditions, while the column efficiency was comparable. The separation results demonstrate that rods-on-sphere silica particles are a type of new and highly promising packing stationary phase for high performance liquid chromatography.

Layer-by-layer assembly of zeolite imidazolate framework-8 as coating material for capillary electrochromatography.

In this work, open-tubular capillary column coated with zeolite imidazolate framework-8 (ZIF-8) nanocrystals was prepared by a layer-by-layer method. The coating was formed by growing ZIF-8 nanocrystals on either bare fused silica capillary wall or the capillary column premodified with amino groups. The shape and the thickness of the coating formed by using these two methods were almost the same. However, the coverage of the ZIF-8 crystals on the bare fused silica capillary wall was higher than that on the capillary column premodified with amino groups. The ZIF-8 coated capillary column was evaluated for open-tubular capillary electrochromatography. The effect of pH value, buffer concentration, and applied voltage on the separation of phenols was investigated. Good separation of nine phenolic isomers was achieved because of the strong interaction between unsaturated Zn sites and phenols. The column performance for o-nitrophenol was as high as 208 860 plates m(-1) . The run-to-run, day-to-day, and column-to-column reproducibility of retention time and resolution for p-nitrophenol and o-nitrophenol were very good with RSDs of less than 6.5%.

Graphene oxide-SiO2 hybrid nanostructure as coating material for capillary electrochromatography.

Graphene oxide (GO) has been considered as a promising stationary phase for chromatographic separation. However, the very strong adsorption of the analytes on the GO surface lead to the severe peak tailing, which in turn resulting in decreased separation performance. In this work, GO and silica nanoparticles hybrid nanostructures (GO/SiO2 NPs@column) were coated onto the capillary inner wall by passing the mixture of GO and silica sol through the capillary column. The successful of coating of GO/SiO2 NPs onto the capillary wall was confirmed by SEM and electroosmotic flow mobilities test. By partially covering the GO surface with silica nanoparticles, the peak tailing was decreased greatly while the unique high shape selectivity arises from the surface of remained GO was kept. Consequently, compared with the column modified with GO (GO@column), the column modified with GO and silica nanoparticles through layer-by-layer method (GO-SiO2 NPs@column), or the column modified with silica nanoparticles (SiO2 NPs@column), GO/SiO2 NPs@column possessed highest resolutions. The GO/SiO2 NPs@column was applied to separate egg white and both acidic and basic proteins as well as three glycoisoforms of ovalbumin were separated in a single run within 36 min. The intra-day, inter-day, and column-to-column reproducibilities were evaluated by calculating the RSDs of the retention of naphthalene and biphenyl in open-tubular capillary electrochromatography. The RSD values were found to be less than 7.1%.

The notch signaling regulates CD105 expression, osteogenic differentiation and immunomodulation of human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a group of multipotent cells with key properties of multi-lineage differentiation, expressing a set of relatively specific surface markers and unique immunomodulatory functions. IDO1, a catabolic enzyme of tryptophan, represents a critical molecule mediating immunomodulatory functions of MSCs. However, the signaling pathways involved in regulating these key properties still remain elusive. To investigate the involvement of Notch signaling as well as other potential signaling pathway(s) in regulating these critical properties of MSCs, we treated human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with γ-secreatase inhibitor I (GSI-I), which inhibits both Notch signaling and ubiquitin-proteasome activities. It was shown that the GSI-I treatment resulted in apoptosis, reduced expression of surface markers CD73, CD90 and CD105, reduced osteogenic differentiation, and reduction of the hUC-MSCs-mediated suppression of Th1 lymphocyte proliferation and the IFN-γ-induced IDO1 expression. Through distinguishing the effects of GSI-I between Notch inhibition and proteasome inhibition, it was further observed that, whereas both Notch inhibition and proteasome inhibition were attributable to the reduced CD105 expression and osteogenic differentiation, but not to the induced apoptosis. However, Notch inhibition, but not proteasome inhibition, only contributed to the significant effect of GSI-I on Th1 proliferation probably through reducing IDO1 promoter activity. In conclusion, the Notch signaling may represent a very important cell signaling capable of regulating multiple critical properties, especially the immunomodulatory functions of MSCs.