Characteristics of T-Cell Receptor Repertoire for Differential Response to Methotrexate Treatment for Rheumatoid Arthritis.

BACKGROUND: Methotrexate (MTX) serves as the initial treatment for rheumatoid arthritis (RA). However, a substantial proportion of RA patients, estimated between 30% and 50%, do not respond positively to MTX. While the T-cell receptor (TCR) is crucial for the immune response during RA, its role in differentiating MTX responsiveness has not been thoroughly investigated. METHODS: This study used next-generation sequencing to analyze the TCR ß-chain complementary determining region sequences in peripheral blood mononuclear cells obtained from RA patients before MTX treatment. This study aimed to compare the characteristics of the TCR repertoire between the MTX responder and non-responder groups. RESULTS: The study identified a significant difference in the TRBV6-6 gene (p = .003) concerning MTX treatment response. Additionally, a significant difference was found in the number of "3" nucleotide deletions at the 5'Jdels site (p = .023) in the VDJ rearrangement. CONCLUSION: These findings suggest distinct TCR repertoire characteristics between MTX responder and non-responder groups among RA patients. This discovery offers new insights into understanding the variable responses of RA patients to MTX therapy.

DNA Origami-Constructed Nanotapes for Sunitinib Adsorption and Inhibition of Renal Clear Carcinoma Cells.

Sunitinib (SUN) is a first-line drug for the treatment of renal clear carcinoma cells by targeting receptor tyrosine kinases (RTK) on the cell membrane. However, the effective delivery of SUN to the cell membrane remains a significant challenge. In this study, we fabricated precisely structured DNA nanotapes with strong surface SUN adhesion, enabling RTK inhibition of renal clear carcinoma cells. In our design, the precisely assembled linear topological six-helical-bundle DNA origami serves as the framework, and positively charged chitosan is adsorbed onto the DNA origami surface, thereby forming DNA nanotapes. The SUN was efficiently loaded onto the surface of the DNA nanotapes by electrostatic interaction. We found that DNA nanotapes exhibit excellent stability in serum. Importantly, DNA nanotapes carrying SUN can achieve prolonged cell membrane retention and inhibit RTK, thereby enhancing cytotoxicity toward 786-0 cells. Taken together, this study provides a promising candidate platform for the efficient delivery of cell membrane receptor inhibitors in anticancer therapy.

Extracellular Vesicle Spherical Nucleic Acids.

Extracellular vesicles (EVs) are naturally occurring vesicles secreted by cells that can transport cargo between cells, making them promising bioactive nanomaterials. However, due to the complex and heterogeneous biological characteristics, a method for robust EV manipulation and efficient EV delivery is still lacking. Here, we developed a novel class of extracellular vesicle spherical nucleic acid (EV-SNA) nanostructures with scalability, programmability, and efficient cellular delivery. EV-SNA was constructed through the simple hydrophobic coassembly of natural EVs with cholesterol-modified oligonucleotides and can be stable for 1 month at room temperature. Based on programmable nucleic acid shells, EV-SNA can respond to AND logic gates to achieve vesicle assembly manipulation. Importantly, EV-SNA can be constructed from a wide range of biological sources EV, enhancing cellular delivery capability by nearly 10-20 times. Compared to artificial liposomal SNA, endogenous EV-SNA exhibited better biocompatibility and more effective delivery of antisense oligonucleotides in hard-to-transfect primary stem cells. Additionally, EV-SNA can deliver functional EVs for immune regulation. As a novel material form, EV-SNA may provide a modular and programmable framework paradigm for EV-based applications in drug delivery, disease treatment, nanovaccines, and other fields.

ECM stiffness affects cargo sorting into MSC-EVs to regulate their secretion and uptake behaviors.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have garnered extensive attention as natural product-based nanomedicines and potential drug delivery vehicles. However, the specific mechanism for regulating MSC-EVs secretion and delivery remains unclear. Here, we demonstrate that extracellular matrix (ECM) stiffness regulates the secretion and delivery of EVs by affecting MSCs' cargo sorting mechanically. Using multi-omics analysis, we found that a decrease in ECM stiffness impeded the sorting of vesicular transport-related proteins and autophagy-related lipids into MSC-EVs, impairing their secretion and subsequent uptake by macrophages. Hence, MSC-EVs with different secretion and uptake behaviors can be produced by changing the stiffness of culture substrates. This study provides new insights into MSC-EV biology and establishes a connection between MSC-EV behaviors and ECM from a biophysical perspective, providing a basis for the rational design of biomedical materials.

Identification of novel protein biomarkers from the blood and urine for the early diagnosis of bladder cancer via proximity extension analysis.

BACKGROUND: Bladder cancer (BC) is a very common urinary tract malignancy that has a high incidence and lethality. In this study, we identified BC biomarkers and described a new noninvasive detection method using serum and urine samples for the early detection of BC. METHODS: Serum and urine samples were retrospectively collected from patients with BC (n = 99) and healthy controls (HC) (n = 50), and the expression levels of 92 inflammation-related proteins were examined via the proximity extension analysis (PEA) technique. Differential protein expression was then evaluated by univariate analysis (p < 0.05). The expression of the selected potential marker was further verified in BC and adjacent tissues by immunohistochemistry (IHC) and single-cell sequencing. A model was constructed to differentiate BC from HC by LASSO regression and compared to the detection capability of FISH. RESULTS: The univariate analysis revealed significant differences in the expression levels of 40 proteins in the serum (p < 0.05) and 17 proteins in the urine (p < 0.05) between BC patients and HC. Six proteins (AREG, RET, WFDC2, FGFBP1, ESM-1, and PVRL4) were selected as potential BC biomarkers, and their expression was evaluated at the protein and transcriptome levels by IHC and single-cell sequencing, respectively. A diagnostic model (a signature) consisting of 14 protein markers (11 in serum and three in urine) was also established using LASSO regression to distinguish between BC patients and HC (area under the curve = 0.91, PPV = 0.91, sensitivity = 0.87, and specificity = 0.82). Our model showed better diagnostic efficacy than FISH, especially for early-stage, small, and low-grade BC. CONCLUSION: Using the PEA method, we identified a panel of potential protein markers in the serum and urine of BC patients. These proteins are associated with the development of BC. A total of 14 of these proteins can be used to detect early-stage, small, low-grade BC. Thus, these markers are promising for clinical translation to improve the prognosis of BC patients.

Directing the Encapsulation of Single Cells with DNA Framework Nucleator-Based Hydrogel Growth.

Encapsulating individual mammalian cells with biomimetic materials holds potential in ex vivo cell culture and engineering. However, current methodologies often present tradeoffs between homogeneity, stability, and cell compatibility. Here, inspired by bacteria that use proteins stably anchored on their outer membranes to nucleate biofilm growth, we develop a single-cell encapsulation strategy by using a DNA framework structure as a nucleator (DFN) to initiate the growth of DNA hydrogels under cell-friendly conditions. We find that among the tested structures, the tetrahedral DFN can evenly and stably reside on cell membranes, effectively initiating hybridization chain reactions which generate homogeneously dense yet flexible single-cell encapsulation for diverse cell lines. The encapsulation persists for up to 72 hours in a serum-containing cell culture environment, representing a ~70-fold improvement compared to encapsulations mediated by single-stranded DNA nucleators. The metabolism and proliferation of the encapsulated cells are suppressed, but can be restored to the original efficiencies upon release, suggesting the superior cell compatibility of the encapsulation. We also find that compared to naked cells, the encapsulated cells exhibit a lower autophagy level after undergoing mechanical stress, suggesting the protective effect of the DNA encapsulation. This method may provide a new tool for ex vivo cell engineering.

Centrifugal Flows Drive Reverse Rotation of Feynman's Sprinkler.

The issue of reversibility in hydromechanical sprinklers that auto-rotate while ejecting fluid from S-shaped tubes raises fundamental questions that remain unresolved. Here, we report on precision experiments that reveal robust and persistent reverse rotation under suction and a model that accounts for the observed motions. We implement an ultralow friction bearing in an apparatus that allows for free rotation under ejection and suction for a range of flow rates and arbitrarily long times. Flow measurements reveal a rocketlike mechanism shared by the reverse and forward modes that involves angular momentum flux, whose subtle manifestation in the reverse case stems from centrifugal effects for flows in curved conduits. These findings answer Feynman's long-standing question by providing quantitatively accurate explanations of both modes, and they suggest further inquiries into flux-based force generation and the roles of geometry and Reynolds number.

Covalently hybridized carbon dots@mesoporous silica nanobeads as a robust and versatile phosphorescent probe for time-resolved biosensing and bioimaging.

Phosphorescence analyses have attracted broad attention due to their remarkable merits of the elimination of auto-fluorescence and scattering light. However, it remains a great challenge to develop novel materials with uniform size and morphology, stability, long lifetime, and aqueous-phase room temperature phosphorescence (RTP) characteristics. Herein, monodisperse and uniform RTP nanobeads were fabricated by an in situ covalent hybridization of carbon dots (CDs) and dendritic mesoporous silicon nanoparticles (DMSNs) via silane hydrolysis. The formation of Si-O-C and Si-C/N covalent bonds is beneficial for the fixation of vibrations and rotations of the luminescent centers. Specially, the nanopores of DMSNs provide a confined area that can isolate the triplet state of CDs from water and oxygen and thus ensure the occurrence of aqueous-phase RTP with an ultra-long lifetime of 1.195 s (seen by the naked eye up to 9 seconds). Through surface modifying folic acid (FA), CDs@DMSNs can serve as a probe to distinguish different cell lines that feature varying FA receptor expression levels. In addition, taking MCF-7 as the model, highly sensitive and quantitative detection (linear range: 103-106 cells per mL) has been achieved via an RTP probe. Furthermore, their potential applications in cellular and in vivo time-gated phosphorescence imaging have been proposed and demonstrated, respectively. This work would provide a new route to design CD-based RTP composites and promote their further applications in the medical and biological fields.

DNA Nanomaterial-Empowered Surface Engineering of Extracellular Vesicles.

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that are critical mediators of intercellular communication. They contain diverse bioactive components, which are promising diagnostic biomarkers and therapeutic agents. Their nanosized membrane-bound structures and innate ability to transport functional cargo across major biological barriers make them promising candidates as drug delivery vehicles. However, the complex biology and heterogeneity of EVs pose significant challenges for their controlled and actionable applications in diagnostics and therapeutics. Recently, DNA molecules with high biocompatibility emerge as excellent functional blocks for surface engineering of EVs. The robust Watson-Crick base pairing of DNA molecules and the resulting programmable DNA nanomaterials provide the EV surface with precise structural customization and adjustable physical and chemical properties, creating unprecedented opportunities for EV biomedical applications. This review focuses on the recent advances in the utilization of programmable DNA to engineer EV surfaces. The biology, function, and biomedical applications of EVs are summarized and the state-of-the-art achievements in EV isolation, analysis, and delivery based on DNA nanomaterials are introduced. Finally, the challenges and new frontiers in EV engineering are discussed.

DNA-Programmed Stem Cell Niches via Orthogonal Extracellular Vesicle-Cell Communications.

Extracellular vesicles (EVs) are natural carriers for intercellular transfer of bioactive molecules, which are harnessed for wide biomedical applications. However, a facile yet general approach to engineering interspecies EV-cell communications is still lacking. Here, the use of DNA to encode the heterogeneous interfaces of EVs and cells in a manner free of covalent or genetic modifications is reported, which enables orthogonal EV-cell interkingdom interactions in complex environments. Cholesterol-modified DNA strands and tetrahedral DNA frameworks are employed with complementary sequences to serve as artificial ligands and receptors docking on EVs and living cells, respectively, which can mediate specific yet efficient cellular internalization of EVs via Watson-Crick base pairing. It is shown that based on this system, human cells can adopt EVs derived from the mouse, watermelon, and Escherichia coli. By implementing several EV-cell circuits, it shows that this DNA-programmed system allows orthogonal EV-cell communications in complex environments. This study further demonstrates efficient delivery of EVs with bioactive contents derived from feeder cells toward monkey female germline stem cells (FGSCs), which enables self-renewal and stemness maintenance of the FGSCs without feeder cells. This system may provide a universal platform to customize intercellular exchanges of materials and signals across species and kingdoms.

Persistent corotation of the large-scale flow of thermal convection and an immersed free body.

Inspired by the superrotation of the Earth's solid core, we investigate the dynamics of a free-rotating body as it interacts with the large-scale circulation (LSC) of the Rayleigh-Bénard thermal convection in a cylindrical container. A surprising and persistent corotation of both the free body and the LSC emerges, breaking the axial symmetry of the system. The corotational speed increases monotonically with the intensity of thermal convection, measured by the Rayleigh number Ra, which is proportional to the temperature difference between the heated bottom and cooled top. The rotational direction occasionally and spontaneously reverses, occurring more frequently at higher Ra. The reversal events follow a Poisson process; it is feasible that flow fluctuations randomly interrupt and reestablish the rotation-sustaining mechanism. This corotation is powered by thermal convection alone and promoted by the addition of a free body, enriching the classical dynamical system.

MTHFR and MTRR Genetic Polymorphism of Methotrexate Therapy Outcomes in Early Rheumatoid Arthritis.

Purpose: Methotrexate (MTX) is used as an anchor drug for the treatment of rheumatoid arthritis (RA) and there may be differences in drug action between genotypes. The purpose of this study was to investigate the relationship between clinical efficacy response and disease activity of MTX monotherapy with methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms. Patients and Methods: In the study, a population of 32 patients in East China with early RA fulfilling the diagnostic standards of the American College of Rheumatology (ACR) were enrolled, all of them received MTX monotherapy. Genotyping of patients MTHFR C677T and A1298C, MTRR A66G using tetra-primer ARMS-PCR method and sanger sequencing to verify its accuracy. Results: The distribution of three polymorphic genotypes that were studied is in accordance with the Hardy-Weinberg genetic equilibrium. The patient pathology variables smoke (OR = 0.088, P = 0.037), drink alcohol (OR = 0.039, P = 0.016) and males (OR = 0.088, P = 0.037) were significantly associated with non-response to MTX. Genotype, allele distribution and genetic statistical models were not found to be related to MTX treatment response and disease activity in both the response groups and non-response groups. Conclusion: Our findings suggest that the MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms may not predict MTX clinical treatment response and disease activity in patients with early RA. The study revealed that smoke, alcohol, and males were possible influential factors for MTX non-response.

3D diversiform dynamic process of microvilli in living cells.

Microvilli are membrane protrusions involved in many membrane-associated physiological processes. Previous studies have focused on the dynamics of individual microvilli, however, the morphological classification of microvilli and the dynamics of microvillar clusters as the basic functional domain remain largely unknown. Here we used atomic force microscopy (AFM) to achieve nanoscale resolution 3D microvilli images of living HeLa cells. We found that there were mainly two subtypes of microvilli with different morphologies and lifecycle that were unequally present on the cell membrane. Employing a strategy to track microvillar cluster movement at nanometer resolution, we further revealed the polymorphic movement of microvillar clusters in 3D. Overall, these data strengthened the morphology and dynamics of cell membranes and associated structures, which provided a new perspective for microvillar function research.

Programmable DNA Hydrogels as Artificial Extracellular Matrix.

The cell microenvironment plays a crucial role in regulating cell behavior and fate in physiological and pathological processes. As the fundamental component of the cell microenvironment, extracellular matrix (ECM) typically possesses complex ordered structures and provides essential physical and chemical cues to the cells. Hydrogels have attracted much attention in recapitulating the ECM. Compared to natural and synthetic polymer hydrogels, DNA hydrogels have unique programmable capability, which endows the material precise structural customization and tunable properties. This review focuses on recent advances in programmable DNA hydrogels as artificial extracellular matrix, particularly the pure DNA hydrogels. It introduces the classification, design, and assembly of DNA hydrogels, and then summarizes the state-of-the-art achievements in cell encapsulation, cell culture, and tissue engineering with DNA hydrogels. Ultimately, the challenges and prospects for cellular applications of DNA hydrogels are delivered.

Exosomes mediate an epithelial-mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy.

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß-2) was used to induce epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co-cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT-promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR-543 was found in exosomes from EMTed RPE cells, and miR-543-enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.

Label-Free and Three-Dimensional Visualization Reveals the Dynamics of Plasma Membrane-Derived Extracellular Vesicles.

Plasma membrane-derived extracellular vesicles (PEVs) are carriers of biological molecules that perform special cell-cell communications. Nevertheless, the characterization of complicated PEV biology is hampered by the failure of current methods, mainly due to lack of specific labels and insufficient resolution. Here, we employed atomic force microscopy and scanning ion conductance microscopy, both capable of three-dimensional nanoscale resolution, for the label-free visualization of the PEV morphology, release, and uptake at the single-vesicle level. Except for classical microvesicles, we observed a cluster-like PEVs subtype in tumor cells. Moreover, both PEV subtype release times positively correlated with size. Through three-dimensional nanoscale imaging, we visualized the multiform PEV-cell interaction behaviors of individual vesicles, which was challenged in conventional PEV imaging. Finally, we developed single-cell manipulation strategies to induce micrometer-sized PEV generation. Collectively, these results revealed the heterogeneous morphology and dynamics of PEVs at the single vesicle level, which provided new insight into the PEV biology.

[Progress in extracellular vesicle imaging methods].

Extracellular vesicles (EVs), including exosomes and microvesicles, are lipid bilayer-enclosed nanovesicles secreted by cells. These EVs are important mediators of intercellular communication by serving as vehicles for transfer of proteins, mRNA, miRNA and lipids between cells. Various visualization methods have been established to explore the characteristics of EVs and their role in physiological and pathological processes. The nanoscale size and high heterogeneity of EVs hamper the identification of their biological characteristics and functions. This review presents a comprehensive overview of EV imaging methods in light of the origin, separation and dynamic tracking of EVs, and the advantages and disadvantages of different imaging strategies are discussed. We believe that studies at the levels of single vesicles and single cells will become the frontier of future researches of EVs.

Spatial high resolution of actin filament organization by PeakForce atomic force microscopy.

OBJECTIVES: To investigate the heterogeneous feature of actin filaments (ACFs) associated with the cellular membrane in HeLa and HCT-116 cells at the nanoscale level. MATERIALS AND METHODS: Fluorescence microscopy coupled with atomic force microscopy (AFM) was used to identify and characterize ACFs of cells. The distribution of ACFs was detected by Fluor-488-phalloidin-labelled actin. The morphology of the ACFs was probed by AFM images. The spatial correlation of the microvilli and ACFs was explored with different forces of AFM loading on cells. RESULTS: Intricate but ordered structures of the actin cytoskeletons associated with cellular membrane were characterized and revealed. Two different layers of ACFs with distinct structural organizations were directly observed in HCT-116 and HeLa cells. Bundle-shaped ACFs protruding the cellular membrane forming the microvilli, and the network ACFs underneath the cellular membrane were resolved with high resolution under near-physiological conditions. Approximately 14 nm lateral resolution was achieved when imaging single ACF beneath the cellular membrane. On the basis of the observed spatial distribution of the ultrastructure of the ACF organization, a model for this organization of ACFs was proposed. CONCLUSIONS: We revealed the two layers of the ACF organization in Hela and HCT-116 cells. The resolved heterogeneous structures at the nanoscale level provide a spatial view of the ACFs, which would contribute to the understanding of the essential biological functions of the actin cytoskeleton.

Atomic force microscopy-based single-molecule force spectroscopy detects DNA base mismatches.

Atomic force microscopy-based single-molecule-force spectroscopy is limited by low throughput. We introduce addressable DNA origami to study multiple target molecules. Six target DNAs that differed by only a single base-pair mismatch were clearly differentiated a rupture force of only 4 pN.

Starvation effect on the morphology of microvilli in HeLa cells.

Microvilli are membrane protrusions enabling the increase of the cell surface area by over hundreds fold, thereby enhancing nutrition absorption. However, the correlation between the morphology of the microvilli and absorption capability of cells remains elusive. Herein, by combining atomic force microscopy with fluorescence microscopy, we explored the effects of starvation on the morphology of microvilli in HeLa cells at the single cell level. We found that there is an increasing signal of dextran absorption after starvation, and importantly, we observed a significant increase in the number of single microvillus and the lamella-shaped microvilli on the surface of HeLa cells. In addition, we also found reversible changes in the morphology of microvilli under starvation stress and after relief from starvation. These phenomena indicate that the morphology of the microvilli plays a crucial role in absorption capacity of HeLa cells. Our finding provides direct evidence for comprehensive understanding the function of microvilli.