Cells secrete extracellular vesicles (EVs) through various biogenesis pathways, resulting in distinct molecular compositions even when originating from the same cells. Analyzing individual EVs is challenging due to the need to overcome issues such as nanoscale size, heterogeneity of EVs, and measurement accuracy. Overcoming these challenges in EV research not only advances the field of biopsy but also enables progress in the biological research of EVs. In this study, we analyze quantifying membrane proteins using a novel methodology based on single-molecule fluorescence spectroscopy. We used TIRF based fluorescence imaging or Fluorescence Correlation Spectroscopy (FCS) to observe antibodies or aptamers binding to single EVs. Our results demonstrate the quantification of various membrane proteins, including CD63 and CD81. Consequently, we develop a membrane protein quantification assay for individual EVs using single-molecule and particle fluorescence imaging spectroscopy. Through this single molecule assay, we will elucidate the distribution of membrane proteins in EVs derived from various tumor cells, potentially enabling their use in future biopsies or quantification studies.
Antibody-based therapies offer significant promise for the treatment of various diseases, including cancer and infectious agents. In cancer immunotherapy, these therapies aim to activate the immune system against tumor cells or inhibit their growth. However, the complex nature of cancer necessitates the exploration of multi-target therapeutic approaches, such as bispecific antibodies. These antibodies offer the potential to target multiple cancer types and variants, potentially overcoming resistance mechanisms. Single-domain antibodies (sdAbs) possess unique characteristics, including a compact size, high stability, and specific antigen recognition, making them ideal building blocks for bispecific antibody development. In this study, we describe the development of a readily manageable and cost-effective sdAb discovery platform utilizing rabbit immunization. Briefly, rabbits were immunized with a recombinant CD3 epsilon domain to elicit an immune response. Subsequently, antigen-specific sdAbs were isolated from the immunoglobulin heavy chain variable region. This accessible sdAb discovery platform has the potential to be applied in a variety of fields, including biological research and therapeutic development.
The development of bispecific antibodies that redirect the cytotoxic activity of CD3+ T cells against tumors is a promising immunotherapy strategy for hematological malignancies and solid tumors. The discovery and development of novel anti-CD3 antibodies are key to the efficacy and safety of the bispecific antibodies. Camelid-derived nanobodies have significant potential in bispecific antibodies as T cell engagers due to their small size, low production cost, high stability, and antigen specificity.
In this study, we identified variable nanobodies specific for the CD3 by immunizing a Vicugna pacos (alpaca) with recombinant human CD3 epsilon domain. In particular, the anti-CD3 Nb 1D10 clone showed a moderate affinity with recombinant human and Macaca fascicularis (cynomolgus monkey) CD3 and binding to CD3+ cell lines. These results highlight the potential of the nanobody for the development of nanobody-based bispecific antibodies as a T cell engager.
Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a critical role in regulating appetite and exhibits increased expression in peripheral insulin-target tissues during obesity. This suggests its potential involvement in obesity-induced pro-inflammatory responses. Selective targeting of peripheral CB1R could offer a novel therapeutic approach to break the link between insulin resistance and metabolic inflammation. However, The widespread distribution of CB1R, including the central nervous system (CNS), presents a challenge. CNS-directed CB1R blockade can lead to severe psychological effects like depression and suicidality. This study investigates the development of peripherally-restricted, high-affinity single-domain antibodies (sdAbs) targeting CB1R for selective appetite modulation. We employed an APG-solubilized, recombinant CB1R for rabbit immunization. Antigen-specific sdAbs were subsequently isolated from the immunoglobulin heavy chain variable region. Biopanning of the resulting phage display library was conducted to identify sdAbs with high binding affinity for CB1R. Our findings demonstrate the development of CB1R-specific sdAbs, potentially offering a novel and targeted strategy for obesity management with minimized CNS exposure and reduced risk of associated psychiatric side effects
Bispecific antibodies (BsAbs) that redirect cytotoxic T lymphocytes (CTLs) via CD3 engagement represent a promising approach in cancer immunotherapy. Here, we explore the development of VNAR-based BsAbs for improved tumor targeting. Shark-derived VNARs possess unique properties such as small size, high stability, and antigen specificity, making them ideal candidates for T cell engagement. We immunized banded houndsharks (Triakis scyllium) with the recombinant human CD3 epsilon domain to isolate VNARs that specifically recognize CD3. The isolated anti-CD3 VNARs showed moderate affinity for recombinant human CD3 and bound to CD3+ cell lines. These results suggest the potential of these VNARs for the construction of VNAR-based BsAbs as T cell engagers, providing a novel avenue for cancer immunotherapy.
The neonatal Fc receptor (FcRn) extends the serum half-life of immunoglobulins G (IgG) through a pH-dependent interaction that protects and recycles IgG during intracellular trafficking. To understand the molecular basis of the prolonged half-life observed with the Fc variant KU-1, this study employs X-ray crystallography and in silico modeling to elucidate the mode of interaction (MOI) between FcRn and KU-1. Deciphering this interaction has the potential to significantly impact the development of KU-1-based biopharmaceuticals, paving the way for enhanced therapeutic efficacy and safety profiles.
CD27, a costimulatory molecule of the TNF receptor superfamily expressed on T lymphocytes, plays a critical role in regulating T cell survival, differentiation, and effector function. Upon binding its ligand, CD70, CD27 signaling enhances T cell proliferation and differentiation into effector and memory T cells. This agonistic activity positions CD27 as a promising target for immunomodulatory cancer therapy. This study investigates the development of therapeutic agents targeting the CD27 for cancer immunotherapy. We employed a recombinant human CD27 extracellular domain to immunize an alpaca (Vicugna pacos), generating antigen-specific single-domain antibodies (nanobodies). Through biopanning using the immunized phage display library, we identified the anti-CD27 cpNb4 clone exhibiting high binding affinity for recombinant human CD27 and specific binding to cell-surface CD27 expressed on CHO-K1 cells. These findings establish cpNb4 as a promising candidate for further investigation, potentially paving the way for its integration into combination immunomodulatory cancer therapy regimens.
Cellular senescence is a permanent cell proliferation arrest compromising cell regeneration and tissue repair process which gradually leads to age-related disorders. The driving factor is considered to be senescence-associated secretory phenotype(SASP) a diverse pro-inflammatory secretory factors exerted to nearby cells resulting in normal cell aging. Targeted therapies with complex drug that can selectively modulate these cells offer promising avenues for anti-aging interventions. In this study, we developed extracellular vesicles(EVs) with specific ligands as a novel drug carrier system aimed to selectively target senescent cells. By attaching ligands on the surface of EVs, we enhanced their affinity for senescent cells over normal cells and the targeting efficiency was assessed using fluorescence-activated cell sorting(FACs). The results demonstrated a significantly higher uptake of modified EVs in senescent cells which suggest that this selective delivery can effectively serve as a precision drug carrier. Future work will focus on loading therapeutic agents that exhibit senolytic or senomorphic activities to these ligand-modified EVs aiming to selectively attenuate cellular senescence.
The accumulation of aggregates of the microtubule-binding protein Tau represents a pathological hallmark in Alzheimer’s disease (AD). While Tau is primarily recognized for its interaction with microtubules, recent findings suggest the presence of Tau clusters near the plasma membrane, potentially serving as binding partners for Axonal Initial Segment (AIS)-related membrane proteins and synaptic proteins. Additionally, during AD, pathogenic tau is known to traverse the membrane via cell-to-cell transport. Furthermore, recently our group identified lipidation as a process enabling Tau’s interaction with the membrane. However, despite tau’s hydrophilic nature, the precise mechanism through which Tau dynamics might fulfill a novel physiological function by facilitating its interaction with hydrophobic lipid membranes remains elusive. In this study, we performed single-molecule imaging with total internal reflection fluorescence microscopy (TIRF) to observe tau dynamics near the plasma membrane of differentiated PC12 cells. Indeed, expression of Tau mutant constructs with inhibited lipidation in PC12 cells resulted in increased mobility of Tau near the plasma membrane. Moreover, treatment with an inhibitor targeting lipidation produced similar effects as observed with Tau mutants, suggesting that lipidation-mediated membrane interaction slows Tau mobility. In primary hippocampal neurons, we observed colocalization of Tau with lipidation-related proteins, and the Proximity Ligation Assay (PLA) confirmed their presence within 40nm proximity. This study introduces a novel post-translational modification mechanism enabling Tau interaction with the membrane. It show that Tau exhibits distinctive dynamic characteristics in close proximity to the plasma membrane, where its interaction with membrane-associated proteins could potentially serve as a potent mechanism for spatially guiding tau towards native membrane-mediated functions.
The trans-activating CRISPR RNA (tracrRNA) is fundamental to the CRISPR/Cas9 system, forming guide RNA with crRNA. Despite its known importance in crRNA maturation and Cas9 RNP-mediated DNA cleavage, the exact function of tracrRNA scaffolds remains unclear. In this investigation, we generated five tracrRNA variants by removing specific scaffolds, including Stem loops 1, 2, and 3, and the Linker. Using a new single-molecule assay, we directly observed target binding and cleavage processes guided by Cas9 RNP. Our findings underscore the vital role of the Linker in initiating R-loops and highlight the significance of Stem loop 2 in identifying PAM-distal mismatches within target DNA. Furthermore, we explored cleavage efficiency by adding tracrRNA segments, indicating that maintaining the integrity of Stem loops 2 and 3 is crucial for potent Cas9 activity. We believe that these results deepen our understanding of Cas9 functionality and offer insights into its detailed mechanism from target binding to cleavage.
Tau, known primarily as a microtubule-binding protein, is also found in the nucleus where it binds to DNA. Recent investigations have focused on its role in stabilizing DNA and chromosomes, but the biophysical understanding of its molecular mechanisms, particularly regarding tau’s phase separation properties, remains limited. In this study, we used in vitro single-molecule assays to show that tau interacts with DNA to form co-condensates, significantly altering the mechanical properties of DNA. Our findings indicate that tau can wet the DNA strand in low-salt conditions, effectively condensing and stiffening the DNA. At high concentrations, tau also forms droplet-shaped condensates on DNA, similar to its interaction with microtubules. Notably, these condensates are mobile and may act as nucleation sites for microtubule growth. This study reveals previously unknown effects of Tau-DNA condensation and suggests that these interactions could influence microtubule dynamics during mitosis.
Extracellular vesicles (EVs) are released from cells and can be taken up by other cells to mediate communication among distant cells. The process of vesicle uptake is initiated by the docking of the vesicles onto membrane proteins on cells, but a generalizable technique for quantitatively observing these vesicle–protein interactions (VPIs) is lacking. Here, we develop a technique that measures VPIs between single vesicles and cell-surface proteins using total internal reflection fluorescence microscopy. We first describe a simple procedure that can effectively label vesicles without complex purification. Subsequently, we quantify the interaction between the labeled vesicles and target proteins either attached to a surface or embedded in a lipid bilayer. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we determine the binding affinity of vesicles toward the ICAM-1 depending on cell types of vesicle origin. Moreover, controlling the surface density of proteins also reveals robust support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle uptake and to guide the development of therapeutic vesicles.
Pharmaceutical and biological researchers consistently explore questions related to protein structures and mutations to better understand virus evolution. In thermodynamics, protein structures are predicted through computational simulations, such as molecular dynamic simulation, which calculates free energy, intermediate states, mutation effects, and protein-protein interactions. However, this novel method has limitations in deciphering complex protein structures. To bridge this gap in protein understanding, machine learning and deep learning are applied to study virus evolution. Notably, escape mutations of SARS-CoV-2 have been predicted using natural language processing techniques, which interpret amino acid sequences in terms of semantic change (antigenic variant) and grammatical quality (viability/fitness). Surprisingly, training models using only amino acid sequences was sufficient to predict escape mutations without additional information on protein structure and function.
Despite the potential of natural language models to suggest possible escape mutations, there is a need to enhance the accuracy of these predictions to minimize the selection of unnecessary candidates. In this study, we evaluated and refined a novel language model by incorporating nucleotide substitutions to improve prediction accuracy. Biologically, amino acid sequences are determined by nucleotide compositions, and most mutations occur at the DNA or RNA level. Although deep learning models might indirectly learn this information from amino acid sequences, integrating direct nucleotide data into the model has resulted in more precise estimations with higher accuracy. This approach has enabled us not only to reduce unnecessary candidates for escape mutations and but also to enhance prediction of characteristic and dominant mutations.
Cellular senescence is a permanent cell proliferation arrest compromising cell regeneration and tissue repair process which gradually leads to age-related disorders. The driving factor is considered to be senescence-associated secretory phenotype(SASP) a diverse pro-inflammatory secretory factors exerted to nearby cells resulting in normal cell aging. Targeted therapies with complex drug that can selectively modulate these cells offer promising avenues for anti-aging interventions. In this study, we developed extracellular vesicles(EVs) with specific ligands as a novel drug carrier system aimed to selectively target senescent cells. By attaching ligands on the surface of EVs, we enhanced their affinity for senescent cells over normal cells and the targeting efficiency was assessed using fluorescence-activated cell sorting(FACs). The results demonstrated a significantly higher uptake of modified EVs in senescent cells which suggest that this selective delivery can effectively serve as a precision drug carrier. Future work will focus on loading therapeutic agents that exhibit senolytic or senomorphic activities to these ligand-modified EVs aiming to selectively attenuate cellular senescence.
Exocytosis is a complex process involving the regulated release of neurotransmitters from presynaptic neurons, and precise control of this process is crucial for neurotransmission. Synapsin and the SNARE (Soluble NSF Attachment Protein Receptor) complex are proteins that play significant roles in regulating exocytosis. Studies have demonstrated that synapsin modulates vesicle release by controlling the movement of vesicles to the active zone, where the SNARE complex facilitates vesicle fusion with the presynaptic membrane. Despite synapsin being the most abundant protein in neurons and both proteins interacting with synaptic vesicles, the role of synapsin in modulating SNARE dynamics remains unclear. In this investigation, we employed magnetic tweezers to probe the interaction between synapsin and the SNARE complex. By exerting controlled forces on individual SNARE complexes in the presence of synapsin, we observed that synapsin can impact the mechanical properties of SNARE, implying a potential role for synapsin in regulating neurotransmitter release through its effects on SNARE dynamics. These findings emphasize the importance of exploring synaspin-SNARE interactions in the nervous system and offer fresh insights into the role of synapsin in neuronal function.
The trans-activating CRISPR RNA (tracrRNA) is fundamental to the CRISPR/Cas9 system, forming guide RNA with crRNA. Despite its known importance in crRNA maturation and Cas9 RNP-mediated DNA cleavage, the exact function of tracrRNA scaffolds remains unclear. In this investigation, we generated five tracrRNA variants by removing specific scaffolds, including Stem loops 1, 2, and 3, and the Linker. Using a new single-molecule assay, we directly observed target binding and cleavage processes guided by Cas9 RNP. Our findings underscore the vital role of the Linker in initiating R-loops and highlight the significance of Stem loop 2 in identifying PAM-distal mismatches within target DNA. Furthermore, we explored cleavage efficiency by adding tracrRNA segments, indicating that maintaining the integrity of Stem loops 2 and 3 is crucial for potent Cas9 activity. We believe that these results deepen our understanding of Cas9 functionality and offer insights into its detailed mechanism from target binding to cleavage.
Audio-to-talking face generation stands at the forefront of advancements in generative AI. It bridges the gap between audio and visual representations by generating synchronized and realistic talking faces. This significantly improves human-computer interaction and content accessibility for diverse audiences. Despite substantial research in this area, critical challenges such as the lack of realistic facial animations, inaccurate audio-lip synchronization, and intensive computational demands continue to restrict the practicality of the talking face generation methods applications. To address these issues, we introduce a novel approach leveraging the emerging capabilities of Stable diffusion models and vision Transformers for Talking face generation (StableTalk). By incorporating the Re-attention mechanism and adversarial loss into StableTalk, we have markedly enhanced the audio-lip alignment and the consistency of facial animations across frames. More importantly, we have optimized computational efficiency by refining operations within the latent space and dynamically adjusting the visual focus based on the given conditions. Our experimental results demonstrate that StableTalk surpasses existing methods in terms of image quality, audio-lip synchronization, and computational efficiency.
The eukaryotic cell cycle, a pivotal biological process, has been extensively studied and
mathematically modelled in recent decades. Despite concerted efforts, identifying the minimal gene set essential for orderly cell cycle progression remains elusive. Synthetic biology, renowned for genetic engineering applications, also provides a pathway for addressing fundamental biological queries through “learning from building.” The Synthetic Yeast Genome (Sc2.0) project exemplifies this by synthesising Saccharomyces cerevisiae’s genome with changes that advance our understanding of eukaryotic genomes.
Expanding from Sc2.0’s groundwork, we aim to pioneer synthetic yeast genomes that are
minimal, modular, and reprogrammable. As a proof-of-concept, we constructed a synthetic
genome module housing nine of the key cell cycle genes. Employing CRISPR, we
systematically deleted these genes from their native loci and reinserted them together as a
synthetic gene cluster. While individually non-essential, the combined absence of all nine
genes renders this synthetic module indispensable.
Through Cre/loxP-mediated recombination, we investigated the gene combinations necessary for yeast cell cycle progression. Cre recombinase facilitated targeted gene deletions between intergenic loxP sites within the module, and rapidly generated diverse strains with combinatorial cluster deletion profiles, covering all potential combinations. Using flow cytometry sorting, we developed a way to isolate hundreds of viable deletion combinations and developed the Pool of Long Amplified Reads (POLAR) sequencing technique to enable the analysis of gene deletion frequency and gene content combinations for hundreds of strains with different cell cycle modules. These experimental findings were compared to computational models of the cell cycle and get us closer to understanding the minimal gene content for this function.
Upon pioneering this work, we now envisage a future where genome designers can predict
gene sets necessary for specialised tasks and can then synthetically arrange these genes on chromosomes and design intergenic regions to regulate their gene expression appropriately.
International commerce is a sphere where well-built customs rules are crucial. Nevertheless, due to existence of illegal acts and fraudulent undertakings, there is an urge for safety and economic soundness in customs controls. India’s customs service and related organizations employ artificial intelligence-based technologies that aid in combating illegal trade globally. The paper examines how AI can be used to identify people who misuse technology for illicit imports or exports. These evaluations also demonstrate how border control has become more dependent on AI, identify major concerns, and predict future trends. AI may provide an opportunity to strengthen border security as well as expedite legal business relations.
People quickly recognise human actions carried out in everyday activities. There is evidence that Minimal Recognisable Configurations (MIRCs) contain a combination of spatial and temporal visual features critical for reliable recognition. For complex activities, observers may have different descriptions varied in their semantic similarity (e.g., washing dishes vs cleaning dishes), potentially complicating the investigation of MIRCs in action recognition. Therefore, we measured the semantic consistency for 128 short videos of complex actions from the Epic-Kitchens-100 dataset (Damen et al., 2022), selected based on poor classification performance by our state-of-the-art computer vision network MOFO (Ahmadian et al., 2023). In an online experiment, participants viewed each video and identified the performed action by typing a description using 2-3 words (capturing action and object). Each video was classified by at least 30 participants (N=76 total). Semantic consistency of the responses was determined using a custom pipeline involving the sentence-BERT language model, which generated embedding vectors representing semantic properties of the responses. We then used adjusted pair-wise cosine similarities between response vectors to compute a ground truth description for each video, a response with the greatest semantic neighbourhood density (e.g., pouring oil, closing shelf). The greater the semantic neighbourhood density was for a ground truth candidate, the more semantically consistent were responses for the associated video. We uncovered 87 videos where semantic consistency confirmed their reliable recognisability, i.e. where cosine-similarity between the ground truth candidate and at least 70% of responses was above a similarity threshold of 0.65. We will use a subsample of these videos to investigate the role of MIRCs in human action recognition, e.g., gradually degrading the spatial and temporal information in videos and measuring the impact on action recognition. The derived semantic space and MIRCs will be used to revise MOFO into a more biologically consistent and better performing model.