Metabolic regulation of homologous recombination repair by MRE11 lactylation.

Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.

SUMOylation of HNRNPA2B1 modulates RPA dynamics during unperturbed replication and genotoxic stress responses.

Replication protein A (RPA) is a major regulator of eukaryotic DNA metabolism involved in multiple essential cellular processes. Maintaining appropriate RPA dynamics is crucial for cells to prevent RPA exhaustion, which can lead to replication fork breakage and replication catastrophe. However, how cells regulate RPA availability during unperturbed replication and in response to stress has not been well elucidated. Here, we show that HNRNPA2B1SUMO functions as an endogenous inhibitor of RPA during normal replication. HNRNPA2B1SUMO associates with RPA through recognizing the SUMO-interacting motif (SIM) of RPA to inhibit RPA accumulation at replication forks and impede local ATR activation. Declining HNRNPA2SUMO induced by DNA damage will release nuclear soluble RPA to localize to chromatin and enable ATR activation. Furthermore, we characterize that HNRNPA2B1 hinders homologous recombination (HR) repair via limiting RPA availability, thus conferring sensitivity to PARP inhibitors. These findings establish HNRNPA2B1 as a critical player in RPA-dependent surveillance networks.

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology.

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Tandem Deubiquitination and Acetylation of SPRTN Promotes DNA-Protein Crosslink Repair and Protects against Aging.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequences of SPRTN deubiquitination remain unclear. Here we report that, in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. Deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model where two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and a healthy lifespan.

Alternative splicing of a potato disease resistance gene maintains homeostasis between growth and immunity.

Plants possess a robust and sophisticated innate immune system against pathogens and must balance growth with rapid pathogen detection and defense. The intracellular receptors with nucleotide-binding leucine-rich repeat (NLR) motifs recognize pathogen-derived effector proteins and thereby trigger the immune response. The expression of genes encoding NLR receptors is precisely controlled in multifaceted ways. The alternative splicing (AS) of introns in response to infection is recurrently observed but poorly understood. Here we report that the potato (Solanum tuberosum) NLR gene RB undergoes AS of its intron, resulting in two transcriptional isoforms, which coordinately regulate plant immunity and growth homeostasis. During normal growth, RB predominantly exists as intron-retained isoform RB_IR, encoding a truncated protein containing only the N-terminus of the NLR. Upon late blight infection, the pathogen induces intron splicing of RB, increasing the abundance of RB_CDS, which encodes a full-length and active R protein. By deploying the RB splicing isoforms fused with a luciferase reporter system, we identified IPI-O1 (also known as Avrblb1), the RB cognate effector, as a facilitator of RB AS. IPI-O1 directly interacts with potato splicing factor StCWC15, resulting in altered localization of StCWC15 from the nucleoplasm to the nucleolus and nuclear speckles. Mutations in IPI-O1 that eliminate StCWC15 binding also disrupt StCWC15 re-localization and RB intron splicing. Thus, our study reveals that StCWC15 serves as a surveillance facilitator that senses the pathogen-secreted effector and regulates the trade-off between RB-mediated plant immunity and growth, expanding our understanding of molecular plant-microbe interactions.

Paracrine Wnt5a-ß-Catenin Signaling Triggers a Metabolic Program that Drives Dendritic Cell Tolerization.

Despite recent advances, many cancers remain refractory to available immunotherapeutic strategies. Emerging evidence indicates that the tolerization of local dendritic cells (DCs) within the tumor microenvironment promotes immune evasion. Here, we have described a mechanism by which melanomas establish a site of immune privilege via a paracrine Wnt5a-ß-catenin-peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway that drives fatty acid oxidation (FAO) in DCs by upregulating the expression of the carnitine palmitoyltransferase-1A (CPT1A) fatty acid transporter. This FAO shift increased the protoporphyrin IX prosthetic group of indoleamine 2,3-dioxgenase-1 (IDO) while suppressing interleukin(IL)-6 and IL-12 cytokine expression, culminating in enhanced IDO activity and the generation of regulatory T cells. We demonstrated that blockade of this pathway augmented anti-melanoma immunity, enhanced the activity of anti-PD-1 antibody immunotherapy, and suppressed disease progression in a transgenic melanoma model. This work implicates a role for tumor-mediated metabolic reprogramming of local DCs in immune evasion and immunotherapy resistance.

PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.

Programmed death ligand 1 (PD-L1, also called B7-H1) is an immune checkpoint protein that inhibits immune function through its binding of the programmed cell death protein 1 (PD-1) receptor. Clinically approved antibodies block extracellular PD-1 and PD-L1 binding, yet the role of intracellular PD-L1 in cancer remains poorly understood. Here, we discovered that intracellular PD-L1 acts as an RNA binding protein that regulates the mRNA stability of NBS1, BRCA1, and other DNA damage-related genes. Through competition with the RNA exosome, intracellular PD-L1 protects targeted RNAs from degradation, thereby increasing cellular resistance to DNA damage. RNA immunoprecipitation and RNA-seq experiments demonstrated that PD-L1 regulates RNA stability genome-wide. Furthermore, we developed a PD-L1 antibody, H1A, which abrogates the interaction of PD-L1 with CMTM6, thereby promoting PD-L1 degradation. Intracellular PD-L1 may be a potential therapeutic target to enhance the efficacy of radiotherapy and chemotherapy in cancer through the inhibition of DNA damage response and repair.

ENL reads histone ß-hydroxybutyrylation to modulate gene transcription.

Histone modifications are typically recognized by chromatin-binding protein modules (referred to as 'readers') to mediate fundamental processes such as transcription. Lysine ß-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drive cell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone ß-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

Elucidating the binding partners of histone post-translational modifications (hPTMs) is key to understanding epigenetic regulatory pathways. Lysine ß-hydroxybutyrylation (Kbhb) is a novel hPTM that couples metabolism to transcription. However, the effectors reading this mark are poorly understood as the Kbhb-mediated protein­protein interactions are weak and transient. Here, we presented a quantitative chemical proteomics approach using multivalent photoaffinity probes to robustly capture interactors of this mark. Thus, we identified ENL as a novel binder of Kbhb of histone H3 lysine 9 (H3K9bhb). Biochemical studies and CUT&Tag analysis further revealed that ENL recognizes H3K9bhb and co-localizes with it on gene promoters to modulate transcription and tumorigenesis. This study highlights ENL as a histone Kbhb reader for the regulation of transcription.

A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells.

At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which were co-localized with molecules in the autophagosome system. A protease inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the aggresome formation of p.G64R-DOK7. To match the differentiation levels between patient-derived and control induced pluripotent stem cells (iPSCs), we corrected c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made aggresome to a less extent compared with p.G64R in transfected COS7 cells. These results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely to compromise MuSK phosphorylation for AChR clustering.

Directed evolution of adeno-associated virus for efficient gene delivery to microglia.

As the resident immune cells in the central nervous system (CNS), microglia orchestrate immune responses and dynamically sculpt neural circuits in the CNS. Microglial dysfunction and mutations of microglia-specific genes have been implicated in many diseases of the CNS. Developing effective and safe vehicles for transgene delivery into microglia will facilitate the studies of microglia biology and microglia-associated disease mechanisms. Here, we report the discovery of adeno-associated virus (AAV) variants that mediate efficient in vitro and in vivo microglial transduction via directed evolution of the AAV capsid protein. These AAV-cMG and AAV-MG variants are capable of delivering various genetic payloads into microglia with high efficiency, and enable sufficient transgene expression to support fluorescent labeling, Ca2+ and neurotransmitter imaging and genome editing in microglia in vivo. Furthermore, single-cell RNA sequencing shows that the AAV-MG variants mediate in vivo transgene delivery without inducing microglia immune activation. These AAV variants should facilitate the use of various genetically encoded sensors and effectors in the study of microglia-related biology.

A modified Agrobacterium-mediated transformation for two oomycete pathogens.

Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.

Origin, loss, and regain of self-incompatibility in angiosperms.

The self-incompatibility (SI) system with the broadest taxonomic distribution in angiosperms is based on multiple S-locus F-box genes (SLFs) tightly linked to an S-RNase termed type-1. Multiple SLFs collaborate to detoxify nonself S-RNases while being unable to detoxify self S-RNases. However, it is unclear how such a system evolved, because in an ancestral system with a single SLF, many nonself S-RNases would not be detoxified, giving low cross-fertilization rates. In addition, how the system has been maintained in the face of whole-genome duplications (WGDs) or lost in other lineages remains unclear. Here we show that SLFs from a broad range of species can detoxify S-RNases from Petunia with a high detoxification probability, suggestive of an ancestral feature enabling cross-fertilization and subsequently modified as additional SLFs evolved. We further show, based on its genomic signatures, that type-1 was likely maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through S-locus deletions or by retaining duplications. Two deletion lineages regained SI through type-2 (Brassicaceae) or type-4 (Primulaceae), and one duplication lineage through type-3 (Papaveraceae) mechanisms. Thus, our results reveal a highly dynamic process behind the origin, maintenance, loss, and regain of SI.

Single-cell transcriptomic profiling of the neonatal oviduct and uterus reveals new insights into upper Müllerian duct regionalization.

The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies.

Syk-dependent homologous recombination activation promotes cancer resistance to DNA targeted therapy.

Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase (PARP) inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase acknowledged for its regulatory roles in immune cell function, cell adhesion, and vascular development. This study presents evidence indicating that Syk expression in high-grade serous ovarian cancer and triple-negative breast cancers promotes DNA double-strand break resection, homologous recombination (HR), and subsequent therapeutic resistance. Our investigations reveal that Syk is activated by ATM following DNA damage and is recruited to DNA double-strand breaks by NBS1. Once localized to the break site, Syk phosphorylates CtIP, a pivotal mediator of resection and HR, at Thr-847 to promote repair activity, particularly in Syk-expressing cancer cells. Inhibition of Syk or its genetic deletion impedes CtIP Thr-847 phosphorylation and overcomes the resistant phenotype. Collectively, our findings suggest a model wherein Syk fosters therapeutic resistance by promoting DNA resection and HR through a hitherto uncharacterized ATM-Syk-CtIP pathway. Moreover, Syk emerges as a promising tumor-specific target to sensitize Syk-expressing tumors to PARP inhibitors, radiation and other DNA-targeted therapies.

MxB inhibits long interspersed element type 1 retrotransposition.

Long interspersed element type 1 (LINE-1, also L1 for short) is the only autonomously transposable element in the human genome. Its insertion into a new genomic site may disrupt the function of genes, potentially causing genetic diseases. Cells have thus evolved a battery of mechanisms to tightly control LINE-1 activity. Here, we report that a cellular antiviral protein, myxovirus resistance protein B (MxB), restricts the mobilization of LINE-1. This function of MxB requires the nuclear localization signal located at its N-terminus, its GTPase activity and its ability to form oligomers. We further found that MxB associates with LINE-1 protein ORF1p and promotes sequestration of ORF1p to G3BP1-containing cytoplasmic granules. Since knockdown of stress granule marker proteins G3BP1 or TIA1 abolishes MxB inhibition of LINE-1, we conclude that MxB engages stress granule components to effectively sequester LINE-1 proteins within the cytoplasmic granules, thus hindering LINE-1 from accessing the nucleus to complete retrotransposition. Thus, MxB protein provides one mechanism for cells to control the mobility of retroelements.

Low-Temperature and Fast-Charging Lithium Metal Batteries Enabled by Solvent-Solvent Interaction Mediated Electrolyte.

Lithium metal batteries utilizing lithium metal as the anode can achieve a greater energy density. However, it remains challenging to improve low-temperature performance and fast-charging features. Herein, we introduce an electrolyte solvation chemistry strategy to regulate the properties of ethylene carbonate (EC)-based electrolytes through intermolecular interactions, utilizing weakly solvated fluoroethylene carbonate (FEC) to replace EC, and incorporating the low-melting-point solvent 1,2-difluorobenzene (2FB) as a diluent. We identified that the intermolecular interaction between 2FB and solvent can facilitate Li+ desolvation and lower the freezing point of the electrolyte effectively. The resulting electrolyte enables the LiNi0.8Co0.1Mn0.1O2||Li cell to operate at -30 °C for more than 100 cycles while delivering a high capacity of 154 mAh g-1 at 5.0C. We present a solvation structure and interfacial model to analyze the behavior of the formulated electrolyte composition, establishing a relationship with cell performance and also providing insights for the electrolyte design under extreme conditions.

Tinnitus classification based on resting-state functional connectivity using a convolutional neural network architecture.

OBJECTIVES: Many studies have investigated aberrant functional connectivity (FC) using resting-state functional MRI (rs-fMRI) in subjective tinnitus patients. However, no studies have verified the efficacy of resting-state FC as a diagnostic imaging marker. We established a convolutional neural network (CNN) model based on rs-fMRI FC to distinguish tinnitus patients from healthy controls, providing guidance and fast diagnostic tools for the clinical diagnosis of subjective tinnitus. METHODS: A CNN architecture was trained on rs-fMRI data from 100 tinnitus patients and 100 healthy controls using an asymmetric convolutional layer. Additionally, a traditional machine learning model and a transfer learning model were included for comparison with the CNN, and each of the three models was tested on three different brain atlases. RESULTS: Of the three models, the CNN model outperformed the other two models with the highest area under the curve, especially on the Dos_160 atlas (AUC = 0.944). Meanwhile, the model with the best classification performance highlights the crucial role of the default mode network, salience network, and sensorimotor network in distinguishing between normal controls and patients with subjective tinnitus. CONCLUSION: Our CNN model could appropriately tackle the diagnosis of tinnitus patients using rs-fMRI and confirmed the diagnostic value of FC as measured by rs-fMRI.

Copper-Catalyzed Asymmetric Remote C(sp3)-H Alkylation of N-Fluorocarboxamides with Glycine Derivatives and Peptides.

Saturated hydrocarbon bonds are ubiquitous in organic molecules; to date, the selective functionalization of C(sp3)-H bonds continues to pose a notorious difficulty, thereby garnering significant attention from the synthetic chemistry community. During the past several decades, a wide array of powerful new methodologies has been developed to enantioselectively modify C(sp3)-H bonds that is successfully applied in asymmetric formation of diverse bonds, including C-C, C-N, and C-O bonds; nevertheless, the asymmetric C(sp3)-H alkylation is elusive and, therefore, far less explored. In this work, we report a direct and robust strategy to construct highly valuable enantioenriched unnatural α-amino acid (α-AA) cognates and peptides by a copper-catalyzed enantioselective remote C(sp3)-H alkylation of N-fluorocarboxamides and readily accessible glycine esters under ambient conditions. The key to success lies in the optically active Cu catalyst generated through the coordination of glycine derivatives to enantiopure bisphosphine/Cu(I) species, which is beneficial to the single electronic reduction of N-fluorocarboxamides and the subsequent stereodetermining alkylation. More importantly, all types (primary, secondary, tertiary, and even α-oxy) of δ-C(sp3)-H bonds could be site- and stereospecifically activated by the kinetically favored 1,5-hydrogen atom transfer (1,5-HAT) step.

Effect of Surface Modification on the Luminescence of Individual Upconversion Nanoparticles.

Lanthanide-doped upconversion nanoparticles (UCNPs) hold promise for single-molecule imaging owing to their excellent photostability and minimal autofluorescence. However, their limited water dispersibility, often from the hydrophobic oleic acid ligand during synthesis, is a challenge. To address this, various surface modification strategies' impact on single-particle upconversion luminescence are studied. UCNPs are made hydrophilic through methods like ligand exchange with dye IR806, HCl or NOBF4 treatment, silica coating (SiO2 or mesoporous mSiO2), and self-assembly with polymer of DSPE-PEG or F127. The studies revealed that UCNPs modified with NOBF4 and DSPE-PEG exhibited notably higher single-particle brightness with minimal quenching (3% and 8%, respectively), followed by SiO2, F127, IR806, mSiO2, and HCl (84% quenching). HCl disrupted UCNPs's crystal lattice, weakening luminescence, while mSiO2 absorbed solvent molecules, causing luminescence quenching. Energy transfer to IR806 also reduced the brightness. Additionally, a prevalence of upconversion red emission over green is observed, with the red-to-green ratio increasing with irradiance. UCNPs coated with DSPE-PEG exhibited the brightest single-particle luminescence in water, retaining 48% of its original emission due to a lower critical micelle concentration and superior water protection. In summary, the investigation provides valuable insights into the role of surface chemistry on UCNPs at the single-particle level.