Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury.

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21-3p, along with the downregulation of hsa-miR-152-3p and hsa-miR-186-5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

Fluorogenic in-situ Labelling of Gelatin Polymer in Aqueous Solution and Hydrogel.

Gelatin polymers made from partially degraded collagen are important biomaterials, but their in-situ analysis suffers from uncontrollable covalent labelling and poor spatio-temporal imaging resolution. Herein, three tetrazolate-tagged tetraphenylethylene fluorophores (TPE-TAs) are introduced for practical fluorogenic labelling of gelatin in aqueous phase and hydrogels. These probes with aggregation-induced emission characteristics offer negligible background and elicit turn-on fluorescence by simply mixing with the gelatin in aqueous phase, giving a detection limit of 0.15 mg/L over a linear dynamic range up to 100 mg/L. This method does not work for collagens and causes minimal interference with gelatin properties. Mechanistic studies reveal a key role for multivalent electrostatic interactions between the abundant basic residues in gelatin (e.g., lysine, hydroxylysine, arginine) and anionic tetrazolate moieties of the lipophilic fluorophore synergistically in spatially rigid macromolecular encapsulation to achieve fluorogenic labelling. The AIE strategy by forming non-covalent fluorophore-gelatin complexes was developed for novel hydrogels that exhibited reversible fluorescence in response to dynamic microstructural changes in the hydrogel scaffold upon salting-in/out treatments, and enabled high spatio-temporal imaging of the fiber network in lyophilized samples. This work may open up avenues for in-situ imaging analysis and evaluation of gelatin-based biomaterials during processes such as in vivo degradation and mineralization.

Bacterial outer membrane vesicle-cancer cell hybrid membrane-coated nanoparticles for sonodynamic therapy in the treatment of breast cancer bone metastasis.

Breast cancer bone metastasis is a terminal-stage disease and is typically treated with radiotherapy and chemotherapy, which causes severe side effects and limited effectiveness. To improve this, Sonodynamic therapy may be a more safe and effective approach in the future. Bacterial outer membrane vesicles (OMV) have excellent immune-regulating properties, including modulating macrophage polarization, promoting DC cell maturation, and enhancing anti-tumor effects. Combining OMV with Sonodynamic therapy can result in synergetic anti-tumor effects. Therefore, we constructed multifunctional nanoparticles for treating breast cancer bone metastasis. We fused breast cancer cell membranes and bacterial outer membrane vesicles to form a hybrid membrane (HM) and then encapsulated IR780-loaded PLGA with HM to produce the nanoparticles, IR780@PLGA@HM, which had tumor targeting, immune regulating, and Sonodynamic abilities. Experiments showed that the IR780@PLGA@HM nanoparticles had good biocompatibility, effectively targeted to 4T1 tumors, promoted macrophage type I polarization and DC cells activation, strengthened anti-tumor inflammatory factors expression, and presented the ability to effectively kill tumors both in vitro and in vivo, which showed a promising therapeutic effect on breast cancer bone metastasis. Therefore, the nanoparticles we constructed provided a new strategy for effectively treating breast cancer bone metastasis.

GLUT1 promotes cell proliferation via binds and stabilizes phosphorylated EGFR in lung adenocarcinoma.

BACKGROUND: While previous studies have primarily focused on Glucose transporter type 1 (GLUT1) related glucose metabolism signaling, we aim to discover if GLUT1 promotes tumor progression through a non-metabolic pathway. METHODS: The RNA-seq and microarray data were comprehensively analyzed to evaluate the significance of GLUT1 expression in lung adenocarcinoma (LUAD). The cell proliferation, colony formation, invasion, and migration were used to test GLUT1 's oncogenic function. Co-immunoprecipitation and mass spectrum (MS) were used to uncover potential GLUT1 interacting proteins. RNA-seq, DIA-MS, western blot, and qRT-PCR to probe the change of gene and cell signaling pathways. RESULTS: We found that GLUT1 is highly expressed in LUAD, and higher expression is related to poor patient survival. GLUT1 knockdown caused a decrease in cell proliferation, colony formation, migration, invasion, and induced apoptosis in LUAD cells. Mechanistically, GLUT1 directly interacted with phosphor-epidermal growth factor receptor (p-EGFR) and prevented EGFR protein degradation via ubiquitin-mediated proteolysis. The GLUT1 inhibitor WZB117 can increase the sensitivity of LUAD cells to EGFR-tyrosine kinase inhibitors (TKIs) Gefitinib. CONCLUSIONS: GLUT1 expression is higher in LUAD and plays an oncogenic role in lung cancer progression. Combining GLUT1 inhibitors and EGFR-TKIs could be a potential therapeutic option for LUAD treatment.

Farnesyltransferase inhibitor lonafarnib suppresses respiratory syncytial virus infection by blocking conformational change of fusion glycoprotein.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators.

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik , a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

Newly formed dust within the circumstellar environment of SN Ia-CSM 2018evt.

Dust associated with various stellar sources in galaxies at all cosmic epochs remains a controversial topic, particularly whether supernovae play an important role in dust production. We report evidence of dust formation in the cold, dense shell behind the ejecta-circumstellar medium (CSM) interaction in the Type Ia-CSM supernova (SN) 2018evt three years after the explosion, characterized by a rise in mid-infrared emission accompanied by an accelerated decline in the optical radiation of the SN. Such a dust-formation picture is also corroborated by the concurrent evolution of the profiles of the Hα emission line. Our model suggests enhanced CSM dust concentration at increasing distances from the SN as compared to what can be expected from the density profile of the mass loss from a steady stellar wind. By the time of the last mid-infrared observations at day +1,041, a total amount of 1.2 ± 0.2 × 10-2 M⊙ of new dust has been formed by SN 2018evt, making SN 2018evt one of the most prolific dust factories among supernovae with evidence of dust formation. The unprecedented witness of the intense production procedure of dust may shed light on the perceptions of dust formation in cosmic history.

Synergistic monitoring of PM2.5 and CO2 based on active and passive remote sensing fusion during the 2022 Beijing Winter Olympics.

Green and low-carbon are the keywords of the 2022 Beijing Winter Olympic Games (WOG) and the core of sustainable development. Beijing's P M 2.5 and C O 2 emissions attracted worldwide attention during WOG. However, the complex emission sources and frequently changing weather patterns make it impossible for a single monitoring approach to meet the high-resolution, full-coverage monitoring requirements. Therefore, we proposed an active-passive remote sensing fusion method to address this issue. The haze layer height (HLH) was first retrieved from vertical aerosol profiles measured by our high-spectral-resolution lidar located near Olympic venues, which provides new insights into the nonuniform boundary layer and the residual aerosol aloft above it. Second, we developed a bootstrap aggregating (bagging) method that assimilates the lidar-based HLH, satellite-based AOD, and meteorological data to estimate the hourly P M 2.5 with 1 km resolution. The P M 2.5 at Beijing region, Bird's Nest, and Yanqing venues during WOG was 23.00±18.33, 22.91±19.48, and 16.33±10.49µg/m 3, respectively. Third, we also derived the C O 2 enhancements, C O 2 spatial gradients resulting from human activities, and annual growth rate (AGR) to estimate the performance of carbon emission management in Beijing. Based on the top-down method, the results showed an average C O 2 enhancement of 1.62 ppm with an annual decline rate of 2.92 ppm. Finally, we compared the monitoring data with six other international cities. The results demonstrated that Beijing has the largest P M 2.5 annual decline rate of 7.43µg/m 3, while the C O 2 AGR is 1.46 ppm and keeps rising, indicating Beijing is still on its way to carbon peaking and needs to strive for carbon neutrality.

Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations.

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target ß-catenin and NF-κB essential modulator. Among the twelve ß-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds ß-catenin with an IC50 of 0.010 ± 0.06 µM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

STEM enables mapping of single-cell and spatial transcriptomics data with transfer learning.

Profiling spatial variations of cellular composition and transcriptomic characteristics is important for understanding the physiology and pathology of tissues. Spatial transcriptomics (ST) data depict spatial gene expression but the currently dominating high-throughput technology is yet not at single-cell resolution. Single-cell RNA-sequencing (SC) data provide high-throughput transcriptomic information at the single-cell level but lack spatial information. Integrating these two types of data would be ideal for revealing transcriptomic landscapes at single-cell resolution. We develop the method STEM (SpaTially aware EMbedding) for this purpose. It uses deep transfer learning to encode both ST and SC data into a unified spatially aware embedding space, and then uses the embeddings to infer SC-ST mapping and predict pseudo-spatial adjacency between cells in SC data. Semi-simulation and real data experiments verify that the embeddings preserved spatial information and eliminated technical biases between SC and ST data. We apply STEM to human squamous cell carcinoma and hepatic lobule datasets to uncover the localization of rare cell types and reveal cell-type-specific gene expression variation along a spatial axis. STEM is powerful for mapping SC and ST data to build single-cell level spatial transcriptomic landscapes, and can provide mechanistic insights into the spatial heterogeneity and microenvironments of tissues.

Cu-Catalyzed Divergent Transformations of Allenylethylene Carbonates with Diboron Reagents.

Divergent transformations of allenylethylene carbonates with diboron reagents catalyzed by copper are disclosed. By using CuCl/IPr·HCl as the catalyst, the allenylethylene carbonates react with B2hex2 to afford 2,4-dien-1-ols as the product in the presence of Cs2CO3 as the base, iPrOH as the additive, and 1,4-dioxane as the solvent. And they react with B2pin2 to form boronic half acids in the presence of NaOtBu as the base, water as the additive, and THF as the solvent. The reactions afford corresponding products in good stereoselectivities and yields, and further derivatizations of boronic half acids and study of the mechanism are also demonstrated.

Macrophage-derived biomimetic nanoparticles enhanced SDT combined with immunotherapy inhibited tumor growth and metastasis.

Combination therapy based on sonodynamic therapy (SDT) combined with immune checkpoint blockers anti-PD-L1 provides effective anti-tumor effects. We designed a combination therapy based on M1/PLGA@IR780/CAT NPs of SDT-enhanced immunity combined with immune checkpoint blockers against PD-L1, which was based on M1 macrophage membrane-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the acoustic sensitizer IR780 and catalase (CAT) to successfully realize it. SDT based on M1/PLGA@IR780/CAT NPs could induce tumor cell death by promoting dendritic cell (DC) maturation and modulating the tumor immune microenvironment. In particular, the systemic anti-tumor immune response and potent immune memory induced upon combination with anti-PD-L1 checkpoint blockade not only alleviated the progression of mammary cancer in 4T1 mice and effectively blocked distant metastasis, but also prevented tumor recurrence, providing a promising new therapeutic strategy for clinical tumor therapy.

Mechanistic insights into radical formation and functionalization in copper/N-fluorobenzenesulfonimide radical-relay reactions.

Copper-catalysed radical-relay reactions that employ N-fluorobenzenesulfonimide (NFSI) as the oxidant have emerged as highly effective methods for C(sp3)-H functionalization. Herein, computational studies are paired with experimental data to investigate a series of key mechanistic features of these reactions, with a focus on issues related to site-selectivity, enantioselectivity, and C-H substrate scope. (1) The full reaction energetics of enantioselective benzylic C-H cyanation are probed, and an adduct between Cu and the N-sulfonimidyl radical (˙NSI) is implicated as the species that promotes hydrogen-atom transfer (HAT) from the C-H substrate. (2) Benzylic versus 3° C-H site-selectivity is compared with different HAT reagents: Cu/˙NSI, ˙OtBu, and Cl˙, and the data provide insights into the high selectivity for benzylic C-H bonds in Cu/NFSI-catalyzed C-H functionalization reactions. (3) The energetics of three radical functionalization pathways are compared, including radical-polar crossover (RPC) to generate a carbocation intermediate, reductive elimination from a formal CuIII organometallic complex, and radical addition to a Cu-bound ligand. The preferred mechanism is shown to depend on the ligands bound to copper. (4) Finally, the energetics of three different pathways that convert benzylic C-H bonds into benzylic cations are compared, including HAT/ET (ET = electron transfer), relevant to the RPC mechanism with Cu/NFSI; hydride transfer, involved in reactions with high-potential quinones; and sequential ET/PT/ET (PT = proton transfer), involved in catalytic photoredox reactions. Collectively, the results provide mechanistic insights that establish a foundation for further advances in radical-relay C-H functionalization reactions.

Comparative Analysis of Differentially Expressed Genes in Chondrocytes from Rats Exposed to Low Selenium and T-2 Toxin.

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD.

Programmable late-stage functionalization of bridge-substituted bicyclo[1.1.1]pentane bis-boronates.

Modular functionalization enables versatile exploration of chemical space and has been broadly applied in structure-activity relationship (SAR) studies of aromatic scaffolds during drug discovery. Recently, the bicyclo[1.1.1]pentane (BCP) motif has increasingly received attention as a bioisosteric replacement of benzene rings due to its ability to improve the physicochemical properties of prospective drug candidates, but studying the SARs of C2-substituted BCPs has been heavily restricted by the need for multistep de novo synthesis of each analogue of interest. Here we report a programmable bis-functionalization strategy to enable late-stage sequential derivatization of BCP bis-boronates, opening up opportunities to explore the SARs of drug candidates possessing multisubstituted BCP motifs. Our approach capitalizes on the inherent chemoselectivity exhibited by BCP bis-boronates, enabling highly selective activation and functionalization of bridgehead (C3)-boronic pinacol esters (Bpin), leaving the C2-Bpin intact and primed for subsequent derivatization. These selective transformations of both BCP bridgehead (C3) and bridge (C2) positions enable access to C1,C2-disubstituted and C1,C2,C3-trisubstituted BCPs that encompass previously unexplored chemical space.

Enhanced Osteolysis Targeted Therapy through Fusion of Exosomes Derived from M2 Macrophages and Bone Marrow Mesenchymal Stem Cells: Modulating Macrophage Polarization.

Aseptic loosening of prostheses is a highly researched topic, and wear particle-induced macrophage polarization is a significant cause of peri-prosthetic osteolysis. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs-Exos) promote M2 polarization and inhibit M1 polarization of macrophages. However, clinical application problems such as easy clearance and lack of targeting exist. Exosomes derived from M2 macrophages (M2-Exos) have good biocompatibility, immune escape ability, and natural inflammatory targeting ability. M2-Exos and BMSCs-Exos fused exosomes (M2-BMSCs-Exos) are constructed, which targeted the osteolysis site and exerted the therapeutic effect of both exosomes. M2-BMSCs-Exos achieved targeted osteolysis after intravenous administration inhibiting M1 polarization and promoting M2 polarization to a greater extent at the targeted site, ultimately playing a key role in the prevention and treatment of aseptic loosening of prostheses. In conclusion, M2-BMSCs-Exos can be used as a precise and reliable molecular drug for peri-prosthetic osteolysis. Fused exosomes M2-BMSCs-Exos  were originally proposed and successfully prepared, and exosome fusion technology provides a new theoretical basis and solution for the clinical application of therapeutic exosomes.

A facile strategy for the large-scale preparation of starch-based AIE luminescent nanoaggregates via host-guest interactions and their versatile applications.

Luminescent nanomaterials with outstanding optical properties have attracted growing interest due to their widespread applications. However, large-scale fabrication of luminescent nanomaterials with desired properties through a simple and economical process remains challenging. As a renewable natural resource, starch is non-toxic, easily accessible, and inexpensive, making it a popular choice for uses in various biomedical fields. In this work, we present a facile assembly strategy for the fabrication of starch-based luminescent nanoaggregates using starch as the host material and aggregation-induced emission luminogens (AIEgens) as guest molecules. By employing simple procedures under mild conditions, highly luminescent nanoparticles with small sizes, high water dispersibility, and low cytotoxicity are prepared on a large scale. The resulting nano-assemblies demonstrate significantly enhanced fluorescence intensities, reduced susceptibility to photobleaching and low cytotoxicity. These fluorescent supramolecular aggregates can be employed in various application fields, including the fabrication of fluorescent hydrogels, fingerprint detection, cell imaging and in vivo lymphatic system imaging. The methodology developed in this work has immense potential to greatly promote the production of high-quality nanoparticles on the industrial scale, offering a cost-effective solution that can meet the needs of various applications and pave the way for wider implementation of nanotechnology.

STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

ABSTRACT: Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Copper-Catalyzed Benzylic C-H Cross-Coupling Enabled by Redox Buffers: Expanding Synthetic Access to Three-Dimensional Chemical Space.

ConspectusCross-coupling methods are the most widely used synthetic methods in medicinal chemistry. Existing reactions are dominated by methods such as amide coupling and arylation reactions that form bonds to sp2-hybridized carbon atoms and contribute to the formation of "flat" molecules. Evidence that three-dimensional structures often have improved physicochemical properties for pharmaceutical applications has contributed to growing demand for cross-coupling methods with sp3-hybridized reaction partners. Substituents attached to sp3 carbon atoms are intrinsically displayed in three dimensions. These considerations have led to efforts to establish reactions with sp3 cross-coupling partners, including alkyl halides, amines, alcohols, and carboxylic acids. As C(sp3)-H bonds are much more abundant that these more conventional coupling partners, we have been pursuing C(sp3)-H cross-coupling reactions that achieve site-selectivity, synthetic utility, and scope competitive with conventional coupling reactions.In this Account, we outline Cu-catalyzed oxidative cross-coupling reactions of benzylic C(sp3)-H bonds with diverse nucleophilic partners. These reactions commonly use N-fluorobenzenesulfonimide (NFSI) as the oxidant. The scope of reactivity is greatly improved by using a "redox buffer" that ensures that the Cu catalyst is available in the proper redox state to promote the reaction. Early precedents of catalytic Cu/NFSI oxidative coupling reactions, including C-H cyanation and arylation, did not require a redox buffer, but reactions with other nucleophiles, such as alcohols and azoles, were much less effective under similar conditions. Mechanistic studies show that some nucleophiles, such as cyanide and arylboronic acids, promote in situ reduction of CuII to CuI, contributing to successful catalytic turnover. Poor reactivity was observed with nucleophiles, such as alcohols, that do not promote CuII reduction in the same manner. This insight led to the identification of sacrificial reductants, termed "redox buffers", that support controlled generation of CuI during the reactions and enable successful benzylic C(sp3)-H cross-coupling with diverse nucleophiles. Successful reactions include those that feature direct coupling of (hetero)benzylic C-H substrates with coupling partners (alcohols, azoles) and sequential C(sp3)-H functionalization/coupling reactions. The latter methods feature generation of a synthetic linchpin that can undergo subsequent reaction with a broad array of nucleophiles. For example, halogenation/substitution cascades afford benzylic amines, (thio)ethers, and heterodiarylmethane derivatives, and an isocyanation/amine-addition sequence generates diverse benzylic ureas.Collectively, these Cu-catalyzed (hetero)benzylic C(sp3)-H cross-coupling reactions rapidly access diverse molecules. Analysis of their physicochemical and topological properties highlights the "drug-likeness" and enhanced three-dimensionality of these products relative to existing bioactive molecules. This consideration, together with the high benzylic C-H site-selectivity and the broad scope of reactivity enabled by the redox buffering strategy, makes these C(sp3)-H cross-coupling methods ideally suited for implementation in high-throughput experimentation platforms to explore novel chemical space for drug discovery and related applications.