Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

Phosphorylation of PDHA by AMPK Drives TCA Cycle to Promote Cancer Metastasis.

Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for full-blown metastasis is largely unknown. Here, we show that AMPK (AMP-activated protein kinase), activated in mouse metastasis models, drives pyruvate dehydrogenase complex (PDHc) activation to maintain TCA cycle (tricarboxylic acid cycle) and promotes cancer metastasis by adapting cancer cells to metabolic and oxidative stresses. This AMPK-PDHc axis is activated in advanced breast cancer and predicts poor metastasis-free survival. Mechanistically, AMPK localizes in the mitochondrial matrix and phosphorylates the catalytic alpha subunit of PDHc (PDHA) on two residues S295 and S314, which activates the enzymatic activity of PDHc and alleviates an inhibitory phosphorylation by PDHKs, respectively. Importantly, these phosphorylation events mediate PDHc function in cancer metastasis. Our study reveals that AMPK-mediated PDHA phosphorylation drives PDHc activation and TCA cycle to empower cancer cells adaptation to metastatic microenvironments for metastasis.

Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway.

Hirschsprung disease (HSCR), one of several neurocristopathies in children, is characterized by nerve loss in the large intestine and is mainly treated by surgery, which causes severe complications. Enteric neural crest-derived cell (ENCC) transplantation is a potential therapeutic strategy; however, so far with poor efficacy. Here, we assessed whether and how fecal microbiota transplantation (FMT) could improve ENCC transplantation in a rat model of hypoganglionosis; a condition similar to HSCR, with less intestinal innervation. We found that the hypoganglionosis intestinal microenvironment negatively influenced the ENCC functional phenotype in vitro and in vivo. Combining 16S rDNA sequencing and targeted mass spectrometry revealed microbial dysbiosis and reduced short-chain fatty acid (SCFA) production in the hypoganglionic gut. FMT increased the abundance of Bacteroides and Clostridium, SCFA production, and improved outcomes following ENCC transplantation. SCFAs alone stimulated ENCC proliferation, migration, and supported ENCC transplantation. Transcriptome-wide mRNA sequencing identified MAPK signaling as the top differentially regulated pathway in response to SCFA exposure, and inhibition of MEK1/2 signaling abrogated the SCFA-mediated effects on ENCC. This study demonstrates that FMT improves cell therapy for hypoganglionosis via short-chain fatty acid metabolism-induced MEK1/2 signaling.

The electrochemistry of stable sulfur isotopes versus lithium.

Sulfur in nature consists of two abundant stable isotopes, with two more neutrons in the heavy one (34S) than in the light one (32S). The two isotopes show similar physicochemical properties and are usually considered an integral system for chemical research in various fields. In this work, a model study based on a Li-S battery was performed to reveal the variation between the electrochemical properties of the two S isotopes. Provided with the same octatomic ring structure, the cyclo-34S8 molecules form stronger S-S bonds than cyclo-32S8 and are more prone to react with Li. The soluble Li polysulfides generated by the Li-34S conversion reaction show a stronger cation-solvent interaction yet a weaker cation-anion interaction than the 32S-based counterparts, which facilitates quick solvation of polysulfides yet hinders their migration from the cathode to the anode. Consequently, the Li-34S cell shows improved cathode reaction kinetics at the solid-liquid interface and inhibited shuttle of polysulfides through the electrolyte so that it demonstrates better cycling performance than the Li-32S cell. Based on the varied shuttle kinetics of the isotopic-S-based polysulfides, an electrochemical separation method for 34S/32S isotope is proposed, which enables a notably higher separation factor than the conventional separation methods via chemical exchange or distillation and brings opportunities to low-cost manufacture, utilization, and research of heavy chalcogen isotopes.

Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Is it time to reassess variant annotation?

Variant annotation is one of the most essential steps in selecting candidates for further investigation. With the advancement in functional genomics, new variant annotation tools focus on annotation based on empirically generated data instead of theoretically based predictions. This is a direct result of the large national and international consortia that generated enormous experiment-based or validated data at multiple omics levels. Here, we highlight the recent empirically based annotation methods and discuss their strengths and weaknesses.

Surveying mutation density patterns around specific genomic features.

Mutation density patterns reveal unique biological properties of specific genomic regions and shed light on the mechanisms of carcinogenesis. Although previous studies reported insightful mutation density patterns associated with certain genomic regions such as transcription start sites and DNA replication origins, a tool that can systematically investigate mutational spatial patterns is still lacking. Thus, we developed MutDens, a bioinformatic tool for comprehensive analysis of mutation density patterns around genomic features, namely, genomic positions, in humans and model species. By scanning the bidirectional vicinity regions of given positions, MutDens systematically characterizes the mutation density for single-base substitution mutational classes after adjusting for total mutation burden and local nucleotide proportion. Analysis results using MutDens not only verified the previously reported transcriptional strand bias around transcription start sites and replicative strand bias around DNA replication origins, but also identified novel mutation density patterns around other genomics features, such as enhancers and retrotransposon insertion polymorphism sites. To our knowledge, MutDens is the first tool that systematically calculates, examines, and compares mutation density patterns, thus providing a valuable avenue for investigating the mutational landscapes associated with important genomic features.

Isotropic super-resolution light-sheet microscopy of dynamic intracellular structures at subsecond timescales.

Long-term visualization of the dynamic interactions between intracellular structures throughout the three-dimensional space of whole live cells is essential to better understand their functions, but this task remains challenging due to the limitations of existing three-dimensional fluorescence microscopy techniques, such as an insufficient axial resolution, low volumetric imaging rate and photobleaching. Here, we present the combination of a progressive deep-learning super-resolution strategy with a double-ring-modulated selective plane illumination microscopy design capable of visualizing the dynamics of intracellular structures in live cells for hours at an isotropic spatial resolution of roughly 100 nm in three dimensions at speeds up to roughly 17 Hz. Using this approach, we reveal the complex spatial relationships and interactions between endoplasmic reticulum (ER) and mitochondria throughout live cells, providing new insights into ER-mediated mitochondrial division. We also examined the motion of Drp1 oligomers involved in mitochondrial fission and revealed the dynamic interactions between Drp1 and mitochondria in three dimensions.

A cardioviral 2C-ATP complex structure reveals the essential role of a conserved arginine in regulation of cardioviral 2C activity.

2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions. IMPORTANCE: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure-function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein.

Attention-based cross domain graph neural network for prediction of drug-drug interactions.

Drug-drug interactions (DDI) may lead to adverse reactions in human body and accurate prediction of DDI can mitigate the medical risk. Currently, most of computer-aided DDI prediction methods construct models based on drug-associated features or DDI network, ignoring the potential information contained in drug-related biological entities such as targets and genes. Besides, existing DDI network-based models could not make effective predictions for drugs without any known DDI records. To address the above limitations, we propose an attention-based cross domain graph neural network (ACDGNN) for DDI prediction, which considers the drug-related different entities and propagate information through cross domain operation. Different from the existing methods, ACDGNN not only considers rich information contained in drug-related biomedical entities in biological heterogeneous network, but also adopts cross-domain transformation to eliminate heterogeneity between different types of entities. ACDGNN can be used in the prediction of DDIs in both transductive and inductive setting. By conducting experiments on real-world dataset, we compare the performance of ACDGNN with several state-of-the-art methods. The experimental results show that ACDGNN can effectively predict DDIs and outperform the comparison models.

A transformer-based genomic prediction method fused with knowledge-guided module.

Genomic prediction (GP) uses single nucleotide polymorphisms (SNPs) to establish associations between markers and phenotypes. Selection of early individuals by genomic estimated breeding value shortens the generation interval and speeds up the breeding process. Recently, methods based on deep learning (DL) have gained great attention in the field of GP. In this study, we explore the application of Transformer-based structures to GP and develop a novel deep-learning model named GPformer. GPformer obtains a global view by gleaning beneficial information from all relevant SNPs regardless of the physical distance between SNPs. Comprehensive experimental results on five different crop datasets show that GPformer outperforms ridge regression-based linear unbiased prediction (RR-BLUP), support vector regression (SVR), light gradient boosting machine (LightGBM) and deep neural network genomic prediction (DNNGP) in terms of mean absolute error, Pearson's correlation coefficient and the proposed metric consistent index. Furthermore, we introduce a knowledge-guided module (KGM) to extract genome-wide association studies-based information, which is fused into GPformer as prior knowledge. KGM is very flexible and can be plugged into any DL network. Ablation studies of KGM on three datasets illustrate the efficiency of KGM adequately. Moreover, GPformer is robust and stable to hyperparameters and can generalize to each phenotype of every dataset, which is suitable for practical application scenarios.

Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.

Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3Cpro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3Cpro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3Cpro. Our lipid-binding assays showed that SVA 3Cpro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3Cpro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3Cpro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3Cpro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3Cpro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3Cpro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme's proteolytic activity during infection.

Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility.

BACKGROUND: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference.

Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2) loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to ∼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.

Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection.

SignificanceThe detection of low-abundance molecular biomarkers is key to the liquid-biopsy-based disease diagnosis. Existing methods are limited by the affinity and specificity of recognition probes and the mass transportation of analyte molecules onto the sensor surfaces, resulting in insufficient sensitivity and long assay time. This work establishes a rapid and ultrasensitive approach by actively tuning binding kinetics and accelerating the mass transportation via nanoparticle micromanipulations. This is significant because it permits extremely sensitive measurements within clinically acceptable assay time. It is incubation-free, washing-free, and compatible with low- and high-affinity probes.

Ultrasensitive Single-Molecule Biosensor by Periodic Modulation of Magnetic Particle Motion.

Ultrasensitive detection of low-abundance biomarkers by modern single-molecule technologies is critical for better diagnosis of severe diseases, but inevitable nonspecific bindings often cause fluctuations in the single-molecule counting results. Here we present an approach to improve the specificity in a single-molecule immunoassay by translating molecular binding signals into periodic nanomotion of magnetic particles. The sandwiched immunoassay is modified by using a long linker to tether one antibody onto a gold-covered substrate and a magnetic particle with another antibody coated as the reporter. By actively oscillating the particles with alternating magnetic fields, we could reliably identify specific binding through intensity fluctuation in plasmonic images of single particles. As a proof of concept, we demonstrate the detection of IFN-γ at the femtomolar level by the digital counting of specifically bound molecules. This active strategy outperforms existing passive motion-based approaches in sensitivity and speed, paving the way for disease diagnosis with low-abundance biomarkers.

MELK aggravates lung adenocarcinoma by regulating EZH2 ubiquitination and H3K27me3 histone methylation of LATS2.

We tried to elucidate the possible roles of maternal embryonic leucine pull chain kinase (MELK) in lung adenocarcinoma (LUAD) growth and metastasis. Differentially expressed genes in LUAD samples were analysed by the GEPIA database. Clinical tissue samples and cells were collected for MELK, EZH2 and LATS2 expression determination. Co-IP assay was used to verify the interaction between EZH2 and MELK; CHX tracking assay and ubiquitination assay detected the degradation of MELK on EZH2 ubiquitination. ChIP assay detected the enrichment of EZH2 and H3K27me3 on the LATS2 promoter region. LUAD cells were selected for in vitro validation, and the tumorigenic ability of LUAD cells was also observed in a transplantation tumour model of LUAD nude mice. MELK and EZH2 were highly expressed in LUAD samples, while LATS2 was lowly expressed. MELK interacted with EZH2 to inhibit its ubiquitination degradation; EZH2 elevated H3K27me3 modification in the LATS2 promoter to lower LATS2 expression. Silencing MELK or EZH2 or overexpressing LATS2 restrained LUAD cell proliferation and invasion, and facilitated their apoptosis. Silencing MELK or EZH2 or overexpressing LATS2 suppressed tumour formation in nude mice. This study demonstrated that MELK aggravated LUAD by upregulating EZH2 and downregulating LATS2.

Colorectal cancer subtyping and prognostic model construction based on interleukin-related genes.

Members of the interleukin (IL) family are closely linked to cancer development and progression. However, research on the prognosis of colorectal cancer (CRC) related to IL is still lacking. This study investigated new CRC prognostic markers and offered new insights for CRC prognosis and treatment. CRC-related data and IL gene data were collected from public databases. Sample clustering was done with the NMF package to divide samples into different subtypes. Differential, enrichment, survival, and immune analyses were conducted on subtypes. A prognostic model was constructed using regression analysis. Drug sensitivity analysis was performed using GDSC database. Western blot analysis was performed to assess the effect of IL-7 on the JAK/STAT signaling pathway. Flow cytometry was used to examine the impact of IL-7 on CD8+ T cell apoptosis. Two CRC subtypes based on IL-associated genes were obtained. Cluster 1 had a higher survival rate than cluster 2, and they showed differences in some immune levels. The two clusters were mainly enriched in the JAK-STAT signaling pathway, T helper 17 cell differentiation, and the IL-17 signaling pathway. An 11-gene signature was built, and risk score was an independent prognosticator for CRC. The low-risk group showed a higher sensitivity to nine common targeted anticancer drugs. Western blot and flow cytometry results demonstrated that IL-7 could phosphorylate STAT5 and promote survival of CD8+ T cells. In conclusion, this study divided CRC samples into two IL-associated subtypes and obtained an 11-gene signature. In addition, targeted drugs that may improve the prognosis of patients with CRC were identified. These findings are of paramount importance for patient prognosis and CRC treatment.NEW & NOTEWORTHY We identified two clusters with significant survival differences in colorectal cancer (CRC) based on interleukin-related genes, constructed an 11-gene risk score model that can independently predict the prognosis of CRC, and explored some targeted drugs that may improve the prognosis of patients with CRC. The results of this study have important implications for the prognosis and treatment of CRC.

PCSK9 inhibition interrupts the cross-talk between keratinocytes and macrophages and prevents UVB-induced skin damage.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that promotes the degradation of low-density lipoprotein receptors. It is involved in hyperlipidemia as well as other diseases, such as cancer and skin inflammation. However, the detailed mechanism for PCSK9 on ultraviolet B (UVB)-induced skin lesions was not clear. Thus, the role and possible action mechanism of PCSK9 in UVB-induced skin damage in mice were studied here using siRNA and a small molecule inhibitor (SBC110736) against PCSK9. Immunohistochemical staining revealed a significant increase in PCSK9 expression after UVB exposure, indicating the possible role of PCSK9 in UVB damage. Skin damage, increase in epidermal thickness, and keratinocyte hyperproliferation were significantly alleviated after treatment with SBC110736 or siRNA duplexes, compared with that in the UVB model group. Notably, UVB exposure triggered DNA damage in keratinocytes, whereas substantial interferon regulatory factor 3 (IRF3) activation was observed in macrophages. Pharmacologic inhibition of STING or cGAS knockout significantly reduced UVB-induced damage. In the co-culture system, supernatant from UVB-treated keratinocyte induced IRF3 activation in macrophages. This activation was inhibited with SBC110736 and by PCSK9 knockdown. Collectively, our findings reveal that PCSK9 plays a critical role in the crosstalk between damaged keratinocytes and STING activation in macrophages. The interruption of this crosstalk by PCSK9 inhibition may be a potential therapeutic strategy for UVB-induced skin damage.

Enantioselective Construction of Quaternary Stereocenters via Cooperative Photoredox/Fe/Chiral Primary Amine Triple Catalysis.

The catalytic and enantioselective construction of quaternary (all-carbon substituents) stereocenters poses a formidable challenge in organic synthesis due to the hindrance caused by steric factors. One conceptually viable and potentially versatile approach is the coupling of a C-C bond through an outer-sphere mechanism, accompanied by the realization of enantiocontrol through cooperative catalysis; however, examples of such processes are yet to be identified. Herein, we present such a method for creating different compounds with quaternary stereocenters by photoredox/Fe/chiral primary amine triple catalysis. This approach facilitates the connection of an unactivated alkyl source with a tertiary alkyl moiety, which is also rare. The scalable process exhibits mild conditions, does not necessitate the use of a base, and possesses a good functional-group tolerance. Preliminary investigations into the underlying mechanisms have provided valuable insights into the reaction pathway.