2D fin field-effect transistors integrated with epitaxial high-k gate oxide.

Precise integration of two-dimensional (2D) semiconductors and high-dielectric-constant (k) gate oxides into three-dimensional (3D) vertical-architecture arrays holds promise for developing ultrascaled transistors1-5, but has proved challenging. Here we report the epitaxial synthesis of vertically aligned arrays of 2D fin-oxide heterostructures, a new class of 3D architecture in which high-mobility 2D semiconductor fin Bi2O2Se and single-crystal high-k gate oxide Bi2SeO5 are epitaxially integrated. These 2D fin-oxide epitaxial heterostructures have atomically flat interfaces and ultrathin fin thickness down to one unit cell (1.2 nm), achieving wafer-scale, site-specific and high-density growth of mono-oriented arrays. The as-fabricated 2D fin field-effect transistors (FinFETs) based on Bi2O2Se/Bi2SeO5 epitaxial heterostructures exhibit high electron mobility (µ) up to 270 cm2 V-1 s-1, ultralow off-state current (IOFF) down to about 1 pA µm-1, high on/off current ratios (ION/IOFF) up to 108 and high on-state current (ION) up to 830 µA µm-1 at 400-nm channel length, which meet the low-power specifications projected by the International Roadmap for Devices and Systems (IRDS)6. The 2D fin-oxide epitaxial heterostructures open up new avenues for the further extension of Moore's law.

Downregulation of the m6A reader YTHDC2 upregulates exosome content in lung adenocarcinoma via inhibiting IFIT and OAS family members.

N6-Methyladenosine (m6A) is the most prevalent mRNA modification. Its biological function primarily relies on its "Reader" protein, such as YTHDC2. Previous studies have shown that YTHDC2 downregulation is a procarcinogenic phenomenon in lung adenocarcinoma (LUAD). However, further investigation is needed to understand the molecular mechanisms of downstream genes and the associated biological phenomena following YTHDC2 downregulation. Here, we found that YTHDC2 knockout upregulated exosome content in LUAD. Following YTHDC2 knockout, the mRNA levels of OAS family members (OASs) and IFIT family members (IFITs) also decreased; and inhibition of OASs and IFITs could promote exosome content. Several m6A modification sites on the NT domain of OASs and the TPR12 domain of IFITs were found to increase the stability of OASs and IFITs in a YTHDC2-dependent manner. OASs and IFITs affected exosome content through target genes including RAB5A, RAB7, and RAB11A, and three arginine (R) amino acids on IFITs were critical for combination IFITs with targeted RAB mRNAs and subsequent degradation. Simultaneously, OASs degraded targeted RABs through RNAseL. Additionally, mutual bindings between OASs and IFITs were critical for their target gene degradation. Collectively, the above findings might provide a theoretical basis for the treatment of LUAD patients with low YTHDC2 expression.

PALMD haploinsufficiency aggravates extracellular matrix remodeling in vascular smooth muscle cells and promotes calcification.

Reduced PALMD expression is strongly associated with the development of calcified aortic valve stenosis; however, the role of PALMD in vascular calcification remains unknown. Calcified arteries were collected from mice to detect PALMD expression. Heterozygous Palmd knockout (Palmd+/-) mice were established to explore the role of PALMD in subtotal nephrectomy-induced vascular calcification. RNA sequencing was applied to detect molecular changes in aortas from Palmd+/- mice. Primary Palmd+/- vascular smooth muscle cells (VSMCs) or PALMD-silenced VSMCs by short interfering RNA were used to analyze PALMD function in phenotypic changes and calcification. PALMD haploinsufficiency aggravated subtotal nephrectomy-induced vascular calcification. RNA sequencing analysis showed that loss of PALMD disturbed the synthesis and degradation of the extracellular matrix (ECM) in aortas, including collagens and matrix metalloproteinases (Col6a6, Mmp2, Mmp9, etc.). In vitro experiments revealed that PALMD-deficient VSMCs were more susceptible to high phosphate-induced calcification. Downregulation of SMAD6 expression and increased levels of p-SMAD2 were detected in Palmd+/- VSMCs, suggesting that transforming growth factor-ß signaling may be involved in PALMD haploinsufficiency-induced vascular calcification. Our data revealed that PALMD haploinsufficiency causes ECM dysregulation in VSMCs and aggravates vascular calcification. Our findings suggest that reduced PALMD expression is also linked to vascular calcification, and PALMD may be a potential therapeutic target for this disease. NEW & NOTEWORTHY We found that PALMD haploinsufficiency causes extracellular matrix dysregulation, reduced PALMD expression links to vascular calcification, and PALMD mutations may lead to the risk of both calcific aortic valve stenosis and vascular calcification.

The transcriptional regulator KLF15 is necessary for myoblast differentiation and muscle regeneration by activating FKBP5.

Successful muscle regeneration following injury is essential for functional homeostasis of skeletal muscles. Krüppel-like factor 15 (KLF15) is a metabolic transcriptional regulator in the muscles. However, little is known regarding its function in muscle regeneration. Here, we examined microarray datasets from the Gene Expression Omnibus database, which indicated downregulated KLF15 in muscles from patients with various muscle diseases. Additionally, we found that Klf15 knockout (Klf15KO) impaired muscle regeneration following injury in mice. Furthermore, KLF15 expression was robustly induced during myoblast differentiation. Myoblasts with KLF15 deficiency showed a marked reduction in their fusion capacity. Unbiased transcriptome analysis of muscles on day 7 postinjury revealed downregulated genes involved in cell differentiation and metabolic processes in Klf15KO muscles. The FK506-binding protein 51 (FKBP5), a positive regulator of myoblast differentiation, was ranked as one of the most strongly downregulated genes in the Klf15KO group. A mechanistic search revealed that KLF15 binds directly to the promoter region of FKBP5 and activates FKBP5 expression. Local delivery of FKBP5 rescued the impaired muscle regeneration in Klf15KO mice. Our findings reveal a positive regulatory role of KLF15 in myoblast differentiation and muscle regeneration by activating FKBP5 expression. KLF15 signaling may be a novel therapeutic target for muscle disorders associated with injuries or diseases.

Single-crystalline van der Waals layered dielectric with high dielectric constant.

The scaling of silicon-based transistors at sub-ten-nanometre technology nodes faces challenges such as interface imperfection and gate current leakage for an ultrathin silicon channel1,2. For next-generation nanoelectronics, high-mobility two-dimensional (2D) layered semiconductors with an atomic thickness and dangling-bond-free surfaces are expected as channel materials to achieve smaller channel sizes, less interfacial scattering and more efficient gate-field penetration1,2. However, further progress towards 2D electronics is hindered by factors such as the lack of a high dielectric constant (κ) dielectric with an atomically flat and dangling-bond-free surface3,4. Here, we report a facile synthesis of a single-crystalline high-κ (κ of roughly 16.5) van der Waals layered dielectric Bi2SeO5. The centimetre-scale single crystal of Bi2SeO5 can be efficiently exfoliated to an atomically flat nanosheet as large as 250 × 200 µm2 and as thin as monolayer. With these Bi2SeO5 nanosheets as dielectric and encapsulation layers, 2D materials such as Bi2O2Se, MoS2 and graphene show improved electronic performances. For example, in 2D Bi2O2Se, the quantum Hall effect is observed and the carrier mobility reaches 470,000 cm2 V-1 s-1 at 1.8 K. Our finding expands the realm of dielectric and opens up a new possibility for lowering the gate voltage and power consumption in 2D electronics and integrated circuits.

Biexcitons-plasmon coupling of Ag@Au hollow nanocube/MoS2 heterostructures based on scattering spectra.

The strong interaction between light and matter is one of the current research hotspots in the field of nanophotonics, and provides a suitable platform for fundamental physics research such as on nanolasers, high-precision sensing in biology, quantum communication and quantum computing. In this study, double Rabi splitting was achieved in a composite structure monolayer MoS2 and a single Ag@Au hollow nanocube (HNC) in room temperature mainly due to the two excitons in monolayer MoS2. Moreover, the tuning of the plasmon resonance peak was realized in the scattering spectrum by adjusting the thickness of the shell to ensure it matches the energy of the two excitons. Two distinct anticrossings are observed at both excitons resonances, and large double Rabi splittings (90 meV and 120 meV) are obtained successfully. The finite-difference time domain (FDTD) method was also used to simulate the scattering spectra of the nanostructures, and the simulation results were in good agreement with the experimental results. Additionally, the local electromagnetic field ability of the Ag@Au hollow HNC was proved to be stronger by calculating and comparing the mode volume of different nanoparticles. Our findings provides a good platform for the realization of strong multi-mode coupling and open up a new way to construct nanoscale photonic devices.

Evaluation of Six eGFR Equations in Predicting Acute Kidney Injury in Patients after Off-Pump Coronary Artery Bypass Grafting: A Case Control Study.

Background: There are six widely used equations to calculate the estimated glomerular filtration rate (eGFR) of patients. We aimed to assess the predictive power of preoperative eGFR calculated by these equations for the occurrence of postoperative acute kidney injury (AKI). Methods: Patients who underwent isolated coronary surgery from January 2016 to January 2021 were continuously enrolled. Serum creatinine and cystatin C used to calculate eGFR were both measured within 1 week before surgery. The eGFR was calculated using six equations: Cockcroft Gault (CG) equation, Chinese abbreviated modification of diet in renal disease (MDRD) equation, chronic kidney disease-epidemiology (CKD-EPI) equation, and full age spectrum (FAS) equation. Postoperative AKI was diagnosed by Kidney Disease Improving Global Outcomes criteria (KDIGO) (① urine volume < 0.5 mL/kg/h for 6 h; ② an increase in serum creatinine by ≥ 26.5 µmol/L within 48 h; ③ an increase in serum creatinine to ≥ 1.5 times baseline levels, which is known or presumed to have occurred within the prior 7 days), and the occurrence of AKI within 7 days after surgery was followed. Results: A total of 1428 patients were included, of which 319 patients (25.5%) developed postoperative AKI. After adjustment, all eGFRs (CG OR = 0.983, MDRD OR = 0.983, CKD-EPI crea OR = 0.97, CKD-EPI cys OR = 0.955, FAS crea OR = 0.978, FAS cys OR = 0. 941, all p < 0.001) were significantly associated with AKI. The area under the receiver operating characteristic curve (AUC) was 0.621 for CG, 0.614 for MDRD, 0.643 for CKD-EPI crea , 0.739 for CKD-EPI cys , 0.643 for FAS crea , 0.744 for FAS cys , respectively. There was no difference in predictive power between FAS cys and CKD-EPI cys (p = 0.33, DeLong's test). Conclusions: Preoperative eGFR calculated by FAS cys and CKD-EPI cys equations have better performance in predicting AKI after off-pump coronary artery bypass grafting than other equations.

Combination of betulinic acid and EGFR-TKIs exerts synergistic anti-tumor effects against wild-type EGFR NSCLC by inducing autophagy-related cell death via EGFR signaling pathway.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of lung cancer patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type EGFR tumors has been shown to be marginal. Methods that can sensitize EGFR-TKIs to EGFR wild-type NSCLC remain rare. Hence, we determined whether combination treatment can maximize the therapeutic efficacy of EGFR-TKIs. METHODS: We established a focused drug screening system to investigate candidates for overcoming the intrinsic resistance of wild-type EGFR NSCLC to EGFR-TKIs. Molecular docking assays and western blotting were used to identify the binding mode and blocking effect of the candidate compounds. Proliferation assays, analyses of drug interactions, colony formation assays, flow cytometry and nude mice xenograft models were used to determine the effects and investigate the molecular mechanism of the combination treatment. RESULTS: Betulinic acid (BA) is effective at targeting EGFR and synergizes with EGFR-TKIs (gefitinib and osimertinib) preferentially against wild-type EGFR. BA showed inhibitory activity due to its interaction with the ATP-binding pocket of EGFR and dramatically enhanced the suppressive effects of EGFR-TKIs by blocking EGFR and modulating the EGFR-ATK-mTOR axis. Mechanistic studies revealed that the combination strategy activated EGFR-induced autophagic cell death and that the EGFR-AKT-mTOR signaling pathway was essential for completing autophagy and cell cycle arrest. Activation of the mTOR pathway or blockade of autophagy by specific chemical agents markedly attenuated the effect of cell cycle arrest. In vivo administration of the combination treatment caused marked tumor regression in the A549 xenografts. CONCLUSIONS: BA is a potential wild-type EGFR inhibitor that plays a critical role in sensitizing EGFR-TKI activity. BA combined with an EGFR-TKI effectively suppressed the proliferation and survival of intrinsically resistant lung cancer cells via the inhibition of EGFR as well as the induction of autophagy-related cell death, indicating that BA combined with an EGFR-TKI may be a potential therapeutic strategy for overcoming the primary resistance of wild-type EGFR-positive lung cancers.

Direct trifluoromethylselenolations of electron-rich (hetero)aromatic rings with N-trifluoromethylselenolating saccharin.

A novel, easily synthesizable, shelf-stable electrophilic trifluoromethylselenolating reagent, N-trifluoromethylselenosaccharin, has been developed. This reagent can be synthesized in good yield by a two-step one-pot reaction from BnSeCF3, SO2Cl2, and silver saccharin. N-Trifluoromethylselenosaccharin proves to be an efficient trifluoromethylselenolating reagent, enabling the direct trifluoromethylselenolation of various electron-rich aromatic and heteroaromatic rings under mild reaction conditions. It exhibits excellent chemoselectivity and excellent compatibility with various functional groups, making it suitable for late-stage trifluoromethylselenolation applications in complex natural product and drug synthesis.

Br-to-Cl Transformation Guided the Formation of Polyhalogenated Dibenzo-p-dioxins/Dibenzofurans.

Polyhalogenated dibenzo-p-dioxins/dibenzofurans (PXDD/Fs) are commonly released into the environment as byproducts of combustion processes, accompanied by flue gases. Chlorinated (Cl) and brominated (Br) precursors play crucial roles in forming PXDD/Fs. However, the specific contributions of Cl-precursors and Br-precursors to PXDD/Fs formation have not been fully elucidated. Herein, we demonstrate that the formation of Br-precursors can increase the fraction of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) congeners substituted at specific positions, such as 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,4,7,8-PeCDF, and 2,3,4,6,7,8-HxCDF. This is attributed to the electrophilic chlorination reaction of the Br-precursors, which includes the Br-to-Cl transformation pathway, following the principle of regioselectivity. The observed formation of polybrominated/chlorinated dibenzo-p-dioxins/benzofurans (PBCDD/Fs) from 1,2-dibromobenzene (1,2-DiBBz) as a Br precursor provides direct evidence supporting the proposed Br-to-Cl transformation. Quantum chemical calculations are employed to discuss the principle of regioselectivity in the Br-to-Cl transformation, clarifying the priority of the position for electrophilic chlorination. Additionally, the concentration of PCDD/Fs formed from 1,2-DiBBz is 1.6 µg/kg, comparable to that of polybrominated dibenzo-p-dioxins/dibenzofurans (PBDD/Fs) (2.4 µg/kg), highlighting the potential of brominated organic pollutants as precursors for PCDD/Fs formation. This study provides three potential pathways for PCDD/Fs formation from Br-precursors, establishing a theoretical foundation for elucidating the formation mechanism of PXDD/Fs in the coexistence of Cl and Br.

Complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice.

Mutations in fibrillin 1 (FBN1) is the main cause of Marfan syndrome (MFS) with thoracic aortic aneurysm (TAA) as the main complication. Activation of the complement system plays a key role in the formation of thoracic and abdominal aortic aneurysms. However, the role of the complement system in MFS-associated aortic aneurysms remains unclear. In this study, we observed increased levels of complement C3a and C5a in the plasma of MFS patients and mouse, and the increased deposition of the activated complement system product C3b/iC3b was also observed in the elastic fiber rupture zone of 3-month-old MFS mice. The expression of C3a receptor (C3aR) was increased in MFS aortas, and recombinant C3a promoted the expression of cytokines in macrophages. The administration of a C3aR antagonist (C3aRA) attenuated the development of thoracic aortic aneurysms in MFS mice. The increased inflammation response and matrix metalloproteinases activities were also attenuated by C3aRA treatment in MFS mice. Therefore, these findings indicate that the complement C3a/C3aR inhibition alleviates the formation of aortic aneurysm in Marfan syndrome mice.

Phthalate exposure and subfecundity in preconception couples: A nested case-control study.

BACKGROUND: Phthalates (PAEs) are endocrine-disrupting chemicals ubiquitously found in the environment. This study aimed to examine the association between exposure of PAEs and subfecundity in preconception couples. METHODS: This is a nested case-control study based on preconception cohort. Preconception couples with intention to conceive were enrolled and followed up until a clinically confirmed pregnancy or 12 menstrual cycles of preparation for conception. A total of 107 couples with subfecundity- time to pregnancy (TTP) more than 12 menstrual cycles, and 144 couples ≤12 cycles were included in the analysis. The levels of PAE metabolites in one spot urine samples were detected and compared between the groups. The weighted quantile sum (WQS) regression model and Bayesian kernel machine regression (BKMR) model were used to examine the joint effects of couples' exposure to PAEs on subfecundity. RESULTS: Using the multivariate binary logistic regression model, compared to the lowest quartile of urinary ∑PAEs concentration group, both preconception females (aOR=2.42, 95% CI: 1.10-5.30, p=0.027) and males (aOR=2.99, 95% CI: 1.36-6.58, p=0.006) in the highest quartile group had an increased risk of subfecundity, and a dose-response relationship was observed between PAEs and the risk of subfecundity. The WQS analyses found that co-exposure to PAE mixture was a risk factor for subfecundity in preconception female (aOR=1.76, 95% CI: 1.38-2.26, p<0.001), male (aOR=1.58, 95% CI: 1.20-2.08, p=0.001), and couple (aOR=2.39, 95% CI: 1.61-3.52, p<0.001). The BKMR model found a positive combined effect of mixed exposure to PAEs on the risk of subfecundity. CONCLUSIONS: PAEs increase the risk of subfecundity in preconception couples. Our research reinforced the need of monitoring PAE exposure for the purpose of improving human reproductive health.

Customized Heteronuclear Dual Single-Atom and Cluster Assemblies via D-Band Orchestration for Oxygen Reduction Reaction.

Advanced oxygen reduction reaction (ORR) catalysts, integrating with well-dispersed single atom (SA) and atomic cluster (AC) sites, showcase potential in bolstering catalytic activity. However, the precise structural modulation and in-depth investigation of their catalytic mechanisms pose ongoing challenges. Herein, a proactive cluster lockdown strategy is introduced, relying on the confinement of trinuclear clusters with metal atom exchange in the covalent organic polymers, enabling the targeted synthesis of a series of multicomponent ensembles featuring FeCo (Fe or Co) dual-single-atom (DSA) and atomic cluster (AC) configurations (FeCo-DSA/AC) via thermal pyrolysis. The designed FeCo-DSA/AC surpasses Fe- and Co-derived counterparts by 18 mV and 49 mV in ORR half-wave potential, whilst exhibiting exemplary performance in Zn-air batteries. Comprehensive analysis and theoretical simulation elucidate the enhanced activity stems from adeptly orchestrating dz 2-dxz and O 2p orbital hybridization proximate to the Fermi level, fine-tuning the antibonding states to expedite OH* desorption and OOH* formation, thereby augmenting catalytic activity. This work elucidates the synergistic potentiation of active sites in hybrid electrocatalysts, pioneering innovative targeted design strategies for single-atom-cluster electrocatalysts.

Detecting Submicromolar Analytes in Mixtures with a 5 min Acquisition on 600 MHz NMR Spectrometers.

Amino compounds are widely present in complex mixtures in chemistry, biology, medicine, food, and environmental sciences involving drug impurities and metabolisms of proteins, biogenic amines, neurotransmitters, and pyrimidine in biological systems. Nuclear magnetic resonance (NMR) spectroscopy is an excellent tool for simultaneously identifying and quantifying these in-mixture compounds but has a limit-of-detection (LOD) over several micromolarities (>5 µM). To break such a sensitivity barrier, we developed a sensitive and rapid method by combining the probe-induced sensitivity enhancement and nonuniform-sampling-based 1H-13C HSQC 2D-NMR (PRISE-NUS-HSQC). We introduced two 13CH3 tags for each analyte to respectively increase the 1H and 13C abundances for up to 6 and 200 fold. This enabled high-resolution detection of 0.4-0.8 µM analytes in mixtures in 5 mm tubes with a 5 min acquisition on 600 MHz spectrometers. The method is much more sensitive and faster than traditional 1H-13C HSQC methods (∼50 µM, >10 h). Using sulfanilic acid as a single reference, furthermore, we established a database covering chemical shifts and relative-response factors for >100 compounds, enabling reliable identification and quantification. The method showed good quantitation linearity, accuracy, precision, and applicability in multiple biological matrices, offering a rapid and sensitive approach for quantitative analysis of large cohorts of chemical, medicinal, metabolomic, food, and other mixtures.

3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.

IL-17A induces valvular endothelial inflammation and aggravates calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is an aging related disease characterized by inflammation and fibrocalcific remodeling. IL-17A is a key cytokine associated with pathophysiology of inflammatory and fibrotic disease. Previous studies showed accumulation of IL-17A-producing T helper lymphocytes in human calcified aortic valves and significantly elevated IL-17RA expression in calcified valves. However, the role of IL-17A signaling in the initiation and development of CAVD is still unclear. In this study, by analyzing public transcriptome databases, we found that IL-17A-IL-17RA signaling is activated in calcified valves. Gene expression analysis revealed significantly increased IL-17A, IL-17RA, and RUNX2 expression in calcified human aortic valves compared to in non-calcified valves, and the expression of IL-17A and IL-17RA were positively correlated with RUNX2 expression. A 5/6 nephrectomy was performed in Apoe-/- (Apoe knockout) mice to establish a CAVD mouse model. IL-17A-neutralizing antibodies significantly reduced valve calcium deposition and decreased expression of RUNX2 in aortic valves. Immunofluorescence staining of human aortic valves and qRT-PCR analysis of primary aortic valve cells revealed abundant expression of IL-17RA in valvular endothelial cells (VECs). RNA sequencing indicated that IL-17A promoted the activation of inflammatory signaling pathways in VECs. Furthermore, qRT-PCR and cytometric bead array analysis confirmed that IL-17A promoted the expression or secretion of inflammatory cytokines IL-6 and IL-1ß, chemokines CXCL2 and CXCL8, and fibrosis-related gene COL16A1. Our findings indicate that elevated IL-17A in CAVD may promote valve inflammation, fibrosis, and calcification by inducing endothelial activation and inflammation. Targeting IL-17A-IL-17RA signaling may be a potential therapeutic strategy for CAVD.

Multivalent adaptor proteins specifically target NK cells carrying a universal chimeric antigen receptor to ErbB2 (HER2)-expressing cancers.

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

Computationally rational design of metal-involving halogen bonds with π-covalency: Structures and bonding analysis.

Traditional π-covalent interactions have been proved in the non-metal halogen bond adducts formed by chloride and halogenated triphenylamine-based radical cations. In this study, we have rationally designed two metal-involving halogen bond adducts with π-covalency property, such as [L1-Pd···I-PTZ]+ (i.e., 1) and [L2-Pd···I-PTZ]+ (i.e., 2), in which the square-planar palladium complexes serve as halogen bond acceptor and 3,7-diiodo-10H-phenothiazine radical cation (i.e., [I-PTZ]•+ ) acts as halogen bond donor. Noncovalent interaction analysis and quantum theory of atoms in molecules analysis revealed that there are notable halogen bond interactions along the Pd···I direction without genuine chemical bond formed in both designed adducts. Energy decomposition analysis together with natural orbital for chemical valence calculations were performed to gain insight into their bonding nature, which demonstrated the presence of remarkable π-covalent interactions and σ-covalent interactions in both 1 and 2. We therefore proposed a new strategy for building the metal-involving halogen bonds with π-covalency property, which will help the further development of new types of halogen bonds.