A second-generation M1-polarized CAR macrophage with antitumor efficacy.

Chimeric antigen receptor (CAR) T cell therapies have successfully treated hematological malignancies. Macrophages have also gained attention as an immunotherapy owing to their immunomodulatory capacity and ability to infiltrate solid tumors and phagocytize tumor cells. The first-generation CD3ζ-based CAR-macrophages could phagocytose tumor cells in an antigen-dependent manner. Here we engineered induced pluripotent stem cell-derived macrophages (iMACs) with toll-like receptor 4 intracellular toll/IL-1R (TIR) domain-containing CARs resulting in a markedly enhanced antitumor effect over first-generation CAR-macrophages. Moreover, the design of a tandem CD3ζ-TIR dual signaling CAR endows iMACs with both target engulfment capacity and antigen-dependent M1 polarization and M2 resistance in a nuclear factor kappa B (NF-κB)-dependent manner, as well as the capacity to modulate the tumor microenvironment. We also outline a mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we provide a second-generation CAR-iMAC with an ability for orthogonal phagocytosis and polarization and superior antitumor functions in treating solid tumors relative to first-generation CAR-macrophages.

Fine-tuning of mTOR signaling by the UBE4B-KLHL22 E3 ubiquitin ligase cascade in brain development.

Spatiotemporal regulation of the mechanistic target of rapamycin (mTOR) pathway is pivotal for establishment of brain architecture. Dysregulation of mTOR signaling is associated with a variety of neurodevelopmental disorders. Here, we demonstrate that the UBE4B-KLHL22 E3 ubiquitin ligase cascade regulates mTOR activity in neurodevelopment. In a mouse model with UBE4B conditionally deleted in the nervous system, animals display severe growth defects, spontaneous seizures and premature death. Loss of UBE4B in the brains of mutant mice results in depletion of neural precursor cells and impairment of neurogenesis. Mechanistically, UBE4B polyubiquitylates and degrades KLHL22, an E3 ligase previously shown to degrade the GATOR1 component DEPDC5. Deletion of UBE4B causes upregulation of KLHL22 and hyperactivation of mTOR, leading to defective proliferation and differentiation of neural precursor cells. Suppression of KLHL22 expression reverses the elevated activity of mTOR caused by acute local deletion of UBE4B. Prenatal treatment with the mTOR inhibitor rapamycin rescues neurogenesis defects in Ube4b mutant mice. Taken together, these findings demonstrate that UBE4B and KLHL22 are essential for maintenance and differentiation of the precursor pool through fine-tuning of mTOR activity.

HK2 in microglia and macrophages contribute to the development of neuropathic pain.

Neuropathic pain is a complex pain condition accompanied by prominent neuroinflammation involving activation of both central and peripheral immune cells. Metabolic switch to glycolysis is an important feature of activated immune cells. Hexokinase 2 (HK2), a key glycolytic enzyme enriched in microglia, has recently been shown important in regulating microglial functions. Whether and how HK2 is involved in neuropathic pain-related neuroinflammation remains unknown. Using a HK2-tdTomato reporter line, we found that HK2 was prominently elevated in spinal microglia. Pharmacological inhibition of HK2 effectively alleviated nerve injury-induced acute mechanical pain. However, selective ablation of Hk2 in microglia reduced microgliosis in the spinal dorsal horn (SDH) with little analgesic effects. Further analyses showed that nerve injury also significantly induced HK2 expression in dorsal root ganglion (DRG) macrophages. Deletion of Hk2 in myeloid cells, including both DRG macrophages and spinal microglia, led to the alleviation of mechanical pain during the first week after injury, along with attenuated microgliosis in the ipsilateral SDH, macrophage proliferation in DRGs, and suppressed inflammatory responses in DRGs. These data suggest that HK2 plays an important role in regulating neuropathic pain-related immune cell responses at acute phase and that HK2 contributes to neuropathic pain onset primarily through peripheral monocytes and DRG macrophages rather than spinal microglia.

On the nature of Cu-carbon interaction through N-modification for enhanced ethanol synthesis from syngas and methanol.

Direct conversion of syngas into ethanol is an attractive process because of its short route and high-added value, but remains an enormous challenge due to the low selectivity caused by unclear active sites. Here, the Cu(111) supported N-modified graphene fragments C13-mNm/Cu(111) (m = 0-2) are demonstrated to be an efficient catalyst for fabricating ethanol from syngas and methanol. Our results suggest that the Cu-carbon interaction not only facilitates CO activation, but also significantly affects the adsorption stability of C2 intermediates and finally changes the fundamental reaction mechanism. The impeded hydrogenation performance of C13/Cu(111) due to the introduced Cu-carbon interaction is dramatically improved by N-doping. Multiple analyses reveal that the promoted electron transfer and the enhanced electron endowing ability of C13-mNm/Cu(111) (m = 1-2) to the co-adsorbed CH3CHxOH (x = 0-1) and H are deemed to be mainly responsible for the remarkable enhancement in hydrogenation ability. From the standpoint of the frontier molecular orbital, the decreased HOMO-LUMO gap and the increased overlap extent of HOMO and LUMO with the doping of N atoms also further verify the more facile hydrogenation reactions. Clearly, the Cu-carbon interaction through N-modification is of critical importance in ethanol formation. The final hydrogenation reaction during ethanol formation is deemed to be the rate-controlling step. The insights gained here could shed new light on the nature of Cu-carbon interaction in carbon material modified Cu-based catalysts for ethanol synthesis, which could be extended to design and modify other metal-carbon catalysts.

The ZSWIM8 ubiquitin ligase regulates neurodevelopment by guarding the protein quality of intrinsically disordered Dab1.

Protein quality control (PQC) is essential for maintaining protein homeostasis and guarding the accuracy of neurodevelopment. Previously, we found that a conserved EBAX-type CRL regulates the protein quality of SAX-3/ROBO guidance receptors in Caenorhabditis elegans. Here, we report that ZSWIM8, the mammalian homolog of EBAX-1, is essential for developmental stability of mammalian brains. Conditional deletion of Zswim8 in the embryonic nervous system causes global cellular stress, partial perinatal lethality and defective migration of neural progenitor cells. CRISPR-mediated knockout of ZSWIM8 impairs spine formation and synaptogenesis in hippocampal neurons. Mechanistic studies reveal that ZSWIM8 controls protein quality of Disabled 1 (Dab1), a key signal molecule for brain development, thus protecting the signaling strength of Dab1. As a ubiquitin ligase enriched with intrinsically disordered regions (IDRs), ZSWIM8 specifically recognizes IDRs of Dab1 through a "disorder targets misorder" mechanism and eliminates misfolded Dab1 that cannot be properly phosphorylated. Adult survivors of ZSWIM8 CKO show permanent hippocampal abnormality and display severely impaired learning and memory behaviors. Altogether, our results demonstrate that ZSWIM8-mediated PQC is critical for the stability of mammalian brain development.

Heat Shock Factor A1s are required for phytochrome-interacting factor 4-mediated thermomorphogenesis in Arabidopsis.

Thermomorphogenesis and the heat shock (HS) response are distinct thermal responses in plants that are regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and HEAT SHOCK FACTOR A1s (HSFA1s), respectively. Little is known about whether these responses are interconnected and whether they are activated by similar mechanisms. An analysis of transcriptome dynamics in response to warm temperature (28°C) treatment revealed that 30 min of exposure activated the expression of a subset of HSFA1 target genes in Arabidopsis thaliana. Meanwhile, a loss-of-function HSFA1 quadruple mutant (hsfa1-cq) was insensitive to warm temperature-induced hypocotyl growth. In hsfa1-cq plants grown at 28°C, the protein and transcript levels of PIF4 were greatly reduced, and the circadian rhythm of many thermomorphogenesis-related genes (including PIF4) was disturbed. Additionally, the nuclear localization of HSFA1s and the binding of HSFA1d to the PIF4 promoter increased following warm temperature exposure, whereas PIF4 overexpression in hsfa1-cq partially rescued the altered warm temperature-induced hypocotyl growth of the mutant. Taken together, these results suggest that HSFA1s are required for PIF4 accumulation at a warm temperature, and they establish a central role for HSFA1s in regulating both thermomorphogenesis and HS responses in Arabidopsis.

Significance of a tumor microenvironment-mediated P65-miR-30a-5p-BCL2L11 amplification loop in multiple myeloma.

Despite significant progress in the treatment of myeloma, multiple myeloma (MM) remains an incurable hematological malignancy due to cell adhesion-mediated drug resistance (CAM-DR) phenotype. However, data on the molecular mechanisms underlying the CAM-DR remains scanty. Here, we identified a miRNA-mRNA regulatory network in myeloma cells that are directly adherent to bone marrow stromal cells (BMSCs). Our data showed that the BMSCs up-regulated miR-30a-5p and down-regulated BCL2L11 at both mRNA and protein level in the myeloma cells. Besides, luciferase reporter genes demonstrated direct interaction between miR-30a-5p and BCL2L11 gene. Moreover, the BMSCs activated NF-ΚB signaling pathway in myeloma cells and the NF-κB P65 was shown to directly bind the miR-30a-5p promoter region. Moreover, suppression of the miR-30a-5p or upregulation of the BCL2L11 promoted apoptosis of the myeloma cells independent of the BMSCs, thus suggesting clinical significance of miR-30a-5p inhibitor and PLBCL2L11 plasmid in CAM-DR. Together, our data demonstrated the role of P65-miR-30a-5p-BCL2L11 loop in CAM-DR myeloma cells. These findings give new insights into the role of tumor microenvironment in the treatment of patients with myeloma.

Transcriptional Profiling Reveals a Time-of-Day-Specific Role of REVEILLE 4/8 in Regulating the First Wave of Heat Shock-Induced Gene Expression in Arabidopsis.

Although much is known about plant responses to heat shock (HS), how plants sense high temperature and the primary HS signal transduction pathway leading to HS-regulated gene expression are still poorly understood. To identify primary transcription factors that mediate HS-regulated gene expression and their target genes, RNA sequencing was performed to detect genes whose expression is rapidly altered by HS in Arabidopsis (Arabidopsis thaliana). The results showed several genes were induced after only 5 min of HS treatment, suggesting that HS signaling occurs very rapidly. Analysis of the cis-elements in the promoters of genes upregulated by 10 min of HS treatment identified HEAT SHOCK FACTOR A1s (HSFA1s) and circadian clock proteins REVEILLE4 (RVE4) and RVE8 as essential transcription factors that independently mediate early HS-induced gene expression. Using hsfa1a/b/d/e and rve4/8 mutants, we identified subsets of HSFA1s- or RVE4/8-dependent early HS-induced genes and showed RVE4/8 regulate plant thermotolerance partially by regulating the expression of downstream transcription factors ETHYLENE RESPONSIVE FACTOR53 (ERF53) and ERF54, specifically around noon. These findings reveal a potential transcriptional regulatory hierarchy governing the first wave of HS-induced gene expression. They also provided important insight into the mechanism by which the circadian clock gates thermotolerance and prepares plants for exposure to high temperatures during the day.

Chemical composition of essential oils from Thymus mongolicus, Cinnamomum verum, and Origanum vulgare and their acaricidal effects on Haemaphysalis longicornis (Acari: Ixodidae).

Chemical acaricides are mainly used in traditional tick control, which leads to the emergence of tick resistance and concurrently results in environmental pollution. In the present study, the chemical constituents of essential oils (EOs) from Thymus mongolicus, Cinnamomum verum, and Origanum vulgare was analyzed, and their potential application was evaluated to control the vector tick Haemaphysalis longicornis, which is widely distributed over vast areas of Eurasia, Australia, and New Zealand. Gas chromatography-mass spectrometry analysis revealed that the phenols thymol and carvacrol accounted for 34.66% and 75.72% of the EOs of T. mongolicus and O. vulgare, respectively, whereas trans-cinnamaldehyde (49.42%) was the main constituent of C. verum EO. Immersion tests showed that the EOs of C. verum and O. vulgare had significant acaricidal activity against larval H. longicornis, with the 50% lethal concentration (LC50) being 16.07 and 18.02 mg/mL, respectively, and the 95% lethal concentration (LC95) being 120.37 and 130.09 mg/mL, respectively. The EOs of O. vulgare and T. mongolicus showed significant acaricidal activity against unfed adult H. longicornis, with LC50 being 43.50 and 44.21 mg/mL, respectively, and LC95 being 113.66 and 137.99 mg/mL, respectively. The fumigant toxicity test showed significant acaricidal activity of the three EOs against both unfed and engorged nymphal and adult H. longicornis. Enzyme assays revealed that the EOs of both C. verum and O. vulgare significantly inhibited glutathione S-transferase activity (P < 0.05). In contrast, the activities of carboxylesterase and multifunction oxidases were significantly inhibited by EOs extracted from all three plants (P < 0.05). Taken together, these findings suggest that plant EOs may serve as an environment-friendly alternative for synthetic acaricides in future tick control.

A novel defensin-like peptide contributing to antimicrobial and antioxidant capacity of the tick Dermacentor silvarum (Acari: Ixodidae).

Defensins are the most diverse groups of antimicrobial peptides in invertebrate animals. In ticks, defensins show great potential as targets for tick control, and display future prospect for therapeutic drug development. In the present study, a novel defensin-like gene (Ds-defensin) contributing to the antimicrobial and antioxidant capacity of the tick Dermacentor silvarum was characterized. The full-length of the Ds-defensin gene was 382 bp, which displayed tissue-specific expression and was highly abundant in the salivary glands and carcasses of the adults. It encodes a 71-amino acid defensin-like protein, and the protein precursor is characterized by a 22-amino acid signal peptide and a 34-amino acid mature peptide. The peptide displayed potent activity against most of the tested gram-positive bacteria, including Staphylococcus aureus, S. carnosus and Nocardia asteroides, and one tested gram-negative bacterium, Psychrobacter faecalis. Scanning electron microscopy revealed that the cell wall and surface of treated bacteria became rough and gradually formed pores after a 30-min exposure to the Ds-defensin peptide. Additionally, the peptide also showed significant antioxidant capacity. The above results implied that the defensin-like peptide may play an important role in tick defense and the interaction with microorganisms.

mTOR-mediated metabolic reprogramming shapes distinct microglia functions in response to lipopolysaccharide and ATP.

Microglia constantly survey the brain microenvironment and rapidly adopt different phenotypes in response to environmental stimuli. Such dynamic functions require a unique metabolism and bioenergetics. However, little is known about the basic metabolism of microglia and how metabolic changes regulate microglia function. Here, we uncover that microglia activation is accompanied by extensive transcriptional changes in glucose and lipid metabolism-related genes. Using metabolic flux assays, we found that LPS, a prototype of the pathogen-associated molecular patterns (PAMPs), significantly enhanced glycolysis but suppressed oxidative phosphorylation (OXPHOS) in primary cultured microglia. By contrast, ATP, a known damage-associated molecular pattern (DAMPs) that triggers sterile activation of microglia, boosted both glycolysis and OXPHOS. Importantly, both LPS and ATP activated the mechanistic target of rapamycin (mTOR) pathway and enhanced the intracellular reactive oxygen species (ROS). Inhibition of mTOR activity suppressed glycolysis and ROS production in both conditions but exerted different effects on OXPHOS: it attenuated the ATP-induced elevation of OXPHOS, yet had no impact on the LPS-induced suppression of OXPHOS. Further, inhibition of mTOR or glycolysis decreased production of LPS-induced proinflammatory cytokines and ATP-induced tumor necrosis factor-α (TNF-α) and brain derived neurotrophic factor (BDNF) in microglia. Our study reveals a critical role for mTOR in the regulation of metabolic programming of microglia to shape their distinct functions under different states and shed light on the potential application of targeting metabolism to interfere with microglia-mediated neuroinflammation in multiple disorders.

CD73-derived adenosine controls inflammation and neurodegeneration by modulating dopamine signalling.

Ectonucleotidase-mediated ATP catabolism provides a powerful mechanism to control the levels of extracellular adenosine. While increased adenosine A2A receptor (A2AR) signaling has been well-documented in both Parkinson's disease models and patients, the source of this enhanced adenosine signalling remains unclear. Here, we show that the ecto-5'-nucleotidase (CD73)-mediated adenosine formation provides an important input to activate A2AR, and upregulated CD73 and A2AR in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease models coordinatively contribute to the elevated adenosine signalling. Importantly, we demonstrate that CD73-derived adenosine-A2AR signalling modulates microglial immunoresponses and morphological dynamics. CD73 inactivation significantly attenuated lipopolysaccharide-induced pro-inflammatory responses in microglia, but enhanced microglia process extension, movement and morphological transformation in the laser injury and acute MPTP-induced Parkinson's disease models. Limiting CD73-derived adenosine substantially suppressed microglia-mediated neuroinflammation and improved the viability of dopaminergic neurons and motor behaviours in Parkinson's disease models. Moreover, CD73 inactivation suppressed A2AR induction and A2AR-mediated pro-inflammatory responses, whereas replenishment of adenosine analogues restored these effects, suggesting that CD73 produces a self-regulating feed-forward adenosine formation to activate A2AR and promote neuroinflammation. We further provide the first evidence that A2A enhanced inflammation by antagonizing dopamine-mediated anti-inflammation, suggesting that the homeostatic balance between adenosine and dopamine signalling is key to microglia immunoresponses. Our study thus reveals a novel role for CD73-mediated nucleotide metabolism in regulating neuroinflammation and provides the proof-of-principle that targeting nucleotide metabolic pathways to limit adenosine production and neuroinflammation in Parkinson's disease might be a promising therapeutic strategy.

Brassinosteroids regulate outer ovule integument growth in part via the control of INNER NO OUTER by BRASSINOZOLE-RESISTANT family transcription factors.

Brassinosteroids (BRs) play important roles in regulating plant reproductive processes. BR signaling or BR biosynthesis null mutants do not produce seeds under natural conditions, but the molecular mechanism underlying this infertility is poorly understood. In this study, we report that outer integument growth and embryo sac development were impaired in the ovules of the Arabidopsis thaliana BR receptor null mutant bri1-116. Gene expression and RNA-seq analyses showed that the expression of INNER NO OUTER (INO), an essential regulator of outer integument growth, was significantly reduced in the bri1-116 mutant. Increased INO expression due to overexpression or increased transcriptional activity of BRASSINAZOLE-RESISTANT 1 (BZR1) in the mutant alleviated the outer integument growth defect in bri1-116 ovules, suggesting that BRs regulate outer integument growth partially via BZR1-mediated transcriptional regulation of INO. Meanwhile, INO expression in bzr-h, a null mutant for all BZR1 family genes, was barely detectable; and the outer integument of bzr-h ovules had much more severe growth defects than those of the bri1-116 mutant. Together, our findings establish a new role for BRs in regulating ovule development and suggest that BZR1 family transcription factors might regulate outer integument growth through both BRI1-dependent and BRI1-independent pathways.

Alternative Splicing of Disabled-1 Controls Multipolar-to-Bipolar Transition of Migrating Neurons in the Neocortex.

Multipolar-to-bipolar transition (MBT) is crucial for the neuronal migration and positioning in the neocortex. Reelin-Disabled-1 (Dab1) signaling plays a pivotal role in neuronal migration, yet how Dab1 coordinatively regulates downstream molecules to affect MBT remains unclear. We have previously found that alternative splicing produces multiple Dab1 isoforms with different tyrosine motifs and differential ability to recruit downstream effectors. Here, we report that splicing of Dab1 exons 7 and 8 and 9bc dynamically regulates the inclusion and activities of Dab1 tyrosine motifs in the neocortex. By in utero electroporation, we show that expression of Dab1 isoforms missing exons 7 and 8 or retaining exons 9bc in WT neurons resulted in neuronal migration defects with attenuated Dab1 tyrosine phosphorylation, disrupted leading process extension, and disorientated multipolar neurons in the multipolar accumulation zone. Introducing the canonical Dab1 form, but not those missing exons 7 and 8 or retaining exons 9bc, into Dab1-deficient neurons promoted MBT and rescued neuronal migration defects, suggesting that alternative splicing of Dab1 modulates the tyrosine motif switch and mediates MBT of cortical neurons. Our study reveals a critical mechanism by which Dab1 alternative splicing coordinately controls MBT and neuronal migration in a spatiotemporal manner.

Proteome-wide Mapping of Endogenous SUMOylation Sites in Mouse Testis.

SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry. Bioinformatics analyses showed that SUMO1-modified proteins were enriched in transcription regulation and DNA repair. Nab1 was validated to be an authentic SUMOylated protein and Lys479 was identified to be the major SUMOylation site. The SUMOylation of Nab1 enhanced its interaction with HDAC2 and maintained its inhibitory effect on EGR1 transcriptional activity. Therefore, we provided a novel approach to investigating endogenous SUMOylation sites in tissue samples.

Implication of microglia activation and CSF-1/CSF-1Rpathway in lumbar disc degeneration-related back pain.

Back pain is common and costly. Although lumbar disc degeneration has long been regarded as a major contributor to back pain, how disc degeneration leads to back pain remains unclear. Recent studies observed microglia activation in the spinal cord after disc degeneration, suggesting activated microglia may be involved in discogenic back pain. To determine whether microglia activation participates in disc degeneration-induced back pain, we used a modified disc puncture-induced degeneration-related back pain mouse model to examine the changes in spinal microglia and investigate the potential link between microglia activation and discogenic back pain. In this study, 46 CX3CR1GFP/+ male mice were used in experimental and sham groups. A modified posterolateral retroperitoneal approach was used to expose the L3/L4 disc to induce the needle puncture in the experimental group. Behavioral tests, including grip force and physical function, were used to measure back pain at pre- and postsurgery. The L3 dorsal root ganglions and lumbar spinal cord were obtained at postoperative weeks 1 to 4 followed by immunofluorescence with different antibodies. Micrographs were obtained by confocal microscopy, and morphometric measurements of microglia were analyzed using Imaris. The punctured disc underwent progressive degeneration and mice with disc degeneration showed impaired grip force and physical function. Compared to the control mice, the number of microglia in the lumbar spinal cord was significantly increased in the disc-punctured animals. Moreover, accumulated microglia exhibited larger soma size and lesser ramification in the disc-injured mice. Immunofluorescence demonstrated colony-stimulating factor 1, a cytokine that promotes microglia repopulation, was significantly increased in L3 dorsal root ganglions, whereas its receptor colony-stimulating factor 1 receptor was upregulated on microglia in the disc-injured mice. In summary, lumbar disc puncture caused progressive disc degeneration which induced microglia activation and back pain in mice. Increased colony-stimulating factor 1/colony-stimulating factor 1 receptor signaling is involved in the disc degeneration-induced microglia activation and back pain.

Nicking-enhanced rolling circle amplification for sensitive fluorescent detection of cancer-related microRNAs.

In this study, a biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) was proposed for the highly sensitive detection of cancer-related let-7a microRNA (miRNA). The sensing system consists of a padlock probe (PP), which contains a target recognition sequence and two binding sites for nicking endonuclease (NEase), and molecular beacon (MB) as reporting molecule. Upon hybridization with let-7a, the PP can be circularized by ligase. Then, the miRNA acted as polymerization primer to initiate rolling circle amplification (RCA). With the assistance of NEase, RCA products can be nicked on the cyclized PP and are displaced during the subsequent duplication process, generating numerous nicked fragments (NFs). These NFs not only induce another RCA reaction but also open the molecular beacons (MBs) via hybridization, leading to significantly amplified fluorescence signal. Under the optimized conditions, this method exhibits high sensitivity toward target miRNA let-7a with a detection limit of as low as 10 pM, a dynamic range of three orders of magnitude is achieved, and its family member is easily distinguished even with only one mismatched base. Meanwhile, it displays good recovery and satisfactory reproducibility in fetal bovine serum (FBS). Therefore, these merits endow the newly proposed N-RCA strategy with powerful implications for miRNA detection. Graphical abstract A biosensing system based on nicking-enhanced rolling circle amplification (N-RCA) for the highly sensitive detection of cancer-related let-7a microRNA.

Carbon chain growth by formyl coupling over the Cu/γ-AlOOH(001) surface in syngas conversion.

Catalytic conversion of syngas to valuable chemicals and fuels such as ethanol is an extremely desirable process route. In the present study, the elementary steps leading to the formation of ethanol via syngas conversion over the Cu/γ-AlOOH(001) surface have been explored using density functional theory (DFT) calculations. The reaction pathway CO + H → CHO, CHO + CHO → OHCCHO → CHCHO + O, CHCHO + 4H → CH2CHO + 3H → CH3CHO + 2H → CH3CH2O + H → C2H5OH is the most favorable; during the whole process, CH3CHO formation needs to overcome the highest activation barrier. Different from the γ-AlOOH(001) surface, carbon chain growth is realized via the formyl coupling mechanism on the Cu/γ-AlOOH(001) surface; this step needs to overcome a 1.07 eV activation barrier and is exothermic by 0.73 eV. Our Bader charge analyses revealed that the addition of the Cu component enhances the electrostatic interaction between the CHO intermediate and the γ-AlOOH(001) surface with the aid of the formed CuOx species; as a result, the initial C-C chain forms in a different way. Moreover, the rate constant results manifest that the formation of the OHCCHO key intermediate can be facilitated by increasing the reaction temperature. We expect the obtained results will be useful for future experimental studies to improve the selectivity of C2 oxygenates in syngas conversion.