Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis.

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.

Dissecting the Enantioselective Neurotoxicity of Isocarbophos: Chiral Insight from Cellular, Molecular, and Computational Investigations.

Chiral organophosphorus pollutants are found abundantly in the environment, but the neurotoxicity risks of these asymmetric chemicals to human health have not been fully assessed. Using cellular, molecular, and computational toxicology methods, this story is to explore the static and dynamic toxic actions and its stereoselective differences of chiral isocarbophos toward SH-SY5Y nerve cells mediated by acetylcholinesterase (AChE) and further dissect the microscopic basis of enantioselective neurotoxicity. Cell-based assays indicate that chiral isocarbophos exhibits strong enantioselectivity in the inhibition of the survival rates of SH-SY5Y cells and the intracellular AChE activity, and the cytotoxicity of (S)-isocarbophos is significantly greater than that of (R)-isocarbophos. The inhibitory effects of isocarbophos enantiomers on the intracellular AChE activity are dose-dependent, and the half-maximal inhibitory concentrations (IC50) of (R)-/(S)-isocarbophos are 6.179/1.753 µM, respectively. Molecular experiments explain the results of cellular assays, namely, the stereoselective toxic actions of isocarbophos enantiomers on SH-SY5Y cells are stemmed from the differences in bioaffinities between isocarbophos enantiomers and neuronal AChE. In the meantime, the modes of neurotoxic actions display that the key amino acid residues formed strong noncovalent interactions are obviously different, which are related closely to the molecular structural rigidity of chiral isocarbophos and the conformational dynamics and flexibility of the substrate binding domain in neuronal AChE. Still, we observed that the stable "sandwich-type π-π stacking" fashioned between isocarbophos enantiomers and aromatic Trp-86 and Tyr-337 residues is crucial, which notably reduces the van der Waals' contribution (ΔGvdW) in the AChE-(S)-isocarbophos complexes and induces the disparities in free energies during the enantioselective neurotoxic conjugations and thus elucidating that (S)-isocarbophos mediated by synaptic AChE has a strong toxic effect on SH-SY5Y neuronal cells. Clearly, this effort can provide experimental insights for evaluating the neurotoxicity risks of human exposure to chiral organophosphates from macroscopic to microscopic levels.

Defect Engineering in CuSx/COF Hybridized Heterostructures: Synergistic Facilitation of the Charge Migration for an Efficacious Photocatalytic Conversion of CO2 into CO.

The photocatalytic CO2 reduction reaction (CO2RR) provides an attractive approach to tackling environmental issues. To actualize the optimal catalytic efficiency, one efficacious strategy is to rationally modulate the charge migration for the adopted heterogeneous catalysts. Herein, by virtue of a one-step hydrothermal method, Cu2S nanospheres and defect-rich Cu2S (CuSx) nanosheets are wrapped by a triazine-containing covalent framework (TP-TA COF), resulting in CuSx/TP-TA and Cu2S/TP-TA. Owing to the heterojunction construction that suppresses the carrier recombination, both hybridized structures present enhanced charge migration in comparison to that of their corresponding sulfides and COF constituents. It is worth emphasizing that CuSx/TP-TA proffers a significantly greater photocurrent than Cu2S/TP-TA. The subsequent photocatalytic reduction of CO2 also exhibits an apparently higher CO evolution rate, about 2.8 times higher than the Cu2S/TP-TA photocatalyst. The above evident improvement owes much to the heterostructure establishment between CuSx and TP-TA COF, as well as the synergistic effect provided by the defect engineering for CuSx, both of which are able to enhance the separation efficiency of photoinduced carriers. Our work sheds light on the rational construction of heterogeneous structures between organic and inorganic photocatalysts, which emphasizes the possible synergistic effect of defect centers for enhancing photocatalytic performance.

Construction of ternary CuO/CuFe2O4/g-C3N4 composite and its enhanced photocatalytic degradation of tetracycline hydrochloride with persulfate under simulated sunlight.

In this study, a graphitic carbon nitride (g-C3N4) based ternary catalyst CuO/CuFe2O4/g-C3N4 (CCCN) is successfully prepared thorough calcination method. After confirming the structure and composition of CCCN, the as-synthesized composites are utilized to activate persulfate (PS) for the degradation of organic contaminant. While using tetracycline hydrochloride (TC) as pollutant surrogate, the effects of initial pH, PS and catalyst concentration on the degradation rate are systematically studied. Under the optimized reaction condition, CCCN/PS is able to give 99% degradation extent and 74% chemical oxygen demand removal in assistance of simulated solar light, both of which are apparently greater than that of either CuO/CuFe2O4 and pristine g-C3N4. The great improvement in degradation can be assignable to the effective separation of photoinduced carriers thanks to the integration between CuO/CuFe2O4 and g-C3N4, as well as the increased reaction sites given by the g-C3N4 substrate. Moreover, the scavenging trials imply that the major oxidative matters involved in the decomposition are hydroxyl radicals (•OH), superoxide radicals (•O2-) and photo-induced holes (h+).

Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

Synergetic Effects of Pd0 Metal Nanoparticles and Pd2+ Ions on Enhanced Photocatalytic Activity of ZnWO4 Nanorods for Nitric Oxide Removal.

Doping and novel metallic nanoparticles loading on the photocatalyst are two effective means to enhance its photocatalytic activity. In our study, Pd0/Pd2+-co-modified ZnWO4 nanorods were fabricated by a two-step hydrothermal process and room-temperature reduction method. The performance of the as-prepared samples was evaluated through the photocatalytic nitric oxide (NOx) removal under simulated solar and visible-light irradiation. Pd0/Pd2+-co-modified ZnWO4 nanorods present a significantly enhanced photocatalytic activity for NOx removal compared with Pd0-loaded or Pd2+-doped ZnWO4 under simulated sunlight irradiation owing to a narrower band gap of Pd2+ doping compared with that of pure ZnWO4. The role of Pd0 nanoparticles is to act as an electron reservoir to restrain the recombination of e-/h+ pairs. According to the trapping measurements, the photoinduced holes and electrons play critical roles during the photocatalytic process. In addition, electron spin resonance (ESR) results further confirm that •O2- and •OH radicals are present and assist in the photocatalysis under simulated solar light irradiation. Stability test demonstrated that 1.5% Pd0/0.5% Pd2+-co-modified ZnWO4 nanorods as photocatalyst have high photocatalytic stability in NOx removal. This work proved that Pd0/Pd2+-co-modified ZnWO4 nanorods can be considered as an efficient photocatalyst for NOx removal.

Rescue of Glaucomatous Neurodegeneration by Differentially Modulating Neuronal Endoplasmic Reticulum Stress Molecules.

UNLABELLED: Axon injury is an early event in neurodegenerative diseases that often leads to retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. The connection of these two obviously sequential degenerative events, however, is elusive. Deciphering the upstream signals that trigger the neurodegeneration cascades in both neuronal soma and axon would be a key step toward developing the effective neuroprotectants that are greatly needed in the clinic. We showed previously that optic nerve injury-induced neuronal endoplasmic reticulum (ER) stress plays an important role in retinal ganglion cell (RGC) death. Using two in vivo mouse models of optic neuropathies (traumatic optic nerve injury and glaucoma) and adeno-associated virus-mediated RGC-specific gene targeting, we now show that differential manipulation of unfolded protein response pathways in opposite directions-inhibition of eukaryotic translation initiation factor 2α-C/EBP homologous protein and activation of X-box binding protein 1-promotes both RGC axons and somata survival and preserves visual function. Our results indicate that axon injury-induced neuronal ER stress plays an important role in both axon degeneration and neuron soma death. Neuronal ER stress is therefore a promising therapeutic target for glaucoma and potentially other types of neurodegeneration. SIGNIFICANCE STATEMENT: Neuron soma and axon degeneration have distinct molecular mechanisms although they are clearly connected after axon injury. We previously demonstrated that axon injury induces neuronal endoplasmic reticulum (ER) stress and that manipulation of ER stress molecules synergistically promotes neuron cell body survival. Here we investigated the possibility that ER stress also plays a role in axon degeneration and whether ER stress modulation preserves neuronal function in neurodegenerative diseases. Our results suggest that neuronal ER stress is a general mechanism of degeneration for both neuronal cell body and axon, and that therapeutic targeting of ER stress produces significant functional recovery.

Exploring the neurotoxicity of chiral dinotefuran towards nicotinic acetylcholine receptors: Enantioselective insights into species selectivity.

Dinotefuran is a chiral neonicotinoid that is widely distributed in environmental matrices, but its health risks to different organisms are poorly understood. This study investigated the neurotoxic responses of honeybee/cotton aphid nicotinic acetylcholine receptors (nAChRs) to chiral dinotefuran at the enantiomeric scale and demonstrated the microscopic mechanism of species selectivity in nAChR-mediated enantioselective neurotoxicity. The findings indicated that (S)-dinotefuran had a higher affinity for honeybee nAChR than (R)-dinotefuran whereas both enantiomers exhibited similar bioactivity toward cotton aphid nAChR. The results of dynamic neurotoxic processes indicated the association of conformational changes induced by chiral dinotefuran with its macroscopic neurotoxicity, and (R)-dinotefuran, which exhibit low toxicity to honeybee, was found to induce significant conformational changes in the enantioselective neurotoxic reaction, as supported by the average root-mean-square fluctuation (0.35 nm). Energy decomposition results indicated that electrostatic contribution (ΔGele) is the critical energy term that leads to substantial enantioselectivity, and both Trp-51 (－2.57 kcal mol-1) and Arg-75 (－4.86 kcal mol-1), which form a hydrogen-bond network, are crucial residues in mediating the species selectivity for enantioselective neurotoxic responses. Clearly, this study provides experimental evidence for a comprehensive assessment of the health hazards of chiral dinotefuran.

An MgO passivation layer and hydrotalcite derived spinel Co2AlO4 synergically promote photoelectrochemical water oxidation conducted using BiVO4-based photoanodes.

MxCo3-xO4 co-catalysed photoanodes with high potential for improvement in PEC water-oxidizing properties are reported. However, it is difficult to control the recombination of photogenerated carriers at the interface between the catalyst and cocatalyst. Here, an ultra-thin MgO passivation layer was introduced into the MxCo3-xO4/BiVO4 coupling system to construct a ternary composite photoanode Co2AlO4/MgO/BiVO4. The photocurrent density of the electrode is 3.52 mA cm-2, which is 3.2 times that of BiVO4 (at 1.23 V vs. RHE). The photocurrent is practically increased by 0.86 mA cm-2 and 1.56 mA cm-2 in comparison with that of Co2AlO4/BiVO4 and MgO/BiVO4 electrodes, respectively. Meanwhile, the Co2AlO4/MgO/BiVO4 electrode has the highest charge separation efficiency, the lowest charge transfer resistance (Rct) and best stability. The excellent PEC performance could be attributed to the inhibitive effect provided by the MgO passivation layer that efficaciously suppresses the electron-hole recombination at the interface and drives the hole transfer outward, which is induced by Co2AlO4 to capture the electrode/electrolyte interface for efficient water oxidation reaction. In order to understand the origin of this improvement, first-principles calculations with density functional theory (DFT) were performed. The theoretical investigation converges to our experimental results. This work proposes a novel idea for restraining the recombination of photogenerated carriers between interfaces and the rational design of efficient photoanodes.

[Recent progress of the aptamer-based antiviral drugs].

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.

Highly efficient photocatalytic hydrogen production by ZnCdS composite catalyst modified with NiCoP nanosheets prepared by LDH precursor.

The unique characteristics and diverse applications of 2D transition metal phosphides have aroused significant interest. In this paper, we successfully prepared 2D NiCoP modified ZnCdS composite. The NiCoP nanosheets were successfully obtained by phosphating layered double hydroxide (LDH) precursor. The results show that the ZnCdS-8%NiCoP has the highest photocatalytic performance among all the composite photocatalysts with the H2 evolution rate of 1370.1 µmol h-1, which is 17.9 folds higher than obtained with pure ZnCdS. Detailed analysis reveal that NiCoP nanosheets functions as an excellent electron acceptor, speeding up the directed migration of electrons. Furthermore, the rational mechanism of photocatalytic has been presented based on density function theory (DFT) calculations, which is well congruent with experimental results. Our research offers a simple, environmentally benign, and scalable technique for making highly effective photocatalysts, as well as a novel perspective on transition metal phosphides rational design.

In Situ N, O-Dually Doped Nanoporous Biochar Derived from Waste Eutrophic Spirulina for High-Performance Supercapacitors.

Sustainable and high-performance energy storage materials are crucial to address global energy and environmental challenges. In this study, Spirulina platensis was used as the carbon and nitrogen source, and Spirulina-based nanoporous biochar (SNPB) was synthesized through chemical activation using KOH as the activating agent in N2 atmosphere. SNPB-800-4 was characterized by N2 adsorption-desorption and XPS, showing a high specific surface area (2923.7 m2 g-1) and abundant heteroatomic oxygen (13.78%) and nitrogen (2.55%). SNPB-800-4 demonstrated an exceptional capacitance of 348 F g-1 at a current density of 1 A g-1 and a remarkable capacitance retention of 94.14% after 10,000 cycles at a current density of 10 A g-1 in 6 M KOH. Notably, symmetric supercapacitors SNPB-800-4//SNPB-800-4 achieved the maximum energy and power densities of 17.99 Wh kg-1 and 162.48 W kg-1, respectively, at a current density of 0.5 A g-1, and still maintained 2.66 Wh kg-1 when the power density was increased to 9685.08 W kg-1 at a current density of 30 A g-1. This work provides an easily scalable and straightforward way to convert waste algae biomass into in situ N, O-dually doped biochar for ultra-high-power supercapacitors.

Massively parallel in vivo Perturb-seq reveals cell type-specific transcriptional networks in cortical development.

Systematic analysis of gene function across diverse cell types in vivo is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell's perturbation in high-throughput single-cell assays. Leveraging AAV's versatile cell type tropism and high labeling capacity, we expanded the resolution and scale of in vivo CRISPR screens: allowing phenotypic analysis at single-cell resolution across a multitude of cell types in the embryonic brain, adult brain, and peripheral nervous system. We undertook extensive tests of 86 AAV serotypes, combined with a transposon system, to substantially amplify labeling and accelerate in vivo gene delivery from weeks to days. Using this platform, we performed an in utero genetic screen as proof-of-principle and identified pleiotropic regulatory networks of Foxg1 in cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct networks essential for cell fate specification. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% (mediated by lentivirus), and achieve analysis of over 30,000 cells in one experiment, thus enabling massively parallel in vivo Perturb-seq. Compatible with various perturbation techniques (CRISPRa/i) and phenotypic measurements (single-cell or spatial multi-omics), our platform presents a flexible, modular approach to interrogate gene function across diverse cell types in vivo, connecting gene variants to their causal functions.

In situ N, O co-doped porous carbon derived from antibiotic fermentation residues as electrode material for high-performance supercapacitors.

With the widespread use of antibiotics, the safe utilization of waste antibiotic fermentation residues has become an urgent issue to be resolved. In this study, in situ N, O co-doped porous carbon was prepared using fresh oxytetracycline fermentation residue under the mild activation of the green activator K2CO3. The optimal sample exhibited a 3D grid carbon skeleton structure, excellent specific surface area (SBET = 948 m2 g-1), and high nitrogen and oxygen content (N = 3.42 wt%, O = 14.86 wt%). Benefiting from its developed morphology, this sample demonstrated excellent electrochemical performance with a high specific capacitance of 310 F g-1 at a current density of 0.5 A g-1 in the three-electrode system. Moreover, it exhibited superior cycling stability with only a 5.32% loss of capacity after 10 000 cycles in 6 M KOH aqueous electrolyte. Furthermore, the symmetric supercapacitor prepared from it exhibited a maximum energy density of 7.2 W h kg-1 at a power density of 124.9 W kg-1, demonstrating its promising application prospects. This study provided a green and facile process for the sustainable and harmless treatment of antibiotic fermentation residues.

N/O Co-doped hierarchical nanoporous biochar derived from waste polypropylene nonwoven for high-performance supercapacitors.

How to efficiently treat municipal solid waste (MSW) has become one of the critical solutions in response to the call for "carbon neutrality". Here, the waste polypropylene nonwoven fabric of waste diapers was converted into hierarchical nanoporous biochar (HPBC) through pre-carbonization and activation processes as an ideal precursor for supercapacitors (SCs) with excellent performance. The prepared HPBC-750-4 with an ultrahigh specific surface area (3838.04 m2 g-1) and abundant heteroatomic oxygen (13.25%) and nitrogen (1.16%) codoped porous biochar structure. Given its structural advantages, HPBC-750-4 achieved a specific capacitance of 340.9 F g-1 at a current density of 1 A g-1 in a three-electrode system. Its capacitance retention rate was above 99.2% after 10 000 cycles at a current density of 10 A g-1, which indicated an excellent rate capability and long-term cycling stability. Furthermore, the HPBC-750-4//HPBC-750-4 symmetric SC exhibited a superb energy density of 10.02 W h kg-1 with a power density of 96.15 W kg-1 in a 6 M KOH electrolyte. This work not only demonstrates the enormous potential of waste polypropylene nonwoven fabric in the SC industry but also provides an economically feasible means of managing MSW.

[Combinatorial RNAi and its application in cancer gene therapy].

RNA interference (RNAi) has been proved as a novel approach for gene therapy. However, RNAi mono-therapy only aims at single gene, it therefore may ultimately fail to cure cancers caused by polygene variation. To overcome the deficiency of RNAi mono-therapy, "combinatorial RNA interference" (coRNAi) was put forward as a new strategy. By co-expressing the inducers of RNAi triggering single or multiple targets directly and other RNA- or protein-based silencers, coRNAi keeps target genes silent, prevents carcinogenic progression and induces apoptosis of tumor cells. This paper mainly reviews the major strategies of coRNAi and their applications in cancer gene therapy.

[Ubiquitination of recombinant adeno-associated viral vector and its application].

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

The hydrophilic treatment of a novel co-catalyst for greatly improving the solar water splitting performance over Mo-doped bismuth vanadate.

In this work, Molybdenum (Mo) doped bismuth vanadate (BiVO4) is carried out by traditional calcination method, while carbon-based cobalt (Co-Ci) is prepared by photoelectric deposition (PED) and used as co-catalyst to decorate the surface, its photocurrent density reached 3.15 mA/cm2 at 1.23 V vs RHE. More importantly, the H-Co-Ci/Mo: BiVO4 photoanode obtained by plasma etching of Co-Ci/Mo: BiVO4 has greatly improved surface hydrophilicity. The photocurrent density of H-Co-Ci/Mo: BiVO4 photoanode is 4.8 times that of BiVO4 photoanode, reaching 3.95 mA/cm2. In addition, the incident photon-current conversion efficiency (IPCE) value of the H-Co-Ci/Mo: BiVO4 photoanode is as high as 60%, and both the injection and separation efficiency have also been enhanced. The enhanced photoelectrochemical (PEC) performance is attributed to the good wettability of the material surface and improvement of water oxidation kinetics. These findings provide a mild and efficient modification method for improving BiVO4 used for water splitting, and are expected to provide new ideas for other photoanodes.

Characterizing the potentially neuronal acetylcholinesterase reactivity toward chiral pyraclofos: Enantioselective insights from spectroscopy, in silico docking, molecular dynamics simulation and per-residue energy decomposition studies.

Chiral organophosphorus agents are distributed ubiquitously in the environment, but the neuroactivity of these asymmetric chemicals to humans remains uncertain. This scenario was to explore the stereoselective neurobiological response of human acetylcholinesterase (AChE) to chiral pyraclofos at the enantiomeric scale, and then decipher the microscopic basis of enantioselective neurotoxicity of pyraclofos enantiomers. The results indicated that (R)-/(S)-pyraclofos can form the bioconjugates with AChE with a stoichiometric ratio of 1:1, but the neuronal affinity of (R)-pyraclofos (K = 6.31 × 104 M-1) with AChE was larger than that of (S)-pyraclofos (K = 1.86 × 104 M-1), and significant enantioselectivity was existed in the biochemical reaction. The modes of neurobiological action revealed that pyraclofos enantiomers were situated at the substrate binding domain, and the strength of the overall noncovalent bonds between (S)-pyraclofos and the residues was weaker than that of (R)-pyraclofos, resulting in the high inhibitory effect of (R)-pyraclofos toward the activity of AChE. Dynamic enantioselective biointeractions illustrated that the intervention of inherent conformational flexibility in the AChE-(R)-pyraclofos was greater than that of the AChE-(S)-pyraclofos, which arises from the big spatial displacement and the conformational flip of the binding domain composed of the residues Thr-64～Asn-89, Gly-122～Asp-134, and Thr-436～Tyr-449. Energy decomposition exhibited that the Gibbs free energies of the AChE-(R)-/(S)-pyraclofos were ΔG° = ï¼37.4/－30.2 kJ mol-1, respectively, and the disparity comes from the electrostatic energy during the stereoselective neurochemical reactions. Quantitative conformational analysis further confirmed the atomic-scale computational chemistry conclusions, and the perturbation of (S)-pyraclofos on the AChE's ordered conformation was lower than that of (R)-pyraclofos, which is germane to the interaction energies of the crucial residues, e.g. Tyr-124, Tyr-337, Asp-74, Trp-86, and Tyr-119. Evidently, this attempt will contribute mechanistic information to uncovering the neurobiological effects of chiral organophosphates on the body.