A novel engineered IL-21 receptor arms T-cell receptor-engineered T cells (TCR-T cells) against hepatocellular carcinoma.

Strategies to improve T cell therapy efficacy in solid tumors such as hepatocellular carcinoma (HCC) are urgently needed. The common cytokine receptor γ chain (γc) family cytokines such as IL-2, IL-7, IL-15 and IL-21 play fundamental roles in T cell development, differentiation and effector phases. This study aims to determine the combination effects of IL-21 in T cell therapy against HCC and investigate optimized strategies to utilize the effect of IL-21 signal in T cell therapy. The antitumor function of AFP-specific T cell receptor-engineered T cells (TCR-T) was augmented by exogenous IL-21 in vitro and in vivo. IL-21 enhanced proliferation capacity, promoted memory differentiation, downregulated PD-1 expression and alleviated apoptosis in TCR-T after activation. A novel engineered IL-21 receptor was established, and TCR-T armed with the novel engineered IL-21 receptors (IL-21R-TCR-T) showed upregulated phosphorylated STAT3 expression without exogenous IL-21 ligand. IL-21R-TCR-T showed better proliferation upon activation and superior antitumor function in vitro and in vivo. IL-21R-TCR-T exhibited a less differentiated, exhausted and apoptotic phenotype than conventional TCR-T upon repetitive tumor antigen stimulation. The novel IL-21 receptor in our study programs powerful TCR-T and can avoid side effects induced by IL-21 systemic utilization. The novel IL-21 receptor creates new opportunities for next-generation TCR-T against HCC.

Keratin Formed Bioadhesive Ophthalmic Gel for the Bacterial Conjunctivitis Treatment.

Keratin has the potential to function as the gel matrix in an ophthalmic formulation for the encapsulation of the macrolide antibiotic azithromycin. The quality of this formulation was thoroughly evaluated through various analyses, such as in vitro release assessment, rheological examination, intraocular retention studies in rabbits, assessment of bacteriostatic efficacy, and safety evaluations. It is worth mentioning that the gel demonstrated shear thinning properties and exhibited characteristics of an elastic solid, thereby confirming its structural stability. The gel demonstrated a notable affinity for mucosal surfaces in comparison to traditional azithromycin aqueous solutions. In vitro release testing revealed that drug release transpired via diffusion mechanisms, following a first-order kinetic release pattern. Additionally, the formulated gel exhibited remarkable antibacterial efficacy against Staphylococcus aureus and Pseudomonas aeruginosa in bacteriostatic evaluations. Lastly, safety assessments confirmed that the gel eye drops induced minimal irritation and displayed no apparent cytotoxicity, indicating their good safety and biocompatibility for ocular application. Thus, these findings indicated that the prepared azithromycin gel eye drops complied with the requisite standards for ophthalmic preparations.

External Validation of the Risk Assessment Model of Venous Thromboembolism in Multicenter Internal Medicine Inpatients.

To external validate the risk assessment model (RAM) of venous thromboembolism (VTE) in multicenter internal medicine inpatients. We prospectively collected 595 internal medical patients (310 with VTE patients, 285 non-VTE patients) were from Beijing Shijitan Hospital, Beijing Chaoyang Hospital, and the respiratory department of Beijing Tsinghua Changgeng Hospital from January 2022 to December 2022 for multicenter external validation. The prediction ability of Caprini RAM, Padua RAM, The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE) RAM, and Shijitan (SJT) RAM were compared. This study included a total of 595 internal medicine inpatients, including 242 (40.67%) in the respiratory department, 17 (2.86%) in the respiratory intensive care unit, 49 (8.24%) in the neurology department, 34 (5.71%) in the intensive care unit, 26 (4.37%) in the geriatric department, 22 (3.70%) in the emergency department, 71 (11.93%) in the nephrology department, 63 (10.59%) in the cardiology department, 24 (4.03%) in the hematology department, 6 (1.01%) in the traditional Chinese medicine department, 9 (1.51%) cases in the rheumatology department, 7 (1.18%) in the endocrinology department, 14 (2.35%) in the oncology department, and 11 (1.85%) in the gastroenterology department. Multivariate logistic regression analysis showed that among internal medicine inpatients, age > 60 years old, heart failure, nephrotic syndrome, tumors, history of VTE, and elevated D-dimer were significantly correlated with the occurrence of VTE (P < .05). The incidence of VTE increases with the increase of D-dimer. It was found that the effectiveness of SJT RAM (AUC = 0.80 ± 0.03) was better than Caprini RAM (AUC = 0.74 ± 0.03), Padua RAM (AUC = 0.72 ± 0.03) and IMPROVE RAM (AUC = 0.52 ± 0.03) (P < .05). The sensitivity and Yoden index of SJT RAM were higher than those of Caprini RAM, Pauda RAM, and IMPROVE RAM (P < .05), but specificity was not significantly different between the 4 models (P > .05). The SJT RAM derived from general hospitalized Chinese patients has effective and better predictive ability for internal medicine inpatients at risk of VTE.

Erratum: [Corrigendum] Macleaya cordata extracts exert antiviral effects in newborn mice with rotavirus­induced diarrhea via inhibiting the JAK2/STAT3 signaling pathway.

[This corrects the article DOI: 10.3892/etm.2020.8766.].

Associations between the cause of amblyopia and pre-treatment contrast sensitivity, stereoacuity, fixation, and nystagmus.

Purpose: To explore the association between the cause of amblyopia and pre-treatment contrast sensitivity, stereoacuity, fixation and nystagmus. Design: Retrospective cohort study. Methods: A retrospective review was conducted for 3408 patients with amblyopia who had not yet started amblyopia treatment utilizing a large amblyopia patient database maintained at Zhongshan Ophthalmic Centre. Six amblyogenic factor subtypes were identified: anisometropia, isoametropia, strabismus, anisometropia and strabismus, monocular visual deprivation, and binocular visual deprivation amblyopia. Monocular best corrected visual acuity (BCVA), the contrast sensitivity function (CSF), fixation, and stereopsis were compared between the subtypes before and after propensity score matching (PSM) for age and sex. Results: The two deprivation groups had poorer BCVA and CSF than the other groups. There were no systematic differences in CSF between the non-deprivation groups. Nystagmus was more common in the bilateral amblyopia groups compared to the monocular amblyopia groups. Eccentric fixation was uncommon with the exception of the anisometropia and strabismus group which had an eccentric fixation rate of 20%. Distance stereoacuity measured without monocular cues was absent for almost all patients. The results were consistent when analyzed using PSM. Conclusion: Visual deprivation causes more severe amblyopia than other amblyogenic factors. For non-deprivation amblyopia subtypes, individual differences such as variation in the severity of the amblyogenic factor might be more important in determining pre-treatment vision than whether amblyopia was caused by refractive error, strabismus or both.

Production of anhydrous hydrogen fluoride from fluorosilicic acid: a review.

Anhydrous hydrogen fluoride (AHF), a critical raw material for industries such as aluminum, pharmaceuticals, and petroleum, has traditionally been sourced from fluorite-a non-renewable mineral. The unsustainable reliance on fluorite has catalyzed the search for alternative AHF production methods. A promising substitute is fluorosilicic acid (FSA), a byproduct of the phosphate fertilizer industry previously deemed waste. Transforming fluorosilicic acid into AHF not only yields a valuable resource but also addresses the environmental and economic challenges associated with waste management. The innovative practice of producing AHF from fluorosilicic acid signals a shift towards sustainable chemical production by capitalizing on waste, potentially diminishing reliance on fluorite and reducing the industry's environmental impact. This review thoroughly dissects the AHF synthesis process from fluorosilicic acid. Despite the acknowledged importance of fluorinated compounds in numerous industrial applications, research on their synthesis from fluorosilicic acid is limited and fragmented. This review seeks to amalgamate this scattered information by closely scrutinizing diverse industrial processing methods. Additionally, it explores the current and future landscape, economic feasibility, and strategies to navigate the obstacles inherent in synthesizing AHF from fluorosilicic acid. It also assesses the environmental impact of these methods, thereby contributing to the dialogue in this emerging field. The primary aim of this manuscript is to foster further research and promote the industrial uptake of this sustainable process. Highlighting the challenges and proposing potential improvements, the review supports the responsible reuse of waste and advocates for advancements in industrial practices.

Efficacy, tolerability, and safety of the oral phosphate binder VS-505 (AP301).

BACKGROUND: VS-505 (AP301), an acacia and ferric oxyhydroxide polymer, is a novel fiber-iron-based phosphate binder. This two-part phase 2 study evaluated the tolerability, safety, and efficacy of oral VS-505 administered three times daily with meals in treating hyperphosphatemia in chronic kidney disease (CKD) patients receiving maintenance hemodialysis (MHD). METHODS: In Part 1, patients received dose-escalated treatment with VS-505 2.25, 4.50, and 9.00 g/day for 2 weeks each, guided by serum phosphorus levels. In Part 2, patients received randomized, open-label, fixed-dosage treatment with VS-505 (1.50, 2.25, 4.50, or 6.75 g/day) or sevelamer carbonate 4.80 g/day for 6 weeks. The primary efficacy endpoint was the change in serum phosphorus. RESULTS: The study enrolled 158 patients (Part 1: 25; Part 2: 133), with 130 exposed to VS-505 in total. VS-505 was well tolerated. The most common adverse events were gastrointestinal disorders, mainly feces discolored (56%) and diarrhea (15%; generally during weeks 1‒2 of treatment). Most gastrointestinal disorders resolved without intervention, and none were serious. In Part 1, serum phosphorus significantly improved (mean change -2.0 mg/dL; 95% confidence interval -2.7, -1.4) after VS-505 dose escalation. In Part 2, serum phosphorus significantly and dose-dependently improved in all VS-505 arms, with clinically meaningful reductions with VS-505 4.50 and 6.75 g/day, and sevelamer carbonate 4.80 g/day (mean change -1.6 (-2.2, -1.0), -1.8 (-2.4, -1.2), and -1.4 (-2.2, -0.5) mg/dL, respectively). In both Parts, serum phosphorus reductions occurred within 1 week of VS-505 initiation, returning to baseline within 2 weeks of VS-505 discontinuation. CONCLUSION: VS-505, a novel phosphate binder, was well tolerated with a manageable safety profile, and effectively and dose-dependently reduced serum phosphorus in CKD patients with hyperphosphatemia receiving MHD. Clinical Trial registration number: NCT04551300.

Renewable lignin-derived heteroatom-doped porous carbon nanosheets as an efficient oxygen reduction catalyst for rechargeable zinc-air batteries.

Lignin upgrading to various functional products is promising to realize high-value utilization of low-cost and renewable biomass waste, but is still in its infancy. Herein, using industry waste lignosulfonate as the biomass-based carbon source and urea as the dopant, we constructed a heteroatom-doped porous carbon nanosheet structure by a simple NaCl template-assisted pyrolytic strategy. Through the synergistic effect of the NaCl template and urea, the optimized lignin-derived porous carbon catalyst with high content of active nitrogen species and large specific surface area can be obtained. As a result, the fabricated catalysts exhibited excellent electrocatalytic oxygen reduction activity, as well as good methanol tolerance and stability, comparable to that of commercial Pt/C. Moreover, rechargeable Zn-air batteries assembled with this electrocatalyst have a peak power density of up to 150 mW cm-2 and prominent long-term cycling stability. This study offers an inexpensive and efficient way for the massive production of highly active metal-free catalysts from the plentiful, inexpensive and environmentally friendly lignin, offering a good direction for biomass waste recycling and utilization.

Recent Advances in the Nutritional Value, Chemical Compositions, Pharmacological Activity, and Application Value of Orychophragmus violaceus: A Comprehensive Review.

Orychophragmus violaceus (L.) O. E. Schulz (Brassicaceae) is widely distributed and plentiful in China and has been widely used for its application in ornamental, oil, ecology, foraging, and food. Recent studies have revealed that the main components of Orychophragmus violaceus include flavonoids, alkaloids, phenylpropanoids, phenolic acids, terpenoids, etc., which have pharmacological activities such as antioxidation, antiradiation, antitumor, hepatic protection, antiferroptosis, anti-inflammatory, and antibacterial. In this paper, the nutritional value, chemical compositions, pharmacological activity, and application value of Orychophragmus violaceus are summarized by referring to the relevant domestic and international literature to provide a reference for further research, development, and utilization of Orychophragmus violaceus in the future.

ICGA-GPT: report generation and question answering for indocyanine green angiography images.

BACKGROUND: Indocyanine green angiography (ICGA) is vital for diagnosing chorioretinal diseases, but its interpretation and patient communication require extensive expertise and time-consuming efforts. We aim to develop a bilingual ICGA report generation and question-answering (QA) system. METHODS: Our dataset comprised 213 129 ICGA images from 2919 participants. The system comprised two stages: image-text alignment for report generation by a multimodal transformer architecture, and large language model (LLM)-based QA with ICGA text reports and human-input questions. Performance was assessed using both qualitative metrics (including Bilingual Evaluation Understudy (BLEU), Consensus-based Image Description Evaluation (CIDEr), Recall-Oriented Understudy for Gisting Evaluation-Longest Common Subsequence (ROUGE-L), Semantic Propositional Image Caption Evaluation (SPICE), accuracy, sensitivity, specificity, precision and F1 score) and subjective evaluation by three experienced ophthalmologists using 5-point scales (5 refers to high quality). RESULTS: We produced 8757 ICGA reports covering 39 disease-related conditions after bilingual translation (66.7% English, 33.3% Chinese). The ICGA-GPT model's report generation performance was evaluated with BLEU scores (1-4) of 0.48, 0.44, 0.40 and 0.37; CIDEr of 0.82; ROUGE of 0.41 and SPICE of 0.18. For disease-based metrics, the average specificity, accuracy, precision, sensitivity and F1 score were 0.98, 0.94, 0.70, 0.68 and 0.64, respectively. Assessing the quality of 50 images (100 reports), three ophthalmologists achieved substantial agreement (kappa=0.723 for completeness, kappa=0.738 for accuracy), yielding scores from 3.20 to 3.55. In an interactive QA scenario involving 100 generated answers, the ophthalmologists provided scores of 4.24, 4.22 and 4.10, displaying good consistency (kappa=0.779). CONCLUSION: This pioneering study introduces the ICGA-GPT model for report generation and interactive QA for the first time, underscoring the potential of LLMs in assisting with automated ICGA image interpretation.

Tuning d-orbitals to control spin-orbit coupling in terminated MXenes.

Theory and experiment have revealed that spin-orbit coupling (SOC) strongly depends on the relativistic effect in topological insulators (TIs), while the influence of orbitals is always ignored. Herein, we provide a direct way of controlling effective SOC with the help of orbital effects, reducing the dependence on elements. Taking 5d W2CO2 and 4d Mo2CO2 MXenes as a specific example, we predict that by decreasing the hybridization strength of W atoms with C or O atoms in 2D W2CO2, the nontrivial bandgaps at the Γ-point are directly enhanced. The weak hybridization of W atoms with ligand elements enhances the electron localization of degenerate d-orbitals of three groups under the triangular prism crystal field, inducing stronger on-site Coulomb repulsion that enhances orbital polarization as well as boosts the SOC effect. Meanwhile, similar results have also been observed in 4d Mo2CO2. This implies that the orbital effects are an efficient and straightforward way to control the nontrivial bandgap in 2D MXene TIs. Our work not only provides an alternative perspective on designing large nontrivial bandgaps but also brings a possibility to control the SOC effect for TI devices.

Perceptual Learning Based on the Lateral Masking Paradigm in Anisometropic Amblyopia With or Without a Patching History.

Purpose: Perceptual learning (PL) has shown promising performance in restoring visual function in adolescent amblyopes. We retrospectively compared the effect of a well-accepted PL paradigm on patients with anisometropic amblyopia with or without a patching therapy history (patching therapy [PT] group versus no patching therapy [NPT] group). Methods: Eighteen PT and 13 NPT patients with anisometropic amblyopia underwent monocular PL for 3 months. During training, patients practiced a Gabor detection task following the lateral masking paradigm by applying a temporal two-alternative forced choice procedure with the amblyopic eye. Monocular contrast sensitivity functions (CSF), visual acuity, interocular differences in visual function metrics, and stereoacuity were compared before and after training. Results: PL improved the visual acuity of the amblyopia eyes by 0.5 lines on average in the PT group and 1.5 lines in the NPT group. A significant reduction in the interocular difference in visual acuity was observed in the NPT group (P < 0.01) but not in the PT group (P = 0.05). Regarding CSF metrics, the area under the log CSF and cutoff in the amblyopic eyes of the NPT groups increased after training (P < 0.05). In addition, the interocular differences of the CSF metrics (P < 0.05) in the NPT group were significantly reduced. However, in the PT group, all the CSF metrics were unchanged after training. A total of 27 of 31 patients in both groups had no measurable stereopsis pretraining, and recovery after training was not significant. Conclusions: PL based on a lateral masking training paradigm improved visual function in anisometropic amblyopia. Patients without a patching history achieved greater benefits. Translational Relevance: PL based on a lateral masking training paradigm could be a new treatment for amblyopia.

Binocular Summation With Quantitative Contrast Sensitivity Function: A Novel Parameter to Evaluate Binocular Function in Intermittent Exotropia.

Purpose: Intermittent exotropia (IXT) is the most common form of strabismus. Surgery can potentially improve binocular function in patients with IXT. We aimed to evaluate binocular function using a novel parameter-binocular summation ratio (BSR), measured using quantitative contrast sensitivity function (CSF) in patients with IXT before and after surgery. Methods: Prospective study of 63 patients with IXT and 41 healthy controls were consecutively enrolled and underwent quantitative CSF testing binocularly and monocularly. BSR was calculated by dividing the CSF of the binocular value by the better monocular value. Forty-eight patients with IXT underwent strabismus surgery. BSR, stereoacuity, fusion ability, and strabismus questionnaires were assessed pre-operatively and 2 months postoperatively. Results: Sixty-three patients with IXT (median age = 9 years) compared with 41 healthy controls showed a worse mean BSR based on all CSF metrics at baseline (the area under the log CSF [AULCSF], spatial frequency [SF] cutoff, and contrast sensitivity at 1.0-18.0 cpd SF). All 48 patients with IXT showed successful alignment after surgery, and there were significant improvements in BSR based on the AULCSF, SF cutoff, and contrast sensitivity at 6.0, 12.0, and 18.0 cpd SF, respectively. The distance stereoacuity and fusion ability also improved after surgery, and a better BSR was associated with better stereoacuity and fusion. For strabismus questionnaires, the psychosocial subscale scores improved postoperatively, whereas the functional subscale scores did not change. Conclusions: BSR based on quantitative CSF can characterize binocular function across a range of spatial frequencies and can be used as a supplemental measurement for monitoring binocularity in patients with IXT in clinical settings.

A continuously efficient O2-supplying strategy for long-term modulation of hypoxic tumor microenvironment to enhance long-acting radionuclides internal therapy.

Radionuclides internal radiotherapy (RIT) is a clinically powerful method for cancer treatment, but still poses unsatisfactory therapeutic outcomes due to the hypoxic characteristic of tumor microenvironment (TME). Catalase (CAT) or CAT-like nanomaterials can be used to enzymatically decompose TME endogenous H2O2 to boost TME oxygenation and thus alleviate the hypoxic level within tumors, but their effectiveness is still hindered by the short-lasting of hypoxia relief owing to their poor stability or degradability, thereby failing to match the long therapeutic duration of RIT. Herein, we proposed an innovative strategy of using facet-dependent CAT-like Pd-based two-dimensional (2D) nanoplatforms to continuously enhance RIT. Specifically, rationally designed 2D Pd@Au nanosheets (NSs) enable consistent enzymatic conversion of endogenous H2O2 into O2 to overcome hypoxia-induced RIT resistance. Furthermore, partially coated Au layer afford NIR-II responsiveness and moderate photothermal treatment that augmenting their enzymatic functionality. This approach with dual-effect paves the way for reshaping TME and consequently facilitating the brachytherapy ablation of cancer. Our work offers a significant advancement in the integration of catalytic nanomedicine and nuclear medicine, with the overarching goal of amplifying the clinical benefits of RIT-treated patients.

Double Carbon Networks Reinforce the Thermal Storage and Thermal Transfer Properties of 1-Octadecanol Phase Change Materials.

Using thermal storage materials with excellent thermal properties in the energy utilization system enables efficient use of renewable energy sources. Organic phase change materials (PCMs) have the advantages of high heat storage density, no corrosion, and low cost, but low thermal conductivity and insufficient heat transfer capacity have always been the bottlenecks in their application. In this paper, melamine foam@ reduction graphene oxide (MF@rGO) and carbon foam@ reduction graphene oxide (CF@rGO) composite foams with double carbon networks were prepared by self-assembly method and further employed in 1-octadecinal (OD) PCMs. The microstructure, chemical composition, phase change behavior, thermal conductivity, and photothermal conversion performance of MF@rGO/OD and CF@rGO/OD were studied in detail using SEM, FTIR, Raman DSC, and LFA. The melting and solidification enthalpies of CF@rGO/OD composite PCMs were 208.3 J/g and 191.4 J/g, respectively, its thermal conductivity increased to 1.54 W/m·K, which is 6.42 times that of pure OD. The porous structure and high thermal conductivity of the double carbon network substantially enhance the efficiency of energy storage and release in composite PCMs. CF@rGO/OD composite PCMs have excellent heat storage performance and heat transfer capacity, and a wide range of application prospects in the fields of low-temperature solar heat storage, precision instrument temperature control, and intelligent buildings.

Visible-Light-Promoted Intermolecular ß-Acyl Difunctionalization of Alkenes via Oxidative Radical-Polar Crossover.

A visible-light-induced ß-acyl difunctionalization of alkenes with acyl oxime esters and various nucleophiles was developed to achieve molecular complexity from readily available raw materials via oxidative radical-polar crossover. A variety of nucleophiles, including NH-sulfoximines, indoles, indazole, and trimethoxybenzene, were all effectively applicable to the sustainable reaction system. The novel synthetic strategy features mild reaction conditions, a broad substrate scope (39 examples), easy scale-up, and excellent regioselectivity.

Highly sensitive and selective detection of triphosgene with a 2-(2'-hydroxyphenyl)benzimidazole derived fluorescent probe.

In this work, a 2-(2'-hydroxyphenyl)benzimidazole derived fluorescent probe, 2-(2'-hydroxy-4'-aminophenyl)benzimidazole (4-AHBI), was synthesized and its fluorescent behavior toward triphosgene were evaluated. The results showed that 4-AHBI exhibited high sensitivity (limit of detection, 0.08 nM) and excellent selectivity for triphosgene over other acyl chlorides including phosgene in CH2Cl2 solution. Moreover, 4-AHBI loaded test strips were prepared for the practical sensing of triphosgene.

Structural characterization and adjuvant action of Paulownia tomentosa flower polysaccharide on the immune responses to classical swine fever vaccine in mice.

Paulownia tomentosa flower polysaccharide (PTFP) from dried cultured P. tomentosa flowers, is widely known for its immunomodulatory activities. Here, PTFP was extracted from Paulownia tomentosa flower using hot water extraction, followed by ethanol precipitation methods. Structural characterization of PTFP was revealed by scanning electron microscope, high-performance anion-exchange chromatography, gel chromatography, ultraviolet and infrared spectral. Meanwhile, adjuvant action of PTFT on the immune responses to classical swine fever vaccine in mice was evaluated to further proclaim the immune regulatory effect of PTFP. The results showed that PTFP was a type of heteropolysaccharide with a dense, rough surface and high molecular weight (667.02 kDa), mainly composed of glucose (30.93%), rhamnose (29.99%), galactose (15.66%), arabinose (6.95%), mannose (5.52%), and xylose (4.80%). The results of gel chromatography suggested that the molecular configuration of PTFP may be a spherical structure. The infrared spectrum results confirmed that the functional groups and chemical bond of PTFP contained -OH, O-H, C-H, C=O, C-O, etc. Moreover, PTFP exhibited obvious immune enhancement effect by improving concanavalin A (ConA), lipopolysaccharide (LPS), and CSFV E2-stimulated splenocyte growth and natural killer cell activity in CSFV-immunized mice. Similarly, the titers of CSFV E2-specific IgG, IgG1, IgG2a, and IgG2b antibodies and IFN-γ and IL-10 levels in CSFV-immunized mice were distinctly increased by PTFP treatment. Overall, PTFP was a macromolecular heteropolysaccharide primarily containing glucose and rhamnose, and possessed the auxiliary effect of immune enhancement on the immune responses to classical swine fever vaccine.

Nitric oxide is involved in the regulation of guard mother cell division by inhibiting the synthesis of ACC.

A stoma forms by a series of asymmetric divisions of stomatal lineage precursor cell and the terminal division of a guard mother cell (GMC). GMC division is restricted to once through genetic regulation mechanisms. Here, we show that nitric oxide (NO) is involved in the regulation of the GMC division. NO donor treatment results in the formation of single guard cells (SGCs). SGCs are also produced in plants that accumulate high NO, whereas clustered guard cells (GCs) appear in plants with low NO accumulation. NO treatment promotes the formation of SGCs in the stomatal signalling mutants sdd1, epf1 epf2, tmm1, erl1 erl2 and er erl1 erl2, reduces the cell number per stomatal cluster in the fama-1 and flp1 myb88, but has no effect on stomatal of cdkb1;1 cyca2;234. Aminocyclopropane-1-carboxylic acid (ACC), a positive regulator of GMC division, reduces the NO-induced SGC formation. Further investigation found NO inhibits ACC synthesis by repressing the expression of several ACC SYNTHASE (ACS) genes, and in turn ACC represses NO accumulation by promoting the expression of HEMOGLOBIN 1 (HB1) encoding a NO scavenger. This work shows NO plays a role in the regulation of GMC division by modulating ACC accumulation in the Arabidopsis cotyledon.