Dynamic Arabidopsis P5CS filament facilitates substrate channelling.

In plants, the rapid accumulation of proline is a common response to combat abiotic stress1-7. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalysing the initial two-step conversion from glutamate to proline8. Here we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS1 (AtP5CS1) and P5CS2 (AtP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of AtP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveal that filament-based substrate channelling is essential for maintaining the high catalytic efficiency of AtP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of AtP5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response.

Influencing factors comparing different vault groups after phakic implantable collamer lens implantation: review and meta-analysis.

BACKGROUND: Studies on the factors affecting vault after posterior chamber phakic Implantable Collamer Lens (ICL) have been carried out, but most of them are single-centered and subjective selections of parameters. The present study aimed to systematically analyze the factors for vault. METHODS: A systematic review of case series, case-control, and cohort studies derived from the articles published in PubMed, the Cochrane Library, Embase, Web of Science, CNKI, CBM, Wanfang and VIP, as well as ClinicalTrials, which were conducted to search for studies on factors of vault using four core terms: phakic intraocular lenses, vault, risk factor and observational study, from January 01, 1997, to February 20, 2023. The included studies were meta-analyzed quantitatively and described qualitatively. Subsequently, meta-regression and subgroup analysis were used. RESULTS: We identified 13 studies (1,607 subjects), and 14 factors were considered. Meta-analysis showed that anterior chamber depth (ACD), horizontal corneal white-to-white (hWTW), ICL-size, and age are dual effects of the abnormal vaults; anterior chamber volume (ACV) and lens thickness (LT) are a one-way effect; while axial length (AL), ICL- spherical equivalent (ICL-SE) and Km are insignificant. In addition, descriptive analysis of anterior chamber angle (ACA), horizontal sulcus to sulcus (hSTS), ciliary processes height (T value), crystalline lens rise (CLR), and gender showed that all factors except gender tend to have significant effects on vault. Sensitivity analysis showed stable combined results. Country and design respectively affect the heterogeneity in ACD and ICL-size at low vault, while design affects the heterogeneity in ACD at high vault. No publication bias exists. CONCLUSIONS: Vault after ICL is related to multiple factors, especially anterior segmental biologic parameters, and they are weighted differently. We hope to provide a reference for the selection and adjustment of ICL.

Comparative analysis of two retinal fractures with ultrabroad­angle fundus photography systems.

The aim of the present study was to compare the performance of the Opel Panorama 200 and Zeiss Clarus 500 (Carl Zeiss AG) systems in diagnosing retinal fractures. Human subjects were selected from 298 fundus examinations (531 eyes) in ophthalmology from February 2021 to June 2021, including 68 patients with retinal fissures (95 eyes). All fundus tests were performed with Opel Panoramic 200. Zeiss Clarus 500 (Carl Zeiss AG) fundus photography, slit-lamp full retinal lens (Ocular Mainster Wide Field; Ocular Instruments), and retinal laser photocoagulation was performed for all affected eyes. The diagnostic sensitivity of the two examination methods was compared, and their sensitivities for posterior retina, peripheral nose, crystal eye, cataract, positive experiment, and myopia testing were compared. In all, 68 patients (95 eyes) were clinically examined and treated 112 laser times. For retinal fractures, the Opel Panorama 200 used a check sensitivity of 89.5%, and the Clarus 500 check had a sensitivity of 94.7%, with the difference being non-significant (P=0.358). Moreover, Clarus 500 diagnosed the sensitivity of the temporal periphery significantly higher than that of Opel Panorama 200 (P=0.048). Opel Panorama 200 displayed statistically significant sensitivity compared with Clarus 500 diagnosis with crystalline and crystal fewer eyes (P>0.05); Clarus 500 sensitivity for cataract diagnosis (crystal turbidity level 3 and above) was significantly higher than that of Opel Panorama 200 (P=0.033). Opel Panoramic 200 displayed significant sensitivity to ocular myopia and medium to moderate myopia (P>0.05). Clarus 500 diagnosed high myopia with a significantly higher sensitivity than Opel Panorama 200 (P=0.045). Opel Panorama 200 and Zeiss Clarus 500 displayed the same level of sensitivity to retinal fissures, with improved sensitivity in refractive turbidity and for retinal fissures located in the far periphery of the temporal side.

Analysis of the improvement in monocular amblyopia visual acuity caused by the changes in non-amblyopia visual acuity in 74 adults.

To observe the clinical phenomenon of amblyopia vision improvement in patients with monocular amblyopia over 18 years old after non-amblyopia diseases, analyze the conditions and causes of vision improvement, explore the plasticity of the adult optic nerve, and provide a clinical basis for the treatment of adult amblyopia. A total of 74 patients with monocular amblyopia combined with non-amblyopia visual acuity decline from 2018 to 2021 were collected. The patient's age, initial best-corrected visual acuity (BCVA), pattern visual evoked potential examination results, and visual acuity regression were recorded. The BCVA of amblyopia was recorded every 3 months using an early treatment of diabetic retinopathy study visual acuity chart. In the 3rd month, BCVA increased by 16.2%, reaching 98% in the 9th month and 100% in the 12th months. According to the age of patients, the group aged 18 to 35 years was better than the group aged 35 to 60 years, whereas the group aged 35 to 60 years was better than the group aged over 60 years (P < .05). According to the comparison of initial visual acuity, the BCVA of the < 5 letter group was lower than that of the other 2 groups (P < .05). According to the pattern visual evoked potential results, the peak time of the < 10 ms group was better than that of the 10 to 20 ms group; the 10 to 20 ms group was better than that of the > 20 ms group; the peak decrease of the < 30% group was better than that of the 30% to 50% group; and the 30% to 50% group was better than that of the > 50% group (P < .05). The visual acuity regression of amblyopia in the 0.5 to 1-year group was higher than that in the other 2 groups (P < .05). This study confirms that adult amblyopia can still be cured under certain conditions. This visual plasticity is related to age, initial visual acuity, and excitability of the visual center. This study provides new clinical evidence and diagnostic ideas for the study of the pathogenesis of adult amblyopia.

Glutaredoxin 2 protects lens epithelial cells from epithelial-mesenchymal transition by suppressing mitochondrial oxidative stress-related upregulation of integrin-linked kinase.

Glutaredoxin 2 (Grx2), a mitochondrial glutathione-dependent oxidoreductase, is crucial for maintaining redox homeostasis and cellular functions in the lens. The oxidative stress-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is related to posterior capsule opacification. In this study, we investigated the effects of Grx2 on oxidative stress-induced EMT in LECs during posterior capsule opacification. We found that Grx2 expression was substantially decreased during the EMT of LECs and in a mouse model of cataract surgery. Deletion of Grx2 aggravated the generation of reactive oxygen species, including those that are mitochondria-derived, and promoted the proliferation and EMT of the LECs. This was reversed by Grx2 overexpression. In vivo, proteomic liquid chromatography-mass spectrometry analysis showed that integrin-linked kinase (ILK) was significantly upregulated in the lens posterior capsule of a Grx2 knockout (KO) mouse model. Compared with that of the wild-type group, the expression of ILK and EMT markers was increased in the Grx2 KO group which was reversed in the Grx2 knock-in group. Inhibition of ILK partially blocked Grx2 knockdown-induced EMT and prevented the increased phosphorylation of Akt and GSK-3ß and the nuclear translocation of ß-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/ß-catenin pathway partially blocked the Grx2 knockdown-induced EMT. In conclusion, we demonstrated that Grx2 protects LECs from oxidative stress-related EMT by regulating the ILK/Akt/GSK-3ß axis.

Structural basis of human PRPS2 filaments.

BACKGROUND: PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1. RESULTS: Order to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity. CONCLUSION: Findings suggest that the regulation of hPRPS2 polymer is distinct from ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.

Cytoophidia and filaments: you must unlearn what you have learned.

The nucleotide CTP can be synthesized de novo from UTP via the metabolic enzyme CTP synthase (CTPS). As a textbook enzyme, CTPS has been extensively studied for seven decades. However, it came as a surprise when CTPS was found to form snake-shaped mesoscale cytoophidia in fruit fly cells. Since 2010, more and more studies have demonstrated that CTPS can form cytoophidia within the cells across all three domains of life. Oligomers of CTPS form filaments that are undetectable under light microscopy. This review summarizes our current understanding of cytoophidia and filaments, highlighting some basic features such as conservation, morphology and functions of the two levels of CTPS structures.

Ultraviolet B radiation induces oxidative stress and apoptosis in human lens epithelium cells by activating NF-κB signaling to down-regulate sodium vitamin C transporter 2 (SVCT2) expression.

Ultraviolet B (UVB) exposure is reported to cause cataract formation by inducing excessive reactive oxygen species (ROS) and apoptosis in human lens epithelial cells (HLECs). Sodium-dependent Vitamin C transports-2 (SVCT2) is a ascorbic acid (AsA) transporter for that can protect cells and tissues from oxidative stress. Here, we focus on the functional characterization and mechanism analysis of SVCT2 in UVB-treated HLECs. The results showed a significant reduction of SVCT2 expression in HLECs treated with UVB. SVCT2 abated apoptosis and Bax expression and increased Bcl-2 expression. Moreover, SVCT2 decreased ROS accumulation and MDA level, but increased the activities of antioxidant enzymes (SOD and GSH-PX). NF-κB inhibitor (PDTC) alleviated ROS production and apoptosis, and promoted SVCT2 expression in UVB-treated HLECs. Additionally, ROS inhibitor (NAC) suppressed oxidative stress, apoptosis, and induced SVCT2 expression in UVB-treated HLECs, while these effects were significantly abated due to the activation of NF-κB signaling. Furthermore, SVCT2 facilitated 14C-AsA absorption in UVB-treated HLECs. Together, our findings demonstrated that UVB exposure-induced ROS generation, which further activated NF-κB signaling to down-regulate SVCT2 expression in HLECs. Then, downregulated SVCT2 promoted ROS accumulation and induced apoptosis by decreasing AsA uptake. Our data reveal a novel NF-κB/SVCT2/AsA regulatory pathway and suggest the therapeutic potential of SVCT2 in UVB-induced cataract.

Vascular endothelial cell-derived exosomal miR-1246 facilitates posterior capsule opacification development by targeting GSK-3ß in diabetes mellitus.

Posterior capsule opacification (PCO) is a serious complication after cataract surgery. Diabetes could increase the occurrence of PCO, but the mechanism is still unclear. The purpose of this study is to investigate the role of small extracellular vesicles (sEVs) derived from diabetic aqueous humor in PCO process. Intraoperatively-derived aqueous humor sEVs from patients with diabetic related cataract (DRC) promoted the epithelial-mesenchymal transition (EMT) and metastasis of human lens epithelial cells (LECs). Via mouse PCO surgical model and DiI labeled fluorescence detection of sEVs, the sEVs derived from vascular endothelium were discovered directly contacting with LECs. Furthermore, we demonstrated that high-glucose-cultured human umbilical vein endothelial cells (HUVEC) -derived sEVs facilitated EMT process of HLE-B3 using co-culture model in vitro. miRNA-seq data and GEO datasets analysis revealed that miR-1246 was essential in EMT process with diabetes. The miR-1246 was highly expressed in diabetic aqueous humor sEVs and high-glucose-treated vascular-endothelial-cell-derived sEVs. Moreover, miR-1246 promoted the metastasis and EMT process of HLE-B3 cells by directly targeting GSK-3ß. Inhibiting miR-1246 could negatively regulated EMT. This finding might serve as a potential therapy for diabetic PCO.

Surgical resection and orbital iodine-125 brachytherapy for orbital malignancy: a novel treatment for orbital lymphoma.

OBJECTIVES: Orbital lymphoma is one of the most common adult orbital malignancies, accounting for approximately 10% of all orbital tumors. This study aimed to analyze the effects of surgical resection and orbital iodine-125 brachytherapy implantation for orbital lymphoma. PATIENTS AND METHODS: This was a retrospective study. Clinical data of 10 patients were collected from October 2016 to November 2018 and followed up to March 2022. Patients underwent the primary surgery for maximal safe removal of the tumor. After a pathologic diagnosis of a primary orbital lymphoma was established, iodine-125 seed tubes were designed based on the tumor size and invasion range, and direct vision was placed into the nasolacrimal canal or/and under the orbital periosteum around the resection cavity during the secondary surgery. Then, follow-up data, including the general situation, ocular condition, and tumor recurrence, were recorded. RESULTS: Of the 10 patients, the pathologic diagnoses included extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (6 cases), small lymphocytic lymphoma (1 case), mantle cell lymphoma (2 cases), and diffuse large B-cell lymphoma (1 case). The number of seeds implanted ranged from 16 to 40. The follow-up period ranged between 40 and 65 months. All patients in this study were alive and well had tumors that were completely controlled. No tumor recurrences or metastases occurred. Three patients had dry eye syndrome and two patients had abnormal facial sensation. No patient had radiodermatitis involving the skin around the eye, and no patient had radiation-related ophthalmopathy. CONCLUSIONS: Based on preliminary observations, iodine-125 brachytherapy implantation appeared to be a reasonable alternative to external irradiation for orbital lymphoma.

Nondestructive Evaluation of Composite Bonding Structure Used in Electrical Insulation Based on Active Infrared Thermography.

Nondestructive testing and evaluation of composite insulating components of electrical equipment is extremely necessary for assuring the safety of a power system. However, most existing nondestructive testing methods are not applicable for fast and effective live detection due to their time-consuming operation, high cost, and contact or near-field measurement. In this work, the effectiveness of active infrared thermography was investigated for detecting defects in silicone rubber (SIR)-fiber-reinforced plastic (FRP) bonding structures, which have been commonly used in insulating components of power equipment. The effectiveness of differential thermal image for enhancing the contrast of defective and sound areas and eliminating additive noise was demonstrated. Particularly, frame difference thermal image obtained by subtracting two differential thermal images extracting from respectively before and after the contrast inversion was proposed to enhance defect identification. The results revealed that defects of various sizes and depths such as voids, cracks, and interface disbonding of the SIR-FRP bonding structure were accurately detected by thermographic data. With the advantages of a quick and simple process, safety, universal applicability, visual results, far-field measurement, and quantitative defect estimation capabilities, active infrared thermography would be quite promising for live detection of electrical equipment.

Filamentation modulates allosteric regulation of PRPS.

Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.

The protective mechanism of Grx2 in ultraviolet-B (UVB)-induced cataract formation.

OBJECTIVE: We established a mouse cataract model by irradiating Grx2 knockout (KO) and knock-in (KI) genetically modified mice with UVB to explore the protective mechanism of Grx2 against UVB lens damage. METHODS: After irradiating Grx2 KO and Grx2 KI mice with UVB lamps, we observed and recorded the general physiological conditions and lens opacity of the mice. The crystalline grading system of the University of Oxford was used to classify the opacity of the lens. Lens reactive oxygen species (ROS) contents were detected using a microplate reader, western blot, and enzyme-linked immunosorbent assay (ELISA) to detect antioxidant and antioxidant enzyme contents. Statistical analysis of the recorded data was performed by using SPSS 19.0 software. RESULTS: After UVB irradiation, the weight of Grx2 KO mice was slightly lower than that of wild-type (WT) mice of the same age. Compared to WT mice, the lens opacity of Grx2 KO mice appeared earlier, the nucleus density of the lens increased, and the opacity increased in the first week after UVB irradiation. Meanwhile, the lenses of Grx2 KI mice remained transparent. The experiment showed that the content of ROS increased, the level of glutathione (GSH) decreased, the content of 8-OHdG increased, and the expression of BCL2 decreased after UVB irradiation. Compared to WT mice, these changes were more significant in Grx2 KO mice. CONCLUSION: This experiment found that knocking out the Grx2 gene accelerated the occurrence and development of UVB-induced cataracts in mice and that Grx2 plays an important role in the oxidative damage caused by UVB radiation by repairing the antioxidant enzymes of the lens. This study provides a new animal model and research ideas for the study of cataract pathogenesis.

GTP-Dependent Regulation of CTP Synthase: Evolving Insights into Allosteric Activation and NH3 Translocation.

Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.

Structural basis of dynamic P5CS filaments.

The bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is vital to the synthesis of proline and ornithine, playing an essential role in human health and agriculture. Pathogenic mutations in the P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants. We have recently found that P5CS forms cytoophidia in vivo and filaments in vitro. However, it is difficult to appreciate the function of P5CS filamentation without precise structures. Using cryo-electron microscopy, here we solve the structures of Drosophila full-length P5CS in three states at resolution from 3.1 to 4.3 Å. We observe distinct ligand-binding states and conformational changes for the GK and GPR domains, respectively. Divergent helical filaments are assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations disturbing those interfaces prevent P5CS filamentation and greatly reduce the enzymatic activity. Our findings reveal that filamentation is crucial for the coordination between the GK and GPR domains, providing a structural basis for the catalytic function of P5CS filaments.

MiR-34a-5p Negatively Regulates Oxidative Stress on Lens Epithelial Cells by Silencing GPX3 - A Novel Target.

PURPOSE: MiR-34a-5p is reported to be related with age-related nuclear cataract. This study investigated the mechanism of miR-34a-5p in the regulation of oxidative stress on lens epithelial cells. METHODS: The three candidate miRNAs were screened by CCK-8 assays after transfection of mimics or inhibitor in H2O2-treated HLE-B3 cells. The apoptosis, ROS level and GPX activity of HLE-B3 cells transfected with miR-34a-5p mimics or inhibitor were analysed by flow cytometry, cellular ROS and GPX activity test. The target genes of miR-34a-5p were predicted by proteomic and bioinformatic analysis. The relationship between miR-34a-5p and GPX3 were internally validated by qRT-PCR and Western blot and externally verified by dual-luciferase reporter assay. The effect of miR-34a-5p-GPX3 axis on regulation of oxidative stress in HLE-B3 cells were conducted by overexpression of GPX3 and tested by flow cytometry analysis, cellular ROS and GPX detection. RESULTS: The viability of H2O2-treated HLE-B3 cells were weakened by up-regulated miR-34a-5p. Cell apoptosis and oxidative damage were also induced by overexpression of miR-34a-5p. GPX3 and SRC were identified as target genes of miR-34a-5p by combined analysis of proteomic and bioinformatics, while GPX3 was selected for further research for its connection with anti-oxidation. Western blot and qRT-PCR tests proved that GPX3 is negatively regulated by miR-34a-5p. Dual-luciferase reporter assay verified that GPX3 is the direct target of miR-34a-5p. The increased oxidative stress induced by transfection of miR-34a-5p mimics in H2O2-treated HLE-B3 cells was attenuated by overexpression of GPX3. CONCLUSIONS: MiR-34a-5p is a negative regulator of oxidative stress on lens epithelial cells and the mechanism is by silencing the expression of GPX3. These data suggest that miR-34a-5p may be a potential novel therapeutic target for the prevention and treatment of age-related cataract.

Reduction of Amides to Amines with Pinacolborane Catalyzed by Heterogeneous Lanthanum Catalyst La(CH2C6H4NMe2-o)3@SBA-15.

Hydroboration of amides is a useful synthetic strategy to access the corresponding amines. In this contribution, it was found that the supported lanthanum benzyl material La(CH2C6H4NMe2-o)3@SBA-15 was highly active for the hydroboration of primary, secondary, and tertiary amides to amines with pinacolborane. These reactions selectively produced target amines and showed good tolerance for functional groups such as -NO2, -halogen, and -CN, as well as heteroatoms such as S and O. This reduction procedure exhibited the recyclable and reusable property of heterogeneous catalysts and was applicable to gram-scale synthesis. The reaction mechanisms were proposed based on some control experiments and the previous literature. This is the first example of hydroborative reduction of amides to amines mediated by heterogeneous catalysts.

Structural basis for ligand binding modes of CTP synthase.

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo-electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Iodine-125 interstitial brachytherapy for malignant lacrimal sac tumours: an innovative technique.

OBJECTIVES: Primary malignant tumours of the lacrimal sac are rare, surgery and radiotherapy may induce substantial side effects for patients. Here, this article reports an innovative technique of interstitial brachytherapy developed for the treatment of malignant lacrimal sac tumours. PATIENTS AND METHODS: Four patients (male 3, female 1), with an average age of 52.7 years (range 41-72 years), were individually diagnosed with squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma and lymphoma. All patients received Iodine-125 interstitial brachytherapy after surgical resection for malignant lacrimal sac tumours. Visual function examination (vision, intraocular tension, fundus photography, fluorescein angiography, and optical coherence tomography) and CT/MRI/PET-MRI were performed to look for signs of recurrent tumours or metastasis. RESULTS: Four patients were followed for an average of 28 months (range, 23-37 months). All patients were free from local disease. Their visual function was normal, and CT/MRI did not reveal any tumour recurrence. CONCLUSIONS: Iodine-125 interstitial brachytherapy can be used as an alternative to wide excision or exenteration of these tumours. There was good local control, reasonable maintenance of vision, and good cosmesis.