TBDCR NPs) that can massively generate both type I and type II ROS under direct X‐ray irradiation is reported for hypoxia‐tolerant X‐PDT. Heteroatoms are introduced to enhance X‐ray harvesting and ROS generation ability, and AIE‐active TBDCR exhibits aggregation‐enhanced ROS especially less oxygen‐dependent hydroxyl radical  generation ability. TBDCR NPs with a distinctive PEG crystalline shell to provide a rigid intraparticle microenvironment show further enhanced ROS generation. Intriguingly, TBDCR NPs show bright near‐infrared fluorescence and massive singlet oxygen and HO•− generation under direct X‐ray irradiation, which demonstrate excellent antitumor X‐PDT performance both in vitro and in vivo. To the best of knowledge, this is the first pure organic PS capable of generating both 1O2 and radicals (HO•−) in response to direct X‐ray irradiation, which shall provide new insights for designing organic scintillators with excellent X‐ray harvesting and predominant free radical generation for efficient X‐PDT.X‐ray induced photodynamic therapy (X‐PDT) circumvents the poor penetration depth of conventional PDT with minimal radio‐resistance generation. However, conventional X‐PDT typically requires inorganic scintillators as energy transducers to excite neighboring photosensitizers (PSs) to generate reactive oxygen species (ROS). Herein, a pure organic aggregation‐induced emission (AIE) nanoscintillator ( TBDCR NPs) is developed for X‐PDT. TBDCR NPs could be directly excited by X‐ray to emit bright fluorescence and massively generate singlet oxygen and less oxygen‐dependent hydroxyl radicals in aggregate for type I and type II X‐PDT, which demonstrate excellent tumor eradication performance to radio‐resistant tumors both in vitro and in vivo.A pure organic aggregation‐induced emission (AIE) nanoscintillator ( RT capitalizes on high‐energy ionizing radiation to damage cellular deoxyribonucleic acid (DNA) or radiolysis water to generate toxic reactive oxygen species (ROS). However, the high X‐ray dosage, unavoidable ionizing irradiation damage to healthy tissues and the tumor hypoxia‐related RT resistance have been major obstacles in RT‐based cancer therapy. To date, numerous approaches have been employed to increase the therapeutic efficacy of RT, including oxygen delivery, increasing radiosensitivity, modulating tumor microenvironment (TME), and so forth. Nonetheless, the state‐of‐art strategy to increase RT performance is still using high‐Z metal‐based radiosensitizers such as Pt, Au or tantalum oxide nanoparticles with high X‐ray attenuation coefficients to increase the intratumoral ionizing irradiation. Recently, several radiosensitizers such as hafnium dioxide (HfO2) nanoparticles (NBTXR3) have been tested in clinical trials in Europe and US for treating hepatocarcinoma and other cancers. Despite the potency of these inorganic radiosensitizers to increase the RT efficacy, a very high X‐ray dosages (50–70 Gy) is still required and the generation of RT‐resistance cannot be circumvented. Hence, more efforts are urgently needed to increase effectiveness of RT without RT‐resistance generation for tumor eradication.Since Roentgen's discovery of X‐ray in 1895 for the first time, X‐ray have received widespread research interests in biomedical imaging and therapeutics.1 Afte with advantages of precise controllability, noninvasiveness, high spatiotemporal accuracy as well as negligible drug resistance, which shows great potential in oncotherpy. Generally, PDT generates cytotoxic ROS utilizing light, oxygen, and photosensitizers (PSs) to deliver direct therapeutic effect, which could also elicit immunogenic cell death to activate the antitumor immunotherapeutic response. PSs can be categorized into two classes, type I PSs produce free radicals like hydroxyl radicals (HO•−), superoxide radicals (O2•−), by electron transfer mechanism from triplet excited PSs, while type II PSs generate singlet oxygen (1O2) through the energy exchange process with surrounding oxygens. The vast majority of developed PSs belong to type II PSs, while very recent advance suggests that type I PSs are less oxygen‐dependent and more promising for hypoxia tumor treatment, benefited from intracellular superoxide dismutase (SOD)‐mediated disproportionation. In this regard, designing effective type I PSs for PDT has attracted great research interests recently. However, most of them  are excited by visible or near‐infrared (NIR) light where the limited light penetration depth due to light scattering and tissue absorption has greatly hampered their applications. Although, utilizing multiphoton‐excitation or upconversion technique could also increase the penetration depth, the light penetration is still very superficial (<1 cm), and the curative impact of PDT on deep tumors is far from satisfactory.Photodynamic therapy (PDT) is a minimally invasive method for clinical cancer treatment,11 wit X‐PDT usually requires a inorganic scintillator as the energy transducer to convert X‐ray to visible light and subsequently excite neighboring PSs via a two‐step energy transfer (EnT) process. Generally, current scintillation systems commonly used for X‐PDT include anchoring organic PSs at scintillator surface or constructing MOFs with heavy metal units and photosensitizing ligands. However, combining these inorganic scintillators and organic PSs not only complicates material synthesis, but also undoubtedly results in tremendous energy loss from the inefficient two‐step EnT processes. On the other hand, organic scintillators which could be directly excited by X‐ray to emit photons have recently been reported by Huang, Liu and others. Organic scintillators could generate 25% singlet excitons and 75% triplet excitons under direct X‐ray irradiation, which is very promising for X‐PDT as ROS is generated from triplet excitons, but such possibility has been less explored. To the best of our knowledge, there is only one report of pure organic scintillator for X‐PDT, which is unfortunately a planar type II PS whose antitumor performance will be hampered by the hypoxia tumor microenvironment. Moreover, the vast majority of those reported PSs (for both type I and type II PSs) for PDT and X‐PDT have planar molecular structures, and they tend to form aggregates with strong π‐π stacking‐related aggregation‐caused quenching (ACQ) effect. Such notorious ACQ effect results in unavoidable fluorescence quenching and low ROS efficiency for these PSs at aggregated states or at high concentrations in anaerobic tumor microenvironments, seriously affecting therapeutic efficacy of X‐PDT and PDT. As opposed to the ACQ effect, fluorogens with aggregation‐induced emission characteristic (AIEgens) display negligible fluorescence in the molecular state but show magnified fluorescence in aggregate as a result of the restriction of intramolecular motion (RIM) that blocks the nonradiative pathway. Additionally, the inhibited molecular motions of the AIEgens in the aggregate could promote the radiative decay and intersystem crossing (ISC) process, offering the possibility of increased fluorescence and ROS generation. Therefore, it is crucial for creating pure organic AIE scintillators that can generate type I ROS under direct X‐ray irradiation for effective X‐PDT to solve all the above serious problems, as they could effectively generate triplet excitons, produce less‐oxygen dependent free radicals, and avoid ACQ compromised ROS generation in aggregate for ultimate tumor treatment.In recent years, X‐ray mediated PDT (X‐PDT) that combines the merits of deep tissue penetration and minimal drug resistance has attracted great research interests.25 X‐PTBDCR NPs) that profoundly generates 1O2 and HO•− under direct X‐ray irradiation to realize effective X‐PDT  as the electron donor, TBDCR showed aggregation enhanced fluorescence, triplet exciton formation and ROS generation. TBDCR was further encapsulated into lipid‐polyethylene glycol (PEG) nanoparticles with sizes of ≈3.8 nm that is beneficial for tumor accumulation, deep tumor penetration and body clearance. Most importantly, TBDCR NPs exhibited a unique PEG crystalline shell, which leads to a rigid microenvironment and a much more compact packing for TBDCR, resulting in further improved ROS generation. Intriguingly, TBDCR NPs showed massive generation of 1O2 and higher toxic HO•− under both light excitation and direct X‐ray irradiation, without the aid of inorganic energy transducers. Thanks to the 1O2 and HO•− induced PDT and deeper tissue penetration ability of X‐ray, TBDCR NPs demonstrated excellent tumor eradication against HeLa cancer cells (Hela) as well as radio‐resistant HelaR tumors both in vitro and in vivo. This work of designing X‐ray activatable AIE nanoscintillator circumvents the limitations of poor penetration depth, low hypoxia tolerance and compromised ROS generation in aggregates for conventional PDT. Our design evidences the enormous potentials of type I AIE organic nanoscintillators in X‐PDT and shall pave new insights for further development of novel PSs as well as nanoscintillators for cancer theranostics.In this work, we report a pure organic AIE nanoscintillator malononitrile (DCR) moiety which contains heteroatoms  was employed as an electron‐withdrawing group, and a benzene ring was selected as a π bridge to increase the π conjugation length and to promote highest occupied molecular orbital (HOMO)‐lowest unoccupied molecular orbital (LUMO) separation to reduce the valance electron repulsion for a better ISC process. Finally, as shown in Figure TBDCR) was obtained from the Suzuki coupling and Knoevenagel reaction with a high yield of 85%. The detailed synthetic routes of TBDCR were displayed in Scheme 1H NMR, 13C NMR), and high‐resolution mass spectrometry (HRMS)  by ultraviolet‐visible spectroscopy and photoluminescence (PL) spectroscopy. As displayed in Figure TBDCR has an absorption maximum at ≈443 nm and an emission peak at 653 nm, respectively, with a large Stokes shift of 210 nm. TBDCR possesses a very high molar absorption coefficient of 2.67 × 104 M−1 cm−1 (at 443 nm)  to induce aggregate formation. As indicated in Figure fw, reaching a minimum fluorescence intensity at fw = 50%, which should be caused by the strong intramolecular charge transfer (ICT) feature of TBDCR as water possesses a higher polarity. However, further increasing fw results in amplified emission intensities along with aggregate formation, which should be attributed to that the AIE effect competes over ICT effect. In addition, TBDCR powder showed distinct bright‐orange red fluorescence under a 365 nm UV lamp, further indicating that TBDCR possess excellent fluorescence in solid state and its AIE tendency  value of ≈2.2. The absorption as well as emission spectra in solvents with various polarity were performed to study its ICT property. As shown in Figure TBDCR in these different polar solvents are in the range of 440 to 470 nm, and it showed a very weak fluorescence in the more polar solvents like methanol and dimethyl sulfoxide, in sharp contrast to those in less polar solvents. Intriguingly, TBDCR showed an approximately 160 nm red‐shift in emission peak from hexane (low‐polar solvent) to dichloromethane (high‐polar solvent), which further suggests the strong ICT characteristics of TBDCR.The optical characteristics of TBDCR was further evaluated. Initially, the solid‐state fluorescence spectrum of TBDCR was measured. Interestingly, as displayed in Figure TBDCR in Figure TBDCR, which was delightfully confirmed by time‐gated photoluminescence spectrum where a sharp PL peak at 680 nm was observed at a delay time of 0.1 ms  of 0.116 eV for TBDCR , 9,10‐anthracenediyl‐bis(methylene) dimalonic acid (ABDA), dihydrorhodamine 123 (DHR 123), and hydroxyphenyl fluorescein (HPF) were selected as total ROS, 1O2, O2•− and HO•− indicators, respectively. As displayed in Figure TBDCR within an ultra‐short exposure time . The fluorescence enhancement factor of DCFH was ≈265.2, suggesting the superior ROS generation capability of TBDCR. To study the AIE effect on ROS generation, the ROS of TBDCR at different fw was evaluated. TBDCR showed negligible ROS generation at fw of 0 and 40%  as well as crystal violet (CV), respectively, as demonstrated by the DCF fluorescence enhancement factors.Encouraged by the aforementioned excellent results, the ROS generation ability of 1O2, O2•− and OH•− generation of TBDCR exposure to white light was evaluated. 1O2 generation was assessed by decomposing ABDA upon light irradiation. As shown in Figure TBDCR upon white light illumination , which was reduced by ≈51.7%, while ABDA alone had almost no significant change. Ce6 and CV showed a minor effect on ABDA absorbance under the same conditions, with the ABDA absorbance reduced by only ≈12.5% and ≈14.7%, respectively. Moreover, ABDA decomposition rates as reflected by the slopes of ln(A0/A) versus irradiation time indicated that 1O2 generation efficiency of TBDCR was 2.1‐ and 15.1‐fold higher than Ce6 and CV . In contrast, the fluorescence of DHR 123 showed nonsignificant changes in the blank, Ce6 and CV groups receiving same light exposure. In addition, the DHR 123 fluorescence enhancement factor for TBDCR was determined to be ≈164.01, much higher than Ce6 (∼6.2) and CV (∼50.5), respectively, suggesting the excellent O2•− generation capacity of TBDCR under white light irradiation. The production of OH•− was evaluated with HPF. As shown in Figure TBDCR caused largely magnified HPF fluorescence intensity, and the HPF fluorescence enhancement factor for TBDCR with white light irradiation was determined to be ≈105.2. At the tested condition, Ce6 and CV only lead to a minimal amplification of HPF fluorescence signature, with the enhancement factors of ≈1.7 and ≈2.6, respectively. Such results clearly demonstrated that TBDCR has a proudly better OH•− generating capacity than Ce6 and CV, which is almost 61.8‐ and 40.4‐fold higher than Ce6 and CV, respectively. The capability of TBDCR to generate 1O2 and OH•− under white light irradiation was further conducted via electron paramagnetic resonance (EPR) with 2,2,6,6‐tetramethyl‐4‐piperidinol (TEMP) as well as 5‐dimethyl‐pyrroline n‐oxide (DMPO) as trapping agents for 1O2 and OH•−, respectively. The characteristic signals of 1O2/TEMP and OH•−/DMPO dramatically increased in the presence of TBDCR after light irradiation −2000 (DSPE‐PEG2000) as the encapsulation matrix. Dynamic light scattering (DLS) measurements indicated that the hydrodynamic diameter of TBDCR NPs was ≈18.4 nm  spectrum of TBDCR NPs also indicated high crystalline phase of TBDCR NPs, and the crystalline was attributed be PEG  shells  by employing DCFH as the total ROS indicator under white light irradiation  of TBDCR was firstly studied, bright red fluorescence from TBDCR was clearly observed under X‐ray irradiation , and the absorbance of ABDA in the presence of TNDCR NPs group reduced significantly under X‐ray irradiation, suggesting that TBDCR NPs under X‐ray could effectively generate both 1O2 and OH•−. In addition, as manifested by DCF fluorescence enhancement factor in Figure TBDCR NPs (40 µg mL−1) was almost 9.1‐fold and 6.6‐fold higher than Ce6 and CV, respectively. With ABDA as 1O2 indicator, TBDCR NPs (40 µg mL−1 based on TBDCR concentration) leads to a ≈87% ABDA absorbance reduction with X‐ray dosage of 4 Gy, much better over Ce6 (≈30%) and CV (≈33%)  and X‐ray irradiation, with a fluorescence enhancement factor of ≈27.4. While, Ce6 and CV only led to much lower HPF fluorescence enhancement factors of ≈7.8 and ≈9.9, respectively. Moreover, the stability of TBDCR NPs under X‐ray irradiation was also evaluated, where the absorbance still maintained above 90% of original ones after exposure to X‐ray (8 Gy)  Figure . As aforr Figure . The flu2.31O2 and HO•− generation ability and NIR emission feature of TBDCR NPs, the X‐PDT related photoactivity experiments were conducted in vitro. Initially, the cellular uptake experiment of TBDCR NPs was performed with human cervical Hela cancer cells. As indicated in Figure TBDCR NPs quickly accumulated inside HeLa cells after 2 h incubation with an intense and well‐defined red fluorescence signal in the cytoplasm, and the optimal cellular uptake was achieved at 6 h incubation. In addition, the fluorescence signal patten hints a possible lipid droplet (LD) staining possibility. The intracellular distribution of TBDCR NPs was evaluated using commercial organelle‐selective tracker BODIPY 493/503 to confirm the proposed conjecture. The observed red fluorescence of TBDCR NPs overlapped well with the green fluorescence of the lipid droplet tracker BODIPY 493/503 with a high Pearson's correlation coefficient of 0.87  assay by using HeLa cells and radiation‐resistant Hela cells (HelaR). As shown in Figure TBDCR NPs demonstrated negligible toxicity toward both Hela and HelaR cancer cells without light or X‐ray irradiation, at all tested concentrations, indicating their excellent biocompatibility. X‐PDT induced cell death was then evaluated at different X‐ray dosages. TBDCR NPs (50 µg mL−1 based on TBDCR concentration) effectively inhibited cancer cell proliferation in a radiation dosage‐dependent manner in both Hela and HelaR cancer cells, with the cell viabilities of ≈11.5% and ≈31.6% for Hela and HelaR, respectively , which demonstrates the initial success of TBDCR NPs in X‐PDT. Notably, as shown in Figure TBDCR NPs (50 µg mL−1) upon X‐ray irradiation (8 Gy) also granted its excellent X‐PDT performance under hypoxia condition (1% O2) that is comparable to those under normoxia conditions. Additionally, the more insights were provided on the X‐ray radiation‐caused cell death brought by TBDCR NPs. As shown in Figure TBDCR NPs mediated X‐PDT could significantly increase cell apoptosis, even for radiation‐resistant HeLaR cancer cells. These findings showed a strong concordance with the MTT phototoxicity findings, indicating that the X‐PDT of TBDCR NPs can effectively induce tumor cell apoptosis and death promising for eradication tumor in vivo.Furthermore, in vitro X‐PDT anticancer potency of  (HelaR).6 As sy Figure . However2.4TBDCR NPs for X‐PDT was further explored. Initially, the intracellular ROS generation upon treatment with TBDCR NPs was investigated by employing 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA), a reputable total ROS hypersensitive fluorescent probe. As displayed in Figure TBDCR NPs after X‐ray irradiation (8 Gy) were significantly higher than their corresponding control groups, and DCF positive area was enhanced by ≈2.1 and ≈5.6 fold when compared to Hela and HelaR cells in X‐ray irradiation without TBDCR NPs incubation  treatment, the relative ROS levels in Hela and HelaR were ≈1.9 and 5.0‐fold higher than these only radiation groups  or radiation‐resistant Hela cells (HelaR) to the right hind leg of mice to assess the tumoricidal activity of TBDCR NPs. When the volume of tumors reached ≈80 mm3 (at day 14), the mice were randomly divided into eight groups (n = 4 mice per group): 1) HeLa + Control; 2) HeLa + TBDCR NPs; 3) HeLaR + Control; 4) HeLaR + TBDCR NPs; 5) HeLa + Control + X‐ray; 6) HeLa + TBDCR NPs + X‐ray; 7) HeLaR + Control + X‐ray; 8) HeLaR + TBDCR NPs + X‐ray. Among them, TBDCR NPs or PBS (Control) was intravenously injected into mice at day 6. The mice in groups 5–8 were irradiated with X‐rays at day 7 and day 9 . Tumor volumes and body weights were monitored twice per week and the results were displayed in Figure TBDCR NPs themselves exhibited negligible suppressive effect to HeLa and HeLaR tumors, and the tumors grow to 922.0 mm3 and 1104.8 mm3, respectively. Furthermore, only X‐ray irradiation (HeLa + Control + X‐ray and HeLaR + Control + X‐ray groups) exhibited partially inhibition of tumor growth in compared with tumors treated with PBS (as control) and TBDCR NPs only, indicating the limited antitumor efficiency of radiotherapy. Additionally, under the same dosages of X‐ray irradiation, the tumor growth in group HeLaR + X‐ray was significantly faster in mice than group HeLa + X‐ray, which confirmed that HelaR has greater resistance to radiation than Hela. In contrast, the tumor tissue growth was significantly inhibited in the presence of X‐ray and TBDCR NPs, where negligible tumor growth was observed for HeLa + TBDCR NPs + X‐ray group, with the final tumor volume was reduced to 66.5 mm3. While the tumors in group HeLaR + TBDCR NPs + X‐ray only increased to 152.0 mm3, ≈24.4% and ≈13.3% of those in groups HeLaR + Control and HeLaR + Control + X‐ray, respectively, indicating that TBDCR NPs mediated X‐PDT could significantly promote the antitumor effect of RT. Moreover, the tumoricidal activity effect of the TBDCR NPs under X‐ray irradiation was validated by the appropriate tumor photographs  to The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes. Fungicides are pervasively utilized; thus, they can easily accumulate in the environment and cause deleterious effects on both human health and environmental safety. Since the 1950s, triazole fungicides (TOFs) have been extensively employed to inhibit fungi in crops, such as fruit, vegetable, and flower crops . Various−3 7], and a−3 . A Frenc−3 . The con−3 .Increasing evidence shows that DFZ has potential cumulative effects , and it Retinoic acid (RA), a metabolite of vitamin A, is irreplaceable for organ development in mammals . It is wThe current study was designed to examine the correlation between DFZ exposure and testicular damage in male mice, and to further investigate the potential participation of the retinoic acid (RA) pathway in this process through in vivo and in vitro studies. Our findings shed light on the environmental risks of DFZ, broaden our understanding of the toxic effects of DFZ on the male reproductive system, and provide a theoretical basis for the safe and rational use of DFZ.Thirty healthy male C57BL/6 mice  were obtained from the Laboratory Animal Center of Chongqing Medical University. Thirty mice were maintained under SPF conditions  and provided with ad libitum access to food and water. The mice were randomly divided into three groups (10 in each): low-dose group (D20), high-dose group (D40), and control group (Ctrl). Corn oil  was administered to the control group, while DFZ  dissolved in corn oil, 20 and 40 mg/kg/d, was orally administered to the D20 and D40 groups for 35 consecutive days, respectively. Twelve hours after administration ceased, the mice were weighed and then anesthetized with phenobarbital sodium. Blood samples were collected from each mouse into a separate tube in order to isolate the serum for further biochemical analysis. Finally, the mice were immediately sacrificed, and the epididymis and testes were harvested from both sides and weighed. Then, the testicular organ coefficient (testis weight/body weight) was calculated.The isolated epididymis tissues were placed in a sterile glass container containing 2 mL of PBS, cut up, and incubated in 37 °C water for 0.5 h. Subsequently, after removing the debris, another 2 mL of PBS was added to the container, and then, 10 µL of diluted sperm suspension was carefully dispensed onto a hemocytometer plate for counting. Another 10 µL of diluted sperm suspension was dispensed onto glass slides, allowed to dry naturally, fixed with methanol, and stained with eosin. After drying again at room temperature, the morphology of the spermatozoa was observed and imaged with an inverted microscope, and the number of normal sperm and abnormal sperm in a 400× field of view was recorded. The % of sperm malformation was then calculated by dividing the number of abnormal sperm by the total number of sperm.Serum testosterone levels were measured with an enzyme-linked immunosorbent assay (ELISA) kit . Briefly, serum was added to a 96-well plate pre-coated with antigen, followed by the addition of biotinylated detection antibodies, incubation with horseradish peroxidase (HRP) conjugate, and the addition of the substrate reagent. The absorbance (OD) of all 96 wells was measured at 450 nm. Six serum samples were randomly selected from each group and tested in triplicate.Left testis tissues were fixed with 4% paraformaldehyde for more than 24 h, followed by dehydration, embedding, and slicing into 4 µm thick sections for hematoxylin and eosin (H&E) staining. Subsequently, all the testicular sections were observed by a light microscope and quantitative analysis was performed using Fiji software. Briefly, six testes’ samples from each group were randomly chosen, six random 200× fields of view were selected for each testicular section, the number of layers of spermatogenic epithelium and damaged seminiferous tubules (ST) in each field was counted, and the average diameter of the ST in each field of view was measured.TUNEL apoptosis assay kit  was used for apoptosis detection. All samples were observed with a fluorescence microscope . The process of the experiment was performed according to the manufacturer’s instructions. Details can be found in our previous reports .Testicular tissues were homogenized in RIPA lysis buffer, and the supernatants were collected after homogenization. Subsequently, the protein concentrations were detected using a BCA kit according to the manufacturer’s instructions. Primary antibodies against STRA8  and SCYP3  were used. Beta-Actin  was used as an internal reference. Quantitative analysis was conducted by Image Lab software .The concentrations of ROL (nmol/L), RE (pg/mL), and RA (pg/mL) in serum, testis, and GC-2 cells were measured using ELISA kits . In short, specimens, standards, and HRP-labeled detection antibodies are added sequentially to the precoated microtiter wells with ROL/RE/RA antibody, and then, the plates were incubated and thoroughly washed. Color was developed with TMB substrate. The OD was measured by a microplate reader at 450 nm. Six sample replicates and three technical replicates were set for this experiment.−ΔΔCt method, with β-actin expression as a reference for normalization. Further details can be found in our previous reports. The primer sequences are provided in Total RNA was extracted from testis and GC-2 cells using a Simply P Total RNA Extraction Kit , and single-strand cDNA was then synthesized by an RT Master Mix Kit . Each reaction was configured with 10 uL of reaction system: 1 uL of cDNA sample, 5 uL of SYBR Green qPCR Master Mix , 0.2 uL of pre/reverse primer, and 3.6 uL of nuclease free water. A total of 3 min of initial denaturation at 95 °C, 15 s of denaturation at 95 °C (40 cycles), and 30 s of extension at 60 °C were used in the RT–qPCR programs. The relative mRNA levels of the target genes were quantified using the 22 and at 37 °C. DMEM  supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin were used for cell culture, the cell was passaged at a ratio of 1:10 when it reached 80–90% confluence.GC-2 cells, a mouse germ cell lines, procured from the American Tissue Culture Collection were cultured in an incubator with 5% CO3 cells/well) were seeded in a 96-well plate and incubated for 24 h. Subsequently, various concentrations of DFZ  were added to the plate and incubated for an additional 24 h. Then, 10 µL of CCK8 solution was added to each well and incubated at 37 °C for 2 h. OD values were measured at 450 nm.CCK8 assays were conducted to evaluate GC-2 cell’s vitality after DFZ exposure. GC-2 cells . Metascape platforms (http://metascape.org/gp/index.html (accessed on 26 March 2023)) were adopted for DEGs functional annotation. We constructed a protein–protein interaction (PPI) network for target-enriched genes using the STRING database with an interaction score ≥ 0.4., cytoHubba plugins of Cytoscape software ) were used to identify the top 5 Hub genes.Three samples of 0 or 20 μM DFZ-treated GC-2 cells were subjected to mRNA sequencing analysis. Differentially expressed genes (DEGs) were screened with log2 (fold change) ≥ 2 or ≤−2 and p < 0.05 was considered to be statistically significant.GraphPad Prism 9.0  was used for statistical analysis. Experimental data were expressed as the mean ± standard deviation. One-way ANOVA with Dunnett’s post hoc test was used to compare multiple groups, and Compared with the control group, the body weight A,B, testH&E staining results revealed significant histopathological changes A. In theTunnel assay showed a significant increase in apoptosis rate in D20 and D40 groups, and apoptosis occurred mainly in spermatocytes A. The prTo investigate the effect of DFZ on RA pathway homeostasis, we measured the levels of RA, RE (the stored form of ROL) and ROL (the substrate of RA synthesis) in testicular tissues. The results revealed that the level of RA, RE, and ROL in testicular tissues were significantly elevated after DFZ treatment A–C.RT–qPCR experiments were conducted to investigate the alteration expression of key enzymes and receptors that are involved in RA synthesis and catabolism. The mRNA expression levels of STRA6, RDH10, and ALDH1A1-3, which are key enzymes involved in ROL, RE, and RA synthesis, were significantly increased after DFZ treatment  were significantly increased while genes involved in RA catabolism, such as CYP26A1 and CYP26B1, were significantly decreased. These results indicate disruption in RA homeostasis after DFZ exposure.GC-2 cell line, derived from immortalized mouse pachytene spermatocytes, is an important tool for studying the cellular effects of spermatocytes . During In vitro, DFZ induced significant cytotoxicity in GC-2 cells, with cell viability decreasing gradually with increased dose and cellular ultrastructure being altered after DFZ exposure. The increased levels of ROL, RE, and RA in GC-2 cells after DFZ exposure are consistent with the detection of ROL, RE, and RA levels in testes in vivo. Analysis of transcriptome results found that multiple terms related to RA signaling were enriched in the transcriptome sequencing analysis of GC-2 cells and were mainly associated with ROL and RA metabolic processes. Terms related to the apoptosis process were also enriched. By integrating the enriched terms, we found an indirect link between the apoptotic signaling pathway and ROL metabolism is established via chemical carcinogenesis receptor activation. PPI results suggest that HMOX1 and FOS are genes linking RA pathway-related genes and apoptosis-related genes, and ALDH1A1, ALDH1A7, AOX1, CYP26B1, and CYP1A1 were identified as hub genes. Previous studies have reported that the RA derivative increases expression of HMOX1, induces apoptosis in ARPE-19 cells  and RA iBased on the above reports and our experimental results, we conjecture that DFZ exposure could enhance RA synthesis through the upregulation of ALDH1A1, ALDH1A7, and AOX1 expression, while reducing RA catabolism through the downregulation of CYP26B1 and CYP1A1, thus leading to increased HMOX1 expression and decreased FOS expression, and eventually mediating apoptosis in GC-2 cells. The specific molecular mechanism underlying RA pathway dysregulation-induced testicular damage after DFZ exposure needs further exploration. DFZ has been reported to induce apoptosis through the induction of oxidative stress in zebrafish and SH-SY5Y cells ,23. TherIn our study, we found that prepubertal DFZ exposure could induce testicular injury in adulthood. In addition, DFZ could increase RA synthesis and decrease cytochrome p450-mediated RA catabolism, thus disrupting RA homeostasis in the testis. The imbalance of RA in the testis is an important cause of DFZ-induced testicular injury, possibly linked to the increased apoptosis of GC-2 cells, which warrants further investigation.


VgVw−1 with heat release of ~18 kJ mol−1 CH4 and the active ice can be easily regenerated by depressurization below the ice point. The active ice undergoes cyclic ice−hydrate−ice phase changes during gas uptake/release, thus removing most critical drawbacks of hydrate-based technologies. Our work provides a green and economic approach to gas storage and gas separation and paves the way to industrial application of hydrate-based technologies.Gas hydrates provide alternative solutions for gas storage & transportation and gas separation. However, slow formation rate of clathrate hydrate has hindered their commercial development. Here we report a form of porous ice containing an unfrozen solution layer of sodium dodecyl sulfate, here named active ice, which can significantly accelerate gas hydrate formation while generating little heat. It can be readily produced via forming gas hydrates with water containing very low dosage (0.06 wt% or 600 ppm) of surfactant like sodium dodecyl sulfate and dissociating it below the ice point, or by simply mixing ice powder or natural snow with the surfactant. We prove that the active ice can rapidly store gas with high storage capacity up to 185  Gas hydrates have promising energy storage applications, a main bottleneck being their slow formation kinetics. Here, the authors demonstrate that by dispersing kinetic promoters in porous ice as active ice for gas hydrate formation, a minute-level formation process can be achieved for hydrate-based technologies. Ideally, each unit volume of hydrate can store 180 volumes of natural gas2; different gases have different equilibrium conditions in hydrate formation; in addition, gas hydrate can be preserved under atmospheric pressure at 268.15 K3. These features make it a promising medium for gas storage and separation. However, the feasibility of these hydrate−based technologies is heavily reliant on the formation rate of gas hydrate, which is naturally slow.Gas hydrates have attracted much attention because not only natural gas hydrates are believed to be the largest natural gas resource on Earth, but also gas hydrates provide alternative and innovative solutions for industrial requirements, e.g., natural gas storage & transportation and gas mixture separation. Gas hydrate forms when water molecules and gas molecules contact at low temperatures and high pressures; as a result, gas molecules are enwrapped in lattices that consisted of hydrogen−bonded water molecules5, leaving a large amount of unconverted water beneath the hydrate film. Besides that, the heat released during hydrate formation due to its exothermic nature also would retard hydrate formation in turn by rising the reactants’ temperature. Thus, the slow hydrate formation results from the retarded mass transfer and the heat generation.Gas hydrate formation is an interfacial process between gas and water. For the hydrate formation without human intervention, a solid hydrate film with low permeability would spread over and seal off the interface, which hinders the contact of gas and water for continued gas hydrate formation. Consequently, a spontaneous hydrate growth always stagnates at film thickness less than 100 μm15. Among them, using kinetic promoters like surfactants, e.g., sodium dodecyl sulfate (SDS), is among the most effective ways known today to accelerate hydrate formation. Another class of kinetic promoters that have gained recent prominence are amino acids16. The fastest hydrate formation in the presence of kinetic promoters reported so far could be accomplished at 11 min with water conversion over 80%6. Despite the conspicuous performance of kinetic promoters accelerating hydrate formation, scaling-up the hydrate production via using kinetic promoters may encounter three major challenges.Efforts have been dedicated to remove or minimize mass transfer resistance and to accelerate hydrate formation17. This effect accelerates hydrate formation by sucking the aqueous solution onto the reactor wall and providing larger contact area for hydrate formation. Due to such hydrate growth pattern, the hydrate formation rate is closely related to the solution load in reactors; with the same reactor configuration and under similar thermodynamic conditions, overall hydrate formation rate decreases with the increase of solution load20, which is unfavorable for scaling-up of the process. The second challenge hindering the scaling-up is the temperature increase due to the exothermic nature of the formation process; because of significant heat released during hydrate formation from water or aqueous solution , the temperature of the liquids could rise by 4 ~ 8 K in the 0.5 ~ 2.7 h of formation processes20; a faster hydrate formation process required by industrial hydrate production would further increase the temperature, and then hamper the hydrate formation in turn. The third challenge is the inadequate utilization of the reactor space; plenty of gas space is required for the upward growth to avoid the blockage of the gas inlet/outlet; for that reason, gas hydrate formation in the presence of surfactants is generally conducted with gas volume of 65% ~ 90% in the reactors. Besides the three major challenges, the hollow appearance of the hydrate22 formed with the wall−climbing effect could decrease the apparent gas storage density; meanwhile, the serious bubble and foam generation during hydrate dissociation is also detrimental to industrial gas hydrate production.The accelerated hydrate formation comes from an upward and wall-adhering growth pattern of gas hydrate induced by kinetic promoters, which is known as wall−climbing effectVgVw−1 at 272.65 K. Beside the rapid formation rate, the active ice exhibits multiple advantages, i.e., good repeatability, recyclability, compressibility, simple storage condition and low heat generation. Characterization results show that the active ice is porous and contains unfrozen SDS solution layer. Gas hydrate formation in this solution layer at temperature slightly lower than the ice point spawns a virtuous circle of ice melt—hydrate formation—ice melt, which hastens gas hydrate formation strikingly. The ice with unfrozen surfactant solution layer seems to be activated for gas hydrate, it is therefore an active ice. Inspired by this, we improve the preparation process of active ice without impairing its performance in gas hydrate formation. With the multiple advantages, the active ice approach is capable of overcoming the obstacles to scaling−up gas hydrate formation process for industrial purpose.In order to break through the limits of mass and heat transfer on hydrate formation rate and reduce the subsequent problems, here, we report an active ice approach and demonstrate its excellence as a formation medium for gas hydrate. We first use a kind of active ice produced by decomposing primary gas hydrate that is formed from SDS solution to form methane hydrate, and the hydrate formation typically completes within 5 min with gas uptake over than 170 4–SDS hydrate), we wonder whether cage structures retain in the solid medium produced after hydrate dissociation below ice point and try to use it for reformation of gas hydrate. An active ice was made by decomposing a primary gas hydrate mass below the ice point, which was previously synthesized with methane and 600−ppm SDS solution  after a 1 h formation process, including an induction time of 18 min. By contrast, the active ice gulps methane very rapidly without retard at 272.65 K  and the ice melting heat ; meanwhile, the gas uptakes (181.10 VgVw−1 and 173.21 VgVw−1) reached 91.19% and 90.97% of their final values, respectively  if directly using the hollow active ice column produced by dissociating CH4 − SDS hydrate displaying in Fig. α, the ratio of the specific volume of active ice packing bed to the specific volume of perfect ice crystal of the same mass, retains higher than 1.398. Therefore, decreasing the apparent volume of the active ice (or gas hydrate) by compression and good regeneration capacity can be realized at the same time. However, this only happens at moderate compression levels. The apparent methane storage capacity of active ice packing bed reached 126.36 VgVbed−1 under the optimized loosening coefficient α = 1.492 and 5.76 MPa 25.Although the porous or powdery morphology of active ice brings high gas uptake rate, it makes the apparent specific volume of active ice packing bed much bigger than that of ice crystal and results in lower apparent storage capacity. For example, the apparent storage capacity is only 83.92 ± 2.6 2–SDS hydrate below the ice point  mode could be adopted because of the smaller heat release26. Because the uncertainty of the scaling-up effect, the above methods are only suggestions for the potential use of the active ice in gas storage and transportation and gas separation, and they must be developed and analyzed in further studies.We propose a conceptual process with respect to the potential application of the active ice in natural gas storage and transportation. The active ice could be prepared by decomposing gas-promoter hydrate below the ice point, or grinding the ice of suitable kinetic promoters’ aqueous solution, or just simply mixing ice powder or natural snow with kinetic promoters. As regards the most economical or feasible preparation method, it depends on specific situations. After being produced and shaped in a factory, the active ice could be loaded in a tank on vehicles, in which built-in heat exchangers should be installed. The on-board tanks will be used for gas storage in the gas fields. Natural gas is precooled and injected into the tank and is expected to form gas hydrate rapidly with the active ice. The formed hydrate then will be degassed to atmospheric pressure and transported to its destination and natural gas is recovered along with the active ice. This kind of charging–discharging mode based on the rapid hydrate formation is similar to compressed natural gas (CNG) method; however, it is superior in the economic benefits, safety benefits and environmental friendliness perspective according to the literatureIn summary, we have discovered a kind of active ice, which is of porous or powdery appearance and contains low-dose kinetic promoters. The promoters locally decrease the freezing point of water while keeping its effect on hydrate formation, thus create extensive active sites in the active ice at temperature slightly lower than the ice point of pure water. The spiral development of ice melting—hydrate formation—ice melting at the active sites enables minute-level and high-conversion gas hydrate formation even at very small driving force. The fast hydrate formation, in association with the small formation heat, the shapable packing bed of active ice, and the recyclability of the active ice, could overcome the main obstacles in realizing hydrate-based technologies. What’s more, the greenness and cheapness of the active ice, make it an excellent alternative sorbent for more extensive gas storage or separation.Methane, carbon dioxide, hydrogen, and ethane with purity of 99.99% were purchased from Beijing AP BAIF Gas Industry CO., Ltd. Sodium dodecyl sulfate (SDS) with ACS reagent grade (>99%) was provided by Sigma-Aldrich. Sodium oleate with purity >97% and N-Carbobenzoxy-DL-leucine with purity >98% were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Dodecylbenzenesulfonic acid (> 90%), Sodium dodecyl benzene sulfonate (>95%), Sodium laurylsulfonate (>98%) were provided by Shanghai Makclin Biochemical Co., Ltd. Deionized water was produced in our laboratory.3 was used as the reactor. A piston was mounted at the bottom of the reactor to compress the active ice. An iron ring reciprocates inside the reactor and acts as a stirrer before gas hydrate formation in aqueous solution, which was driven by a group of magnets beside the reactor. A stainless-steel gas reservoir with an internal volume of 130.23 cm3 was used to precool gas. The reactor and the gas reservoir were mounted in an air bath (temperature stability = ±0.1 K). Two pressure transducers  were used to measure the pressures of the gas reservoir and the reactor, respectively. The pressure data were logged by a data acquisition system for every 1 min. A buffer tank was used to precool the pressurizing fluid of the pump, and the inner volume of the buffer tank is 80.10 cm3. A stainless-steel reactor with internal diameter of 5.07 cm and 220.00 cm3 was used to conduct the scale-up hydrate formation experiments in Supplementary Fig. 3 was used to precool the gas.Supplementary Fig. 3 is surrounded by the windows and the stainless-steel wall, and the in situ hydrate formation and dissociation for Raman characterization were performed in the chamber. An annular passage surrounding the chamber was designed as the flow channel for coolant to regulate the temperature in the chamber. A pt100 temperature sensor with accuracy of 0.05 K was inserted into the chamber. A pressure transducer with accuracy of 0.02 MPa was used to measure the pressure in the reactor.Another reactor was used coupled with a Raman spectrometer for in situ characterization of the active ice. The schematic of the reactor is shown in Supplementary Fig. 27. About 3 h later, the pressure drop caused by hydrate formation decreased to less than 20 kPa h−1. Then, the formation of the hydrate (primary hydrate) is considered to be accomplished. The active ice was produced by dissociating the primary hydrate below the ice point. Before the hydrate dissociation, the temperature of the air bath was reset to 272.65 K. 3 h later, the primary hydrate was depressurized to atmospheric pressure within 20 s. Then, the primary hydrate dissociated at 272.65 K and under atmospheric pressure. The temperature and pressure change in the preparation of the active ice via dissociating methane hydrate are shown in Supplementary Fig. 1) methane hydrate formation-dissociation route. The active ice was prepared following a hydrate formation-dissociation method shown in Supplementary Fig.  methane In the experiments shown in Fig. The hydrate formation experiments in the active ice were mainly conducted in the sapphire reactor depicted in Supplementary Fig. The gas separation experiments were also conducted in the apparatus depicted in Supplementary Fig. Both the gas storage and separation results were calculated based on the molar balance of gas in the gas and hydrate phase. The detailed treatment is shown in Supplementary Information.The methods of Raman, PXRD, SEM, and DSC characterization are shown in Supplementary Information.Supplementary informationPeer Review FileSource Data


In vitro, RmlA activates a range of common sugar-1-phosphates to produceNDP-sugars for biochemical and synthetic applications. However, ourability to probe bacterial glycan biosynthesis is hindered by limitedchemoenzymatic access to rare NDP-sugars. We postulate that naturalfeedback mechanisms impact nucleotidyltransferase utility. Here, weuse synthetic rare NDP-sugars to identify structural features requiredfor regulation of RmlA from diverse bacterial species. We find thatmutation of RmlA to eliminate allosteric binding of an abundant rareNDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphatesubstrates, as products no longer affect turnover. In addition topromoting an understanding of nucleotidyltransferase regulation bymetabolites, this work provides new routes to access rare sugar substratesfor the study of important bacteria-specific glycan pathways.Bacterial glycomesare rich in prokaryote-specific or“rare”sugars that are absent in mammals. Like common sugars found acrossorganisms, rare sugars are typically activated as nucleoside diphosphatesugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferaseRmlA initiates the production of several rare NDP-sugars, which inturn regulate downstream glycan assembly through feedback inhibitionof RmlA via binding to an allosteric site.  Rare nucleotide sugars that contain β-anomericcenters are challenging to access synthetically, perhaps due to stabilityissues.22 While a recent one-pot, multistep enzymatic strategy produces nucleotiderare sugars from simple monosaccharides, these routes require severalproteins, some of which suffer from product inhibition.24 Similarly, nucleotidyltransferases like RmlA are often modulatedby intermediates that bind to an allosteric site to control flux.29 Despite several studies on RmlA,31, the structural features of nucleotide sugars involvedin allosteric recognition are still unknown, which restricts our abilityto devise new strategies to rare nucleotide sugars via nucleotidyltransferaseactivity and impedes the study of bacterial glycan biosynthetic pathwaysthat represent potential antibiotic and antivirulence targets.34Bacterial monosaccharides are enriched in other6-deoxy-35 we now evaluate if mutated RmlA can producerare deoxynucleoside diphosphate (dNDP)-sugars that typically inhibitRmlA. We find that 1 is the most potent regulator ofRmlA across species and that RmlA mutants insensitive to feedbackinhibition can activate various l-sugar substrates. In additionto promoting an understanding of nucleotidyltransferase regulation,which ultimately controls surface glycan assembly, this work providesalternative routes to nucleotide l-sugars.Here, we synthesize rare nucleotide sugar analoguesto examinestructural features that are required for allosteric regulation ofRmlA. Since we have shown that mutation of the allosteric pocket ofRmlA affects substrate selectivity at the active site,l-Rha-1P (3) and selectS-1P analogues that could be activated to generate a collection ofnucleotide sugars for structure–function studies , α-l-Rha-1P, and β-l-6dTal-1P (5).These S-1Ps would allow us to evaluate the consequences of hydroxylationof the C6-position and altered anomeric or hydroxyl stereochemistryon resulting nucleotide sugar function. Additionally, α-l-Rha and β-l-6dTal are present in bacterialnucleotide sugars21 that could potentially impact cellular nucleotidyltransferase activity.Inspired by stereoselective syntheses of α- or β-l-Rha-1P using temperature-controlled phosphorylation,39 we prepared partially acetylated precursors for l-Rha (6a), l-Man (6b), and l-6dTal(6c). 6a and 6b were obtainedfrom commercial l-Rha and l-Man, respectively, followingacetylation and benzylamine-mediated selective deprotection of theanomeric position  with appropriate stereochemistry. Weaimed to obtain β- studies 1C. To doposition 2A.40,41 c effect 2B.42,43 l-Rha, l-Man,and l-6dTal (6a–6c) precursorswere then phosphorylated (9a–9c).14 Notably, we could neverisolate α-l-6dTal-1P. Following mild deprotection toyield free S-1P-triethylammonium salts (3–5), we confirmed the anomeric configuration of each using 1H and 13C NMR , which was prepared in 10 stepswith an overall yield of 11%.To produce desired S-1Ps, acetylated orylated 2C. For e 13C NMR 2D. HeterN,N-carbonyldiimidazole(CDI)-activated (deoxy)nucleosidemonophosphates ((d)NMPs) have been coupled to S-1Ps to improve uponslower methods (t > 1 week).52 Since both l-Rha and l-6dTal are activated inbacteria as dTDP-β-l-sugars, we first produced dTMP-imidazolide(dTMP-Im) as a stable adduct to enable coupling to S-1Ps without requiringrepeated in situ generation.53 For optimization of dTMP-Im and S-1P couplings ,54 and reactions were monitored by 31P NMR . Of the nine catalyststested, only N-methyl imidazole-HCl (NMI-HCl) ledto formation of considerable dNDP-sugar (t = 18 h).Similar results have been observed in phosphate couplings betweenCDI-activated S-1Ps and lipids.54To convert S-1Ps to nucleotidesugars, ouplings 3A, the ml-S-1Ps todNMP-Im reactants, as yields of nucleotide sugar syntheses typicallyvary widely.19 Over a short time course,a commercial l-deoxyhexose-1P, β-l-Fuc-1P,underwent nearly maximal nucleotide sugar formation at t = 3 h with NMI-HCl added, similar to α-Glc-1P . Coupling of β-l-Rha-1P (3) afforded 1 only in thepresence of catalyst (S4). Activation of theremaining synthetic l-S-1Ps , β-l-6dTal-1P (5)) were similarly accomplished. Since uridine diphosphate(UDP) and guanosine diphosphate (GDP) are common activating groupsfor sugar metabolites,563 was also activated with appropriate NMP-Im substratesto generate additional NDP-sugar analogues for structure–functionstudies NDP-l-sugarsto define structural features necessary for RmlA regulation of bothGram-negative , Salmonella enterica LT2 (SALTY)) and Gram-positive, acid-fast (Mycobacteriumtuberculosis (Mtb)) species, which have similar allostericsite structures . We reconstituted the activity ofeach RmlA to measure the half-maximal inhibitory concentration (IC50) of each nucleotide sugar (S6). Alltested RmlA homologues were inhibited by 1 in the mid-to high-micromolar range. Notably, the Hill slopes differed for each,with Mtb and SALTY RmlA tetramers producing the highest and lowestslope values, respectively ,indicating that intact nucleotide sugars were required. Comparisonto the α-anomer 10 resulted in a >7-fold IC50 increase across species. dTDP-β-l-Man (11), which contains a hydroxyl at the C6-position, behavedsimilarly. Inversion of stereochemistry at C4 in dTDP-β-l-6dTal (2) only showed inhibition against Pa RmlA.While thymidine analogues can inhibit RmlA,30 these results highlight the importance of the sugar in forming contactswith RmlA, as seen in solved cocomplexes (l-Rha (12) and GDP-β-l-Rha (13) led to loss of inhibitory activity, exceptfor weak inhibition of Pa RmlA by 12. Hence, NDPs, especiallythose with purines, are not tolerated in the dNDP binding site.In bacterial NDP-sugar biosynthetic pathways, RmlA is feedback-inhibitedby binding of tetramer 4A.12 We de sugar 4A & S6. ructures S5. We reructures S5. We re63 we recently demonstrated that engineering of SALTY RmlA’sallosteric site leads to enhanced substrate promiscuity.35 Incorporation of the allosteric mutation E256Din a known active site mutant (Y146F) carrying the stabilizing alterationD104N also led to improvements in catalytic activity. As 1 tightly regulates all tested RmlA proteins and makes contacts withSALTY RmlA E256 . Hence,we postulated that engineered RmlA may activate l-S-1Ps withoutexperiencing feedback inhibition by dNDP-l-sugar products.Expanding on past work,mlA E256 4C, left,presence 4C, rightl-S-1Ps, we evaluated the substratescope of engineered RmlA. As expected,35 incubation of RmlA* with α-Glc-1P, α-Man-1P, or β-l-Fuc-1P and dTTP (2 mM each) led to formation of the correspondingdTDP-sugars (l-S-1Ps (3–5) or corresponding α-l-S-1Pswere incubated with dTTP under similar conditions, only β-l-Man-1P (4) was activated withRmlA proteins,63 we next tested synthetic l-S-1Ps at a higher concentration (10 mM) and found that bothβ-l-Rha-1P (3) and β-l-6dTal-1P(5) could be activated by RmlA* using dTTP, as confirmedby LC-MS analysis of reactions and relevant standards . Under these conditions, α-l-Man-1P and only trace α-l-Rha-1P could be activated(traces iii-iv). Notably, the yield of dTDP-β-l-Man(11) was ∼10× higher than that of 1 . Wild-type enzyme does notpromote activation of β-l-S-1P; even mutation of H117,which contacts the sugar on 1 . While RmlA* can activateα-Glc-1P with structurally diverse dNTPs and NTPs,354  or 3  is unique in its ability to inhibit RmlA proteinsacrossspecies at mid-micromolar concentrations. As there is an estimated20–600 μM of nucleotide sugars in bacteria, these inhibitoryconcentrations are physiologically relevant.64 Since dTDP-β-l-6dTal (2) is producedby a cellular pathway that relies on RmlA, these results suggest thatdirect product inhibition may dominate in some species,26 unless high micromolar concentrations are reached.Further, RmlA proteins from different species displayed distinct degreesof cooperativity. The observed differences between Mtb and Pa RmlAinhibition help rationalize the finding that a potent Pa RmlA allostericinhibitor showed only weak activity against Mtb,13 even though rmlA is essential in Mtb.65 Hence, these findings may aid in the futuredesign of species-selective inhibitors of RmlA.In conclusion, we found that dTDP-β-l-S-1Ps. It has been hypothesized that RmlAmight accept β-l-S-1Ps in the slightly higher-energy 4C1 conformation, which resembles the native α-Glc-1Psubstrate63 . Alternatively, β-l-S-1Ps might assume a flipped 1C4 conformationin the active site, as observed when β-l-Man-1P wasdocked into SALTY RmlA . Whilenot all substrates could be utilized, such as α-l-Rha-1Pand GTP, dTDP-β-l-sugar production was likelyenhanced due to lowered allosteric inhibition by the product as wellas expanded substrate tolerance35 in theactive site. Hence, allosteric regulation does restrict the substratescope of this model nucleotidyltransferase. These findings serve asa foundation for improved chemoenzymatic syntheses of rare sugar substratesto study bacterial glycan assembly.Using engineeredSALTY RmlA that is nearly insensitive to allostericregulation, we demonstrated the first RmlA-mediated activation ofrare β-


Moreover, the rat gastric ulcer index and mucosal section experiments further confirmed the relief of gastrointestinal irritation and the adverse reaction rate of the indobufen-treated group compared to those of the aspirin group. Furthermore, indobufen was verified to exert reversible inhibitory activity on the heme group of COX-1 and thus reversibly inhibit COX-1 activity. In general, compared with aspirin, the long-term oral administration of indobufen yields a lower risk of gastrointestinal symptoms, such as ulcers.Clinically, indobufen is widely used for the treatment of antiplatelet aggregation and anticoagulation. Prior studies have discovered that abnormal platelet function can be promptly restored to normal when the drug is stopped. Herein, through the study of the enzyme reaction kinetics, we demonstrated that the inhibitory effect of indobufen on cyclooxygenase-1 (COX-1) was reversible and non-competitive. Specifically, the cyclooxygenase inhibition experiment showed that the level of 6-keto-PGF1α in the gastric mucosa of the indobufen-treated groups was significantly higher than that of the aspirin group ( When a reversible inhibitor was added to the system, the concentration of the enzyme was constant, and the enzyme activity was proportionally inhibited, resulting in a line with a lower slope compared to that of the control group. When an irreversible inhibitor was added to the system, the inhibitor inactivated a certain amount of enzyme. If the amount of enzyme was greater than a certain amount, enzyme activity was shown, and the plotted line was parallel to the control group . IbuprofBased on the determination of indobufen as a reversible inhibitor of COX-1, we investigated the reversible inhibition type through enzymatic reaction kinetics. The kinetic characteristics of competitive inhibition are an increase in the Michaelis constant (Km), an unchanged maximum (Vm), an increase in the slope of the straight line, and the intersection of the straight lines on the vertical axis when an inhibitor is present. In contrast, the kinetics of non-competitive inhibition are characterized by a constant Michaelis constant (Km), a decreasing maximum (Vm), and an increasing slope of the straight line, which will intersect on the transverse axis when inhibitors are present . As showIndobufen is a reversible, non-competitive inhibitor of COX-1. Non-competitive inhibitors do not directly interact with the catalytic active center of the enzyme but exert the inhibitory effect by acting on other groups in the enzyme, such as prosthetic groups . As showIndobufen is effective in antiplatelet aggregate and anticoagulate. Indobufen can inhibit ADP-induced platelet aggregation, in addition to AA-induced platelet aggregation (COX-1 pathway). In addition, indobufen can effectively inhibit the production of coagulation factor IIa, and the production of coagulation factor Xa is also suppressed at high doses. Therefore, indobufen not only inhibits platelet aggregation through the COX-1 and ADP-induced platelet aggregation pathways, but also shows obvious effects on anticoagulation.The incidence of gastrointestinal irritation and adverse reactions was significantly lower in the low-dose and medium-dose indobufen groups than in the aspirin group, but higher in the high-dose indobufen group than in the aspirin group. This may be because aspirin irreversibly inhibits COX-1 in cells of the gastrointestinal tract and significantly reduces the prostaglandins of the gastric mucosa. We experimentally verified that the inhibitory effect of indobufen on COX-1 was reversible and non-competitive, and thus, the level of prostaglandins in the gastrointestinal cells was higher in the indobufen group than in the aspirin group.Non-competitive inhibitors do not interact directly with the catalytic active center of the enzyme, but exert their inhibitory effect by acting on other groups in the enzyme, such as prosthetic groups. The heme group is a critical prosthetic group in the process of COX-1-catalyzed AA generation to form PG, and the iron ions in heme play a role in electron transfer during AA oxidation. Our experiments confirm that indobufen may interact with COX-1 through the heme group.In summary, our research indicates that indobufen possesses stronger antiplatelet effects compared to aspirin. This may be attributed to the ability of indobufen to exert its antiplatelet aggregation effects through multiple pathways. Unlike aspirin, which does not exhibit significant inhibitory effects on ADP-induced platelet activation, indobufen does show such effects. This action may offer an alternative treatment for patients who demonstrate low responsiveness to aspirin in clinical practice. Previously conducted studies have demonstrated that aspirin resistance or low responsiveness increases the risk of major adverse events such as death, myocardial infarction, and cerebrovascular accidents by more than threefold compared to the risk experienced by aspirin-sensitive individuals .Furthermore, indobufen exhibits anticoagulant activity by reducing coagulation factors IIa and Xa. Investigating and elucidating the anticoagulant mechanism of indobufen are beneficial for its application in patients with nephrotic syndrome and atrial fibrillation. Patients with nephrotic syndrome present a prothrombotic state, significantly increasing the risk of venous and arterial thrombosis . Atrial Simultaneously, through the gastric ulcer index and gastric mucosal section confirmation, in clinical settings, it was demonstrated that indobufen induced less gastrointestinal irritation than aspirin. The increased propensity of aspirin to cause gastrointestinal irritation primarily arises from its irreversible inhibition of COX-1 in gastrointestinal cells and its significant reduction in gastric-mucosa-protective prostaglandin PGI2 compared to indobufen. This suggests that indobufen exhibits minimal gastrointestinal reactions and a lower bleeding risk, making it an optimized treatment option for individuals at high risk of gastrointestinal injury and bleeding. Ge Junbo et al. conducted the OPTION study, which focused on coronary heart disease patients receiving drug-eluting stent implantation and testing negative for myoglobin. Their study revealed that indobufen significantly reduced the one-year net adverse clinical event risk by 27% compared to aspirin, mainly due to the substantial reduction in bleeding risk without increasing the risk of ischemia .In patients with acute ischemic stroke, indobufen significantly reduced the rate of platelet aggregation and was comparable to aspirin in the overall clinical response rate, while exhibiting fewer gastrointestinal bleeding events, especially in patients with a history of gastrointestinal ulcers . These fOur research also confirms that the inhibitory effect of indobufen on COX-1 is reversible. This feature allows for the restoration of platelet function within 24 h after discontinuation of the medication, thereby reducing the bleeding risk during antiplatelet therapy in clinical practice. Even in cases where bleeding occurs, it is easier to control . ParticuWe believe that our research has certain clinical implications. Indobufen exhibits both antiplatelet and anticoagulant effects, while causing minimal gastrointestinal damage and carrying a lower risk of bleeding compared to aspirin. It possesses higher safety profiles and may serve as a new treatment option in clinical practice for the prevention and management of coronary heart disease and stroke.


Large mammals’ behavioral responses to humans as predators may be impacted by hunting intensity. Using a playback experiment, we found that two wild ungulates exhibited reactive (flee) rather than proactive responses (decrease habitat use) to human vocalizations at a hunting-prohibited site in North China. The wild ungulates had equal or even higher flight probabilities upon hearing vocalizations of humans than the native extant large carnivore (leopard). We also found habituation-type responses featured as progressively decreased responses to the vocalizations in both ungulates.Capreolus pygarus and wild boar Sus scrofa) to the sounds of humans, an extant predator (leopard Panthera pardus) and a control (wind), and examined their flight responses and detection probabilities when hearing different type of sounds. Both species showed higher flight probabilities when hearing human vocalization than wind, and wild boar were even more likely to flee upon hearing human vocalization than leopard roar, suggesting the behavioral response to humans can equal or exceed that of large carnivores in these two ungulates even in an area without hunting practices. Recorded sounds had no effect on detection probability of both ungulates. Additionally, with repeated exposure to sounds, regardless of treatment, roe deer were less likely to flee and wild boars were more likely to be detected, indicating a habituation-type response to sound stimuli. We speculate that the immediate flight behavior rather than shifts in habitat use of the two species reflect the low hunting/poaching pressure at our study site and suggest further examination of physiological status and demographic dynamics of the study species to understand human influence on their long-term persistence.Large mammals can perceive humans as predators and therefore adjust their behavior to achieve coexistence with humans. However, lack of research at sites with low hunting intensity limits our understanding of how behavioral responses of animals adapt to different predation risks by humans. At Heshun County in North China, where hunting has been banned for over three decades and only low-intensity poaching exists, we exposed two large ungulates (Siberian roe deer  Humans can function as an apex predator in ecosystems, killing wildlife at rates that equal or exceed that of non-human predators . Fear ofPuma concolor ; searched by “Chinese reading” in Chinese, “leopard roaring” and “wind blowing”, respectively) and edited all for consistency of amplitude and clipped them to 30 s. The sound clips were broadcasted at a consistent mean sound pressure of 70 dB , following the settings used in previous studies [We used an Automated Behavioral Response (ABR) system ,17 to me studies ,23,24.2 and 1.7 km2, respectively [We selected 36 experimental sites in mixed forests and placed one ABR  at each site c. Each Aectively . Thus, sWe used a logistic regression model with mixed effects to examine the effects of different types of recorded sounds on the immediate behavior and habitat use of the study species during the study period. For the immediate behavior, we measured the flight probability of the individual after hearing the sound. We grouped detections of the same species at the same site within 30 min into one independent detection. For each independent detection, we determined whether the ungulate fled away from the experimental site  in response to the broadcast. A social group was defined as fleeing if any individual demonstrated a flight response.We measured the habitat use of the study species by dividing the experiment period into sequential one-week periods from each site when the ABR system was operational  and assessed whether roe deer or wild boar were detected or not in each site-week . We hypothesized that repeated exposure to the recordings would alter a species’ use of the experimental site, leading to changes in their overall detection probability. We used the logistic regression for our analysis as formal occupancy model did not meet convergence due to the high naïve occupancy rate of the study species . For our dependent variable we used detection probability during each site-week, rather than the count of independent detections, as the study species were rarely detected more than once during a site-week.For both analyses , we incorporated three variables as fixed effects, including type of recorded sounds (Treatment), number of days (for flight response) or weeks (for site-week detection probability) from the beginning of each experimental period (Length), and the ID of the experimental period (Period). We used leopard call rather than wind sound as the reference level for Treatment to compare whether human effects differed from leopard effects. As the univariate additive models (GAMM) indicated no nonlinear correlation between Length and the probability of flight and detection (estimated degree of freedom = 1), we did not incorporate any higher order of terms for Length in the subsequent analysis. For each analysis, we formulated five candidate models, each with a different hypothesis , and alsWe fitted all candidate models for both flight and detection probability in the two study species separately using the package “lme4”  in the RThree ABR units was lost during the experiment , and we also excluded records when the ABR unit malfunctioned during the site-week. In total, we obtained data from 5909 camera-days in which the ABR was operational . During the entire study period we obtained 255 and 224 independent detections for roe deer and wild boar, respectively.p < 0.001), and exhibited a similar probability of flight upon hearing sounds of human and leopards  (p < 0.001) (p = 0.031), and also were more likely to flee upon hearing sounds of a leopard than the sounds of wind   . The fli< 0.001) . For wil= 0.002) .p = 0.57; p = 0.006) .For shifts in habitat use during the course of the study, no model was a better predictor of detection of roe deer than the null model. Model 2 had similar performance to the null model (△AIC = 1.87), and indicated that Length had no significant effect (= 0.006) . Model 3The experimental period and its interaction term with treatment had no significant effect on either the flight or detection probability of roe deer and wild boar , indicatDespite numerous studies focusing on human effects on ungulate spatio-temporal behaviors, as well as vigilance and foraging intensity ,8,13,43,Our study also showed that flight response to human cues was a more sensitive response than spatial avoidance of humans as we found no variation in site use between treatments for the two ungulates, which is also consistent with our previous findings that roe deer and wild boar exhibit no spatial avoidance to sites frequented by humans but avoid direct encounters with humans in our study area . Anti-prRoe deer and wild boar also exhibited increased flight probability when hearing leopard sounds as compared to hearing wind sounds. Moreover, wild boar were more likely to flee when they heard human sounds than when they heard leopard sounds, while roe deer exhibited similar flight probabilities when they heard both sounds. Since there was no evidence of different hunting pressure by poachers on these two species in our study area, we speculate that this difference was due to wild boar experiencing higher hunting pressure from humans than leopards, as wild boars are not a preferred prey of leopards compared to roe deer . AdditioAs human disturbances within ecosystems increase ,55, unde


Intertwined association between infectious gastroenteritis (IGE) and inflammatory bowel disease (IBD) has not been investigated clearly. We aimed to examine the bidirectional association between IGE and IBD.A bidirectional study using the Taiwan National Health Insurance Research Database was designed. Through a case-control design, we identified 2899 new IBD cases during 2006–2017 and matched to 28,990 non-IBD controls. We used conditional logistic regression model to estimate odds ratios (OR) of IBD for previous IGE in different exposure time-windows within 5-years before IBD diagnosis and Poisson regression model to estimate incidence rate ratio (IRR) of subsequent IGE for IBD group to non-IBD group.The mean age at the initial IBD diagnosis was 41 years. More IBD patients (21.49%) than controls (12.60%) had been exposed to IGE during > 6 months to 5 years before IBD diagnosis, the OR of IBD for IGE was 1.89 [95% confidence interval: 1.69–2.11]. Excess OR decreased as IGE exposure time before the index date increased. More IGE episodes were associated with additional increase in IBD risk . The IRR of having IGE for IBD group to non-IBD group was 2.42 before IBD diagnosis and increased to 5.74 after IBD diagnosis.These findings suggested an IGE-IBD bidirectional association. More attention is needed for physicians to develop preventive strategies and be aware of the higher risk of subsequent IGE in IBD patients.The online version contains supplementary material available at 10.1186/s40001-023-01324-y. Inflammatory bowel disease (IBD), including ulcerative colitis (UC), Crohn’s disease (CD), and unclassified IBD, results from genetic susceptibility and environmental factors that cause an imbalance in immune response and gut microbiota . IBD hasInfectious gastroenteritis (IGE), which causes dysbiosis, plays an important role in the pathogenesis of IBD . An assoClostridium difficile, Campylobacter species, E.coli, and Salmonella species have been commonly reported in the flare-up IBD in previous studies . Physicians have to submit patients’ histopathological and endoscopic (or radiologic) examination data to the NHI, and IBD patients shall then be registered in RCI after approval by the NHI’s Expert Committee. We retrospectively reviewed outpatient and inpatient medical records for each IBD case and determined the initial IBD diagnosis date which was defined as the index date in our study.Controls were selected by the incidence density sampling method and were matched to IBD cases by birth date, age, and sex. For each newly diagnosed IBD patient, we randomly selected 10 concurrent controls from NHI beneficiaries who were still alive and free of IBD on the index date of the matched case. The index date of each case was assigned as the index date of the matched controls. Using the dynamic sampling method, once a control was diagnosed with IBD later in life, this control was classified as an IBD case from the day of the initial diagnosis. To restrict confounding effects, we excluded individuals with a history of colorectal cancer before the index date in both cases and controls. The selection process of the study subjects was depicted in the flowchart in Additional file The same study subjects in the case-control study were also included in the cohort study. We classified IBD cases as the exposed group and non-IBD controls as the non-exposed group. We retrospectively and prospectively calculated the incidence of antecedent and subsequent IGE. The follow-up period started from the index date of IBD diagnosis and ended on December 31, 2017.Medical visits for IGE were indicated by relevant diagnosis codes (ICD9-CM/ICD10-CM codes: 001-009/A00-A09) in the medical records. Inpatient and outpatient medical records for both cases and controls were traced back to identify IGE episodes that occurred within five years before their index date. Since multiple IGE episodes rarely occurred in a considerably short period, we collapsed multiple medical visits with an IGE diagnosis into one episode if the time interval between any two visits was ≤ 30 days. We investigated IGE episodes in five exposure time-windows to determine the critical time that might be most relevant to IBD risk. The five time-windows were ≤ 1 year, > 6 months to 1 year, > 1–3 years, > 3–5 years, and > 6 months to 5 years before the index date.In this cohort study, we recorded subsequent IGE episodes after IBD (or index date) during the follow-up period and antecedent IGE episodes before IBD (or index date).Other characteristics that potentially contributed to the risk of IBD and were likely to change the probability of IGE were considered potential confounding factors. In addition to demographic characteristics (age and sex), we collected socioeconomic data on monthly NHI-insured salaries, median family-income levels, and urbanization levels of residence districts to reflect individual and neighborhood socioeconomic status, clinical features including the Charlson Comorbidity Index (CCI) , and hisCategorical variables were described with counts and percentages and compared using the Chi-squared test between cases and controls as well as between the UC and CD groups. Continuous variables were described with mean and standard deviation (SD) and compared using Student’s t-test. In the case–control design, we constructed univariable and multivariable conditional logistic regression analyses to estimate the crude and adjusted odds ratios (ORs) of IGE for cases compared to their matched controls in each exposure time-window. The ORs in multivariable analyses were adjusted for potential confounding factors, including monthly insured salary, median family income level, urbanization level, CCI, and history of selected immune-related diseases. Since age at index date and sex were tightly matched and excellently balanced in the case and control groups, these two variables were not adjusted. Subgroup analyses were performed for patients with CD and UC. As CCI is a composite index of overall illness severity, we also adjusted for certain specific CCI-component diseases based on statistical and clinical significance. Because of the low prevalence, the selected immune-related diseases were managed as a composite variable, demonstrating the history of having at least one immune-related disease.© 2002–2012 .For the analysis of the association of IBD with the risk of subsequent IGE, we estimated the incidence rates (IR) and 95% confidence intervals (CI) of IGE before (pre-IBD) and after (post-IBD) IBD diagnosis in IBD and non-IBD groups, assuming that IR followed a Poisson distribution. We adopted a Poisson regression model with an offset of observed person-years to estimate the adjusted incidence rate ratio (IRR) between the IBD-group and non-IBD groups for pre-IBD and post-IBD IGE, respectively. The Wald test was used to estimate the CIs of the regression coefficients and determine statistical significance. The IRRs in the multivariable analyses were also adjusted for potential confounding factors, as mentioned above in the logistic regression analyses. Data analyses in this study were performed using SAS/STAT software, Version 9.4 of the SAS system for WindowsTable Table Table We further examined the relationship between the cumulative frequency of IGE and IBD risk during the period > 6 months to 5 years before the index date. The ORs for IBD increased linearly with the frequency of antecedent IGE. Compared with people without a history of IGE, those who had experienced 1, 2, 3, 4, and 5 IGE episodes had increasing ORs of 1.64, 2.19, 2.57, 3.50, and 4.57, respectively. . Table We used the national health claims database to identify IBD cases in Taiwan, most of whom had UC (71.4%). To our best knowledge, this is the first longitudinal study to suggest a bidirectional association between IGE and IBD. IBD patients had higher odds of experiencing IGE within five years before IBD diagnosis than controls, the OR increased with an increased cumulative frequency of IGE episodes. However, IBD cases had an even higher IRR of subsequent IGE after IBD diagnosis.Clostridium difficile, Cytomegalovirus, Campylobacter spp., and other pathogenic bacteria [We found that the excess incidence and mean number of IGE episodes in IBD cases were magnified after IBD diagnosis. Compared with the controls, the IRR increased from 2.42 before IBD diagnosis to 5.74 after IBD diagnosis. In the aftermath of IBD, various factors such as gut dysbiosis, compromised repairment of the gut epithelial barrier, dysregulated immune responses, and persistent intestinal inflammation may collectively contribute to the rising incidence of IGE . Retrospbacteria , 24, 26.Our study revealed an association between previous IGE episodes and IBD development. A history of IGE occurring within 1 year before the index date was significantly associated with an increased IBD risk. This association diminished after excluding IGE within 6 months before IBD diagnosis, at which point the diagnosed IGE might be the initial presentation of IBD, according to the estimation from CHAD trial . The assSalmonella, Campylobacter, and Clostridium difficile, while specific pathogens could only be identified in quite limited IGE episodes in IBD cases [Our study also revealed that IBD risk increased with cumulative IGE episodes during > 6 months to 5 years before the index date. Compared to the control group, aOR increased from 1.64 to 4.57 in people experiencing 1 to ≥5 IGE episodes. These findings are similar to those from the study of Axelrad et al. , which rBD cases , 10, 15,BD cases .Intestinal microbiota are important and necessary for humans, as they participate in the process of intestinal homeostasis, intestinal immune development, host metabolism, and protection of the host from pathogenic infection via colonization resistance –36. The The present study had certain limitations. First, this study lacked sufficient culture data or specific pathogen diagnoses for most gastroenteritis events, which may limit our study to further associate IBD risk with specific pathogens and distinguish between IGE episodes and IBD-related symptoms. Second, there are also some limitations that may cause misclassification of IGE in our study. The IGE diagnosis was based on ICD codes in our study. Without information on the clinical presentations of each IGE episode, there may be misclassification of IGE in both the IBD group and the non-IBD group. In addition to that, the number of IGE cases may be underestimated because many people with IGE did not seek medical care. Moreover, many people who were eventually diagnosed with IBD initially presented with some digestive symptoms and were diagnosed as having IGE. Therefore, we compared the frequency of IGE between two groups in several time-windows as early as possible before IBD was diagnosed . This minimized the possibility of unequal frequency of seeking medical care for IGE between IBD cases and controls, it also largely excluded the IGE-like symptoms which were essentially early manifestations of IBD. As a result, the non-differential misclassification of IGE between the two groups could only lead to the association between IBD and IGE being underestimated and towards the null.In conclusion, our study suggested a bidirectional association between IGE and IBD. The patients with incident IBD had experienced more IGE previously and were more likely to have subsequent IGE after IBD development. These findings highlight the potential impact of IGE and alert physicians to be aware of the increased risk of subsequent IGE in IBD patients, especially in newly industrialized countries where IBD incidence has been markedly increased in the past decades.Additional file 1: Figure S1. Flow chart for study subject selection. Table S1. ICD codes of immune-related diseases.