Disrupting reconsolidation of drug memories leads to the attenuation of drug-seeking behavior in both animal models and people with addictions. Additionally, epigenetic mechanisms regulated by DNA methyltransferase (DNMT) are involved in the reconsolidation of fear and cocaine reward memory. In the present study, we investigated the role of DNMT in the reconsolidation of heroin reward memory. In the heroin self-administration model in rats, we tested the effects of DNMT inhibition during the reconsolidation process on cue-induced reinstatement, heroin-priming-induced reinstatement, and spontaneous recovery of heroin-seeking behavior. We found that the bilateral infusion of 5-azacytidine (5-AZA) inhibiting DNMT into the basolateral amygdala (BLA) immediately after heroin reward memory retrieval, but not delayed 6 h after retrieval or without retrieval, decreased subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior. These findings demonstrate that inhibiting the activity of DNMT in BLA during the reconsolidation of heroin reward memory attenuates heroin-seeking behavior, which may provide a potential strategy for the therapeutic of heroin addiction.The persistence of drug memory contributes to relapse to drug seeking. The association between repeated drug exposure and drug-related cues leads to cravings triggered by drug-paired cues. The erasure of drug memories has been considered a promising way to inhibit cravings and prevent relapse. The re-exposure to drug-related cues destabilizes well-consolidated drug memories, during which a  A major challenge in treating heroin addiction is relapse and is closely related to the persistence of drug reward memories . Drug mede novo protein synthesis is required during the reconsolidation process and the other is that reconsolidation occurred within a 6 h time window , a subregion of the amygdala, is closely implicated in learning, memory, and emotional behavior . Substane window . Therefoe window . Moreovee window . In shorDNA methyltransferase (DNMT), widely expressed in the nervous system of mammalians, is an enzyme catalyzing DNA methylation that is critical for the formation of amygdala-dependent memory and the maintenance of long-term memory . In the In this study, we investigated the effect of intra-BLA DNMT inhibition during reconsolidation on the subsequent heroin-seeking behavior of heroin reward memory. We found that a bilateral infusion of the 5-azacytidine (5-AZA) into the basolateral amygdala (BLA) to inhibit the activity of DNMT immediately after heroin reward memory retrieval, but no longer than 6 h after retrieval or with a 5-AZA infusion without retrieval, decreased subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior.The male Sprague Dawley rats weighed 280–300 g on arrival and were placed in a climate-controlled environment with a constant 22 ± 2°C temperature and 60% humidity. Food and water were freely accessible to the rats and they were under a 12-h light/dark cycle. Leading up to the surgeries, the operator handled the rats for 3 min/d for 5 day so that they would be more accustomed to the operator. The current study and all animal procedures were performed following the Guide of Hunan Province for the Care and Use of Laboratory Animals. The experiments were approved by the Local Committee on Animal Care and Use and Protection of the Hunan Normal University. The Dark phase was the time all the experiments were performed.A sodium pentobarbital anesthesia  was administered to the rats (300–320 before surgery) using catheters inserted into the right jugular vein and terminating at the opening of the right atrium . The guiAs reported by A 10-day training program was conducted to train the rats to self-administer heroin (0.05 mg/kg/infusion) in three 1-h training processes, separated by 5 min breaks. During training, the fixed-ratio 1 reinforcement schedule was implemented at the beginning of each dark cycle. There was a 40-s timeout period following every infusion. The house light was on when each session began. During the training sessions, the rats were deprived of food. To protect the rats from an overdose, the number of heroin infusions was limited to 20 per hour . The herAfter the SA training, rats received a 9-day nosepoke extinction training for 3 h per day with no illumination or any stimulus in the original training chamber (experiments 1–4). A nosepoke in either operandum would result in no consequences .After 24 h, following the last nosepoke extinction , the rats were subjected to conditioned stimulus in the original training chamber for 15 min to reactivate heroin reward memory. The retrieval conditions were similar to the heroin SA training, but a nosepoke in the active operandum caused no heroin infusions.In experiments 1 and 2, immediately after the reactivation session, the rats received bilateral infusions of the 5-AZA  intra-BLA to inhibit the activity of DNMT. The syringe pump used the 10 μl Hamilton syringes. The syringes were linked to the infusion cannula  by the polyethylene tubing, while the controls had an equal volume infusion of vehicle (0.5% DMSO). The syringes were kept at the injection site for at least 2 min after completing the injection and then slowly withdrawn. In experiment 3, the rats received an infusion of the 5-AZA or vehicle with no light/tone stimuli reactivation. Finally, in experiment 4, the rats received infused 5-AZA or the vehicle 6 h after retrieval. The rats were placed in their home cage after infusion manipulation.Daily cue extinction was performed on the rats in experiments 1, 3, and 4 for 3 h. The conditions were the same as the heroin SA session but without heroin infusions following the tone/light cue.This test was carried out on the rats 24 h after the 5-AZA or the vehicle intra-BLA infusion. The testing conditions were the same as the heroin SA session except that the active nosepoke did not have any tone-light cues, nor was it reinforced with heroin. The number of nosepokes was recorded for 1 h and the houselight was on for the whole session.Heroin  was systematically injected into the rats for 5 min before the reinstatement test. The conditions of the test were the same as the SA training except that a nosepoke in the active operandum led to the delivery of a cue (tone/light) but not the heroin. A reinstatement test was performed for 1 h and the number of active nosepokes was recorded.After withdrawal (28 days), the number of active nosepokes was recorded for 1 h in the spontaneous recovery test with the same conditions as in the reactivation.The role of immediate post-reactivation 5-AZA treatment intra-BLA on subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior.The rats were subjected to the heroin SA training for 10 days in 1 h sessions, 3 times per day. They were then trained for 9 days of daily nosepoke extinction in the original chamber. After 24 h following the last nosepoke extinction, the rats were reactivated for 15 min with heroin reward cues in the training context. After reactivation, the rats were divided into two groups: (1) infusing 5-AZA intra-BLA bilaterally 1 μg in 0.5 μl/side (5-AZA group); (2) infusing vehicle intra-BLA bilaterally 0.5 μl/side (vehicle group). The rats received a bilateral infusion of either 5-AZA or vehicle intra-BLA immediately after the reactivation. After 24 h, we tested the heroin-seeking behavior of cue-induced reinstatement in rats. A priming-induced reinstatement test was conducted 2 days after the cue extinction session see .The long-term role of immediate post-reactivation 5-AZA treatment intra-BLA on cue-induced reinstatement and spontaneous recovery.For the heroin self-administration session and nosepoke extinction session, the conditions and tone/light reactivation in Experiment 2 were the same as in Experiment 1. The rats were subjected to the 5-AZA treatment of 1 μg in 0.5 μl/side and the controls received the vehicle immediately after a 15-min tone/light reactivation. The cue-induced reinstatement test was performed 24 h later to assess the heroin-seeking behavior. After 28 days of withdrawal, the spontaneous recovery was tested .The role of immediate post-reactivation 5-AZA infusion intra-BLA on subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior without reactivation.During Experiment 3, the experimental protocol was similar to Experiment 1, except that the rats were subjected to the infusion of 5-AZA or vehicle intra-BLA bilaterally without reactivation see .The role of delayed 5-AZA treatment intra-BLA post-reactivation on subsequent cue- and heroin-priming-induced reinstatement of heroin-seeking behavior.During Experiment 4, the experimental protocol was the same as in Experiment 1 except that the rats were subjected to the infusion of 5-AZA or vehicle intra-BLA bilaterally 6 h after the 15-min retrieval see .post hoc tests to analyze the two-way ANOVAs for specific pair-wise comparisons and examine any significant main effects or interactions .The results were reported as mean ± SEM and analyzed using the two-way/repeated measures ANOVAs in GraphPad, v.9.0. Each experiment had a between-subjects factor for the infusion treatment (5-AZA vs. vehicle) and a within-subjects factor for the test  (see section “Results”). We used Tukey’s Immediate post-reactivation 5-AZA treatment intra-BLA reduced subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior.N = 10) and the rats in the vehicle group (N = 10), indicated by the total number of heroin infusions , a main effect of test day , and a significant treatment × test day interaction  during the cue-induced reinstatement test. The post hoc analysis revealed that the number of active nose pokes of the 5-AZA group was significantly decreased compared with the vehicle group during the cue-induced reinstatement test (p < 0.01)  = 4.570, p = 0.0465], a main effect of test day , and a significant treatment × nosepokes interaction  during the heroin reinstatement test. The post hoc analysis showed that the number of active nosepokes in the 5-AZA group was significantly lower than the vehicle group during the heroin-induced reinstatement test (p < 0.01) (We found that the active nosepokes of the 5-AZA group significantly differed from the vehicle group in both the cue-induced and heroin-priming-induced reinstatement tests. A two-way ANOVA with the treatment (5-AZA vs. vehicle) as the between-subjects factor and test day (last extinction day vs. cue reinstatement day) as the within-subjects factor revealed a main effect of treatment [These findings suggest that inhibiting the activity of DNMT with infusions of 5-AZA in the BLA immediately following the heroin cue retrieval significantly reduced cue-induced and heroin-priming-induced heroin-seeking behavior.Immediate post-reactivation 5-AZA treatment intra-BLA reduced subsequent cue-induced heroin seeking and the spontaneous recovery of heroin-seeking behavior.N = 8) or the vehicle (N = 8) [main effect of acquisition date: F = 22.31, p < 0.0001; infusion treatment: F = 2.671, p = 0.1245; acquisition date × infusion treatment: F = 0.8167, p = 0.6018; F = 23.59, p < 0.0001; infusion treatment: F = 0.08584, p = 0.7738; extinction date × infusion treatment: F = 0.4240, p = 0.9045; In experiment 2, we aimed to test the effect of immediate post-reactivation 5-AZA treatment intra-BLA on cue-induced heroin seeking reinstatement and the long-term effect on heroin reward memory in two groups of rats . No diffF = 19.98, p = 0.0005; infusion treatment: F = 6.843, p = 0.0203; test × infusion treatment: F = 7.339, p = 0.0170; post hoc analysis showed that active nosepokes were significantly reduced in the 5-AZA group compared with the vehicle group in the cue-induced reinstatement test (p < 0.01)  = 63.00, p < 0.0001; infusion treatment: F = 8.866, p = 0.0100; test × infusion treatment: F = 16.31, p = 0.0012; post hoc analysis revealed that drug-seeking in the 5-AZA group was significantly reduced compared to the vehicle group in the spontaneous recovery test (p < 0.01)  and vehicle group (N = 8) of rats. Following the acquisition session of heroin self-administration and extinction session which is the same as experiments 1, rats were infused with 5-AZA or the vehicle intra-BLA immediately after exposing to the training chamber for 15 min with no cue exposure  = 25.84, p < 0.0001; infusion treatment: F = 0.7240, p = 0.4091; acquisition date × infusion treatment: F = 0.6614, p = 0.7423; F = 37.20, p < 0.0001; infusion treatment: F = 0.2503, p = 0.6246; extinction date × infusion treatment: F = 0.2094, p = 0.9887; In experiment 3, we examined whether the effect of DNMT in the BLA on the reconsolidation of heroin reward memory depended on retrieval by using the 5-AZA group  = 43.38, p < 0.0001; infusion treatment: F = 0.2478, p = 0.6264; test × infusion treatment: F = 0.08683, p = 0.7726; F = 71.04, p < 0.0001; infusion treatment: F = 0.04099, p = 0.8425; test × infusion treatment: F = 0.002420, p = 0.9615; In cue-induced reinstatement test, we did not find a difference in active nosepokes between the groups [main effect of test: Thus, these results indicated that the 5-AZA treatment intra-BLA without reactivation had no effects on the subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior, indicating that the effect of DNMT in the BLA on the reconsolidation of heroin reward memory was reactivation-dependent.Delayed 5-AZA treatment intra-BLA following reactivation had no effect on subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior.N = 9) or vehicle (N = 9)  = 35.09, p < 0.0001; infusion treatment: F = 0.4635, p = 0.5057; acquisition date × infusion treatment: F = 0.5624, p = 0.8260; F = 38.71, p < 0.0001; infusion treatment: F = 0.03292, p = 0.8583; extinction date × infusion treatment: F = 0.6184, p = 0.7612; Finally, in experiment 4, we investigated whether the role of 5-AZA treatment intra-BLA in the reconsolidation of heroin reward memory had a time window by using two groups of rats infused with either 5-AZA ( (N = 9) . In lineF = 48.29, p < 0.0001; infusion treatment: F = 0.08935, p = 0.7689; extinction date × infusion treatment: F = 0.01919, p = 0.8915; F = 72.48, p < 0.0001; infusion treatment: F = 0.1839, p = 0.6737; test × infusion treatment: F = 0.2131, p = 0.6506; However, intra-BLA 5-AZA treatment 6 h after the reactivation did not affect the subsequent cue-induced reinstatement of heroin seeking behavior, indicated by the number of active nosepokes had no difference between groups [maineffect of test: These experiments indicated that the role of 5-AZA on heroin-seeking behavior was time-specific, and inhibiting the activity of DNMT should be within 6 h after reactivation to suppress the heroin-seeking behavior.Our study examined the role of DNA methyltransferase (DNMT) in the BLA on the reconsolidation of heroin reward memory. The main findings are as follows: (1) DNMT inhibition in the BLA immediately after light/tone cue reactivation reduces subsequent cue-induced and heroin-priming-induced reinstatement of heroin-seeking behavior; (2) a 5-AZA infusion in the BLA without reactivation has no effect on the subsequent cue-induced reinstatement of heroin-seeking behavior; (3) the inhibitory effect of a 5-AZA infusion in the BLA immediately after the reactivation session on heroin-seeking behavior lasts at least 28 days. These findings indicate that the activity of DNMT in the BLA is required for the reconsolidation of the heroin reward memory and inhibiting the DNMT in the BLA attenuates heroin-seeking behavior by disrupting the reconsolidation of heroin reward memory.A relapse caused by persistent heroin reward memory is a major challenge to the therapy of heroin addiction . Studiesde novo protein synthesis (It has been reported that the memory reconsolidation process involves DNA transcription and ynthesis . DNA traynthesis . DNMT, aynthesis , suppresynthesis . Thus, Dynthesis . Howeverynthesis . One of ynthesis . Moreoveynthesis . This maynthesis . Our preynthesis . Thus, tynthesis . Howeverynthesis , other pynthesis .In conclusion, our study demonstrated that DNA methylation regulates the reconsolidation of heroin reward memory in the BLA. Our study also highlights the significant effect of epigenetic regulation, specifically DNA methylation, in the reconsolidation of heroin reward memory. Our findings provide theoretical support for the molecular mechanisms of reconsolidation of drug memory in the BLA and the development of potential therapies for heroin addiction.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Local Committee on Animal Care and Use and Protection of the Hunan Normal University.SQ, CY, and YL: conceptualization. CS and SH: data curation. SQ and CS: writing of original draft preparation. CS and CY: review and editing. YL: funding acquisition. All authors contributed to the article and approved the submitted version.


Identifying opportunities to safely reduce antibiotic prescribing is necessary for prescribers and antibiotic stewardship teams to minimize unwarranted antibiotic use. We aimed to quantify excess antibiotic use in General Surgery.We retrospectively audited the antibiotic prescribing for patients discharged from the General Surgery specialty in an acute hospital in the south-west of England between 01/08/21 and 31/08/21 using an audit tool developed by Public Health England. The appropriateness of prescribing was determined for each patient at three antibiotic decision time-points: at initiation, the pre-72h antibiotic review, and treatment duration. Two infection specialists and a general surgeon reviewed each patient. Indication and excess days of therapy (DOTs) were calculated at each decision time-point and expressed as a proportion of total DOTs.Eighty-six patients were prescribed 1162 DOTs; 192 (16.5%) excess DOTs were prescribed in 38 patients (44%), with zero excess days identified in the remaining 48 patients (56%). Seventy-five of 192 (39%) excess DOTs occurred at initiation; 55/192 (29%) after the pre-72hour antibiotic review; and 62/192 (32%) due to protracted antibiotic courses. There was concordance between the general surgeon and infection specialist for the majority of apportioned excess DOTs. However, the surgeon apportioned fewer excess DOTs 160/1162 (13.8%). Overall IV antibiotics accounted for 53.4% of total DOTs. Seventy-two of 86 (83.7%) patients received 620 intravenous DOTs; of these, 79 (12.7%) IV DOTS were unnecessary.We have identified excess antibiotic prescribing in General surgery with comparable excess DOTs at all three time-points.


Immunotherapy plus chemotherapy has been approved for the first-line treatment of extensive-stage small cell lung cancer . Recently, the 2023 version of the National Comprehensive Cancer Network Guidelines recommended immunotherapy plus chemotherapy as the neoadjuvant regimen in patients with resectable non-small cell lung cancer (NSCLC). However, it is still unclear whether the combination regimen of immunotherapy plus chemotherapy is also beneficial for SCLC in the neoadjuvant context. Here, we report the case of a patient with stage IIIB SCLC who showed long-term survival and good tolerance to the neoadjuvant chemoimmunotherapy consisting of tislelizumab  plus etoposide-carboplatin. The patient achieved pathological complete response after receiving two cycles of neoadjuvant tislelizumab and chemotherapy followed by surgery. Two courses of post-operative tislelizumab and etoposide-carboplatin treatment were performed. The patient has survived for more than 23 months with no recurrence or metastases after neoadjuvant therapy. Multiplexed immunofluorescence and immunohistochemistry staining showed that the post-treatment specimens had remarkable immune cells infiltration, including CD3+ T cells, CD4+ T cells, and CD8+ T cells, which contrasted with very low levels of these cells in the pre-treatment samples. This study is, to the best of our knowledge, the first attempt to present the neoadjuvant chemoimmunotherapy of tislelizumab in combination with etoposide-carboplatin in SCLC. Our study suggested that neoadjuvant tislelizumab plus chemotherapy may facilitate radical resection and benefit patients with locally advanced (stage IIB-IIIC) SCLC. It is generally acknowledged that surgery is limited to very early stages (I–IIA) in small cell lung cancer (SCLC). The standard therapies for locally advanced (stage IIB-IIIC) SCLC have shown limited benefits. Therefore, improvements in treatment regimens are still needed. Neoadjuvant treatment showed promise with respect to the R0 resection  rate and pathological complete response  rate, which are related to clinical benefits –4. HowevTislelizumab is a monoclonal antibody with the high affinity for PD-1. A phase II study revealed potent antitumor effects of tislelizumab plus platinum-etoposide in extensive-stage small cell lung cancer , with a median PFS of 6.9 months and 1-year overall survival rate of 76% . Here, we report a patient with stage IIIB (cT3N2M0) SCLC who achieved pCR after receiving neoadjuvant tislelizumab plus etoposide-carboplatin.In January 2021, a 46-year-old Chinese man was admitted to our hospital for cough. The patient was in good health and had no history of smoking, family history of hereditary disease, or tumor. However, the chest computed tomography (CT) and positron emission tomography (PET)-CT scan presented a mass of 5.4 cm × 4.5 cm, centrally located in the left lower lobe, with hilar and mediastinal lymph nodes metastasis , carboplatin , and tislelizumab  every 3 weeks. After two cycles of neoadjuvant treatment, a repeated CT scan manifested the mass obviously shrank to a nodule with a diameter of 0.8 cm (The treatment of the patient was discussed by the multi-disciplinary team (MDT). The tumor was considered potentially resectable; thus, neoadjuvant therapy followed by surgery might be an ideal option. Our patient chose the neoadjuvant regimen of etoposide  in patients with ES-SCLC  in combination with chemotherapy is considered the standard of care in the first-line setting in SCLC. However, in January 2021, the patient finally selected tislelizumab (anti–PD-1 antibody), mainly due to financial distress. Of note, serplulimab, an anti–PD-1 antibody, combined with chemotherapy have recently come under intense focus after a phase 3 study showed improvement in overall survival compared with chemotherapy alone  SCLC without impeding the feasibility of surgery or increasing the incidence of adverse events.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participant was reviewed and approved by The Clinical Research Ethics Committee of West China Hospital Sichuan University. The patient/participant provided his written informed consent to participate in this study. Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.NZ: Conceptualization, Writing- Original draft preparation. YC: Methodology, Software. QH: Investigation. GC, HG: Visualization, Investigation. LJ: histopathological findings. YL, HL: Software, Validation. YZ: Writing- Reviewing and Editing, Supervision. All listed authors participated meaningfully in the study and that they have seen and approved the final manuscript.


Helicobacter pylori (Hp). However, Hp is easy to resistant, making Met unable to be widely used. How to overcome Hp’s Met resistance is still an issue. In this study, Met was used as the primary raw material with linolenic acid to prepare a novel compound-linolenic acid-metronidazole (Lla-Met). The MIC, minimum bactericidal concentration (MBC), colonization amount of Hp in gastric mucosa, etc., were evaluated, respectively. Lla-Met was successfully prepared by the detection of nuclear magnetic resonance, etc., and its MIC and MBC to Hp were 2~4 μg/mL, 8~16 μg/mL. Moreover, in vivo experiments, Lla-Met significantly reduced the colonization of drug-resistant Hp in gastric mucosa. In the toxicity test, Lla-Met inhibited rate to GES-1 and BGC823 cells were 15% at 128 μg/mL; the mice were administered 10 times treatment Lla-Met treatment (240 mg/kg), have no difference significant injuries were found in their stomach, liver, spleen, kidney, and weight. In addition, Hp G27 continued for 18 days in vitro with sub-Lla-Met concentration, G27 did not show drug resistance to Lla-Met; Lla-Met did not exert an effect on non-Hp species with 128 μg/mL; Compared with a neutral environment, when the acid concentration is 3.0, Lla-Met is not decomposed and has better stability. Conclusion: Lla-Met, a newly prepared compound, has relatively well antibacterial of Met-resistant and sensitive Hp, with a capability of overcoming the metronidazole resistance of Hp.Metronidazole (Met) is the first choice for treating  Helicobacter pylori (Hp), a Gram-negative microaerophilic pathogen that colonizes the epithelial cells of the gastric mucosa  lymphoma  remains the treatment of choice for Hp in regions and countries where the clarithromycin resistance rate is less than 15% . The HE and TUNEL stainings of mice gastric mucosal tissue showed that the degree of inflammatory cells and inflammatory factors infiltration in the OPZ+ Lla-Met treatment group was significantly lower than that the standard triple, OPZ+ Lla +Met; implying that OPZ+ Lla-Me could not only significantly reduce the amount of Hp colonization in the gastric mucosae, but alleviate the inflammatory response in the stomach of mice  concentration to observe whether Hp was prone to Lla-Met resistance. As a result, the MIC multiple of Lla-Met did not change when exposed to 0.25 μg/mL Lla-Met for serial passaging for 18 days; metronidazole was induced under the same conditions, with its MIC multiple increases 2-fold on the 6th day, and increases by 16 times on the 15th day compared with the first day. This result, however, noted that Hp was susceptible to resistance under condition of prolonged exposure to metronidazole, while it was less susceptible to Lla-Met, as shown in To evaluate Hp to antibiotics increasing year-on-year, the effectiveness of Hp treatment decreases accordingly . On the other hand, in vivo toxicity experiments in mice showed that after the administration of high-dose Lla-Met, the weights, livers, spleens, and kidneys of the mice did not change significantly, indicating that Lla-Met exerted a better antibacterial effect in vivo and high biosafety.After the acute gastritis infection of mice was treated with Lla-Met, the amount of Hp both in vivo and in vitro, a strong specificity, and high safety. Under acidic conditions, it is more stable and has a significantly better antibacterial effect and can overcome the shortcomings of metronidazole, such as easily making Hp with drug resistance, making it a new, clinically ideal, anti-Hp preparation.In summary, Lla-Metcompound we prepared has a good antibacterial effect on Met-resistant Hp 16sRNA specific primers, CCK8 kit ; UV spectrophotometer 90 (UH5300), multiscan spectrum (Synergy H1), three-gas incubator (Huaxi YCP-100S), three-gas shaker, GES-1 (RRID: CVCL_EQ22), and BGC-823  were used. .Linolenic acid (batch number: 463-40-1) was purchased from Aladdin Biochemical Technology Co., Metronidazole , dicyclohexyl carbodiimide condensing agent , diethylamine, 4-dimethylamino pyridine catalyst , absolute ethanol (AR99%), methanol (AR99%), dichloromethane , concentrated hydrochloric acid (AR99%), glycerin was purchased from Maclean Biochemical Technology Co., Ltd. Amoxicillin , Metronidazole , levofloxacin , Omeprazole , Brain Heart infusion , Columbia blood agar base  were purchased from the United Kingdom, Oxoid biological company, nutrient broth medium , nutrient agar , Sabouraud medium, soy peptone, beef extract, calf serum, Fetal bovine serum , PBS Buffer , RPMR medium1640 medium , Gram stain, bacterial genomic DNA extraction kit , Hp strains: Hp standard strains 26695, G27, and Gastric mucosal colonization strain Hp159 in mice with multiple drug resistance to levofloxacin, clarithromycin, ; 6 clinically Met-resistant strains ; all Hp strains were stored in a brain heart infusion containing 30% glycerin at −80°C.Hp strains: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Candida albicans, Cryptococcus neoformans, Saccharomyces cerevisiae, Bacillus subtilis, Stenotrophomonas maltophilia, Lactobacillus acidophilus, Acetobacter pasteurianus, Candida tropicalis, Lactobacillus Curvatus, Morganella morganii, Campylobacter jejuni, Bacteroides Fragilis, Bifidobacterium longum, Enterobacter hormaechei, Staphylococcus Haemolyticus (purchased from Guangdong Microorganism Conservation Center).Non-Hp were inoculated on a Columbia agar plate containing 10% calf serum, the precipitate was evenly coated with the inoculation loop and placed in a microaerophilic  at 37°C for 72 h, after which small colorless, transparent, small bacterial colonies were observed with the naked eye.Before each experiment, the aforementioned strains were removed from the freezer where they were stored at −80°C, equilibrated to room temperature, and centrifuged to remove the preservation solution. After the remaining Hp bacterias were cultivated in the broth medium and the special medium. Fungi were cultivated in the Sabouraud medium in an incubator at 37°C for 48 h, and allowed steady passage, they were then inoculated on the corresponding medium and cultured in an incubator at 37°C for 24 h.After two stable passages, an appropriate number of colonies were scraped from the agar plate, inoculated in a fresh brain heart infusion (BHI) medium containing 10% calf serum, and shaken at 170 rpm at 37°C under microaerobic conditions for 72 h. On the other hand, other non-(i) Preparation of Lla-Met compound. 1 mM linolenic acid, 5 mL dichloromethane, and DCC condensing agent that had been stirred at room temperature for a certain time, were dosed with 1 mM metronidazole, 5 mg DMAP, and 2 mL diethylamine, stirred overnight, after which a white precipitate was observed in the reaction solution. Thereafter, this solution was analyzed through thin-layer chromatography (TLC). After the linolenic acid was observed to have disappeared completely, the solution was filtered through diatomaceous earth. With the filtrate spin-dried through column chromatography, the target product Lla-Met was collected, which was a compound that was a yellowish oily substance and kept at −20°C away from light. Eventually, after Lla-Met was obtained, the carbon and hydrogen elements therein were detected by the magnetic resonance imaging, the mass spectrum, and the infrared spectroscopy, to infer its purity .(ii) Detection of MIC of Lla-Met by microdilution. Microdilution was used to detect the MIC of linolenic acid-metronidazole compound to standard Hp strains G27, Hp 26695 and clinically drug-resistant strains. With reference to the previous experimental method (Hp in the logarithmic phase were diluted 10-fold in a cell suspension with OD600 nm = 0.3 (about 1 × 108 CFU/mL), which were inoculated on a 96-microwell plate, so that the final concentration of G27 was 1 × 106 CFU/mL, and shaken at 170 rpm at 37°C under microaerobic conditions for 72 h. Metronidazole and linolenic acid were used as positive controls with PBS as a negative control. The MIC was the lowest drug concentration that inhibited 90% bacterial growth. Three biological repeats were performed for each experiment. However, other non-Hp strains were used to prepare a 96-microwell plate in the same way .l method , correspHp through spread plate method.(iii) Detection of MBC of Lla-Met to Met-resistant  Referenced to the previous experimental method  for serial dilution (1:1 to 1:1000), and the 100 μL diluted solution was coated evenly over the Columbia medium containing 10% serum and cultured at a microaerophilic incubator at 37°C for 72 h. Eventually the number of single bacterial colonies on the plate was counted, and MBC of Lla-Met was estimated by drawing a time-colony number (log 10) curve. MBC is the minimum drug concentration required to kill 99.9% of Hp (log 103) within a certain time; PBS and metronidazole were used as controls. Three biological repeats were performed for each experiment.l method . Serial in vivo.(iv) Construction of models of mice with acute gastritis and evaluation of therapeutic effects of Lla-Met  SPF C57BL/6 mice were purchased from Changsha Tianqin Biological Co., Ltd., as approved by the Institutional Animal Care and Use Committee of You jiang Medical College and taken as experimental animals in accordance with animal welfare standards and institutional guidelines, under mice ethics review number: 2019112501. The tested Hp strain was Gastric mucosal colonization strain Hp159 in mice. The mice modeling was established specifically with reference to the experimental methods of Thamphiwatana, Xie et al. (600 nm = 3 (1 × 109) CFU/mL, which was then given to each mouse in a dose of 0.5 mL through oral-nasal feeding, then fasted and were deprived of water for 4 h. The gavage was given to the mice once every other day on five consecutive occasions. At the end, three mice were randomly sacrificed and their gastric tissue taken for identification; identification followed the protocol described above.e et al. , with apAfter the modeling was confirmed as having been established, the mice were randomly divided into four treatment groups: PBS, metronidazole + amoxicillin + Omeprazole , linolenic acid-metronidazole compound + Omeprazole (OPZ + Lla-Met), and metronidazole + linolenic acid + Omeprazole group (Met + Lla + OPZ), with uninfected mice serving as negative controls. The dosage was 138.2 mg/kg omeprazole, 28.5 mg/kg amoxicillin, 30 mg/kg metronidazole + Linolenic acid and 24 mg/kg linolenic acid metronidazole compound. All treatment groups were given a Omeprazole (PPI) 30 min before the administration, after which they were deprived of food and water for 4 h and were administered treatment once a day for three consecutive days. 2 weeks after the last gavage, the mice were weighed, and the mean average body weight calculated. Then mice were fasting for 12 h, sacrificed through cervical dislocation and dissected to acquire stomach tissue. One part of the stomach tissue was stored in formalin while the other part was weighed, homogenized, diluted, coated and inoculated on the Columbia medium containing 10% serum. The medium was placed at a microaerophilic incubator at 37°C for 72 h, in which the colony morphology was observed and acicular transparent colonies were counted using the method described in Section 1.3.4 and identified through gram staining and 16S following the protocol described in Section 1.2.in vivo and in vitro.(v) Evaluation of Lla-Met toxicity (a) Evaluation of the cytotoxicity of Lla-Met compound in vitro. The CCK8 assay was employed to evaluate the Cytotoxicity of Lla-Met on immortalized human gastric epithelial cell lines (GES-1) and human gastric cancer cells (BGC823). GES-1 and BGC823 cells were cultured in a 1640 medium containing 10% fetal bovine serum at 37°C in an incubator with 5% CO2. When cell confluence reached 90%, 1 × 105 cells were laid on a 96-microwell plate and cultured overnight, with different concentrations of Lla-Met acting thereon for 24 h. These cells, however, were treated with CCK8 reagent, and finally detected by the multiscan spectrum at 450 nm to measure the absorbance, with each concentration of cells placed in three holes. Three biological repeats were performed for the each experiment. PBS was used as a negative control. Cell viability was expressed through cell inhibition rate. The formula for calculating cell inhibition rate is as follows: drug treatment group/PBS group × 100%.(b) Evaluation of the cytotoxicity of Lla-Met compound in vivo. To evaluate the cytotoxicity of Lla-Met compound, the uninfected C57BL/6 mice were divided into two groups: the drug and PBS group. The administration group was given 10 times the treatment dose for three consecutive days, once a day. On the other hand, PBS was used as a control group, with the same dose and times of administration as the other group. The mice were weighed the day before administration of treatment and were weighed after administration for 7 days. Three days after withdrawal of treatment, mice in the administration group were weighed, subjected to cervical dislocation, with tissue taken from liver, spleen, and kidney for hematoxylin and eosin (HE) staining.Hp’s drug resistance to Lla-Met compound.(vi) Detection of  Specifically, with reference to the previous experimental method . Thereafter, the given cell suspension was shaken on a shaker for 3 days, after which the optical density value of the cells in each well was detected at OD600 nm with a microplate reader. At the same time, the MIC of the cell culture was detected and recorded with the induction lasting for 18 days. When there were changes in MIC, the concentration of Lla-Met was adjusted over time. Metronidazole was used as a control.l method , G27 was(vii) Stability testing of Lla-Met in an acidic environment.(a) Analysis of the stability of Lla-Met through thin-layer chromatography (TLC). Absolute ethyl alcohol was used to dissolve Lla-Met; solutions of HCl/KCl buffer (0.2 mM) at pH 3.0 and 7.0 were prepared and the above prepared Lla-Met was added to pH 3.0 and 7.0 and placed at room temperature. DCM and methanol were prepared in the ratio of 20:1 (vol/vol) and saturated at room temperature for 2 min. A horizontal line was lightly drawn 10 mm from the bottom of a TLC plate and a capillary dropper was employed to absorb Lla-Met solutions with pH 3.0 and 7.0 and dot them in different positions on the horizontal line. The TLC plate was placed in the developing tank and removed after the developing solvent advanced on the TLC plate and reached a certain distance. The spots formed by the same substances were found to be at the same level after being observed on the plate under a 254 nm UV lamp.(b) Analysis of the stability and bactericidal activity of Lla-Met through plate coating method. Fresh BHI containing 10% serum was prepared into pH 3.0, pH 4.5 and pH 6.0 bacterial culture medium with sterile HCl/KCl (0.2 mM) and NaOH/KCl (0.2 mM) buffer solution. Thereafter, nutrient solutions were distributed across a 96-microwell plate. Meanwhile, serial dilutions of Lla-Met compound of different concentrations were prepared  and the BHI was adopted to dilute the G27 bacteria in the logarithmic phase. The rest steps were referenced the above PBS and metronidazole were used as controls. Three biological repeats were performed for each experiment. MBC is the minimum drug concentration required to kill 99.9% of Hp (log 103) within a certain time.t test; the comparison between multiple sample means was carried out by the single factor variance analysis, with P < 0.05 considered to represent a statistically significant difference.The statistical analysis was performed using the SPSS 17.0 statistical software. The measurement data were expressed as “mean ± standard deviation.” The comparison between two sample means was conducted by the


We have previously demonstrated significant differences in the metabolism of L-methionine (L-Met) and its precursor DL-2-hydroxy-4-methylthiobutyric acid (DL-HMTBA) in IPEC-J2 cells. When DL–HMTBA is added to the diet, intracellular methionine (Met) sources also contain the natural form of L-Met. Then, what is the effect on Met metabolism when these two Met sources exist simultaneously? Moreover, the effects of metabolic differences on cell function remain unclear. In this study, it was found that when the proportion of L-Met to DL–HMTBA was ≤ 40%:60%, Met transmethylation was promoted and when the proportion of L-Met to DL-HMTBA was ≤ 85%:15%, Met trans-sulfuration and regeneration were improved. In addition, DL-HMTBA improved the cell barrier function when the ratio of L-Met to DL-HMTBA was ≤ 40%:60%. This finding may be due to the decrease in the proportion of S-adenosylmethionine to S-adenosylhomocysteine and mRNA N6-methyladenosine (m6A) levels, which increase the mRNA stability and protein expression of tight junction zona occludens-1. To sum up, the effects of L-Met and DL–HMTBA on Met metabolism, especially transmethylation, suggest that DL–HMTBA has the potential to influence the intestinal barrier function of animals through epigenetic processes.Methionine is a substrate for protein synthesis and participates in many other biological events  The intestinal tract is the leading site for digestion and absorption of nutrients, and it is also a crucial defense barrier for the body. The intestinal epithelial layer forms a physical barrier, and its integrity and barrier properties are mainly regulated by tight junctions, which are the critical junction complex at the cell apex and primarily of zona occludens-1 (ZO-1), claudin, occludin, and other proteins . Clinicavia its metabolism. Met and its metabolites taurine (Tau) and glutathione (GSH) can exert antioxidant efficacy (S-Adenosylmethionine (SAM), a metabolite of Met, is a vital methyl donor and participates in DNA, RNA, and proteins methylation (S-adenosylhomocysteine (SAH) and the proportion of SAM to SAH in IPEC-J2 cells , 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) were supplied by Sigma–Aldrich . Fetal bovine serum, antibiotics (penicillin and streptomycin), and Dulbecco's modified Eagle's medium mixed with Ham's F-12 were provided by Gibco . DL–HMTBA was offered by Macklin .Methionine, 5-methylthioadenosine (MTA), cystathionine (Cysta), cysteine (Cys), SAM, homocysteine (Hcy), SAH, GSH, 5-methyltetrahydrofolate (5-MTHF), vitamin B6 (VB6), vitamin B9 (VB9), vitamin B6 (VB12), acetonitrile, formic acid, methanol, ascorbic acid, ammonium acetate, tris-(2-carboxyethyl) phosphine, hydrogen peroxide  and antibiotics (penicillin and streptomycin) at 37°C in a humidified incubator with 5% CO2. Cells were cultured without fetal bovine serum for 18 h and then without Met for 6 h, at last, different ratios of L-Met and DL–HMTBA were given for 2 h.IPEC-J2 cell line was a kind gift from Dr. He Qigai . Cells were used between passages 70 and 90 and the cell inoculating density was 5,000/well or 3 × 102O2 for 24 h or different ratios of L-Met and DL–HMTBA for 2 h and then 0.8 mM H2O2 for 24 h. Then, 10 μl MTT was added to each well, and the cells continued to incubate for 4 h. The supernatant was discarded, and formazan crystals were resuspended with 150 μl dimethyl sulfoxide. Finally, absorbance was read at 490 nm by a spectrophotometer. Results were presented as a percentage of surviving cells compared with control cells.Cells  were seeded in 96-well microplates overnight, then dealt with different concentrations of H5/well) were seeded in the upper room of Transwell plates , then 2.6 ml medium was added into the basal chamber. Millicell-ERS instrument  was used to measure TEER values. The measured values in the blank wells of uninoculated cells were background TEER. The final TEER values were corrected for background and displayed as Ω cm2.Cells . TaqMan Reverse Transcription Kit  was used to transcribe 2 μg RNA into cDNA. Bio-Rad CFX Connect™ Real-Time PCR Detection System  was used to measure the relative mRNA expression. Results were calculated by the 2 primers  were syn6 cells.Impacts of different ratios of L-Met and DL–HMTBA on Met metabolites and coenzyme was measured by LC–MS/MS, the specific experimental steps were shown as described previously . Contentg at 4°C, and supernatants were detected for the total protein contents. A total of 30 μg proteins were treated with 10% SDS-PAGE and then transferred to nitrocellulose membranes. They were then incubated with 5% non-fat milk for 2 h and incubated with the primary antibodies: anti-ZO-1 , anti-FTO , anti-YTHDF2 , anti-METTL3 , and anti-β-actin . After washing three times, blots were incubated with the secondary antibody : anti-rabbit or anti-mouse IgG for another 2 h. Signals were detected with chemiluminescence. Band intensities were measured by Image J .Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors  for 30 min, the products were centrifuged for 15 min at 12,000 × After the test, the culture solution was discarded, the cells in the Transwell chamber were washed with D-Hank's solution preheated at 37°Cthen 1.5 ml of fluorescent yellow solution (100 μg/ml) was added into the upper room. Next, the Transwell plates were placed in a cell culture incubator for 2 h. After treatment, the liquid in the lower chamber was collected for testing. The absorbance was measured with a fluorescence spectrophotometer, the excitation and emission wavelength of which were 427 and 536 nm, respectively. The ratio of fluorescent yellow contents in the lower and upper room was the fluorescent yellow flux rate (%).g, 4°C, and then the supernatants were collected to determine total protein content. Concentrations of H2S in the remaining supernatant were determined using ELISA Kit . Briefly, samples were incubated in the microplates, which were pre-coated with monoclonal antibodies, then a specific enzyme-linked antibody for H2S was added. After washing for 5 times, the substrate solution was added to develop color. Results were measured using a spectrophotometer at a 450 nm wavelength.Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors  for 30 min at 4°C, the products were centrifuged for 15 min at 12,000 × 5C) and results were measured using a spectrophotometer at a 450 nm wavelength. Results were displayed as the percentage of m5C (%) in the total cell DNA.Total DNAs from IPEC-J2 cells were extracted by using a DNA Extraction kit . MethylFlash Methylated DNA Quantification Kit  was used for the detection of DNA methylation. Briefly, genomic DNA was denatured and then incubated with capture and detection antibodies for 5-methylcytosine  was applied to detect the specific m6A modification levels of the target genes, the specific experimental steps were shown in the previous study   with normal culture or H < 0.05) .2O2 injury was investigated. According to the results of the MTT assay, treatment with 0.8 mm H2O2 for 24 h was selected to build the oxidative stress model . Further studies on the gene and protein expression levels of tight junctions have shown that when only DL–HMTBA is present, mRNA levels of ZO-1, claudin, and occludin were significantly higher . Besides, the contents of Cys, GSH, and H2S and the cofactors VB6, VB9, and VB12 involved in metabolism in each treatment group were not significantly different  was significantly higher; when the ratio was ≤ 40%:60%, the mRNA levels of S-adenosylhomocysteine hydrolase (AHcy) and Cystagamma-lyase (CTH) were significantly higher (p < 0.01), in addition, only in 100% DL–HMTBA group, the mRNA levels of 5-methyltetrahydrofolate-Hcy methyltransferase (MTR) and methylenetetrahydrofolate reductase (MTHFR) were significantly higher (p < 0.05).We then detected changes in the mRNA levels of pivotal enzymes associated with the metabolism of Met. Compared with the 100% L-Met group, when the proportion of L-Met to DL–HMTBA was ≤ 85%:15%, the mRNA level of methionine adenosyltransferase 2A (MAT2A) was significantly higher; when the ratio was ≤ 70%:30%, the mRNA level of Cysta-beta-synthase (y higher  (p < 0.0p < 0.05). Notably, a significant negative correlation was between the proportion of SAM to SAH and each gene expression level (p < 0.01), and the correlation coefficient with the mRNA level of MAT2A was 0.884.The aforementioned studies showed that DL–HMTBA significantly affects Met metabolism and gene expression in IPEC-J2 cells. Then, is there a specific correlation between changes in metabolites and changes in gene expression levels? Therefore, we next analyzed the correlation between metabolites of Met and the gene expression of Met metabolic enzymes and tight junction proteins. As shown in 5C and m6A, the major methylation modifications of DNA and mRNA, respectively. As shown in 5C content in the cell DNA, whereas DL–HMTBA significantly reduced m6A mRNA levels (p < 0.01).We considered that the difference in mRNA expression levels caused by DL–HMTBA and L-Met might be due to the difference in the metabolism between them, which leads to the difference in the contents of SAM and SAH and then changes in intracellular methylation modification. Therefore, we next tested the contents of mp < 0.01). Among those rations, different ratios had no obvious impact on the protein level of METTL3 or YTHDF2. However, when the ratio was ≤ 40%:60%, the protein level of FTO was obviously higher than that in the 100% L-Met group (p < 0.01).Therefore, we examined the mRNA levels of the major RNA MTRs, demethylases, and recognition proteins and further detected the protein levels of the genes with significant changes. As shown in 6A modification of mRNA in IPEC-J2 cells. DL–HMTBA may affect the metabolic fate of mRNA in IPEC-J2 cells, for example, affecting the stability of mRNA. Therefore, actinomycin was used to investigate the influence of L-Met and DL–HMTBA on the mRNA stability of these genes. The results showed that the mRNA stability of MAT2A after 2 h and ZO-1 after 1 h of actinomycin treatment was significantly improved by DL–HMTBA (p < 0.05).L-Met and DL–HMTBA had significantly different impacts on the mRNA levels of MAT2A, CBS, and ZO-1. The aforementioned findings also showed that DL–HMTBA could reduce mDL–HMTBA   and China Agriculture Research System of MOF and MARA.FZ was employed by Wuhan Sun HY Biology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


IgG4-related disease (IgG4-RD) is an emerging immune-mediated disease that can involve any organ. The involvement of the pancreas and biliary tract is the most common and well-studied in the literature. It is characterized by a non-specific presentation, mimicking a malignant process. The goal was to look at the different clinical and paraclinical aspects of this disease, as well as the challenges that come from its management. It was made up of three observations of patients with IgG4-RD involving the biliary tract and pancreas. The first observation concerned intrahepatic biliary cholangitis that was accompanied by porto-mesenteric thrombosis, which was discovered by cholestatic jaundice on the 15th day after an appendectomy, and the patient improved under corticosteroids and anticoagulants. The second observation concerned an acute revelation of the disease. It was an acute attack of chronic pancreatitis of IgG4-RD. The main symptoms were pancreatic pain and exocrine pancreatic insufficiency, and corticosteroid therapy allowed remission. The third observation was related to autoimmune pancreatitis (AIP) and IgG4-related sclerosing cholangitis, revealed by jaundice with cholestasis. The patient acquired corticosteroid resistance and an adverse progression to decompensated cirrhosis, and liver transplantation was indicated. The clinical presentation of IgG4-RD is heterogeneous, as evidenced by our three clinical observations. There are still significant gaps in our understanding, particularly in terms of pathogenesis and factors that influence therapy response. Further observational and interventional research is needed to better manage this disease. The discovery of IgG4-related disease (IgG4-RD) has revolutionized pancreatic and biliary autoimmune disease management. It is a recently discovered disease characterized by fibro-inflammatory attacks that can affect one or several organs in a synchronous or metachronous way . The epiThe pancreatic localization of igG4 disease was the first to be described, and thus the most investigated. It usually manifests as a chronic pseudotumor; however, acute manifestations have been described. IgG4-RD can be mistaken for a variety of conditions, including pancreatic adenocarcinoma.Although biliary tract involvement is most often associated with pancreatic involvement, isolated sclerosing cholangitis has been observed in certain cases. It shares certain similarities with cholangiocarcinoma. IgG4-RD's biliopancreatic involvement is known to be steroid-sensitive with a significant risk of relapse or cortico-resistance. Immunomodulatory therapies such as thiopurines, methotrexate, and rituximab have been shown to be necessary in some cases .The purpose of this case series is to focus on the current status of biliary and pancreatic IgG4-RD, as well as the challenges in diagnosing and managing it. We also want to remind specialists that they should rule out pancreatic or biliary cancers first to avoid unnecessary surgery for a steroid-sensitive disease.Case 1A 60-year-old male patient was admitted to our gastroenterology unit on the 15th day of an appendectomy. He presented with jaundice for four months with pruritus, abdominal pain, and asthenia. His medical history only included transurethral resection of the prostate in 2021.On examination, the patient presented with generalized mucocutaneous jaundice with scratch marks. An abdominal examination showed tenderness in the right iliac fossa. Laboratory tests showed leukocytosis and an elevated C-reactive protein, cholestasis with unconjugated hyperbilirubinemia, and without cytolysis Table .An abdominal ultrasound showed thickening and intrahepatic bile duct (IHBD) dilatation in the left hepatic lobe, a portal and mesenteric thrombosis, as well as an abscess in the right iliac fossa. The gallbladder and the main bile duct were normal Figure .Magnetic resonance cholangiopancreatography (MRCP) confirmed the dilatation of the IHBD of the left liver lobe with regular wall thickening without any visible obstruction Figure .The patient underwent ultrasound-guided drainage for the abscess in the right iliac fossa, and then he received antibiotics and anticoagulants for the porto-mesenteric thrombosis. For cholestasis, the initial etiological assessment was negative, including hepatitis B and C virus serology, and autoimmune tests including antinuclear antibodies (ANA), anti-native DNA antibodies (anti-DNA), anti-smooth muscle antibodies (ASMA), anti-mitochondrial antibodies (AMA), anti-soluble liver antigen autoantibodies (SLA), and anti-liver-kidney microsomal antibody (anti-LKM). The biopsy of bile duct thickening showed non-specific lymphoplasmacytic inflammatory infiltrates with no signs of malignancy, while the serum level of IgG4 was elevated to 1.820 g/l (0.040-0.870g/l), carbohydrate antigen19-9 was slightly elevated at 55 U/ml (˂37 U/mL).Ultimately, the diagnosis of IgG4-related sclerosing intrahepatic cholangitis was withheld. Corticosteroid was started from 0.6 mg/kg/day, which is equivalent to 50 mg of prednisolone daily for one month, tapered gradually (a decrease of 5 mg every two weeks). At present, after six months of withdrawing the corticosteroids, the patient is asymptomatic with complete resolution of jaundice, normalization of the liver biochemical tests, and a normal IgG4 serum level. The follow-up abdominal Doppler ultrasound showed a normal liver without any dilatation or thickening of the IHBD with complete re-permeabilization of the portal veins and superior mesenteric veins. A regular follow-up will be performed every six months by clinical examination, abdominal ultrasound, liver biochemical tests, and IgG4 serum level.Case 2A 35-year-old male male patient was referred to the emergency department for acute epigastric pain with diarrhea. His medical history includes recurrent acute pancreatitis in 2015 and 2020. On the physical exam, there was epigastric tenderness. Lipasemia was elevated to 190 U/l, the liver biochemical tests were normal, the lipidic tests showed slight hypercholesterolemia at 2.2 g/l, and the phosphocalcic test was normal. The fecal elastase was low at 109 μg/g of stool .We first suspected an acute flare of chronic pancreatitis. The autoimmune origin was the most probable, given the absence of alcoholism, biliary stones, and metabolic abnormalities. The Ig4 serum level was high at 1.71 g/l (0.040-0.870 g/l), so we withheld the diagnosis of autoimmune pancreatitis (AIP) type 1 with exocrine pancreatic insufficiency. A biopsy was not performed because of the normality of the imaging . Corticosteroid therapy was started with 40 mg of prednisolone for one month, then progressive degression was started. The patient is on the 12th week of corticosteroids with the disappearance of epigastric pain and signs of pancreatic insufficiency. The IgG4 serum level was normal.Case 3A 63-year-old male patient was referred to our department for cholestatic jaundice evolving for one month with chronic diarrhea associated with asthenia and weight loss. He had no known past medical history. The clinical examination revealed generalized jaundice with right upper quadrant tenderness.Initial labs showed cholestasis with elevated bilirubin (40.4 mg/L), conjugated bilirubin 39 mg/l), GGT (640U/l), ALP (787U/l), and cytolysis . Abdominal CT showed a body enlarged pancreas. MRCP revealed a normal common bile duct and dilated intrahepatic ducts with an enlarged pancreas , alpha-1 antitrypsin was normal, and fecal elastase was less than 50 μg/g of stool. The patient underwent a liver biopsy, which showed extensive portal chronic inflammation with ductular proliferation. The portal inflammation was composed mostly of lymphocytes with focal scattered plasma cells and a few eosinophils. The bile ducts showed focal injuries with ductular proliferation, prominent interface hepatitis, and scattered lobular inflammatory infiltrates with hepatocyte damage were also noted.We finally retained the diagnosis of IgG4 sclerosing cholangitis with autoimmune pancreatitis. Corticosteroid therapy at 0.6 mg/kg/day of prednisolone was started for four weeks, then tapered to 5 mg/two weeks. After three months of corticosteroid therapy, jaundice disappeared, but the patient kept asthenia with cytolysis and cholestasis. The decision was to add azathioprine at a dose of 2 mg/kg/day and to maintain corticosteroid therapy at a dose of 5 mg/day. After three months of azathioprine, the patient developed cirrhosis with ascitic decompensation. We decided to stop corticosteroids and azathioprine and discussed liver transplantation.IgG4 disease is a recently discovered immune-mediated condition affecting several organ systems. The characteristics of this entity have been the subject of an international consensus adopted in 2012  to the AIts epidemiology is still poorly described. In Japan, the prevalence is estimated at 1/600,000 and the annual incidence at 0.28-1.08/100,000 inhabitants [The pancreatic involvement was the first described manifestation. It causes an enlarged pancreas with progressive parenchymal destruction due to the extension of fibrosis . The aveHistology is the gold standard for diagnosis. Characteristic pathologic features are lymphoplasmacytic infiltrates with prominent IgG4+ plasma cells, storiform fibrosis, and obliterative phlebitis. In practice, it is difficult to obtain a representative pancreatic sample with fine needles by transparietal approach or guided by echoendoscopy.The IgG4 serum level is of major interest in establishing the diagnosis; a threshold of 135 mg/dl has been retained for the positive diagnosis because it distinguishes between pancreatic cancers and chronic calcifying pancreatitis; this threshold has a sensitivity of 90% and a negative predictive value of 96%, but the specificity and positive predictive value are low at 60% and 34%, respectively .In our series, we reported two cases of IgG4 pancreatitis with diverse clinical presentations. The first observation concerned acute recurrent pancreatitis with normal imaging, a high IgG4 level, and exocrine pancreatic insufficiency. Similar cases have been reported as single clinical cases . We consIgG4-related cholangitis is second only to pancreatic involvement in IgG4 disease, which is associated in 83% of cases. Several criteria have been established for the diagnosis of IgG4 cholangitis; the most widely used are the Japanese ones published in 2012 . The preIgG4 cholangitis is characterized mostly by jaundice, as well as cytolysis and cholestasis. Imaging frequently shows biliary tract stenosis with thickenings. It primarily affects the extrahepatic bile ducts. Isolated intrahepatic involvement is possible but uncommon. A radiological classification has been established, individualizing four forms based on the levels of biliary stenosis. These features are not unique to IgG4 cholangitis .Elevated IgG4 levels are important but inconstant criteria for the diagnosis. It can be normal in 10% to 20% of cases of IgG4 cholangitis. A threshold of 135 mg/dl seems to be useful to distinguish IgG4 cholangitis from pancreatic cancer and primary sclerosing cholangitis (PSC). However, a threshold of 182 mg/dl has been recommended with a specificity of 96.6% for distinguishing CS-IgG4 from cholangiocarcinoma . A new qIn histopathology, IgG4 cholangitis presents the same common histological characteristics as IgG4-RD, including lymphoplasmacytic infiltrates with prominent IgG4+plasma cells, storiform fibrosis, and obliterative phlebitis. However, these criteria are difficult to demonstrate in the case of intrahepatic involvement .When we did liver biopsies on two of our patients who had intrahepatic lesions on imaging, we discovered extensive lymphoplasmacytic infiltrates but no malignant cells. Because the immunohistochemistry study was inconclusive, we relied on other clinical, biological, radiological, and therapeutic responses to establish the diagnosis.IgG4-RD is a corticosteroid-sensitive disorder with a 97% to 100% response rate. Western teams start with 0.6 to 0.8 mg/kg/day for four weeks, then reduce it by 5 mg every two weeks for three to six months. To limit the risk of relapse, most Japanese teams continue low-dose corticosteroids (2.5 to 5 mg/day) for three years . MonitorIgG4-RD is a recurring pathology, with a relapse incidence ranging from 26% to 70% after stopping corticosteroids . RelapseIn case of relapse, immunosuppressive drugs are recommended along with corticosteroids. Azathioprine is the most commonly used, although the level of evidence is low. Rituximab is a promising treatment for the management of relapse and maintenance of remission .In our case series, two patients have responded well to corticosteroid therapy without relapse to date, but the third patient has developed decompensated cirrhosis despite the addition of azathioprine.The IgG4-related disease is an emerging and poorly understood disease. Nearly two decades ago, it was identified as a distinct systemic disease entity. Pancreatic and biliary involvement are the most frequent involvement. They mainly mimic pancreatic cancer and cholangiocarcinoma. To avoid diagnostic errors, several diagnostic criteria have been validated by international consensus to avoid diagnostic errors. More prospective studies are needed to better define IgG4 illness and fill gaps in our understanding of pathophysiology and diagnosis. Furthermore, international consensus management of this disease in its early stages in the event of relapse is required. We hope our cases contribute to the literature pool. Ultimately, a complete understanding of the pathophysiology and evidence-based management guidelines awaits further studies.


In recent decades, researchers have begun to investigate innovative sustainable construction materials for the development of greener and more environmentally friendly infrastructures. The main purpose of this article is to investigate the possibility of employing date palm tree waste as a natural fiber alternative for conventional steel and polypropylene fibers (PPFs) in concrete. Date palm fibers are a common agricultural waste in Middle Eastern nations, particularly Saudi Arabia. As a result, this research examined the engineering properties of high-strength concrete using date palm fibers, as well as the performance of traditional steel and PPF concrete. The concrete samples were made using 0.0%, 0.20%, 0.60%, and 1.0% by volume of date palm, steel, and polypropylene fibers. Ten concrete mixtures were made in total. Compressive strength, flexural strength, splitting tensile strength, density, ultrasonic pulse velocity (UPV), water absorption capability, and water permeability tests were performed on the fibrous-reinforced high-strength concrete. With a 1% proportion of date palm, steel, and polypropylene fibers, the splitting tensile strength improved by 17%, 43%, and 16%, respectively. By adding 1% fiber, flexural strength was increased by 60% to 85%, 67% to 165%, and 61% to 79%. In addition, date palm fibers outperformed steel and PPFs in terms of density, UPV, and water permeability. As a result, date palm fibers might potentially be employed in the present construction sector to improve the serviceability of structural elements. Environmental pollution is a crucial cause of climate change that may lead to a risk to human health. Hence, the efficient management of waste materials is essential for a smooth industrial revolution ,2. ReseaPalm fibers have strand textures with distinct properties such as being low-cost, available in some regions, lightweight, and durable . Fibers Since a lack of information on the engineering characteristics of the agro-waste date palm fiber-reinforced high-strength concrete found in the literature, the objective of this study is to examine the engineering characteristics of the date palm fiber-reinforced concrete in all aspects and make comparisons with conventional steel and polypropylene fibrous concrete.2/kg and 3.15, respectively. The cement contained 59% C3S, 12.10% C2S, 10.60% C3A, and 10.4% C4AF, as reported by the manufacturer [This study used ordinary Portland cement Type-I for fabricating the high-strength date palm, steel, and polypropylene fibrous concrete specimens. The fineness and specific gravity of the cement were 410 mfacturer  and conffacturer . The cheThe fine aggregate consists of natural dune sand with maximum particles passing through sieve No.4. More information about the fine aggregate used herein can be seen in previous work by the authors . The coaThe filtered tap water was utilized as a fundamental element in manufacturing the high-strength date palm, steel, and polypropylene fibrous concrete and curing concrete. The properties of the water have accomplished the requirement of fabricating high-strength fibrous concrete with ASTM C1602/C1602M . Super PThe date palm fibers were obtained from around Najran city, Saudi Arabia’s date palm trees are aged between 15 and 25 years old, which signifies one of the best available diversities and are accountable for significant agricultural waste production. These date palm fibers were manually collected from the palm trees. The date palm fibers are sited around the tree’s trunk in a bidirectional form, comprised of two or three layers packed and superimposed. The collected raw date palm fibers had different diameters and lengths. The steel fibers were hooked at both ends, which were bundles glued as demonstrated in This study used polypropylene fiber  to fabri3 prisms to compute different hardened characteristics of the high-strength palm fibrous concrete. The configurations of the high-strength fibrous concrete mixtures are revealed in Using different fractions  by binder weight for date palm, steel, and polypropylene fibers, a total of 10 mixtures were fabricated. The experimental program was carried out on 100 mm cubes, 150 mm diameter × 300 mm height cylinders and 100 × 100 × 500 mm3. The specimens were tested after the 28-day curing regimen. Prior to testing, the specimens were kept at ambient temperature (23 °C ± 2 °C) for 24 h. Three specimens were tested for each mixture, and the average values were reported.The high-strength date palm, steel, and polypropylene fibrous concrete specimens were tested according to BS EN12390-3  under th3 high-strength fibrous concrete prism specimens was computed utilizing a Linear variable displacement transducer (LVDT) attached to the center of the prism specimens. The applied load and displacement were automatically recorded in the TDS-530 data logger during the execution of the experiment on the samples. The recorded results were transferred from the data logger to a computer to analyze the load-displacement graphs.The flexural strength of the high-strength date palm, steel, and polypropylene fibrous concrete specimens was executed under a four-point loading arrangement that was implemented as per the BS EN 12390-5 , which wThe splitting tensile strengths of the high-strength date palm, steel, and polypropylene fibrous concrete were assessed at 28-days. The cylindrical high-strength fibrous concrete specimens were used for splitting tensile strength. The cylindrical specimens with dimensions of 100 mm diameter and 200 mm height and experiments were executed following BS EN 12390-6 .The density of the high-strength date palm, steel, and polypropylene fibrous concrete specimens was measured after the curing period of 28-days. This experiment was carried out on the samples before the mechanical compression test. These experiments contained the determination of the weight and volume of the fibrous concrete specimens. The density of the fibrous concrete samples is determined, conferring to the formula: density = weight/volume.The integrity and homogeneity of the high-strength date palm, steel, and polypropylene fibrous concrete specimens were confirmed by the ultrasonic pulse velocity (UPV) test ,40. The The superior quality of concrete is labeled by encompassing trivial pores changed by excessive water. Therefore, the water absorption capacity measuring experiment is usually applied to compute concrete quality, such as density, rigidity, and durability. The water absorption test for high-strength date palm, steel, and polypropylene fibrous concrete was conducted by conferring to BS 2011 Part 122  having t2 for an irreparable period of 72 h. After the specified time, the fibrous concrete samples were removed from water pressure and divided into two halves, while the water-exposed specimen’s face was shown to be face down. The intensity of water penetration into the fibrous concrete samples was recorded and assessed as the permeability.The water penetration intensity into the high-strength date palm, steel, and polypropylene fibrous concrete specimens was evaluated using a Zwick machine . This exThe compressive strength of date palm, steel, and polypropylene fibrous-reinforced high-strength concrete with different volume fractions of fibers is revealed in cdf, csf, and cpf are the compressive strengths of the date palm, steel, and polypropylene fibrous-reinforced high-strength concrete, respectively, and fdV, fsV and fpV are the volume fractions of date palm, steel, and polypropylene fibers, respectively.The correlation between the compressive strength and the addition of date palm, steel, and polypropylene fibers to the high-strength concrete is revealed in The flexural strength and impact resistance of fiber-reinforced concrete is very significant when infrastructures are exposed to the marine environment or extreme weathering conditions ,48. The rdf, rsf and rpf is the flexural strength of the date palm, steel, and polypropylene fibrous-reinforced high-strength concrete, respectively.The correlation between the flexural strength and volume fractions of the date palm, steel, and polypropylene fibrous concrete is represented in The splitting tensile strength characteristics of the date palm, steel, and polypropylene fibrous high-strength concrete are exhibited in tdf, tsf, and tpf is the splitting tensile strength of the date palm, steel, and PPFs reinforced high-strength concrete, respectively.The densities of the high-strength fibrous concrete incorporating 0%, 0.20%, 0.60%, and 1.0% of date palm, steel, and polypropylene fibers at 28 days are demonstrated in dγ, sγ, and pγ are the density of the high-strength date palm, steel, and PPFs, respectively.The correlation between the density of high-strength concrete and volume fractions of date palm, steel, and polypropylene fibers is represented in Numerous studies have revealed that concrete density increases by increasing steel fiber’s contents in the concrete ,49. MoreThe relationship between the UPV and volume fraction of the date palm, steel, and polypropylene fibers in the concrete is presented in The water absorption capability for date palm, steel, and polypropylene fibrous-reinforced high-strength concrete is shown in The relationship between the water permeability of high-strength concrete and volume fractions of date palm, steel, and polypropylene fibers is shown in Μd, µs, and µp are the water permeability of the high-strength date palm, steel, and polypropylene fibrous concrete, respectively.The load-displacement characteristics of fiber-reinforced high-strength concrete are very important as it implies the serviceability of structural elements. The load-displacement relationship is usually affected by the initial cracking, yield and ultimate loads, and stiffness of the elements. Ductility is expressed as the competence of a material or infrastructure to tolerate substantial plastic deformation without significant loss in strength proficiency . Hence, This study examined the distinct engineering characteristics of high-strength concrete incorporating agro-waste date palm fibers and compared it with conventional steel and polypropylene fibrous concrete. Hence, this experimental investigation emphasized the compressive strength, flexural strength, splitting tensile strength, density, ultrasonic pulse velocity (UPV), water absorption capability, and water permeability of the date palm, steel, and polypropylene fiber-reinforced high-strength concrete. The following conclusions can be drawn from this study.The impact of steel fibers on the compressive strength was superior compared with the date palm and PPF-reinforced concrete due to the better interconnecting bonds between the fibers and concrete matrix.The flexural strength of the fibrous-reinforced high-strength concrete was significantly improved due to the addition of date palm, steel, and polypropylene fibers. Compared with the reference specimens, the flexural strength was enhanced by about 60% to 85%, 67% to 165%, and 61% to 79% with fibers.The splitting tensile strength of the fibrous-reinforced high-strength concrete was substantially enhanced with the volume fractions of date palm, steel, and polypropylene fibers. The splitting tensile strength was increased by 17%, 43%, and 16% when the percentage of date palm, steel, and polypropylene fibers was 1.0% compared to the reference specimen since the tensile strength of steel fibers was much higher than the date palm and polypropylene fibers.3, 2543.20 to 2589.40 kg/m3, and 2502.40 to 2460.60 kg/m3 with the addition of fibers.The density of the fibrous-reinforced high-strength concrete improved to 2514.50 to 2496.33 kg/mThe UPV of the fibrous-reinforced high-strength concrete revealed a declining trend with increasing volume fractions of date palm, steel, and polypropylene fibers.The smallest increase in water permeability was remarked for the steel fibers compared with date palm and polypropylene fibers because the steel fiber’s physio-chemical characteristics did not permit water penetration into the concrete.The ductility of the fibrous-reinforced high-strength concrete increased by up to 129% over the reference specimen. This enhancement was correlated with increasing the fiber volume in the concrete.


Intracanal separation of nickel titanium files hinders complete shaping, cleaning, and filling of the root canal system and ultimately influences the endodontic treatment outcome. In this case report, we presented a successful broken instrument retrieval from the middle third of the mesiobuccal root canal of tooth #30 with the assistance of cone-beam computed tomography based preoperative computer-assisted simulation, micro-trepan bur and micro-tube from Micro-Retrieve & Repair system and dental operative microscope. The involved tooth was then successfully cleaned, shaped and obturated followed by coronal restoration. At the three-year follow-up, tooth #30 was asymptomatic and functioned well without radiographic changes. The present case provides an example to show the robustness of computer-assisted technology in dental procedures and to show how the combination of advanced techniques can facilitate root canal therapy. In the present study, we presented a case report, in which the broken Ni-Ti instrument was removed from the mandibular right first molar with the aid of cone-beam computed tomography (CBCT) based preoperative computer-assisted simulation, Micro-Retrieve & Repair system as well as dental operating microscope (DOM).Instrument separation of nickel titanium (Ni-Ti) files within the root canal is an unfortunate procedural accident. When it comes to fractured instrument removal, computer-assisted technology has its powerful potentials. To the best of our knowledge, the case reports published,A 32-year-old male without contributory medical history was referred to the department of endodontics and the chief complaint was a broken instrument in lower right posterior teeth region. The patient's dental history indicated that there was a fractured instrument in the mandibular right posterior tooth for one week. Intraoral examination revealed that a temporary restoration was placed on tooth #30. The percussion test produced a slight response, and the patient complained of pain during the bite test. In addition, mobility of tooth #30 scored as 1° (less than 1mm), and periodontal probing depth was less than 3mm. No sinus tract was observed around tooth #30.The radiograph confirmed that tooth #30 had intracanal medicament and a separated endodontic instrument (approximately 3-4 mm in length) located at the middle third of the mesial root canal . There w4 the following steps were included in the framework . The CBCT data was imported into Mevislab to build an environment to perform an interactively virtual canal path preparation, which simulates the process of broken instrument removal under actual condition. Similar to the previously published study,ramework :A 3D data set from the scanned broken instrument model CBCT image was build.A virtual micro-trepan bur (a cylinder) in the canal was created and the size for the micro-trepan  was set.Then the virtual micro-trepan bur was interactively placed to a proper position around the broken instrument  to simulate removing dentin to form a narrow parallel cylindrical space.Then the image with the cylinder for subsequent remaining dentin thickness analysis and measurement was saved . AccordiThen micro-trepan bur and micro-tube for the removal of the broken instrument were planned, and the risk and prognosis were explained to the patient. Informed consent was obtained from the patient before the operation.5 was prepared at the coronal end of the broken instrument using modified GG burs (no. 3) Then, a 20-G micro-trepan bur from Micro-Retrieve & Repair system with a 0.9-mm outside diameter and a 0.6-mm inside diameter was selected as per the simulation and operated with an endodontic motor  in a counterclockwise direction (500 rpm) to expose a ~1 mm length of the broken fragment by removing the surrounding dentin. After removing the dentin surrounding the broken instrument, the micro-tube was used to withdraw the fragment from the mesiobuccal canal . After complete removal of calcium hydroxide intracanal medicament which was previously placed in the root canal by the former clinician, the broken instrument could be observed in the mesiobuccal canal with the help of the DOM . In ordeal canal . The lenal canal . Completal canal . Then thOn the second visit, the patient was asymptomatic and the temporary filling was intact. The working length was confirmed using an apex locator  for all four root canals. The canals were prepared with ProTaper Universal rotary file system . After completion of chemo-mechanical preparation, the canals were irrigated with 1% NaOCl and dried with paper points. The master cone was fitted and confirmed by radiograph . AH Plus8 instrument removal procedures were simulated successfully, and the remaining dentin thickness was measured. In the present report, the framework used in Mevislab helped to perform advanced simulation and analysis including virtual canal preparation using the micro-trepan bur shape to simulate the dentin removal path around the broken instrument for the retrieval of the broken instrument and perform preoperative analysis of benefit and risk factors. Pre-operative computer-assisted simulation of broken instrument removal provided a powerful platform in measuring remaining dentin wall thickness in the present study. According to Lim and Stock,9 200-300|jm dentin thickness should be retained after preparation in order to withstand compaction forces during obturation and to prevent perforation or fracture. Also, the attempt of retrieval may be risky if the value falls below a certain value. The remaining dentin thickness measured in the present report 868μm was greater than 200-300μm, thus it was relatively safe and significant to preserve tooth integrity.In the present case report, the micro-trepan/microtube technique was chosen to remove the broken instrument in both preoperative simulation and clinically in tooth #30. In previous studies,10 Secondly, even if the ultrasonics is able to trough the dentin around the coronal fragment it cannot be retrieved out on its own. In this scenario, micro-tube can be a helpful aid to retrieve the broken instrument out from the root canal. Thus the use of micro-trepan bur and micro-tube helps to save the dentin and preserve the integrity of the tooth. However, on the other hand, excess removal of dentin during trephine may result in perforation or vertical fracture. The clinician should perform a sufficient preoperative assessment of the root canal morphology and the location of broken fragment followed by selection of the removal technique which helps in the minimum removal of dentin preserving tooth integrity.Ultrasonics when used during the retrieval process to trough the coronal portion of dentin around the broken instrument then it leads to loss of more dentin surface when compared with micro-trepan bur.The fractured instrument especially a broken Ni-Ti file leads to obstruction in the root canal and blocks thorough cleaning and shaping procedures during the root canal therapy. In the context of broken instrument removal, location of the broken instrument, remaining dentin thickness, possible complications should be assessed prior thoroughly for proper decision making and treatment planning.The present case report showed that combined with software, CBCT data can help to simulate the removal of broken instrument technique and also determine the remaining dentin thickness. Therefore, this type of preoperative assessment can be a safe and conservative method for better treatment outcome in broken instrument retrieval.