A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome.

BACKGROUND: G-quadruplex DNA (G4) is a non-canonical structure forming in guanine-rich regions, which play a vital role in cancer biology and are now being acknowledged in both nuclear and mitochondrial (mt) genome. However, the impact of G4-based targeted therapy on both nuclear and mt genome, affecting mt function and its underlying mechanisms remain largely unexplored. METHODS: The mechanisms of action and therapeutic effects of a G4-binding platinum(II) complex, Pt-ttpy, on mitochondria were conducted through a comprehensive approaches with in vitro and in vivo models, including ICP-MS for platinum measurement, PCR-based genetic analysis, western blotting (WB), confocal microscope for mt morphology study, extracellular flux analyzer, JC1 and Annexin V apoptosis assay, flow cytometry and high content microscope screening with single-cell quantification of both ROS and mt specific ROS, as well as click-chemistry for IF study of mt translation. Decipher Pt-ttpy effects on nuclear-encoded mt related genes expression were undertaken via RNA-seq, Chip-seq and CUT-RUN assays. RESULTS: Pt-ttpy, shows a highest accumulation in the mitochondria of A2780 cancer cells as compared with two other platinum(II) complexes with no/weak G4-binding properties, Pt-tpy and cisplatin. Pt-ttpy induces mtDNA deletion, copy reduction and transcription inhibition, hindering mt protein translation. Functional analysis reveals potent mt dysfunction without reactive oxygen species (ROS) induction. Mechanistic study provided first evidence that most of mt ribosome genes are highly enriched in G4 structures in their promoter regions, notably, Pt-ttpy impairs most nuclear-encoded mt ribosome genes' transcription through dampening the recruiting of transcription initiation and elongation factors of NELFB and TAF1 to their promoter with G4-enriched sequences. In vivo studies show Pt-ttpy's efficient anti-tumor effects, disrupting mt genome function with fewer side effects than cisplatin. CONCLUSION: This study underscores Pt-ttpy as a G4-binding platinum(II) complex, effectively targeting cancer mitochondria through dual action on mt and nuclear G4-enriched genomes without inducing ROS, offering promise for safer and effective platinum-based G4-targeted cancer therapy.

BCKDK modification enhances the anticancer efficacy of CAR-T cells by reprogramming branched chain amino acid metabolism.

Altered branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, are frequently observed in patients with advanced cancer. We evaluated the efficacy of chimeric antigen receptor (CAR) T cell-mediated cancer cell lysis potential in the immune microenvironment of BCAA supplementation and deletion. BCAA supplementation increased cancer cell killing percentage, while accelerating BCAA catabolism and decreasing BCAA transporter decreased cancer cell lysis efficacy. We thus designed BCKDK engineering CAR T cells for the reprogramming of BCAA metabolism in the tumor microenvironment based on the genotype and phenotype modification. BCKDK overexpression (OE) in CAR-T cells significantly improved cancer cell lysis, while BCKDK knockout (KO) resulted in inferior lysis potential. In an in vivo experiment, BCKDK-OE CAR-T cell treatment significantly prolonged the survival of mice bearing NALM6-GL cancer cells, with the differentiation of central memory cells and an increasing proportion of CAR-T cells in the peripheral circulation. BCKDK-KO CAR-T cell treatment resulted in shorter survival and a decreasing percentage of CAR-T cells in the peripheral circulation. In conclusion, BCKDK-engineered CAR-T cells exert a distinct phenotype for superior anticancer efficiency.

Present situation of minimally invasive surgical treatment for early gastric cancer.

Minimally invasive surgery is a kind of surgical operation, which is performed by using professional surgical instruments and equipment to inactivate, resect, repair or reconstruct the pathological changes, deformities and wounds in human body through micro-trauma or micro-approach, in order to achieve the goal of treatment, its surgical effect is equivalent to the traditional open surgery, while avoiding the morbidity of conventional surgical wounds. In addition, it also has the advantages of less trauma, less blood loss during operation, less psychological burden and quick recovery on patients, and these minimally invasive techniques provide unique value for the examination and treatment of gastric cancer patients. Surgical minimally invasive surgical techniques have developed rapidly and offer numerous options for the treatment of early gastric cancer (EGC): endoscopic mucosal resection (EMR), underwater EMR (UEMR), endoscopic submucosal dissection (ESD), endoscopic full-thickness resection (EFTR), endoscopic submucosal excavation (ESE), submucosal tunnel endoscopic resection), laparoscopic and endoscopic cooperative surgery (LECS); Among them, EMR, EFTR and LECS technologies have a wide range of applications and different modifications have been derived from their respective surgical operations, such as band-assisted EMR (BA-EMR), conventional EMR (CEMR), over-the-scope clip-assisted EFTR, no-touch EFTR, the inverted LECS, closed LECS, and so on. These new and improved minimally invasive surgeries are more precise, specific and effective in treating different types of EGC.

Transcranial Direct Current Stimulation for Anxiety During Laparoscopic Colorectal Cancer Surgery: A Randomized Clinical Trial.

Importance: Perioperative anxiety is prevalent among patients undergoing surgical treatment of cancer and often influences their prognosis. Transcranial direct current stimulation (tDCS) has shown potential in the treatment of various anxiety-related disorders, but data on the impact of tDCS on perioperative anxiety are limited. Objective: To evaluate the effect of tDCS in reducing perioperative anxiety among patients undergoing laparoscopic colorectal cancer (CRC) resection. Design, Setting, And Participants: This randomized clinical trial was conducted from March to August 2023 at the Affiliated Hospital of Xuzhou Medical University. Patients aged 18 years or older undergoing elective laparoscopic radical resection for CRC were randomly assigned to either the active tDCS group or the sham tDCS group. Intention-to-treat data analysis was performed in September 2023. Interventions: Patients were randomly assigned to receive 2 sessions of either active tDCS or sham tDCS over the left dorsolateral prefrontal cortex on the afternoon of the day before the operation and in the morning of the day of operation. Main Outcomes and Measures: The main outcome was the incidence of perioperative anxiety from the day of the operation up to 3 days after the procedure, as measured using the Hospital Anxiety and Depression Scale-Anxiety (HADS-A) subscale (range: 0-21, with higher scores indicating more anxiety). Secondary outcomes included postoperative delirium (assessed by the Confusion Assessment Method or Confusion Assessment Method intensive care unit scale); pain (assessed by the 10-point Numeric Rating Scale [NRS], with scores ranging from 0 [no pain] to 10 [worst pain]); frailty (assessed by the Fatigue, Resistance, Ambulation, Illness and Loss of Weight [FRAIL] Index, with scores ranging from 0 [most robust] to 5 [most frail]; and sleep quality (assessed by the Pittsburgh Sleep Quality Index [PSQI], with scores ranging from 0 to 21 and higher scores indicating worse sleep quality) after the 2 sessions of the tDCS intervention. Results: A total of 196 patients (mean [SD] age, 63.5 [11.0] years; 124 [63.3%] men) were recruited and randomly assigned to the active tDCS group (98 patients) or the sham tDCS group (98 patients). After the second tDCS intervention on the day of the operation, the incidence of perioperative anxiety was 38.8% in the active tDCS group and 70.4% in the sham tDCS group (relative risk, 0.55 [95% CI, 0.42-0.73]; P < .001). Patients in the active tDCS group vs the sham tDCS group were less likely to have postoperative delirium (8.2% vs 25.5%) and, at 3 days after the operation, had lower median (IQR) pain scores (NRS, 1.0 [1.0-1.0] vs 2.0 [2.0-2.0]), better median (IQR) sleep quality scores (PSQI, 10.5 [10.0-11.0] vs 12.0 [11.0-13.0]), and lower median (IQR) FRAIL Index (2.0 [1.0-2.0] vs 2.0 [2.0-3.0]). Conclusions and Relevance: Findings of this randomized clinical trial indicate that administration of 2 preoperative sessions of tDCS was associated with a decreased incidence of perioperative anxiety in patients undergoing elective CRC resection. Active tDCS was also associated with better anxiety scores, pain levels, and sleep quality as well as reduced postoperative delirium and frailty. The findings suggest that tDCS may be a novel strategy for improving perioperative anxiety in patients undergoing CRC resection. Trial Registration: Chinese Clinical Trial Register Identifier: ChiCTR2300068859.

Ultrasound­targeted microbubble destruction technology delivering ß­klotho to the heart enhances FGF21 sensitivity and attenuates heart remodeling post­myocardial infarction.

Fibroblast growth factor (FGF)21 is a peptide hormone that improves mitochondrial function and energy metabolism, and the deficiency of its co­receptor ß­klotho (KLB) causes decreased FGF21 sensitivity. The present study examined whether the cardiac delivery of plasmids containing the KLB gene via ultrasound­targeted microbubble destruction (UTMD) enhances the efficacy of FGF21 against heart failure post­acute myocardial infarction (AMI). For this purpose, the levels of FGF21 in patients and rats with heart dysfunction post­infarction were determined using ELISA. Sprague­Dawley rats received the 3X UTMD­mediated delivery of KLB@cationic microbubbles (KLB@CMBs) 1 week following the induction of AMI. Echocardiography, histopathology and biochemical analysis were performed at 4 weeks following the induction of AMI. The results revealed that patients with heart failure post­infarction had higher serum FGF21 levels than the healthy controls. However, the downstream signal, KLB, but not α­klotho, was reduced in the heart tissues of rats with AMI. As was expected, treatment with FGF21 did not substantially attenuate heart remodeling post­infarction. It was found that decreased receptors KLB in the heart may result in the insensitivity to FGF21 treatment. In vivo, the UTMD technology­mediated delivery of KLB@CMBs to the heart significantly enhanced the effects of FGF21 administration on cardiac remodeling and mitochondrial dysfunction in the rats following infarction. The delivery of KLB to the heart by UTMD and the administration of FGF21 attenuated mitochondrial impairment and oxidative stress by activating nuclear factor erythroid 2­related factor 2 signals. On the whole, the present study demonstrates that the cardiac delivery of KLB significantly optimizes the cardioprotective effects of FGF21 therapy on adverse heart remodeling. UTMD appears a promising interdisciplinary approach with which to improve heart failure post­myocardial infarction.

Development and validation of an interpretable model integrating multimodal information for improving ovarian cancer diagnosis.

Ovarian cancer, a group of heterogeneous diseases, presents with extensive characteristics with the highest mortality among gynecological malignancies. Accurate and early diagnosis of ovarian cancer is of great significance. Here, we present OvcaFinder, an interpretable model constructed from ultrasound images-based deep learning (DL) predictions, Ovarian-Adnexal Reporting and Data System scores from radiologists, and routine clinical variables. OvcaFinder outperforms the clinical model and the DL model with area under the curves (AUCs) of 0.978, and 0.947 in the internal and external test datasets, respectively. OvcaFinder assistance led to improved AUCs of radiologists and inter-reader agreement. The average AUCs were improved from 0.927 to 0.977 and from 0.904 to 0.941, and the false positive rates were decreased by 13.4% and 8.3% in the internal and external test datasets, respectively. This highlights the potential of OvcaFinder to improve the diagnostic accuracy, and consistency of radiologists in identifying ovarian cancer.

Programmed Remodeling of the Tumor Milieu to Enhance NK Cell Immunotherapy Combined with Chemotherapy for Pancreatic Cancer.

Natural killer (NK) cell-based adoptive immunotherapy has demonstrated encouraging therapeutic effects in clinical trials for hematological cancers. However, the effectiveness of treatment for solid tumors remains a challenge due to insufficient recruitment and infiltration of NK cells into tumor tissues. Herein, a programmed nanoremodeler (DAS@P/H/pp) is designed to remodel dense physical stromal barriers and for dysregulation of the chemokine of the tumor environment to enhance the recruitment and infiltration of NK cells in tumors. The DAS@P/H/pp is triggered by the acidic tumor environment, resulting in charge reversal and subsequent hyaluronidase (HAase) release. HAase effectively degrades the extracellular matrix, promoting the delivery of immunoregulatory molecules and chemotherapy drugs into deep tumor tissues. In mouse models of pancreatic cancer, this nanomediated strategy for the programmed remodeling of the tumor microenvironment significantly boosts the recruitment of NK92 cells and their tumor cell-killing capabilities under the supervision of multiplexed near-infrared-II fluorescence.

Multichannel-optical imaging for in vivo evaluating the safety and therapeutic efficacy of stem cells in tumor model in terms of cell tropism, proliferation and NF-κB activity.

Stem cell-based cancer treatment has garnered significant attention, yet its safety and efficacy remain incompletely understood. The nuclear factor-kappa B (NF-κB) pathway, a critical signaling mechanism involved in tumor growth, angiogenesis, and invasion, serves as an essential metric for evaluating the behavior of stem cells in tumor models. Herein, we report the development of a triple-channel imaging system capable of simultaneously monitoring the tropism of stem cells towards tumors, assessing tumor proliferation, and quantifying tumor NF-κB activity. In this system, we generated a CRISPR-Cas9 gene-edited human glioblastoma cell line, GE-U87-MG, which provided a reliable readout of the proliferation and NF-κB activity of tumors by EF1α-RFLuc- and NF-κB-GLuc-based bioluminescent imaging, respectively. Additionally, near infrared-II emitting Tat-PEG-AgAuSe quantum dots were developed for tracking of stem cell tropism towards tumor. In a representative case involving human mesenchymal stem cells (hMSCs), multichannel imaging revealed no discernible effect of hMSCs on the proliferation and NF-κB activity of GE-U87-MG tumors. Moreover, hMSCs engineered to overexpress the necrosis factor-related apoptosis-inducing ligand were able to inhibit NF-κB activity and growth of GE-U87-MG in vivo. Taken together, our imaging system represents a powerful and feasible approach to evaluating the safety and therapeutic efficacy of stem cells in tumor models.

Development and validation of ultrasound-based radiomics model to predict germline BRCA mutations in patients with breast cancer.

BACKGROUND: Identifying breast cancer (BC) patients with germline breast cancer susceptibility gene (gBRCA) mutation is important. The current criteria for germline testing for BC remain controversial. This study aimed to develop a nomogram incorporating ultrasound radiomic features and clinicopathological factors to predict gBRCA mutations in patients with BC. MATERIALS AND METHODS: In this retrospective study, 497 women with BC who underwent gBRCA genetic testing from March 2013 to May 2022 were included, including 348 for training (84 with and 264 without a gBRCA mutation) and 149 for validation(36 patients with and 113 without a gBRCA mutation). Factors associated with gBRCA mutations were identified to establish a clinicopathological model. Radiomics features were extracted from the intratumoral and peritumoral regions (3 mm and 5 mm) of each image. The least absolute shrinkage and selection operator regression algorithm was used to select the features and logistic regression analysis was used to construct three imaging models. Finally, a nomogram that combined clinicopathological and radiomics features was developed. The models were evaluated based on the area under the receiver operating characteristic curve (AUC), calibration, and clinical usefulness. RESULTS: Age at diagnosis, family history of BC, personal history of other BRCA-related cancers, and human epidermal growth factor receptor 2 status were independent predictors of the clinicopathological model. The AUC of the imaging radiomics model combining intratumoral and peritumoral 3 mm areas in the validation set was 0.783 (95% confidence interval [CI]: 0.702-0.862), which showed the best performance among three imaging models. The nomogram yielded better performance than the clinicopathological model in validation sets (AUC: 0.824 [0.755-0.894] versus 0.659 [0.563-0.755], p = 0.007). CONCLUSION: The nomogram based on ultrasound images and clinicopathological factors performs well in predicting gBRCA mutations in BC patients and may help to improve clinical decisions about genetic testing.

Transport and transformations of cadmium in water-biofilm-sediment phases as affected by hydrodynamic conditions.

Hydrodynamic conditions play a crucial role in governing the fate, transport, and risks of metal elements. However, the contribution of hydrodynamic conditions to the fate and transport of heavy metals among water, sediment, and biofilm phases is poorly understood. In our study, we conducted experiments in controlled hydrodynamic conditions using a total of 6 two-phase and 9 three-phase mesocosms consisting of water, biofilm, and sediment. We also measured Cd (cadmium) specification in different phases to assess how hydrodynamic forces control Cd bioavailability. We found that turbulent flow destroyed the surface morphology of the biofilm and significantly decreased the content of extracellular polymeric substances (p < 0.05). This led to a decrease in the biofilm's adsorption capacity for Cd, with the maximum adsorption capacity (0.124 mg/g) being one-tenth of that under static conditions (1.256 mg/g). The Cd chemical forms in the biofilm and sediment were significantly different, with the highest amount of Cd in the biofilm being acid-exchangeable, accounting for up to 95.1% of the total Cd content. Cd was more easily released in the biofilm due to its weak binding state, while Cd in the sediment existed in more stable chemical forms. Hydrodynamic conditions altered the migration behavior and distribution characteristics of Cd in the system by changing the adsorption capacity of the biofilm and sediment for Cd. Cd mobility increased in laminar flow but decreased in turbulent flow. These results enhance our understanding of the underlying mechanisms that control the mobility and bioavailability of metals in aquatic environments with varying hydrodynamic conditions.

A novel entire traction method with clip-anchored nylon ring facilitating endoscopic submucosal dissection (ESD) of early gastric cancer.

A 48-year-old woman with a high-grade intraepithelial neoplasia of the gastric antrum was referred for ESD treatment.The difficulty of ESD is to expose the submucosa,especially in difficult sites and lesions with severe fibrosis.Adequate submucosal exposure is the most critical technology to reduce complications and improving efficiency.Here we report a novel entire traction method to facilitate safe and efficient ESD.

The pleiotropic enhancer enh9 promotes cell proliferation and migration in non-small cell lung cancer via ERMP1 and PD-L1.

Enhancers, cis-acting DNA elements for transcriptional regulation, are important regulators of cell identity and disease. However, of the hundreds of thousands of enhancers annotated in the human genome, only a few have been studied for their regulatory mechanisms and functions in cancer progression and therapeutic resistance. Here, we report the pleiotropy of one enhancer (named enh9) in both cell proliferation and migration in non-small cell lung cancer (NSCLC) cells. By integrating multi-genomic data, ERMP1 and PD-L1 were screened out as potential targets of enh9. CUT&Tag sequencing demonstrated that enh9 was involved in the genomic interactions between the transcription factor RELA and the promoters of ERMP1 and PD-L1. In addition, ERMP1 and PD-L1 were validated to be involved in cell proliferation and migration, respectively. Our study fully elucidated the function and transcriptional regulation mechanisms of enh9 in NSCLC. The exploration on enhancers is promising to provide new insights for cancer diagnosis and therapy.

Mapping Online Community Spaces Through Online Focus Group Discussions Among Gay, Bisexual, and Other Men Who Have Sex With Men in Guangdong, China: Implications for Human Immunodeficiency Virus/Sexually Transmitted Disease Prevention Services.

BACKGROUND: Chinese gay, bisexual, and other men who have sex with men (GBMSM) face discrimination in many facility-based health services, thus increasing the importance of online engagement. The purpose of this study was to examine online GBMSM community spaces and implications for HIV/sexually transmitted disease prevention services. METHODS: We conducted a total of 6 online focus group discussions with Chinese GBMSM from Guangdong province on the chat-based platform WeChat in 2021. Focus group discussions were asynchronous, and participants were able to provide and map out online spaces that they had participated in and share their perspectives on online engagement. Data were analyzed through framework analysis. RESULTS: Overall, 48 participants participated. Most were mainly sexually attracted to men (n = 43; 90.0%) and never participated in in-person LGBTQ-related events (n = 29; 60.4%). Participants articulated a typology of online spaces along the axes of whether such spaces were Chinese platforms (vs. non-Chinese) or whether they were GBMSM-specific (vs. non-GBMSM-specific). Participants articulated several advantages of online spaces, including greater anonymity, opportunities for community building, sharing of sexual health information, and being able to meet other GBMSM more efficiently. Drawbacks included the lack of personal connection, lack of safety measures for youth, encountering deception and the use of fake profile pictures, and needing a virtual proxy network to access some websites. Participants provided suggestions to further improve their experiences of online spaces. CONCLUSIONS: Although broad-based, GBMSM-specific messaging can be implemented in Chinese, GBMSM-specific spaces, sexual health messaging may also reach niche GBMSM communities in a variety of non-GBMSM spaces.

Intrathecal injection of methotrexate and dexamethasone for vasculitis granuloma of the fourth ventricle: a case report and literature review.

Granulomatosis with polyangiitis (GPA) is a pauci-immune small vessel vasculitis characterised by neutrophil-mediated vasculitis and granuloma. The presence of intracranial parenchymal space-occupying lesions is rarely seen in GPA patients. In this manuscript, we report a case of GPA with granuloma of the fourth ventricle accompanied by obstructive hydrocephalus. Treatment with glucocorticoids (GCs) and multiple immunosuppressants cyclophosphamide (CYC), mycophenolate mofetil (MMF), and rituximab (RTX) showed poor efficacy in this case. After removal of the granuloma by craniotomy, GPA relapsed within 3 months. Under the premise of GC and MMF treatment combined with intrathecal injection of dexamethasone (DXM) and methotrexate (MTX), the intracranial granuloma gradually shrank, and the patient's general condition was alleviated, showing that this is an effective treatment method. Key Points • To date, there are few reports of granulomatous vasculitis combined with granuloma of the fourth ventricle, and our case is the second. • In this case, multiple immunosuppressants and rituximab were ineffective treatments, and the intracranial granuloma was effectively controlled by intrathecal injection of dexamethasone (DXM) and methotrexate (MTX). • Based on this report, it can be suggested that intrathecal injection is effective in treating patients with GPA and central nervous system involvement, but large-scale sample studies are needed.

A near-infrared fluorescent probe for viscosity: Differentiating cancer cells from normal cells and dual-modal imaging in tumor mice.

As a basic parameter of the intracellular microenvironment, viscosity is closely related to the development of cancer. Thus, it is necessary to utilize a sensitive tool to visualize the viscosity in tumor cells and mice, which is helpful for the diagnosis of cancer. Herein, a novel dual-modal probe (IX-V) that has a near-infrared fluorescence (NIRF) and photoacoustic (PA) response to viscosity is synthesized. In low viscosity media, the probe has no fluorescence. With the increase of viscosity, the fluorescence is produced in the near-infrared region due to the inhibition of the TICT process. At the same time, the probe shows different photoacoustic (PA) signals in different viscosity media. Most notably, the viscosity in tumor cells has been imaged successfully by the application of IX-V, and the probe can effectively distinguish cancer cells from normal cells co-cultured in one dish by the difference of fluorescence intensity. In addition, the probe has been used for dual-modal imaging (NIRF and PA) of viscosity in tumor mice, which provides a tool for exploring the relationship between viscosity and diseases. That is to say, IX-V can achieve complementary imaging effects and has great application prospects in the tumor diagnosis.

Associations of fruit, whole grain, and total energy intakes with gut microbiome diversity and composition.

Background: The relationship between fruit, whole grain, and total energy consumption and the gut microbiome in the Chinese population remains unclear. Objective: We investigated the relationship between intakes of fruits, whole grains, and energy, and the diversity and composition of gut microbiota. Design: This cross-sectional study included 167 subjects aged 40-75 years who underwent colonoscopy at Nankai Hospital in Tianjin, China. Each of the participants completed a personal history questionnaire, a 7-day dietary record, and donated a fecal sample. The V3-V4 hypervariable region of the bacterial 16S rRNAgene was amplified and sequenced using Illumina Novaseq. The relationship between diet and gut microbiota was evaluated in terms of both the overall composition and the abundance of specific taxon. Results: Fruits intake was positively related to the abundance of Bacilli, Porphyromonadaceae, Streptococcaceae, Parabacteroides, Streptococcus, and Bilophila in fecal samples. Higher whole grains intake was associated with higher microbial diversity, as measured by Shannon, Simpson, and Chao1 indices. Specifically, there was a significant increase inthe relative abundance of Lachnospiraceae and a decrease in Actinobacteria with increased whole grains intake. Moreover, higher intake of total energy was associated with a lower abundance of Anaerostipes and a higher abundance of Lactobacillales and Acidaminococcus. Conclusion: Whole grains intake was positively associated with gut microbial diversity. Fruits and total energy intake were related to the abundance of specifictaxon (e.g., Bacilli and Acidaminococcus). These findings highlight the potential importance of dietary interventions for modulating gut microbiota composition and promoting overall health.

Research protocol of the efficacy of probiotics for the treatment of alcohol use disorder among adult males: A comparison with placebo and acceptance and commitment therapy in a randomized controlled trial.

BACKGROUND AND AIM: Primarily, this study compares the efficacy of probiotic and acceptance and commitment therapy (ACT) in alleviating the severity of alcohol craving and alcohol use disorder (AUD) among patients who had undergo two weeks of in-patient detoxification. Secondarily, this study compares the efficacy of probiotic and ACT in mitigating the severity of comorbid depression and anxiety symptoms; decreasing serum level of pro-inflammatory cytokines, such as interleukin 1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α); changing the event-related potential in electroencephalogram (EEG) and restoring microbiota flora in the gut of AUD patients. METHODS AND ANALYSIS: Initially, during Phase I of the study, the serum level of IL-1ß, IL-6 and TNF-α; ERP changes in the EEG and fecal microbiota content will be compared between 120 AUD patients and 120 healthy controls. Subsequently in Phase II of the study, 120 AUD patients will be randomized by stratified permuted block randomization into the probiotic, ACT and placebo groups in a 1:1:1 ratio. Participants in the probiotic and placebo groups will be administered one sachet per day of Lactobacillus spp. probiotic and placebo, respectively for 12 weeks. While those in the ACT group will receive one session per week of ACT for 8 weeks. Outcome measures will be administered at four timepoints, such as t0 = baseline assessment prior to intervention, t1 = 8 weeks after intervention began, t2 = 12 weeks after intervention and t3 = 24 weeks after intervention. Primary outcomes are the degrees of alcohol craving, alcohol withdrawal during abstinence and AUD. Secondary outcomes to be assessed are the severity of co-morbid depression and anxiety symptoms; the serum levels of IL-1ß, IL-6 and TNF-α; changes in ERP and fecal microbiota content. TRIAL REGISTRATION NUMBER: NCT05830708 (ClinicalTrials.gov). Registered on April 25, 2023.