CD3ζ-Mediated Signaling Protects Retinal Ganglion Cells in Glutamate Excitotoxicity of the Retina.

Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.

Oral Administration of Deer Bone Collagen Peptide Can Enhance the Skin Hydration Ability and Antioxidant Ability of Aging Mice Induced by D-Gal, and Regulate the Synthesis and Degradation of Collagen.

Skin problems caused by aging have attracted much attention, and marine collagen peptides have been proved to improve these problems, while mammalian collagen peptides are rarely reported. In this study, fermented deer bone collagen peptide (FCP) and non-fermented deer bone collagen peptide (NCP) were extracted from fermented and non-fermented deer bone, respectively, and their peptide sequences and differential proteins were analyzed using LC-MS/MS technology. After they were applied to aging mice induced with D-gal, the skin hydration ability, antioxidant ability, collagen synthesis, and degradation ability of the mice were studied. The results show that FCP and NCP are mainly peptides that constitute type Ⅰ collagen, and their peptide segments are different. In vivo experiments show that FCP and NCP can improve the richness of collagen fibers in the skin of aging mice; improve the hydration ability of skin; promote the activity of antioxidant-related enzymes; and also show that through the TGF-ß and MAPK pathways, the synthesis and degradation of collagen in skin are regulated. These results show that deer bone collagen peptide can improve skin problems caused by aging, promote skin hydration and antioxidant capacity of aging mice, and regulate collagen synthesis and degradation through the MAPK pathway.

Polypeptide-PNP2 in Corn Cervi Pantotrichum Ameliorates Cognitive Impairment in Alzheimer's Disease Mice by Inhibiting Microglial Cell Activation.

We isolated a polypeptide PNP2 from Corn Cervi Pantotrichum and investigated its effect and mechanism on cognitive impairment in Alzheimer's disease (AD) mice. Morris water maze was used to assess the degree of cognitive impairment in mice. Histopathological changes were detected by H&E staining; the expressions of inflammatory cytokines were assayed by ELISA. Western blotting was employed to detect the protein expressions. PNP2 could improve cognitive impairment, central inflammatory response, and NLRP3 signaling in AD mice. In vitro experiments revealed that PNP2 could suppress the inflammatory response of microglial cells and reduce the activation of NLRP3 in microglial cells, while MCC950 could antagonize the effects of PNP2. Polypeptide component PNP2 in Corn Cervi Pantotrichum can ameliorate central nervous inflammation and cognitive impairment in AD mice by suppressing NLRP3 signaling.

Choline Phosphate-Grafted Nanozymes as Universal Extracellular Vesicle Probes for Bladder Cancer Detection.

Urinary extracellular vesicles (uEVs) are regarded as highly promising liquid-biopsy biomarkers for the early diagnosis and prognosis of bladder cancer (BC). However, detection of uEVs remains technically challenging owing to their huge heterogeneity and ultralow abundance in real samples. We herein present a choline phosphate-grafted platinum nanozyme (Pt@CP) that acts as a universal EV probe for the construction of a high-throughput and high-sensitivity immunoassay, which allowed multiplex profiling of uEV protein markers for BC detection. With the Pt@CP-based immunoassays, three uEV protein markers (MUC-1, CCDC25, and GLUT1) were identified for BC, by which the BC cases (n = 48), cystitis patients (n = 27), and healthy donors (n = 24) were discriminated with high clinical sensitivity and specificity (area under curve = 98.3%). For the BC cases (n = 9) after surgery, the Pt@CP-based immunoassay could report the postoperative residual tumor that cannot be observed by cystoscopy, which is clinically significant for assessing BC recurrence. This work provides generally high sensitivity for EV detection, facilitating the discovery and clinical use of EV-based biomarkers.

Paeoniflorin alleviates depression by inhibiting the activation of NLRP3 inflammasome via promoting mitochondrial autophagy.

Depression ranks among the most common neuropsychiatric disorders globally. Current studies examining the roles of inflammation and mitochondrial autophagy in the antidepressant efficacy of paeoniflorin (PF) are sparse. This study aimed to elucidate PF's antidepressant mechanism by promoting autophagy and inhibiting NLRP3 inflammasome activation using chronic unpredictable mild stimulation (CUMS)-induced C57BL/6 mouse models in vivo and corticosterone (CORT)-induced HT22 cell models in vitro. Results demonstrated that PF enhanced the viability of HT22 cells following CORT exposure, restored mitochondrial membrane potential (MMP), reduced reactive oxygen species accumulation, increased LC3 fluorescence intensity, and suppressed inflammatory cytokine secretion and inflammation activation. Additionally, PF ameliorated depressive behaviors induced by CUMS and improved damage in hippocampal neurons. It also reduced the expression of NLRP3, ASC, Caspase-1, IL-1ß, and the assembly of the NLRP3 inflammasome. Moreover, PF upregulated the expression of autophagy-related proteins in the hippocampus, facilitating the clearance of damaged mitochondria and enhancing autophagy. The role of autophagy in PF's antidepressant effects was further confirmed through the use of the autophagy inhibitor 3-methyladenine (3-MA), which reduced the efficacy of PF. In conclusion, PF effectively improved depressive behaviors in CUMS-induced mice and reduced NLRP3-mediated inflammation both in vivo and in vitro, likely via the induction of autophagy.

Analysis of the mechanism of fibrauretine alleviating Alzheimer's disease based on transcriptomics and proteomics.

The dried rattan stem of the Fibraurea Recisa Pierre plant contains the active ingredient known as fibrauretine (FN). Although it greatly affects Alzheimer's disease (AD), the mechanism of their effects still remains unclear. Proteomics and transcriptomics analysis methods were used in this study to determine the mechanism of FN in the treatment of AD. AD model is used through bilateral hippocampal injection of Aß1-40. After successful modeling, FN was given for 30 days. The results showed that FN could improve the cognitive dysfunction of AD model rats, reduce the expression of Aß and P-Tau, increase the content of acetylcholine and reduce the activity of acetylcholinesterase. The Kyoto Encyclopedia of Genes and Genomes enriched differentially expressed genes and proteins are involved in signaling pathways including metabolic pathway, AD, pathway in cancer, PI3K-AKT signaling pathway, and cAMP signaling pathway. Transcriptomics and proteomics sequencing resulted in 19 differentially expressed genes and proteins. Finally, in contrast to the model group, after FN treatment, the protein expressions and genes associated with the PI3K-AKT pathway were significantly improved in RT-qPCR and Western blot and assays. This is consistent with the findings of transcriptomic and proteomic analyses. Our study found that, FN may improve some symptoms of AD model rats through PI3K-AKT signaling pathway.

PM2.5 exposure promotes the progression of acute kidney injury by activating NLRP3-mediated macrophage inflammatory response.

AIM: We reveal the mechanism of action whereby ambient PM2.5 promotes kidney injury. METHODS: Using C57BL/6 mice, the effects of PM2.5 exposure on the acute kidney injury (AKI) were investigated, including renal function changes, expression of inflammatory cytokines, histopathological changes, as well as activation of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3（NLRP3）. The effects of PM2.5 on renal injury after NLRP3 inhibition were explored using NLRP3 inhibitor (MCC950) and NLRP3 knockout mice. The effects of PM2.5 on the inflammatory response of renal macrophages were investigated at the cellular level. RESULTS: PM2.5 exposure could promote kidney injury, NLRP3 activation and inflammatory response in mice. After using MCC950 and NLRP3 knockout mice, the effects of PM2.5 and the kidney injury could be inhibited. The cellular-level results also suggested that MCC950 could inhibit the effects of PM2.5. CONCLUSION: PM2.5 can promote the progression of AKI and aggravate tissue inflammation through NLRP3, which is an important environmental toxicological mechanism of PM2.5.

Temporomandibular Joint Disorder: An integrated study of the pathophysiology, neural mechanisms, and therapeutic strategies.

OBJECTIVE: The study aims to investigate Temporomandibular Joint Disorder (TMJD) through a interdisciplinary lens, integrating insights from neuroscience, dentistry, and psychology to dissect its complex pathophysiology and neural mechanisms. It focuses on exploring the neurobiological underpinnings of TMJD, emphasizing the role of pain perception, modulation, and the impact of neurophysiological changes on the disorder. DESIGN: This is a comprehensive narrative review of the literature. RESULTS: Research findings pinpoint altered pain perception and modulation processes as central neural mechanisms contributing to TMJD, highlighting the importance of personalized treatment approaches due to the disorder's complexity and patient variability. The study recognizes advances in neuroscience offering new treatment avenues, such as neuromodulation and biofeedback, which provide non-invasive and personalized options. However, it also addresses the challenges in TMJD research, such as the multifaceted nature of the disorder and the need for more comprehensive, interdisciplinary strategies in research and clinical practice. CONCLUSIONS: TMJD is a multifaceted disorder requiring an interdisciplinary approach for effective management. The study stresses the crucial role of neuroscience in understanding and treating TMJD, facilitating the development of innovative treatment strategies. It emphasizes the need for further research, advocating an integrated approach that combines neuroscience, dentistry, and psychology to address TMJD's complexities comprehensively and improve patient care, thereby enhancing the quality of life for affected individuals.

Unlocking the potential of biochar: an iron-phosphorus-based composite modified adsorbent for adsorption of Pb(II) and Cd(II) in aqueous environments and response surface optimization of adsorption conditions.

In this work, iron-phosphorus based composite biochar (FPBC) was prepared by modification with potassium phosphate and iron oxides for the removal of heavy metal ions from single and mixed heavy metal (Pb and Cd) solutions. FTIR and XPS characterization experiments showed that the novel modified biochar had a greater number of surface functional groups compared to the pristine biochar. The maximum adsorption capacities of FPBC for Pb(II) and Cd(II) were 211.66 mg·g-1 and 94.08 mg·g-1 at 293 K. The adsorption of Pb(II) and Cd(II) by FPBC followed the proposed two-step adsorption kinetic model and the Freundlich isothermal adsorption model, suggesting that the mechanism of adsorption of Pb(II) and Cd(II) by FPBC involved chemical adsorption of multiple layers. Mechanistic studies showed that the introduction of -PO4 and -PO3 chemisorbed with Pb(II) and Cd(II), and the introduction of -Fe-O increased the ion exchange with Pb(II) and Cd(II) during the adsorption process and produced precipitates such as Pb3Fe(PO4)3 and Cd5Fe2(P2O7)4. Additionally, the abundant -OH and -COOH groups also participated in the removal of Pb(II) and Cd(II). In addition, FPBC demonstrated strong selective adsorption of Pb(II) in mixed heavy metal solutions. The Response Surface Methodology(RSM) analysis determined the optimal adsorption conditions for FPBC as pH 5.31, temperature 26.01 °C, and Pb(II) concentration 306.30 mg·L-1 for Pb(II). Similarly, the optimal adsorption conditions for Cd(II) were found to be pH 5.66, temperature 39.34 °C, and Cd(II) concentration 267.68 mg·L-1. Therefore, FPBC has the potential for application as a composite-modified adsorbent for the adsorption of multiple heavy metal ions.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

The Structure of Hypersexuality and Its Relation to Impulsivity.

Among the multiple controversies surrounding hypersexuality is the important issue of whether it constitutes a univocal construct. Although an initial study supported its homogeneity, more resent research has identified two separate subcomponents-problematic sexuality and sexual drive. The present survey study addressed this issue in a sample that included both in-person tested college students (n = 69) and online respondents (n = 339). A factor analysis of scales attempting to capture the indicators of each subcomponent of hypersexuality yielded two correlated, but separate factors. Whereas Problematic Sexuality (PS) comprised scales measuring sexual compulsivity, using sex as a coping mechanism, and the negative consequences of sexual behavior, Sexual Drive (SD) was defined by frequent sexual activity, preoccupation with sexual fantasies, a predilection for impersonal sexual behavior, and facile sexual arousal. These two subcomponents of hypersexuality were found to covary with different types of impulsivity, further supporting their discrimination and providing external validation for their differentiation. Contrary to a priori hypotheses, however, PS correlated highly with Callous/Manipulative/Risk-Taking as well as with a predicted Affective Instability/Behavioral Disinhibition factor, suggesting that PS may constitute an equifinality of separate developmental trajectories for those high on both subtypes of hypersexuality.

Palmatine improves cognitive dysfunction in Alzheimer's disease model rats through autophagy pathway and regulation of gut microbiota.

Alzheimer's disease (AD) is a primary degenerative encephalopathy that first appeared as a decline in memory and learning skills. Over time, the condition's severity grew. Palmatine (Pal) alleviates Alzheimer's disease symptoms, which has neuroprotective benefits. Numerous investigations have demonstrated a close relationship among AD and gut structure changes. The aim of the research was investigating whether the improvement of Pal on AD is linked to regulating gut flora and autophagy. First, we used Aß1-40 to induce apoptosis in HT22 cells. After Pal treatment, apoptosis can be improved. Then, We used bilateral intracranial hippocampal injection of Aß1-40 for establishing the AD model, after treatment with Pal, the morris water maze experiment and eight-arm maze test demonstrated that Pal enhanced the AD rats' capacity for learning and memory, HE staining illustrated that Pal improved the morphological abnormalities of brain cells and gut tissue damage. Pal reduced the death of hippocampus neurons, as shown by Nissl staining. Pal substantially reduced Tau hyperphosphorylation and Aß accumulation in the brain, according to immunohistochemical labelling. Pal improved the expression of LC3, Beclin 1, AMPK, and suppressed the expression of mTOR and P62, as validated by RT-qPCR and immunofluorescence labelling. This suggests that Pal's treatment of AD may be associated with the control of the AMPK/mTOR autophagy signalling system. 16S rRNA sequencing and short-chain fatty acids (SCFAs) content detection analysis illustrated that Pal has the potential to enhance the content of SCFAs, reverse the alterations in gut microorganisms. It has been showed by the study that Pal could improve AD by activating autophagy signaling pathway and improving gut barrier changes.

Sortilin-Mediated Inhibition of TREK1/2 Channels in Primary Sensory Neurons Promotes Prediabetic Neuropathic Pain.

Neuropathic pain can occur during the prediabetic stage, even in the absence of hyperglycemia. The presence of prediabetic neuropathic pain (PDNP) poses challenges to the management of individuals with prediabetes. However, the mechanisms underlying this pain remain unclear. This study aims to investigate the underlying mechanism and identify potential therapeutic targets of PDNP. A prediabetic animal model induced by a high-energy diet exhibits both mechanical allodynia and thermal hyperalgesia. Furthermore, hyperexcitability and decreased potassium currents are observed in the dorsal root ganglion (DRG) neurons of these rats. TREK1 and TREK2 channels, which belong to the two-pore-domain K+ channel (K2P) family and play an important role in controlling cellular excitability, are downregulated in DRG neurons. Moreover, this alteration is modulated by Sortilin, a molecular partner that modulates the expression of TREK1. The overexpression of Sortilin negatively affects the expression of TREK1 and TREK2, leading to increased neuronal excitability in the DRG and enhanced peripheral pain sensitivity in rats. Moreover, the downregulation of Sortilin or activation of TREK1 and TREK2 channels by genetic or pharmacological approaches can alleviate PDNP. Therefore, targeting the Sortilin-mediated TREK1/2 pathway may provide a therapeutic approach for ameliorating PDNP.

The risk factors for postoperative temporary vocal cord paralysis after thyroid cancer surgery: an observational retrospective cohort study.

OBJECTIVE: To analyze the potential factors influencing new-onset postoperative transient vocal cord paralysis (VCP) in thyroid cancer patients. METHODS: Case information of 8,340 thyroid cancer patients hospitalized at XX from January 2018 to December 2020 was collected retrospectively and analyzed. The possible influencing factors were analyzed using a chi-square test, rank-sum test, and multiple logistic regression analysis. A nomogram was used to construct the clinical prediction model that was validated in the validation set by ROC, calibration curves, and DCA. RESULTS: The STROCSS guideline was followed to conduct a retrospective cohort study. A total of 8,340 patients, including 1,817 (21.8%) men and 6,523 (78.2%) women, were enrolled in this study. The rate of temporary VCP was 3.6% (308/8,340). Based on the results of postoperative laryngoscopy, the patients were divided into VCP group and non-VCP group. Comparative analysis between the groups revealed that potential factors associated with postoperative transient VCP were tumor location on the dorsal side of the gland (P = 0.042), ultrasound showing a maximum nodal diameter > 1 cm (P = 0.002), multifocal carcinoma (P < 0.001), invasion of surrounding tissue (P = 0.005), lymph node metastases in the central compartment (P = 0.034), lateral cervical lymph node metastasis (P < 0.001), and prolonged operation (P < 0.001). A multiple logistic regression analysis showed that the independent risk factors in postoperative transient VCP were T stage (OR = 1.411, P = 0.013, 95% CI: 1.075-1.853), multifocal carcinoma (OR = 1.532, P = 0.013, 95% CI: 1.095-2.144), and duration of surgery (OR = 1.009, P < 0.001, 95% CI: 1.006-1.012). Finally, a clinical prediction model was established via a nomogram and was validated in the validation set, although its diagnostic efficacy needs to be improved further. CONCLUSION: High T stage, multifocal carcinoma, and prolonged operation time may be independent risk factors for the occurrence of postoperative transient VCP in patients undergoing initial surgery for thyroid cancer.

A review of dose escalation for FDA-approved products treating solid tumors and hematological malignancies in first-in-human trials.

First-in-human (FIH) dose-escalation trials on oncology should prioritize safety and emphasize efficacy. We reviewed the FIH trials of 67 anti-tumor products approved by the Food and Drug Administration between 2018 and 2023 and found that the "3 + 3" design remains the predominant dose-escalation method (66.2%). The number of patients receiving sub-therapeutic doses is positively correlated with the maximum tolerated dose (MTD) or maximum dose (MD) to starting dose ratio (P = 0.056) and the number of dose levels in trials (P < 0.001). In addition, the proportion of products with a high ratio in antibody drugs is higher than that in small molecules (P < 0.001). The MTD or MD exceeded the label dose by three or more doses in 22.03% of the products. In conclusion, optimizing the starting dose selection method, refining the way of determining doses, and finding alternative indicators to replace toxicity as the endpoints will increase the effectiveness and broaden the beneficiary scope.

[Effectiveness of vertical compression of locking plate combined with hollow screws in treatment of Sanders type â ¡ and â ¢ calcaneal fractures].

Objective: To probe into the effectiveness of vertical compression of locking plate combined with hollow screws in the treatment of Sanders type Ⅱ and Ⅲ calcaneal fractures. Methods: The clinical data of 128 patients with Sanders type Ⅱ and Ⅲ calcaneal fractures who were admitted between March 2019 and April 2022 and met the selection criteria were retrospectively analyzed. Among them, 65 patients were treated with locking plate combined with hollow screw vertical compression (study group), and 63 patients were treated with simple locking plate (control group). There was no significant difference in baseline data between the two groups ( P>0.05), such as gender, age, fracture side and Sanders classification, cause of injury, time from injury to operation. The operation time, intraoperative blood loss, hospital stay, and fracture healing time were recorded and compared between the two groups. Before operation and at 12 months after operation, the American Orthopaedic Foot and Ankle Association (AOFAS) score (including total score, pain score, functional score, and alignment score) was used to evaluate the recovery of foot function, and imaging indicators such as calcaneal width, calcaneal height, calcaneal length, Böhler angle, and Gissane angle were measured on X-ray films. Results: All patients were followed up 12 months after operation. There was no significant difference in operation time, intraoperative blood loss, hospital stay, and fracture healing time between the two groups ( P>0.05). Poor wound healing occurred in 1 case in the study group and 2 cases in the control group. At 12 months after operation, there was no significant difference between the two groups in the pre- and post-operative difference of calcaneal length, calcaneal height, Gissane angle, and Böhler angle ( P>0.05). However, the pre- and post-operative difference in calcaneal width in the study group was significantly higher than that in the control group ( P<0.05). The pre- and post-operative difference of AOFAS total score in the study group was significantly higher than that in the control group ( P<0.05), and further analysis showed that the pre- and post-operative difference of pain and function scores in the study group were significantly higher than those in the control group ( P<0.05), while there was no significant difference in the pre- and post-operative difference of force score between the two groups ( P>0.05). Conclusion: Compared with simple locking plate treatment, the treatment of Sanders type Ⅱ and Ⅲ calcaneal fractures with vertical compression of locking plate combined with hollow screws can more effectively improve the width of the subtalar calcaneal articular surface, avoid peroneal longus and brevis impingement, reduce pain, and increase the range of motion of the subtalar joint, and the effectiveness is better.

Stable continuous flow CANDAN process transitioning from anammox UASB reactor by facilitating indigenous nitrite-producing denitrification community.

The emerging nitrogen removal process known as CANDAN (Complete Ammonium and Nitrate removal via Denitratation-Anammox over Nitrite) has been developed in Sequencing Batch Reactors (SBRs). Yet, starting up and maintaining stability in continuous-flow reactors remain challenging. This study explores the feasibility of transitioning the CANDAN process from an anammox-dominated process by introducing appropriate external organics to facilitate indigenous nitrite-producing denitrification community in an Upflow Anaerobic Sludge Blanket (UASB) reactor. 150-day operation results indicate that under feeding rates of domestic wastewater at 0.54 L/h and nitrate-containing wastewater at 1.08 L/h, excellent N removal was achieved, with effluent TN below 10.0 mg N/L. Adding external sodium acetate at a COD/NO3--N = 2.0 triggered denitratation, ex-situ denitrification activity tests showed increased nitrite production rates, maintaining the nitrate-to-nitrite transformation ratio (NTR) above 90 %. Consequently, anammox activity was consistently maintained, dominating Total Nitrogen (TN) removal with a contribution as high as 78.3 ± 8.0 %. Anammox functional bacteria, Brocadia and Kuenenia were identified and showed no decrease throughout the operation, indicating the robustness of the anammox process. Notably, the troublesome of sludge flotation, did not occur, also contributing to sustained outstanding performance. In conclusion, this study advances our understanding of the synergistic interplay between anammox and denitrifying bacteria in the Anammox-UASB system, offering technical insights for establishing a stable continuous-flow CANDAN process for simultaneous ammonium and nitrate removal.