Application of individual brain connectome in chronic ischemia: mapping symptoms before and after reperfusion.

How brain functions in the distorted ischemic state before and after reperfusion is unclear. It is also uncertain whether there are any indicators within ischemic brain that could predict surgical outcomes. To alleviate these issues, we applied individual brain connectome in chronic steno-occlusive vasculopathy (CSOV) to map both ischemic symptoms and their postbypass changes. A total of 499 bypasses in 455 CSOV patients were collected and followed up for 47.8 ± 20.5 months. Using multimodal parcellation with connectivity-based and pathological distortion-independent approach, areal MR features of brain connectome were generated with three measurements of functional connectivity (FC), structural connectivity, and PageRank centrality at the single-subject level. Thirty-three machine-learning models were then trained with clinical and areal MR features to obtain acceptable classifiers for both ischemic symptoms and their postbypass changes, among which, 11 were deemed acceptable (AUC > 0.7). Notably, the FC feature-based model for long-term neurological outcomes performed very well (AUC > 0.8). Finally, a Shapley additive explanations plot was adopted to extract important individual features in acceptable models to generate "fingerprints" of brain connectome. This study not only establishes brain connectomic fingerprint databases for brain ischemia with distortion, but also provides informative insights for how brain functions before and after reperfusion.

Taurine ameliorates sensorimotor function by inhibiting apoptosis and activating A2 astrocytes in mice after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a form of severe acute stroke with very high mortality and disability rates. Early brain injury (EBI) and delayed cerebral ischemia (DCI) contribute to the poor prognosis of patients with SAH. Currently, some researchers have started to focus on changes in amino acid metabolism that occur in brain tissues after SAH. Taurine is a sulfur-containing amino acid that is semi-essential in animals, and it plays important roles in various processes, such as neurodevelopment, osmotic pressure regulation, and membrane stabilization. In acute stroke, such as cerebral hemorrhage, taurine plays a neuroprotective role. However, the role of taurine after subarachnoid hemorrhage has rarely been reported. In the present study, we established a mouse model of SAH. We found that taurine administration effectively improved the sensorimotor function of these mice. In addition, taurine treatment alleviated sensorimotor neuron damage and reduced the proportion of apoptotic cells. Furthermore, taurine treatment enhanced the polarization of astrocytes toward the neuroprotective phenotype while inhibiting their polarization toward the neurotoxic phenotype. This study is the first to reveal the relationship between taurine and astrocyte polarization and may provide a new strategy for SAH research and clinical treatment.

Attenuation of neuronal ferroptosis in intracerebral hemorrhage by inhibiting HDAC1/2: Microglial heterogenization via the Nrf2/HO1 pathway.

AIM: The class I histone deacetylases (HDACs) implicate in microglial heterogenization and neuroinflammation following Intracerebral hemorrhage (ICH). Ferroptosis has also been reported in the ICH model. However, the relationship between HDAC1/2's role in microglial heterogenization and neuronal ferroptosis remains unclear. METHODS: In both in vivo and in vitro models of ICH, we used Romidepsin (FK228), a selective HDAC1/2 inhibitor, to investigate its effects on microglial heterogenization and neuronal ferroptosis. In the in vitro ICH model using Hemin, a transwell system was utilized to examine how microglia-driven inflammation and ICH-triggered neuronal ferroptosis interact. Immunostaining, Western blotting and RT-qPCR were used to evaluate the microglial heterogenization and neuronal ferroptosis. Microglial heterogenization, neuronal ferroptosis, and neurological dysfunctions were assessed in vivo ICH mice model performed by autologous blood injection. RESULTS: HDAC1/2 inhibition altered microglial heterogenization after ICH, as showing the reducing neuroinflammation and shifting microglia towards an anti-inflammatory phenotype by immunostaining and qPCR results. HDAC1/2 inhibition reduced ferroptosis, characterized by high ROS and low GPx4 expression in HT22 cells, and reduced iron and lipid deposition post-ICH in vivo. Additionally, the Nrf2/HO1 signaling pathway, especially acetyl-Nrf2, activated in the in vivo ICH model due to HDAC1/2 inhibition, plays a role in regulating microglial heterogenization. Furthermore, HDAC1/2 inhibition improved sensorimotor and histological outcomes post-ICH, offering a potential mechanism against ICH. CONCLUSION: Inhibition of HDAC1/2 reduces neuro-ferroptosis by modifying the heterogeneity of microglia via the Nrf2/HO1 pathway, with a particular focus on acetyl-Nrf2. Additionally, this inhibition aids in the faster removal of hematomas and lessens prolonged neurological impairments, indicating novel approach for treating ICH.

Switching of brain networks across different cerebral perfusion states: insights from EEG dynamic microstate analyses.

The alteration of neural interactions across different cerebral perfusion states remains unclear. This study aimed to fulfill this gap by examining the longitudinal brain dynamic information interactions before and after cerebral reperfusion. Electroencephalogram in eyes-closed state at baseline and postoperative 7-d and 3-month follow-ups (moyamoya disease: 20, health controls: 23) were recorded. Dynamic network analyses were focused on the features and networks of electroencephalogram microstates across different microstates and perfusion states. Considering the microstate features, the parameters were disturbed of microstate B, C, and D but preserved of microstate A. The transition probabilities of microstates A-B and B-D were increased to play a complementary role across different perfusion states. Moreover, the microstate variability was decreased, but was significantly improved after cerebral reperfusion. Regarding microstate networks, the functional connectivity strengths were declined, mainly within frontal, parietal, and occipital lobes and between parietal and occipital lobes in different perfusion states, but were ameliorated after cerebral reperfusion. This study elucidates how dynamic interaction patterns of brain neurons change after cerebral reperfusion, which allows for the observation of brain network transitions across various perfusion states in a live clinical setting through direct intervention.

First application of the novel anaerobic/aerobic/anoxic (AOA) process for advanced nutrient removal in a wastewater treatment plant.

The stringent effluent quality standards in wastewater treatment plants (WWTPs) can effectively mitigate environmental issues such as eutrophication by reducing the discharge of nutrients into water environments. However, the current wastewater treatment process often struggles to achieve advanced nutrient removal while also saving energy and reducing carbon consumption. The first full-scale anaerobic/aerobic/anoxic (AOA) system was established with a wastewater treatment scale of 40,000 m3/d. Over one year of operation, the average TN and TP concentration in the effluent of 7.53 ± 0.81 and 0.37 ± 0.05 mg/L was achieved in low TN/COD (C/N) ratio (average 5) wastewater treatment. The post-anoxic zones fully utilized the internal carbon source stored in pre-anaerobic zones, removing 41.29 % of TN and 36.25 % of TP. Intracellular glycogen (Gly) and proteins in extracellular polymeric substances (EPS) served as potential drivers for post-anoxic denitrification and phosphorus uptake. The sludge fermentation process was enhanced by the long anoxic hydraulic retention time (HRT) of the AOA system. The relative abundance of fermentative bacteria was 31.66 - 55.83 %, and their fermentation metabolites can provide additional substrates and energy for nutrient removal. The development and utilization of internal carbon sources in the AOA system benefited from reducing excess sludge production, energy conservation, and advanced nutrient removal under carbon-limited. The successful full-scale validation of the AOA process provided a potentially transformative technology with wide applicability to WWTPs.

Synchronous Achievement of Advanced Nitrogen Removal and N2O Reduction in the Anoxic Zone in the AOA Process for Low C/N Municipal Wastewater.

Continuous flow processes for the in situ determination of N2O emissions during low C/N municipal wastewater treatment have rarely been reported. The anaerobic/aerobic/anoxic (AOA) process has recently shown promising potential in energy savings and advanced nitrogen removal, but it still needs to be comprehensively explored in relation to N2O emissions for its carbon reduction advantages. In this study, a novel gas-collecting continuous flow reactor was designed to comprehensively evaluate the emissions of N2O from the gas and liquid phases of the AOA process. Additionally, the measures of enhancing endogenous denitrification (ED) and self-enriching anaerobic ammonium oxidation (Anammox) were employed to optimize nitrogen removal and achieve N2O reduction in the anoxic zone. The results showed that enhanced ED coupled with Anammox led to an increase in the nitrogen removal efficiency (NRE) from 67.65 to 81.96%, an enhancement of the NO3- removal rate from 1.76 mgN/(L h) to 3.99 mgN/(L h), and the N2O emission factor in the anoxic zone decreased from 0.28 to 0.06%. Impressively, ED eliminated 91.46 ± 2.47% of the dissolved N2O from the upstream aerobic zone, and the dissolved N2O in the effluent was reduced to less than 0.01 mg/L. This study provides valuable strategies for fully evaluating N2O emissions and N2O reduction from the AOA process.

Disclosing the effect of exogenous betaine on growth of Suaeda salsa (L.) Pall in the Liaohe coastal wetland, North China.

Liaohe coastal wetland has experienced severe degradation of Suaeda salsa (L.) Pall (S. salsa) in recent years. However, the impact of exogenous betaine (GB) on S. salsa growth remains unclear. Therefore, we conducted a natural simulated cultivation in soils of coastal wetland to investigate the effects of GB on S. salsa growth. The results showed that GB increased the height and weight of S. salsa, and meanwhile stimulated the synthesis of endogenous betaine and amino acids, increased soluble sugars and elevated the activity of Na+, K+-ATPase (enhancing osmotic stability). In addition, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased, and malondialdehyde (MDA) and H2O2 decreased correspondingly, thereby improving the antioxidant capacity. Overall, GB application significantly alleviated salt stress and effectively promoted S. salsa growth. This study first indicated the important role of GB in influencing S. salsa growth, offering potential strategies for remediation in coastal wetlands.

Safety and efficacy of remote ischemic conditioning in adult moyamoya disease patients undergoing revascularization surgery: a pilot study.

Background and purpose: Revascularization surgery for patients with moyamoya disease (MMD) is very complicated and has a high rate of postoperative complications. This pilot study aimed to prove the safety and efficacy of remote ischemic conditioning (RIC) in adult MMD patients undergoing revascularization surgery. Methods: A total of 44 patients with MMD were enrolled in this single-center, open-label, prospective, parallel randomized study, including 22 patients assigned to the sham group and 22 patients assigned to the RIC group. The primary outcome was the incidence of major neurologic complications during the perioperative period. Secondary outcomes were the modified Rankin Scale (mRS) score at discharge, at 90 days post-operation, and at 1 year after the operation. The outcome of safety was the incidence of adverse events associated with RIC. Blood samples were obtained to monitor the serum concentrations of cytokines (VEGF, IL-6). Results: No subjects experienced adverse events during RIC intervention, and all patients could tolerate the RIC intervention in the perioperative period. The incidence of major neurologic complications was significantly lower in the RIC group compared with the control group (18.2% vs. 54.5%, P = 0.027). The mRS score at discharge in the RIC group was also lower than the control group (0.86 ± 0.99 vs. 1.18 ± 1.22, P = 0.035). In addition, the serum IL-6 level increased significantly at 7 days after bypass surgery in the control group and the serum level of VEGF at 7 days post-operation in the RIC group. Conclusion: In conclusion, our study demonstrated the neuroprotective effect of RIC by reducing perioperative complications and improving cerebral blood flow in adult MMD patients undergoing revascularization surgery. Thus, RIC seems to be a potential treatment method for MMD. Clinical trial registration: ClinicalTrials.gov, identifier: NCT05860946.

18F-FDG PET and a classifier algorithm reveal a characteristic glucose metabolic pattern in adult patients with moyamoya disease and vascular cognitive impairment.

Vascular cognitive impairment (VCI) is a critical issue in moyamoya disease (MMD). However, the glucose metabolic pattern in these patients is still unknown. This study aimed to identify the metabolic signature of cognitive impairment in patients with MMD using 18F-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) and establish a classifier to identify VCI in patients with MMD. One hundred fifty-two patients with MMD who underwent brain 18F-FDG PET scans before surgery were enrolled and classified into nonvascular cognitive impairment (non-VCI, n = 52) and vascular cognitive impairment (VCI, n = 100) groups according to neuropsychological test results. Additionally, thirty-three health controls (HCs) were also enrolled. Compared to HCs, patients in the VCI group exhibited extensive hypometabolism in the bilateral frontal and cingulate regions and hypermetabolism in the bilateral cerebellum, while patients in the non-VCI group showed hypermetabolism only in the cerebellum and slight hypometabolism in the frontal and temporal regions. In addition, we found that the patients in the VCI group showed hypometabolism mainly in the left basal ganglia compared to those in the non-VCI group. The sparse representation-based classifier algorithm taking the SUVr of 116 Anatomical Automatic Labeling (AAL) areas as features distinguished patients in the VCI and non-VCI groups with an accuracy of 82.4%. This study demonstrated a characteristic metabolic pattern that can distinguish patients with MMD without VCI from those with VCI, namely, hypometabolic lesions in the left hemisphere played a more important role in cognitive decline in patients with MMD.

Diagnosis of Unruptured Intracranial Aneurysm by High-Performance Serum Metabolic Fingerprints.

Unruptured intracranial aneurysm (UIA) is a high-risk cerebrovascular saccular dilatation, the effective medical management of which depends on high-performance diagnosis. However, most UIAs are diagnosed incidentally during neurovascular imaging modalities, which are time-consuming and harmful (e.g., radiation). Serum metabolic fingerprints is a promising alternative for early diagnosis of UIA. Here, nanoparticle enhanced laser desorption/ionization mass spectrometry is applied to obtain high-performance UIA-specific serum metabolic fingerprints. Diagnostic performance with an area-under-the-curve (AUC) of 0.842 (95% confidence interval (CI): 0.783-0.891) is achieved by the constructed machine learning (ML) model, including ML algorithm selection and feature selection. Lactate, glutamine, homoarginine, and 3-methylglutaconic acid are identified as the metabolic biomarker panel, which showed satisfactory diagnosis (AUC of 0.812, 95% CI: 0.727-0.897) and effective growth risk assessment (p<0.05, two-tailed t-test) of UIAs. This work aims to promote the diagnostics of UIAs and metabolic biomarker screening for medical management.

Modified exosomal SIRPα variants alleviate white matter injury after intracerebral hemorrhage via microglia/macrophages.

BACKGROUND: Despite limited efficiency, modulation of microglia/macrophages has shown to attenuate neuroinflammation after intracerebral hemorrhage (ICH). In this context, we evaluated the efficacy of modified exosomal signal regulatory protein α (SIRPα) variants (SIRPα-v Exos) in microglia/macrophages and neuroinflammation-associated white matter injury after ICH. METHODS: SIRPα-v Exos were engineered to block CD47-SIRPα interactions. After obtaining SIRPα-v Exos from lentivirus-infected mesenchymal stem cells, C57BL/6 mice suffering from ICH underwent consecutive intravenous injections of SIRPα-v Exos (6 mg/kg) for 14 days. Afterwards, the volume of hematoma and neurological dysfunctions were assessed in mice continuously until 35 days after ICH. In addition, demyelination, electrophysiology and neuroinflammation were evaluated. Furthermore, the mechanisms of microglial regulation by SIRPα-v Exos were investigated in vitro under coculture conditions. RESULTS: The results demonstrated that the clearance of hematoma in mice suffering from ICH was accelerated after SIRPα-v Exo treatment. SIRPα-v Exos improved long-term neurological dysfunction by ameliorating white matter injury. In addition, SIRPα-v Exos recruited regulatory T cells (Tregs) to promote M2 polarization of microglia/macrophages in the peri-hematoma tissue. In vitro experiments further showed that SIRPα-v Exos regulated primary microglia in a direct and indirect manner in synergy with Tregs. CONCLUSION: Our studies revealed that SIRPα-v Exos could accelerate the clearance of hematoma and ameliorate secondary white matter injury after ICH through regulation of microglia/macrophages. SIRPα-v Exos may become a promising treatment for ICH in clinical practice.

Involvement of the gut-brain axis in vascular depression via tryptophan metabolism: A benefit of short chain fatty acids.

Cerebral hemodynamic dysfunction and hypoperfusion have been found to underlie vascular depression, but whether the gut-brain axis is involved remains unknown. In this study, a rat model of bilateral common carotid artery occlusion (BCCAO) was adopted to mimic chronic cerebral hypoperfusion. A reduced sucrose preference ratio, increased immobility time in the tail suspension test and forced swim test, and compromised gut homeostasis were found. A promoted conversion of tryptophan (Trp) into kynurenine (Kyn) instead of 5-hydroxytryptamine (5-HT) was observed in the hippocampus and gut of BCCAO rats. Meanwhile, 16S ribosomal RNA gene sequencing suggested a compromised profile of the gut SCFA-producing microbiome, with a decreased serum level of SCFAs revealed by targeted metabolomics analysis. With SCFA supplementation, BCCAO rats exhibited ameliorated depressive-like behaviors and improved gut dysbiosis, compared with the salt-matched BCCAO group. Enzyme-linked immunosorbent assays and quantitative RT-PCR suggested that SCFA supplementation suppressed the conversion of Trp to Kyn and rescued the reduction in 5-HT levels in the hippocampus and gut. In addition to inhibiting the upregulation of inflammatory cytokines, SCFA supplementation ameliorated the activated oxidative stress and reduced the number of microglia and the expression of its proinflammatory markers in the hippocampus post BCCAO. In conclusion, our data suggested the participation of the gut-brain axis in vascular depression, shedding light on the neuroprotective potential of treatment with gut-derived SCFAs.

Balance nitrogen and phosphorus efficient removal under carbon limitation in pilot-scale demonstration of a novel anaerobic/aerobic/anoxic process.

Nutrient removal in carbon limited wastewater with high efficiency and energy saving remains a bottleneck for wastewater treatment plants (WWTPs). This study established a pilot-scale anaerobic/aerobic/anoxic (AOA) system with processing capacity of 100 m3/d for the first time. During almost 300 days of stable operation, enhanced nitrogen and phosphorus removal at a C/N of 5 was achieved, and the concentrations of total nitrogen (TN) and total phosphorus (TP) in effluent were 3.60 ± 1.55 and 0.24 ± 0.13 mg/L. Tetrasphaera and Candidatus Competibacter were the dominant phosphorus accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) in the AOA system. Moreover, the low phosphorus release ensured sufficient intracellular carbon storage by endogenous denitrification, which was the critical factor for nitrogen and phosphorus removal in carbon limited wastewater. The denitrification phosphorus removal (DPR) ability further removed phosphorus and prevented secondary phosphorus release to maintain a low phosphorus concentration in effluent. Finally, rapid start-up, high nutrient removal efficiency and low energy consumption make the proposed AOA process suitable for application in newly constructed and renovated WWTPs.

A novel process for anammox pretreatment of municipal wastewater: semi-partial nitrification, biological phosphorus removal and recovery.

Achieving simultaneous semi-partial nitrification and deep phosphorus removal is a preferred process technology for Anammox pretreatment. In this study, semi-partial nitrification combined with in-situ phosphorus recovery (PNPR) was used to treat municipal wastewater. The SRT conflict between the nitrification and phosphorus removal was resolved by in-situ phosphorus recovery every 20 cycles of Anaerobic/Oxid, and a supernatant with more than 10 times the influent phosphorus concentration was obtained, thus achieving bio-enhanced phosphorus removal and recovery with satisfactory semi-partial-nitrification effluent. Interestingly, the results showed that phosphorus removal and recovery process could improve the activity of AOB. The PNPR system's nitrite accumulation rate (NAR) and phosphorus removal rate (PRR) were more than 90% each, whereas the relative abundance of AOB and PAOs increased from 0.04% to 0.74% and from 0.25% to 0.70%, respectively (P < 0.01). Furthermore, on average, the NO2--Neff/NH4+-Neff value was 1.96, which laid the foundation for the subsequent anammox treatment.

Minocycline Alleviates White Matter Injury following Intracerebral Hemorrhage by Regulating CD4+ T Cell Differentiation via Notch1 Signaling Pathway.

Neuroinflammation is a major reason for white matter injury (WMI) after intracerebral hemorrhage (ICH). Apart from microglia/macrophage activation, T cells also play an important role in regulating immune responses after ICH. In a previous study, we have revealed the role of minocycline in modulating microglia/macrophage activation after ICH. However, the exact mechanisms of minocycline in regulating T cells differentiation after ICH are still not well understood. Hence, this study explored the relationship between minocycline and CD4+ T cell differentiation after ICH. Piglet ICH model was used to investigate naive CD4+ T cell differentiation and T cells signal gene activation after ICH with immunofluorescence and whole transcriptome sequencing. Naive CD4+ T cells and primary oligodendrocyte coculture model were established to explore the effect and mechanism of minocycline in modulating CD4+ T cell differentiation after ICH. Flow cytometry was used to indicate CD4+ T cell differentiation after ICH. The mechanism of minocycline in modulating CD4+ T cell differentiation was demonstrated with immunofluorescence and western blot. Double immunostaining of representative CD4+ T cell marker CD3 and different subtype CD4+ T cell assisted proteins (IL17, IL4, Foxp3, and IFNγ) demonstrated naive CD4+ T cell differentiation in piglet after ICH. Whole transcriptome sequencing for perihematomal white matter sorted from piglet brains indicated T cell signal gene activation after ICH. The results of luxol fast blue staining, immunofluorescent staining, and electron microscopy showed that minocycline alleviated white matter injury after ICH in piglets. For our in vitro model, minocycline reduced oligodendrocyte injury and neuroinflammation by regulating CD4+ T cell differentiation after ICH. Moreover, minocycline increased the expression of NOTCH1, ACT1, RBP-J, and NICD1 in cultured CD4+ T cell when stimulated with hemoglobin. Hence, minocycline treatment could modulate naive CD4+ T cell differentiation and attenuate white matter injury via regulating Notch1 signaling pathway after ICH.

Temperature compensated fiber optic magnetic sensor based on the combination interference principle.

In this paper, a highly sensitive temperature compensated fiber optic magnetic field sensor by Sagnac and Mach-Zehnder combination interference (SMZI) is proposed and verified. The sensing structure relies on microstructured exposed core fiber (ECF) filled with ethanol and magnetic fluid (MF). The refractive index of MF and ethanol is affected by the magnetic field and temperature (MFT). SMZI is based on the multimode and birefringence characteristics of ECF. The measurement principle is that the spectra of Sagnac interference and Mach-Zehnder interference have respective sensitivities to the MFT. The magnetic sensitivity can reach 1.17 nm/mT, and the temperature sensitivity is up to -1.93 nm/°C. At the same time, the sensor has good repeatability and low detection limits of 0.41 mT and 0.25°C, respectively. It not only solves the cross-influence of temperature but also makes the spectral analysis more intuitive. The sensor has a broad development prospect in the application of MFT detection.

The microbiota-gut-brain axis participates in chronic cerebral hypoperfusion by disrupting the metabolism of short-chain fatty acids.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) underlies secondary brain injury following certain metabolic disorders and central nervous system (CNS) diseases. Dysregulation of the microbiota-gut-brain axis can exacerbate various CNS disorders through aberrantly expressed metabolites such as short-chain fatty acids (SCFAs). Yet, its relationship with CCH remains to be demonstrated. And if so, it is of interest to explore whether restoring gut microbiota to maintain SCFA metabolism could protect against CCH. RESULTS: Rats subjected to bilateral common carotid artery occlusion (BCCAO) as a model of CCH exhibited cognitive impairment, depressive-like behaviors, decreased gut motility, and compromised gut barrier functions. The 16S ribosomal RNA gene sequencing revealed an abnormal gut microbiota profile and decreased relative abundance of some representative SCFA producers, with the decreased hippocampal SCFAs as the further evidence. Using fecal microbiota transplantation (FMT), rats recolonized with a balanced gut microbiome acquired a higher level of hippocampal SCFAs, as well as decreased neuroinflammation when exposed to lipopolysaccharide. Healthy FMT promoted gut motility and gut barrier functions, and improved cognitive decline and depressive-like behaviors by inhibiting hippocampal neuronal apoptosis in BCCAO rats. Long-term SCFA supplementation further confirmed its neuroprotective effect in terms of relieving inflammatory response and hippocampal neuronal apoptosis following BCCAO. CONCLUSION: Our results demonstrate that modulating the gut microbiome via FMT can ameliorate BCCAO-induced gut dysbiosis, cognitive decline, and depressive-like behaviors, possibly by enhancing the relative abundance of SCFA-producing floras and subsequently increasing SCFA levels. Video abstract.

Anaerobic duration optimization improves endogenous denitrification efficiency by glycogen accumulating organisms enhancement.

Without additional carbon sources, a low endogenous denitrification rate (EDNR) is the critical factor limiting its application in postdenitrification systems. This study optimized the quantitative distribution of anaerobic carbon source removal pathways based on chemometrics for the first time and explored the effect of anaerobic carbon conversion on anoxic endogenous denitrification. Results showed that enhancing the intracellular carbon storage of glycogen accumulating organisms (GAOs) by optimizing anaerobic duration can effectively improve the EDNR. The anaerobic stage was proposed to end at the peak concentration of polyhydroxyalkanoates (PHAs). A two-stage endogenous denitrification system was established to explore the long-term operating performance before and after optimizing anaerobic duration. Results showed that the average NO3- removal rate increased by 25%. qPCR and optimized stoichiometric analyses indicated that the relative abundance and intracellular carbon storage proportion of GAOs increased by 67% and 25%, respectively. This study provided an effective strategy to improve postdenitrification efficiency.