Tetramethylpyrazine nitrone delays the aging process of C. elegans by improving mitochondrial function through the AMPK/mTORC1 signaling pathway.

Aging is characterized as the process of functional decline in an organism from adulthood, often marked by a progressive loss of cellular function and systemic deterioration of multiple tissues. Among the numerous molecular, cellular, and systemic hallmarks associated with aging, mitochondrial dysfunction is considered one of the pivotal factors that initiates the aging process. During aging, mitochondria undergo varying degrees of damage, resulting in impaired energy production and disruption of the homeostatic regulation of mitochondrial quality control systems, which in turn affects cellular energy metabolism and results in cellular dysfunction, accelerating the aging process. AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin complex 1 (mTORC1) are two central kinase complexes responsible for sensing intracellular nutrient levels, regulating metabolic homeostasis, modulating aging and play a crucial role in maintaining the homeostatic balance of mitochondria. Our previous studies found that the novel compound tetramethylpyrazine nitrone (TBN) can protect mitochondria via the AMPK/mTOR pathway in many animal models, extending healthy lifespan through the Nrf2 signaling pathway in nematodes. Building upon this foundation, we have posited a reasonable hypothesis, TBN can improve mitochondrial function to delay aging by regulating the AMPK/mTORC1 signaling pathway. This study focuses on the C. elegans, exploring the impact and underlying mechanisms of TBN on aging and mitochondrial function (especially the mitochondrial quality control system) during the aging process. The present studies demonstrated that TBN extends lifespan of wild-type nematodes and is associated with the AMPK/mTORC1 signaling pathway. TBN elevated ATP and NAD+ levels in aging nematodes while orchestrating mitochondrial biogenesis and mitophagy. Moreover, TBN was observed to significantly enhance normal activities during aging in C. elegans, such as mobility and pharyngeal pumping, concurrently impeding lipofuscin accumulation that were closely associated with AMPK and mTORC1. This study not only highlights the delayed effects of TBN on aging but also underscores its potential application in strategies aimed at improving mitochondrial function via the AMPK/mTOR pathway in C. elegans.

TBN improves motor function and prolongs survival in a TDP-43M337V mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are serious neurodegenerative diseases. Although their pathogenesis is unclear, the abnormal accumulation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological feature that exists in almost all patients. Thus far, there is no drug that can cure ALS/FTLD. Tetramethylpyrazine nitrone (TBN) is a derivative of tetramethylapyrazine, derived from the traditional Chinese medicine Ligusticum chuanxiong, which has been widely proven to have therapeutic effects on models of various neurodegenerative diseases. TBN is currently under clinical investigation for several indications including a Phase II trial of ALS. Here, we explored the therapeutic effect of TBN in an ALS/FTLD mouse model. We injected the TDP-43 M337V virus into the striatum of mice unilaterally and bilaterally, and then administered 30 mg/kg TBN intragastrically to observe changes in behavior and survival rate of mice. The results showed that in mice with unilateral injection of TDP-43M337V into the striatum, TBN improved motor deficits and cognitive impairment in the early stages of disease progression. In mice with bilateral injection of TDP-43M337V into the striatum, TBN not only improved motor function but also prolonged survival rate. Moreover, we show that its therapeutic effect may be through activation of the Akt/mTOR/GSK-3ß and AMPK/PGC-1α/Nrf2 signaling pathways. In summary, TBN is a promising agent for the treatment of ALS/FTLD.

Celastrol attenuates hepatitis C virus translation and inflammatory response in mice by suppressing heat shock protein 90ß.

Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90ß isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R2 = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 µM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90ß, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90ß. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90ß and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90ß. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90ß in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90ß, which could be developed as a lead for the treatment of HSP90ß positive HCV-associated HCC.

Tetramethylpyrazine nitrone TBN extends the lifespan of C. elegans by activating the Nrf2/SKN-1 signaling pathway.

SKN-1, the ortholog of mammalian Nrf2 proteins, is a transcription factor that plays an important role in oxidative stress resistance and longevity. Similar to other defense systems, the Nrf2-mediated stress response is compromised in aging and neurodegenerative diseases. Our previous studies demonstrated that tetramethylpyrazine nitrone (TBN), a derivative of tetramethylpyrazine armed with a potent free radical-scavenging nitrone moiety, exerted multifunctional neuroprotection in neurological and other diseases. However, the ability of TBN to extend a healthy lifespan and its underlying mechanisms of action are not yet clear. C. elegans have become a popular animal model in aging research. Herein, we demonstrate that TBN can extend the lifespan, promote age-associated health indicators, and restore mitochondrial function in C. elegans. TBN also significantly reduced ROS levels and superoxide accumulation in C. elegans. We show that TBN-mediated lifespan extension is SKN-1dependent. The present study provides valuable insights into the mechanisms by which TBN inhibits aging via the Nrf2/SKN-1 pathway in C. elegans.

Design, Synthesis, and Biological Evaluation of Novel Tetramethylpyrazine Derivatives as Potential Neuroprotective Agents.

Oxidative stress plays a crucial role in neurological diseases, resulting in excessive production of reactive oxygen species, mitochondrial dysfunction and cell death. In this work, we designed and synthesized a series of tetramethylpyrazine (TMP) derivatives and investigated their abilities for scavenging free radicals and preventing against oxidative stress-induced neuronal damage in vitro. Among them, compound 22a, consisted of TMP, caffeic acid and a nitrone group, showed potent radical-scavenging activity. Compound 22a had broad neuroprotective effects, including rescuing iodoacetic acid-induced neuronal loss, preventing from tert-butylhydroperoxide (t-BHP)-induced neuronal injury. Compound 22a exerted its neuroprotective effect against t-BHP injury via activation of the phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway. Furthermore, in a rat model of permanent middle cerebral artery occlusion, compound 22a significantly improved neurological deficits, and alleviated the infarct area and brain edema. In conclusion, our results suggest that compound 22a could be a potential neuroprotective agent for the treatment of neurological disease, particularly ischemic stroke.

Synthesis and Biological Evaluation of Danshensu and Tetramethylpyrazine Conjugates as Cardioprotective Agents.

Myocardial ischemia is a primary cause of sudden death worldwide. Numerous active ingredients of traditional Chinese medicines including danshensu (DSS) and tetramethylpyrazine (TMP) have been widely used for the treatment of myocardial ischemia. To enhance their therapeutic efficacy and improve their drugability, in this work, we designed new DSS and TMP conjugates. Their water solubility and protective effects were studied in vitro and in experimental animal models. The new compounds demonstrated higher activities than the positive control agents acetylated danshensu and tetramethylpyrazine conjugate (ADTM) and salvianolic acid B (SAB) in preventing cells from oxidative insult. Among the new compounds, 14, bearing two glycine moieties, was more water soluble. In addition, compound 14 was much more potent in preventing cells from oxidative injury, at least 10- and 20-fold as potent as ADTM and SAB, respectively. The protective effects of compound 14 may be attributed to its anti-radical activity and anti-apoptotic activity. These results suggest that compound 14 is a promising candidate for the treatment of myocardial ischemia.

Tetramethylpyrazine Nitrone Improves Neurobehavioral Functions and Confers Neuroprotection on Rats with Traumatic Brain Injury.

Oxidative stress is one of the major secondary injury mechanisms after traumatic brain injury (TBI). 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical-scavenging nitrone moiety, has been demonstrated promising therapeutic efficacy in ischemic stroke and Parkinson's models. The present study aims to investigate the effects of TBN on behavioral function and neuroprotection in rats subjected to TBI. TBN (90 mg/kg) was administered twice daily for 7 days by intravenous injection following TBI. TBN improved neuronal behavior functions after brain injury, including rotarod test and adhesive paper removal test. Compared with the TBI model group, TBN treatment significantly protected NeuN-positive neurons, while decreased glial fibrillary acidic protein (GFAP)-positive cells. The number of 4-hydroxynonenal (4-HNE)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells around the damaged area after TBI were significantly decreased in the TBN treatment group. In addition, TBN effectively reversed the altered expression of Bcl-2, Bax and caspase 3, and the down-regulation of nuclear factor erythroid-derived 2-like 2 (Nrf-2) and hemeoxygenase-1 (HO-1) proteins expression stimulated by TBI. In conclusion, TBN improves neurobehavioral functions and protects neurons against TBI. This protective effect may be achieved by anti-neuronal apoptosis, alleviating oxidative stress damage and up-regulating Nrf-2 and HO-1 expression.

Synthesis and preliminary biologic activity evaluation of nitric oxide-releasing andrographolide derivatives in RIN-m cells.

Pancreatic ß-cell dysfunction and death are important feature of diabetes mellitus. Beta-cell protection has demonstrated clinical benefits in the treatment of this disease. In the present study, andrographolide derivatives with nitric oxide (NO)-releasing capability were synthesized and their protective effects against tert-butyl hydroperoxide (t-BHP) induced cell damage were investigated in RIN-m cells. Compound 6b was found to release a moderate amount of NO and was more potent than its natural parent andrographolide in inhibiting cell apoptosis. These findings suggested that andrographolide derivatives with NO-releasing capacity may be a potential therapy for diabetes.

Tetramethylpyrazine Nitrone Promotes the Clearance of Alpha-Synuclein via Nrf2-Mediated Ubiquitin-Proteasome System Activation.

Aggregation of α-synuclein (α-syn) and α-syn cytotoxicity are hallmarks of sporadic and familial Parkinson's disease (PD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent enhancement of the expression of the 20S proteasome core particles (20S CPs) and regulatory particles (RPs) increases proteasome activity, which can promote α-syn clearance in PD. Activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) may reduce oxidative stress by strongly inducing Nrf2 gene expression. In the present study, tetramethylpyrazine nitrone (TBN), a potent-free radical scavenger, promoted α-syn clearance by the ubiquitin-proteasome system (UPS) in cell models overexpressing the human A53T mutant α-syn. In the α-syn transgenic mice model, TBN improved motor impairment, decreased the products of oxidative damage, and down-regulated the α-syn level in the serum. TBN consistently up-regulated PGC-1α and Nrf2 expression in tested models of PD. Additionally, TBN similarly enhanced the proteasome 20S subunit beta 8 (Psmb8) expression, which is linked to chymotrypsin-like proteasome activity. Furthermore, TBN increased the mRNA levels of both the 11S RPs subunits Pa28αß and a proteasome chaperone, known as the proteasome maturation protein (Pomp). Interestingly, specific siRNA targeting of Nrf2 blocked TBN's effects on Psmb8, Pa28αß, Pomp expression, and α-syn clearance. In conclusion, TBN promotes the clearance of α-syn via Nrf2-mediated UPS activation, and it may serve as a potentially disease-modifying therapeutic agent for PD.

Tetramethylpyrazine Nitrone alleviates D-galactose-induced murine skeletal muscle aging and motor deficits by activating the AMPK signaling pathway.

Tetramethylpyrazine nitrone (TBN), a novel derivative of tetramethylpyrazine (TMP) designed and synthesized by our group, possesses multi-functional mechanisms of action and displays broad protective effects in vitro and in animal models of age-related brain disorders such as stroke, Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Parkinson's disease (PD). In the present report, we investigated the effects of TBN on aging, specifically on muscle aging and the associated decline of motor functions. Using a D-galactose-induced aging mouse model, we found that TBN could reverse the levels of several senescence and aging markers including p16, p21, ceramides, and telomere length and increase the wet-weight ratio of gastrocnemius muscle tissue, demonstrating its efficacy in ameliorating muscle aging. Additionally, the pharmacological effects of TBN on motor deficits (gait analysis, pole-climbing test and grip strength test), muscle fibrosis (hematoxylin & eosin (HE), Masson staining, and αSMA staining), inflammatory response (IL-1ß, IL-6, and TNF-α), and mitochondrial function (ATP, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were also confirmed in the D-galactose-induced aging models. Further experiments demonstrated that TBN alleviated muscle aging and improved the decline of age-related motor deficits through an AMPK-dependent mechanism. These findings highlight the significance of TBN as a potential anti-aging agent to combat the occurrence and development of aging and age-related diseases.

Metagenomic Analysis of a Continuous-Flow Aerobic Granulation System for Wastewater Treatment.

Aerobic granulation is an emerging process in wastewater treatment that has the potential to accelerate sedimentation of the microbial biomass during secondary treatment. Aerobic granulation has been difficult to achieve in the continuous flow reactors (CFRs) used in modern wastewater treatment plants. Recent research has demonstrated that the alternation of nutrient-abundant (feast) and nutrient-limiting (famine) conditions is able to promote aerobic granulation in a CFR. In this study, we conducted a metagenomic analysis with the objective of characterizing the bacterial composition of the granular biomass developed in three simulated plug flow reactors (PFRs) with different feast-to-famine ratios. Phylogenetic analyses revealed a clear distinction between the bacterial composition of aerobic granules in the pilot simulated PFRs as compared with conventional activated sludge. Larger and denser granules, showing improved sedimentation properties, were observed in the PFR with the longest famine time and were characterized by a greater proportion of bacteria producing abundant extracellular polymeric substances (EPS). Functional metagenomic analysis based on KEGG pathways indicated that the large and dense aerobic granules in the PFR with the longest famine time showed increased functionalities related to secretion systems and quorum sensing, which are characteristics of bacteria in biofilms and aerobic granules. This study contributes to a further understanding of the relationship between aerobic granule morphology and the bacterial composition of the granular biomass.

Tetramethylpyrazine nitrone exerts neuroprotection via activation of PGC-1α/Nrf2 pathway in Parkinson's disease models.

INTRODUCTION: Parkinson's disease (PD) is common neurodegenerative disease where oxidative stress and mitochondrial dysfunction play important roles in its progression. Tetramethylpyrazine nitrone (TBN), a potent free radical scavenger, has shown protective effects in various neurological conditions. However, the neuroprotective mechanisms of TBN in PD models remain unclear. OBJECTIVES: We aimed to investigate TBN's neuroprotective effects and mechanisms in PD models. METHODS: TBN's neuroprotection was initially measured in MPP+/MPTP-induced PD models. Subsequently, a luciferase reporter assay was used to detect peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) promoter activity. Effects of TBN on antioxidant damage and the PGC-1α/Nuclear factor erythroid-2-related factor 2 (Nrf2) pathway were thoroughly investigated. RESULTS: In MPP+-induced cell model, TBN (30-300 µM) increased cell survival by 9.95 % (P < 0.05), 16.63 % (P < 0.001), and 24.09 % (P < 0.001), respectively. TBN enhanced oxidative phosphorylation (P < 0.05) and restored PGC-1α transcriptional activity suppressed by MPP+ (84.30 % vs 59.03 %, P < 0.01). In MPTP-treated mice, TBN (30 mg/kg) ameliorated motor impairment, increased striatal dopamine levels (16.75 %, P < 0.001), dopaminergic neurons survival (27.12 %, P < 0.001), and tyrosine hydroxylase expression (28.07 %, P < 0.01). Selegiline, a positive control, increased dopamine levels (15.35 %, P < 0.001) and dopaminergic neurons survival (25.34 %, P < 0.001). Additionally, TBN reduced oxidative products and activated the PGC-1α/Nrf2 pathway. PGC-1α knockdown diminished TBN's neuroprotective effects, decreasing cell viability from 73.65 % to 56.87 % (P < 0.001). CONCLUSION: TBN has demonstrated consistent effectiveness in MPP+-induced midbrain neurons and MPTP-induced mice. Notably, the therapeutic effect of TBN in mitigating motor deficits and neurodegeneration is superior to selegiline. The neuroprotective mechanisms of TBN are associated with activation of the PGC-1α/Nrf2 pathway, thereby reducing oxidative stress and maintaining mitochondrial function. These findings suggest that TBN may be a promising therapeutic candidate for PD, warranting further development and investigation.

Therapeutic effects of tetramethylpyrazine nitrone in rat ischemic stroke models.

Free radical-mediated neuronal cell damage is an important pathological process in ischemic stroke. We have previously reported a novel dual-functional agent, 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a derivative of tetramethylpyrazine armed with anitrone moiety. In this report, we further evaluate TBN'stherapeutic parameters in a rat middle cerebral artery occlusion (MCAO) model. Its abilities to cross the blood-brain barrier, scavenge free radicals, and inhibit Ca(2+) influx were also investigated. TBN showed significant activity in both the transient MCAO (t-MCAO) and permanent MCAO (p-MCAO) stroke models in the rat. The therapeutic time window is 8 hr in the t-MCAO model. TBN readily crossed the blood-brain barrier and in vitro had strong activity in neutralizing ·OH, O(-)(2)·, and ONOO(-) and significantly decreased intracellular Ca(2+) concentration. TBN is a promising new treatment forischemic stroke, with multiple mechanisms of action. It blocks Ca(2+) overload and neutralizes ·OH, O(-)(2)·, and ONOO(-).

Novel multi-functional nitrones for treatment of ischemic stroke.

Ischemic stroke resulting from obstruction of blood vessels is an enormous public health problem with urgent need for effective therapy. The co-administration of thrombolytic/antiplatelet agent and neuroprotective agent improves therapeutic efficacy and agent possessing both thrombolytic/antiplatelet and antiradical activities provides a promising strategy for the treatment of ischemic stroke. We have previously reported a novel compound, namely TBN, possessing both antiplatelet and antiradical activities, showed significant neuroprotective effect in a rat stroke model. We herein report synthesis of a series of new pyrazine derivatives, and evaluation of their biological activities. Their mechanisms of action were also investigated. Among these new derivatives, compound 21, armed with two nitrone moieties, showed the greatest neuroprotective effects in vitro and in vivo. Compound 21 significantly inhibited ADP-induced platelet aggregation. In a cell free antiradical assay, compound 21 was the most effective agent in scavenging the three most damaging radicals, namely (·)OH, O(2)(·-) and ONOO(-).

Memantine nitrate MN-08 suppresses NLRP3 inflammasome activation to protect against sepsis-induced acute lung injury in mice.

Sepsis is a life-threatening organ dysfunction with devastating consequences, prominent among which is lung damage. Memantine, an N-methyl-D-aspartic acid receptor (NMDAR) antagonist, is able to alleviate acute lung injury (ALI). Nitric oxide (NO) suppresses NLRP3 inflammasome activation against lipopolysaccharide (LPS)-induced septic shock. MN-08, a novel nitrate derivative of memantine, possesses both the ability to antagonize NMDAR and release NO. In the present study, we aimed to investigate the protective effects of MN-08 against LPS-induced systemic inflammation and septic lung injury in mice, and to explore the underlying mechanisms of MN-08 in LPS-induced mice and THP-1 macrophages. MN-08 significantly increased the survival rate of septic mice, alleviated LPS-induced sepsis in mice via improving systemic inflammatory response syndrome and immune dysfunction, and attenuated pulmonary injury and inflammatory infiltration. More importantly, the therapeutic benefit of MN-08 for sepsis was greater than that of memantine and dexamethasone. Mechanistically, MN-08 exerted anti-inflammatory activity through inhibiting nuclear translocation of NF-κB, activation of the MAPK signaling pathway and the signaling transduction of STAT3/NF-κB. In addition, MN-08 suppressed NLRP3 inflammasome activation. Taken together, our studies demonstrate that MN-08 may be a promising therapeutic agent for sepsis-induced acute lung injury.

Tetramethylpyrazine nitrone activates hypoxia-inducible factor and regulates iron homeostasis to improve renal anemia.

Renal anemia is one of the most common complications of chronic kidney disease and diabetic kidney disease. Despite the progress made in recent years, there is still an urgent unmet clinical need for renal anemia treatment. In this research, we investigated the efficacy and mechanism of action of the novel tetramethylpyrazine nitrone (TBN). Animal models of anemia including the streptozotocin (STZ)-induced spontaneously hypertensive rats (SHR) and the cisplatin (CDDP)-induced C57BL/6J mice are established to study the TBN's effects on expression of hypoxia-inducible factor and erythropoietin. To explore the mechanism of TBN's therapeutic effect on renal anemia, cobalt chloride (CoCl2) is used in Hep3B/HepG2 cells to simulate a hypoxic environment. TBN is found to increase the expression of hypoxia-inducible factor HIF-1α and HIF-2α under hypoxic conditions and reverse the reduction of HIFs expression caused by saccharate ferric oxide (SFO). TBN also positively regulates the AMPK pathway. TBN stimulates nuclear transcription and translation of erythropoietin by enhancing the stability of HIF-1α expression. TBN has a significant regulatory effect on several major biomarkers of iron homeostasis, including ferritin, ferroportin (FPN), and divalent metal transporter-1 (DMT1). In conclusion, TBN regulates the AMPK/mTOR/4E-BP1/HIFs pathway, and activates the hypoxia-inducible factor and regulates iron homeostasis to improve renal anemia.

Feast/famine ratio determined continuous flow aerobic granulation.

Plug flow reactors (PFRs) made of multiple completely stirred tank reactors (CSTRs) in series were used to cultivate aerobic granules in real domestic wastewater. Theoretically, changing the number of CSTR chambers in series will change the nature of plug flow, and thus alter the pattern of the feast/famine condition and impact the aerobic granulation progress. Therefore, PFRs were operated in 4-, 6-, and 8-chamber mode under the same gravity selection pressure (a critical settling velocity of 9.75 m h-1) and hydraulic retention time (6.5 h) until steady states were reached to evaluate the effect of the feast/famine condition on continuous flow aerobic granulation. The sludge particle size, circularity, settleability, specific gravity, zone settling velocity, and extracellular polymeric substance contents were analyzed to evaluate the role that a feast/famine regime plays in aerobic granulation. It was found that aerobic granulation failed whenever the feast/famine ratio was greater than 0.5. The results support a conclusion that the feast/famine condition is likely a prerequisite for continuous flow aerobic granulation.

Free ammonia resistance of nitrite-oxidizing bacteria developed in aerobic granular sludge cultivated in continuous upflow airlift reactors performing partial nitritation.

Free ammonia (FA) inhibition has been taken advantage as a strategy to suppress the growth of nitrite-oxidizing bacteria (NOB) in aerobic granules stabilized in a continuous upflow airlift reactor to achieve partial nitritation. However, after nearly 18 months of continuous exposure of aerobic granules to FA in the reactor, the FA inhibition of NOB was proven ineffective, and the partial nitritation gradually shifted to partial nitrification even though the long-term granule structural stability remained excellent under the continuous-flow mode. The extent of NOB resistance to FA inhibition was quantified based on the kinetic response of NOB to various FA concentrations in the form of an uncompetitive inhibition coefficient. It was confirmed that the NOB immobilized in larger granules under longer term exposure to FA tend to become more resistant to FA. Thereby, it was concluded that NOB can develop strong resistance to FA after continuous exposure, and thus, FA inhibition is not a reliable strategy to achieve partial nitritation in mainstream wastewater treatment. PRACTITIONER POINTS: Nitrifying aerobic granules can remain structurally stable in continuous-flow bioreactors. NOB developed free ammonia resistance after 6-month continuous exposure. Larger aerobic granules tended to develop stronger free ammonia resistance. Free ammonia inhibition is not a reliable strategy for mainstream anammox.

Dynamic response of aerobic granular sludge to feast and famine conditions in plug flow reactors fed with real domestic wastewater.

Plug flow reactors (PFRs) approximated by the connection of multiple completely stirred tank reactors (CSTRs) in series were used to achieve continuous flow aerobic granulation in real domestic wastewater. This study revealed, possibly for the first time, that the morphology and characteristics of aerobic granular sludge transformed in the course of a mixed liquor flow through a PFR. The feast zone, located at the front end of the PFR, can quickly develop filamentous structure on the surface of aerobic granular sludge which later disappeared in the famine zone at the back end of the PFR. Detention time from the front to the back end of the PFR was only 6.5 h. During this period the observed sludge morphological change led to sludge settleability fluctuation as much as 66% in zone settling velocity, 16% in specific gravity, and 40% in settled sludge volume. Further analysis revealed these types of sludge morphologies and characteristics were closely related to the specific substrate removal rate profiles of the PFR, i.e., the feast zone might have encouraged filamentous bacteria to extend outward into the bulk solution for soluble substrate, and the famine zone appeared to play an essential role in solidifying the structure of granular sludge structure prior to subjecting it to the gravity selection pressure. It can be inferred from this study that the lack of a famine zone in aerobic granulation reactors can loosen the granule structure and in turn deteriorate granule settleability. For a PFR, a famine zone following the feast zone is essential for maintaining the structural integrity of aerobic granular sludge in a continuous flow wastewater treatment system.

Neuroprotection against 1-Methyl-4-phenylpyridinium-induced cytotoxicity by naturally occurring polydatin through activation of transcription factor MEF2D.

Polydatin is the major active ingredient of Polygonum cuspidatum Sieb. Et Zucc. A recent study indicated that polydatin could protect against substantia nigra dopaminergic degeneration in rodent models associated with Parkinson's disease. However, mechanisms that underlie the neuroprotection of polydatin have not been fully elucidated. In the current study, the neuroprotective effects and detailed mechanisms of action of polydatin were investigated in Parkinson's disease-related cellular models. Polydatin dose- and time-dependently prevented neurotoxicity caused by 1-methyl-4-phenylpyridinium ion (MPP+) in primary cerebellar granule neurons. Moreover, we found that polydatin enhanced the activity of the transcription factor myocyte enhancer factor 2D (MEF2D) at both basal and pathological conditions using luciferase reporter gene assay. Additionally, western blot analysis revealed that polydatin could downregulate glycogen synthase kinase 3ß (GSK3ß), which is a negative regulator of MEF2D. Molecular docking simulations finally suggested an interaction between polydatin and a hydrophobic pocket within GSK3ß. All these results suggest that polydatin prevents MPP+-induced neurotoxicity via enhancing MEF2D through the inhibition of GSK3ß and that treatment with polydatin is worthy of further anti-Parkinson's disease study in future.