Evaluation of various carbon sources on ammonium assimilation and denitrifying phosphorus removal in a modified anaerobic-anoxic-oxic process from low-strength wastewater.

A pilot-scale continuous-flow modified anaerobic-anoxic-oxic (MAAO) process examined the impact of external carbon sources (acetate, glucose, acetate/propionate) on ammonium assimilation, denitrifying phosphorus removal (DPR), and microbial community. Acetate exhibited superior efficacy in promoting the combined process of ammonia assimilation and DPR, enhancing both to 50.0 % and 60.0 %, respectively. Proteobacteria and Bacteroidota facilitated ammonium assimilation, while denitrifying phosphorus-accumulating organisms (DPAOs) played a key role in nitrogen (N) and phosphorus (P) removal. Denitrifying glycogen-accumulating organisms (DGAOs) aided N removal in the anoxic zone, ensuring stable N and P removal and recovery. Acetate/propionate significantly enhanced DPR (77.7 %) and endogenous denitrification (37.9 %). Glucose favored heterotrophic denitrification (29.6 %) but had minimal impact on ammonium assimilation. These findings provide valuable insights for wastewater treatment plants (WWTPs) seeking efficient N and P removal and recovery from low-strength wastewater.

Integration of ammonium assimilation with denitrifying phosphorus removal for efficient nutrient management in wastewater treatment.

Nutrient removal from sewage is transitioning to nutrient recovery. However, biological treatment technologies to remove and recover nutrients from domestic sewage are still under investigation. This study delved into the integration of ammonium assimilation with denitrifying phosphorus removal (DPR) as a method for efficient nutrient management in sewage treatment. Results indicated this approach eliminated over 80 % of the nitrogen in the influent, simultaneously recovering over 60 % of the nitrogen as the activated sludge through ammonia assimilation, and glycerol facilitated this process. The nitrification/denitrifying phosphorus removal ensured the stability of both nitrogen and phosphorus removal. The phosphorus removal rate exceeded 96 %, and the DPR rate reached over 90 %. Network analysis highlighted a stable community structure with Proteobacteria and Bacteroidota driving ammonium assimilation. The synergistic effect of fermentation bacteria, denitrifying glycogen-accumulating organisms, and denitrifying phosphorus-accumulating organisms contributed to the stability of nitrogen and phosphorus removal. This approach offers a promising method for sustainable nutrient management in sewage treatment.

Integrated Network Pharmacology and Proteomic Analyses of Targets and Mechanisms of Jianpi Tianjing Decoction in Treating Vascular Dementia.

Background: Vascular dementia (VD), associated with cerebrovascular injury, is characterized by severe cognitive impairment. Jianpi Tianjing Decoction (JTD) has been widely used to treat VD. However, its molecular targets and mechanisms of action in this treatment remain unclear. This study integrated network pharmacology and proteomics to identify targets and mechanisms of JTD in the treatment of VD and to provide new insights and goals for clinical treatments. Methods: Systematic network pharmacology was used to identify active chemical compositions, potential targets, and mechanisms of JTD in VD treatment. Then, a mouse model of VD was induced via transient bilateral common carotid artery occlusion to verify the identified targets and mechanisms of JTD against VD using 4D label-free quantitative proteomics. Results: By screening active chemical compositions and potential targets in relevant databases, 187 active chemical compositions and 416 disease-related compound targets were identified. In vivo experiments showed that JTD improved learning and memory in mice. Proteomics also identified 112 differentially expressed proteins in the model and sham groups and the JTD and model groups. Integrating the network pharmacology and proteomics results revealed that JTD may regulate expressions of cytochrome c oxidase subunit 7C, metabotropic glutamate receptor 2, Slc30a1 zinc transporter 1, and apolipoprotein A-IV in VD mice and that their mechanisms involve biological processes like oxidative phosphorylation, regulation of neuron death, glutamate secretion, cellular ion homeostasis, and lipoprotein metabolism. Conclusions: JTD may suppress VD development via multiple components, targets, and pathways. It may thus serve as a complementary treatment option for patients with VD.

Performance and mechanism of modified biological nutrient removal process in treating low carbon-to-nitrogen ratio wastewater.

For solving the challenge of difficult nutrient removal, high running cost and CO2 emission at low carbon-to-nitrogen (C:N) ratio, Bi-Bio-Selector for nitrogen and phosphorus removal (BBSNP) process was developed. Under parallel operation conditions, full-scale BBSNP was less influence by low C:N ratio (3.5-2) than Anaerobic-anoxic-aerobic (AAO) and achieved better nitrogen removal performance. The mechanism of performance advantage in BBSNP was analyzed by mass balance and high throughout sequencing. It demonstrated BBSNP developed unique microbial community at C:N ratio of 2. Higher abundance of Saccharibacteria, Ferruginibacter, Ottowia, Dokdonella, Candidatus_Nitrotoga and Nitrospira in BBSNP was responsible for better chemical oxygen demand (COD) utilization efficiency, denitrification, denitrifying phosphorus removal and nitrification. Meanwhile, under low C:N ratio, BBSNP could save 10% organic carbon and 15% oxygen requirement, reduce 53% running cost and 21% CO2 emission, which had practical value in relieving energy crisis and carbon emission of wastewater treatment plants (WWTPs).

Effect of voltage intensity on the nutrient removal performance and microbial community in the iron electrolysis-integrated aerobic granular sludge system.

The effects of voltage intensity on the nutrient removal performance and microbial community in the iron electrolysis-integrated aerobic granular sludge (AGS) system were investigated over a period of 15 weeks. Results revealed that the application outcomes of iron electrolysis for AGS systems relied on voltage intensity. When a constant voltage of 1.5 V was applied, the sludge granulation was most obviously accelerated with a specific growth rate of the sludge diameter of 0.078 day-1, and the removal efficiencies of total nitrogen (TN) and total phosphorus (TP) increased by 14.1% and 20.2%, respectively, compared to the control reactor (without the iron electrolysis-integration). Moreover, the AGS developed at different voltages included different microbial communities, whose shifts were driven by the Fe content and the average diameter of AGS. Both heterotrophic nitrifiers and mixotrophic denitrifiers were significantly enriched in the AGS developed at 1.5 V, which effectively enhanced TN removal. Together with the response of the functional genes involved in Fe, N, and P metabolism, the electrolytic iron-driven nutrient degradation pathway was further elaborated. Overall, this study clarified the optimum voltage condition when iron electrolysis was integrated into the AGS system, and revealed the enhancement mechanism of this coupling technology on nutrient removal during the treatment of low-strength municipal wastewater.

Effect of fire needle therapy on mild-moderate benign prostatic hyperplasia: Protocol for a randomized controlled pilot trial.

BACKGROUND: Benign prostatic hyperplasia (BPH) is the most common non-cancerous disease of the prostate and leads to lower urinary tract symptoms in middle-aged and elderly males. Fire needle therapy could improve the lower urinary tract symptoms associated with mild-moderate BPH in clinical practice. The aim of the present pilot study is to assess the preliminary effects of fire needle therapy on mild-moderate BPH. METHODS: The present study is a prospective parallel randomized controlled pilot trial. A total of 60 eligible participants will be randomly assigned to a treatment or control group at a 1:1 ratio. The treatment group will receive fire needle therapy and the control group will receive watchful waiting with lifestyle advice and education regarding BPH. Participants will receive intervention for 4 weeks, with a follow-up period of 4 additional weeks. Adverse events will be recorded to assess the safety and tolerability of fire needle therapy for mild-moderate BPH. The primary outcome will be the change in the International Prostate Symptom Score. The secondary outcomes will include the change in the mean number of nightly urinations, the maximum urinary flow rate, the average flow rate, and the prostate volume as measured by a B-mode ultrasound device. All outcome measures will be observed at baseline and at 4 and 8 weeks following the beginning of treatment. DISCUSSION: The present study will provide evidence of the preliminary effects of fire needle therapy on mild-moderate BPH and indicate an optimal sample size for future studies.

Exogenous acyl-homoserine lactones adjust community structures of bacteria and methanogens to ameliorate the performance of anaerobic granular sludge.

Quorum sensing (QS) signalling has been extensively studied in single species populations, activated sludge, biofilm and aerobic granular sludge. However, ecological roles of QS in anaerobic granular sludge, particularly in the content of the relationship between QS signalling and microbial community composition and function, have been rarely reported. Herein, five acyl-homoserine lactones (AHLs) molecules were added in the anaerobic granular sludge system for treating traditional Chinese medicine (TCM) wastewater respectively. The results indicated that the introduction of specific AHLs could enhance the abilities of organic matters removal and methanation in anaerobic granular sludge, and meanwhile, exogenous AHLs played an important role to regulate the concentration of EPS. Sequencing analysis indicated that microbial community structures of bacteria and methanogens changed to varying degrees by adding AHLs. This study suggested that exogenous AHLs could play a role in mediating microbial community structure, thereby enhancing the performance of anaerobic granular sludge. The regulatory mechanism of AHLs on community structure was discussed, and a speculative action model was established. Exogenous regulation by selective enhancement of AHLs-mediated QS in anaerobic granular sludge provided an innovative and attractive strategy for strengthening wastewater treatments.

Evaluation of phase separation in a single-stage vertical anaerobic reactor: Performance and microbial composition analysis.

In order to explore whether the acidogenic phase and methanogenic phase could be separated vertically into a single-stage anaerobic reactor, a controlled double circulation (CDC) anaerobic reactor was proposed for treating traditional Chinese medicine (TCM) wastewater in this study. The results showed that most of the organic pollutants and refractory were removed in the first reaction area where most of the amount of sludge existed. The organic acids were accumulated in the first reaction area, and larger specific methanogenic activity (SMA) and coenzyme F420 values were found in the second reaction area. Bacterial and archaeal community structures in the two reaction areas of the CDC reactor were analyzed by Illumina MiSeq Sequencing, which revealed that the archaeal community showed larger difference compared with the bacterial community. Differences in the performance and microbial composition of the two reaction areas confirmed that phase separation was implemented in the CDC reactor.

Predicting acute toxicity of traditional Chinese medicine wastewater using UV absorption and volatile fatty acids as surrogates.

In this study, the applicability of UV absorbance at 254 nm (UV254) and volatile fatty acids (VFAs) to serve as reliable surrogates to predict acute toxicity of traditional Chinese medicine (TCM) wastewater was investigated. The medicine residues and VFAs were identified as main components of the TCM wastewater, and their individual and joint toxicity assays were operated with luminescent bacteria. The median effective concentration (EC50) values of medicine residues and VFAs were in the range of 26.46-165.55 mg/L and 11.45-20.58 g/L, respectively. The joint toxicity action modes of medicine residues, VFAs and medicine residues-VFAs were identified as additive, additive and synergistic respectively. UV254 and VFAs showed better correlations with acute toxicity according to the correlation analysis, compared with other conventional parameters. The regression model was a good fit for toxic unit (TU50) as a function of UV254 and VFAs according to the stepwise regression method (adjusted R2 = 0.836). Validation of the model to the pilot-scale samples provided satisfactory prediction results in the influent and hydrolysis acidification effluent samples tests, but for EGSB effluent and final effluent samples, the model needed further optimization. Surrogates prediction using UV254 and VFAs provided a valuable and cost-saving tool for rapid or on-line monitoring of acute toxicity of TCM wastewater.

Curcumol allosterically modulates GABA(A) receptors in a manner distinct from benzodiazepines.

Inhibitory A type γ-aminobutyric acid receptors (GABAARs) play a pivotal role in orchestrating various brain functions and represent an important molecular target in neurological and psychiatric diseases, necessitating the need for the discovery and development of novel modulators. Here, we show that a natural compound curcumol, acts as an allosteric enhancer of GABAARs in a manner distinct from benzodiazepines. Curcumol markedly facilitated GABA-activated currents and shifted the GABA concentration-response curve to the left in cultured hippocampal neurons. When co-applied with the classical benzodiazepine diazepam, curcumol further potentiated GABA-induced currents. In contrast, in the presence of a saturating concentration of menthol, a positive modulator for GABAAR, curcumol failed to further enhance GABA-induced currents, suggesting shared mechanisms underlying these two agents on GABAARs. Moreover, the benzodiazepine antagonist flumazenil did not alter the enhancement of GABA response by curcumol and menthol, but abolished that by DZP. Finally, mutations at the ß2 or γ2 subunit predominantly eliminated modulation of recombinant GABAARs by curcumol and menthol, or diazepam, respectively. Curcumol may therefore exert its actions on GABAARs at sites distinct from benzodiazepine sites. These findings shed light on the future development of new therapeutics drugs targeting GABAARs.

Microbial community composition and function in a pilot-scale anaerobic-anoxic-aerobic combined process for the treatment of traditional Chinese medicine wastewater.

Biodegradation of traditional Chinese medicine (TCM) wastewater was investigated in a pilot-scale anaerobic-anoxic-aerobic combined process, which was composed of an expanded granular sludge blanket (EGSB) reactor, a hydrolysis acidification (HA) reactor and a biological contact oxidation (BCO) reactor. In stable stage, the average values of COD and color in the combined process effluent were 45.7mgL-1 and 13 times, respectively. Excellent linear relations (R2>0.915) were achieved between color and UV254 at three color levels. Comprehensive community structures of the combined process were analysed by Illumina MiSeq Sequencing, which revealed that microbial community in the aerobic reactor had the greatest diversity and richness. Bacteroidetes, Firmicutes and Proteobacteria were dominant phyla in the three reactors, and Bacteroidales, Geobacter, ZB2 were the predominant functional microorganisms in the anaerobic, anoxic and aerobic reactors, respectively. Good removal efficiencies and presence of core microorganisms confirmed that the combined process was feasible for treating TCM wastewater.

Effect of heterogeneous Fenton-like pre-treatment on anaerobic granular sludge performance and microbial community for the treatment of traditional Chinese medicine wastewater.

The effect of a heterogeneous Fenton-like pre-treatment on the anaerobic processes, characteristics and microbial community of sludge was investigated for traditional Chinese medicine (TCM) wastewater containing rhein. When the concentrations of rhein were 50mg/L and 100mg/L, the toxic effect was physiological toxicity for anaerobic granular sludge. Using a single double circle (DC) reactor for the treatment of TCM wastewater containing rhein at concentrations of 15-20mg/L, the chemical oxygen demand (COD) removal rate was 69%, and coenzyme F420 was nearly undetectable in the 3D-excitation-emission matrix (EEM) spectra of soluble microbial products (SMP). The abundances of Methanoregula, Methanobacterium, Methanosphaerula were only 5.57%, 2.39% and 1.08% in the DC reactor, respectively. TCM wastewater containing rhein could be successfully treated by the combination of the heterogeneous Fenton-like pre-treatment and the DC reactor processes, and the COD removal rate reached 95%. Meanwhile, the abundances of Methanoregula, Methanobacterium, Methanosphaerula increased to 22.5%, 18.5%, and 13.87%, respectively. For the bacterial community, the abundance of Acidobacteria_Gp6 decreased from 6.99% to 1.07%, while the abundances of Acidobacteria_Gp1 and Acidobacteria_Gp2 increased from 1.61% to 6.55% and from 1.28% to 5.87%, respectively.

Downregulation of nitric oxide by electroacupuncture against hypoxic­ischemic brain damage in rats via nuclear factor­κB/neuronal nitric oxide synthase.

The present study aimed to investigate the role of nitric oxide (NO) against perinatal hypoxic­ischemic brain damage (HIBD) in rats by electroacupuncture (EA) and to examine its potential neuroprotective mechanism. NO content, the number of positive cells, neuronal nitric oxide synthase (nNOS) and nuclear factor­κB (NF­κB) in rat cortex cells were determined. The results demonstrated that treatment with EA significantly downregulated the NO content in the cortex cells (*P<0.05, **P<0.01, compared with the control groups) and alleviated cell damage in the cortex of rats with HIBD. The activator, S­adenosyl­L­methionine and the inhibitor, hydroxylamine of cystathionine­ß­synthase (CBS), aggravated and remitted the hypoxic damage in the cortex cells, respectively. In addition, treatment with EA significantly downregulated the expression of nNOS and NF­κB in the rat cortex cells (*P<0.05, **P<0.01, compared with the control groups). The results also indicated that treatment with EA downregulated the NO content of cortical cells against HIBD via the NF­κB/nNOS pathway and further implied that the hydrogen sulfide/CBS system may be involved in the process. The present study provided a significant reference for the prevention and treatment of HIBD using the EA technique and also described a novel protective mechanism.

Start-up procedures and analyses of sludge characteristics in a novel double circle anaerobic reactor for treating traditional Chinese medicine wastewater.

The start-up procedures and characteristics of the granular sludge from a novel double circle (DC) anaerobic reactor for treating traditional Chinese medicine (TCM) wastewater were investigated. Following a start-up period, the DC reactor demonstrated good chemical oxygen demand removal at 96.87%. The Fourier transform infrared spectra and excitation-emission matrix (EEM) fluorescence spectra from the soluble microbial products of the effluent showed that amino acid and aromatic compounds in TCM wastewater were effectively removed by the DC reactor. The peak of the particle size distribution from the granular sludge in the first reaction area of the DC reactor ranged from 700 to 1500 µm with a mean size of 513 µm, and the mean size of the granular sludge in the second reaction area was approximately 250 µm. The protein-like peak in the EEM fluorescence clearly decreased and the coenzyme F420 of the fluorescence peak clearly increased for the extracellular polymeric substances of the granular sludge.

Neuroprotection of up-regulated carbon monoxide by electrical acupuncture on perinatal hypoxic-ischemic brain damage in rats.

This study investigated the neuroprotection and potential mechanism of carbon monoxide (CO) against perinatal hypoxic-ischemic brain damage in rats by electrical acupuncture (EA). Animal behavior, morphological changes, cystathionine beta-synthase (CBS), hypoxia-inducible factor-1α (HIF-1α), and heme oxygenase-1 (HO-1) expression levels, and CO content in rat cortex cells were determined. Results demonstrated that EA treatment decreased the slope behavior and increased the overhang behavior of perinatal rats. The treatment also decreased the number of positive cells. The activator and inhibitor of CBS aggravated and remitted the hypoxic damage in cortex cells, respectively. EA treatment decreased CBS expression level and increased HO-1 and HIF-1α expression levels in perinatal rat cortex cells. Compared with the control groups, the CO content of cortex cells in the EA treatment group significantly increased (**p < 0.01). We hypothesized that EA treatment increases cortical CO content to protect against hypoxic damage via the hydrogen sulfide/CBS-CO/HO-1-HIF-1α system. This study provided a significant reference for EA therapy and cued a novel protective mechanism for cerebral palsy.

Influence of the organic loading rate on the performance and the granular sludge characteristics of an EGSB reactor used for treating traditional Chinese medicine wastewater.

The effects of the organic loading rate (OLR) on the performance and the granular sludge characteristics of an expanded granular sludge bed (EGSB) reactor used for treating real traditional Chinese medicine (TCM) wastewater were investigated. Over 90% of the COD removal by the EGSB reactor was observed at the OLRs of 4 to 13 kg COD/(m(3) day). However, increasing the OLR to 20 kg COD/(m(3) day) by reducing the hydraulic retention time (HRT 6 h) reduced the COD removal efficiency to 78%. The volatile fatty acid (VFA) concentration was 512.22 mg/L, resulting in an accumulation of VFAs, and propionic acid was the main acidification product, accounting for 66.51% of the total VFAs. When the OLR increased from 10 to 20 kg COD/(m(3) day), the average size of the granule sludge decreased from 469 to 258 µm. There was an obvious reduction in the concentration of Ca(2+) and Mg(2+) in the granular sludge. The visible humic acid-like peak was identified in the three-dimensional excitation-emission matrix (EEM) fluorescence spectra of the soluble microbial products (SMPs). The fatty acid bond, amide II bond, amide III bond, and C-H bond bending were also observed in the Fourier transform infrared (FTIR) spectra of the SMPs. Methanobacterium formicicum, Methanococcus, and Bacteria populations exhibited significant shifts, and these changes were accompanied by an increase in VFA production. The results indicated that a short HRT and high OLR in the EGSB reactor caused the accumulation of polysaccharides, protein, and VFAs, thereby inhibiting the activity of methanogenic bacteria and causing granular sludge corruption.

Maternal obesity caused by overnutrition exposure leads to reversal learning deficits and striatal disturbance in rats.

Maternal obesity caused by overnutrition during pregnancy increases susceptibility to metabolic risks in adulthood, such as obesity, insulin resistance, and type 2 diabetes; however, whether and how it affects the cognitive system associated with the brain remains elusive. Here, we report that pregnant obesity induced by exposure to excessive high fatty or highly palatable food specifically impaired reversal learning, a kind of adaptive behavior, while leaving serum metabolic metrics intact in the offspring of rats, suggesting a much earlier functional and structural defects possibly occurred in the central nervous system than in the metabolic system in the offspring born in unfavorable intrauterine nutritional environment. Mechanically, we found that above mentioned cognitive inflexibility might be associated with significant striatal disturbance including impaired dopamine homeostasis and disrupted leptin signaling in the adult offspring. These collective data add a novel perspective of understanding the adverse postnatal sequelae in central nervous system induced by developmental programming and the related molecular mechanism through which priming of risk for developmental disorders may occur during early life.

Occurrence of PAOI in a low temperature EBPR system.

The occurrence of Accumulibacter Type I (a known phosphorus-accumulating organism, PAO) has received increased attention due to the potential operating benefits associated with their denitrifying activity in enhanced biological phosphorus removal (EBPR) wastewater treatment plants. In this study, after a shift from an enriched glycogen-accumulating organism (GAO) culture (competitors of PAO) to a PAO-enriched system, Accumulibacter Type I (PAO I) became dominant in an anaerobic-aerobic EBPR system fed with acetate and operated at 10°C with a net aerobic solids retention time (SRT) of 6 d. Since Accumulibacter Type II (PAO II) were not detected, the low temperature in combination with the net aerobic SRT applied appeared to have suppressed their growth as well. The stoichiometry of PAO I was in agreement with previous metabolic models, suggesting that it was the main PAO organisms present in previous studies operated under similar conditions. Moreover, under poly-P limiting conditions, PAO I were unable to switch to a GAO-like metabolism at low temperatures. These results contribute to increase the understanding of the physiology, microbial metabolism and microbial ecology of PAO I.

α-asarone from Acorus gramineus alleviates epilepsy by modulating A-type GABA receptors.

Alpha (α)-asarone is a major effective compound isolated from the Chinese medicinal herb Acorus gramineus, which is widely used in clinical practice as an antiepileptic drug; however, its mechanism of action remains unclear. In this study, we have characterized the action of α-asarone on the excitability of rat hippocampal neurons in culture and on the epileptic activity induced by pentylenetetrazole or kainate injection in vivo. Under cell-attached configuration, the firing rate of spontaneous spiking was inhibited by application of α-asarone, which was maintained in the Mg(2+)-free solution. Under whole-cell configuration, α-asarone induced inward currents in a concentration-dependent manner with an EC(50) of 248 ± 33 µM, which was inhibited by a GABA(A) receptor blocker picotoxin and a competitive GABA(A) receptor antagonist bicuculline but not a specific glycine receptor inhibitor strychnine. Measurement of tonic GABA currents and miniature spontaneous inhibitory postsynaptic currents indicated that α-asarone enhanced tonic GABAergic inhibition while left phasic GABAergic inhibition unaffected. In both pentylenetetrazole and kainate seizure models, α-asarone suppressed epileptic activity of mice by prolonging the latency to clonic and tonic seizures and reducing the mortality as well as the susceptibility to seizure in vivo presumably dependent on the activation of GABA(A) receptors. In summary, our results suggest that α-asarone inhibits the activity of hippocampal neurons and produces antiepileptic effect in central nervous system through enhancing tonic GABAergic inhibition.

Subunit-specific inhibition of glycine receptors by curcumol.

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of ß subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.