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1.
J Food Sci ; 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32458467

RESUMO

The eight biogenic amines (BA), which are histamine (HIS), tryptamine, putrescine, 2-phenylethylamine, cadaverine, tyramine, spermidine, and spermine, were determined in Chub Mackerel under different storage conditions after being freshly caught. The storage time and temperature were varied and the guts either present or removed. This study describes a reverse-phase high-performance liquid chromatography (HPLC) with a fluorescence detector following precolumn derivation with dansyl chloride method for the determination of BA in in Chub Mackerel samples. HIS represented the highest content of BA regardless of the storage temperature and time. During a 24-hr period, the content of HIS reached its highest level of 6,466.63 mg/kg at the storage temperature of 30 °C, whereas it only reached 28.73 mg/kg in 24 hr when the storage temperature was 0 °C, which is way below the standard acceptable threshold level (400 mg/kg). The storage times for the content of HIS to exceed the standard threshold level for HIS at different storage temperatures of 0, 4, 10, 15, 20, and 25 °C were 12, 5, 4 days, 48, 36, 15, and 14 hr, respectively. However, these times were significantly shortened when the viscera was removed from freshly-caught fish before storage. This study not only provides data for monitoring the content of BA, especially HIS, to ensure the safe storage and consumption of freshly-caught Chub Mackerel, but also presents a cost-effective method to extend the storage time of freshly-caught Chub Mackerel. PRACTICAL APPLICATION: This study not only provides data for monitoring the content of BA, especially HIS, to ensure the safe storage and consumption of freshly-caught Chub Mackerel, but also presents a cost-effective method, which is gut-removal during storage, to extend the storage time of freshly-caught Chub Mackerel.

2.
Mol Med Rep ; 22(1): 566-574, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32377702

RESUMO

BRAF inhibitors are some of the most effective drugs against melanoma; however, their clinical application is largely limited by drug resistance. Juglone, isolated from walnut trees, has demonstrated anti­tumour activity. In the present study, it was investigated whether juglone could enhance the responses to a BRAF inhibitor in melanoma cells (A375R and SK­MEL­5R) with an acquired resistance. These cells were treated with juglone alone, BRAF inhibitor (PLX4032) alone, or juglone combined with PLX4032. It was demonstrated that the combination of juglone and PLX4032 had synergistic effects on BRAF inhibitor­resistant melanoma cells. Juglone potentiated PLX4032­induced cytotoxicity and mitochondrial apoptosis in both A375R and SK­MEL­5R cells, which was accompanied by a decline in mitochondrial membrane potential and a decrease in Bcl­2/Bax ratio. Moreover, juglone combined with PLX4032 markedly increased the intracellular level of reactive oxygen species (ROS) and activated p38 and p53, as compared with juglone alone or PLX4032 alone. Pre­treatment with N­acetyl­L­cysteine, a ROS scavenger, completely reversed the cytotoxicity induced by juglone combined with PLX4032. In conclusion, juglone potentiated BRAF inhibitor­induced apoptosis in resistant melanoma cells, and these effects occurred partially through ROS and the p38­p53 pathway, suggesting the potential of juglone as a sensitizer to BRAF inhibitors in the treatment of melanoma.

3.
Aging (Albany NY) ; 122020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32445555

RESUMO

Articular cartilage defects are common in the clinic but difficult to treat. Exploring the chondrogenic molecular mechanisms of mesenchymal stem cells (MSCs) is of great theoretical interest and industrial significance. Bone morphogenetic protein 2 (BMP2) is a key factor that induces cartilage differentiation and can induce stem cell chondrogenic differentiation. However, the oxidative stress in the microenvironment during cartilage injury and degeneration inhibits cartilage regeneration and homeostasis. Silent mating type information regulator 2 homolog-1 (SIRT1) is an important histone deacetylase that regulates proliferation, differentiation, aging, and inflammation processes; moreover, it is an essential factor for chondrogenesis. The specific mechanism of SIRT1 in cartilage differentiation and homeostasis is still unclear. First, we investigated whether SIRT1 could coordinate BMP2-induced chondrogenic differentiation. Second, we investigated the protective effect of SIRT1 on BMP2-induced MSCs under oxidative stress. The results showed that SIRT1 could promote BMP2-induced chondrogenic differentiation of MSCs, and reduce the apoptosis and decomposition of extracellular matrix under oxidative stress. In summary, these results suggested that SIRT1 plays an important coordination role in BMP2-induced chondrogenic differentiation of stem cells and cartilage maintenance under oxidative stress, establishing the experimental basis for exploring the use of SIRT1 in cartilage defect repair.

4.
J Phys Chem Lett ; : 3594-3600, 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32310664

RESUMO

The strong spin-orbital coupling and intense optical transition in lead halide perovskites enable facile optical injection and manipulation of spin states potentially useful for spintronics, one manifestation of which is the optical Stark effect (OSE). A strong OSE would benefit from discrete energy levels and a strong band edge transition with concentrated oscillator strength, which can be realized in three-dimensionally confined quantum dots (QDs). This idea, however, has not been explored yet for perovskite materials. Here we report the observation of strong OSE in perovskite colloidal QDs using circularly polarized transient absorption spectroscopy. The large OSE shifts correspond to transition dipoles as large as 52 D, which compares favorably to those recently reported for higher-dimensional perovskites as well as transition metal dichalcogenide monolayers. Colloidal synthesis also allows for facile tuning of the OSE spectral ranges of perovskite QDs via sizes and compositions, and the impact of these parameters on the OSE is examined, providing fundamental insights into the band edge transition of lead halide perovskites.

5.
Biosci Trends ; 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238673

RESUMO

During the COVID-19 outbreak, China made great progress in controlling the epidemic, and the number of confirmed and suspected cases continues to decrease thanks to the various efforts employed. Mobile field hospitals have played a huge role in the centralized management of patients and they have effectively reduced transmission. This article describes some of our experiences operating mobile field hospitals in order to provide a reference and to better inform countries that are dealing with this crisis.

6.
Gene ; 749: 144679, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330536

RESUMO

BACKGROUND: Hepatocellular carcinoma is one of the most common cancers worldwide. HBV-related HCC has characteristics of faster progression and worse prognosis. Previous studies have confirmed that HBx protein plays numbers of important roles in development of HBV-HCC. However, the molecular mechanism of carcinogenicity of HBx is still not well documented. METHODS: Firstly, a HCC cell line over-expressing HBx was established and its function was verified. Subsequently, the differentially expressed genes were detected by transcriptome sequencing technology and use the Western Blot technology to detect the up-regulated genes in HBx overexpressed cells, and the functional correlation of the genes was analyzed. Finally, tissue microarray was used to correlate up-regulated gene with clinical follow-up data to verify correlation with clinical prognosis. RESULTS: Over-expression of HBx could promote cell proliferation, and over-expression of HBx could up-regulate the expression of S100A4 protein. ShRNA experiments showed that HBx promoted cell proliferation by upregulating the expression of S100A4. IFN-α2b can down-regulate the expression of S100A4 and inhibit the proliferation of HCC cells. The expression of S100A4 in cancer was significantly up-regulated compared with adjacent tissues, and was also significantly associated with tumors volume, the expression of PD-L1 and the survival time of patients with HCC. CONCLUSION: In general, S100A4 may be an effective therapeutic target for HBV-HCC. And the connection between S100A4 and HBV are not clear yet. This study may play a guiding role in the future clinical treatment of HCC.

7.
Chem Biodivers ; 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32347603

RESUMO

The in situ application of iChip cultivation in mangrove sediment from Hainan province, China, led to the isolation of a novel bacterial species Gallaecimonas mangrovi HK-28. The extract of G. mangrovi HK-28 exhibited antibiotic activity against the aquatic pathogen Vibrio harveyi, and its chemical constituents were further investigated by bioactivity-guided isolation. Three new diketopiperazines, gallaecimonamides A-C (1-3), were accordingly isolated from the EtOAc extract of the fermentation broth of G. mangrovi HK-28. The planar structures of 1-3 were determined using HRESIMS together with 1D and 2D NMR. The absolute configurations of 1-3 were assigned by optical rotation, NOESY experiment and TDDFT ECD calculations. The in vitro antibacterial and antimalarial activities of 1-3 were assessed. Compound 1 was found to display antibacterial activity against V. harveyi with a MIC value of 50 µM. However, 2 and 3 showed no antibacterial activity against V. harveyi (MIC > 300 µM). In addition, all the isolates did not exhibit any inhibitory activities against V. parahaemolyticus (MIC > 300 µM) and Plasmodium falciparum W2 (EC50 > 100 µg/mL).

8.
Oncol Rep ; 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32319656

RESUMO

Metastatic melanoma is the most aggressive skin cancer. Although BRAF inhibitor treatment has achieved great success in melanoma, resistance develops within 12 months. Icariside II (IS), a natural compound extracted from Herba Epimedii, exerts anticancer properties. In the present study, we determined by MTT, flow cytometry and western blotting, respectively that IS potentiated the PLX4032­induced downregulation of cell viability and increase in apoptosis and autophagy in BRAF inhibitor­resistant melanoma. In addition, we also revealed by flow cytometry and western blotting, respectively, that IS combined with PLX4032 increased mitochondrial and intracellular reactive oxygen species (ROS) generation and subsequently promoted depolarization of mitochondria and release of apoptotic proteins. N­acetyl cysteine (NAC) and glutathione (GSH), ROS scavengers, reversed the IS­induced enhancement of the response to PLX4032. Microphthalmia­associated transcription factor (MITF) and tyrosine­protein kinase Met (c­Met) are well­known factors that contribute to BRAF inhibitor resistance. Furthermore, c­Met is a direct transcriptional target of MITF in melanocytes and melanoma cells. It was also revealed that IS markedly inhibited MITF and c­Met expression partially by increasing ROS production in BRAF inhibitor­resistant melanoma cells.

9.
Sleep Breath ; 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32277395

RESUMO

STUDY OBJECTIVES: Because dexmedetomidine (DEX)-induced sedation mimics non-rapid eye movement (NREM) sleep, its utility in sedating children with REM-predominant disease is unclear. We sought to determine the effectiveness of pediatric drug-induced sleep endoscopy (DISE) using DEX and ketamine for children with REM-predominant OSA, specifically whether or not at least one site of obstruction could be identified. METHODS: A retrospective case series of children without tonsillar hypertrophy undergoing DISE at a tertiary pediatric hospital from 10/2013 through 9/2015 who underwent subsequent surgery to address OSA with polysomnography (PSG) before and after. RESULTS: We included 56 children, mean age 5.6±5.4 years, age range 0.1-17.4 years, mean BMI 20.3±7.4 kg/m2 (76±29 percentile). At least one site of obstruction was identified in all patients, regardless of REM- or NREM-predominance. The mean obstructive apnea-hypopnea index (oAHI) improved (12.6 ± 10.7 to 9.0 ± 14.0 events/h) in children with REM-predominant (P = 0.013) and NREM-predominant disease (21.3 ± 18.9 to 10.3 ± 16.2 events/h) (P = 0.008). The proportion of children with a postoperative oAHI < 5 was 53% and 55% for REM- and NREMpredominant OSA, respectively. Unlike children with NREM-predominant disease, children with REM-predominant disease had significant improvement in the mean saturation nadir (P < 0.001), total sleep time (P = 0.006), and sleep efficiency (P = 0.015). CONCLUSIONS: For children with OSA without tonsillar hypertrophy, DISE using DEX/ketamine was useful to predict at least one site of obstruction, even for those with REM-predominant OSA. DISE-directed outcomes resulted in significant improvements in mean oAHI, total sleep time, sleep efficiency, saturation nadir, and the proportion with oAHI < 5, after surgery for some children with REM-predominant disease.

10.
J Phys Chem Lett ; : 2247-2255, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32119553

RESUMO

Two-dimensional (2D) organic-inorganic hybrid perovskites are promising materials for next-generation optoelectronic devices owning to their structural and functional versatility and enhanced ambient stability. Recent studies have started to focus on engineering the molecular properties of the organic cations to induce inorganic-to-organic energy/charge transfer for new functionalities, yet many puzzles regarding the inorganic-organic interaction mechanisms remain to be resolved. Here we fabricate 2D lead halide perovskites containing naphthalene methylamine (NMA) cations to study naphthalene triplet sensitization by inorganic excitons. We find that triplet sensitization proceeds via a two-step mechanism initiated by subpicosecond hole transfer from the inorganic layer to naphthalene. We also provide spectroscopic evidence for triplet excimer formation, i.e., the association between triplet and ground state molecules. The intensity ratio between the excimer and triplet emissions can be tuned via the percentage of the NMA cations in the organic layer, offering a route to tunable white-light emitters using 2D hybrid perovskites.

11.
Mol Cell Probes ; 51: 101559, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32151764

RESUMO

Intervertebral disc degeneration (IDD) is a main cause of diseases such as discogenic low back pain, cervical and lumbar disc herniation, degenerative spinal stenosis, and lumbar spondylolisthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcription factor, regulates antioxidant genes and induces cellular defense mechanisms against oxidative stress. In this study, the protective effect of plant antioxidant lycopene on nucleus pulposus cells (NPCs) under oxidative stress was investigated. The results indicated that Nrf2 expression decreased in degenerated NPCs. We further found that lycopene was protective in NP tissue under oxidative stress and alleviated oxidative stress-induced apoptosis of degenerative human NPCs via Nrf2. The results also showed that lycopene reduced H2O2-induced decomposition of cartilage extracellular matrix in NPCs. In conclusion, our findings suggested that lycopene may alleviate disc degeneration under oxidative stress through the Nrf2 signaling pathway.

12.
EMBO Rep ; 21(5): e48904, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32212315

RESUMO

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T-cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical "brake" on T-cell activation. Hence, TCR engagement of EGR4-/- T cells leads to enhanced Ca2+ responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca2+ entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti-tumor immunity in EGR4-/- mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T-cell differentiation and function.

13.
Curr Med Sci ; 40(1): 145-154, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166677

RESUMO

Developing the methodologies that allow for safe and effective delivery of therapeutic drugs to target sites is a very important research area in cancer therapy. In this study, polyethylene glycol (PEG)-coated magnetic polymeric liposome (MPL) nanoparticles (NPs) assembled from octadecyl quaternized carboxymethyl chitosan (OQC), PEGylated OQC, cholesterol, and magnetic NPs, and functionalized with epithelial growth factor receptor (EGFR) peptide, were successfully prepared for in-vivo liver targeting. The two-step liver targeting strategy, based on both magnetic force and EGFR peptide conjugation, was evaluated in a subcutaneous hepatocellular carcinoma model of nude mouse. The results showed that EGFR-conjugated MPLs not only accumulated in the liver by magnetic force, but could also diffuse into tumor cells as a result of EGFR targeting. In addition, paclitaxel (PTX) was incorporated into small EGFR-conjugated MPLs (102.0±0.7 nm), resulting in spherical particles with high drug encapsulation efficiency (>90%). The use of the magnetic targeting for enhancing the transport of PTX-loaded EGFR-conjugated MPLs to the tumor site was further confirmed by detecting PTX levels. In conclusion, PTX-loaded EGFR-conjugated MPLs could potentially be used as an effective drug delivery system for targeted liver cancer therapy.

15.
Stem Cell Res Ther ; 11(1): 62, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059748

RESUMO

BACKGROUND: The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs. METHODS: We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3'-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation. RESULTS: We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3'-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone. CONCLUSIONS: Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

16.
Artigo em Inglês | MEDLINE | ID: mdl-32011261

RESUMO

Searching for active modules, i.e., regions showing striking changes in molecular activity in biological networks is important to reveal regulatory and signaling mechanisms of biological systems. Most existing active modules identification methods are based on protein-protein interaction networks or metabolic networks, which require comprehensive and accurate prior knowledge. On the other hand, weighted gene co-expression networks (WGCNs) are purely constructed from gene expression profiles. There is an urgent need to develop an active module identification algorithm for WGCNs to discover regulatory and signaling mechanism associating with a given cellular response. To address this urgent need, we propose a novel algorithm called active modules on the multi-layer weighted network, based on a continuous optimization approach (AMOUNTAIN). The algorithm is capable of identifying active modules not only from single-layer WGCNs but also from multilayer WGCNs such as cross-species and dynamic WGCNs. We AMOUNTAIN to WGCNs constructed from Th17 differentiation gene expression datasets of human and mouse, which include a single layer, a cross-species two-layer and a multilayer dynamic WGCNs. The identified active modules from WGCNs are enriched by known protein-protein interactions, and more importantly, they reveal some interesting and important regulatory and signaling mechanisms of Th17 cell differentiation.

17.
Sensors (Basel) ; 20(3)2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-32012830

RESUMO

The paper presents a review of some of the significant research done on 3D printed mold-based sensors performed in recent times. The utilization of the master molds to fabricate the different parts of the sensing prototypes have been followed for quite some time due to certain distinct advantages. Some of them are easy template preparation, easy customization of the developed products, quick fabrication, and minimized electronic waste. The paper explains the different kinds of sensors and actuators that have been developed using this technique, based on their varied structural dimensions, processed raw materials, designing, and product testing. These differences in the attributes were based on their individualistic application. Furthermore, some of the challenges related to the existing sensors and their possible respective solutions have also been mentioned in the paper. Finally, a market survey has been provided, stating the estimated increase in the annual growth of 3D printed sensors. It also states the type of 3D printing that has been preferred over the years, along with the range of sensors, and their related applications.

18.
Int J Nanomedicine ; 15: 633-645, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32099356

RESUMO

Background: With the increased application of Silver nanoparticles (AgNP), its potential concerns to the health of human beings remain to be defined. This study aims to explore the harmful effects of AgNP on lung tissue in animals and to examine the mechanisms of protection achieved by sodium selenite. Methods: Sprague-Dawley(SD) rats were exposed to AgNP (200 µL,1mg/mL) through a single intratracheal instillation. Sodium selenite (0.2mg/kg) was i.p. injected. Malondialdehyde (MDA) and glutathione (GSH) were measured using a spectrophotometer. Histological outcomes and ultrastructural changes were assessed by hematoxylin and eosin (HE) staining and electronic microscopy. Caspases and mitochondrial fission and fusion markers were measured by Western blotting. Results: The histopathologic findings showed that AgNP significantly increased the thickness of alveolar septa, accumulation of macrophage, and the formation of pulmonary bullae and pulmonary consolidation. Ultrastructural studies showed localization of AgNP inside the mitochondria, hyperplasia and vacuolation of type I and type II alveolar cells, lysis of osmiophilic lamellar bodies, and swollen of the mitochondria. AgNP elevated MDA and reduced GSH levels. AgNP activated caspases-3, increased mitochondrial fission markers Dynamin-related protein 1 (Drp1) and phospho-Drp1(p-Drp1), and decreased fusion proteins optic atrophy 1 (Opa1) and mitofusins 2 (Mfn2). Treatment with sodium selenite for 7 days corrected the AgNP-caused alterations in morphological, ultrastructural, oxidative stress, caspase-3 activation and mitochondrial dynamic imbalance. Conclusion: We conclude that the exposure of AgNP causes lung tissue damage by enhances oxidative stress, activates caspases-3, and triggers mitochondrial dynamic imbalance towards fission. Sodium selenite effectively detoxifies the AgNP-induced damage to the lung tissue by preventing the above alterations.

19.
BMC Evol Biol ; 20(1): 25, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046636

RESUMO

BACKGROUND: Taste is fundamental to diet selection in vertebrates. Genetic basis of sweet taste receptor in the shaping of food habits has been extensively studied in mammals and birds, but scarcely studied in fishes. Grass carp is an excellent model for studying vegetarian adaptation, as it exhibits food habit transition from carnivory to herbivory. RESULTS: We identified six sweet taste receptors (gcT1R2A-F) in grass carp. The four gcT1R2s (gcT1R2C-F) have been suggested to be evolved from and paralogous to the two original gcT1R2s (gcT1R2A and gcT1R2B). All gcT1R2s were expressed in taste organs and mediated glucose-, fructose- or arginine-induced intracellular calcium signaling, revealing they were functional. In addition, grass carp was performed to prefer fructose to glucose under a behavioral experiment. Parallelly, compared with gcT1R2A-F/gcT1R3 co-transfected cells, gcT1R2C-F/gcT1R3 co-transfected cells showed a higher response to plant-specific fructose. Moreover, food habit transition from carnivory to herbivory in grass carp was accompanied by increased gene expression of certain gcT1R2s. CONCLUSIONS: We suggested that the gene expansion of T1R2s in grass carp was an adaptive strategy to accommodate the change in food environment. Moreover, the selected gene expression of gcT1R2s might drive the food habit transition from carnivory to herbivory in grass carp. This study provided some evolutional and physiological clues for the formation of herbivory in grass carp.

20.
Sci Total Environ ; 712: 136409, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-31935543

RESUMO

Analyzing the instream environmental flow demand by coupling the hydrological cycle and the hydrodynamic process with aquatic ecological processes at watershed scale remains one of the most important yet most difficult issues. One or two of the above processes have been the focus in the evaluation of intra-annual ecological water demand in recent studies. In this study, a hydrology-hydrodynamic-habitat model was developed and applied to the Huangshui River basin. A new classification method for instream ecological water demand (IEWD), which considered sensitive species was proposed. The suitable level of IEWD and crucial values with different flow frequencies were analysed, including runoff, water level, water surface width and weighted usable areas (WUA). The results of the study indicated that monthly IEWD had an increasing trend during the flood season and a decreasing trend during the non-flood season in three sections at different suitable levels. With the increase of suitable levels, the range of IEWD in three sections also increased. The IEWD and crucial values were the lowest in March with the smallest range and were the highest from July to October because the amount of precipitation during that period accounted for nearly 84.3% of that of the entire year. Furthermore, the lower the flow frequency in three sections, the higher the suitable levels of IEWD, as well as water level and water surface width every month. When the flow frequency of 90% decreased to 75%, the value of IEWD increased by at least 55% during the wet season and doubled during the dry season. The WUA with the lowest or highest flow frequencies were relatively poor, especially reproduction period. The IEWD and crucial values at different suitable levels agreed with the actual situations. Thus, this study provided a new method for implementing river ecosystem restoration and aquatic ecosystem management.

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