Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG

Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody

AAV-Delivered Antibody Mediates Significant Protective Effects against SIVmac239 Challenge in the Absence of Neutralizing Activity

Long-term delivery of potent broadly-neutralizing antibodies is a promising approach for the prevention of HIV-1 infection. We used AAV vector intramuscularly to deliver anti-SIV monoclonal antibodies (mAbs) in IgG1 form to rhesus monkeys. Persisting levels of delivered mAb as high as 270 mug/ml were achieved. However, host antibody responses to the delivered antibody were observed in 9 of the 12 monkeys and these appeared to limit the concentration of delivered antibody that could be achieved. This is reflected in the wide range of delivered mAb concentrations that were achieved: 1-270 mug/ml. Following repeated, marginal dose, intravenous challenge with the difficult-to-neutralize SIVmac239, the six monkeys in the AAV-5L7 IgG1 mAb group showed clear protective effects despite the absence of detectable neutralizing activity against the challenge virus. The protective effects included: lowering of viral load at peak height; lowering of viral load at set point; delay in the time to peak viral load from the time of the infectious virus exposure. All of these effects were statistically significant. In addition, the monkey with the highest level of delivered 5L7 mAb completely resisted six successive SIVmac239 i.v. challenges, including a final challenge with a dose of 10 i.v. infectious units. Our results demonstrate the continued promise of this approach for the prevention of HIV-1 infection in people. However, the problem of anti-antibody responses will need to be understood and overcome for the promise of this approach to be effectively realized

Automated radiofrequency-based US measurement of common carotid intima-media thickness in RA patients treated with synthetic vs synthetic and biologic DMARDs

Objective. To compare the carotid intima-media thickness (IMT) assessed with automated radiofrequency-based US in RA patients treated with synthetic vs synthetic and biologic DMARDs and controls. Methods. Ninety-four RA patients and 94 sex-and age-matched controls were prospectively recruited at seven centres. Cardiovascular (CV) risk factors and co-morbidities, RA characteristics and therapy were recorded. Common carotid artery (CCA)-IMT was assessed in RA patients and controls with automated radiofrequency-based US by the same investigator at each centre. Results. Forty-five (47.9%) RA patients had been treated with synthetic DMARDs and 49 (52.1%) with synthetic and biologic DMARDs. There were no significant differences between the RA patients and controls in demographics, CV co-morbidities and CV disease. There were significantly more smokers among RA patients treated with synthetic and biologic DMARDs (P = 0.036). Disease duration and duration of CS and synthetic DMARD therapy was significantly longer in RA patients treated with synthetic and biologic DMARDs (P<0.0005). The mean CCA-IMT was significantly greater in RA patients treated only with synthetic DMARDs than in controls [591.4 (98.6) vs 562.1 (85.8); P = 0.035] and in RA patients treated with synthetic and biologic DMARDs [591.4 (98.6) vs 558.8 (95.3); P = 0.040). There was no significant difference between the mean CCA-IMT in RA patients treated with synthetic and biologic DMARDs and controls (P = 0.997). Conclusion. Our results suggest that radiofrequency-based measurement of CCA-IMT can discriminate between RA patients treated with synthetic DMARDs vs RA patients treated with synthetic and biologic DMARDs

Anti-drug Antibody Responses Impair Prophylaxis Mediated by AAV-Delivered HIV-1 Broadly Neutralizing Antibodies

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs

Molecular evidence of adenosine deaminase linking adenosine A2A receptor and CD26 proteins

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits

Glycoengineering of AAV-delivered monoclonal antibodies yields increased ADCC activity

The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo, current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies. [Display omitted] Termini et al. describe the creation and validation of a novel glycoengineered-AAV vector for the delivery of anti-HIV antibodies. These AAV are capable of knocking down fucosyltransferase-8 following transduction and, when combined with antibody-Fc mutations, produce broadly neutralizing antibodies with 40- to 60-fold higher ADCC

Water resources sustainability model for wetland conservation based on anonymous expert elicitation

[EN] Wetlands play a key role in preserving biodiversity and preventing climate change. Their conservation poses an important and pressing challenge. In the Mediterranean region, one of the key threats to wetland survival is the lack of water due to competition for resources. The selection of the most sustainable water resources for wetland conservation is a complex elicitation problem. A novel Water Resources Sustainability Model (WRSM) focused on water quality has been developed to support the decision-making. This collaborative elicitation model is based on the analytical hierarchy process and uses the reference environmental status of the wetland. The model can be used to discriminate which water resources are more sustainable for the conservation of the wetland. The WRSM has been applied successfully to Las Tablas de Daimiel National Park. The framework enables establishing priorities when analyzing in terms of water quality any surface, recycled or underground water resources.Canto-Perello, J.; Benitez-Navio, A.; Martín Utrillas, MG.; Martinez-Leon, J.; Curiel Esparza, J. (2021). Water resources sustainability model for wetland conservation based on anonymous expert elicitation. Environmental Modelling & Software. 136:1-12. https://doi.org/10.1016/j.envsoft.2020.104952S112136Aguilera, H., & Merino, L. M. (2018). Data on chemical composition of soil and water in the semiarid wetland of Las Tablas de Damiel National Park (Spain) during a drought period. Data in Brief, 19, 2481-2486. doi:10.1016/j.dib.2018.04.085Aguilera, H., Moreno, L., Wesseling, J. G., Jiménez-Hernández, M. E., & Castaño, S. (2016). Soil moisture prediction to support management in semiarid wetlands during drying episodes. CATENA, 147, 709-724. doi:10.1016/j.catena.2016.08.007Alafifi, A. H., & Rosenberg, D. E. (2020). Systems modeling to improve river, riparian, and wetland habitat quality and area. Environmental Modelling & Software, 126, 104643. doi:10.1016/j.envsoft.2020.104643Alvarez Etxeberria, I., Garayar, A., & Calvo Sánchez, J. A. (2015). Development of sustainability reports for farming operations in the Basque Country using the Delphi method. Revista de Contabilidad, 18(1), 44-54. doi:10.1016/j.rcsar.2014.03.004Bilotta, G. S., & Brazier, R. E. (2008). Understanding the influence of suspended solids on water quality and aquatic biota. Water Research, 42(12), 2849-2861. doi:10.1016/j.watres.2008.03.018Bilotta, G. S., Burnside, N. G., Cheek, L., Dunbar, M. J., Grove, M. K., Harrison, C., … Davy-Bowker, J. (2012). Developing environment-specific water quality guidelines for suspended particulate matter. Water Research, 46(7), 2324-2332. doi:10.1016/j.watres.2012.01.055Blaas, H., & Kroeze, C. (2016). Excessive nitrogen and phosphorus in European rivers: 2000–2050. Ecological Indicators, 67, 328-337. doi:10.1016/j.ecolind.2016.03.004Caen, A., Latour, D., & Mathias, J. D. (2019). Dynamical effects of retention structures on the mitigation of lake eutrophication. Environmental Modelling & Software, 119, 309-326. doi:10.1016/j.envsoft.2019.06.012Camargo, J. A., & Alonso, Á. (2006). Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environment International, 32(6), 831-849. doi:10.1016/j.envint.2006.05.002Canto-Perello, J., Martinez-Leon, J., Curiel-Esparza, J., & Martin-Utrillas, M. (2017). Consensus in prioritizing river rehabilitation projects through the integration of social, economic and landscape indicators. Ecological Indicators, 72, 659-666. doi:10.1016/j.ecolind.2016.09.004Canto-Perello, J., Morera-Escrich, J. L., Martin-Utrillas, M., & Curiel-Esparza, J. (2018). Restoration prioritization framework for roadway high cut slopes to reverse land degradation and fragmentation. Land Use Policy, 71, 470-479. doi:10.1016/j.landusepol.2017.11.020Cirujano, S., Casado, C., Bernués, M., & Camargo, J. A. (1996). Ecological study of Las Tablas de Daimiel National Park (Ciudad Real, central Spain): Differences in water physico-chemistry and vegetation between 1974 and 1989. Biological Conservation, 75(3), 211-215. doi:10.1016/0006-3207(95)00079-8Curiel-Esparza, J., Gonzalez-Utrillas, N., Canto-Perello, J., & Martin-Utrillas, M. (2015). Integrating climate change criteria in reforestation projects using a hybrid decision-support system. Environmental Research Letters, 10(9), 094022. doi:10.1088/1748-9326/10/9/094022Curiel-Esparza, J., Mazario-Diez, J. L., Canto-Perello, J., & Martin-Utrillas, M. (2016). Prioritization by consensus of enhancements for sustainable mobility in urban areas. Environmental Science & Policy, 55, 248-257. doi:10.1016/j.envsci.2015.10.015Curiel-Esparza, J., Reyes-Medina, M., Martin-Utrillas, M., Martinez-Garcia, M. P., & Canto-Perello, J. (2019). Collaborative elicitation to select a sustainable biogas desulfurization technique for landfills. Journal of Cleaner Production, 212, 1334-1344. doi:10.1016/j.jclepro.2018.12.095Dong, Y., Zhang, G., Hong, W.-C., & Xu, Y. (2010). Consensus models for AHP group decision making under row geometric mean prioritization method. Decision Support Systems, 49(3), 281-289. doi:10.1016/j.dss.2010.03.003Forman, E., & Peniwati, K. (1998). Aggregating individual judgments and priorities with the analytic hierarchy process. European Journal of Operational Research, 108(1), 165-169. doi:10.1016/s0377-2217(97)00244-0Gu, S., Gruau, G., Dupas, R., Petitjean, P., Li, Q., & Pinay, G. (2019). Respective roles of Fe-oxyhydroxide dissolution, pH changes and sediment inputs in dissolved phosphorus release from wetland soils under anoxic conditions. Geoderma, 338, 365-374. doi:10.1016/j.geoderma.2018.12.034Haas, M. B., Guse, B., & Fohrer, N. (2017). Assessing the impacts of Best Management Practices on nitrate pollution in an agricultural dominated lowland catchment considering environmental protection versus economic development. Journal of Environmental Management, 196, 347-364. doi:10.1016/j.jenvman.2017.02.060Thi Minh Hanh, P., Sthiannopkao, S., The Ba, D., & Kim, K.-W. (2011). Development of Water Quality Indexes to Identify Pollutants in Vietnam’s Surface Water. Journal of Environmental Engineering, 137(4), 273-283. doi:10.1061/(asce)ee.1943-7870.0000314Hes, E. M., & van Dam, A. A. (2019). Modelling nitrogen and phosphorus cycling and retention in Cyperus papyrus dominated natural wetlands. Environmental Modelling & Software, 122, 104531. doi:10.1016/j.envsoft.2019.104531Juston, J. M., & Kadlec, R. H. (2019). Data-driven modeling of phosphorus (P) dynamics in low-P stormwater wetlands. Environmental Modelling & Software, 118, 226-240. doi:10.1016/j.envsoft.2019.05.002Juwana, I., Muttil, N., & Perera, B. J. C. (2012). Indicator-based water sustainability assessment — A review. Science of The Total Environment, 438, 357-371. doi:10.1016/j.scitotenv.2012.08.093Kløve, B., Allan, A., Bertrand, G., Druzynska, E., Ertürk, A., Goldscheider, N., … Schipper, P. (2011). Groundwater dependent ecosystems. Part II. Ecosystem services and management in Europe under risk of climate change and land use intensification. Environmental Science & Policy, 14(7), 782-793. doi:10.1016/j.envsci.2011.04.005Koskiaho, J., & Puustinen, M. (2019). Suspended solids and nutrient retention in two constructed wetlands as determined from continuous data recorded with sensors. Ecological Engineering, 137, 65-75. doi:10.1016/j.ecoleng.2019.04.006Lefebvre, G., Redmond, L., Germain, C., Palazzi, E., Terzago, S., Willm, L., & Poulin, B. (2019). Predicting the vulnerability of seasonally-flooded wetlands to climate change across the Mediterranean Basin. Science of The Total Environment, 692, 546-555. doi:10.1016/j.scitotenv.2019.07.263Zhuang, L.-L., Yang, T., Zhang, J., & Li, X. (2019). The configuration, purification effect and mechanism of intensified constructed wetland for wastewater treatment from the aspect of nitrogen removal: A review. Bioresource Technology, 293, 122086. doi:10.1016/j.biortech.2019.122086Liu, Z., Tai, P., Li, X., Kong, L., Matthews, T. G., Lester, R. E., & Mondon, J. A. (2019). Deriving site-specific water quality criteria for ammonia from national versus international toxicity data. Ecotoxicology and Environmental Safety, 171, 665-676. doi:10.1016/j.ecoenv.2018.12.078Lobanova, A., Liersch, S., Tàbara, J. D., Koch, H., Hattermann, F. F., & Krysanova, V. (2017). Harmonizing human-hydrological system under climate change: A scenario-based approach for the case of the headwaters of the Tagus River. Journal of Hydrology, 548, 436-447. doi:10.1016/j.jhydrol.2017.03.015Martin-Utrillas, M., Reyes-Medina, M., Curiel-Esparza, J., & Canto-Perello, J. (2014). Hybrid method for selection of the optimal process of leachate treatment in waste treatment and valorization plants or landfills. Clean Technologies and Environmental Policy, 17(4), 873-885. doi:10.1007/s10098-014-0834-4Man, Y., Hu, Y., & Ren, J. (2019). Forecasting COD load in municipal sewage based on ARMA and VAR algorithms. Resources, Conservation and Recycling, 144, 56-64. doi:10.1016/j.resconrec.2019.01.030Martinez-Martinez, E., Nejadhashemi, A. P., Woznicki, S. A., Adhikari, U., & Giri, S. (2015). Assessing the significance of wetland restoration scenarios on sediment mitigation plan. Ecological Engineering, 77, 103-113. doi:10.1016/j.ecoleng.2014.11.031Mayo, A. W., Muraza, M., & Norbert, J. (2018). Modelling nitrogen transformation and removal in mara river basin wetlands upstream of lake Victoria. Physics and Chemistry of the Earth, Parts A/B/C, 105, 136-146. doi:10.1016/j.pce.2018.03.005Moreno, L., Jiménez, M.-E., Aguilera, H., Jiménez, P., & de la Losa, A. (2010). The 2009 Smouldering Peat Fire in Las Tablas de Daimiel National Park (Spain). Fire Technology, 47(2), 519-538. doi:10.1007/s10694-010-0172-yNagisetty, R. M., Flynn, K. F., & Uecker, D. (2019). Dissolved oxygen modeling of effluent-dominated macrophyte-rich Silver Bow Creek. Ecological Modelling, 393, 85-97. doi:10.1016/j.ecolmodel.2018.12.009Navarro, V., García, B., Sánchez, D., & Asensio, L. (2011). An evaluation of the application of treated sewage effluents in Las Tablas de Daimiel National Park, Central Spain. Journal of Hydrology, 401(1-2), 53-64. doi:10.1016/j.jhydrol.2011.02.008Norouzian-Maleki, S., Bell, S., Hosseini, S.-B., & Faizi, M. (2015). Developing and testing a framework for the assessment of neighbourhood liveability in two contrasting countries: Iran and Estonia. Ecological Indicators, 48, 263-271. doi:10.1016/j.ecolind.2014.07.033Novakowski, N., & Wellar, B. (2008). Using the Delphi Technique in Normative Planning Research: Methodological Design Considerations. Environment and Planning A: Economy and Space, 40(6), 1485-1500. doi:10.1068/a39267Okoli, C., & Pawlowski, S. D. (2004). The Delphi method as a research tool: an example, design considerations and applications. Information & Management, 42(1), 15-29. doi:10.1016/j.im.2003.11.002O’Neil, G. L., Goodall, J. L., Behl, M., & Saby, L. (2020). Deep learning Using Physically-Informed Input Data for Wetland Identification. Environmental Modelling & Software, 126, 104665. doi:10.1016/j.envsoft.2020.104665Pérez-Martín, M. A., Estrela, T., & del-Amo, P. (2016). Measures required to reach the nitrate objectives in groundwater based on a long-term nitrate model for large river basins (Júcar, Spain). Science of The Total Environment, 566-567, 122-133. doi:10.1016/j.scitotenv.2016.04.206Pottinger, T. G. (2017). Modulation of the stress response in wild fish is associated with variation in dissolved nitrate and nitrite. Environmental Pollution, 225, 550-558. doi:10.1016/j.envpol.2017.03.021Prăvălie, R., Patriche, C., & Bandoc, G. (2017). Quantification of land degradation sensitivity areas in Southern and Central Southeastern Europe. New results based on improving DISMED methodology with new climate data. CATENA, 158, 309-320. doi:10.1016/j.catena.2017.07.006Restuccia, F., Huang, X., & Rein, G. (2017). Self-ignition of natural fuels: Can wildfires of carbon-rich soil start by self-heating? Fire Safety Journal, 91, 828-834. doi:10.1016/j.firesaf.2017.03.052Rivers-Moore, N. A., Dallas, H. F., & Morris, C. (2013). Towards setting environmental water temperature guidelines: A South African example. Journal of Environmental Management, 128, 380-392. doi:10.1016/j.jenvman.2013.04.059Rusydi, A. F. (2018). Correlation between conductivity and total dissolved solid in various type of water: A review. IOP Conference Series: Earth and Environmental Science, 118, 012019. doi:10.1088/1755-1315/118/1/012019Sánchez-Montoya, M. del M., Arce, M. I., Vidal-Abarca, M. R., Suárez, M. L., Prat, N., & Gómez, R. (2012). Establishing physico-chemical reference conditions in Mediterranean streams according to the European Water Framework Directive. Water Research, 46(7), 2257-2269. doi:10.1016/j.watres.2012.01.042Sanchez-Ramos, D., Sánchez-Emeterio, G., & Florín Beltrán, M. (2015). Changes in water quality of treated sewage effluents by their receiving environments in Tablas de Daimiel National Park, Spain. Environmental Science and Pollution Research, 23(7), 6082-6090. doi:10.1007/s11356-015-4660-ySapriza-Azuri, G., Jódar, J., Carrera, J., & Gupta, H. V. (2015). Toward a comprehensive assessment of the combined impacts of climate change and groundwater pumping on catchment dynamics. Journal of Hydrology, 529, 1701-1712. doi:10.1016/j.jhydrol.2015.08.015Singh, S., Ghosh, N. C., Krishan, G., Galkate, R., Thomas, T., & Jaiswal, R. K. (2015). Development of an Overall Water Quality Index (OWQI) for Surface Water in Indian Context. Current World Environment, 10(3), 813-822. doi:10.12944/cwe.10.3.12Singh, S., Ghosh, N. C., Gurjar, S., Krishan, G., Kumar, S., & Berwal, P. (2017). Index-based assessment of suitability of water quality for irrigation purpose under Indian conditions. Environmental Monitoring and Assessment, 190(1). doi:10.1007/s10661-017-6407-3Sperotto, A., Molina, J. L., Torresan, S., Critto, A., Pulido-Velazquez, M., & Marcomini, A. (2019). A Bayesian Networks approach for the assessment of climate change impacts on nutrients loading. Environmental Science & Policy, 100, 21-36. doi:10.1016/j.envsci.2019.06.004Sun, B., Tang, J., Yu, D., Song, Z., & Wang, P. (2019). Ecosystem health assessment: A PSR analysis combining AHP and FCE methods for Jiaozhou Bay, China1. Ocean & Coastal Management, 168, 41-50. doi:10.1016/j.ocecoaman.2018.10.026Sutadian, A. D., Muttil, N., Yilmaz, A. G., & Perera, B. J. C. (2015). Development of river water quality indices—a review. Environmental Monitoring and Assessment, 188(1). doi:10.1007/s10661-015-5050-0Sutadian, A. D., Muttil, N., Yilmaz, A. G., & Perera, B. J. C. (2017). Using the Analytic Hierarchy Process to identify parameter weights for developing a water quality index. Ecological Indicators, 75, 220-233. doi:10.1016/j.ecolind.2016.12.043Tooth, S. (2018). The geomorphology of wetlands in drylands: Resilience, nonresilience, or …? Geomorphology, 305, 33-48. doi:10.1016/j.geomorph.2017.10.017Tyagi, S., Sharma, B., Singh, P., & Dobhal, R. (2020). Water Quality Assessment in Terms of Water Quality Index. American Journal of Water Resources, 1(3), 34-38. doi:10.12691/ajwr-1-3-3Viaroli, S., Mastrorillo, L., Lotti, F., Paolucci, V., & Mazza, R. (2018). The groundwater budget: A tool for preliminary estimation of the hydraulic connection between neighboring aquifers. Journal of Hydrology, 556, 72-86. doi:10.1016/j.jhydrol.2017.10.066Wang, H.-J., Xiao, X.-C., Wang, H.-Z., Li, Y., Yu, Q., Liang, X.-M., … Jeppesen, E. (2017). Effects of high ammonia concentrations on three cyprinid fish: Acute and whole-ecosystem chronic tests. Science of The Total Environment, 598, 900-909. doi:10.1016/j.scitotenv.2017.04.070Xu, Y., Wang, Y., Li, S., Huang, G., & Dai, C. (2018). Stochastic optimization model for water allocation on a watershed scale considering wetland’s ecological water requirement. Ecological Indicators, 92, 330-341. doi:10.1016/j.ecolind.2017.02.019Yuan, L., Ge, Z., Fan, X., & Zhang, L. (2014). Ecosystem-based coastal zone management: A comprehensive assessment of coastal ecosystems in the Yangtze Estuary coastal zone. Ocean & Coastal Management, 95, 63-71. doi:10.1016/j.ocecoaman.2014.04.005Zhang, R., Zhang, X., Yang, J., & Yuan, H. (2013). Wetland ecosystem stability evaluation by using Analytical Hierarchy Process (AHP) approach in Yinchuan Plain, China. Mathematical and Computer Modelling, 57(3-4), 366-374. doi:10.1016/j.mcm.2012.06.014ZHANG, L. (2016). CALCULATION OF WETLANDS ECOLOGICAL WATER REQUIREMENT IN CHINA’S WESTERN JILIN PROVINCE BASED ON REGIONALIZATION AND GRADATION TECHNIQUES. Applied Ecology and Environmental Research, 14(3), 463-478. doi:10.15666/aeer/1403_463478Zhang, B., Zhao, D., Zhou, P., Qu, S., Liao, F., & Wang, G. (2020). Hydrochemical Characteristics of Groundwater and Dominant Water–Rock Interactions in the Delingha Area, Qaidam Basin, Northwest China. Water, 12(3), 836. doi:10.3390/w1203083