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Streamflow components (e.g., young water vs old water) and hydrological transit times play an important role in water resource and water quality management. We collected a four-year record of stable isotopes of oxygen and hydrogen (δ18O and δ2H) in precipitation, groundwater and stream water for six catchments in the Yongan watershed of eastern China. The stable isotope records were used to identify spatio-temporal variations in the young water fraction (Fyw, defined as the proportion of the transit-time distribution younger than a threshold age) and mean transit time (MTT) based on sine-wave fitting and convolution integral methods, respectively. The Fyw ranged from 14 to 35% in the Yongan watershed. Cumulative transit time showed contrasting distributions, suggesting considerable heterogeneity and a complex interplay between catchment characteristics. Estimated MTTs ranged from 3.2 to 6.3 years and may be explained by catchment characteristics (e.g., elevation and topographic gradient). Observed spatial trends in MTTs likely result from contrasting contributions of different-aged subsurface water flows across the six catchments. The use of Fyw constraints in estimating MTTs reduced uncertainty in some catchments, suggesting the potential benefits of combining multiple approaches (e.g., Fyw) to optimize the results of traditional calibration methods. The relatively low Fyw and long MTTs highlight the importance of groundwater contributions to streamflow generation and imply a considerable lag time in river water quantity and quality responses to catchment-scale water resource management. Coupling multiple metrics (e.g., Fyw and MTT) and isotope models enhances our understanding of watershed-scale hydrologic processes and hydrograph separation.

Quantitative information on long-term net anthropogenic phosphorus inputs (NAPI) and its relationship with riverine phosphorus (P) export are critical for developing sustainable and efficient watershed P management strategies. This is the first study to address long-term (1980-2015) NAPI and riverine P flux dynamics for the Yangtze River basin (YRB), the largest watershed in China. Over the 36-year study period, estimated NAPI to the YRB progressively increased by ∼1.4 times, with NAPIA (chemical fertilizer input + atmospheric deposition + seed input) and NAPIB (net food/feed imports + non-food input) contributing 65% and 35%, respectively. Higher population, livestock density and agricultural land area were the main drivers of increasing NAPI. Riverine total phosphorus (TP), particulate phosphorus (PP) and suspended sediment (SS) export at Datong hydrological station (downstream station) decreased by 52%, 75% and 75% during 1980-2015, respectively. In contrast, dissolved phosphorus (DP) showed an increase in both concentration (∼7-fold) and its contribution to TP flux (∼16-fold). Different trends in riverine P forms were mainly due to increasing dam/reservoir construction and changes in vegetation/land use and NAPI components. Multiple regression models incorporating NAPIA, NAPIB, dam/reservoir storage capacity and water discharge explained 84% and 92% of the temporal variability in riverine DP and PP fluxes, respectively. Riverine TP flux estimated as the sum of DP and PP fluxes showed high agreement with measured values (R2 = 0.87, NSE = 0.84), indicating strong efficacy for the developed models. The model forecasted an increase of 50% and 7% and a decrease of 15% and 22% in riverine DP flux from 2015 to 2045 under developing, dam building, NAPIA and NAPIB reduction scenarios, respectively. This study highlights the importance of including enhanced P transformation from particulate to bioavailable forms due to river regulation and changes in land-use, input sources and legacy P pools in development of P pollution control strategies.

Legacy phosphorus (P) accumulated in watersheds from excessive historical P inputs is recognized as an important component of water pollution control and sustainable P management in watersheds worldwide. However, little is known about how watershed P buffering capacity responds to legacy P pressures over time and how long it takes for riverine P concentrations to recover to a target level, especially in developing countries. This study examined long-term (1960–2010) accumulated legacy P stock, P buffering capacity and riverine TP flux dynamics to predict riverine P-reduction recovery times in the Yongan watershed of eastern China. Due to a growing legacy P stock coupled with changes in land use and climate, estimated short- and long-term buffering metrics (i.e., watershed ability to retain current year and historically accumulated surplus P, respectively) decreased by 65% and 36%, respectively, resulting in a 15-fold increase of riverine P flux between 1980 and 2010. An empirical model incorporating accumulated legacy P stock and annual precipitation was developed (R² = 0.99) and used to estimate a critical legacy P stock of 22.2 ton P km⁻² (95% CI 19.4–25.3 ton P km⁻²) that would prevent exceedance of a target riverine TP concentration of 0.05 mg P L⁻¹. Using an exponential decay model, the recovery time for depleting the estimated legacy P stock in 2010 (29.3 ton P km⁻²) to the critical level (22.2 ton P km⁻²) via riverine flux was 456 years (95% CI 353–560 years), 159 years (95% CI 57–262 years) and 318 years (95% CI 238–400 years) under scenarios of a 4% reduction in annual P inputs, total cessation of P inputs, and 4% reduction of annual P inputs with a 10% increase in average annual precipitation, respectively. Given the lower P buffering capacity and lengthening recovery time, strategies to reduce P inputs and utilize soil legacy P for crop production are necessary to effectively control riverine P pollution and conserve global rock P resources. A long-term perspective that incorporates both contemporary and historical information is required for developing sustainable P management strategies to optimize both agronomic and environmental benefits at the watershed scale.

Accurate source identification is critical for optimizing water pollution control strategies. Although the dual stable isotope (15N-NO3-/18O-NO3-) approach has been widely applied for differentiating riverine nitrogen (N) sources, the relatively short-term (75% of river discharge and were likely the major hydrological pathways for N delivery to the river. Riverine NO3- sources varied with dominant land use (p 30%) were the dominant riverine NO3- sources, implying considerable potential for N pollution legacy effects. Results were consistent with observed nitrous oxide dynamics and N sources identified in previous modeling studies. As the first attempt to apply multiple isotope tracers for exploring and quantifying N transformation and transport pathways, this study provides an integrated approach for verifying and understanding the N pollution legacy effects observed in many watersheds worldwide. This study highlights that river N pollution control in many watersheds requires particular attention to groundwater restoration and soil N management in addition to N input control strategies.

Legacy nutrient accumulation from excess anthropogenic inputs has become a serious environmental issue in many watersheds worldwide. In this review, we provide a systematic overview of sources, legacy nutrient pools, watershed-scale water quality models, and agronomic and environmental implications of legacy nitrogen and phosphorus pools. Hydrological, biogeochemical, and anthropogenic factors exert interacting controls on legacy nutrient dynamics in soils, sediments, and vadose zone/groundwater. Most current watershed models do not effectively incorporate legacy nutrient dynamics, while models that consider legacy effect often have high uncertainty in their treatment of legacy nutrient dynamics. In many intensively managed watersheds, legacy nutrients are a dominant and long-term (> 10 years) source of nutrients to receiving waters, as well as a potentially important nutrient source for crop production. Many existing beneficial management measures have limitations for reducing legacy nutrient losses to surface waters due to appreciable differences in legacy nutrient forms, watershed storage locations, and temporal dynamics compared to those of contemporary nutrient inputs. Recognizing the importance of legacy nutrients is necessary for developing sustainable watershed nutrient management plans for future food, bioenergy, and water security. These plans require strategies to maximize use of legacy nutrient resources to minimize their loss to the atmosphere (e.g., N2O emissions) and surface waters. Finally, this synthesis identified future research needs for improving the understanding, utilization, and mitigation of watershed legacy nutrient pools.

AIM: To investigate the oncolytic effect of human umbilical cord mesenchymal stem cells (hUMSCs) delivering reovirus type 3 (Reo3) on chronic myeloid leukemia (CML) K562 cells. METHODS: The expression of junctional adhesion molecule-A (JAM-A), a receptor susceptible to Reo3, on the surface of hUMSCs and K562 cells was assessed by flow cytometry. Intracellular viral inclusion body distribution 72 h after Reo3 infection in hUMSCs was observed by electron microscopy. The hUMSCs were infected with various multiplicities of infection (MOI) of Reo3 (M01=0, 1, 2 and 3) for 24, 48, 72, 96 and 120 h, and the most suitable MOI was identified by CCK-8 assay. Subsequently, hUMSCs were infected with the optimal titer of Reo3 for the same durations, and supernatants were collected. The titer of Reo3 in the supernatant from each group was measured using mouse fibroblast L929 cells combined with median tissue culture infectious dose (TCID50) method, determining the optimal infection time. The K562 cells were divided into 4 groups: control group, hUMSCs group, Reo3 group, and hUMSCs-Reo3 group. Ratios of hUMSCs to K562 cells in hUMSCs group and hUMSCs-Reo3 group were set at low, medium and high (5^1, 10^1 and 20: 1). The changes of K562 cell viability after co-cultured with hUMSCs-Reo3 for 24, 48 and 72 h were analyzed by CCK-8 assay. The apoptosis of K562 cells was evaluated by flow cytometry. The half maximal effective concentration (EC50) of anti-Reo3 monoclonal antibody was determined using L929 cells. The oncolytic effect of hUMSCs-Reo3 on K562 cells with antibody present in vitro was verified. Western blot analysis was used to detect the protein levels of Bcl-2, Bax, survivin and cleaved caspase-3 in K562 cells after treatment. A BALB/c nude mouse subcutaneous tumor model was constructed with K562 cells (n=6) to analyze the in vivo anti-tumor effect of hUMSCs-Reo3. RESULTS: The expression levels of JAM-A on the surfaces of hUMSCs and K562 cells were found to be 11. 0% and 99. 0%, respectively. Electron microscopy revealed a significant presence of viral inclusion bodies within hUMSCs 72 h following infection with Reo3. Within 120 h, no statistically significant difference was observed in the viability of hUMSCs between Reo3 (MOI=1) group and uninfected group, establishing the optimal MOI. The TCID50 results indicated that the highest virus titer in the lysate of hUMSCs in Reo3 (MOI=1) group occurred 48 h after infection, determining 48 h as the optimal infection time. The K562 cells co-cultured with hUMSCs-Reo3 for 24, 48 and 72 h showed a dose- and time-dependent inhibition of cell viability. The EC50 of the anti-Reo3 monoclonal antibody was found to be 1: 34. Even in the presence of antibodies at various concentrations (1: 34, 1 • 300 and 1: 600), hUMSCs were capable of transporting Reo3 to inhibit K562 cell viability and induce apoptosis in vitro. Compared with control group, significant down-regulation of Bcl-2 and survivin expression levels in K562 cells was noted after 48 h of co-culture with hUMSCs-Reo3 (P<0. 05), while Bax and cleaved caspase-3 expression levels were significantly up-regulated (P< 0. 05 or P<0. 01). In the BALB/c nude mouse tumor-bearing model, determination of tumor volume changes, pathological examination of tumor tissue and major organs, and assessment of cathepsin B/L activity using a small animal live imaging system confirmed the oncolytic effect of hUMSCs-Reo3 on K562 cells in vivo without adverse effects on normal tissues. CONCLUSION: The hUMSCs are effective in transporting Reo3, and this delivery system is capable of releasing sufficient quantities of Reo3 in both in vivo and in vitro settings to inhibit the malignant proliferation of K562 cells and promote apoptosis, thereby exerting an oncolytic effect. [ABSTRACT FROM AUTHOR]

AIM: To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant (hUMSC-CM) combined with temozolomide (TMZ) on various glioma cell lines, and to elucidate the underlying mechanisms. METHODS: The hUMSC-CM was harvested using two different serum deprivation techniques at 24 and 48 h, and was converted into freeze-dried powder, which was then given to rat malignant glioma cell line RG-2, human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations (0, 1, 3, 6 and 9 g/L). The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24, 48 and 72 h were assessed using CCK-8 assay. Hematoxylin-eosin (HE) staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations (0, 25, 50, 100, 200 and 400 µmol/L). Two concentrations (3 and 9 g/L) of hUMSC-CM and 3 concentrations (50, 100 and 200 µmol/L) of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations. TUNEL staining was utilized to detect apoptosis. Flow cytometry was utilized to analyze cell cycle modifications. The expression alterations of apoptosis-inducing proteins, cleaved caspase-3, cleaved caspase-8 and cleaved PARP1, as well as autophagy-inducing proteins beclin-1 and LC3, were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro. RESULTS: The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied, with RG-2 showing the highest sensitivity, followed by U251, and then LN-428. The inhibitory effect of hUMSC-CM (3 and 9 g/L) and TMZ (50, 100 and 200 µmol/L) combined treatment on glioma cells was significantly greater than that that of single-agent treatments (P<0. 05), demonstrating a dose- and concentration-dependent enhancement. Notably, the combination of 9 g/L hUMSC-CM (C9) with 50 µmol/L TMZ (T50) effectively suppressed glioma cell growth. CCK-8 assay indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone (P<0. 05). HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50. Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells. Furthermore, compared with control group, the levels of cleaved caspase-3, cleaved caspase-8, cleaved PARP1, beclin-1, and LC3-II/LC3-I were significantly elevated in the C9+T50-treated glioma cells (P<0. 01). CONCLUSION: (1) The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells, with a synergistic sensitization observed across different cell lines. (2) The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells. [ABSTRACT FROM AUTHOR]