Negatively charged nanodiscs for the reduction of toxicity and enhanced efficacy of polymyxin B against Acinetobacter baumannii sepsis.

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. STATEMENT OF SIGNIFICANCE: Multidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii (CRAB), currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Polymyxin resistance caused by large-scale genomic inversion due to IS26 intramolecular translocation in Klebsiella pneumoniae.

Polymyxin B and polymyxin E (colistin) are presently considered the last line of defense against human infections caused by multidrug-resistant Gram-negative organisms such as carbapenemase-producer Enterobacterales, Acinetobacter baumannii, and Klebsiella pneumoniae. Yet resistance to this last-line drugs is a major public health threat and is rapidly increasing. Polymyxin S2 (S2) is a polymyxin B analogue previously synthesized in our institute with obviously high antibacterial activity and lower toxicity than polymyxin B and colistin. To predict the possible resistant mechanism of S2 for wide clinical application, we experimentally induced bacterial resistant mutants and studied the preliminary resistance mechanisms. Mut-S, a resistant mutant of K. pneumoniae ATCC BAA-2146 (Kpn2146) induced by S2, was analyzed by whole genome sequencing, transcriptomics, mass spectrometry and complementation experiment. Surprisingly, large-scale genomic inversion (LSGI) of approximately 1.1 Mbp in the chromosome caused by IS26 mediated intramolecular transposition was found in Mut-S, which led to mgrB truncation, lipid A modification and hence S2 resistance. The resistance can be complemented by plasmid carrying intact mgrB. The same mechanism was also found in polymyxin B and colistin induced drug-resistant mutants of Kpn2146 (Mut-B and Mut-E, respectively). This is the first report of polymyxin resistance caused by IS26 intramolecular transposition mediated mgrB truncation in chromosome in K. pneumoniae. The findings broaden our scope of knowledge for polymyxin resistance and enriched our understanding of how bacteria can manage to survive in the presence of antibiotics.

Protective effect of the novel cyclic peptide ASK0912 on mice with sepsis induced by Acinetobacter baumannii.

BACKGROUND: Sepsis has become a global health concern owing to its increasing incidence and high mortality rate. In the present study, we investigated a novel drug candidate ASK0912 on its protective effects in mice with Acinetobacter baumannii 20-1-induced sepsis, and studied the related mechanisms. MATERIAL AND METHODS: To analyze the protective effect of ASK0912 on septic mice, survival rates, body temperature, organ and blood bacterial loads, white blood cell and platelet counts, organ damage, and cytokine levels were determined. RESULTS: ASK0912 remarkably increased the survival rate of mice with sepsis induced by A. baumannii 20-1 at a low dose of 0.6 mg/kg. Rectal temperature measurements showed that ASK0912 treatment prevented the body temperature decrease of septic mice to some extent. Treatment with ASK0912 can notably reduce the organ and blood bacterial loads and alleviate platelet count reduction due to sepsis. ASK0912 attenuated organ damage, including reduced levels of total bile acids, urea, and creatinine, aggregation of inflammatory cells, and mitigation of structural changes in septic mice, as demonstrated by biochemical analysis and hematoxylin & eosin staining. Additionally, multiplex assay showed that abnormally increased cytokine levels (IL-1ß, IL-3, IL-5, IL-6, IL-10, IL-13, MCP-1, RANTES, KC, MIP-1α, MIP-1ß, and G-CSF) in septic mice decreased after ASK0912 treatment. CONCLUSIONS: ASK0912 can not only improve the survival rate, hypothermia, lower the bacterial loads in the organs and blood, but also alleviate the pathophysiological manifestations such as intravascular coagulation abnormalities, organ damages, and immune system disorder of sepsis mice induced by A. baumannii 20-1.

In vivo pharmacodynamic study of contezolid acefosamil, a prodrug of contezolid for oral and intravenous administration.

OBJECTIVES: Contezolid acefosamil is a novel O-acyl phosphoramidate prodrug of contezolid. In the current study, we aimed to systemically evaluate the efficacy of contezolid acefosamil against infections caused by multiple Gram-positive pathogens, and compare the efficacy of the prodrug by oral and intravenous administrations. METHODS: The in vivo pharmacodynamic efficacy of contezolid acefosamil was evaluated in mouse models of systemic (with five S. aureus, three S. pneumoniae and two S. pyogenes bacterial isolates) and thigh (with two S. aureus isolates) infections using linezolid as the reference agent. RESULTS: In both models, contezolid acefosamil administrated either orally or intravenously, demonstrated high antibacterial efficacy similar to linezolid, and the antibacterial efficacy of oral and intravenous contezolid acefosamil were comparable. CONCLUSIONS: The high aqueous solubility and great efficacy of contezolid acefosamil support its clinical development as an injectable and oral antibiotic suitable for serious Gram-positive infections.

Identification and Quantification of S-Sulfenylation Proteome of Mycobacterium tuberculosis under Oxidative Stress.

The ability to maintain redox homeostasis is critical for Mycobacterium tuberculosis (Mtb) to survive the redox stress of the host. There are many antioxidant systems in Mtb to ensure its normal replication and survival in the host, and cysteine thiols are one of them. S-sulfenylation is one of the reversible modifications of cysteine thiols to resist oxidative stress. In the study, we investigated the total cysteine thiols modification and S-sulfenylation modification of Mtb proteome under the oxidative stress provided by hydrogen peroxide. To determine and quantify the S-sulfenylation modified proteins, high specific IodoTMT6plex reagents and high resolution mass spectrometry were used to label and quantify the peptides and proteins modified. There are significant differences for the total cysteine modification levels of 279 proteins and S-sulfenylation modification levels of 297 proteins under hydrogen peroxide stress. Functional enrichment analysis indicated that these cysteine-modified proteins were involved in the oxidation-reduction process, fatty acid biosynthetic process, stress response, protein repair, cell wall, etc. In conclusion, our study provides a view of cysteine modifications of the Mtb proteome under oxidative stress, revealing a series of proteins that may play a role in maintaining redox homeostasis. IMPORTANCE With the continuous spread of drug-resistant tuberculosis, there is an urgent need for new antituberculosis drugs with new mechanisms. The ability of Mtb to resist oxidative stress is extremely important for maintaining redox homeostasis and survival in the host. The reversible modifications of cysteine residues have a dual role of protection from irreversible damage to protein functions and regulation, which plays an important role in the redox homeostasis system. Thus, to discover cysteine modification changes in the proteome level under oxidative stress is quintessential to elucidate its antioxidant mechanism. Our results provided a list of proteins involved in the antioxidant process that potentially could be considered targets for drug discovery and vaccine development. Furthermore, it is the first study to determine and quantify the S-sulfenylation-modified proteins in Mtb, which provided better insight into the Mtb response to the host oxidative defense and enable a deeper understanding of Mtb survival strategies.

Curdione Relieved Isoproterenol-Induced Myocardial Damage through Inhibiting Oxidative Stress and Apoptosis.

Isoproterenol (ISO) is widely used to treat bronchial asthma, cardiogenic or septic shock, complete atrioventricular block, and cardiac arrest. However, it can also cause myocardial damage owing to infarct-like necrosis. Curdione, an extract of the Chinese herb Rhizoma Curcumae, has a variety of pharmacological activities, including cardioprotective effects. In this study, we investigated the protective effects of curdione and its underlying mechanisms in an ISO-induced myocardial injury model. Our results showed that curdione attenuated ISO-induced H9c2 cell proliferation inhibition and lactic dehydrogenase (LDH) release. Curdione ameliorated morphological damage and reduced the ISO-induced elevation of serum creatine kinase-MB isoenzyme (CK-MB) and LDH. Furthermore, curdione inhibited ISO-induced cell apoptosis, modulated the expression of Bcl-2 and Bax proteins, repealed the accumulation of ISO-induced reactive oxygen species (ROS), prevented mitochondrial dysfunction, and activated the Nrf2/SOD1/HO-1 signaling pathway. The above results show that curdione exerts a protective effect against ISO-induced myocardial damage by inhibiting apoptosis and oxidative stress, suggesting that curdione is a potential therapeutic strategy to prevent ISO-induced myocardial damage.

Total knee arthroplasty in patients with haemophilic arthropathy is effective and safe according to the outcomes at a mid-term follow-up.

BACKGROUND: Haemophilic arthropathy (HA), a common complication of haemophilia, is secondary to recurrent joint bleeding and increases the prevalence of end-stage osteoarthritis (OA). Total knee arthroplasty (TKA) is a reliable treatment for haemophilia patients. This study was performed to evaluate the mid-term outcomes of TKA for end-stage HA. We hypothesized that the rate of complications of TKA is higher for patients with haemophilia than for patients without haemophilia. METHODS: Patients with HA undergoing TKA from January 2015 to December 2016 in our centre were retrospectively reviewed. All patients were managed by a multidisciplinary team. The improvements in flexion contracture, range of motion (ROM), Knee Society Score (KSS; clinical and functional), Visual Analogue Scale (VAS) score, and satisfaction at final follow-up were analysed to evaluate the effectiveness of TKA in HA. The complications were analysed to evaluate the safety of TKA in HA. RESULTS: Twenty-eight patients (32 knees) were included in the study. The follow-up was 69.1 ± 5.1 months. Significant differences between the preoperative and final follow-up values of flexion contracture (which changed from 21.1 ± 6.5° to 14.3 ± 4.1°, P < 0.001), ROM (from 53.9 ± 15.0° to 70.3 ± 16.3°, P < 0.001), clinical KSS (from 33.5 ± 14.4° to 62.7 ± 9.5°, P < 0.001), functional KSS (from 46.1 ± 15.5° to 62.9 ± 9.7°, P < 0.001), and VAS score (from 6.8 ± 1.4 to 4.9 ± 1.3, P < 0.01) were observed. Importantly, the incidence of complications was 15.6% and the satisfaction was 100% in our mid-term study. CONCLUSION: Under elaborative and comprehensive management, TKA is effective and safe in patients with advanced HA on the basis of mid-term follow-up outcomes.

ML364 exerts the broad-spectrum antivirulence effect by interfering with the bacterial quorum sensing system.

Antivirulence strategy has been developed as a nontraditional therapy which would engender a lower evolutionary pressure toward the development of antimicrobial resistance. However, the majority of the antivirulence agents currently in development could not meet clinical needs due to their narrow antibacterial spectrum and limited indications. Therefore, our main purpose is to develop broad-spectrum antivirulence agents that could target on both Gram-positive and Gram-negative pathogens. We discovered ML364, a novel scaffold compound, could inhibit the productions of both pyocyanin of Pseudomonas aeruginosa and staphyloxanthin of Staphylococcus aureus. Further transcriptome sequencing and enrichment analysis showed that the quorum sensing (QS) system of pathogens was mainly disrupted by ML364 treatment. To date, autoinducer-2 (AI-2) of the QS system is the only non-species-specific signaling molecule that responsible for the cross-talk between Gram-negative and Gram-positive species. And further investigation showed that ML364 treatment could significantly inhibit the sensing of AI-2 or its nonborated form DPD signaling in Vibrio campbellii MM32 and attenuate the biofilm formation across multi-species pathogens including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The results of molecular docking and MM/GBSA free energy prediction showed that ML364 might have higher affinity with the receptors of DPD/AI-2, when compared with DPD molecule. Finally, the in vivo study showed that ML364 could significantly improve the survival rates of systemically infected mice and attenuate bacterial loads in the organs of mice. Overall, ML364 might interfere with AI-2 quorum sensing system to exert broad-spectrum antivirulence effect both in vitro and in vivo.

Perioperative Blood Loss Can Be Reduced If Total Knee Arthroplasty Was Performed in the Si Hour-Period, Compared with the Wei Hour-Period: A Retrospective Cohort Study.

OBJECTIVE: To investigate the efficacy and safety of performing primary unilateral total knee arthroplasty (TKA) in the "Si hour-period" meaning 09:00 a.m. to 11:00 a.m. (one of the 12 two-hour periods into which the day was traditionally divided, each being given the name of one of the 12 earthly branches), compared with the "Wei hour-period" (13:00-15:00). METHODS: Patient documentations were studied for those who underwent a primary unilateral TKA performed by the same surgical team with a tourniquet between January 2018 and January 2021 at our medical center. Eighty-four patients were enrolled and assigned into group A (in Si hour-period) and group B (in Wei hour-period). The main outcomes were total blood cell loss (TBL), hidden blood loss (HBL), visible blood loss (VBL), maximum hemoglobin (Hb) drop, and transfusion rate. Secondary outcomes were length of hospital stay (LOS), postoperative femorotibial mechanical axis (FTMA), FTMA correction, platelet count, plasma D-dimer (D-D), prothrombin time (PT), international normalized ratio (INR), and the incidence of postoperative complications. RESULTS: Group A showed statistical significance lower at the mean TBL, the mean HBL, and the maximum Hb drop (95% CI: -352.8 to -46.1,P=0.011, 95% CI: -348.0 to -40.1,P=0.014, and 95% CI: -9.5 to -0.7,P=0.023, respectively) after TKA than group B. The postoperative platelet count of group A was more significant than that of group B (95% CI:3.1 to 52.9, P=0.028). The VBL, transfusion rate, the LOS, postoperative FTMA, FTMA correction, plasma D-D, PT, INR, and the incidence of postoperative complications (wound complications, calf muscular vein thrombosis, infection, pulmonary embolism, and deep vein thrombosis) were similar between the two groups (P > 0.05, respectively). CONCLUSION: Our study shows that blood loss can be reduced when TKA is performed in the "Si hour-period," which may be due to increasing platelet count, and postoperative complications did not increase, compared with the Wei hour-period. We recommend that the selective operation, such as TKA, should be performed in the "Si hour-period" in clinical practice between the two hour-period.

[Progress on femorotibial mechanical axis of total knee arthroplasty].

Femorotibial mechanical axis (FTMA) is one of important factors influencing clinical effect after total knee arthroplasty (TKA). It is generally believed that the range of lower limb alignment after TKA is controlled within neutral FTMA ± 3 °, which has more advantages in improving joint function, prolonging prosthesis survival rate and reducing revision rate, and obtain better clinical results. Therefore, neutral FTMA is also considered to be the gold standard for TKA. However, with the application of computer-assisted surgery and other technologies, the alignment of FTMA is more accurate than before, but the clinical effect after surgery has not significantly improved. Some scholars have begun to question the necessity of neutral alignment of FTMA, and proposed alignment methods such as kinematics and retained residual deformity, which could achieve better clinical effects. In recent years, it has been reported that FTMA might not be the most important factor influencing postoperative clinical effects, and it is suggested that the arrangement and measurement of lower limbs and the effects on adjacent joint functions could affect clinical effect after TKA. The paper reviews neutral FTMA alignment is still an important factor for success of TKA. After a thorough evaluation according to the patient's condition, it should be appropriately applied in the case of neutral FTMA alignment; the operator should explore other factors which affect clinical outcome after TKA, and improve it to achieve the best therapeutic effect.

The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus.

BACKGROUND: Infections caused by drug-resistant Staphylococcus aureus, especially vancomycin-intermediate Staphylococcus aureus (VISA), leave clinicians with limited therapeutic options for treatment. Persister cells is a leading cause of recalcitrant infection and antibiotic treatment failure, and there is no drug in clinical use that specifically targets persister cells currently. Here, we report a promising combination therapy of sodium new houttuyfonate (SNH) and berberine chloride (BBR) which is able to eradicate both growing and persistent drug-resistant Staphylococcus aureus. RESULTS: The susceptibility test showed SNH exhibited anti-MRSA activity with MIC90 at 64 µg/mL, while BBR showed weak anti-MRSA activity with MIC90 at 512 µg/mL. MICs of BBR in combination with 1/2 MIC SNH decreased by 4 to 64 folds compared with MICs of BBR alone. The results of time-killing assays revealed that the combined use of sub-MIC SNH and BBR offered an in vitro synergistic action against growing MRSA (including pathogenic MRSA) and VISA strains. More importantly, the combination of SNH and BBR was able to eradicate VISA Mu50 and pathogenic MRSA persister cells. The synergistic effect is likely related to the interruption of the cell membrane caused by SNH, which is confirmed by scanning electron microscope and membrane potential and permeability analysis. CONCLUSIONS: Our study provide a promising clinical curative strategy for combating drug-resistant S. aureus infections, especially for recalcitrant infections caused by persister cells.

Effects of Oenanthe javanica on Nitrogen Removal in Free-Water Surface Constructed Wetlands under Low-Temperature Conditions.

To investigate the role and microorganism-related mechanisms of macrophytes and assess the feasibility of Oenanthe javanica (Blume) DC. in promoting nitrogen removal in free-water surface constructed wetlands (FWS-CWS) under low temperatures (<10 °C), pilot-scale FWS-CWS, planted with O. javanica, were set up and run for batch wastewater treatment in eastern China during winter. The presence of macrophytes observably improved the removal rates of ammonia nitrogen (65%-71%) and total nitrogen (41%-48%) (p < 0.05), with a sharp increase in chemical oxygen demand concentrations (about 3-4 times). Compared to the unplanted systems, the planted systems not only exhibited higher richness and diversity of microorganisms, but also significantly higher abundances of bacteria, ammonia monooxygenase gene (amoA), nitrous oxide reductase gene (nosZ), dissimilatory cd1-containing nitrite reductase gene (nirS), and dissimilatory copper-containing nitrite reductase gene (nirK) in the substrate. Meanwhile, the analysis of the microbial community composition further revealed significant differences. The results indicate that enhanced abundances of microorganisms, and the key functional genes involved with nitrogen metabolism in the planted systems played critical roles in nitrogen removal from wastewater in FWS-CWS. Furthermore, abundant carbon release from the wetland macrophytes could potentially aid nitrogen removal in FWS-CWS during winter.

New natural inhibitors of hexokinase 2 (HK2): Steroids from Ganoderma sinense.

Hexokinase 2 (HK2), a rate-limiting enzyme in the first step of glycolysis pathway, expresses at high level in cancer cells compared with normal cells. HK2 provides a new target for cancer therapy due to its pivotal role in tumor tumourigenic and metastatic process. The structure-based virtual ligand screening in a small in-house database of natural products predicted that a new steroid, (22E,24R)-6ß-methoxyergosta-7,9(11),22-triene-3ß,5α-diol (2) from Ganoderma sinense has high binding affinity to HK2 with significant calculated binding free energy. Based on this prediction, compound 2, together with the other 12 steroid analogues (1, 3-13) from this plant were selected for further in vitro microscale thermophoresis (MST), enzyme inhibition, and cell-based assays based on the HK2 target. And compound 2 was finally identified as an HK2 inhibitor. As the first natural HK2 inhibitor, compound 2 can be considered as a potential drug candidate targeting at HK2 for cancer therapy.

Are the traditional large-scale drought indices suitable for shallow water wetlands? An example in the Everglades.

Numerous drought indices have been developed over the past several decades. However, few studies have focused on the suitability of indices for studies of ephemeral wetlands. The objective is to answer the following question: can the traditional large-scale drought indices characterize drought severity in shallow water wetlands such as the Everglades? The question was approached from two perspectives: the available water quantity and the response of wetland ecosystems to drought. The results showed the unsuitability of traditional large-scale drought indices for characterizing the actual available water quantity based on two findings. (1) Large spatial variations in precipitation (P), potential evapotranspiration (PE), water table depth (WTD) and the monthly water storage change (SC) were observed in the Everglades; notably, the spatial variation in SC, which reflects the monthly water balance, was 1.86 and 1.62 times larger than the temporal variation between seasons and between years, respectively. (2) The large-scale water balance measured based on the water storage variation had an average indicating efficiency (IE) of only 60.01% due to the redistribution of interior water. The spatial distribution of variations in the Normalized Different Vegetation Index (NDVI) in the 2011 dry season showed significantly positive, significantly negative and weak correlations with the minimum WTD in wet prairies, graminoid prairies and sawgrass wetlands, respectively. The significant and opposite correlations imply the unsuitability of the traditional large-scale drought indices in evaluating the effect of drought on shallow water wetlands.

A Hardy Plant Facilitates Nitrogen Removal via Microbial Communities in Subsurface Flow Constructed Wetlands in Winter.

The plants effect in subsurface flow constructed wetlands (SSF-CWs) is controversial, especially at low temperatures. Consequently, several SSF-CWs planted with Iris pseudacorus (CWI) or Typha orientalis Presl. (CWT) and several unplanted ones (CWC) were set up and fed with secondary effluent of sewage treatment plant during the winter in Eastern China. The 16S rDNA Illumina Miseq sequencing analysis indicated the positive effects of I. pseudacorus on the bacterial community richness and diversity in the substrate. Moreover, the community compositions of the bacteria involved with denitrification presented a significant difference in the three systems. Additionally, higher relative abundances of nitrifying bacteria (0.4140%, 0.2402% and 0.4318% for Nitrosomonas, Nitrosospira and Nitrospira, respectively) were recorded in CWI compared with CWT (0.2074%, 0.0648% and 0.0181%, respectively) and CWC (0.3013%, 0.1107% and 0.1185%, respectively). Meanwhile, the average removal rates of NH4(+)-N and TN in CWI showed a prominent advantage compared to CWC, but no distinct advantage was found in CWT. The hardy plant I. pseudacorus, which still had active root oxygen release in cold temperatures, positively affected the abundance of nitrifying bacteria in the substrate, and accordingly was supposed to contribute to a comparatively high nitrogen removal efficiency of the system during the winter.

Decreasing but still significant facilitation effect of cold-season macrophytes on wetlands purification function during cold winter.

To identify the facilitation effect of a cool-season aquatic macrophyte (FEam) for use in effluent purification via constructed floating wetlands (CFWs) and to determine the possible pathways used during a winter period with an average temperature of less than 5 °C, pilot-scale CFWs were planted with the cold-season macrophyte Oenanthe clecumbens and were operated as batch systems. Although some leaves withered, the roots retained relatively high levels of activity during the winter, which had average air and water temperatures of 3.63 and 5.04 °C, respectively. The N and P removal efficiencies in CFWs decreased significantly in winter relative to those in late autumn. The presence of cool-season plants resulted in significant improvements in N and P removal, with a FEam of 15.23-25.86% in winter. Microbial N removal accounted for 71.57% of the total N removed in winter, and the decrease in plant uptake was the dominant factor in the wintertime decrease in N removal relative to that in late autumn. These results demonstrate the importance of cold-season plants in CFWs for the treatment of secondary effluent during cold winters.

Growth and physiological responses of submerged plant Vallisneria natans to water column ammonia nitrogen and sediment copper.

Background. The decline of submerged plant populations due to high heavy metal (e.g., Cu) levels in sediments and ammonia nitrogen (ammonia-N) accumulation in the freshwater column has become a significant global problem. Previous studies have evaluated the effect of ammonia-N on submerged macrophytes, but few have focused on the influence of sediment Cu on submerged macrophytes and their combined effects. Methods. In this paper, we selected three levels of ammonia-N (0, 3, and 6 mg L(-1)) and sediment Cu (25.75 ± 6.02 as the control, 125.75 ± 6.02, and 225.75 ± 6.02 mg kg(-1)), to investigate the influence of sediment Cu and ammonia-N on submerged Vallisneria natans. We measured the relative growth rate (RGR), above- and below- ground biomass, chlorophyll, non-protein thiol (NP-SH), and free proline. Results and Discussion. The below-ground biomass of V. natans decreased with increasing Cu sediment levels, suggesting that excessive sediment Cu can result in significant damage to the root of V. natans. Similarly, the above-ground biomass significantly decreased with increasing ammonia-N concentrations, indicating that excessive water ammonia-N can cause significant toxicity to the leaf of V. natans. In addition, high ammonia-N levels place a greater stress on submerged plants than sediment Cu, which is indicated by the decline of RGR and chlorophyll, and the increase of (NP-SH) and free proline. Furthermore, high sediment Cu causes ammonia-N to impose greater injury on submerged plants, and higher sediment Cu levels (Cu ≥ 125.75 mg kg(-1)) led to the tolerant values of ammonia-N for V. natans decreasing from 6 to 3 mg L(-1). This study suggests that high sediment Cu restricts the growth of plants and intensifies ammonia-N damage to V. natans.