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IκBα is a critical protein that inhibits NF-κB nuclear translocation and impairs NF-κB–mediated signaling. The abundance of IκBα determines the activation and restoration of the inflammatory response. However, posttranslational regulation of IκBα remains to be fully understood. In this study, we identified ubiquitin-specific protease 39 (USP39) as a negative regulator in the NF-κB inflammatory response by stabilizing basal IκBα. The expression of USP39 in macrophages was reduced under LPS-induced inflammation. Knockdown or knockout of USP39 in macrophages significantly increased the expression and secretion of proinflammatory cytokines upon exposure to LPS or Escherichia coli, whereas reexpression of exogenous USP39 in USP39-deficient macrophages rescued the effect. Moreover, USP39-defective mice were more sensitive to LPS or E. coli–induced systemic sepsis. Mechanistically, USP39 interacted with and stabilized IκBα by reducing K48-linked polyubiquination of IκBα. Taken together, to our knowledge, our study for the first time revealed the inhibitory function of USP39 in the NF-κB inflammatory response, providing a previously unknown mechanism for control of inflammatory cytokine induction in the cellular anti-inflammatory response.

Crocetin is a bioactive ingredient in saffron, derived from the Crocus sativus stigmas of the Iridaceae family. As a chemically carotenoid derivative, crocetin exhibites effects like anti-inflammatory, antioxidant, neuroprotective, etc. However, the protective effect of crocetin on glaucoma and its mechanism remains unclear. The current study assesed the neuroprotective and anti-inflammatory effects of crocetin on retinal neurons in glaucoma rats which were induced by 0.3% carbomer injection into the anterior chamber.The pathological structures on the retina and optic nerve were observed and examined by HE staining and transmission electron microscopy. Immunohistochemical staining was used to detect the expression of TNF-α, IL-1β, and IL-6 of the retina and the expression of a brain-derived neurotrophic factor (BDNF) in the primary visual cortex (PVC). Western blot was carried out to detect the expression of PI3K, Akt, and NF-κB in the retina. It was found that crocetin ameliorated the pathological changes of the retina and ON and reduced the number of apoptotic retinal ganglion cells. Immunohistochemical staining showed that crocetin could decrease the contents of TNF-α, IL-1β, and IL-6 and increase the contents of BDNF. Western blot showed that crocetin was found to suppress the expression of PI3K, Akt, and NF-κB.The results obtained in this study have indicated that crocetin showes neuroprotective effects on retinal ganglion cells in glaucoma rats and inhibits retinal dysfunction. Meanwhile, crocetin exerted an anti-inflammatory effect to protect the retina by inhibiting the expression of the PI3K/Akt/NF-κB signaling pathway. This work provides substantial evidence that crocetin may be a potential drug for the treatment of glaucoma.

Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect.This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS).In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB).According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group.Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Inflammation is a biological process closely related to different kinds of diseases, such as cancer and metabolic diseases. Therefore, effective control of the occurrence and development of inflammation is of great significance for disease prevention and control. Recently, 2-substituted indoles have gradually become a research hotspot because of their stability and pharmacological activity. Here we synthesized a series of compound containing 2-substituted indoles and investigated XCR-7a's role in inflammatory response. Our data show that XCR-7a can inhibit the production of inflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and inflammatory mediator cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS) in mouse peritoneal macrophages. Also, XCR-7a has a protective effect on LPS-induced inflammatory response in mice. Mechanically, we found that XCR-7a could inhibit the phosphorylation of c-Fos induced by LPS, which suggested that the protective effect of XCR-7a on inflammation was related to its negative regulation to phosphorylation of c-Fos. Briefly, our results demonstrated that XCR-7a could be expected to be a potential drug for controlling inflammation.

Background Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. Results Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. Conclusions These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene–lignan pathway network analysis highlighted polyploidy–responsive key functional genes and regulators.

MoO 2 and MoO 3 with specific morphologies are synthesized on flexible carbon cloth through a facile hydrothermal method as supercapacitor electrodes. The prepared prismatic MoO 3 shows a specific capacitance of 54.7 F g −1 in 1 M LiOH electrolyte. Moreover, the prepared MoO 2 presents a spherical profile with hierarchical porous structure. Each MoO 2 sphere is stacked with several layers of MoO 2 “spindle” which is further built by amounts of MoO 2 nanoparticles. A high specific capacitance of 381.0 F g −1 is achieved for the MoO 2 sphere electrode, 7 times higher than that of MoO 3 .

SELEX is the cornerstone for aptamer research with broad applications in biosensors and medicine. To improve the affinity of selected aptamers, we propose a structure-guided post-SELEX approach, an optimization method based on the precise secondary structure of the aptamer–ligand complex. We demonstrate this approach using the Ochratoxin A (OTA) aptamer. Guided by the structure, we designed a new aptamer whose affinity is improved by more than 50-fold. We also determined the high-resolution NMR structure of the new aptamer-OTA complex and elucidated the discriminatory recognition mechanism of one atomic difference between two analogs, OTA and OTB. The aptamer forms an unusual hairpin structure containing an intramolecular triple helix, which is not seen in the previously determined aptamer complex. The π–π stacking, the hydrophobic interaction, hydrogen bonds and halogen bonds between OTA and the aptamer contribute to the recognition of OTA, and the halogen bonds play an important role in discriminating between OTA and OTB. Our results demonstrate that the structure-guided post-SELEX approach improves aptamers affinity. An improved OTA biosensor system might be developed using this new strategy.

Pt-free counter electrode (CE) composed of La2MoO6 (La2O3-MoO3) was successfully synthesized by simple pyrolysis of lanthanum acetate (C6H9O6La·xH2O) and hexaammonium heptamolybdate tetrahydrate ((NH4)6Mo7O24·4H2O). Furthermore, three proportions composites catalysts of La2MoO6@MWCNTs based on La2MoO6 and multiwall carbon nanotubes (MWCNTs) were prepared and characterized as Pt-free catalyst for CE in dye-sensitized solar cells (DSSCs). The morphology and structure of La2MoO6@MWCNTs composites were determined by scanning electron microscopy, transmission electron microscope and X-ray diffraction. The electrochemical performance of La2MoO6@MWCNTs composite catalysts for CEs was determined by photocurrent-voltage measurements, cyclic voltammetry, electrochemical impedance spectroscopy, and Tafel polarization. The power conversion efficiencies of 4.68%, 4.87% and 5.06% are obtained for La2MoO6:MWCNTs with the mass ratios of 5:1, 3:1 and 1:1 towards the reduction of I3– to I– under the same conditions, respectively, which are superior to those of MWCNTs (3.94%) and La2MoO6 (1.71%) electrodes. The experimental results reveal that the presence of MWCNTs results in an augmented active catalytic surface area and enhanced charge transfer from CE to the electrolyte.

Background: Polycystic ovary syndrome (PCOS), one of the most common endocrine diseases in women of childbearing age, has been found to be accompanied by changes in the gut microbiota. The Bu Shen Yang Xue formula (BSYXF) is a traditional Chinese medicine widely used for the treatment of PCOS. This study aimed to investigate whether the protective effects of β-sitosterol, the main active ingredient of BSYXF, on PCOS was mediated by regulating gut microbiota.Methods: The presence of β-sitosterol in BSYXF was detected by liquid chromatography-mass spectrometry. The PCOS-like mouse model was induced by dehydroepiandrosterone. The fecal supernatant of β-sitosterol-treated mice was prepared for fecal microbiota transplantation (FMT). Body weight and wet weight of the uterus and ovary of the mice were recorded for organ index calculation. Hematoxylin and eosin stain was used to assess the endometrial morphology and microenvironment changes. Expression of endometrial receptivity markers cyclooxygenase-2 (COX-2), Integrin ανβ3, leukemia inhibitory factor (LIF), and homeobox A10 (HOXA10) in the endometrium were determined by immunohistochemistry and western blot analysis. Enzyme-linked immunosorbent assay was employed to detect the expression of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), and testosterone (T) in the serum. The diversity of gut microbiota was examined by 16S rDNA gene sequencing.Results: With the treatment of β-sitosterol and β-sitosterol-FMT, the uterine index of PCOS-like mice increased, the ovarian index decreased, levels of COX-2, LH and T decreased, and levels of Integrin ανβ3, LIF, HOXA10, FSH, and P increased. Under β-sitosterol treatment, the structure of the gut microbiota in PCOS-like mice was also changed.Conclusion: β-sitosterol regulates the endometrial receptivity of PCOS and harmonizes the sex hormone balance, which may be related to the changes in the structure and composition of gut microbiota, thus affecting the pathological process of PCOS.

Fe3+ doping is used to enhance the capacity of polythiophene as supercapacitor electrode and the flexible Fe3+ doped PTh electrode on carbon cloth is prepared with an electrochemical deposition method. The successful doping of Fe3+ is confirmed by IR, XPS, and EDS measurements. The electrochemical performance of the PTh electrode is evaluated using cyclic voltammetry, galvanostatic charge-discharge technique, and electrochemical impedance spectroscopy in acidic aqueous electrolyte. The pristine PTh exhibits specific capacitance of 77.2 F g−1 at a current density of 0.5 A g−1. After Fe3+ doping, the specific capacitance is improved to 108.1 F g−1 and the retention rate also improved from 7.8% to 21.2% after 1000 charge-discharge circles.

Pt-free composite counter electrode (CE) composed of La2O3 was successfully synthesized by a simple pyrolysis of Lanthanum acetate hydrate (La(CH3COO)3·xH2O) in a high-temperature solid phase. Furthermore, three proportion composite catalysts of La2O3@MWCNTs based on La2O3 and multiwall carbon nanotubes (MWCNTs) were prepared and characterized as Pt-free catalysts for CE in dye-sensitized solar cells (DSSCs). The morphology and structure of La2O3@MWCNT composites were determined by scanning electron microscopy and X-ray diffraction. The electrochemical performance of La2O3@MWCNT composite catalysts for CEs was determined by photocurrent-voltage measurements, cyclic voltammetry, electrochemical impedance spectroscopy, and Tafel polarization. The experimental results showed that the power conversion efficiencies of 5.20, 5.12, and 4.70% were obtained for La2O3: MWCNTs with mass ratios of 5:1, 3:1, and 1:1 as counter electrode towards the reduction of I3−/I− ions respectively in the encapsulation of DSSC batteries, which were higher than that of MWCNTs (3.94%) and La2O3 (0.77%) electrode under the similar conditions. The enhanced electrode performance was attributed to relatively larger surface area and higher conductivity of La2O3@MWCNT composite catalysts.

A deterministic Extreme Value Analysis method is of particular importance in an engineering-oriented context. In this paper, three extreme value analysis (EVA) methods, in which annual maxima, monthly maxima or the Peaks-Over-Threshold are fitted into Generalized Extreme Value distribution (GEV) function or Generalized Pareto distribution (GP) function, are used to estimate return values of significant wave height. Sensitivity of return levels on water depth and sampling length is vigorously investigated, based on a 40-year long and high-resolution wave hindcast for the South Yellow Sea (SYS). A spatially-and-temporally varied POT sampling method combined with GP function produces most conservative and confident estimates of return levels for a large return period, but produces wider confidence level than the other two EVA approaches based on GEV function for short return period. In the SYS, the return level estimates are significantly reduced with a longer sample length. However, we find that the reduction is closely related to the long-term trend in extreme wave heights, rather than due to the sampling effect. From deep to shallow waters, spatial inhomogeneity of return levels increases, which should be considered in the engineering practice.

Non‑alcoholic fatty liver disease (NAFLD), which affects approximately one‑third of the general population, has become a global health problem. Thus, more effective treatments for NAFLD are urgently required. In the present study, high levels of C‑C motif ligand 19 (CCL19), signaling pathways such as Toll‑like receptor 4 (TLR4)/nuclear factor‑κB (NF‑κB), and proinflammatory factors including interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) were detected in NAFLD patients, thereby indicating that there may be an association between CCL19 and these factors in NAFLD progression. Using a high‑fat diet (HFD), the present study generated a Sprague‑Dawley rat model of NAFLD, which displayed dyslipidemia with increased levels of plasma aspartate aminotransferase, alanine aminotransferase, total cholesterol and triglyceride. Dyslipidemia, liver histopathology and gene expression analyses indicated that the NAFLD model was successfully induced by HFD, and metformin and berberine (BBR) were effective treatments for NAFLD. HFD‑induced CCL19 levels and associated factors were markedly reduced by the two drug treatments. In addition, metformin or BBR alone significantly promoted adenosine monophosphate‑activated protein kinase (AMPK) phosphorylation, which was inhibited by HFD. These results demonstrated that metformin and BBR could improve NAFLD, which may be via the activation of AMPK signaling, and the high expression of CCL19 in NAFLD was significantly reduced by metformin and BBR. It could be inferred that inhibition of CCL19 may be an effective treatment for NAFLD.

There is still no resolution for arterial remodeling related with hypertension, though hypertension treatment has access to a number of pharmacological agents. The present study aimed at investigating the prevention of Cyathula officinalis Kuan's roots (C. officinalis Kuan) against in arterial remodeling in vitro. Spontaneously hypertensive rats (SHRs) were intragastrically administered 3, 6 or 12 g/kg C. officinalis Kuan or normal saline or enalapril (2.5 mg/kg) once a day for 8 weeks. Hematoxylin and eosin were used to measure blood pressure and stain carotid and arota. The serum concentration of nitric oxide (NO) was measured by NO assay kit (nitrate reductase method). The endothelin-1 transcriptional level, endothelial NO synthase of endothelium as well as angiotensin II receptor type 1 (AT1R) of aorta and carotid was tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the protein level in aorta was also measured by western blotting. The blood pressure in SHR+enalapril, SHR+3 g/kg, SHR+6 g/kg and SHR+12 g/kg C. officinalis Kuan groups was significantly decreased at 4, 6 and 8 weeks post-treatment compared with SHR group. Different doses of C. officinalis Kuan and enalapril treatment showed aortic wall thinness and strengthened NO serum level, but made no impact on the transcriptional level of AT1R in aorta or endothelial NO synthase in carotid. It is suggested by such results that therapy by C. officinalis Kuan is able to fight against arterial remodeling, thus may provide a new means to treat arterial remodeling caused by hypertension.

There is still no resolution for arterial remodeling related with hypertension, though hypertension treatment has access to a number of pharmacological agents. The present study aimed at investigating the prevention of Cyathula officinalis Kuan's roots (C. officinalis Kuan) against in arterial remodeling in vitro. Spontaneously hypertensive rats (SHRs) were intragastrically administered 3, 6 or 12 g/kg C. officinalis Kuan or normal saline or enalapril (2.5 mg/kg) once a day for 8 weeks. Hematoxylin and eosin were used to measure blood pressure and stain carotid and arota. The serum concentration of nitric oxide (NO) was measured by NO assay kit (nitrate reductase method). The endothelin-1 transcriptional level, endothelial NO synthase of endothelium as well as angiotensin II receptor type 1 (AT1R) of aorta and carotid was tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the protein level in aorta was also measured by western blotting. The blood pressure in SHR+enalapril, SHR+3 g/kg, SHR+6 g/kg and SHR+12 g/kg C. officinalis Kuan groups was significantly decreased at 4, 6 and 8 weeks post-treatment compared with SHR group. Different doses of C. officinalis Kuan and enalapril treatment showed aortic wall thinness and strengthened NO serum level, but made no impact on the transcriptional level of AT1R in aorta or endothelial NO synthase in carotid. It is suggested by such results that therapy by C. officinalis Kuan is able to fight against arterial remodeling, thus may provide a new means to treat arterial remodeling caused by hypertension.

Investigating the structure, modification, interaction, and function of biomolecules in their native cellular environment leads to physiologically relevant knowledge about their mechanisms, which will benefit drug discovery and design. In recent years, nuclear and electron magnetic resonance (NMR) spectroscopy has emerged as a useful tool for elucidating the structure and function of biomacromolecules, including proteins, nucleic acids, and carbohydrates in living cells at atomic resolution. In this review, we summarize the progress and future of in-cell NMR as it is applied to proteins, nucleic acids, and carbohydrates.

Macromolecular crowding and confinement are two factors that potentially affect protein structure and function in a complex cellular environment. The confinement effect on the structure and function of holoCaM [Ca2+ -loaded calmodulin (CaM)], a two-domain protein involved in many calcium-mediated signaling pathways, has been investigated previously. However, little is known about how macromolecular crowding affects holoCaM structure and function. Here, the structure-function correlations of holoCaM are investigated in macromolecular crowded environments. It was found that macromolecular crowding impacts its structure and function mildly. The major conformational states are still extended conformation with inter-domain separation in crowded environment as well as those in dilute solution, but the population of transiently compact conformation increases compared to dilute solution. Furthermore, macromolecular crowding facilitates the binding of CaM with AcN19 peptide (CaM-bind domain of α-syn). This study provides a direct comparison for macromolecular crowding and confinement effects on protein structure and function, which helps to understand chemistry regulation in living cells.

One kind of potentially biodegradable cationic sorbent, which bears hydroxyl groups of phosphoric acid as its functional groups, with high sorption capacity of copper was prepared by thermochemically esterifying phosphoric acid (PA) onto soybean hull. Sorption of Cu(II) from aqueous solution onto modified soybean hull (MSH) was investigated in a batch system. The sorption experiments were performed under various conditions such as different initial pH, copper concentration, MSH dosage, and contact time. The maximum copper sorption was obtained when initial solution pH≥3.5. The isothermal data of copper sorption fitted the Langmuir model and the sorption process could be described by the pseudo-first-order kinetic model. The maximum sorption capacity (Qm) of MSH for Cu(II) was 31.55 mg/g. For 100 mg/l of Cu(II) solution, a sorption ratio above 91% could be achieved by 5.0 g/l of MSH. The equilibrium of Cu(II) sorption was reached within 50 min. The foreign cation and chelator in Cu(II) solution caused decline of Cu(II) sorption.

In this paper, waste sawdust was functionalized by monosodium glutamate for improving its cationic sorption capacity. The functionalized sawdust (FS) and crude sawdust (CS) were compared for their malachite green (MG) sorption behaviors with a batch system. The effects of various experimental parameters (e.g. initial pH, sorbent dose, dye concentration, contact time, and temperature etc.) were investigated and the sorption kinetic and thermodynamic characteristics were understood. The MG removal ratios on FS and on CS increased with increasing initial pH and came up to the maximum value beyond pH 6 for FS and pH 8 for CS, respectively. The ratio of sorbed MG kept above 95% for 250 mg/l of MG solution when 2.0 g/l or more of FS was used. The MG removal percentage decreased more on CS than on FS with increasing initial MG concentration. The isothermal data of MG sorbed on FS and on CS followed the Langmuir model. By functionalizing, the sorption capacity (Q(m)) of sawdust for MG was increased from 85.47 to 196.08 mg/g and the sorption equilibrium time of MG was shortened from 23 to 4.5 h. The MG sorption processes on FS and on CS followed the pseudo-second-order rate kinetics. The sorptions of MG on FS and on CS were spontaneous and exothermic processes and lower temperatures were favorable for the sorption processes.

In this paper, a new cation exchanger is prepared by activating sawdust with epichlorohydrin, followed by coupling the epoxy-activated sawdust with aspartic acid. The sawdust cation exchanger (SCE) was investigated for its methylene blue (MB) removal capacity from aqueous solution. The removal experiments were performed under various conditions such as different initial pH, SCE dosage, dye concentration, ionic strength, and contact time in a batch system. The highest sorption capacity of MB was obtained beyond pH 4. The 2.0 g/l or more of SCE could remove above 95% of MB from 250 mg/l of dye solution. Increasing the ionic strength of the solution caused decline of MB sorption. The sorption isotherm conformed well to the Langmuir model. The sorption process could be described by the pseudo-second-order kinetic model.

A cationic sorbent, which bears carboxyl groups of citric acid (CA) derived from esterified wheat straw, was prepared using the solid-phase esterifying technique. Copper (Cu) removal from aqueous solution with modified wheat straw (MWS) was investigated to evaluate its capacities of cation exchange and metal chelation. The effects of various experimental parameters (e.g. ,s olution pH, Cu concentration, sorbent dose, contact time, and presence of light metal) are studied in batch experiments. The maximum value of Cu removal appeared in the range of pH 4t o5 .T he percentage of Cu removal decreased with increasing initial Cu concentration. The isothermal data of Cu sorption conformed well to the Langmuir model, and the maximum sorption capacity (Qm )o f MWS fo rC u was 78.13 mg/g. For 100 mg/l of Cu solution, a removal ratio of greater than 96% could be achieved with 2.0 g/l or more of MWS. The equilibrium of Cu removal was reached within 90 min. The presence of univalent and bivalent light metal ions (Na + ,K + ,M g 2+ ,a nd Ca 2+ )d idnot significantly interfere with Cu removal by MWS.