Systemic juvenile idiopathic arthritis (sJIA) is a complex disease with an autoinflammatory component of unknown etiology related to the innate immune system. A major role in the pathogenesis has been ascribed to proinflammatory cytokines like interleukin-6 (IL-6), and effective drugs inhibiting their signaling are being developed. This study evaluates sJIA patients treated with the IL-6 inhibitor tocilizumab (TCZ) concerning clinical response rate, disease course and adverse effects in a real-life clinical setting.https://aid-register.de). Data for this retrospective TCZ study were documented by 13 centers.In 2009 a clinical and research consortium was established, including an online registry for autoinflammatory diseases (AID)  received therapy with TCZ. Long term treatment (median 23 months) has been documented in 24/46 patients who were evaluated according to Wallace criteria . Under observation co-medication were used in 40/46 cases. Adverse events were reported in 11/46 patients. The clinical response rate  within the first 12 weeks of treatment was calculated to be 35%.Out of 200 sJIA children reported in the German AID-registry, 46 were treated with TCZ, showing a clinical response rate of 35% during the first 12 weeks, and inactive disease and/or remission under medication in 75% after one year. Adverse events were seen in 24% and severe adverse events in 4%.The AID-Registry is funded by the BMBF . The prevalence of amyloidosis was approximately 1–2% until the 1990s and decreased continuously in the last years [Systemic juvenile idiopathic arthritis (sJIA) is a complex disease with an autoinflammatory component of unknown origin. sJIA is characterized by daily spiking fever which persists for at least 2 weeks, arthritis, rash, serositis, lymphadenopathy or hepato(spleno)megaly . Currentst years .sJIA is based on abnormalities in the innate immune system leading to an activation of immunocompetent cells, e.g. phagocytes, with the release of proinflammatory interleukins (ILs) like IL-1, IL-6, IL-18 and proinflammatory S100-proteins . TCZ is https://aid-register.de); additionally, patient material is collected and stored in a biomaterial bank for serum and plasma [The AID-registry is part of the AID-Net (Network for autoinflammatory diseases), a research initiative funded by the German Federal Ministry of Education and Research. Patient data are documented following a pseudonymisation procedure in an online registry  for biomarkers and genetic analysis are collected at study enrollment and follow-up visits. Generally one visit per quarter was scheduled for each patient accounting for an average of 4 visits per year in all sJIA patients. A total of 46/200 (23%) sJIA children were treated with TCZ and longitudinally documented in follow-up. Patients treated with TCZ provided follow-up visits at week 5 and week 12 after treatment start. These patients were available for analysis of the clinical response at week 12 after start of TCZ treatment. The clinical response rate under TCZ at week 12 measured the effectiveness of TCZ and was defined as follows: no symptoms and normal inflammation parameters at that time. Remission after 12 months was evaluated based on Wallace criteria, and therefore we differentiate between active disease (AD), inactive disease (ID) and clinical remission on medication (CRM). Wallace et al. defined ID by meeting the following criteria: no active arthritis, no fever, no exanthema, no serositis, no splenomegaly, no lymphadenopathy, no active uveitis, normal ESR and CRP, no disease activity in physician’s report. CRM was defined as ID for at least 6 months. Active disease (AD) described a visit with symptoms (see above) and increased inflammatory parameters . CRP, ESn = 23), Essen (n = 3), Heidelberg (n = 3), St. Augustin (n = 3), Aachen, Berlin, Bochum, Dresden, Duesseldorf, Hamburg, Krefeld, Landshut and Muenster (each n = 1–2).We included sJIA patients who fulfilled ILAR classification criteria , but alsDescriptive analyses included mean and range for continuously distributed parameters as well as absolute and relative frequencies for categorical data. The number of adverse events was reported by 100 years under TCZ treatment. The confidence interval for the rate per 100 treatment years was estimated by exact Poisson intervals. The likelihood to achieve a clinical response under TCZ at week 12 was analyzed by logistic regression analyses. Additionally, clinical responses at various visits  were correlated with disease activity at the last available visit in 2014. Logistic regression analyses were conducted in STATA 12.1 .The AID-registry has been approved by the ethics committees and the data protection responsibles at the University of Duisburg-Essen and Muenster, as well as the Medical Association Nordrhein in Duesseldorf. Parents, children between 9 and 13 years of age and young patients aged ≥14 years provided informed consent.Forty-six children  with a median age of 11 (range 2–21) years at their last documented visit up to 04/2014) were included in the analyses. Age of first symptoms was 4 (range 0.6–16) years. Altogether, 680 visits were analyzed. Therapy with TCZ was started at the age of 9 range 1–18) years. Duration from diagnosis to TCZ start was 24.5 (1–188) months. 28/46 (61%) had a Caucasian origin , 4/46 (9%) were from Turkey and 6/46 13%) from other East European  and Asian countries and 8/46 (17%) of unknown or mixed ethnicity. 34/46 (74%) patients fulfilled ILAR classification criteria, 12/46 (26%) patients did not fulfill ILAR criteria but other definitions for sJIA and diagnosis was confirmed by pediatric rheumatologists [% from ot years. D14 were iSymptoms at diagnosis of sJIA and at the start of TCZ treatment did not differ significantly. Fever, lymphadenopathy, abdominal pain, hepatomegaly, nausea and vomiting were seen less often at TCZ start, whereas morning stiffness, myalgia, diarrhea, arthralgia and arthritis were observed more frequently. Before TCZ CRP was increased (90 (range 0.1–320) mg/l). Laboratory parameters  before diagnosis and under IL-6 inhibition revealed that monocyclic (MC) courses of sJIA, before initiating therapy with TCZ, showed higher CRP and ESR levels than polycyclic (PC) and polyarticular (PA) courses. After TCZ initiation CRP and ESR levels declined irrespective of the disease course. In fact, 27/46 (59%) patients receiving TCZ showed no measurable CRP during therapy or during a clinically diagnosed flare. 10/46 (22%) showed no measurable CRP within the first 4 weeks after starting TCZ.The median time under TCZ exposure was 14.9 (range 1–48) months for the 46 sJIA patients. In total, 18 (39.1%) patients achieved an inactive disease and 14 (30.4%) a state of remission on medication according to the Wallace criteria. Therefore, a favourable outcome under TCZ treatment was reported for 32/46 (69.6%) of the patients in the registry. Remission on medication was additionally analyzed in a subgroup of patients who were treated with TCZ for at least 12 months. 24/46 sJIA patients (52%) who received TCZ for a median of 23 (range 12–48) months were available for this analysis. The state of remission on medication was achieved for 13/24 (54.2%) sJIA patients, and additional 5/24 (20.8%) were in a state of inactive disease, amounting 18/24 (75%) patients with favourable outcome.Additionally, clinical response rates within the first 12 weeks of treatment have been estimated in all 46 patients. 16/46 (35%) patients showed an effectiveness of TCZ with no clinical manifestation and normalized inflammation parameters after 12 weeks of treatment. Comparison of initial response  and disease activity at last visit suggest that a rapid response to TCZ seems to be related to long term inactivity of sJIA. 7/17 (41%) patients showing inactive disease at the last visit in 2014 had a response to TCZ within 5 weeks. Active disease in the current visit in 2014 was mostly seen in patients who did not show any response within an observation time of > 24 weeks .p = 0.005) followed by the MC courses  compared to the worst outcome for patients with PA courses (29%).Forty-six sJIA children were categorized into monocyclic (MC) 12/46 (26%), polycyclic (PC) 16/46 (35%) and polyarticular (PA) 18/46 (39%) disease courses. Comparison of these courses revealed significant differences in the outcome; PC showed the highest clinical response rate  = 7.0, 95%CI: 1.8–27.2, n = 3, SAEs n = 2.Adverse events (AE) were analyzed for 46 patients contributing to 63 TCZ exposure years. Fourteen adverse events  were reported in 11/46 (24%) patients including leukopenia, infections and elevated transaminases (at least two-fold increased), 2 serious adverse events  were one Hodgkin’s lymphoma  and one Only 4/46 (9%) patients  had to be defined as non-responders over the whole time. In 3/4 patients, symptoms got worse. They subsequently received canakinumab (CANA)/nonsteroidal anti-inflammatory drugs (NSAID), etanercept (ETA)/NSAID and anakinra (ANA)/MTX, respectively. Follow-up data from the fourth non-responder was not available.Before starting TCZ therapy, 33/46 (72%) patients received disease-modifying antirheumatic drugs (DMARDs) (cyclosporine A (CSA), MTX, azathioprine (AZA), abatacept (ABA), colchicine (COL)), 24/46 (52%) biologicals , CANA, infliximab (IFX)), 23/46 (50%) corticosteroids (CO) and 21/46 (46%) NSAIDs. 6/46 (13%) children received no co-medication before TCZ.at the initiation of TCZ were DMARDs  in 35/46 (76%) patients, NSAID in 16/46 (35%) and corticosteroids (CO) in 18/46 (39%) who received steroids in high- or low-dose and 3/18 (17%) who required additional steroid-pulses. 2/46 (4%) patients received ANA or rituximab (RTX) additionally to TCZ. In our patient population 21/46 (46%) had received TCZ as first biological treatment, 25/46 (54%) as second, third, fourth or fifth line therapy. Among those receiving TCZ as first line treatment, 14/21 (67%) showed ID or CRM after 12 months. The switch of biological agents to TCZ resulted in 14/25 (56%) additional cases in ID or CRM. In 9/46 patients a switch from TCZ to other biological agents was seen because of non-response (n = 4) or adverse effects (n = 5).Co-medications Over the whole period of TCZ therapy corticosteroids (CO) were discontinued in 50% (9/18) children. Under observation co-medication were used in 40/46 cases. Co-medications like DMARDs, corticosteroids (CO) and others were very variable and depended on the participating center. Monotherapy with TCZ was rare in responder (n = 2 and n = 1 according to response after 12 weeks and 12 months). After 12 weeks clinical responders (16/46) received TCZ + CO n = 3, TCZ + DMARD etc. n = 11, TCZ + DMARD etc. + CO n = 0 and only TCZ n = 2. After 12 months clinical responders (18/24) received TCZ + CO n = 1, TCZ + DMARD etc. n = 15, TCZ + DMARD etc. + CO n = 1 and only TCZ n = 1 .sJIA is a severe multi-organ disease of unknown etiology. Classification and diagnostic criteria are currently under discussion because sJIA is not a classical autoimmune disease like most other subtypes of JIA but more related to AID. Only 74% of the patients included into the AID-registry fulfilled the ILAR criteria for sJIA while 26% were classified as sJIA based on other criteria , 17–20. Long term follow-up in the AID-registry enables us to report first results on treatment, clinical response rate and tolerance of TCZ in a real-life setting. Another source of real-life data is the German Biker registry . For thin = 4) or adverse effects (n = 5). Kostik et al. showed that during TCZ treatment 40 out of 48 cases (83.3%) achieved remission within 5 months. Patients who achieved remission had milder disease courses [Woerner et al. describe an achievement of inactive disease with maintenance in follow-up in 37/77 (48%) patients who had received biologicals without switching. This was observed in 33/61 patients on anti-IL-1 treatment and 2/2 on TCZ therapy. A switch to a second, third or fourth biological agent resulted in inactive disease in further 13/77 (17%) patients. Median follow-up duration on biological drug was 33.8 months for patients who did not experience a switch and 6.7 months for switchers . In our  courses .Possible courses of sJIA are monocyclic (MC), polycyclic (PC) and polyarticular (PA) (arthritis > 4 joints) activity . PatientA large proportion of patients received MTX 89%) and CO (66%) before using TCZ . In the 9% and COThese findings have led to a shift in treating sJIA, with a recent tendency to use ANA, CANA and TCZ as first line therapy . WoernerAs adverse effects, nasopharyngitis, respiratory tract infections, gastroenteritis, pharyngitis and elevation of transaminases have been described previously , 36, 37.IL-6 induces elevation of acute phase reactants like CRP but also serum amyloid A, fibrinogen and ferritin , 40. It Reports on IL-6 blockade in sJIA are limited by relatively small patient numbers, diversity of study populations, highly heterogeneous treatment regimes, variable co-medications, and relatively short follow-up periods. Despite these shortcomings, TCZ appears to be effective in most patients. In our real-life clinical setting, we observed a clinical response rate of 35% after 12 weeks, a remission on medication in 54% and inactive disease in 21% after 12 months. If evaluated over the entire observation time, remission on medication was achieved in 30% and inactive disease in 39% of cases. It should be noted, however, that patients often require additional treatment. Fine-tuning of dosage and co-medication will need to be addressed by future long-term studies on larger patient cohorts.


Carbonate and borate fluxes provide the largest and structurally best crystals at significantly lower growth temperatures of 1533 K compared to flux-free synthesis (1623 K). The maximum synthesis temperature can be further reduced by the application of PbO-containing fluxes (down to 1223 K upon use of 80 at % PbO), however, Pb-substituted crystals Bax1–PbxFe12O19 with Pb contents in the range of 0.23(2) ≤ x ≤ 0.80(2) form, depending on growth temperature and flux PbO content. The degree of Pb-substitution has only a minor influence on unit cell and magnetic parameters, although the values for Curie temperature, saturation magnetization, as well as the coercivity of these samples are significantly reduced in comparison with those from samples obtained from the other fluxes. Due to the lowest level of impurities, the samples from carbonate flux show superior quality compared to materials obtained using other methods.Barium hexaferrite powder samples with grains in the μm-range were obtained from solid-state sintering, and crystals with sizes up to 5 mm grown from PbO, Na The increase in electromagnetic pollution due to the rapid development of gigahertz (GHz) electronic systems and telecommunication has resulted in a growing and intense interest in electromagnetic absorber technology. Electromagnetic interference (EMI) can cause severe interruption of electronically controlled systems. It can produce device malfunctions, generate false images, increase clutter on radar, and reduce performance because of system-to-system coupling. These are some of the reasons why the use of self-generated electromagnetic radiation apparatuses, which include cellular telephones and wireless computers, are strictly prohibited in certain areas, for example, in hospitals, banks, petrol stations and inside airplanes. To overcome the problems created by EMI, electromagnetic wave absorbers with the capability of absorbing unwanted-undesirable electromagnetic signals are used, and research on their electromagnetic and absorption properties is in progress ,2. RecenHexaferrites with planar magnetic anisotropy are widely used as electromagnetic wave absorbers in the GHz range. Barium hexaferrite powders are ideally suited fillers for the development of electromagnetic attenuation materials at microwave frequencies, due to their low cost, low density, high stability, large electrical resistivity, and high microwave magnetic loss. Thus, they find applications as electromagnetic emission absorber materials ,13,14,15Hа of M-type  Ba hexaferrite can be changed by substituting Fe3+ ions, resulting in a shift in resonance frequency [It is well known that the magnetic properties and, in particular, the anisotropy field requency ,16,17. Trequency ,19,20. S12O19 of 1580 ± 50 °С [2+ by Pb2+, since the eponym of the material with a magnetoplumbite-type crystal structure is the Pb-containing mineral PbFe12O19 [In recent years, the technology of barium hexaferrite synthesis via various techniques has greatly improved, but the main obstacle in crystal growth continues to be the high melting temperature of BaFe ± 50 °С . Current ± 50 °С , or from ± 50 °С . An impo ± 50 °С ,25, bora ± 50 °С ,27, and  ± 50 °С ,28, whicbFe12O19 .2O3 and BaCO3 were heated to 1623 K and held for 3 h.Binary iron and lead oxide, barium oxoborate, as well as barium and sodium carbonate were used as initial components for single crystal growth. For the initial compositions of the different experiments, see Kα radiation . To determine the crystallographic details of the Pb-substituted samples, single crystal X-ray diffraction intensities were collected with monochromatic Mo Kα radiation (NONIUS κ-CCD). This information is presented in the TC  and a vibrating sample magnetometer .To investigate the composition, structure, and properties of the samples, they were examined by the following methods: The chemical compositions of the grown samples were determined with use of an electron scanning microscope with an energy dispersive spectrometer for elemental analysis . X-ray powder diffraction analysis was carried out with filtered Cu 2CO3, BaB2O4, and PbO fluxes to analyze the crystal quality, size, and the resulting magnetic properties. In the case of the PbO flux, a significant substitution of Ba by Pb depending on the growth temperature and flux concentration was observed. x = 0.21, 0.45, and 0.81 for a general formula Bax1–PbxFe12O19, compared to 0.23, 0.44, and 0.80, respectively, from refinements of X-ray diffraction data obtained on single crystals.Barium hexaferrite crystals were grown in the absence of a flux as well as from NaThe optimal temperature of the solid-state synthesis of Ba hexaferrite, which ensures a stable formation and crystal growth, was earlier established experimentally to be 1623 K . Figure 2CO3 and BaB2O4 fluxes were carried out at 1533 K, thus 90 K below the solid-state sintering experiment. Under these conditions, Na2CO3 eventually decomposes to form Na2O. Still, crystal sizes and visual quality were significantly improved  = 1.61 Å; r(Pb2+) = 1.49 Å with CN = 12 [x is expected. However, upon lower PbO concentrations in the flux (60 at %) the unit cell is even very slightly enlarged, but almost does not change with increasing Pb content. Due to the rigid iron oxide framework with larger Ba2+ ions as spacers, the unit cell generally hardly changes upon partial substitution by Pb2+. However, the pure lead compound has a significant about 1.5% smaller unit cell [0.77(2)Pb0.23Fe12O19 at 60 at % PbO over Ba0.56(2)Pb0.44Fe12O19 at 70 at % PbO to Ba0.20(3)Pb0.80Fe12O19 at 80 at % PbO. Earlier, an approximate composition of Ba0.9Pb0.1Fe12O19 was discussed for a PbO flux proportion of 50 at % at 1473 K [As was earlier observed , the PbOfraction  already  CN = 12 ), a decrnit cell . In ordet 1473 K .x1–PbxFe12O19 with no indication for any Ba/Pb order. Close inspection of the refinement results revealed a large flat displacement ellipsoid for the mixed occupied Ba/Pb site  to the 12j site  within the space group P63/mmc is discussed, at which Pb is equally distributed over six split-positions resulting in an occupancy of 1/6  and BaB2O4 fluxes show the highest values of Hc, which are in agreement with earlier derived numbers for materials obtained from such fluxes [The samples grown from Nah fluxes  and are 12O19 with sizes up to 5 mm grown from PbO, Na2CO3, and BaB2O4 fluxes as well as μm-size powder-sintered samples were investigated. Fluxes are able to reduce the growth temperature compared to solid-state sintering, but may supply impurities or additional challenges due to evaporation. The Na2CO3 flux method provides the largest crystals and can be used to obtain materials of superior quality, since coercivity and magnetization suffer from a strong dependence on impurity values. This study shows the possibility to control the magnetic properties of BaFe12O19 in a wide range by using different synthesis techniques.Barium hexaferrite samples BaFeFor PbO-containing fluxes, the lowest melting temperature of 1223 K was observed for a solution with 80 at % PbO, which is significantly lower than the melting temperature of pure barium hexaferrite (1853 K) . However2O flux are optimal for the production of spherical isolator elements. The crystals showed diameters up to 7 mm and thicknesses up to 5 mm. BaB2O4 and PbO fluxes can be used for the production of single crystals with large aspect ratios for flat/plane attenuator manufacturing, since crystal diameters ranged up to 5 mm, while typically exhibiting thicknesses below 1 mm. However, lead oxide shows a high evaporation rate at the applied growth temperatures, and thus presents environmental issues during its use as a flux as well as in the final material lead content. The solid-state sintering synthesis is ideally suited for the cost-effective production of powder for absorbing coatings.From the materials grown in this study, the largest single crystals obtained from Na


Alzheimer’s disease (AD) is a multi-factorial neurodegenerative disorder with abnormal accumulation of amyloid-β (Aβ) plaques, neuroinflammation and impaired neurogenesis. Mounting evidences suggest that single-target drugs have limited effects on clinical treatment and alternative or multiple targets are required. In recent decades, natural compounds and their derivatives have gained increasing attention in AD drug discovery due to their inherently enormous chemical and structural diversity. In this study, we demonstrated that naringin dihydrochalcone (NDC), a widely used dietary sweetener with strong antioxidant activity, improved the cognitive function of transgenic AD mice. Pathologically, NDC attenuated Aβ deposition in AD mouse brain. Furthermore, NDC reduced periplaque activated microglia and astrocytes, indicating the inhibition of neuroinflammation. It also enhanced neurogenesis as investigated by BrdU/NeuN double labeling. Additionally, the inhibition of Aβ level and neuroinflammation by NDC treatment was also observed in an AD cell model or a microglia cell line. Taken together, our study indicated that NDC might be a potential therapeutic agent for the treatment of AD against multiple targets that include Aβ pathology, neuroinflammation and neurogenesis. Multiple factors are involved in the pathogenesis of AD: (1) the accumulation of amyloid-β (Aβ) and excessive Aβ . WT and APP/PS1 mice chronically administered 200 μl of NDC (100 mg/kg) or vehicle per 20 g mouse body weight body weight by oral administration once a day from 3–4 to 6–7 months of age (n = 10–12 mice per group).The APPswe/PS1ΔE9 (APP/PS1) transgenic mice (JAX Stock No. 004462) expressing mouse/human amyloid precursor protein (Mo/HuAPP695swe) and human Presenilin1 (PS1ΔE9) were used in our investigation and mice were maintained and genotyped according to the guidelines of Jackson Laboratory. The wild type (WT) littermates were used as age- and gender-matched controls. NDC  once a day, from days 73 to 79 during drug administration, as described by Encinas et al. .The Morris Water Maze (MWM) were carried out as previously described Morris, . The appThe Novel Object Recognition test (NOR) is also widely used to evaluate recognition memory in mice. The detailed protocol with modifications is as previously described  stored in −80°C were homogenized in 1 ml 2% SDS (dissolved in PBS), then centrifuged at 1,20,000 g for 60 min at room temperature. The supernatant was collected as the soluble fraction and quantified with human Aβ ELISA kits according to the users’ guidelines (ExCell Bio). Total Aβ levels in HEK293/APPswe cell culture medium were also quantified with ELISA.AβSupernatant is also used for Western blot analyses. Proteins in supernatant were separated by SDS-PAGE, and transferred onto membrane. Proteins were labeled with β-actin  and IL-1β rabbit polyclonal antibody  and the immunoreactive bands were detected by chemiluminescent detection (Bio-Rad) of peroxidase-conjugated antibody . The intensity of each band was quantified by ImageJ and normalized to β-actin.After behavioral tests, the mice were anesthetized with chloral hydrate and transcardially perfused with phosphate-buffered saline (PBS) buffer and then with 4% paraformaldehyde (PFA) in PBS. The half brain tissue were serially sectioned at 30 μm thickness and stained using Thioflavin S  and anti-Aβ antibody 6E10  for amyloid plaques, a polyclonal rabbit antibody against GFAP (DAKO) for astrocytes, a mouse antibody against Iba1(WAKO) for microglia and anti-NeuN antibody for neurons.According to previous studies . Each section was captured by confocal microscopy in z stack, covered all layers of cells and all positive-staining cells were counted. For counting the NeuNHEK293 cells were purchased from ATCC. HEK293/APPswe cells were transfected, selected and maintained in our lab. BV2 cell lines were cultured and maintained in Dulbecco’s Minimal Essential Medium (DMEM), with 10% fetal bovine serum and 100 U/ml penicillin and 0.1 mg/ml streptomycin. HEK293/APPswe cells were cultured in MEM under the same condition. BV2 cells were treated with 0.3 μg/ml lipopolysaccharides  and various concentrations of NDC for 6 h. HEK293/APPswe cells were treated with various concentrations of NDC for 24 h.® (Sigma) and used NanoDrop 1000 Spectrophotometer (Thermo Scientific) to assess the RNA purity and integrity. TIANScript M-MLV kit (TIANGEN) was used to synthesis cDNA according to protocols, and rRNasin®  was used in the synthesis. The expression level of mRNA was measured by quantitative Real-time PCR (qPCR) using the 2× HotStart SYBR Green qRT-PCR Master Mix kit from ExCell. The reaction parameters were: 95°C for 10 min; 95°C for 30 s, 40 cycles; 60°C for 30 s; 72°C for 30 s. An additional cycle was performed for evaluation of primer’s dissociation curve: 95°C for 1 min, 60°C for 30 s and 95°C for 30 s. Each cDNA sample was amplified in duplicates. Primer sequences are listed in Table The analysis of mRNA expression was performed as previously reported  according to the manufacturer’s instructions.t-test to determine the statistical significance of treatment sets. For multiple comparisons, one-way ANOVA or two-way ANOVA were performed, followed by Bonferroni test. Differences were considered significant when p < 0.05.All data are presented as mean ± SEM. Statistical analysis was performed with GraphPad Prism 6 Software . Results were analyzed by two-tailed F = 18.65, p < 0.001). We found that compared with WT mice, APP/PS1 mice spent more time in locating the platform (p < 0.001), indicating it exhibited significant cognitive decline in learning, and there was no significant difference between NDC-treated mice and WT mice (p > 0.05), indicating that the cognitive function of spatial memory was significantly improved by treatment of NDC  = 8.650, p < 0.005; Figure F = 0.000097, p > 0.05), compared with WT mice, APP/PS1 mice spent less time in target quadrant  = 1.340, p > 0.05) or distance  = 1.334, p > 0.05) of swimming, suggesting that NDC treatment did not influence locomotor activity of mice . After drug administration, the behavioral tests were performed to investigate the cognitive function of these mice. MWM was carried out to assess spatial learning and memory ability Figure . The latC Figure . On day C Figure  and spen Figures . These rF = 5.412, p < 0.05), the recognition index of NDC-treated mice was similar with that of WT mice  = 5.459, p < 0.001; Figure F = 0.3613, p > 0.05; Figure To identify whether NDC could reduce Aβ level Abnormal neuroinflammation, including activated astrocytes and microglia, is a typical hallmark of neurodegenerative disease, and amyloid plaques are surrounded by activated microglia and astrocytes in the brains of AD patients  = 13.04, p < 0.001), Interleukin 6  = 6.350, p < 0.001), and tumor necrosis factor alpha (TNF-α) expression  = 4.825, p < 0.01; Figures We then investigated the effect of NDC on the LPS-induced microglia activation. The BV2 cells were incubated with various concentrations of NDC in the presence of LPS (0.3 ug/mL) for 6 h. qPCR results demonstrated that NDC inhibited LPS-induced proinflammatory cytokines, interleukin 1 beta  for 7 days and then euthanized after behavioral tests. Brain sections were stained with BrdU/NeuN Figure . CompareIn this study, NDC significantly ameliorated cognitive impairment and neuropathology in transgenic AD mice through reducing amyloid plaque burden and Aβ levels, suppressing neuroinflammation and promoting neurogenesis, which is in line with previous findings on other natural compounds, such as curcumin and Naoling decoction (Ono et al., In current AD drug research, many targets or strategies are considered, such as preventing Aβ accumulation or tau phosphorylation, inhibition of secretase activity that directly modulates Aβ generation, OS, neuroinflammation, as well as mitochondrial damage (Tundis et al., in vitro assays including treatment of NDC and Aβ in primary microglia culture should be carried out.In previous studies, the relationship between neuroinflammation and Aβ is complicated. On the one hand cerebral inflammation could increase Aβ production (Iadecola, In summary, the current study first demonstrates the protective effects of NDC on AD pathology by targeting multiple processes in an AD mouse model, which suggests that NDC may serve as a promising therapeutic agent for AD.in vitro experiments. KZ and WY contributed to the manuscript preparation. SH and JL contributed to critical revision of the manuscript. All the authors reviewed and commented on the manuscript.GP substantially designed and controlled the study. WY and YZ performed the behavioral tests. KZ, WY and YZ performed histopathological and data analysis. YA and TH performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dolutegravir (DTG) plus darunavir/ritonavir (DRV/r) is a simple combination of drugs that has the best genetic barrier to HIV-1 resistance and may be fit for salvage therapy.All HIV-1-infected subjects treated with DTG plus DRV/r between March 2014 and September 2015 in eight Italian centres were included in the analysis. The main metabolic data, efficacy parameters and safety data routinely collected were provided. This observational study is aimed to assess the efficacy of such approach. The primary end-point was the proportion of subjects achieving or maintaining virologic suppression <50 copies/mL at week 24. Secondary end points were maintaining virologic suppression in the follow-up (weeks 48 and 96) and safety.One hundred and thirty subjects were followed for a median of 56 months. Reasons for switching were simplification (44.6%), viral failure (30%), toxicity (16.9%), non-adherence (4.6%), persistent low-level viremia (3.1%), and drug-drug interaction (0.8%). At baseline, 118 subjects had documented resistance to 1 to 5 antiretroviral classes while 12 had viral rebound at a time when genotypic tests were not yet available.Seventeen and 14 subjects took DRV/r and DTG twice daily, respectively. One subject was lost to follow-up, one discontinued for liver enzymes’ elevation, one died of illicit drug abuse and one of cancer-related complications.The proportion of subjects with ongoing HIV replication dropped from 40% to 6.1%. Those with undetectable viral load increased from 38.5% to 76.2%. At week 48, 17.7% had HIV RNA between 1 and 49 copies/mL.The number of subjects with altered serum glucose, creatinine, ALT, AST, total-, HDL- and LDL-cholesterol, triglycerides and MDRD <90 mL/min decreased by week 48, while those having MDRD <60 mL/min remained 4.6%. Overall 90/283 baseline laboratory alterations returned to normality.Switching to DTG plus DRV/r proved to be safe, suppressing viral replication without metabolic impact.The online version of this article (10.1186/s12879-017-2755-4) contains supplementary material, which is available to authorized users. The present work is the 48-week update of a previously reported study , concernThe combination of elvitegravir/cobicistat/tenofovir alafenamide/emtricitabine plus 800 mg DRV (two pills once daily) demonstrated superiority towards the continuation of baseline therapy in a randomized (2:1), open-label, switch trial in treatment-experienced virologically suppressed subjects . The basIn this context some authors were looking forward for nucleoside-sparing regimens in the long-term care of HIV infection, welcoming the possible positive contribution of DTG .The present study reports what the clinical practice had already realised, aiming for convenience, simplicity, potency and high genetic barrier, prescribing this combination to several patients and controlling them closely until viral suppression was achieved.Tivicay plus Prezista Observational cohort). After approval by Ethics Committees (EC) no further enrolment was allowed. Since the last publication two centres that were waiting for EC approval joined the group, increasing the population size. For homogeneity no data were collected after week 48 . The follow-up was censored at November, 30, 2016. Subjects were not required to be naive to DRV or DTG, but regimens containing both drugs were not included.All subjects who had started DTG plus DRV/r between March 1, 2014 and September 30, 2015 were included in an Italian observational multicenter cohort named Tivista  Guidelines.The aim of the study is to assess the efficacy of this approach. The primary end-point was the proportion of subjects achieving or maintaining virologic suppression <50 copies/mL at week 24. Secondary end points were maintaining virologic suppression in the follow-up (weeks 48 and 96) and safety, as proportion of drop-outs for any reason and grade 3–4 adverse events.In this update it was decided to document the impact of the switch on some routine safety analyses. Investigators retrospectively reported to the coordinating center serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) and triglycerides (TG). The estimated glomerular filtration rate (e-GFR) was calculated at baseline and at follow-up according to the Modification of Diet in Renal Disease (MDRD) equation . Plasma Viremia was measured depending on centers with Abbot HIV-1 RT-PCR, threshold 37 copies, COBAS TaqMan HIV-1 test, threshold 20 copies, and Single Copy Assay. TruGene™ was used for resistance testing, validated on the Stanford Algorithm, and cumulative data was considered. Data were collected from electronic or hand-written patients’ case record forms, according to each centre’s organization and sent as pre-specified excel files. Available data from routine therapeutic monitoring of darunavir and dolutegravir trough plasma concentration were analysed.For the toxicity analyses the Common Terminology Criteria for Adverse Events  was considered . Adverse10 copies/mL in those who do not reach suppression at each time point. The other analyses consider the on-treatment population. The sample size was obtained from the participants’ adhesion.The statistical analysis was limited to routine parameters  and to the paired t-test for the comparison between the baseline and 48-week metabolic and immunologic values. The virologic response is reported as the number of patients in each viral suppression category and as the median and interquartile range of logn = 63), followed by drug abuse, 26.9% (n = 35) and heterosexual intercourse, 24.6% (n = 32). Reasons that led to the switch were simplification, 44.6% (n = 56), viral failure 30% (n = 39) toxicity 16.9% , non-adherence, 4.6% (n = 6), persistent low-level viremia, 3.1% (n = 4), and drug-drug interaction, 0.8% (n = 1). The algorithm of the study population is represented in Fig. One hundred and thirty subjects were followed for a median of 56 months, mean 64 months. The median age was 52 years, females were 25.4% and non-Caucasians 8.9%. The median baseline CD4+ T-cell count was 526/mmc IQR 329–718). Fifty-nine subjects had been exposed to INSTIs and all had been exposed to PIs. Eighty-one had darunavir in their pre-switch regimen and one had dolutegravir. The main risk factor for acquiring HIV was being male homosexual, 48.5%  had never experienced virologic failure but had transmitted drug resistance mutations.One hundred and sixteen patients (89.2%) had NRTI resistance-associated mutations (RAMs) at baseline, 98 (75.4%) had NNRTI RAMs, 91 (70%) had PI RAMs and 12 (10.6%) had integrase strand transfer inhibitors (INSTI) RAMs , and 12 had reduced sensitivity to INSTIs  but none rebounded. None developed new drug resistance mutations during the study.n = 70), 35 were not failing at baseline, but rather simplified from raltegravir and boosted darunavir-based three- (n = 31) or four-drugs regimens (n = 9).Of those subjects who had resistance to 3–4 drug classes (10 HIV-1 RNA copies/mL). All of them suppressed viremia to <50 copies/mL during the follow-up.One hundred and thirteen have started the combination of DRV 800 mg plus RTV 100 mg plus DTG 50 mg every 24 h, 3 DRV 600 mg plus RTV 100 mg every 12 h and 14 DRV 600 mg plus RTV 100 mg plus DTG 50 mg every 12 h. All of those who were on BID therapy had active HIV-1 replication at baseline  At week 48, 19 subjects  had HIV RNA between 1 and 49 copies/mL and eight subjects  had ≥50 HIV-1 RNA copies/mL. Of the latter, 3 had a slow but constant decay from median baseline 5.12 to 2.30 log10 HIV-1 RNA copies/mL, 3 had at least three-class drug resistance with a Stanford DRV score > 60 and median viral decay from 4.39 to 1.75 log10 HIV-1 RNA copies/mL, and 2 had dropped out of therapy 4 and 7 days before blood sampling. Considering separately two populations, those who started with active HIV replication (≥ 50 copies/mL) and those who started with 0–49 copies/mL, at week 48 the success rate was 88.5% in those who had started with active replication, as 3 with high baseline viral load and 3 with extensive baseline resistance, had still very low but detectable viremia, while in those who started with 0–49 copies/mL the success rate was 97.4% as in this group two subjects were not on therapy on the sampling day, as described above. Of interest, both were in the subgroup starting with 1–49 copies/mL, as some authors suggest that this low but persistent viremia may sometime hide adherence problems. The mean viral load in this population at week 48 was 1.89 log10 copies/mL  increased from 38.5% to 76.2%. Overall, at baseline 78 subjects had <50 HIV-1 RNA copies/mL (60%) and this proportion increased at week 48 to 90,8% (trough was 579 ng/mL (range 275–5036), while DRV median Ctrough was 3007 ng/mL (range 678–8053) and no values were found to be below the safety threshold. Five subjects were taking DRV 600 mg twice daily and had Cmin above the threshold for resistant strains, and three were taking DTG twice daily.A single-point pharmacokinetic analysis in a subgroup of 32 subjects confirmed the safety of the association: DTG median CThe proportion of subjects having any metabolic alterations at baseline decreased for serum glucose, creatinine, MDRD <90 mL/min, ALT, AST, total-, HDL- and LDL-cholesterol and triglycerides. The proportion of subjects having MDRD <60 remained stable at 4.6%. Overall 84/175 (48%) baseline laboratory alterations returned to normality. Considering each parameter in the whole population, none varied significantly Fig. .Fig. 3MeIn particular, the median variation in serum creatinine was −0,03 and the eGFR decrease on average by 2.66 mL/min. None of the subjects who had altered serum creatinine at baseline showed progression and 6 out of 8 improved, having discontinued tenofovir.Overall this strategy yielded mean savings for € 169,428.00 considering the whole population, the observation period and the dropouts. The figure was generated only from the cost of the previous and current antiretroviral regimen.min in the subjects tested nor leaving space for plasma HIV-1 RNA rebound in any subject. In particular, 70% of our patients would not have been suitable for the combination of DTG plus atazanavir, which has a favourable interaction profile [While awaiting the results of the randomized DUALIS study , our dat profile  due to b profile  PossibleThe study limitations include poor patient selection (the only two inclusion criteria were being HIV-1 antibody positive and having started DTG plus DRV/r by the end of September 2015), leading to the inclusion of a heterogeneous population, made out of failures and simplification, once- and twice-daily regimens, adherent and poorly adherent subjects and subjects harbouring treatment-resistant and treatment-sensitive strains. The set of metabolic parameters furthermore reflects standard follow-up data shared by centres and does not include bone density measures nor inflammatory markers.Switching to DTG plus DRV/r provided a simple and safe rescue regimen to all subjects, controlling viral replication in a high proportion of patients. The metabolic impact was favourable in a proportion of subjects who had baseline alterations, while no significant modifications were observed in mean and median values when also normal baseline values were included.


Vascular calcification is a significant predictor of coronary heart disease events, stroke, and lower-limb amputation. Advanced glycation end-products (AGEs) play a key role in the development of vascular calcification. However, the role of Nε-carboxymethyl-lysine (CML), a major active ingredient of heterogeneous AGEs, in the development of atherosclerotic calcification in diabetic patients and the underlying mechanism remain unclear. Hence, the role and the mechanism of CML in the transmission pathway of diabetic calcification cascade were investigated in the present study.In vivo and in vitro investigations were performed. In study I, 45 diabetic patients hospitalized for above-knee amputation in the Department of Orthopedics, Affiliated Hospital of Jiangsu University were recruited from February 2010 to June 2015. The patients were categorized based on the severity of anterior tibial artery stenosis, which was assessed by color Doppler ultrasound, into mild stenosis , moderate stenosis , and severe stenosis/occlusion groups . In study II, the specific mechanism of CML in the transmission pathway of the diabetic calcification cascade signal was investigated in A7r5 aortic smooth muscle cells under high-lipid, apoptosis-coexisting conditions. ELISA (for serum CML concentration of patients), ultrasound , H&E staining (for plaque morphology), vonKossa staining , calcium content assay , and Western blot analyses of CML, receptor for advanced glycation end products (RAGE), NADPH oxidase 4, phosphorylated p38, core-binding factor α1 (cbfα1), alkaline phosphatase (ALP) and β-actin were then performed.2 = 0.6141, P < 0.0001). Semi-quantitative Western blot analysis suggested that the intensity of CML/RAGE signal increased with progression of atherosclerotic calcification in diabetic patients. In subsequent in vitro study, the related pathway was blocked by anti-RAGE antibody, NADPH oxidase inhibitor DPI, p38MAPK inhibitor SB203580, and anti-cbfa1 antibody in a step-wise manner to observe changes in calcium deposition and molecular signals. Results suggested that CML may play a key role in atherosclerotic calcification mainly through the CML/RAGE- reactive oxygen species (ROS)-p38MAPK-cbfα1-ALP pathway.Morphological analysis revealed extensive calcification lesions in the intima and media of the anterior tibial artery. The extent and area of calcium deposition in the intima significantly increased with disease progression. Interestingly, spotty calcification was predominant in the atherosclerotic plaques of diabetic patients with amputation, and macrocalcification was almost invisible. Pearson correlation analysis revealed that serum CML level exhibited a significant positive correlation with calcium content in the arterial wall (RSpotty calcification was predominant in the atherosclerotic plaques of amputated diabetic patients. CML/RAGE signal may induce the calcification cascade in diabetes via ROS-p38MAPK. The International Diabetes Federation reported that the number of diabetic patients worldwide was 387 million in 2014 and is predicted to reach 592 million by 2035 , blood urea nitrogen (BUN) and serum creatinine (SCr) were collected before the amputation. All patients were divided into three groups based on the severity of anterior tibial artery stenosis, which was assessed by color Doppler ultrasound: mild stenosis group , moderate stenosis group , and severe stenosis/occlusion group . Written informed consents were obtained from all patients. This study was approved by the Ethical Committee of Jiangsu University and conducted in agreement with the institutional guidelines.Related indices of lower extremity arteries were detected by Philips HDll color Doppler ultrasound equipped with a linear array transducer (7–12 MHz variable frequency). The angle between the direction of the sound beam and the blood flow was less than 60°. The patients were positioned in supine position or prone position for all measurements. The femoral artery (FA), popliteal artery (PA), anterior tibial artery (ATA), and dorsalis pedis artery (DPA) were successively surveyed. Two-dimensional ultrasound was used to display the vessel long/short axis section images and detect plaque size, number, and vascular diameter. Color Doppler flow imaging (CDFI) was used to observe blood flow filling, vascular stenosis, and occlusion. The degree of artery stenosis was calculated by dividing the residual diameter (N) by the vessel diameter at a point distal to the stenosis where the normal vessel caliber was restored (D). The formula is presented as follows:Serum CML concentration of patients who underwent amputation was measured by CML ELISA Kit  according to the manufacturer’s instructions. The mean minimum detectable concentration is 0.126  ng/mL. The antibodies in the CML kit are highly specific for CML adducts and do not exhibit cross reactivity to non-CML proteins in human plasma. The intra- and inter-assay coefficients of variation (CV) for CML are 5.2–7.4% and 4.7–15.2%, respectively.Anterior tibial artery samples were obtained from the foot of diabetic patients who underwent lower extremity amputation. After perfusion with cold phosphate buffer saline (PBS), each artery was cleaned to remove connective and adipose tissues and then divided longitudinally into three pieces. One piece was immediately frozen and stored at −80 °C until protein extraction for Western blot and ALP activity detection. One piece was used for calcium content detection, and the other piece was fixed in 10% neutral buffered formalin overnight and embedded in paraffin. From the paraffin blocks, 4 μm-thick serial sections were examined for hematoxylin–eosin (H&E) and von Kossa staining.l-glutamine, 110 mg/L sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. After RAW264.7 apoptosis induced by 50 μg/mL oxLDL for 48 h, the culture flasks were tapped to completely harvest the apoptotic bodies (ABs) as described previously [g (4 °C) for 20 min to remove cell debris. The resultant cell-free supernatant was re-centrifuged at 150,000×g (4 °C) for 1 h to obtain a pellet containing ABs. The pellet was washed three times with Hank’s Balanced Salt Solution . Protein content was determined using Bradford method.RAW264.7 macrophages and A7r5 aortic VSMCs were cultured in DMEM/LOWGLUCOSE, supplemented with 10% fetal bovine serum (FBS), 4.0 mmol/L eviously . The supThe specific mechanism of CML in the transmission pathway of diabetic calcification cascade signal was also investigated using in vitro experiments. A7r5 VSMCs were divided into the following groups: control group (A7r5 medium supplemented with 80 μg/mL RAW264.7-derived-ABs plus 50 μg/mL oxLDL), CML group , anti-RAGE group , NADPH oxidase inhibitor group , p38MAPK inhibitor group , and anti-cbfα1 group . Cells in each group were cultured for 7 days, and the medium was replaced every 2 days.2O). The paraffin sections of the isolated anterior tibial artery were dewaxed and hydrated. Two slices were immersed in 1% silver nitrate for 30 min under intense sunbeam or ultraviolet light. The samples were then washed in distilled water to remove excess reagent, incubated with 5% sodium thiosulfate for 5 min, washed once in tap water and several times in distilled water, and finally counterstained with eosin (tissue) or neutral red (cell) for 10 min. After several washes, the slices were observed under an Olympus microscope.The A7r5 cell climbing slices were fixed in 4% paraformaldehyde for 30 min and then washed twice with double-distilled water  was determined as previously described . Dried aThe ALP activity assay kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate, which turns yellow (λmax = 405 nm) when dephosphorylated by ALP. As previously described , the totTotal proteins of anterior tibial artery and cells were initially extracted by centrifugation in RIPA lysis buffer. Equal amounts of protein samples were loaded into 10 or 12% SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane. The nonspecific proteins were blocked with 5% nonfat dried milk for 1 h. The membranes were incubated with the primary antibodies anti-CML (1:5000), anti-RAGE (1:500), anti-Nox4 (1:500), anti-P-p38 (1:500), anti-cbfα1 (1:500), anti-ALP (1:500), and anti-β-actin (1:1000) overnight at 4 °C as well as with secondary antibody (HRP-conjugated IgG) for 1 h. HRP-conjugated secondary antibodies were used in conjunction with an ECL chemiluminescence detection system. Protein expression was analyzed by Gel-Pro Analyzer 4 software and normalized to that of β-actin.t test was used. Comparison among more than two groups was conducted using one-way analysis of variance (ANOVA), followed by post hoc LSD test. Pearson correlation analysis was used to detect the correlation between serum CML level and calcium content in the arterial wall. A two-tailed P < 0.05 was considered statistically significant.Data were expressed as mean ± SD and analyzed by SPSS 13.0 software. For comparison between two variables, unpaired Student’s Descriptive information regarding baseline characteristics and laboratory results is listed in Table In gross observation, different degrees of gangrene, swelling, skin ulcers, and infection in diabetic foot patients were observed among the three groups Fig. . The extIn contrast to the lesions in the mild/moderate stenosis group, advanced atherosclerotic lesions with abundant cholesterol crystals and loss of integrity of the intern elastic lamina occurred in the severe stenosis/occlusion group Fig. . von KosP < 0.001) compared with those in the mild stenosis group. The index in the severe stenosis/occlusion group was increased by 1.57-fold  compared with that in the moderate stenosis group. Pearson correlation analysis revealed a significant positive correlation between serum CML level and calcium content in the arterial wall  . Intercellular calcium deposition was reduced by 50.6% . The expression levels of Nox4, P-p38, cbfα1, and ALP were decreased by 39.6, 32.5, 53.0, and 55.2%, respectively.Compared with the indices in the CML-oxLDL-Abs group, the anti-RAGE blocking group under high-lipid, apoptosis-coexisting conditions induced the following changes. ALP activity was inhibited by 51.8%  and 33.0% , respectively, comparing with CML-oxLDL-Abs group. Meanwhile, the expression levels of Nox4, P-p38, cbfα1, and ALP in western blot were decreased by 86.8, 22.4, 49.4, and 33.2%, respectively, but there were no significant changes in the expression of RAGE.When pretreated with NADPH oxidase inhibitor DPI, ALP activity and Intercellular calcium deposition were reduced by 39.7%  and intercellular calcium deposition was reduced by 43.7% . However, the expression levels of P-p38, cbfα1, and ALP were downregulated by 88.7, 82.2, and 53.8%, respectively. Interestingly, no significant changes in the expression of RAGE and Nox4 were observed.Subsequently, the treatment with p38MAPK inhibitor SB203580 induced similar changes with DPI: ALP activity was inhibited by 44.6%  and reduced intercellular calcium deposition by 49.0% . And importantly, anti-cbfa1 antibody reduced the expression level of ALP by 57.1%. However, it had no significant effects on the expression of RAGE, Nox4, P-p38, and cbfα1. Hence, our data above suggested that CML may play a key role in atherosclerotic calcification mainly through CML/RAGE-ROS-p38MAPK-cbfα1-ALP pathway.Moreover, compared with CML-oxLDL-Abs group, the treatment with anti-cbfa1 antibody could inhibite ALP activity by 54.5% , the Nox family of NADPH oxidases including seven different enzymes is the principal source [The interaction between AGEs and RAGE enhances intracellular oxidative stress and promotes the production of reactive oxygen species (ROS) , 25. ROSl source –29. Theil source , 31. Furl source , 33. GivEarly studies revealed that AGEs could mediate the activation of p38MAPK signals through ROS .In additFirstly, in the model of CML-accelerated VSMC calcification under high-lipid, apoptosis-coexisting conditions, anti-RAGE antibody, NADPH oxidase inhibitor DPI, p38MAPK inhibitor SB203580 and anti-cbfα1 antibody significantly inhibited ALP activity and intercellular calcium deposition, but the specific signal stream was unclear. Then the expressions of related signal protein were further established. The expression levels of Nox4, P-p38, cbfα1 and ALP were significantly decreased when pretreated with NADPH oxidase inhibitor DPI, but no significant changes in the expression of RAGE, indicating that CML/RAGE signal was the upstream signals of Nox-derived ROS, p38MAPK, cbfα1 and ALP. Subsequently, our data showed that the treatment with p38MAPK inhibitor SB203580 downregulated the expression of P-p38, cbfα1 and ALP. However, no significant changes in the expression of RAGE and Nox4 were observed, suggesting p38MAPK, cbfα1 and ALP were the downstream molecules of Nox-derived ROS. Now that the expression of cbfα1 could be significantly inhibited at different extents by DPI and SB203580, respectively, blocking experiment with anti-cbfα1 antibody was performed. The ALP expression was significantly reduced by anti-cbfα1 antibody. However, no significant changes in the expression of RAGE, Nox4, P-p38, and cbfα1 were observed, which shed light on that cbfα1 was the upstream molecule of ALP, but downstream molecule of p38MAPK. Considering these results, we conclude that CML/RAGE–ROS-p38MAPK-cbfα1- ALP calcification may be the most important cascade of CML/RAGE in diabetic calcification.In conclusion, these findings suggest that spotty calcification was predominant in the atherosclerotic plaques of amputated diabetic patients and that CML/RAGE signal may induce the calcification cascade in diabetes via ROS-p38MAPK. On the basis of the target signal of the calcification cascade, related drug development and subsequent optimization of an intervention strategy would provide new opportunities for clinical treatment of chronic vascular complications of diabetes.


The microalgae–fungi pellets are deemed to be a viable option because of their high specific growth rate and nutrient and CO2 removal efficiency under the photoperiod of 14 h light:10 h dark. The order of both the biogas slurry purification and biogas upgrading is ranked the same, that is Chlorella vulgaris–Ganoderma lucidum > Chlorella vulgaris–activated sludge > Chlorella vulgaris under different light intensities. For all cultivation methods, the moderate light intensity of 450 μmol m−2 s−1 is regarded as the best choice. This research revealed that the control of photoperiod and light intensity can promote the biological treatment process of biogas slurry purification and biogas upgrading using microalgal-based technology.Co-cultivation of microalgae and microbes for pollutant removal from sewage is considered as an effective wastewater treatment method. The aim of this study is to screen the optimal photoperiod, light intensity and microalgae co-cultivation method for simultaneously removing nutrients in biogas slurry and capturing CO Therefore, CO2 should be removed to enhance CH4 concentration to meet the efficient combustion standard  [2 is always discharged into the atmosphere as greenhouse gas in these conventional techniques [2 uptake by microalgae is an alternative technique to upgrade biogas, as they have high carbon fixation capacity, rapid specific growth rate, and strong environmental adaptability. In addition, biogas slurry could provide most of the required nutrients for microalgae growth as a readily available nutrient medium [2 from raw biogas by culturing microalgae in biogas slurry is a highly prospective technique for simultaneous biogas upgrading and biogas slurry decontamination, characterized as renewable, sustainable and environment-friendly energy.Owing to fossil fuel combustion during anthropogenic activities, greenhouse gas (GHG) emissions are ever increasing at present, which induces an increased interest in searching for renewable, sustainable and environment-friendly energies as an alternative to fossil fuel due to increased concern over the energy crisis, global warming and climate changes . Biogas 2%, v/v) . Obvious CO2, 15–0%, v/v, chniques . Accordird CH4 > 0%, v/v [4 enrichment based on several crucial factors, such as microalgae species, light wavelength, light intensity, initial influent CO2 concentration, light photoperiod, etc. [v/v) were 93.68 ± 3.25% CH4, 1.57 ± 0.42% CO2, and the highest removal efficiencies of chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) of biogas slurry were 78.91 ± 2.75%, 73.05 ± 2.04%, and 67.54 ± 1.46%, respectively. However, harvesting of the microalgae cell from industrial cultivation for biofuel production, wastewater treatment or value-added chemicals cultivation have always been the major obstacles for the algae-to-fuel approach, as the addition of chemicals or excessive energy demand were required [Chlorella vulgaris, Scenedesmus obliquus and Neochloris oleoabundans mixed with activated sludge) and high levels of biomass productivity were also observed in the experiment [Recently, mono-cultivation of microalgae has already been successfully utilized for simultaneous biogas slurry treatment and CHod, etc. ,11,12. Fod, etc.  reportedrequired ,15. To rrequired . It has required . For co-required ,18,19. Aperiment . SimilarLight wavelength and intensity are essential parameters for microalgae growth and photosynthesis, which are connected with the microalgae carbon fixation . However2 uptake by different microalgal-based cultivation approaches, i.e., mono-cultivation of microalgae, co-cultivation of microalgae with fungi, and co-cultivation of microalgae with activated sludge under various light intensities and photoperiods in photobioreactors. The lighting control strategy and appropriate cultivation technology were optimized by analyzing the biomass reproduction, as well as the removal efficiencies of CO2, COD, TN and TP under various light intensities and photoperiods using three microalgal-based cultivation approaches, i.e., algal, algal–fungal and algal–bacterial cultivation systems.Aiming at these unsolved problems, this research focused on nutrient and COChlorella vulgaris  was selected based on its high biogas tolerance and fast specific growth rate in high nutrient concentration wastewater [3 (1500 mg L−1), K2HPO4·3H2O (40 mg L−1), MgSO4·7H2O (75 mg L−1), CaCl2·2H2O (36 mg L−1), ferric ammonium citrate citric acid·H2O (6 mg L−1), EDTA-Na2 (1 mg L−1), Na2CO3 (20 mg L−1), and A5 (1 mg L−1). The content of A5 consisted of H3BO3 (2860 mg L−1), MnCl2·H2O (1860 mg L−1), ZnSO4·7H2O (222 mg L−1), CuSO4·5H2O (79 mg L−1), NaMoO4·2H2O (390 mg L−1), and CoCl2·6H2O (49 mg L−1) [−2 s−1, photoperiod of light 12 h:dark 12 h, temperature at 25 ± 0.5 °C, 7 days of experimental duration time and artificial oscillation three times a day  at speed of approximately 200 rpm. Such mono-cultivation was carried out in an illuminating incubator . LED lamps were installed on three sides of the incubators. The dry weight (DW) of selected microalgal strains in the stock culture was nearly 135.28 ± 12.09 mg·L−1.stewater ,25. ThenGanoderma lucidum  was obtained from China General Microbiological Culture Collection Center and selected for this experiment because of its high specific growth rate and high pelletization performance with C. vulgaris based on our preliminary studies. For pelletization, every 5 mL spore solution was cultivated at 25 ± 0.5 °C for 7 days in 100 mL synthetic growth medium . Afterwards, algal cultures (100 mL) were centrifuged, washed and resuspended to achieve a final total suspended solid (TSS) concentration of about 153.45 ± 13.77 mg L−1, and then mixed with G. lucidum suspension  for further co-cultivation in the photobioreactors. The algal–fungal mixtures were shaken at 160 rpm for 48 hat constant light intensity of 200 μmol m−2 s−1, light-dark cycle of 12 h:12 h, and temperature of 25 ± 0.5 °C [−1.−1) was derived from a wastewater treatment plant of Shanghai and mixed with 1 L cultured microalgae broth. The photoperiod, light intensity and temperature are the same as Culture 2. The initial TSS concentration in the co-cultivation broth of the biogas slurry was about 135.51 ± 9.06 mg L−1.The 200 mL of nitrifying-denitrifying activated sludge  CH4, 33.79 ± 1.56% (v/v) CO2, 0.38 ± 0.03% (v/v) O2, and 1.23 ± 0.11% (v/v) H2O. Raw biogas slurry was pretreated by passing through a glass microfiber filter  and ultraviolet sterilized  Water Treatment Co., Ltd., Shenzhen, China) to avoid potential interference from impurities sediment and microorganisms. The characteristics of biogas slurry before and after pretreatment are listed in Crude biogas and biogas slurry were obtained from a farm biogas plant in Jiayuan green meadow. Firstly, raw biogas was desulfurized by chemical absorption reactors until the HThe photobioreactor comprises two interconnected 16.8 L  glass-made cylinder blocks  filled with 14 L raw biogas and 2.8 L biogas slurry. For details on the experimental device, refer to our previously published work . The exp2 in biogas using microalgal mono-cultivation and co-cultivation. Firstly, three photoperiods  were settled for each culture . Other constant parameters of cultivation conditions were as follows: cool-white light with light intensity of 200 μmol m−2 s−1, temperature at 25 ± 0.5 °C and 10 days of experimental time. Secondly, the two relatively optimal co-cultivation methods were selected based on the specific growth rate as well as nutrient and CO2 removal efficiency. The different light intensity  was screened, and illuminated with the optimal photoperiod which was determined as the optimal photoperiod in the first step. During 10 days of the operation time, the experimental groups were periodically sampled for evaluation of biomass grow rates, mean daily productivity, biogas purification and the biogas slurry nutrients removal efficiency. Lastly, both the light intensity and photoperiod strategy were optimized by analyzing the economic efficiencies of the biogas CO2 upgrading and the biogas slurry nutrient removal.Batch experiments were carried out to remove nutrient in biogas slurry and CO4, CO2, O2 and H2O) using a circulating gas analyzer . Each 50 mL co-cultivated suspensions were filtered by a glass microfiber filter , and dried at 100 °C for 24 h before being cooled to room temperature in a desiccator. The DW of the microalgae was calculated as the difference between the filter weight before and after filtration. The filtrates were analyzed for COD, TN, and TP according to a standard method [The biogas was sampled from the port of the photobioreactor for components analysis , mean daily productivity , biogas CO2 and total biogas slurry nutrient removal efficiency  and the economic efficiency of the biogas CO2 or biogas slurry nutrient removal  were experimentally estimated by following Equations (1)–(4) [iDW and DW0 are the biomass DW (g L−1) at time it and t0 , d is experimental duration time (days), iC and C0 are biogas CO2 content  or biogas slurry nutrient concentration (g L−1) at time it and t0, k is the electric power charge per unit of power consumption (USD kW−1 h−1), T is the illumination time (h), and p is the LED electrical power consumption (W). Normally, the electric power charge per unit of power consumption k in local is around 0.645 RMB kW−1 h−1, equivalent to 0.096 USD kW−1 h−1, based on the price level in Jiaxing City, China.Special growth rate ( (1)–(4) ,11,20.. One-way analysis of variance was used to determine whether there was a significant difference between the variables, such as light intensity, photoperiod, and microalgal-based cultivation approaches. Duncan’s multiple range tests was used to analyze the difference between groups. The threshold for significance was set at Specific growth rates and mean daily productivity in these cultures were measured under different photoperiods as 12 h light:12 h dark cycle (short), 14 h light:10 h dark cycle (middle) and 16 h light:8 h dark cycle (long), and the results shown in There are three probable reasons for these special experimental phenomena. Firstly, light was the only energy source in photoautotrophic conditions in this study. Furthermore, sufficient light can enhance the growth of photoautotrophic biomass significantly, as the light photons are absorbed by biomass as their nutrients ,28. Neve2 sequestration in the bioreactor, which was the key mechanism for biomass reproduction [2 through photosynthesis than mono-cultivation [2 uptake process [2 uptake and nutrients adsorption by these microalgal–fungal and microalgal–bacterial cells played a key role in microalgae cell growth [2S and CO2 from biogas by three microalgal strains  mixed with activated sludge using biogas slurry as nutrient medium [Secondly, microbial cells in these three cultures have different abilities for metabolism and photosynthesis based on COtivation . Besides process . Such COl growth ,31. Nowal growth ,32. Furtl growth , who revl growth ,34,35. St medium . In concC. vulgaris and revealed that optimal growth of the microalgae occurred when the pH of the medium was adjusted to values of 6.9–8.0, while acidic (3.0–6.2) and alkaline (8.3–9.0) pH values retarded the growth of this alga. In this study, variations of pH under photoperiod with the three cultures are shown in p > 0.05). Consequently, it is of great importance for pH to affect strains growth in the cultivation for biogas upgrading and simultaneously biogas slurry nutrients removal, but all the variation of pH remains in the optimal domain for biomass reproduction under all the culture condition in this operation time. Therefore, pH was not the upmost important factor that affects the microalgal growth and mean daily productivity in this study. Thirdly, as is well-known, the pH is an important factor for cultivation condition in the photobioreactor. Normally, common problems associated with culture media are the use of an inadequate pH and high levels of precipitate resulting from incorrectly formulated media, including omission of vital ingredients . RecentlN. palea and C. vulgaris during the moderate photoperiod averagely. Similarly, Yan and Zheng (2013) reported that Chlorella sp. can remove 36–86% (average 52%) of COD from biogas slurry within 24 h under short (12 h light:12 h dark), moderate (14 h light:10 h dark), and long (16 h light:8 h dark) photoperiods [2 as the sole carbon source and induced COD removal in the biogas slurry subsequently. The carbon existed in the biogas and biogas slurry accounts for approximately 50% of microalgal biomass and acts as fundamental element in microalgal cells [The COD% removal efficiencies of the three cultures were evaluated at different photoperiods, and the results are displayed in operiods . Zhao etoperiods  also indoperiods , who repoperiods . Microalnd Zheng 13 report2 concentration. Our previous work revealed that enhanced average TN% removal efficiency was observed as 61.12–73.24% by co-cultivation of C. vulgaris and G. lucidum [Besides, TN in biogas slurry was also removed significantly in the three cultures separately under the three photoperiods . HoweverC. vulgaris and G. lucidum [As shown in  lucidum . TherefoTo sum up, the results of microalgae DW growth partly agreed with biogas slurry nutrients removal. Specifically, the biogas slurry nutrients  were reduced efficiently by Culture 1, Culture 2 and Culture 3 under the photoperiod treatments. Culture 2 seems to be the optimal procedure for COD, TN and TP removal efficiency in the biogas slurry under 14 h light:10 h dark cycle. Both microalgal–fungal and microalgal–bacterial co-cultivation systems are superior to mono-cultivation approaches for biogas slurry nutrients removals under 14 h light:10 h dark cycle.2 is the vital carbon source of microalgal, fungal and/or bacterial cells in this experiment: approximately half of the microbial cells DW can be attributed to CO2-derived carbon [2, CH4, H2O and O2) were investigated over time, using three different microalgal-based cultivation approaches under three different photoperiods. During the experimental operation time, CO2% removal efficiency increased significantly with all microalgal, microalgal–fungal and microalgal–bacterial cultivation technologies under photoperiods , followed by the long photoperiod (16 h light:8 h dark cycle) and short photoperiod (12 h light:12 h dark cycle). With Culture 2, for example, average and maximum of CO2 remove efficiency achieved 52.26 ± 5.37% and 74.54 ± 5.13%, respectively, at the end of the operation time under middle photoperiod, followed by 50.68 ± 4.75% and 73.28 ± 3.47% with long photoperiod, and finally 51.66 ± 4.65% and 70.28 ± 3.97% with short photoperiod, which were in agreement with results from Zhao et al. [2 removal efficiencies of 81.68 ± 3.28% at low light intensity with long photoperiods, 86.15 ± 3.94% at moderate light intensity with middle photoperiods and 53.25 ± 3.21% at high light intensity with short photoperiods. In the present study, the efficiency of biogas upgrading in different cultivation systems agreed with trends observed for specific growth rate (d−1) and mean daily productivity (g L−1 d−1)  enrichment in upgraded biogas, irrespective of the microalgal strains present, which further confirm the prominent role of co-cultivation with fungi or bacteria [2 can be reduced by 49.95–62.31% by microalgal–bacterial co-cultivation. Similarly, Zhang et al. [2% removal efficiency using microalgal–activated sludge ranged between 66.93% and 88.27%, which is higher than using mono-cultivated microalgae (54.79–74.65%) or co-cultivated microalgae and fungi (56.15–83.31%). In this study, the methane content was enriched significantly because the CO2 content in the biogas was effectively reduced by the photosynthesis of microalgae when co-cultivated with fungi or activated sludge in the photobioreactor with middle photoperiod (14 h light:10 h dark cycle). Hence, CO2 removal during the biogas upgrading provides an additional strategy for GHG emission control by reincorporation it into the biogas production process. COd carbon . Therefooperiods . Furthero et al. , who repL−1 d−1) , partly bacteria , who rep2, COD, TN, and TP removal efficiencies had the same trend. The Economic efficiency of the energy consumption for COD% removal efficiency, TN% removal efficiency, TP% removal efficiency and CO2% removal efficiency by co-cultivated microalgae–fungi (Cultures 2) was significantly higher than other two cultures. These findings agreed with the results of microalgae specific growth rate, mean daily productivity, removal efficiency of CO2 in biogas and biogas slurry nutrient. As a result, the optimal cultivation approach for removal of CO2 in biogas according to economic efficiency was co-cultivation of C. vulgaris with G. lucidum under photoperiod of 14 h light:10 h dark cycle.Using three different microalgal-based cultivation approaches, the economic efficiency of the energy consumption for both biogas and nutrient removal under various photoperiods were evaluated according to the calculation of Equation (4), and the results are shown in −2 s−1. Therefore, it was still unclear whether such selected light intensity was the most optimal one for microalgal–fungal and microalgal–bacterial system. Aiming at such unsolved problems, the effect of light intensity on nutrient and CO2 removal for the two co-cultivation approaches under the optimal photoperiods (14 h light:10 h dark) were evaluated and the results are displayed in According to above-mentioned conclusions, Culture 2 and Culture 3 have significant effects on biogas upgrading and biogas slurry nutrients removal under optimal photoperiods as 14 h light:10 h dark cycle during 10-day operation schedule. However, they were obtained under stationary light intensity of 250 μmol m−2 s−1 achieved higher mean daily productivity, CO2 removal efficiency and biogas slurry nutrient removal efficiency than those under light intensity of 200 μmol m−2 s−1 . In short, the optimal light intensity for nutrients and CO2 removal were 450 μmol m−2 s−1 and 250 μmol m−2 s−1, respectively. Ouyang et al. [−2 s−1 was the most suitable light intensity. Similarly, Ho et al. [−2 s−1 is optimal for microalgal growth (indigenous S. obliquus). This inconsistency between our results and other research might be attributed to the different species, different photoperiod or different cultivation condition applied in its corresponding experiments. Based on  m−2 s−1 . Furtherg et al.  investigo et al.  reported−2 s−1 in this study, during 10 days, the microalgal–fungal and microalgal–bacterial cells reproduced rapidly, possiblu because the nutrients in the biogas slurry culture were sufficient and the metabolic waste had not accumulated thickly. Anyway, the nutrient in the biogas slurry was efficiently reduced during the biogas upgrading process under moderate light intensity of 450 μmol m−2 s−1.Generally, adequate illumination is the most important factor affecting microalgal, fungal, and bacterial growth. Thus, an increase in the light intensity can enhance biomass reproduction of the microbial cells at a certain range. However, once the light intensity exceeds the light saturation limit level, the microalgae would be damaged by excessive light energy because high light intensities may lead to growth inhibition, photosystems overload, photosystem damage, photo oxidation and/or photo inhibition . InverseC. vulgaris with G. lucidum or activated sludge promoted the biogas upgrading and biogas slurry nutrient removal under various light intensities and photoperiods in photobioreactors. Removing nutrient and CO2 with Co-culturing of C. vulgaris and G. lucidum under the photoperiod of 14 h light:10 h dark and moderate light intensity of 450 μmol m−2 s−1 were regarded as the optimal strategy. More than 70% of mean COD, TN and TP in addition to more than 52% of mean CO2 were removed. The analyses of economic efficiency also showed the economic superiority of the selected strategy for biogas slurry purification and biogas upgrading in this study. Co-cultivation of


Several papers have previously reviewed the coupling of Cu and Zn isotope systematics, and we give here a tentative review of the recent publications only on Cu isotopesin variable surface repositories, animals and human beings, with a goal to attract much attention to research on Cu  behaviors in the environment and biological systems.Copper (Cu) is a transition metal and an essential micronutrient for organisms, but also one of the most widespread toxic inorganic contaminants at very high content. The research on Cu isotopes has grown rapidly in the last decade. Hitherto, a large number of studies have been published on the theoretical fractionation mechanisms, experimental data and natural variations of Cu isotopes in variable environments and ecosystems. These studies reported a large variation of δ Cu isotopes are proven to be helpful for understanding sources of Cu and processes that Cu undergoes in surface environments. Cu isotope studies have an apparently longer history, back to the 1940s [Copper (Cu) is a transition metal and a ubiquitous biolimiting micronutrient that involves in many enzymes and regulatory proteins as a cofactor in almost all living organisms . On the 65Cu 69.1% and 63C65Cu 69.1% and 63C 63Cu 30.3%. Cu ishe 1940s ,12,13,14he 1940s ,13,15, a65Cu 69.% and 63Che 1940s ,19,20,21Preceded this work, Albarède has reviewed some earlier papers about the isotope geochemistry of Cu (and Zn), with a focus on the analytical techniques and Cu isotope variations in biological and geological samples . It must+ was oxidized to Cu2+ on the resin and then Cu2+ was extracted into the elution, which allowed for a higher recovery (100 ± 2%) and lower procedure blanks. Since the ocean plays an important role in the global biogeochemical cycle of Cu, two separation protocols were developed for purifying trace Cu from seawater [2 coprecipitation followed by anion exchange chromatography can efficiently separate and purify Cu from seawater samples (recovery of about 99 ± 9%). Later, in 2013, Takano et al. [Before isotope determination by MC-ICP-MS, Cu should be extracted and purified from sample matrix, especially from Zn, Co and Cr that might interfere Cu isotopes during instrumental analysis. Practically, different anion-exchange resins were examined and used to purify Cu from natural samples, including AG1×2 , AG1×4 , AG 1×8 , AG 1×10 , and AG MP-1  ,11,23,24seawater ,26. Bermseawater  firstly 65Cu (‰) notation, with a comparison to the 65Cu/63Cu ratio of the Cu standard solution, calculated by the formula:Nowadays, Cu isotope determinations are generally performed on MC-ICP-MS . The Cu isotope data for a sample are expressed as δ SRM 976 ,23,24, h SRM 976 ,27,28, a SRM 976 ,30,31.65Cu values of +0.01 ± 0.05‰, +0.21 ± 0.05‰, +0.44 ± 0.04‰, +0.91 ± 0.03‰ and δ65Cu = −0.05 ± 0.03‰ relative to the NIST SRM 976 [The NIST SRM 976 Cu solution was the commonly recommended standard for Cu isotopic analysis and widely used to test the instrument stability. Unfortunately, the NIST SRM 976 is no longer available; an alternative reference material is thus needed to replace this standard. This additional standard should be pure and sufficient to meet the global need for long term investigations on Cu isotopes and be (inter-) calibrated against NIST SRM 976. This also helps for testing and verifying accurate Cu isotope analysis ,13,27. R SRM 976 . Thus, tSince instrumental fractionation may eventually be produced in MC-ICP-MS measurements, the raw data  must be corrected to obtain reliable isotope ratio ,28. Norm65CuP-I) was determined for describing the enrichment or depletion of Cu isotope (63Cu or 65Cu) in the product compared to the initial materials, and was calculated as following:65Cup and δ65CuI were the isotope ratio in the product P and initial material I, respectively. 65Cu (−1.46 ~ +4.40‰) in low temperature processes. Studies on leaching of different ores or minerals indicated that, in general, heavy Cu isotope (65Cu) was preferentially released into the acidic solutions (with Δ65Cusolution-solid as large as +3‰), while the lighter remained in the mineral or residual phases [65Cu values to the chalcopyrite, close to an equilibrium fractionation in a closed system. Large isotopic fractionation was observed in aqueous Cu(II) reactions with CuS and FeS with heavy isotope (65Cu) enriched in solution  [65Cucomplex-free: +0.14 to +0.84‰) [65Cusolution-solid [Many experiments have been conducted in laboratory to better understand the Cu isotopic fractionation mechanisms in potential migration and transformation processes involved in the surface biogeochemical cycling of Cu. Previous studies showed that Cu isotope fractionation could be induced by various low temperature processes, such as adsorption, mineral dissolution, redox reaction, complexation with minerals and/or organic matters, and biotic incorporation ,45,46,47l phases ,49,50,51l phases . In a chl ~ +3‰) ,53. Comp +0.84‰) ,40. Durion-solid ,50,54,55on-solid  that Cu 65Cu by up to +3.0‰ in Cu sink on bacteria surfaces in contrast to the aqueous phase. Navarrete et al. [65Cusolution–solid) produced by live bacterial cells ranging from +0.20 to +2.60‰, and live-bacteria intracellular incorporation preferred the lighter Cu isotope even produced a significant larger fractionation of Cu isotopes (apparent Δ65Cusolution-solid ranging from ~+1.0 to +4.4‰). They suggested that the cell enrichment of the light Cu isotope was probably a metabolically-driven phenomenon rather than caused by reversible surface adsorption, a result consistent with the previous study [Cu isotopic fractionation may also be triggered by biological processes. It is worth mentioning that Mathur et al.  observede et al.  performeus study . + and Cu2+ in soils and Cu2+ was the major form [65Cu with Fe oxide proportion at different crystallization stage, and suggested an important effect of redox condition (caused by water saturation) on Cu isotope variation. This was further confirmed by the sequential chemical extraction study of the same group on a soil core from a freshwater intertidal mudflat, which showed δ65Cu values increased with depth while the redox conditions changed from oxic to strongly anoxic conditions [4NO3-extractable, NaOAc-extractable, NH4Ox-extractable, hot H2O2/NH4OAc-extractable and residual fractions, respectively). They observed a strong effect of redox conditions on Cu partitioning and isotope composition in soils and a Cu availability declining under anoxic conditions. In addition, Cu isotope ratios might display seasonal variations with organic matter and redox condition concomitantly changes [65Cu values with depth. The transportation and bioavailability of Cu in soils were strongly controlled by the chemical Cu forms and affected by water-induced alterations under variable soil redox conditions. Cu could exist in three forms as elemental Cu, Cujor form ,60. Sevejor form ,61,62,63nditions  studied  changes  also fou65Cu (average δ65Cu = −0.5 ± 0.2‰) during shale weathering compared to the parent rocks (average δ65Cu = +0.03 ± 0.15‰), while the pore waters were enriched in 65Cu (average δ65Cu = +1.14 ± 0.44‰) [Other studies reported also large variations of Cu isotope ratio during surface weathering of shales. Black shales, one of the main Cu pools that can release Cu to water systems, displayed large isotope variation (between −6.42 and +19.73‰), with heavy Cu isotope significantly preferred to release into fluids, which was in line with leaching experimental results . For ins± 0.44‰) .65Cu (−16.49 to +20.04‰) was observed in ores and minerals [65Cu variation around zero, whereas leached  was isotopically lighter and supergene minerals  isotopically heavier [65Cu values (−0.64 to +1.82‰) than non-metasomatized peridotites (from −0.15 to +0.18‰), indicating metasomatism might trigger significant isotopic fractionation.Lithosphere is an important Cu reservoir on the earth. About 75% of the world’s copper are supplied by porphyry copper systems . There aminerals ,69,70, a heavier ,72,73,74 heavier . It sugg heavier ,78,79. I65Cu variations in native Cu, malachite, azurite and chalcopyrite at a single site, suggesting that the δ65Cu variations were mainly caused by low temperature aqueous process fractionation rather than source heterogeneity. In the sedimentary mineralization, the Cu isotopic ratio of Cu sulphide concretions exhibited abimodal distribution (δ65Cu = −0.91 ± 0.51‰ and −3.32 ± 0.29‰), and redox and salinity variations were likely the major regional controlling parameters affecting the Cu isotopic ratio [65Cu (+5.63‰) of their Cu crystal specimen was significantly different from Michigan Cu samples with a tight cluster of Cu isotope ratio (averaging δ65Cu ~ +0.30‰), but in accordance with the Namibian chalcopyrite (δ65Cu: +1.73~ +9.27‰), signifying a possible origin of these minerals from Namibia.Cu isotope can be significantly fractionated (~36‰) in low-temperature ore deposits, and this was mainly attributed to the processes such as redox reactions, mineral dissolution and precipitation ,49,53,66ic ratio . Interesic ratio  measuredLens culinaris), and two monocots, virginia wild rye (Elymusvirginicus) and hairy-leavedsedge (Carexhirsutella), the shoots were isotopically lighter than the germinated seeds and the Cu isotopic signature of leaves had a correlation with the plant heights [As a redox-active metal, Cu functions as an enzyme-activator and is a significant prosthetic-group-part of many enzymes . Given t heights . Oat pla heights . In addi heights . 65Cu) during plant–soil and within-plant processes are illustrated in 65Cu variation between plant organs and rhizosphere environment (soil/solution) are −1.43 to −0.11‰, while the in-plant Δ65Cu ranged from −0.90 to +1.35‰. The largest variation of Δ65Cuplant-rhizosphere environment (−1.43‰) was observed between root and solution in tomato, while the maximum in-plant Δ65Cu (+1.35‰) was found between stem and root of tomato.Summary on fractionation factors  in aquatic environment  displayed a complementary light pool  [65Cu (+0.31‰), similar to the SPM (δ65Cu = +0.26‰), and higher than the local bedrock (+0.07‰), due to the significant contribution from long-term application of coppery fungicides [65Cu values of the surface water were found increasingly lighter along the distance from the Pebble Porphyry Cu-Au-Mo deposit, indicating a gradually varied contribution from high-temperature-formed mineral weathering [Studies carried out in last decade have shown that Cu isotope approach could provide new insights into fingerprinting sources and processes (and mechanisms) that Cu involved in various aquatic environments, including rivers, streams and oceans ,99,100. ‰ ± 2SD) ,88,89,99‰ ± 2SD) . For exaathering . 65Cu values, and attributed this to the high isotopic exchange rate between the colloidal and ionic Cu. Another interesting study on wetland showed that the dissolved Cu was depleted in 65Cu when traversing the wetland (Δ65Cuinlet−outlet between +0.03 and +0.77‰), probably caused by Cu adsorption onto organic matters and aluminum minerals [Up to now, the fractionation of Cu isotopes triggered by in-river processes  has rarely been reported. Ilina et al.  showed tminerals .65Cu variation (−0.44‰ to +24.4‰) in stream water , which could be stemmed from pyrite tailings and chalcopyrite weathering. Both results were consistent with the leaching experiments that heavy Cu was preferentially enriched in solutions [65Cu values possibly influenced by the atmospheric deposition of smelter exhaust is about +0.94 ± 0.1‰  between 1900 and 1979.Cu isotopes have also been used to trace the impact of mining or smelting activities on aquatic systems. Sulfide-bearing tailing profiles showed that light Cu isotope was enriched in the oxidized zone than in the original materials . Song etolutions ,44,45,48olutions  performe65Cu variation of +0.38 to +1.44‰, averaging +0.70‰ [65Cu in deep seawater, Takano et al. [65Cu (+0.44 ± 0.23‰) than the bulk isotopic ratios (δ65Cu ~ +0.90‰) of dissolved Cu in seawater, comparable to other studies [Except for continental aquatic environments, researchers also conducted studies on Cu isotope compositions in sea water sampled from the English Channel, Atlantic, Pacific, Indian oceans, andarctic gyre and determined a large δg +0.70‰ ,25,83,89g +0.70‰ ,85,89. Io et al.  also demo et al.  compared studies ,89.65Cu variation of the atmospheric precipitation  was −1.44 ~ +0.40‰, averaging −0.12‰ [Up to now, only few studies reported Cu isotopes in the atmosphere, which could however be an important tracer to trace atmospheric Cu contamination and transportation. The reported δg −0.12‰ ,82. More65Cu values  varied significantly from −1.5‰ to +1.5‰, and it was implied that Cu isotope signature could potentially be modified by the prion protein expression [65Cu was enriched by about 1.0‰ in bones of carnivores than those of herbivores [Recently, isotope geochemists have made efforts to investigate metal  isotopes in organisms of animals and human beings and to link isotopic fractionations to the metabolism processes and thus relative health effects. Significantly, Cu is a micronutrient, a catalytic and structural cofactor for various important enzymes referred to tumor development, and also behave as a modulator of inflammation. It is worth mentioning that Cu isotopic signatures have been reported in organs and body fluids of mammals including human beings, though data from human material were very limited for ethical reasons. Oxidized cations such as Cu(II) and low coordination numbers were found to favor heavy isotopes contrast to the reduced product (Cu(I)) and high coordination numbers . For thepression ,102. Morrbivores . 65Cu value in serum (−0.7‰) and the highest in erythrocytes (+0.9‰); Blood of men and women displayed visibly different Cu isotope compositions and with 0.2‰ lighter for women. More interestingly, B-type men erythrocytes could fractionate Cu to a larger extent than all the other blood types, and light Cu was mainly enriched in blood and lymphatic circulation rather than in erythrocytes [65Cu values in human blood decreased with age, and Cu isotope ratios in the Yakut’s blood were significantly lighter than those in blood of European and Japanese populations. In an archeological human bone study, the male’s bones exhibited heavier Cu isotopes than the females’ bones, implying Cu isotopes could be used as a valid tool to distinguish sexes of incomplete human remains [Cu isotopic ratio has been investigated in materials from human bodies both for healthy people and cancer patients . Cu was hrocytes . Jaouen hrocytes  found th remains .65Cu values were observed in the serum of patients with breast, liver, and colon cancer in contrast to those in samples from healthy individuals [63Cu-enriched by about 0.4‰ compared with that of the control group, and more concentrated Cu was observed in red blood cells and serum of HCC patients [65Cu decreased in the serum while increased in cancer cells, which was likely attributed to the extensive oxidative chelation of Cu by cytosolic lactate, and most patients in their study with serum δ65Cu below the threshold level (−0.35‰) did not survive [65Cu decline preceded biomarkers for several months, thus suggested a strong potential of Cu isotope composition as a new diagnostic tool at least for breast and colorectal cancers. In addition to that of healthy individuals, Cu isotopic characteristics were also studied for cancer patients. Significantly lower δividuals . In the patients . For col survive . Particu65Cu (−16.49 to +20.04‰), which might result from many processes including especially the redox reaction. The surface weathering process  would modify the initial Cu isotopic signature of bedrocks, by, for example, enriching heavy Cu isotope (65Cu) in water, and thus light Cu isotope (63Cu) preferentially remains in the parent materials. The input into the ocean through continental rivers is thus generally characterized by higher Cu isotope composition. However, the anthropogenic contribution may neutralize this Cu isotopic signature by adding Cu with lower δ65Cu value. Though the riverine input  could impact the Cu isotopic compositionin offshore seawater, the biological processes may play an important role on the budget of Cu and its isotopes in pelagic water of the open sea. Interestingly, large variation of δ65Cu (−1.45~+1.24‰) has been observed in animal and human materials, implying that the diet intake and metabolism processes would also induce Cu isotope variation. Importantly, several studies reported that healthy individuals and cancer patients possess different Cu isotopic ratios in the same organism, demonstrating a great potential of Cu isotopes in human health studies especially as a new diagnostic toolfor cancers. Though significant progress has been made in the last decade, more studies should be carried out on Cu isotopic fractionation in surface environmental, biogeochemical, and metabolism processes, in order to better understand the transformation of Cu  from the environment to ecosystem and even human bodies and the relative toxicity effect of Cu .Cu isotopes are increasingly studied and applied in surface biogeochemistry in recent years. As a main Cu reservoir on the earth, the lithosphere displays a large variation of δ