Only 8% of these cities had population-weighted mean concentrations below the World Health Organization guideline for annual average PM2.5. City-level PM2.5-attributable mortality rates ranged from 13–125 deaths per 100,000 people. PM2.5 mortality rates and carbon dioxide (CO2) emission rates were weakly positively correlated, with regional influences apparent from clustering of cities within each region. Across 82 cities globally, PM2.5 concentrations and mortality rates were negatively associated with city gross domestic product (GDP) per capita, but we found no relationship between GDP per capita and CO2 emissions rates. While results provide only a cross-sectional snapshot of cities worldwide, they point to opportunities for cities to realize climate, air quality, and health co-benefits through low-carbon development. Future work should examine drivers of the relationships  uncovered here and explore uncertainties to test the robustness of our conclusions.Urban air pollution is high on global health and sustainability agendas, but information is limited on associated city-level disease burdens. We estimated fine particulate matter (PM The world’s urban population is expected to grow from >50% of today’s global population to 66% by 20504, with urban areas projected to absorb all population growth. Efforts to address urban air pollution by intergovernmental organizations, global networks , national governments, and individual cities can benefit from quantitative estimates of urban air pollution-related health impacts. Such estimates can help prioritize mitigation actions in cities  and can motivate national scale policies . Furthermore, since combustion is a major source of greenhouse gases and air pollution5, cities can reap immediate and local health benefits while also contributing to reductions of combustion-related climate-forcing pollutants6. Air pollution disease burdens by source sector have been quantified at the national level8 and city level for individual cities11 but information is limited for cities globally.Urban air pollution is high on the global sustainable development agenda2.5 is considered the leading environmental health risk factor globally and is a top 10 risk factor in countries across the economic development spectrum12. Early studies estimating the global burden of disease from air pollution focused on cities, where most of the world’s ground-based monitors were located13. Currently the most comprehensive global burden of disease studies report estimates at the national scale 14, enabled by the full global coverage and high resolution of satellite remote sensing of aerosol optical depth15. Here, we exploit these global, highly resolved PM2.5 concentrations to estimate the burden of disease attributable to PM2.5 in 250 major cities worldwide. Unlike previous estimates of air pollution disease burdens among subsets of cities18, our globally consistent methods enable comparisons across cities worldwide and are compatible with the Global Burden of Disease 2016 (GBD 2016) Study12.Ambient PM2.5-attributable mortality in 2016 for the 250 most populous urban areas (see Methods regarding the city definition). The median population-weighted PM2.5 concentration was 29 µg/m3 [standard deviation (sd) = 43 µg/m3, range 5–365 µg/m3; Fig. 2.5 (10 µg/m3). Among the 250 cities, only 21  had population-weighted mean concentrations below the guideline, whereas 104 (42%) exceeded the WHO Interim Target 1 (35 µg/m3). The median rate of PM2.5-attributable deaths was 39 deaths per 100,000 people  have exported PM2.5 and health impacts to other places (e.g. Asian cities) which manufacture consumption goods that are then imported elsewhere, as explored previously e.g. 20. To identify cities that are performing better or worse than predicted by the linear per capita GDP-PM2.5 deaths relationship, we examined the regression residuals. Mexico City, Monterrey, Rio de Janeiro, Sao Paolo and Melbourne had lower PM2.5 mortality rates compared with their predicted values, while Wuxi, Tianjin, Wuhan, Moscow and Warsaw had higher mortality rates than expected based upon GDP per capita.To further elucidate why PMons Fig. . The opp2.5 disease burden in urban areas worldwide using methods that are globally consistent  and compatible with the Global Burden of Disease 2016 Study. Estimated PM2.5-attributable deaths per 100,000 people varied by a factor of 10 across the 250 most populous cities worldwide, indicating that some cities are achieving far lower levels of air pollution-related health impacts than others. We found a weakly positive correlation between PM2.5 mortality and CO2 emission rates, which suggests that there may be opportunities for cities to achieve climate and air quality co-benefits through mitigation measures that address both PM2.5 and CO2. In contrast, we found that while regions with wealthier cities have reduced their PM2.5 concentrations and mortality burdens considerably, CO2 emissions have not declined in parallel. This first cross-sectional snapshot of cities globally does not allow for drawing strong conclusions as to the factors driving these relationships. However, we suspect that several explanations for these relationships may be occurring in concert: (1) historical tendency in developed countries to address air quality by implementing end-of-pipe emission controls that reduce air pollution but not carbon ; (2) movement of industry and power generation out of cities, while the relatively “clean” energy sources remaining in cities still produce CO2 emissions; (3) “out-sourcing” manufacturing and associated pollution from wealthy cities to other locations around the world, where lax environmental regulations may result in more emissions per unit energy consumed. While the first factor reduces PM2.5 levels, the second two simply move pollution from one place to another without necessarily improving air quality overall. Future research could examine these and other characteristics of cities, such as development stage, fuel mix for electricity generation and transportation, and sector-specific emissions of PM2.5 and CO2, in more detail and over time, to further elucidate the drivers of the relationships uncovered here.These analyses provide the first estimates of the PM2.5 levels are also mostly trending upward21. This initial analysis of city air pollution burdens using globally consistent methods paints a salient yet still emerging lesson: to slow climate change, improve air quality, and protect public health simultaneously, historically “successful” air quality management programs may not be enough. Low carbon development, however, can avoid the fossil fuel combustion that releases both air pollution and greenhouse gases. As air pollution remains a top 10 risk factor for most countries globally, all cities, even those with relatively low PM2.5 mortality rates, can improve local public health by transitioning away from fossil fuels. Thus, the challenge of urban PM2.5 can also be viewed as an opportunity – reducing fossil fuel combustion offers local and immediate air quality and public health benefits, in addition to slowing climate change globally and over centuries. This opportunity can be realized in many ways, including by improving building energy efficiency, displacing vehicular traffic with active transportation, electrifying public transportation, and transitioning to renewables for power generation. Several of these approaches would have additional co-benefits from fewer road traffic collisions, more physical activity, less noise pollution, and other improvements.The world faces a challenge as urbanization rapidly expands populations mainly in Asian and African cities, where PM2.5 mortality estimates for many cities which otherwise would have none), bottom-up and local data could improve estimates for individual cities. For example, though we used national disease rates, subnational disease rates can vary by ±20–40% or more compared to national average rates22. This additional heterogeneity is not captured here, but is small relative to the global differences we estimate. We neglected uncertainty in the input variables, though PM2.5 concentrations, relative risks, CO2 emissions, carbon footprints, and city GDP are each uncertain and may vary between existing datasets and inventories23. PM2.5 concentrations are uncertain because much of the world still lacks ground monitoring networks, though most monitors included by Shaddick et al.15 were in cities. Beyond PM2.5, urban populations are also exposed to ground-level ozone, nitrogen dioxide, and other combustion-related air pollutants. PM2.5 is also associated with other health outcomes, including asthma24, excluded here for consistency with the 2016 GBD. Our analysis is cross-sectional and could be supplemented with future longitudinal analysis to identify determinants of PM2.5-CO2 relationships  and consider other climate warming pollutants. Exploring uncertainties and their influences on city-level PM2.5-attributable mortality estimates could also test the robustness of these results and conclusions.Several limitations may affect the strength of our conclusions. While our top-down, globally consistent approach offers consistency and broad coverage 15, population, national baseline disease rates, and concentration-response relationships from the GBD 201625. Annual average PM2.5 concentrations were estimated by combining satellite-derived aerosol optical depth with vertical aerosol distribution from a chemical transport model, calibrated to 6,003 measurements from 117 countries. Gridcell concentrations ranged from 0.9 to 990 μg/m3 globally. Gridded population counts aggregated to 0.1° × 0.1° are from the CIESIN Gridded Population of the World v4 . We downloaded country-, age-, and cause- specific baseline deaths in 2016 from the GBD Data Exchange .We estimated PM2.5 concentration using Integrated Exposure Response (IER) curves25. The shape of the IERs depends on the health endpoint, and flattens at very high concentrations, particularly for cardiovascular endpoints. We created lookup tables in 0.1 µg/m3 increments of PM2.5 concentration, following previous studies27. Central estimates of PM2.5-attributable health impacts were calculated using the mean of the 1000 IER parameter draws for each health endpoint, and 95% confidence intervals were calculated using the 2.5th and 97.5th percentiles. We applied theoretical minimum risk exposure levels included with the IER parameter dataset from a uniform distribution of 2.4 to 5.9 µg/m3. All calculations were performed in MATLAB r2013b and R v3.4.2.We calculated age- and cause-specific relative risk of disease for each gridcell PM2.5 in 2016 was associated with 4.1 million deaths , within 0.3% of GBD 2016 results25. Approximately 20%, 39%, 19%, 7%, and 16% were from stroke, ischemic heart disease, chronic obstructive pulmonary disease, lung cancer, and lower respiratory infections, respectively.Globally, we estimate that ambient annual average PM2.5 mortality, we summed gridded PM2.5 mortality estimates within urban spatial extents from the Global Human Settlement grid (GHS-SMOD) for 2015 at 1 km resolution 28. We defined cities following the “urban centers or high density clusters” definition, with ≥1,500 inhabitants per km2 or a density of built-up ≥50% and ≥50,000 inhabitants. We matched GHS-SMOD city identifiers to city names in ArcGIS. GHS-SMOD city definitions treat patches of dense contiguous urban fabric (e.g. Tokyo-Kawasaki-Kawagoe-Hachioji-Yokohama) as one large “city”. Scaling the 1 km urban definition grid to the 0.1° × 0.1° resolution of our disease burden estimates resulted in loss of urban spatial extent, population, and air pollution-attributable deaths compared with the finer resolution. Therefore, to retain as much data as possible, we multiplied our estimated air pollution-attributable deaths in each urban area at 0.1° × 0.1° by the ratio of population in each urban area calculated at high-resolution  versus low resolution (0.1° × 0.1°).For city-specific PM2 emissions in 2016 are from the Open-source Data Inventory for Anthropogenic CO2 (ODIAC), a globally gridded (1 km) satellite-derived dataset29. City carbon footprints (for 2013) are from recently published estimates for 13,000 cities using the same GHS-SMOD city definitions 19. Briefly, national carbon footprints were spatially allocated based on population, purchasing power, and existing subnational estimates from the U.S., China, the European Union, and Japan. CO2 emissions are production-based, while carbon footprints are consumption-based. GDP estimates for 2015 are from a Brookings Institution report30. Statistical associations are indicated for a significance level of p < 0.05.City fossil fuel CO2.5 mortality, except carbon footprints which were estimated with GHS-POP population. Fig. Population-normalized rates were calculated using the GBD population dataset used to calculate PMSupplemental MaterialSupplemental Data


These results demonstrate that MTX-loaded PLGA NPs are good candidates for targeted delivery of MTX to the lymphatic system.Methotrexate (MTX) is a folic acid antagonist used as an effective drug to treat various kinds of cancers. However, MTX has limited use in cancer chemotherapy due to its adverse effects such as poor bioavailability, low specificity, drug resistance, and dose-dependent side effects. To improve lymphatic delivery and reduce toxicity of MTX, MTX-loaded nanoparticles (NPs) were prepared in the present study. NPs were prepared with double emulsion solvent evaporation method using poly(lactide-co-glycolide) (PLGA). NPs were assessed for size, encapsulation efficiency, morphology, Fourier-transform infrared spectroscopy, X-ray diffraction, and thermal characterization. In vitro release profiles and cytotoxicity of these NPs were also evaluated. Prepared NPs and free MTX were administered orally or intravenously (5 mg/kg as MTX) to rats to evaluate their pharmacokinetic characteristics and lymphatic delivery effects. Mean particle size and encapsulation efficiency of NPs were 163.7 ± 10.25 nm and 93.3 ± 0.5%, respectively. Prepared NPs showed a sustained release profile of MTX in vitro and may be effective to cancer cells. Area under the blood concentration-time curve, total clearance, half-life, and lymphatic targeting efficiency were significantly different ( Nanomedicines have recently become an object of great interest in the area of drug delivery systems. Nanoparticles (NPs) are tiny materials of nanometer scale and have unique properties in the body in relation to their size. In addition, NPs can have a relatively large loading capacity due to their high surface area-to-volume ratio . NPs leaMethotrexate   is a folAlthough MTX is used frequently as an effective drug for treating various tumors, autoimmune diseases, and inflammatory diseases, it has some limitations that restrict its use. First, bioavailability of MTX is low due to its poor water solubility and low permeability ,13 as meIn order to overcome these limitations of MTX, various attempts using drug delivery systems have been carried out. Pignatello et al.  have preThe lymphatic system plays an important role in cancer metastasis and immune response ,28. As mResearches have studied polymer-based NP in a variety of ways. Various studies have demonstrated that polymeric NPs of drugs have better cytotoxic effects than free drugs ,33,34. Hd,l-lactide-co-glycolide) (PLGA) is one of the most used biodegradable polymers. It is hydrolyzed to lactic acid and glycolic acid. These two monomers can be metabolized easily in the body by Krebs cycle. Owing to its minimal systemic toxicity, PLGA is widely used for drug delivery or biomaterial applications + in positive electrospray ionization mode. The most abundant fragment ion on multiple reaction monitoring (MRM) was m/z 454.7→308.1 for MTX and m/z 180.0→110.1 for IS. Quantitative analysis for MTX in rat biological samples was performed using a Shimadzu Nexera X2 Series UPLC system  coupled with a Shimadzu 8040 mass spectrometer . Optimized chromatographic separation of MTX was conducted with a KINETEX core-shell C18 column  at a column temperature of 40 °C. The mobile phase was prepared with 0.1% (2) exceeding 0.99. In regression equations, y was the peak-area ratio of MTX to IS and x (ng/mL) was the concentration of MTX.Linearities for MTX in rat biological samples were excellent over the concentration range of 0.1–1000 ng/mL. Regression equations from five replicate calibration curves for rat biological samples were: y = (0.0215 ± 0.0016)x−(0.0071 ± 0.0101) for rat plasma, y = (0.0203 ± 0.0013)x−(0.0022 ± 0.0009) for axillary lymph node, y = (0.0217 ± 0.0021)x + (0.0053 ± 0.0001) for mesenteric lymph node, y = (0.0209 ± 0.0018)x + (0.0036 ± 0.0012) for spleen, y = (0.0213 ± 0.0019)x + (0.0008 ± 0.0069) for thymus, y = (0.0227 ± 0.0022)x + (0.0034 ± 0.0017) for kidney, and y = (0.0223 ± 0.0019)x−(0.0018 ± 0.0002) for liver. These calibration curves fitted well, with the correlation coefficient . Stabilities of MTX in rat samples at various conditions were also evaluated, showing 97.52–101.01%. n = 5) and MTX-loaded PLGA NPs to rats . When MTX-loaded NPs were administered, MTX was absorbed more and remained longer in blood than that when free MTX was administered. PK parameters obtained by noncompartmental analysis are presented in 1/2), maximum blood concentration (Cmax), systemic clearance (CL) and volume of distribution (Vd) of MTX-loaded NPs were significantly different (p < 0.05) from those of free MTX. MTX-loaded NPs showed almost two times higher AUC, longer half-life, and two times lower CL than free MTX. These PK parameter differences were due to the fact that MTX-loaded NPs were particle-type molecules surrounded by PLGA, thus exhibiting different PK characteristics from free MTX which had drug-specific properties. The absolute oral bioavailability (Fab) value of PLGA NPs was 20.99%, different from that of free MTX at 16.04%. Relative bioavailability (Frb) value of PLGA NPs was 254.09% for oral administration and 194.19% for IV administration.max (Tmax) of MTX at oral dose was very short, ranging from 0.80 ± 0.12 h to 1.6 ± 0.7 h, similar to our experimental results (0.75 ± 0.25 h) of free MTX.Our results of bioavailability value for free MTX were similar those of a previous study . The repp < 0.05). Concentrations of MTX from PLGA NPs were increased in spleen and thymus, but decreased in kidney and liver compared to those from free MTX (p < 0.05). These results indicated that improved lymphatic delivery of MTX from PLGA NPs was attributed to prolonged blood retention and decreased renal and hepatic uptake associated with metabolism and excretion in the body.Concentrations of MTX in axillary and mesenteric lymph nodes, spleen, thymus, kidney and liver after oral or IV administration are shown in p < 0.05). Lymphatic targeting efficiencies of prepared NPs were 5.3 to 8.5 times higher than those of free MTX. However, targeting efficiencies of MTX from PLGA NPs in thymus, kidney and liver were lower than those from free MTX (p < 0.05).Lymphatic targeting efficiencies of MTX into axillary and mesenteric lymph nodes, spleen, thymus, kidney and liver were obtained . TargetiThese results suggest that prepared MTX-loaded PLGA NPs are effective for lymphatic delivery of MTX. They are also useful for decreasing tissue toxicity such as nephrotoxicity and hepatotoxicity as major side effects of MTX for both administration routes.As shown in The size of NP is the most important factor in the lymphatic delivery. In various studies, it has been reported that the delivery of particle-type agents to the lymphatic system is highly dependent on the particle size . As mentFinally, we conclude that polymeric NPs give physicochemical stability to protect MTX from excretion or degradation, thereby increasing blood retention time, which allows the appropriate sized NPs to migrate further to the lymphatic system.Formulation studies of MTX using PLGA NPs have been actively conducted recently. However, many researches have focused on the physicochemical properties of formulation ,63 or inHowever, evaluation of therapeutic aspects such as lowering cancer cell transfer rate and increasing cancer survival rate lacked in this study. How increased lymphatic delivery of MTX might affect other side effects of MTX such as neurotoxicity and immunotoxicity was not assessed either. Therefore, further studies are needed to evaluate not only side effects, but also pharmacodynamic aspects using tumor-induced animal models.MTX (purity ≥ 99%), PLGA with a molecular weight 30,000–60,000 g/mol, PVA with molecular weight of 31,000–50,000, phosphate buffered saline (PBS), DHF (purity > 90%) and THF (purity > 90%) were purchased from Sigma-Aldrich . UPLC-MS/MS grade methanol, acetonitrile, water (18.2 mΩ), and HPLC grade ethyl acetate were purchased from Fisher Scientific . UPLC-MS/MS grade formic acid was supplied by Tokyo Chemical Industry . All other reagents and solvents were in analytical grades.Roswell Park Memorial Institute (RPMI) 1640 medium and Trypsin-EDTA were obtained from Welgene Inc. . Fetal bovine serum (FBS) was supplied from Merck Millipore . Penicillin/streptomycin was purchased from Thermo Fisher Scientific . Trypan blue was purchased from Sigma-Aldrich . MCF-7 cells were purchased from Korean Cell Line Bank . CWR22Rv1 cells were purchased from ATCC .g for 15 min. The suspension was deep-frozen at −80°C for 12 h and lyophilized with vacuum pressure less than 50 mTorr at a temperature of −50°C for 48 h. The supernatant was also collected for analyzing free drug.MTX-loaded PLGA NPs were prepared by double emulsion solvent evaporation method of previously reports ,69 with w/v). Tests were conducted under the condition that other variables were fixed (PVA 10% (w/v), oil−water phase volume ratio 1:3 (v/v)).The influence of PLGA concentration on particle size was determined by increasing its concentration from 0.5% to 8% (w/v). Tests were conducted under the condition that other variables were fixed (PLGA 4% (w/v), oil−water phase volume ratio 1:3 (v/v)).The influence of PVA concentration on particle size was determined by increasing its concentration from 1% to 15% (v/v). Concentrations of PLGA and PVA were selected from the prior test.The influence of volume ratio of oil−water phase on particle size was determined by using different ratios ranging from 1:1 to 1:6 (w/v) and placed in measurement cell for analysis (n = 5).Mean particle size and size distribution analysis were performed using the dynamic light scattering technique  at 25°C. Zeta potentials  were measured to evaluate surface charge and stability of particles. These prepared NPs were diluted in deionized water . FTIR spectra showed chemical group of each component and interaction between components. FTIR assays were conducted with scanning at 380–4000 cmXRD assays of MTX, PLGA, physical mixture of MTX/PLGA, and MTX-loaded PLGA NPs were performed using an X-ray diffractometer . The scanning range of 2θ was from 5° to 90°.DSC studies of MTX, PLGA, physical mixture of MTX/PLGA, and MTX-loaded PLGA NPs were conducted using a differential scanning calorimeter . DSC thermograms provided information for physical characteristics of samples including crystalline or amorphous form. Samples were scanned from 0°C to 300 °C at a rate of 10 °C/min.The morphology of MTX-loaded NPs was studied with a field-emission scanning electron microscope . Prepared NPs samples were coated with platinum (~20 nm thick) using an Ion Sputter  for 5 min at 20 mA. These NPs were observed at an accelerating voltage of 15 kV.For in vitro release studies of MTX, prepared NPs and free MTX as control formulation were investigated at different pH  with dialysis method ,71. Brie2 at 37 °C.MCF-7 cells (human breast cancer cell line) and CWR22Rv1 cells (human prostate cancer cell line) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified atmosphere of 5% COCytotoxic effects were determined by trypan blue dye exclusion method. Cells were seeded into 9 cm Petri dishes and allowed to adhere for 24 h. Cells were incubated with free MTX, blank NPs, MTX-loaded NPs, or PBS alone as control. After 96 h, each plate was treated with 0.4% trypan blue in PBS. The morphology of each cell was observed using an optical microscope.® AQueous One Solution . Cells were seeded in 96-well plates and incubated for attachment. After incubation for 24 h, cells were treated with free MTX, blank NPs, or MTX-loaded NPs. Cells incubated with PBS alone were used as controls. Cell viability was observed before treatment and at 24, 48, 96, 108, and 144 h after treatment of each sample. The assay was carried out according to the manufacturer’s protocol. Briefly, 20 μL of MTS reagent was added to each well at the end of the incubation time and cells were incubated at 37 °C for 2 h. Absorbance was measured at 490 nm using a microplate reader . Background absorbance at 650 nm was subtracted. Cell viability was calculated with the following equation:Cell proliferation was quantified with MTS assay using CellTiter 96v/v) containing 25 mM sodium ascorbate, 0.1% (v/v) 2-mercaptoethanol and 25 mM ammonium acetate at pH 7.0. Plates were kept on ice (2 °C) for 30 min. Cells were scraped and the resulting mixture of cells and solvent were transferred to centrifuge tubes. A 10 μL of IS solution (10 μg/mL of phenacetin dissolved in methanol) was also added to each tube. Tubes were heated to 70°C for 5 min to fully denature proteins, and precipitates were removed by centrifugation at 20,000× g for 5 min at 5 °C. Supernatants were dried under nitrogen gas flow and resuspended in 450 μL of potassium phosphate buffer (50 mM with 30 mM ascorbic acid and 0.5% 2-mercaptoethanol at pH 7.0). Rat serum containing folate conjugase was treated with activated charcoal to remove endogenous folate. Then 25 μL of rat serum pretreated with activated charcoal was added to the samples in order to deconjugate polyglutamates of intracellular folates followed by incubation at 37 °C for 2 h. After that, samples were placed in a heated water bath (70 °C) for 5 min and then centrifuged at 20,000 × g for 15 min at 5 °C. To clean up samples, supernatants were transferred to a 10 kDa molecular weight cut-off membrane filter  and centrifuged at 20,000× g for 30 min at 20 °C. The solution at the bottom of the tube was then injected into a UPLC-MS/MS system.As mentioned above, MTX can inhibit intracellular DHFR which catalyzes the conversion of DHF to THF, subsequently leading to cancer cell death. To assess the cytotoxic effect of MTX-loaded PLGA NPs, we quantified cellular concentration of DHF and THF after treating cells with free MTX, blank NPs, or MTX-loaded NPs. Control cells were treated with PBS alone. The assay was conducted with modification of previously reported protocols ,73 usingAnimal experiments were approved by Chonnam National University Animal Experimental Ethics Committee, Republic of Korea (CNU IACUC-YB-2017–47). Sprague−Dawley male rats  were purchased from Damul Science . All rats were housed separately in metabolic cages in an environmentally controlled room  with free access to food and water before experiments. Each rat was fasted overnight before drug administration. These rats were divided into four groups (five rats in each group): (1) free MTX for oral administration, (2) MTX-loaded NPs for oral administration, (3) free MTX for IV administration, and (4) MTX-loaded NPs for IV administration. For IV administration, groups 3 and 4 were cannulated at femoral vein and artery with polyethylene tube  under light ether anesthesia. Prior to experiment, anesthetized rats were kept in restraining cages under normal housing conditions for 1 h until recovery from anesthesia. Single dose (5 mg/kg as MTX) of each formulation was given to rats orally or intravenously. The dose of MTX used in clinical trials varied according to various factors, including the type of disease, age, concomitant medication, and so on . The dosw/v) of PBS (pH 7.4). Remaining biological samples were placed in 1 mL of PBS. Biological samples were homogenized for 1 min. Suspensions were then stored at −70 °C until analysis.To evaluate lymphatic and tissue delivery of MTX, rats were divided into four groups (five rats in each group) and administered with each formulation as mentioned above. At 2.5 h after administration, whole blood was taken via the abdominal aorta. Targeted lymphatic system (mesenteric and axillary lymph nodes), immune tissues (spleen and thymus), and non-targeted normal tissues (kidney and liver) were then isolated and weighed. Liver samples were put in an equal ratio (v/v) formic acid aqueous solution (mobile phase A) and 100% acetonitrile (mobile phase B) with gradient elution at flow rate of 0.3 mL/min. Elution program consisted of 0–0.5 min (10% B), 0.5–0.8 min (10–90% B), 0.8–3.0 min (90% B), 3.0–3.01 min (90–10% B), and 3.01–4.0 min (10% B). Total run time was 4 min per sample. All analytical procedures were evaluated with positive electrospray ionization. Quantification was achieved using MRM modes at m/z 454.7→308.1 for MTX and at m/z 180.0→110.10 for IS.Quantitative analysis of MTX in rat biological matrices was conducted using a Shimadzu Nexera X2 Series UPLC system  coupled with a Shimadzu 8040 mass spectrometer. Optimized chromatographic separation of MTX was performed with a Phenomenex KINETEX core-shell C18 column at a column temperature of 40 °C. The mobile phase consisted of 0.1% (v/v) and 100% methanol, respectively, prior to make each working solution and stored at −20 °C. Standard working solutions of MTX  and IS (100 ng/mL) were diluted with methanol/water  and 100% methanol, respectively, from its standard stock solution. Calibration standards were prepared by adding and mixing each diluted working solution into blank rat biological samples to obtain final concentrations of MTX ranging from 0.1 to 1000 ng/mL. To examine accuracy and precision of UPLC-MS/MS method, quality control samples at four levels  were similarly prepared.To prepare standard stock solutions at 1 mg/mL, MTX and IS were weighed accurately and dissolved in methanol/water , vortexed for 5 min and centrifuged at 13,500× g for 5 min. Then 1000 μL of the supernatant organic layer was dried gently with a nitrogen centrifugal evaporator at 40 °C. The dried residue was reconstituted with 50 μL of mobile phase and vortexed for 5 min. After centrifugation for 5 min at 13,500× g, 5 μL of aliquot was injected for UPLC-MS/MS analysis.Samples were extracted by liquid-liquid extraction method using ethyl acetate. Protein was precipitated by protein precipitation method using acetonitrile. Then 100 μL of rat biological sample was added to 10 μL of IS solution (phenacetin of 100 ng/mL). The mixed sample was added to 1000 μL acetonitrile-ethyl acetate . Cmax and Tmax were individually calculated using plasma concentration-time curve. AUC from 0 to t h after administration (AUC0-t) was calculated by the linear trapezoidal rule. AUC from 0 to infinity (AUC0-∞) was calculated as AUC0-t + Ct/k, where Ct was the last measurable concentration and k was the elimination rate constant at terminal phase. The half-life was calculated to be 0.693/k and systemic clearance was determined as dose/AUC0-∞, IV. Vd was calculated as dose/k·AUC0-∞, IV. Absolute oral bioavailability for each formulation was calculated as AUC0-∞, oral/AUC0-∞, IV × 100 and relative bioavailability of PLGA NPs was calculated as AUC0-∞, PLGA NP/AUC0-∞, free-MTX × 100. Fab was the amount of the drug available to the body or system in the same formulation and Frb was used to compare bioavailability between the formulation and the standard. Targeting efficiency of MTX to the lymphatic system was calculated as the ratio of MTX concentration in axillary and mesenteric lymph node to MTX concentration in rat plasma at 2.5 h after administration of each formulation. All data are expressed as mean ± standard deviation.PK parameters of MTX formulations after oral or intravenous administration to rats were estimated by noncompartmental analysis using WinNonlint-test. Statistical significance was set at p < 0.05. The Statistical Package for the Social Sciences (SPSS) software version 23  was used for all statistical analyses.We analyzed data for statistical significance using Student’s w/v) of PLGA, 1% (w/v) of PVA, 1:3 (v/v) of oil−water phase volume ratio), mean particle size, zeta potential, and drug EE of MTX-loaded NPs were 163.7 ± 10.25 nm, −18.5 ± 2.28 mV, and 93.34 ± 0.51%, respectively. In vitro cytotoxicity studies resulted that prepared NPs had higher antiproliferative effects against cancer cells compared to the control group. These NPs exhibited sustained in vitro release profiles relative to free MTX at various pH conditions. In vivo studies showed that these prepared PLGA NPs had distinctly different PK characteristics from free MTX, with high lymphatic efficiency but low migration and accumulation in kidney and liver. Therefore, we could conclude that these prepared MTX-loaded NPs with both administration routes can be used to improve lymphatic targeting delivery of MTX. Also, it seems possible to reduce their delivery to normal tissues such as kidney and liver known to be related to major adverse effects of MTX.MTX-loaded PLGA NPs could be prepared easily by double emulsion solvent evaporation method. At optimal conditions (0.5% (


Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance. Candida glabrata is today the second or third most common cause of candidiasis, most likely because of its resistance to antifungal drugs, particularly azole drugs, which are used as prophylaxis and first- or second-line therapy , is often involved in the acquisition of fluconazole resistance in C. glabrata isolates  mutations in the CgPDR1 gene (C. glabrata (+ antiporters (DHA) CgQdr2, CgTpo1_1, CgTpo1_2, and CgTpo3  superfamily and the major facilitator superfamily (MFS). One particular ABC transporter, isolates , 8. AddiDR1 gene . Severalglabrata . For exad CgTpo3 –14. In tand Mdr1 , suggestC. albicans clinical isolates, azole resistance arises from point mutations that lead to conformational changes in Erg11, the primary target of azoles -trienol . The populations obtained at these time points were designated 044Fluco21 (posaconazole resistant), 044Fluco31 (posaconazole and clotrimazole resistant), and 044Fluco45 .The 044 clinical isolate was found to exhibit susceptibility to all the azole drugs tested. Specifically, the MIC values obtained for fluconazole, voriconazole, posaconazole, and clotrimazole are 4 µg/ml, 0.25 µg/ml, 0.5 µg/ml, and 0.125 µg/ml, respectively. After exposure to therapeutic serum fluconazole concentrations, the 044 yeast population evolved in a stepwise manner toward multiazole resistance. Specifically, after 21 days, posaconazole resistance was reached , followed by clotrimazole resistance  after 31 days and, finally, fluconazole and voriconazole resistance upon 45 days of induction  , considering a 1.5-fold threshold, with an associated P value of <0.05. Overall, the expression of 355 genes was downregulated, whereas the expression of 299 genes was upregulated, in the posaconazole-resistant population obtained after 21 days of fluconazole induction . At day 31, when the population had further acquired clotrimazole resistance, the expression of 73 genes was downregulated, whereas that of 199 genes was found to be upregulated, relative to expression in the parental clinical isolate (Tables S4 and S5). Finally, at day 45, upon the acquisition of fluconazole/voriconazole resistance, the expression of 6 genes was downregulated, whereas that of 27 genes was found to be upregulated, relative to expression in the parental clinical isolate (Tables S6 and S8). There is very little overlap between the differentially expressed genes in the different populations, with only 44 genes whose altered expression was maintained from day 21 to day 31, and 9 upregulated genes on day 31 whose activated expression was maintained until day 45 . GOToolBox was used to identify the Gene Ontology (GO) biological-process terms overrepresented in each data set .In order to gain insights into the molecular mechanisms underlying the phenotypic changes leading to differential acquisition of resistance to four azoles, the transcriptome-wide changes occurring on the 21st, 31st, and 45th days of fluconazole induction were analyzed by microarray hybridization. It should be noted that the design of the microarrays used was based on the available CBS138 genome; thus, it is possible that specific characteristics of the transcriptome of the clinical isolate analyzed may have been missed in this study. To make sure that the transcriptome observations reflected the evolving population, and not just a specific isolate, a large number of colonies were mixed and analyzed as a whole. Total RNA was extracted from cell populations growing exponentially in YPD medium in the absence of fluconazole in order to ensure that the transcriptome changes observed corresponded to stable transcriptional modifications. Each sample was compared to the azole-susceptible ERG11, whose expression was almost 3-fold higher in the 044Fluco31 strain than in the parental strain but returned to basal levels in the 044Fluco45 strain  mutation identified in other azole-resistant isolates of C. glabrata , confirming their transient upregulation, which reached maximal levels in the 044Fluco31 population . Overexpression of CgEPA3 in the wild-type strain L5U1 was consistently found to increase C. glabrata resistance to miconazole, ketoconazole, tioconazole, clotrimazole, and fluconazole, confirming the role of this gene as a determinant of azole drug resistance  mutation in the multidrug transcription factor Pdr1. Although the point mutation observed in the PDR1 sequence, leading to a Y372C substitution, has not been described before, it is indeed very likely to constitute a GOF mutation, given that it is in the same position as the Pdr1 Y372N GOF mutation identified previously , 044Fluco31 (resistant to pozaconazole and clotrimazole), and 044Fluco45  were obtained in this study through the directed evolution of the 044 clinical isolate, as described bellow. Additionally, the wild-type KUE100 and CBS138 C. glabrata strains were used. Cells were batch-cultured at 30°C with orbital agitation (250 rpm) in the following growth media: yeast extract-peptone-dextrose (YPD) growth medium, containing, per liter, 20 g glucose (Merck), 20 g yeast extract (Difco), and 10 g bacterial peptone (LioChem); BM minimal growth medium, containing, per liter, 20 g glucose (Merck), 2.7 g (NH4)2SO4 (Merck), and 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4 (Difco); Roswell Park Memorial Institute (RPMI) 1640 medium, containing 18 g glucose (Merck), 10.4 g RPMI 1640 (Sigma), and 34.53 g morpholinepropanesulfonic acid  per liter; and Sabouraud’s dextrose broth (SDB), containing 40 g glucose (Merck) and 10 g peptone (LioChem) per liter.The 044 clinical isolate of 2.The VK2/E6E7 human epithelial cell line (ATCC CRL-2616) was used for adhesion assays. This cell line is derived from the vaginal mucosa of a healthy premenopausal female subjected to vaginal repair surgery and was immortalized with human papillomavirus 16/E6E7. Cells maintenance was achieved with keratinocyte–serum-free medium, containing 0.1 ng/ml human recombinant epidermal growth factor (EGF), 0.05 mg/ml bovine pituitary extract, and an additional 44.1 mg/liter calcium chloride. Cells were maintained at 37°C, with 95% air and 5% CO6 blastoconidia, was transferred to different vials, each containing 9 ml of culture medium with or without 16 mg/ml fluconazole, a concentration of the drug that corresponds to therapeutic levels in serum obtained during antifungal treatment  . Interprconazole , 41. EveΔcgepa1, Δcgepa3, and Δcgepa10 deletion mutants , 044Fluco31 (pozaconazole- and clotrimazole-resistant), and 044Fluco45  strains were harvested in the mid-exponential phase of growth in YPD medium. Three independent cultures from each strain were used for transcriptional profiling. RNA extraction was performed as described elsewhere (C. glabrata (C. glabrata CBS138 available at the Yeast Gene Order Browser in 2014 (www.bioconductor.org), as described before . Only genes exhibiting a log2 FC of >1 and a P value of ≤0.05 for at least one probe were selected for further analysis. Gene Ontology enrichment analysis was performed with the GOToolBox Web server  CAGL0E06688g in S. cerevisiae, as described before for other heterologous genes (GAL1) and the yeast selectable marker URA3. The CgEPA3 gene was cloned in two sections, both generated by PCR, using CBS138 genomic DNA and the primers listed in EPA3 gene in C. glabrata, the GAL1 promoter was replaced by the constitutive C. glabrataPDC1 promoter. The PDC1 promoter DNA was generated by PCR using the primers listed in CgEPA3 and pGREG576_PDC1_CgEPA3 were obtained through homologous recombination in S. cerevisiae and were verified by DNA sequencing.The pGREG576 plasmid from the Drag&Drop collection  was usedus genes . pGREG57CgEPA1, CgEPA3, CgEPA9, CgEPA10, CgAWP12, and CgAWP13 genes was carried out in the parental strain KUE100, using the method described in reference CgHIS3 gene. The pHIS906 plasmid including CgHIS3 was used as a template, and transformation was performed as described previously , following the manufacturer’s instructions, and using 10 ng of cDNA per reaction. The RT-PCR step was carried out using SYBR green reagents. Primers for the amplification of the CgEPA1, CgEPA3, CgCDR1, and CgACT1 cDNA were designed using Primer Express software (Applied Biosystems)  . The RT-3H]clotrimazole transport assays were carried out as described before (3H]clotrimazole), yeast cells were grown in BM medium until the mid-exponential phase and were harvested by filtration. Cells were washed and resuspended in BM medium to obtain dense cell suspensions . After a 5-min incubation at 30°C, with agitation (150 rpm), 0.1 µM [3H]clotrimazole  and 30 mg/liter of unlabeled clotrimazole were added to the cell suspensions. The intracellular accumulation of labeled clotrimazole was followed for 30 min, as decribed elsewhere (−1)  at 30°C. Samples were eluted in 100% methanol at a flow rate of 1 ml/min. Ergosterol was detected at 282 nm with a retention time of 12.46 ± 0.24 min, while cholesterol was detected at 210 nm with a retention time of 15.36 ± 0.35 min. Results are expressed in micrograms of ergosterol per milligram (wet weight) of cells.Ergosterol was extracted from cells using the following method adapted from reference C. glabrata strains were tested for their capacity for biofilm formation by use of the crystal violet method. For that, the C. glabrata strains were grown in SDB medium and harvested by centrifugation at mid-exponential phase. The cells were inoculated with an initial OD600 of 0.05 ± 0.005 in 96-well polystyrene microtiter plates (Greiner) in either SDB (pH 5.6) or RPMI (pH 4) medium. Cells were cultivated at 30°C for 15 ± 0.5 h or 4 ± 0.5 h with mild orbital shaking (70 rpm). After the incubation time, each well was washed three times with 200 µl of deionized water to remove cells not attached to the biofilm matrix. Then 200 µl of a 1% crystal violet (Merck) alcoholic solution was used to stain the biofilm present in each well. Following 15 min of incubation with the dye, each well was washed with 250 µl of deionized water. The stained biofilm was eluted in 200 µl of 96% (vol/vol) ethanol, and the absorbance of each well was read in a microplate reader at a wavelength of 590 nm .5 cells/ml a day prior to use. Additionally, C. glabrata cells were inoculated with an initial OD600 of 0.05 ± 0.005 and were cultivated at 30°C for 16 ± 0.5 h with orbital shaking (250 rpm) in YPD medium. In order to initiate the assay, the culture medium of mammalian cells was removed and replaced by new culture medium in each well, and, subsequently, Candida glabrata cells were added to each well, with a density of 12.5 × 105 CFU/well, corresponding to a multiplicity of infection (MOI) of 10. Then the plate was incubated at 37°C under 5% CO2 for 30 min. Afterwards, each well was washed 3 times with 500 µl of 1× phosphate-buffered saline (PBS) (pH 7.2), followed by the addition of 500 µl of 0.5% Triton X-100 and incubation at room temperature for 15 min. The cell suspension in each well was then recovered, diluted, and spread onto agar plates to determine the CFU count, which represents the proportion of cells adherent to the human epithelium.For the adhesion assays, VK2/E6E7 human epithelial cells were grown and inoculated in 24-well polystyrene plates (Greiner) with a density of 2.5 × 10E-MTAB-6787.The data sets were deposited at the Array Express Database with reference number


The control of senescence has economic importance due to its effects on parameters such as herbal product quality and shelf life. This study is on the control of induced senescence in Triticum aestivum L. 'Gün-91' plants with silver nitrate (AgNO3) treatments. It was observed that some changes that occurred with dark and indole-1-acetic acid (IAA) treatments could be reduced with AgNO3 treatments. After dark-induced senescence, it was observed in plants that seedling length, relative water content (RWC), chlorophyll, β-carotene, xanthophylls, total antioxidant capacity, soluble phenol, total soluble protein, catalase (CAT), total superoxide dismutase (SOD), copper-zinc superoxide dismutase (Cu/Zn-SOD) activities, and expression of genes encoding these enzymes declined. After IAA treatments, seedling length, RWC, chlorophyll, β-carotene, xanthophylls, total antioxidant capacity, soluble phenolics, and soluble protein levels declined, whereas activities of CAT, total SOD, and Cu/Zn-SOD enzymes and expression of Cu/Zn-SOD and CAT genes increased. AgNO3 (200 mg L-1 ) applied by spraying onto leaves led to an increase in seedling length, RWC, chlorophyll, β-carotene, xanthophylls, total antioxidant capacity, soluble phenolics, soluble protein levels, and expression of Cu/Zn-SOD, CAT genes, CAT, SOD, and Cu/Zn-SOD enzyme activities compared to controls. Findings obtained from this study showed that the senescence process was related to changes in the levels of antioxidant compounds and enzymes. It was defined that the role of silver ions in slowing senescence was related to antioxidant defense capacity. Senescence effects many different agriculturally importantproperties such as seed count, yield and quality, fruitmaturation, seed germination time, and postharvest shelflife2O2), hydroxyl radical(OH), singlet oxygen (1O2), and other harmful free radicalscauses membrane lipid peroxidation, cellular damage, andprogrammed cell deathReactive oxygen species (ROS) rapidly increase withthe onset of leaf senescence. The increase in superoxideanion (O2-.), hydrogen peroxide activity in plants and subsequent increases in ACS andethylene levels lead to growth inhibition and induction ofsenescenceThe inhibiting effect of Ag + ions on ethylene has beenreported by many researchersIn this study, the concentration of antioxidantcompounds, activity of antioxidant enzymes, and changein expression of genes encoding these enzymes wereinvestigated in indole-3 acetic acid (IAA)- anddark induced senescence processes in wheat. Changes inantioxidant compounds, antioxidant enzyme activities,and antioxidant gene expressions in the senescence processwere investigated with AgNO3 treatment in wheat. Ag+ ionsare known to be effective on senescence. However, there isnot adequate research on effects of these treatments on theantioxidant defense system. According to the hypothesisconstituting the basis of this study, senescence inducerstrigger oxidative stress, whereas senescence inhibitorssuppress oxidative stress. This may be determined withchanges in activities of antioxidant compounds andenzymes and the expression of genes encoding theseenzymes. Findings obtained in this study may provideinformation on the senescence-antioxidant relation andalso contribute to the source of knowledge necessary forresearch on control of the senescence process, agriculturalproduction, and plant biotechnology.In this study, bread wheat (Triticum aestivum L.), which isa species with economic value, was used. Since wheat grainproductivity is closely related to monocarpic senescenceprocesses, it is a suitable plant for senescence studies.Gün-91, widely grown in Turkey, is a culture varietythat is resistant to cold and drought. Wheat seeds wereobtained from the Central Research Institute for FieldCrops in Ankara, Turkey. After germinating wheat seedswith soaked filter paper in a petri dish, they were takento pots containing soil/turf/manure (2/1/1 ratio). Plantswere cultivated in a climate chamber under controlledconditions: 16/8 day/night cycle, 26 ± 2 °C day and 18 ±2 °C night temperatures, 480 µmol m–2 s–1 light intensity,and 65 ± 5% relative humidity. After growing seedlings for3 weeks, a total of 40 pots each containing 25 seedlingswere divided into eight treatment groups of five. Thesegroups were treated with light (control), light + AgNO3(200 mg L–1), dark, dark + AgNO3, light + IAA (50 and100 mg L–1), or light + IAA + AgNO3 (Table Five seedlings were randomly picked from each pot andcut from soil level for length measurement. Fresh weight(FW) of the tissue sample was determined. The sampletissue was kept floating in pure water for 2 h and allowedto absorb water until saturation. After drying the surfacewith filter paper, turgid weight (TW) was measured, andafter drying the sample in an oven at 85–90 °C, dry weight(DW) was measured. RWC was calculated using theformula RWC = 100 × [(FW – DW) / (TW – DW)].Chlorophyll extraction from fresh leaf material was carriedout with 80% acetone (buffered to pH 7.8 with phosphatebuffer). Chlorophyll measurements were done with aspectrophotometer. Chlorophyll contents were calculatedaccording toFresh leaf materials (0.5 g) were ground in a prechilledmortar in 5 mL of acetone containing 200 mg of Na2SO4and then filtered through glass fiber disks (WhatmanGF/A). The volume of the acetone extracts was reducedin a rotary evaporator and then resuspended in 1 mL ofchloroform. Fifty microliters of extracts and standardswere applied to silica gel TLC plates . Hexane, diethyl ether, and acetone were usedat 60:30:20, v:v:v, as the mobile phasePlant samples of 0.5 g were crushed in a mortar with 5 mLof methanol (96%) for determination of  total antioxidativecapacity. The extract was centrifuged for 5 min at 5000× g and the supernatant  was taken. A reactive solutioncontaining 6 M sulfuric acid, 28 mM sodium phosphate,and 4 mM ammonium molybdate was prepared, and then150 µL of supernatant was mixed with the reactive solutionin a test tube so that the final volume would be 3 mL. Thetubes was maintained at 95 °C for 90 min and then cooledto room temperature and their absorbances were measuredat 695 nm.  Total antioxidative capacity was calculated asthe equivalent of ascorbic acidThe frozen leaf samples (0.5 g) were rapidly plunged into20 mL of 80% aqueous ethanol and boiled for 5 min. Afterfiltration through Whatman No. 1 filter paper, ethanol waseliminated from the filtrate by evaporation in vacuum.Total soluble phenolics in the remaining water phasewere determined spectrophotometrically with the Folin–Ciocalteu reagent (prepared by 1:1 dilution with distilledwater), against the chlorogenic acid standardA sample of 0.5 g of frozen leaf material was shakenin 10 mL of 80% cold acetone for 10 min and centrifugedat 10,000 × g for 10 min. The same process was repeatedwith precipitate in 5 mL of 80% acetone, and the pelletwas suspended in 2 mL of Tris-HCl buffer ,shaken for 5 min, and centrifuged at 3000 × g for 5 min.The supernatants were collected and stored at –20 °Cfor soluble protein determination. Soluble protein wasdetermined according toFrozen leaf material (0.5 g) was homogenized in 6 mLof 0.1 M potassium phosphate extraction buffer with an Ultra-Turrax. The homogenate was centrifuged for5 min at 6000 × g and 4 °C. The supernatant was filteredthrough a Whatman GF/A glass fiber disk with a vacuumfiltration system and stored at –70 °C2O2. Activity was determinedby UV spectrophotometer at 240 nm, measuring thedecrease in absorbance for 30 sCAT activity was assayed at 20 °C in a reaction volume of3 mL containing 2.8 mL of 50 mM potassium phosphatebuffer , 120 µL of enzymeextract, and 80 µL of 0.5 M HSOD activity was determined according toTotal RNA from plant tissues was isolated using TRIzolreagent  according to themanufacturer’s instruction. RNA quality was tested byagarose gel electrophoresis using standard protocols. Twomicrograms of total RNA was used to synthesize cDNAusing a First Strand cDNA Synthesis Kit  following the manufacturer’s protocol. AftercDNA synthesis, the amount of cDNA was quantified byNanoDrop ND-1000 spectrophotometer. The cDNAsamples were used as templates to quantify target geneexpression levels.The T. aestivum CAT gene (accession number:D86327.1), and the T. aestivum Cu/Zn SOD gene (accessionnumber U69632.1), and the housekeeping gene T. aestivumGAPDH (accession number: AF251217.1) were used for real-timequantitative PCR analysis. The primer pairs for CAT,Cu/Zn SOD, and GAPDH were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/).Genespecific primers were chosen so that the resulting PCRproduct had approximately the size of 180–230 bp.The primer sequences used were as follows.GAPDH: (F) 5’-GGAGGAGTCTGAGGGAAACC-3’(R) 5’-GTGCTGTATCCCCACTCGTT-3’Cu/Zn SOD: (F) 5’-CTCCTGGACTTCATGGCTTC-3’(R) 5’-CATTAGGGCCAGTCAAAGGA-3’CAT: (F) 5’-TATGAGGAGCGGTTCGACTT-3’(R) 5’-GCGTGTCGGAGTAGGAGAAG-3’Real-time quantitative PCR amplifications wereperformed using the Light Cycler 480 (Roche). Threebiological replications and at least 3 technical replication foreach gene region were performed. The PCR was performedin a reaction mixture of 10 µL composed of 2–3 µL ofcDNA, 0.4–0.8 µL of forward primer and reverse primer(10 pmol), 5 µL of LightCycler 480 SYBR Green I Master(Roche), and ddH2O. The standard curve was preparedfrom serial dilutions of control cDNAs. The followingprogram was applied: initial polymerase activation: 95 °Cfor 10 min, then 45 cycles at 95 °C for 10 s, Tm °C for 10 s,and 72 °C for 5 s. The specificity of the PCR amplificationwas checked with a melt curve analysis (from 95 °C to Tm)following the final cycle of the PCR. PCR conditions wereoptimized for high amplification efficiency of >95% for allprimer pairs used. The Ct (cycle threshold) values of theamplification curves were obtained between the 20th and35th cycles. In melting curve analyses, overlapping singlepeak images were obtained and dimer presence was notdetected.Ct values were established by using the peak profiles.According to results obtained from Ct values, the relativeexpression levels were calculated by using REST 2009software (http://www.gene-quantification.de/rest-2009.html) according to the 2–ΔΔCT algorithm. Reaction efficiency(RE) was considered as 1. Confidence interval (CI) wasconsidered as 95%. The results of CAT and Cu/Zn SODgene expression levels with the 2–ΔΔCT methodData obtained from analyses were evaluated statistically atthe factorial level by means of variance analyses (ANOVA)and their significance levels (P ≤ 0.05 and P ≤ 0.01)were determined. All analyses and measurements wereperformed repetitively at least three times.Compared to the seedlings grown in the light as the controlgroup, dark caused a considerable decrease in seedlinglength. Concentrations of 50 and 100 mg L–1 IAA sprayedonto leaves as a senescence inducer caused an increase inseedling length, while 200 mg L–1 AgNO3 did not cause asignificant change in seedling length for seedlings grown inlight and 200 mg L–1 AgNO3 treatment caused a significantincrease in seedling length for seedlings withdarkinduced senescence (P ≤ 0.05). AgNO3 treatment in thedark resulted in higher seedling length values comparedto the control group. For seedlings treated with 50 and 100mg L–1 IAA, AgNO3 treatment did not have a significanteffect on seedling length .AgNO3 treatment led to an increase in terms of β-caroteneamount in all groups, whereas the most significant increasewas observed in seedlings treated with 50 mg L–1 IAA.However, in terms of β-carotene values, the differencebetween plants treated with AgNO3 and plants not treatedwith AgNO3 was found to be statistically insignificant.AgNO3 treatment was observed to significantly effectsoluble phenolics amount. AgNO3 increased solublephenolics levels significantly in all controls left in the lightand the plants with dark- and IAA-induced senescencecompared to plants not treated with AgNO3 (P ≤ 0.05).AgNO3 increased soluble phenolics levels by 15% forplants left in the light, 33% for plants left in the dark, 31%for plants treated with 50 mg L–1 IAA, and 24% for plantstreated with 100 mg L–1 IAA . AgNO3 treatment increased CAT enzyme activityin plants left in the dark and plants treated with 50 and100 mg L–1 IAA. Differences between controls, 50 and 100mg L–1 IAA-treated seedlings, and seedlings left in thedark and seedlings treated with AgNO3 in terms of CATenzyme activity were found to be statistically significant(P ≤ 0.05). AgNO3 led to an increase of 54% in terms ofCAT activity in seedlings left in the dark. AgNO 3 led toan increase of about 16% in seedlings treated with 50mg L–1 IAA, whereas it led to an increase of about 11%in seedlings treated with 100 mg L–1 IAA. That being said,the highest CAT activity was observed in seedlings treatedwith 100 mg L–1 IAA and AgNO3 Table .Total SOD activity values decreased in plants left in thedark compared to controls and increased in plants treatedwith 50 and 100 mg L–1 IAA. The differences betweentreatment groups in terms of SOD enzyme activity inleaves were found to be statistically significant (P ≤ 0.05).SOD activity of 349 units in controls reached 421 unitswith 100 mg L–1 IAA treatment . Dark treatment led to a significantdecrease in Cu/Zn-SOD gene expression compared tocontrols Figure . IAA treLeaf senescence is a crucial and obscure process in the lifeperiod of plants, throughout which cells undergo majorchanges at structural and functional levels2,4-D-induced leaf senescence in beans was inhibitedwith supplementation of Ag+ ions in the form of AgNO3 orAgNPs. 2,4-D mitigated growth and stimulated senescenceat high concentrations. 2,4-D treatment together with 100µM AgNO3 produced a seedling length close to that of thecontrolIn a study conducted with two different maize varietieswith 80-day and 100-day lifespans, RWC decreased in bothvarieties after tasseling; however, the variety with 100-daylifespan had higher RWCThe decrease in total chlorophyll content was higher inthe early aging line compared to a normal aging line in theleaf senescence process in wheat .In French bean leaves during natural and dark-inducedsenescence, chlorophyll content decreasedIAA sprayed onto beans caused an increase in theamount of carotenoidsTotal antioxidant capacity is used to refer to allantioxidants for removing free radicals in plant samplesIncreasing concentrations of silver nanoparticlescaused a decrease in phenol and tannin content in plantcells due to the toxic effect of silver . In this study, AgNO3 treatment was observed tosignificantly effect soluble phenolics amount. AgNO 3treatment increased soluble phenolics levels significantlyin controls left in the light and the plants with dark- andIAA-induced senescence compared to plants not treatedwith AgNO . AgNO3 increased soluble phenolics levels3by 15% for plants left in the light, 33% for plants left inthe dark, 31% for plants treated with 50 mg L–1 IAA, and24% for plants treated with 100 mg L–1 IAA . Proteincontent decreased in Tropaeolum majus leaves kept in thedark and treated with IAA during senescence . In FreROS concentration increases together with the decreasein SOD and CAT activities; thus, lipid peroxidation,senescence, and cell death increaseAlthough total SOD activity increased at the beginningof senescence in leaves of tobaccoAfter 20 h of incubation in the dark, expression ofmitochondrial Mn-SOD, chloroplastic Fe-SOD, andcytoplasmic Cu/Zn-SOD enzyme transcripts decreased inboth young and aging barley leavesThe decrease in CAT gene expression was more rapidin early aging lines compared to normal aging lines inwheat with the onset of senescence .The CAT gene family consists of three genes called CAT1,CAT2, and CAT3 in the Arabidopsis genomeCAT activity increased at the beginning of senescence;however, a decrease was observed in CAT enzymeactivity as senescence progressed .Cu/Zn-SOD and Mn-SOD activity increased at thebeginning of senescence in bean cotyledonsFindings obtained from this study showed that thesenescence process was related to changes in the level ofantioxidant compounds and enzymes. Dark inductioncauses a decrease in all antioxidant parameters, while withIAA application some antioxidant parameters increased while some  decreased inthe senescence process. AgNO3 treatment showed asenescence-delaying effect. However, conducting a similarstudy with different concentrations of IAA and AgNO 3and performing time-dependent analyses with harvestmay produce more detailed findings about effects of IAAand AgNO3 ions on the senescence process.This study was supported by the Scientific ResearchProjects Unit of Mersin University (Project No. BAP2015TP2-1045).


Serum Golgi protein 73 (GP73) is a promising alternative biomarker of chronic liver diseases, but most data are from patients with HBV infection rather than HCV. n = 174) and Beijing Youan Hospital (n = 120) with different histories of HCV infection were enrolled. The correlations between serum GP73 and other biochemical indices, as well as its correlations with different stages of liver disease progression, were investigated. The receiver operating characteristic (ROC) curve was employed to evaluate the diagnostic potential of serum GP73 for liver necroinflammation and fibrosis, and comparisons of the diagnostic efficiency with traditional indices of hepatic liver injuries were further investigated.  Two independent cohorts of chronic hepatitis C (CHC) patients from the 5th Medical Centre of the Chinese PLA General Hospital (G ≥ 2) and/or with advanced fibrotic stages (F ≥ 3) in both cohorts , as compared to those with a normal or mild liver lesion. Further ROC analysis demonstrated that serum GP73 was comparable to serum ALT and AST in diagnosing the liver necroinflammation grade at G ≥ 2, but its diagnostic values for advanced fibrosis (F ≥ 3) and cirrhosis (F = 4) were limited when compared to APRI and FIB-4, and FIB-4 exhibited the best performance. Notably, an obvious elevation of serum GP73 was observed after patients received PEG-IFN and ribavirin treatment.  Levels of serum GP73 were found significantly elevated in patients with moderate to severe inflammatory grade ( Serum GP73 is an important biomarker in evaluating and monitoring the disease progression including liver necroinflammation and fibrosis in patients with chronic HCV infection, but the value is limited for diagnosing advanced fibrosis and cirrhosis in comparison with APRI and FIB-4. About 80~150 million persons are chronically infected with hepatitis C virus (HCV) worldwide . ChronicAt present, though liver biopsy remains to be the gold standard for grading the activity of inflammation and histological lesions of the disease simultaneously , 13, it Golgi protein 73 (GP73) is a 73 kDa transmembrane glycoprotein mainly expressed in biliary epithelial cells but rarely in hepatocytes in normal liver . The expIn the present study, we aimed to explore the correlations between serum GP73 and other biochemical indices among the chronic hepatitis C patients. Then, the diagnostic potential of serum GP73 for liver lesions was evaluated. Its performance was compared with that of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for identifying hepatic necroinflammation, as well as with that of APRI and FIB-4 models for fibrosis in different cohorts.Two independent cohorts (Cohort A and Cohort B) with different histories of HCV infection were included in this retrospective study. Cohort A is composed of 174 inpatients from the 5th Medical Centre of the Chinese PLA General Hospital (PLAGH) between 2012 and 2017, including 96 patients with precirrhotic CHC, 35 cases with compensated liver cirrhosis (CLC), and 43 cases with decompensated liver cirrhosis (DLC). The demographics, biopsy results, and laboratory data including levels of serum GP73 of these patients were collected . Cohort All participants had accepted liver biopsy to assess the progression of liver disease, and the biopsies of 10 patients with CLC and 38 patients with DLC from Cohort A were achieved when they were undergoing splenectomy because of low thrombocytopenia, esophageal varices, or bleeding resulting from liver cirrhosis. All of them were treatment-naïve patients. The diagnosis of CHC was in accordance with established criteria , 28, andInformed consent was obtained from all participants above. All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The liver specimens were fixed in formalin and embedded in paraffin and then stained with hematoxylin-eosin (HE) and Masson stains. Pathological analyses of the liver biopsies from all the two cohort patients were performed. The Scheuer scoring system (G0-4) was used for the evaluation of histological necroinflammatory activity. To evaluate the hepatic fibrosis stage, the METAVIR scoring system was used which was assessed on a five-point scale: F0, no fibrosis; F1, portal fibrosis without septa; F2, few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. ≥F2 was defined as significant fibrosis, and ≥F3 was defined as advanced fibrosis. All liver specimens were graded and staged by the same experienced liver pathologist.Notably, because of having serious liver cirrhosis in 26 cases with CLC and all DLC cases from the 5th Medical Centre of PLAGH , they weSerum GP73 levels were quantified by using commercially available enzyme-linked immunosorbent assay (ELISA) kits , according to the manufacturer's protocol.In Cohort A, there were 24 patients who got a standardized antiviral therapy by PEGylated interferons (PEG-IFN) plus ribavirin and were followed up in the 5th Medical Centre of PLAGH. Serum GP73 levels were monitored from 6 months to 12 months after the treatment; most of them got complete early or partial virological response.t-test, ANOVA, or Kruskal-Wallis rank sum test according to the data's distribution. In addition, the chi-square test was applied to compare the rates of the classification data. Pearson and Spearman's rank correlation coefficient tests were used to describe the association between two variables. The diagnostic effectiveness of variables was assessed by the area under the receiver operating characteristic (AUROC) curve with 95% confidence interval (CI), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and the differences were tested by Hanley and McNeil. All tests of significance were two-tailed, and P < 0.05 was considered statistically significant.Statistical analyses were performed by SPSS 22.0 software  and GraphPad Prism version 5.0 . Continuous variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR). The differences of groups were analyzed by P > 0.05). P < 0.05) were observed in BMI, HCV RNA, total bilirubin (TBIL), AST, gamma glutamyltransferase (GGT), albumin (ALB), globulin (GLB), red blood cell (RBC), platelet (PLT), prothrombin time (PT), necroinflammatory activity grade, and fibrosis stage between two groups which had absolutely different background of epidemiology. Besides, the 87 healthy individuals enrolled in this study included 14 males and 73 females; their mean age, BMI, and GP73 were 47.08 ± 7.89 years, 23.31 ± 2.36, and 31.37 ± 14.46 ng/mL, respectively, and the median of ALT was 11.0 U/L.The clinical characteristics of the Cohort A patients are shown in M ± SD) were 92.22 ± 57.26 ng/mL in Cohort A without DLC patients and 85.80 ± 29.09 ng/mL in Cohort B, and all were significantly higher than 31.37 ± 14.46 ng/mL in the healthy controls , the serum GP73 levels were significantly higher in the CLC subgroup (131.51 ± 61.97 ng/mL) and DLC subgroup (140.22 ± 55.02 ng/mL) than in the precirrhotic CHC subgroup (77.90 ± 48.34 ng/mL)  of Cohort A were 62.42 ± 35.33 ng/mL (G0-1), 101.47 ± 60.39 ng/mL (G2), and 104.28 ± 63.74 ng/mL (G3-4) in each grade of necroinflammation and were 62.80 ± 32.51 ng/mL (F0-1), 74.72 ± 53.42 ng/mL (F2), 98.02 ± 52.42 ng/mL (F3), and 131.51 ± 61.97 ng/mL (F4) in each stage of fibrosis, which indicates that serum GP73 was correlated tightly with the grade of liver necroinflammation (n = 105) and with the stage of fibrosis (n = 131)   in each inflammatory grade were 61.26 ± 20.91 ng/mL (G0-1), 87.51 ± 25.30 ng/mL (G2), and 94.22 ± 32.42 ng/mL (G3-4) , in the two cohorts.In order to further investigate the potential capability of serum GP73 in predicting liver necroinflammation and fibrosis of chronic hepatitis C patients, the levels of serum GP73 in patients with different necroinflammatory grades and fibrotic scores were analyzed in patients without DLC. The serum GP73 levels , in the two cohorts, then the AUC analysis was conducted to explore the relevant diagnostic potential of this serum marker. As expected, serum GP73 exhibited potential ability to identify patients with liver necroinflammatory grade (G ≥ 2) and/or with advanced fibrosis (F ≥ 3) and cirrhosis (F = 4). However, such diagnostic efficiency in chronic hepatitis C patients was not superior to the traditional biomarkers and models, such as ALT, AST, APRI, and FIB-4. In Cohort A, the AUROC values for serum GP73, ALT, and AST to identify liver necroinflammatory grade 2 or beyond (G ≥ 2) were 0.717, 0.685, and 0.782 (P > 0.05). Meanwhile, the AUROC values for serum GP73, APRI, and FIB-4 were 0.761, 0.796, and 0.848, respectively, when predicting advanced fibrosis (F ≥ 3), and were 0.779, 0.836, and 0.904, respectively, when predicting cirrhosis (F = 4) (G ≥ 2) were 0.794, 0.777, and 0.769, respectively (F ≥ 3)  , and alsectively ; and the (F ≥ 3) , and theP < 0.001, Since the level of serum GP73 was closely correlated with liver inflammation and interferon is an immune modulator, therefore, the influence of PEG-IFN and ribavirin treatment on serum GP73 was investigated. For these, indeed, after accepting PEG-IFN and ribavirin treatment, the serum GP73 levels increased remarkably among the 24 patients who got complete early or partial virological response in Cohort A, increasing from 80.21 ± 52.82 ng/mL at baseline to 122.95 ± 50.55 ng/mL after receiving a median of 6 months of treatment, and the changes were statistically significant  of chronic hepatitis C patients, but its power in diagnosing advanced fibrosis (F ≥ 3) and cirrhosis (F = 4) showed no advantage, as compared to APRI and especially FIB-4. These results differ from those of certain studies in the literature whose study groups had HBV-related infection. It was reported that GP73 is upregulated by HCV infection and mainly enhances HCV secretion through upregulating apolipoprotein E (APOE) [Though some studies have proven that serum GP73 was a valuable candidate for diagnosing advanced fibrosis and cirrhosis , 38, 40 E (APOE) . If the At the same time, our results also revealed that serum GP73 levels were obviously elevated when treatment-naïve patients accepted persistent PEG-IFN and ribavirin treatment and would then decrease to a certain level, which still remained higher than those in healthy individuals, after they achieved SVR and stopped treatment. The result suggests that serum GP73 could not truly reflect the situation of liver injury when used in clinical practice for patients with HCV infection treated by PEG-IFN and ribavirin and clinicians should be more cautious.G ≥ 2) between Cohort A and Cohort B, which were 103.5 ng/mL and 64.43 ng/mL, respectively; such difference was also observed in diagnosing advanced fibrosis (F ≥ 3), which were 63.89 ng/mL and 93.74 ng/mL, respectively. As shown in Limitations of our study are related to the following: (A) It is a retrospective cohort for data from the 5th Medical Centre of PLAGH which might be influenced by unmeasured potential biases. (B) This study included only two centers and the number of cases was limited. (C) There was an obvious difference of the cutoff values of GP73 for diagnosing severe necroinflammatory activities (G ≥ 2). However, it is limited for serum GP73 to diagnose advanced fibrosis (F ≥ 3) and cirrhosis (F = 4) in comparison with APRI and FIB-4.Serum GP73 is an important indicator in evaluating and monitoring the disease progression including liver necroinflammation and fibrosis in patients with chronic HCV infection. Serum GP73 has a certain value in diagnosing liver necroinflammation (