In vitro drug release studies revealed an initial burst for LDD devices. In vivo, slow drug release of PTX was found, whereas it already completed 1 week postoperatively for CAPE and PFD LDD devices. Histological examinations revealed a massive cell infiltration in the periphery of the test specimens. Compact fibrotic capsules around the LDD devices were detectable at 4–36 weeks and least pronounced around PFD-coated specimens. Capsules stained positive for extracellular matrix (ECM) components. The presented model offers possibilities to investigate release kinetics and the antifibrotic potential of drugs in vivo as well as the identification of more effective agents for a novel generation of drug-eluting glaucoma microstents.To prevent implant failure due to fibrosis is a major objective in glaucoma research. The present study investigated the antifibrotic effects of paclitaxel (PTX), caffeic acid phenethyl ester (CAPE), and pirfenidone (PFD) coated microstent test specimens in a rat model. Test specimens based on a biodegradable blend of poly(4-hydroxybutyrate) biopolymer and atactic poly(3-hydroxybutyrate) (at.P(3HB)) were manufactured, equipped with local drug delivery (LDD) coatings, and implanted in the subcutaneous white fat depot. Postoperatively, test specimens were explanted and analyzed for residual drug content. Fat depots including the test specimens were histologically analyzed.  Glaucoma is a leading cause of irreversible blindness worldwide and is mostly characterized by an increased intraocular pressure (IOP), which results from disturbances in the outflow of aqueous humor from the eye. Permanently increased pressure conditions lead to damage of the retinal ganglion cells and degeneration of the optic nerve fibers, resulting in a loss of vision and finally blindness, if untreated . Until nAs a further approach to conventional surgical interventions, alloplastic devices for the drainage of aqueous humor from the anterior chamber into different outflow areas, particularly into Tenon’s capsule, have been used . ClinicaFibrotic and scarring processes are mainly driven by the proliferation of fibroblasts and exuberant extracellular matrix (ECM) expression. It was shown that fibroblasts from different ocular tissues differ in their mRNA profiles  and in tTo address the problem of fibrosis, alloplastic drainage devices can be coated with antifibrotic drugs as it is done with drug-eluting stents in cardiovascular treatment ,24. The Another specific antifibrotic agent is pirfenidone (PFD), which has been shown to decrease the expression of fibrosis relevant cytokines (TGF-β1–3)  and ECM in vitro and in vivo in terms of drug release behavior and tissue response to address the postoperative problem of scarring and fibrosis around the stent in the outflow area. The concept of our implant-based regenerative approach for the drainage of aqueous humor into the retro-orbital intraconal fat tissue is shown in In the present study, a local drug delivery (LDD) coating for a novel biodegradable glaucoma microstent was developed and analyzed As an alternative to conventional permanent, biostable GDDs, the presented biodegradabale microstent serves as temporary pathway for controlled drainage of aqueous humor. Our initial experiments using these biodegradable polymers as GDD provided promising results . An LDD The aim of the current study was the evaluation of drug release and antifibrotic effects of different LDD coatings, containing the drugs PTX and the more specific agents CAPE and PFD, in a rat model. Therefore, GDD test specimens with different LDD coatings were implanted subcutaneously into the white fat depots in front of the right hind leg of rats. After explantation at different postoperative points in time, tissue samples including the test specimens were analyzed with regard to residual drug-loading and fibrotic responses.Test specimens were composed of tubing and an LDD coating, both manufactured based on biodegradable polymeric materials. A polymer blend from poly(4-hydroxybutyrate) (P(4HB); P4HB biopolymer, Tepha, Inc., Lexington, MA, U.S.A.), and amorphous atactic poly(3-hydroxybutyrate) (at.P(3HB)) in a blend ratio of 50/50% (w/w) was used to prepare the tubing and the coatings. Synthesis of at.P(3HB) was conducted according to Jedlinski et al.  and Hubb−1 was used. After each repetition, the mandrels were dried for 20 min at ambient temperature and rotated at 180°. Tubing diameter was measured in 0.5 mm increments along the longitudinal axis using a biaxial laser scanner  after the mandrels were removed.Tubing with a wall thickness of 75 µm was manufactured in a semiautomatic process using a dip-coating robot . Stainless steel mandrels  were dipped repeatedly into the polymer solution prepared from 1150 mg chloroform  and 100 mg of the polymer blend P(4HB)/at.P(3HB) 50/50% (w/w). A withdrawal speed of 300 mm.min−2, was the desired nominal coating weight. Measurement of the coating mass was conducted using an ultramicrobalance .The LDD coatings of the tubular test specimens (length: 10 mm) were applied with an airbrush process using a polymer solution prepared from 28.5 g chloroform  and 100 mg of the polymer blend/drug mixture. A mass of 126 µg, corresponding to a drug loading of 1.4 µg.mmAfter preparation, the test specimens were dried for 7 days in vacuum at ambient temperature. Test specimens from the control, the PTX and the CAPE groups were sterilized by means of ethylene oxide as described previously . For PFDMorphological analysis of the manufactured test specimens was performed using SEM  in environmental mode (ESEM) at a vacuum pressure of 0.5 mbar and an accelerating voltage of 5 kV.in vitro in 2 ml of 0.9% saline solution at 37°C. To protect CAPE from oxidation, 0.2% of ascorbic acid  was added to the saline solution.Analysis of temporal drug release from the test specimens was performed in vitro drug release studies, the test specimens were incubated on a rotating platform-shaking device  at 100 rpm. After a defined time of incubation iΔt, the saline solution was collected and drug content i (Δti)m was analyzed by means of HPLC . Prior to HPLC analysis, the saline extracts were diluted 1:1 (v/v) with methanol. Cumulative released drug mass mj after j cycles was calculated by summation of individual values.During After drug release stagnated, the residual drug in the test specimen was extracted by methanol and analyzed using HPLC. Individual test specimens additionally were taken after extraction and completely dissolved in chloroform again and analyzed using HPLC to find residual drug amounts. A summary of HPLC conditions used for analysis of the different drugs PTX, CAPE, and PFD is shown in in vivo, test specimens explanted at different postoperative points in time it were analyzed for residual drug loading res (ti)m by means of HPLC. Prior to HPLC, the drugs were extracted from test specimens by methanol. Released drug mass i (ti)m at the time it was calculated by subtracting res (ti)m from the initial drug loading initm.For the analysis of temporal drug release All animal studies were approved by the local authorities ) and conducted in accordance with the German Animal Welfare Act .Adult male Wistar rats  aged approximately 3 months were used and individually housed in standard cages at 22 ± 2°C under a 12-h light/dark cycle in a specific pathogen-free housing, with free access to tap water and a standard diet.Rats weighing 280–300 g were anesthetized by intraperitoneal injection of ketamine  and xylazine . The implantation area was shaved and disinfected with Betaisodona . Cutis was carefully opened with a scalpel and by a minimally invasive surgical intervention, the test specimens were implanted into the subcutaneous white fat depots in front of the right hind leg using a PICO-ID-Chip-Injector  B. This rHistological sections of 5 µm thicknesses were AZAN stained using a standard protocol . BrieflyAs a second histological observation, sections were stained with Hematoxylin–Eosin (H&E) . ShortlyFor detailed investigation of the fibrotic and inflammatory responses, immunohistochemistry using the avidin–biotin complex immunoperoxidase method  was carried out as described before . BrieflyThe following primary and secondary antibodies were used for IHC staining .The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Average wall thickness and drug mass of the manufactured test specimens are summarized in in vitro studies of PTX, CAPE, and PFD LDD devices was 70.0 ± 6.5, 75.9 ± 0.9, and 81.4 ± 5.4%, respectively (n=4). After dissolution of extracted microstents in chloroform, no residual drug was found. Slow in vitro drug release within a time frame of 5–6 weeks was found for PTX and CAPE LDD devices.On the contrary, PFD LDD devices showed fast in vitro drug release within 10 h /at.P(3HB)) 50/50% (w/w) into the white fat depots of rats. This polymer blend is characterized by low cytotoxicity and good tibility . The tesThe manufactured test specimens yielded reproducible quality in terms of morphology, initial drug loading and drug recovery after processing. Nevertheless, for PTX and CAPE groups a more porous surface morphology was found when compared with the PFD group. This is probably due to different crystallinities of the used drugs. Irrespective of the drug used, an initial loading and recovery of 20 µg and 76% was found, respectively.in vitro and in vivo conditions, and the different drugs PTX, CAPE, and PFD. Due to the relatively slow degradation of the used semicrystalline P(4HB) within up to 52 weeks, we assume that drug release primary is diffusion controlled [in vitro degradation studies with different P(4HB)/at.P(3HB) blends suggests, that degradation accelerates with increasing content of amorphous at.P(3HB) and therefore, might play a minor role in the current case.Within the present study, we could show distinct differences in drug release behavior between the ntrolled . Neverthin vitro drug release profiles are relatively slow compared with PFD. The solubility of PFD in aqueous solutions is approximately five orders of magnitude higher (20.000 mg.l−1), compared with PTX (0.3 mg.l−1), exemplary [in vitro drug release of PFD is considerably faster  is greater by factors 3–4.6 than CAPE (284.1 g mol−1) and PFD (185.2 g mol−1) and appears to be the dominating parameter, decelerating diffusion controlled drug release of PTX in vivo (Sigma–Aldrich Corp. 2017). PTX release in vivo may be also decelerated due to its lower lipid solubility and saturation effects and therefore, delayed drug diffusion inside the rat fat tissue. Differences in in vitro and in vivo release behavior possibly may be influenced through different drug crystallinity. Kind of solvent and drying conditions like time and temperature within the drug-coating process are significant for the drug crystallinity. This may be an explanation for the different morphologies shown in  release C. The moin vivo following implantation of biologically derived hydroxybutyric acid based polymers in our previous studies [Fibrotic encapsulation following GDD implantation is a known process and one of the most important reasons for implant failure ,53. In c studies ,35. Here studies . Additio studies . Both ciThe formation of a fibrotic capsule around the implant could be delayed by the antifibrotic drug PTX in our examinations. In early postoperative stages (1–2 weeks), a disturbance in the organization of connective and fat tissues and lesions peripherally to the PTX-coated test specimens were obvious. The appearance of lesions and skin ulcers has been described after intradermal injections of PTX in a mouse model , and degin vivo [Test specimens coated with CAPE showed only negligible antifibrotic effects. CAPE has been described with a broad spectrum of biological activities, including anti-inflammatory, antifibrotic, antioxidant, and immune-modulatory properties . Mia andin vivo ,60,61.in vitro studies demonstrated inhibition of cell proliferation, myofibroblast differentiation, collagen contractility, cytokine suppression, decreased collagen and fibronectin expression, and migratory ability of cardiac, renal, lung, hepatic, and ocular fibroblasts [in vivo has also been described in a trabeculectomy model in rabbits, where postoperative application of PFD was associated with less scarring and improved bleb survival [The same applies for PFD, where roblasts ,32,62–66survival  and follsurvival . In our Based on the presented data, the influence of the surface morphology of the test specimens on fibrotic encapsulation behavior could not be clearly distinguished. However, it is obvious that the use of an antifibrotic drug (i.e. PFD) retards and reduces fibrotic encapsulation compared with the control group with the same surface morphology as the PFD group but with no drug incorporated . It can In wound-healing processes, directly after blood clotting and vasoconstriction, the infiltration of leukocytes and an immune response is initiated . Most imTo summarize our findings, postoperative fibrotic and immune responses can be attenuated by glaucoma implants coated with antifibrotic drugs. The management of controlled drug release by avoiding a burst release to guarantee a long-term medication can increase the pharmaceutical effects of antifibrotic drugs with low risk of side effects to sensitive ocular structures. As a further developmental step, the combination of different antifibrotic pharmaceuticals is also conceivable to potentially increase effectiveness. Further investigations will help to develop GDDs with drug delivery characteristics to create a permanent drainage path to lower IOP and prevent glaucoma progression.in vivo, which can be compared with the retrobulbar fat depot in humans. The antifibrotic potential as well as drug-induced side effects were analyzed by histological examinations. PTX delayed the formation of a fibrotic capsule until 12 weeks after implantation. However, the PTX coating caused side effects and could not maintain its antifibrotic activity over the entire observation period of 6 months. The agents CAPE and PFD delayed the formation of a fibrotic capsule around the implants by 2 weeks in comparison with the control group. The PFD coating was overall most effective with regard to capsule thickness and growth of the capsule throughout the observation period.Implantation of drug-coated test specimens into the subcutaneous white fat depot in rats opens up new possibilities to investigate the release kinetics of antifibrotic agents In conclusion, these investigations help to identify new antifibrotic agents,which can be used as medicinal drug coatings for microstents in the treatment of glaucoma draining aqueous humor into the intra-orbital fat depot or tenon space. Our examinations allow the analysis of side effects and the quantitation of drug-specific antifibrotic potential.


A group of linear-transpiration response (LTR) lines exhibited better performance in grain yield and biomass compared to segmented-transpiration response (STR) lines, particularly in the wetter environments, whereas the reverse occurred only in the most stressed trial. LTR lines generally exhibited better water status  and larger green biomass (vegetation indices) during the reproductive stage than STR lines. In both groups, the responses to growing conditions were associated with the expression levels of dehydration-responsive transcription factors (DREB) leading to different performances of primary metabolism-related enzymes. Thus, the response of LTR lines under fair to good conditions was associated with higher transcription levels of genes involved in nitrogen (GS1 and GOGAT) and carbon (RCBL) metabolism, as well as water transport (TIP1.1). In conclusion, modern durum wheat lines differed in their response to water loss, the linear transpiration seemed to favor uptake and transport of water and nutrients, and photosynthetic metabolism led to higher grain yield except for very harsh drought conditions. The transpiration response to VPD may be a trait to further explore when selecting adaptation to specific water conditions.The regulation of plant transpiration was proposed as a key factor affecting transpiration efficiency and agronomical adaptation of wheat to water-limited Mediterranean environments. However, to date no studies have related this trait to crop performance in the field. In this study, the transpiration response to increasing  Additional efforts are required to increase yield gains for the coming decades by selecting traits for higher productivity under high temperatures and water limitation  coincides with the grain filling period. Therefore, an increase in grain yield does not necessarily require crops with a higher water use efficiency but instead with a more effective use of water Blum, . This coIn wheat, large genetic variability has been reported in transpiration sensitivities to evaporative demand and leaf areas  wheat cultivars widely grown in Spain during the past four decades. Several categories of lines that exhibited different transpirative responses were determined. Further, the agronomical responses of these different subsets of lines in terms of grain yield and physiological characteristics were evaluated under a wide range of environmental conditions provided by different locations, years and water regimes (rainfed and support irrigation). Besides grain yield, crop growth was evaluated using vegetation indices acquired at the plot level  and locations (with different temperatures and evaporative demand) in Spain. The agronomic, physiological and molecular measurements performed in the two groups of experiments are summarized in Supplementary Table Triticum turgidum L. ssp. durum (Desf.)] lines released in Spain during the last four decades : Mexa, Vitron, Simeto, Regallo, Gallareta, Bolo, Don Pedro, Sula, Bólido, Iride, Dorondón, Burgos, Claudio, Amilcar, Pelayo, Avispa, Don Sebastián, Don Ricardo, Kiko Nick, and Ramirez. These cultivars represented high-yielding lines at the time they were released and some of them are still cultivated across the Mediterranean basin. They represent a comprehensive set of the durum wheat germplasm cultivated in Spain after the Green Revolution  50%. The plants were uniformly irrigated every 2 days with 50% Hoagland's nutrient solution . To rule out the effect of plant size variation, for each plant the transpiration was normalized to its corresponding leaf area, all transpiration and biomass measures were recorded in the five biological replicates mentioned above.The experiment was conducted from August to October 2015 at the Experimental Facilities of the Faculty of Biology at the University of Barcelona. For each line, ten plants were grown in a greenhouse; two seeds were sown in 2 L pots  containing a mixture  of standard substrate and perlite. Photosynthetic photon flux density (PPFD) at midday of a sunny day inside the greenhouse was 800 μmol m−2. During both experimental campaigns the fertilization was applied in two steps, a first basal application and then a second top dressing application  was measured in two flag leaves per plot using a Decagon SC-1 Leaf Porometer . The canopy temperature of each plot was measured with an infrared thermometer . Ambient temperature was measured simultaneously above each plot using a thermo-hygrometer . Canopy temperature depression (CTD) was then calculated as the difference between canopy temperature and air temperature. The plant anthesis time was counted in days since the sowing date. The vegetation indices were estimated using digital RGB (red-green-blue) pictures taken above the plot, holding the camera at 0.8–1.0 m above the plant canopy in the zenithal plane and focusing near the center of each plot. Pictures were taken with Olympus EM-10 and Nikon D90 digital cameras, with a focal length of 18 and 14 mm, and fields of view (FOV) of 66° 43′ and 46° 51′, during the 2013–2014 and 2014–2015 crop seasons, respectively, with a shutter speed of 1/125 for both cameras. No flash was used and the aperture remained in automatic mode. Photographs were saved in JPEG format with a size of 4608 × 3456 pixels and 4288 × 2848 pixels for the Olympus and Nikon cameras, respectively. Subsequently, pictures were analyzed with open source Breedpix 0.2 software , according to the method of Vergara-Díaz et al.  on sunny days at midday (10 a.m.−2 p.m.). Pools of five flag leaves per plot were frozen into liquid nitrogen and stored at −80°C for laboratory analysis during the 2013–2014 experiments  ratio, together with the total C and N content. Measurements were carried out in an elemental analyser  coupled with an isotope ratio mass spectrometer , operated in continuous flow mode, at the Scientific Facilities of the University of Barcelona as described elsewhere  and WCOR719 (WCOR), superoxide dismutase (SOD), chloroplastic ATP synthase β-subunit (ATPase), cytosolic (GS1), and chloroplastic (GS2) glutamine synthetases, ferredoxin-dependent glutamate synthase (GOGAT), phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase (PK), the Rubisco large subunit (RBCL), and aquaporin TIP1.1. The relative expression was analyzed using the comparative Ct method  using the fold expression ECt−Δ, where E is the corrected efficiency of each primer. For the comparison within categories of lines or environments, gene expression was described in EΔCt−Δ values. Then the target gene relative expression was calculated for each group of lines in higher yield  and lower yield  scenarios. The expression values for each gene in every one of the 20 lines within atrial were the average of three different biological replicates .The frozen flag leaf samples from five plants per plot were ground with liquid nitrogen and subsequently RNA was isolated from 100 mg of this material using Ribozol RNA Extraction Reagents  as described in Medina et al. . RNA quap > 0.05, and the slopes were compared. This analysis was performed with GraphPad Prism software . The slope variation (Δslope) was calculated and used to classify the lines according to its sensitiveness to increasing VPD. In addition, the slope of the linear increase in transpiration as VPD was augmented from around 1 to 4 kPa was also calculated to compare with the range of VPD values usually tested in wheat  and VPD levels, we applied a segmented linear regression (model Y1 = Slope1 × X + Intercept1 and Y2 = Slope2 × X + Intercept2) or a linear regression (model Y1 = Slope1 × X + Intercept1) with 1,000 interactions; these algorithms fitted the better model depending of the data, accounting for a 95% confidence interval and significance of p < 0.001). For gene expression data in particular, a log2 transformation was needed. When the differences between treatments were significant (p < 0.05), the mean comparison was assessed by LSD (least significant differences). The correlation analysis was performed with the Pearson method (p < 0.001). All tests were performed with the R package for statistical computing . Heat maps of relative gene expression were generated using a log transformation of the real-time PCR data presented as ΔCT  with GraphPad Prism version 7.00 . The network analyses for all traits were carried out using significant correlations (p < 0.001) with higher Pearson's coefficients (r > 0.8 and r < −0.8), then the representation was performed by Cytoscape v3.4.0  and linear model comparisons (0) or a higher significant slope variation. The STR- group included the lines Amilcar, Bólido, Don Ricardo, Don Pedro, Don Sebastián, Iride, Kiko Nick, and Vitron and the STR+ group the lines Avispa, Bolo, Gallareta, Mexa, Simeto, and Sula. The slope variation (Δslope) within these three subgroups was significantly different, with values close to zero in LTR lines, and decreasing progressively for STR− and STR+ lines (Table H2O m−2 s−1) (Table The transpiration response to increasing VPD under controlled conditions showed significant differences in the slopes of the 20 durum wheat lines in this experiment Table . The siges Table . Further1) Table . The LTRIn addition, the transpiration response from 1.07 to 4.10 kPa was also calculated  and chlorophyll content (measured at post-anthesis) increased as agronomical conditions improved, while saturation, a* and u* decreased and intensity and b* (which are biomass indicators as well as NDVI) were not affected by growing conditions (Table 13C), canopy temperature depression (CTD) and leaf stomatal conductance (gs) were also evaluated. Thus, grain δ13C decreased and CTD and flag leaf gs increased as growing conditions improved. Finally the N content in grains, which is an indicator of the capacity of the grains to accumulate nitrogen, decreased as growing conditions improved.As expected, lower grain yields for the 20 wheat lines were observed in the rainfed relative to supported irrigation trials (Table p < 0.001) between the LTR and STR fitting lines were observed, especially under high-yielding conditions. In addition, the comparison between the linear fit of yield for the most contrasting groups (LTR and STR+) was also significantly different  and lower values of other indices  plus lower leaf chlorophyll content and carbon content than STR lines  against the average δ13C value for the whole set of 20 lines in each field trial, was compared , highlighting that higher (less negative) δ13C values were achieved for the STR+ than the LTR groups of lines in the environment  with the highest average δ13C values (around −24 %0), whereas in the trial with the lowest  average δ13C (near −28 %0), differences were absent between the two categories of lines.We observed differences in grain yield and physiological traits between groups, according to their capacity to manage water loss Table . LTR lin−1) and HY  were downregulated, whereas the GS2 and TIP1.1 genes were overexpressed  relative to high yield (HY) conditions, dehydrin genes (ed Table . Consider > 0.75) and p values (p < 0.05)  and yielding scenarios . Network analysis was performed using significant correlations between parameters based on Pearson correlation coefficients (−0.75 > DREB1 with the vegetation index (lightness), grain δ13C, and the expression of the GOGAT, PK, and SOD genes; (ii) the expression of dehydrin DNH16 with N content of grains and the expression of PK and (iii) the expression of WCOR with ATPase expression. With regard to N metabolism, it evidenced negative relationships (gray edges): (i) the expression of GS1 with biomass (NDVI) and (ii) the expression of both the GS2 and GOGAT genes with gs. Concerning C metabolism, the expression of RBCL correlated positively with the vegetation index u*, as well as the expression of PEPC with C content. Vegetation indices also correlated against yield and physiological traits; for example gs correlated negatively with biomass greenness (GA), while Hue correlated positively against grain yield and negatively with chlorophyll content.In the LTR lines under high-yielding conditions Figure , there wDREB1 expression was positively correlated with some vegetation indices (v* and b*), as well as with the expression of the DREB2, GOGAT, and PEPC genes. Both GS1 and GOGAT expression were negatively associated with grain C content, while the expression of GS2 was positively correlated with RBCL and DHN16 gene expression. PEPC expression was positively associated with DREB2 and GOGAT expression and also with some vegetation indices (v* and b*). PK and PEPC expression were positively correlated with δ13C and CTD respectively, while the expression of RBCL was negatively correlated with gs. TIP1.1 expression was negatively correlated with GA and positively correlated with DREB2 and WCOR expression. Vegetation indices such as NDVI and chlorophyll content exhibited a negative correlation with SOD gene expression.For the STR+ lines under high-yielding scenarios Figure , DREB1 eDREB2 was positively correlated with ATPase gene expression, and negatively correlated with GS1 and DNH16 expression. The amount of WCOR transcripts was positively correlated with the expression of PEPC, NDVI and GA, while the vegetation indices intensity and a* were negatively correlated. GS2 expression was negatively correlated with biomass (NDVI). PEPC expression correlated positively with RBCL expression and also with the vegetation index lightness, while the expression of PK and SOD were positively associated with each other. Grain δ13C was negatively associated with yield and positively associated with TIP1.1 expression, while gs was positively correlated to DREB1 expression. Furthermore, chlorophyll content and grain C content showed a positive correlation between them.In LTR lines under low-yielding conditions Figure , the expGS2 gene. DREB2 expression was positively correlated with gs and negatively correlated with grain δ13C. GS2 expression was positively associated with TIP1.1 expression and negatively correlated with grain N and C content, whereas GS1 expression was positively correlated with PEPC expression. Moreover, PK expression and grain δ13C were positively correlated.Lastly, for the STR+ lines under low-yielding conditions Figure  grain yis, SPAD, and most of gene expression  traits had positive loading on the main vector (Dim 1) for both LTR and STR+. Similarly, traits like anthesis time (flowering), water status (δ13C) and b*, as well as the expression of TIP1.1 and GS2 had negative loading in the vector lines  at similar but significantly different breakpoint values close to 1 KPa. These VPD values are clearly lower than the 2 KPa or greater reported previously in wheat, which included lines selected for the Mediterranean conditions of Australia  δ13C of mature grains , the STR lines had higher grain yields as well as higher vegetation indices  than the LTR lines. Moreover, δ13C was higher in the STR than the LTR lines, which indicates a higher water use efficiency in the STR lines  , indicating that the more negative the δ13C the better the water status of the crop  Table  than theDREB1 and DREB2) and dehydrins  influencing the expression of key genes in primary metabolism and water transport . DREB1 and DREB2 seemed to be central to the integration of plant responses to the growing-conditions, and appeared to be co-regulated with water status traits  in both the LTR and STR+ groups , the provision of carbon skeletons (PK and PEPC), and the water status (δ13C and CTD) as well as canopy vegetation (lightness and v* indices) , and DNH16 dehydrin under low yield conditions  in low yielding wheat lines  expression was higher in high-yielding conditions, which contrasts with reports of enhanced dehydrin signals under drought conditions  and N (GS2) metabolism enzymes, which suggests that this dehydrin may play a protective role in ensuring greater N assimilation (GS2) and carbon skeleton transformation (PK) . In the case of GS2, grain nitrogen yield was upregulated in STR+ lines, particularly under high-yielding conditions, which may indicate a need for N assimilation due to a clear inhibition of N remobilization and/or a low content of end products.Concerning the N metabolism genes Table , GS2 tras Figure ; this pa2 fixation and may benefit plant growth by diversifying the high amount of N invested in Rubisco. In that regard, Rubisco upregulation in STR+ lines was accompanied by overexpression of GS2. This was probably a response to a higher demand for N supply to synthesize more Rubisco enzyme, which agrees with previous reports about co-ordinated regulation of CO2 fixation and N assimilation during grain filling in wheat , which may also be associated with a more efficient distribution of N within the plant as it is suggested by the GS1 overexpression. This is in line with the importance of GS enzymes for yield production  Figure  showed tThis study provides evidence of how the ability of wheat lines to limit or not limit transpiration during vegetative stage may affect agronomical performance at further stages of the crop cycle and under a wide range of environmental conditions in the Mediterranean. Thus, LTR lines performed better in terms of yield than the STR lines, with the exception of the very dry environmental conditions. Moreover, the study highlights the complexity of physiological and molecular mechanisms associated with these different transpiration responses to high VPD. At the gene expression level both groups of lines are regulated by DREB transcription factors and dehydrins. However, the results suggest that the higher grain yields of LTR lines is in line with their superior water status, and in turn this is associated with more active aquaporins and specific adaptations to carbon and nitrogen metabolism driven by regulation of genes that encode key enzymes. The negative but marginal correlation between plant leaf area and leaf transpiration suggests that the trade-offs between these traits are minor and supports further studies to explore the feasibility of using transpiration for selecting wheat lines better adapted to Mediterranean conditions.SM and JA designed the experiments. SM conducted the experimental work. MN-T and NA grew the field crops during both seasons. SM, RV, MN-T, NA, FC, and OV-D recollected the field data. SM, RV, and JA contributed to the data analysis and interpreted the results. SM wrote the paper under the supervision of RV and JA. All authors revised the manuscript and read and approved the final version.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.


These results highlighted the importance of submerged plant litter in nutrient-rich wetland ecosystems and provide potential tools for managers to improve the ecosystem functions and/or services via altering microbial diversities.Plant litter is an important component in wetland ecosystems, and the role of plant litter decomposition is considered to be important for wetland ecosystem functions and services. However, the consequences of litter inputs have seldom been experimentally tested in real ecosystems such as constructed wetlands (CWs). The enriched nutrients in CWs might weaken the role of litter inputs on soil carbon and nitrogen cycling. Here, we conducted a two-month field experiment to examine the effects of litter inputs on the soils in CWs. Our results showed that litter inputs significantly affected soil microbial  diversities and properties , and litter species with higher stoichiometry ratios, i.e. C/N, C/P and N/P led to higher microbial diversities. However, litter species had no or weak effects on microbial activities (CO Litter decomposition in wetland ecosystems refers to the respiration and assimilation of plant litter by microbes and invertebrates6, and it can be divided into three interlinked processes, i.e. leaching, fragmentation and microbial decay8. Moreover, litter decomposition are commonly considered to be affected by the quality of litter, associated soil microbes and invertebrates and the corresponding environmental factors13, but in wetlands such as stream and other freshwater ecosystems, litter inputs and biotic or abiotic factors are proven to be paramount14. Given the large variation in plant functional traits among plant species, litter decomposition rate can vary significantly among species due to the various ‘afterlife’ effects of litter traits 17, and this interspecific variation in decomposition rates might lead to uncertainties in the effects of wetland plant litter on the soil or water qualities19, and thereby other organism in wetland ecosystems22.Plant litter as the end of primary production entering into detritus food chains plays an important role in wetland ecosystems, and its decomposition can recycle carbon and multiple nutrients, alter environmental variables, and affect wetland ecosystem functions and services28, and there were plenty of evidence either from a specific ecosystem type at local scale or from different biomes at the global scale30. It has been proved that soil ecological processes, including soil C, N cycling, flux of CO2, CH4 are closely related to litter decomposition32. There are multiple pathways that plant litter can affect the soils in wetland ecosystems: (1) litter inputs can have various effects on soil invertebrates or microbes via different physical and chemical traits; (2) litter inputs can also provide food, microhabitat or shelter for soil microbes or other soil fauna34; (3) litter inputs can have negative effects via releasing leachate from litter which contains detrimental organic carbon and/or other compounds37. All above mechanisms indicated that plant litter traits as litter qualities can strongly influence the chemical and physical composition of litter inputs, and thereby their decomposability15 and lead to substantial consequences to wetland ecosystems. However, among various litter traits, litter stoichiometry might form the most important constraints of carbon: nitrogen ratios on soil microbial communities39 and hence act as the key trait to predict the effects of litter inputs on soil properties and microbial communities in wetlands40.Previous studies have proved the significant and diverse effects of plant litter on soil physio-chemical properties and microbial communities25; or subsequently set up new controlled experiments in the lab with the litterbag method or soil-litter mixing method41. For the latter case, knowledge about effects of litter mixing on soil is derived mostly from ‘indoor’ experiments carried out in strictly controlled environments, or via quantifying the litter mass loss and nutrient release43. However, very few investigators have addressed such effects of litter mixing on the wetland soils in relatively dynamic and unstable environments, such as constructed wetlands (CWs) with irregular waste-water inputs19. In such kind of constructed wetlands, other environmental factors might weaken the effects of litter inputs on the soil and thereby other soil organism, such as bacteria and fungi.Moreover, the main methodology to test those effects of plant litter was firstly to sample soils from the field, and then either directly quantify the soil properties and microbial community composition or activity2/CH4 fluxes, total carbon (TC), total nitrogen (TN), carbon and nitrogen isotope changes , as well as microbial diversities. The purpose of our work was to fully understand the role of plant litter in constructed wetlands and develop potential tools for the maintenance, improvement and management of constructed wetlands using plant litter materials.Now in this study, we set up a soil-litter mixing experiment (two months) in an ongoing constructed wetland located in Hanshiqiao wetland, Beijing, China . Our hypothesis is that the effects of litter inputs in such a constructed wetland might not be as overwhelming as those proven in indoor laboratory experiments, in which the related environmental factors are strictly controlled and relatively stable. We explored the effects of litter traits and incubation time on the soil properties of a constructed wetland including soil CO2, but no significant difference in the other variables between initial soil and the control treatment . However, litter species had no significant effects on either CO2 or CH4 . There were no significant differences in the soil fungi composition or the other phyla of soil bacteria before and after incubation . Overall, different species litter did not drive significant differences in the relative abundances of soil bacteria or fungi, except for some phyla (Firmicutes and Ciliophora) with lower relative abundance (<5%) .There were significant or marginally significant relationships between soil microbial diversity and litter stoichiometry ratios, i.e. C/N, C/P and N/P, but no significant relationships between soil microbial diversity and any other litter trait Table . Moreove44. We indeed observed significant effects of litter inputs on soil properties  and microbial diversities, and those effects to some extent depended on litter species identity  to lighter element (12C and 14N), to some extent determining the decomposition rates of plant species litter49. Moreover, we also observed the highest microbial diversities and the highest 15N in the soil mixed with P. crispus litter after two-month incubation, and this might also result from the stoichiometric or isotope ratios of initial P. crispus litter  for constructed wetlands, plant litter of submerged plants should not have been always considered as wastes and being directly refloated from the CWs. Instead, it is possible to shift the ‘unfavored’ submerged plant litter to ‘useful’ tools to improve the ecosystem functions and services of constructed wetlands; (3) plant litter might be a feasible and economic materials for improving the microbial diversities of CWs, and it is worth to comprehensively study the role of wetland plants in constructed wetlands, especially for the role of plant litter, and this might provide valuable suggestions for managers about the maintenance and management of constructed wetlands.In conclusion, our results provided empirical evidence for the effects of submerged plant litter on the soil properties and microbial diversities in a constructed wetland. These findings might have multiple implications for the design, maintenance and management of constructed wetlands: (1) when designing a constructed wetland, it is better not only take the species identity into consideration, but also for the stoichiometry of different growth forms2. The water average depth is about 2.5 m. The dominant plant species in section III are Iris wilsonii, Zizania latifolia, Typha orientalis and Sagittaria sagittifolia, and there were irregular waste water inputs flowing into our study site through early April to late November every year.Our study site was located in the Hanshiqiao wetland, Beijing, China. There was a constructed wetland, which was used to purify the waste water from the pleasure boat area, restaurant and the public toilet in the Hanshiqiao wetland Park Fig. . The CW Ceratophyllum demersum, Myriophyllum verticillatum, Hydrilla verticillata, Potamogeton crispus) were collected from Hanshiqiao wetland park in July 2017 (but not from the constructed wetland). All plant litter  and all the buckets were incubated in the same environment only with different litter input treatments. The distances between each bucket was around 30 cm. The whole experiment last from August 31, 2017 to November 2, 2017, which is also the main senesced period in the study region.Plant litter of four submerged species . Total C and Total N content of were assessed using the VarioMAX CN element analyzer . The TP concentration was analyzed by inductively coupled plasma emission spectroscopy  and the isotopes of C and N were subsequently analyzed using an isotope ratio mass spectrometer .Before incubation, three soil samples Fig. , A2 and Initial soil microbe community, i.e. bacteria and fungi, was a measured using the second generation next throughput sequencing technology . Microbial DNA was extracted from soil samples  using the E.Z.N.A.® soil DNA Kit  according to manufacturer’s protocols. The bacteria 16S and fungi 18S ribosomal RNA gene were amplified by PCR using primers 515F 5′-GTGCCAGCMGCCGCGG-3′, 907R 5′-CCGTCAATTCMTTTRAGTTT-3′and SSU0817F 5′-TTAGCATGGAATAATRRAATAGGA-3′ and 1196R 5′-TCTGGACCTGGTGAGTTTCC-3′ respectively, where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and quantified using QuantiFluor™ -ST . We calculated the Shannon diversity indices to represent the diversity of soil bacteria and fungi.2 and CH4 fluxes of soil was also measured. Concentrations of CO2 and CH4 were measured using a gas chromatograph . At first, we collected soils samples (at the beginning of experiment) or mixtures  (about 10 g dry weight) from the CW by shovel or buckets by self-zip plastic bag. And they were putted in the glass bottle (100 ml). Before gas sampling, we sealed the glass bottle with airtight butyl rubber stoppers. After 24 h of incubation, the headspace gas of the glass bottle were sampled using airtight syringes. All gas samples were measured within 24 h after sampling.In addition, the CO2 and CH4 fluxes. For the last sampling, the properties and microbial communities of litter-soil mixtures were again measured using the methods mentioned above. The whole experiment last two months from August 31, 2017 to November 2, 2017. After incubation, all the plastic buckets were removed from the CW to avoid the continuous disturbance for the CW.During incubation, litter-soil mixtures were sampled every two weeks and then brought to the lab for the measurement of CO13C, 15N, among four submerged plant species. Secondly, we conducted one way ANOVAs to examine the differences of soil microbes (relative abundance and Shannon diversity index), TC, TN, 13C, 15N, CO2 and CH4 between the initial soil (before incubation) and the control treatment after two-month incubation in the constructed wetland  and the change value of soil characters  before and after two-month incubation using regression analysis respectively. In the end, the effects of plant species on microbial respiration  during two-month litter mixing were analyzed using repeated measure ANOVA. Differences between means were tested with Fisher LSD tests; effects were considered significant at P < 0.05. All the ANOVA analyses were conducted in SPSS Statistics , and regression analyses were conducted in R software 3.5.2 (R core Team)54.All data were checked for assumptions of normality and homogeneity of variance before analysis. We firstly compared the differences of initial litter traits, such as TN, TC, TP, C/N, C/P, N/P, Supporting information


Durio zibethinus seed and determined its antibacterial, photocatalytic and cytotoxic effects. Surface plasmon resonance confirmed the formation of DSAgNPs with a maximum absorbance (λmax) of 420 nm. SEM and TEM images revealed DSAgNPs were spherical and rod shaped, with a size range of 20 nm and 75 nm. The zeta potential was found to be −15.41 mV. XRD and EDX analyses confirmed the nature and presence of Ag and AgCl. DSAgNPs showed considerable antibacterial activity, exhibited better cytotoxicity against brine shrimp, and shown better photocatalytic activity against methylene blue. Based on the present research work, it can be concluded that DSAgNPs could be used in the field of water treatment, pharmaceuticals, biomedicine, biosensor and nanotechnology in near future.In the present study, we have developed a green approach for the synthesis of silver nanoparticles (DSAgNPs) using aqueous extract of  Nanoparticles have been extensively explored in the fields of optics, catalysis, engineering, biology, medicine and pharmaceutical sciences because of their unique chemical and physical properties. Different approaches have been practiced for preparing nanoparticles, including chemical or electrochemical methods. Generally, the utilized chemicals or the by-products formed during the process are hazardous that not only increase difficulty in purification stages but these also require high energy for the preparation ,2. ApartMost of the reported silver nanoparticles synthesis methods are found to be associated with various problems, including lack of stability, growth of crystals and particle aggregation. Silver-based products have been utilized over the centuries to prevent and treat numerous diseases, especially infections, due to strong growth inhibitory and bactericidal effects of silver . MoreoveDurio zibethinus (durian) plant, a member of Malvaceae family, has been reported to possess antioxidant, anti-diabetic, anti-hyperlipidemic and anti-proliferative activities [tivities ,16. Moretivities ,18. Its tivities ,20. Toletivities .Durio zibethinus fruit waste (seed) for the biosynthesis of silver nanoparticles. Moreover, the antibacterial potential of DSAgNPs against various bacterial strains was evaluated and photocatalytic activities of DSAgNPs were tested against methylene blue. Additionally, brine shrimp cytotoxicity assay was conducted to analyze preliminary toxicity which could be extrapolated for cell-line toxicity and anti-tumor activities [Durio zibethinus for the biological reduction of silver nanoparticles using under sunlight, and the synthesized silver nanoparticles showed better antimicrobial, cytotoxic and photocatalytic activity with good stability.The main objective of our study was to utilize aqueous extract of tivities ,23. The Durio zibethinus seed aqueous extract as reducing and capping agent. The selection criteria for durian seed was based on several factors, viz. the presence of saccharides, which are responsible for the reduction of Ag+ in reaction solution, amino acids for stabilization of the formed silver nanoparticles, and preference in this area of research, and as it is a waste that causes environmental pollution [In the present study, green synthesis of silver nanoparticles was achieved by bio-reduction of silver nitrate utilizing ollution ,26,27,283 to Ag0 where the observed color change from colorless to yellowish brown confirms the reduction. The maximum absorbance exhibited by the reaction solution at 420 nm  and seed extract (1 mL) at different conditions like room temperature, at 60 °C, in the dark and under sunlight. The reactant solution which was kept at room temperature for 72 h, heated at 60 °C for 1 h and kept in dark for 72 h, did not demonstrate significant color change or absorbance peak in UV-Vis spectral studies.3 concentration, 1.5:8.5 ratio of seed extract and silver nitrate, and 30 min reaction time in sunlight were found to be the optimized conditions for synthesis of DSAgNPs. The sunlight exposure time for the formation of silver nanoparticles was optimized as 30 min (A significant change in the color and absorbance of the reactant solution was only observed when it was kept in the sunlight (approximately 4000 ± 200 lux) for 10 min. Subsequently, parameters viz. concentration of silver nitrate, time of reaction and volume of durian seed extract required for synthesis of DSAgNPs were optimized. Based on the results, it could be deduced that 1.5 mL extract of 10% durian seed in deionized water, 1.5 mM AgNOs 30 min A since tThe present study dealt with a waste-mediated silver nanoparticles synthesis using durian seed aqueous extract, which might be instigated by the presence of saccharides and sterols in the extract. In this study, the −OH and O present in side chains of the saccharides might be involved in the reduction, as mentioned by Nadezhda et al. who stated that the −COO−, −OH and O present in hydrolyzed products  of pectin fragments were involved in the development of silver nanoparticles . The capThe SEM results indicated the size of synthesized nanoparticles was in a range of 20–72 nm, with an average size of 64 nm A.Moreover, the SEM image clearly showed polydisperse spherical and rod-shaped particles, however, the TEM images showed that the particle size ranged from 20−75 nm, with an average size of 60 nm B and SAEThe DLS analysis was carried out for determination of hydrodynamic diameters of DSAgNPs whereby the mean hydrodynamic diameter of DSAgNPs was found to be 176.5 d.nm with intercept 0.809, and a low polydispersity index (PDI) of 0.290 B was obsThe DLS technique measures the hydrodynamic radii of the nanoparticles which include the solvent layer and capping at the interface. Determination of zeta potential (−15.42 mV) confirmed the stability of nanoparticles C. The ne−1 (phenolic OH stretching), 3436 cm−1 (aromatic N–H stretching), 3312 cm−1 , 1630 cm−1 (amide I bond of amino acids due to carbonyl stretch), 1515 cm−1 (aromatic ring stretching), 1375 cm−1 (C–C bond of aromatic ring or amide group-II), 1316-1285 cm−1 (aromatic secondary amine CN stretching) and 650 cm−1 . The results clearly indicate that the O–H, C–C and C–N vibration stretches in DSAgNPs could be due to the presence of saccharides and amino acids in the extract of durian seeds which might be involved in the formation of DSAgNPs by acting as capping and stabilizing agents.The explicit mechanism behind biological extract assistance in the synthesis of nanoparticles is yet to be elucidated . HoweverThe stability of the synthesized DSAgNPs in aqueous dispersion was evaluated based on the changes in absorption maxima of SPR band, over a period of 120 days. The SPR bands remained almost symmetrical within the tested period. Moreover, no significant shift in the surface plasmon absorption peak was observed, which indicates that the biosynthesized DSAgNPs were not aggregated during the storage period, under ambient conditions.S. aureus and S. typhimurium was found to be 4 mg/mL, and MIC against E. coli, B. subtilis and S. typhi was found to be 2 mg/mL. The minimum bactericidal concentration (MBC) of DSAgNPs against E. coli, B. subtilis, S. typhimurium and S. typhi was found to be 4 mg/mL . The study results are analogous with other studies reported by fellow researchers [The synthesized DSAgNPs showed moderate antibacterial action on the tested bacterial pathogens. The minimum inhibitory concentration (MIC) of DSAgNPs against  4 mg/mL . Based o 4 mg/mL , it can earchers ,38,39,40The mechanism of the bactericidal effect of silver nanoparticles is not well-known. Silver nanoparticles may attach to the surface of cell membrane and eventually this may disrupt power functions, viz. permeability and respiration of bacteria. The particles bind to the bacteria based on the availability of surface area for interaction. Moreover, smaller particles tend to possess larger surface area which will exhibit more bactericidal effect as compared to larger particles . MoronesThe dye degradation of MB under sunlight irradiation was performed to assess the photocatalytic activities of biosynthesized DSAgNps. The dye degradation was detected by a gradual change in the solution color from deep blue to colorless.0, where C0 is the initial concentration of MB and C is the concentration of dye solution after “t” min under sunlight irradiation) in the presence and absence of photocatalyst  indicated about 3% mortality in 24 h which clearly validated that the experimental mortalities were not due to the deprivation from food. Concurrently, the effect of durian seed extract on survival of brine shrimp larvae was also evaluated for 24 h, during which it did not show any effect. The highest mortality of 76.0% was observed at 5 mg/mL and the calculated LC50 value of DSAgNPs was found to be 3.03 mg/mL (3030 mg/L). This indicated a direct proportional relationship between the concentration of DSAgNPs and lethality. In this study, we have evaluated the cytotoxicity of DSAgNPs against brine shrimp. The brine exposures to DSAgNPs were conducted in the absence of food. The positive control  concentration [Artemia than the reported result of 331.08 mg/L of neem leaf extract mediated silver nanoparticles reported by Avinash et al. [Arulvasu et al. investigated the cytotoxic effect of chemically synthesized silver nanoparticles against ntration  which ish et al. .50 of 33920 mg/L for green synthesized silver nanoparticles from crude extract of Bergenia ciliate against brine shrimp [Moreover, Phull et al. reported a LCe shrimp  which isArtemia in the present study, they could be further tested for anti-tumor activities and may be further used in the management of cancer along with other anticancer drugs as carrier and active principle ingredient. Moreover, it is safer for aquatic ecology as the synthesized DSAgNPs concentration may be less as compared to the toxic concentration of industrial waste or any environmental pollution present in the aquatic environment.Since DSAgNPs showed enhanced lethal toxicity against Among the advantages of this study we may mention the following. First, this study provies many advantages form the viewpoint of an environmentally friendly approach: (1) waste (durian seed) is used, which is a renewable source; (2) it is a green synthesis approach we have not used any hazardous chemicals to prepare the silver nanoparticles; (3) the application of solar irradiation as a catalyzing agent in the photochemical reaction of the extract and silver salt solution is cost free; (4) photocatalytic studies of methylene blue degradation by the phytosynthesized silver nanoparticles can be envisaged to degrade organic dyes in the textile industry, or to detoxify organic toxins in waste waters.Stapylococcus aureus ATCC 43300, Staphylococcus heamolyticus ATCC 29970 and Bacillus subtilis ATCC 6633) and Gram negative—Salmonella typhi ATCC 7251, Salmonella typhimurium ATCC 14028 and Escherichia coli ATCC 25922)) were obtained from Faculty of Applied Sciences, AIMST University, Malaysia.Fresh and healthy durian seeds were acquired from a local market . Silver nitrate (is ≥99.0%), nutrient broth , Muller Hinton Agar (beef infusion form 300.0 gm/L), casein acid hydrolysate 17.5 gm/L, starch 1.5 gm/L, agar 17.0 mg/L, final pH (at 25 °C) were procured from Himedia Laboratories Pvt. Ltd. . The bacterial cultures  and durian seed extracts (1 mL) were mixed in a 10 mL volumetric flask which was kept in the sunlight for 30 min. The formation of DSAgNPs was corroborated by observing the change in the solution color to brown and by spectrophotometric determination.AgNO3 to silver nanoparticles depends on certain important parameters, viz. time, silver nitrate concentration and durian seed extract volume needed for the reaction. However, these parameters are equally important to determine the size, shape, yield and agglomeration state of DSAgNPs. Hence, the parameters were optimized for the green synthesis of DSAgNPs by altering one parameter at a time while the other parameters were kept fixed. Aqueous dispersion of DSAgNPs was prepared and stored at ambient temperature in a 50 mL volumetric flask which was protected with aluminium foil. Sample aliquots were withdrawn repeatedly at 1 week intervals for a period of 120 days to assess the stability of the synthesized DSAgNPs by measuring changes in UV-Vis spectra.The bioreduction of AgNO−1 to 400 cm−1). The crystallinity of DSAgNPs was confirmed by XRD . The zeta potential of DSAgNPs was determined by a Zetasizer Ver. 7.03 .The Surface Plasmon Resonance (SPR) shift and plasmon intensity of the colloidal DSAgNPs were characterized by UV-Visible spectroscopy  with a wavelength range of 400–700 nm. The morphology and size of the DSAgNPs were confirmed by SEM , FE-TEM , elemental composition by EDX analysis , and surface capping functional groups by FT-IR  at 4000 cmSynthesized DSAgNPs were investigated for antibacterial activities against Gram positive and Gram negative bacteria by Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and disc diffusion assay.The MIC assay was carried out by using the broth microdilution assay. Serial dilutions of DSAgNPs was added to 48-well microtiter plates, then 0.1 mL of suspension of Muller-Hinton (MH) broth medium with the bacterial cultures adjusted to 0.5 McFarland was added to each well and the plates were incubated at 37 °C for 24 h. The MIC value was determined by detecting the lowest concentration of DSAgNPs that inhibited bacterial growth after 24 h.About 50 µL culture suspensions from all the wells of 48-well microtiter plates which used for MIC were inoculated in MH agar medium and the plates were incubated at 37 °C for 24 h. The MBC value was determined by detecting the concentration that killed 100% bacteria.The selected bacteria were inoculated in LB broth. Then sterile discs of 6 mm diameter of Whatman No. 1 filter paper impregnated with 4 mg/mL of DSAgNPs in water, gentamycin (1 mg/mL) (positive control), durian seed extract (used for synthesis of silver nanoparticles), and silver nitrate (1.5 mM) and deionized water (negative control) were placed on the agar surface. The plates were incubated for 24 h at 37 °C. The test was performed in triplicate and the zones of inhibition (ZoIs) were calculated to determine the susceptibility of bacteria.0 is the initial concentration of MB solution and Ct is the concentration of dye solution after “t” h under sunlight irradiation. All dye concentration absorption peaks at 662 nm were measured by UV-Vis spectroscopy [To determine the photocatalytic property of DSAgNPs, 10 mg of DSAgNPs was dispersed in 100 mL of distilled water and the dispersion was sonicated for 2 h in order to determine the photocatalytic properties of DSAgNPs. Methylene blue (MB) powder (1 mg) was added to the above aqueous DSAgNPs solution. It was stirred magnetically for 30 min in the shade and upon completion the colloidal suspension was exposed to solar irradiation. During the experiment, the average atmospheric temperature was found to be 30 °C with 3 h mean sunshine duration. Suspension samples (5 mL) were repeatedly collected from the colloidal mixer at every 30 min and scanned spectrophotometrically at 200 to 800 nm for determination of the degradation of MB. The dye degradation (%) was calculated by using the following equation:troscopy .A. salina were transferred from the brighter part of hatching chamber to 24 well plates containing various concentrations (1–5 mg/mL) of DSAgNPs in 1 mL of sterilized seawater. As a negative control, sterilized seawater without DSAgNPs was used and maintained at 25 °C for 24 h (16 h light and 8 h dark). After incubation, the larval mortality percentage was calculated from the number of larvae surviving in each test concentration. Assays were carried out in triplicate and LC50 values were determined using a regression plot.With the help of capillary glass tubes, ten live and healthy larvae of In this study, silver nanoparticles were synthesized by utilizing renewable sources such as durian seeds and sunlight. As it is a green synthesis approach, the reported method meets the requirement of environmental safety. In this study we have investigated the impact of silver nanoparticles on activities against human pathogens that are major sources of bacterial infections, as catalysis for the degradation of organic dyes which are prime sources of environmental pollution, and cytotoxic activities against brine shrimp which is a preliminary test to analyze the anticancer potential of the tested compounds. Various properties of the synthesized silver nanoparticles were also characterized by UV, SEM, TEM, EDX, XRD and zeta potential. SEM and TEM images confirmed the spherical nature of the silver nanoparticles. XRD images showed the presence of silver chloride nanoparticles and EDX images indicated the presence of chlorine. The extra ring in SAED of TEM indicated the presence of unreduced silver nitrate. DSAgNPs showed antibacterial activity as well as cytotoxicity against brine shrimp. The study results indicated that the green synthesis of DSAgNPs yielded an effective nanoparticle preparation that could be used against human pathogens as a potential therapeutic agent and also for the purification of wastewater in the future. This is evident since the synthesized DSAgNPs displayed good photocatalytic activities for the degradation of methylene blue and were also found to be non-toxic. The present study suggested that greenly synthesized DSAgNPs can prevent many environmental hazards in an eco-friendly way and can also be used as therapeutic agents in the pharmaceutical industry.


We report a customized gene panel assay based on multiplex long-PCR followed by third generation sequencing on nanopore technology (MinION), designed to analyze five frequently mutated genes in chronic lymphocytic leukemia (CLL): TP53, NOTCH1, BIRC3, SF3B1 and MYD88. For this purpose, 12 patients were selected according to specific cytogenetic and molecular features significantly associated with their mutational status. In addition, simultaneous analysis of the targets genes was performed by molecular assays or Sanger Sequencing. Data analysis included mapping to the GRCh37 human reference genome, variant calling and annotation, and average sequencing depth/error rate analysis. The sequencing depth resulted on average higher for smaller amplicons, and the final breadth of coverage of the panel was 94.1%. The error rate was about 6% and 2% for insertions/deletions and single nucleotide variants, respectively. Our gene panel allows analysis of the prognostically relevant genes in CLL, with two PCRs per patient. This strategy offers an easy and affordable workflow, although further advances are required to improve the accuracy of the technology and its use in the clinical field. Nevertheless, the rapid and constant development of nanopore technology, in terms of chemistry advances, more accurate basecallers and analysis software, offers promise for a wide use of MinION in the future. They may help to predict the potential evolution of the disease or the response to therapy. Identifying the most clinically and biologically relevant clones is crucial for evaluating CLL disease progression; in this context, NGS targeted gene sequencing allows driver mutations to be detected, at diagnosis and/or responsible for progression and relapse3, which could be used as predictive or prognostic biomarkers in CLL4.Chronic lymphocytic leukemia (CLL) is a disease with a highly heterogeneous clinical course, characterized by the accumulation and proliferation of clonal B cells in the blood, bone marrow and lymph nodes. Next–generation sequencing (NGS) technologies applied to genome-wide association studies (GWAS) have revealed several recurrent gene mutations associated with CLL that, in association with the high level of genetic heterogeneity among patients, is consistent with the large degree of clinical variability characteristic of this disease. In fact, a number of recurrent gene mutations have been reported in 2–10% of newly diagnosed CLL casesTP53 and ABL1 genes in CLL and in chronic myeloid leukemia (CML) patients, respectively6. Moreover, the very low costs, the ease of use, and the length of the reads make MinION an ideal tool for targeted sequencing of genes7.However, the introduction of NGS technologies in clinical diagnostics requires a high initial investment when purchasing the sequencer, that is a limitation for local research facilities in developing countries and small research centers and hospitals. A valid alternative could be the use of MinION, the first commercially available sequencer based on nanopore technology. MinION has already been successfully used to detect mutations of the In this report we describe the development of a customized gene panel, MinION-based, for targeted sequencing of the five genes TP53, BIRC3, NOTCH1, SF3B1 and MYD88, recurrently mutated in CLL, and analysis of its performance.In our experiment, the library consisting of 12 barcoded samples was loaded for sequencing on MinION, employing a flowcell with 1,170 active pores. A total of 106,543 fast5 total files was produced, containing raw electric signals, for a total of about 65 Mb. Fast5 files were then uploaded in Metrichor for basecalling and demultiplexing during the run. Of the total reads produced, 48,599 passed 2D filters, and 46,451 had a recognizable barcode. Only “pass” reads were used in the study; the number of 2D passed reads for each sample is illustrated in Supplementary Table Thanks to the real-time analysis of the fast5 files produced, after 24 hours from the start of the sequencing we observed that the minimum sequencing depth of the targets for case #7 was lower, on average, than for the other CLL samples. Therefore, we decided to prepare and reload a new pool comprising all the libraries at different weight ratios according to the preliminary results obtained with real-time sequencing monitoring. Employing this expedient, the total pass 2D reads count was greater than 2,000 for each sample except for case #7 Table . Plottin10.NanoOK analysis was performed to calculate the error rate for each amplicon. In Table Analysis of NanoOK data showed that the range of sequencing depth was more uniform for longer amplicons, and, notably, inversely related to the amplicon size, the smaller amplicons having a greater depth (up to about 2100x) . In detail, we created and used a complete workflow , including read mapping, variant calling and annotation. Similarly, the Nanopolish pipeline was applied starting from fast5 files to variant calling, and then the ANNOVAR annotation tool16 from Galaxy was used to annotate them. For both the workflows, SNV and indels analysis was performed for each CLL case.We performed two parallel analyses of MinION sequencing data, uploading the fastq files extracted from fast5 files to Galaxy platform or analyzing them with the Nanopolish variant calling pipeline17. The filtered variants were also analysed in silico by the pathogenicity prediction program SIFT incorporated in ANNOVAR.SNV and indels were filtered for hotspot mutations or genomic location (exonic and splicing variants), functional effect (nonsynonymous variants), and frequency based on minor allele frequency (maf) from the 1000 Genomes Project (<1%). Variant allele frequency cut-off was set at 10% and 15% for SNV and indels, respectively, in agreement with the recent results of serial dilution for MinION somatic mutation analysis9. To reduce the risk of producing false positive results due to sequencing errors, we checked if the same variants were called in different patients. To this aim, we calculated the recurrence of all SNV and indels and found some recurrent variants in several samples, and even in all 12 CLL cases. Except for the known CLL mutation hotspots, and the polymorphisms identified, considering the low likelihood of finding the same variant simultaneously in a small cohort, we decided to exclude the variants occurring in multiple samples from further validation analyses. In total 256 genomic recurrently mutated positions were therefore determined; most of them (91%) were sites of indels, in line with the results of the error rate analysis , whereas 9 were sites of both SNV/indels and 14 only of SNV .We also verified whether these critical positions are known to be sites of experimentally observed mutations in haematopoetic and lymphoid tissue and found that about 7% of them were COSMIC (v81) variants annotated in haematological malignancies Fig. . These fWe also retrieved the nucleotide sequences flanking of all the SNV/indels called and observed that most of these mutations suggested a context-specific error frequently caused by homopolymer sequences  in real-timeTo the best of our knowledge, this is the first report of targeted gene sequencing based on a custom panel of pre-pooled primers for multiplex long-PCR on MinION.Multiplex gene panels are one of the strategies most commonly used to enrich the genomic regions to be sequenced, and are widely employed in NGS technologies. This approach, applied to MinION, allowed us to enrich all the gene regions of interest, setting up only 2 PCR for each sample and thus simplifying the experimental procedures.Thanks to MinION real-time sequencing, reads can be analysed as they are generated, significantly speeding up the analysis of results and modifying the experimental conditions if necessary; this advantage allowed us to intervene in real-time on the quality of case #7 sequencing. Other advantages of MinION include its portability and easy setting for library preparation and sequencing as compared to second-generation sequencers, as well as its cost-effectiveness.et al. described their 13-gene panel for investigating CLL NGS, proposing it in routine diagnostics; the authors declared that using their panel the detection of the main NOTCH1 hotspot p.P2514fs*4 was successful, but that it was not able to identify larger indels26. Currently, only one commercial amplicon-based CLL gene panel is available which allows the detection of both SNV and copy number variant analysis for the nine most prognostically relevant genes in CLL2. Nonetheless, attempts to design a comprehensive gene panel useful for CLL prognostication and therapy-effect prediction, are still continuing2.Thanks to analyses of the utilization and impact of NGS in CLL, in the last years several custom/academic or commercial CLL target panels have been developed, most of them based on the amplicon sequencing approach. For example, in 2013 Kolhman 27. Secondly, some weaknesses related to the general short-read sequencing approach are well known: the technical limit of detecting larger indels and other structural variations (SV), and the impossibility of conducting the phasing analysis of mutations. In fact, SV larger than the read length cannot be detected and, using genomic DNA, the mutations may be too distant to be detected on the same read.All these approaches have surely proven more accurate than nanopore sequencing, allowing even low allele frequency mutations  to be detected, because of their lower error rate. Furthermore, higher throughput allows the analysis of more samples and/or more genes per sample. However, some considerations about short-read sequencing platforms should be made. Firstly, although their accuracy is very high (>99.5%), they are still affected by specific sequencing errors: Illumina displays a tendency towards substitution errors, whereas Ion Torrent technology fails when measuring homopolymersTP53 and del(17p)12, phasing analysis of multiple mutations would allow the physician to establish whether both TP53 alleles are mutated or not28.By exploiting the long reads produced by MinION sequencing, it is possible to evaluate the phasing of mutations, clarifying the allelic context of mutations affecting the same gene, but too geographically distant to be detected with short reads sequencing; this analysis may be particularly useful for CLL patients in the context of disease progression and therapy resistance. For example, in the case of no co-occurrence between mutated In perspective, we could also consider implementing our panel to identify even the structural alterations frequently identified in CLL patients, obtaining the complete CLL mutational spectrum in a single MinION experiment.30. In a recent report our group demonstrated that nanopore technology shows a correlation with SS results but is more sensitive, and has therefore proven to be a useful tool for TP53 gene mutation detection5. NOTCH1 gene mutations in CLL mainly cluster within the p.P2514fs*4 hotspot, and have independently conferred adverse overall survival (OS) in multiple studies, whereas their impact on progression-free survival has been inconsistent33. Moreover, NOTCH1 mutations have also been identified as a predictive marker for patients who are less likely to respond to the addition of rituximab to therapeutic regimens34. Mutations in the SF3B1 gene cluster in the selected HEAT repeats of the SF3B1 protein, and have been identified in 4–12% of CLL patients, with a higher frequency at the time of disease progression13; SF3B1 mutations are correlated with a shorter duration of remission after treatment, fludarabine-refractory disease, and poorer OS36. BIRC3 and MYD88 gene mutations have been detected in up to 4% of CLL cases37; while gene mutations in the former are associated with other adverse prognostic factors that predict chemo-refractoriness and poorer prognosis37, the MYD88 gene mutations, mostly L265P, identify CLL patients with a more favorable outcome14.All genes included in our panel have a prognostic significance in CLL. Mutations of TP53 are found in 4–37% of CLL patients, generally occur within the DNA-binding motifs (exons 4–8), and have been associated with very poor prognosis in several studies38, and new bioinformatic pipelines are being tested, also for variant calling analysis39.Our proof-of-concept demonstration of targeted gene sequencing in CLL using the MinION sequencer reveals the feasibility of this approach but identifies specific challenges to be dealt with in future projects. Indeed, in the last 2 years five different MinION chemistry versions , and several bioinformatics tools have been presented, improving MinION’s performance, but, despite these efforts and progress, the main weak point of MinION, namely its high error rate especially in homopolymeric sequences, still persists. In this context, basecalling is a crucial step, because it can hugely affect MinION sequencing data. To date, several basecallers have been developed to improve read accuracyRegarding the MinION error rate, we approached this issue by excluding from the analysis those genomic positions recurrently detected as mutated in the samples analyzed, admitting the inability to evaluate the mutational status of these genomic positions with the data analysis tools currently available. By retrieving the adjacent bases of these genomic positions, most of the false positive were within or next to homopolymer sequences. This strategy clearly reduced the total actual breadth of coverage, but produced more reliable results, and did not interfere with the detection of the CLL mutation hotspots. The main NOTCH1 hotspot p.P2514fs*4 is a particular case because it involves an “AG” dinucleotide deletion followed by a “G” homopolymer which does not seem to affect its correct identification.41. In the future, the implementation of these innovative approaches in MinION sequencing may contribute to better discriminate between mutated and wild-type DNA molecules and will be extended to achieve the concurrent detection of multiple biomarker mutations.Today, other single-molecule platforms are also being developed in order to overcome the limit of the nanopore error rate and detect very low-frequencies point mutationsIn conclusion, although the sample number in our study is relatively small, and an extension of the case series is warranted to correctly estimate the sensitivity and specificity of our assay, our data showed that the CLL gene panel provides a satisfactory performance. We propose a workflow that can potentially enable laboratories equipped with only basic molecular biology techniques to perform detailed targeted gene sequencing analysis in CLL patients. In the next future, we plan to evaluate how we can scale up the target panel, including more amplicons in a larger patients sample size using MinION. The constant improvement of nanopore technology will likely result inits widespread application in clinical practice.In Situ Hybridization (FISH), as previously reported44, and the immunoglobulin heavy-chain variable (IgHV) mutational status. These clinical parameters are significantly associated with the mutational status of the genes included in our panel35. Only one case, CLL#12, showed none of the frequent genetic aberrations analyzed in FISH analysis. All samples included in the study were characterized for the NOTCH1 hotspot p.P2514fs*4 and MYD88 hotspot p.L265P by molecular assays. For the exons of BIRC3, TP53 and SF3B1 included in the panel, mutational status was verified by Sanger Sequencing (SS). These evaluations were performed in concomitance with the MinION sequencing analysis. All 12 samples were analyzed by MinION and SS or molecular assays in a blinded manner.The study included 12 newly diagnosed CLL patients. Patients were selected according to the presence of the genetic aberrations 11q, 13q and 17p deletions (del(11q), del(13q), del(17p)), and trisomy 12, frequently associated with CLL and identified by Fluorescent Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen) and the DNA concentration and purity were checked using the Qubit 2.0 fluorometer (Life Technologies) and Nanodrop UV-Vis spectrometer (Thermo Fisher Scientific).Patients clinical and biological characteristics are summarized in Table The study complies with the current ethical guidelines, and has been approved by the Azienda Ospedaliero-Universitaria Consorziale Policlinico of Bari . The written informed consent was obtained from the patients included in this study, and patient records/information was anonymized and de-identified prior to analysis.13, NOTCH132 and MYD8814, and the genomic regions of the five genes reported in Table http://primer3.ut.ee/) with the following non-default parameters , to yield product sizes greater than 800 bp. The FastPCR  and Multiple Primer Analyzer (Thermo Fisher Scientific) tools were used to analyze and compare multiple primer sequences simultaneously. Overall, seven pairs of primers were selected for the selected target genes and analyzed with the Multiplex 2.1 tool (http://bioinfo.ut.ee/multiplx/) using the default parameters, to evaluate primers compatibility and find the best primers pooling solution. Two primers pools were thus identified: pool 1 included primers for BIRC3 exons 7–9, TP53 exons 10–11, SF3B1 exons 14–16 and NOTCH1 exon 34 including 3′UTR, while pool 2 was assembled with primers for TP53 exons 2–9, BIRC3 exon 6 and MYD88 exons 3–5. The total panel size was about 15 kb. In Table Our customized CLL panel included the known mutation hotspots of SF3B1For each CLL sample two multiplex long-PCRs were performed with the two primer pools using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.), 70 ng of genomic DNA, in a final volume of 25 uL. Thermal cycling conditions were 98 °C for 10 minutes, 60 °C for 15 seconds, 68 °C for 4 minutes (35 cycles) and 12 °C hold for both the two long-PCR.Since 3 amplicons of pool 1 had a very similar size of about 1.3 kb and were not easily distinguishable by 1% agarose gel electrophoresis, a restriction enzyme of these critical amplicons was made, and the BglII restriction enzyme  was finally selected to verify their successful amplification and discriminate them. In detail, 5 uL of the primer pool 1 PCR products were incubated with 0.5 units of BglII and 1uL of NEBuffer 3.1 in 1 hour at 37 °C. Digestion products were visualized by SYBR Safe on agarose gel 1%.PCR products from both the long PCR were purified with the QIAquick PCR Purification Kit (Qiagen) in an elution volume of 30 uL, and the DNA concentration and purity was measured with a Qubit 2.0 fluorometer (Life Technologies) and Nanodrop UV-Vis spectrometer (Thermo Fisher Scientific).Two uL of pool 1 and pool 2 purified amplicons were visualized by SYBR Safe on agarose gel 1.0%  protocol, the amplicons were end-prepared using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs Inc.) and barcoded with the ligation of nanopore-specific Native Barcodes (NB01-NB12) using Blunt/TA Ligase Master Mix (New England Biolabs Inc.). Equimolar amounts of each barcoded amplicon were then pulled and 540 ng of the final pool were diluted to 58 μl in nuclease-free water and prepared for sequencing with the ligation of Native Barcoding adapters and the tether using NEBNext Quick Ligation Module (New England Biolabs Inc.). All purifications were performed with Agencourt AMPure XP beads (Beckman Coulter Inc.). Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) were used to elute the library in the pre-sequencing mix.After the Platform QC run and the priming of the flowcell, the sequencing mix  was loaded and the MAP_48Hr_Sequencing_Run.py protocol was started (MinION flowcell: FLO-MAP106).Real-time analysis of the sequencing files generated by MinION was performed using a custom script including fast5 extraction, mapping with the BWA-MEM aligner and counting of the mapped reads; for each amplicon, the minimum number of reads per amplicon considered for the real-time analysis was 100x.https://www.ncbi.nlm.nih.gov/sra) under accession n° SRP133465.The sequence data from this study have been submitted to the NCBI Short Read Archive , generating FAST5 files, from which FASTQ files were extracted with the Poretools toolkit 0.6.0.46.The NanoOK tool (v.1.14) was employed to analyze the read depth and error rate, using the FASTA sequences of the target amplicons as reference. Depth of coverage and error rates of the known hotspots were calculated with bam-readcount program and by visual inspection of bam files with the Integrative Genomics Viewer (IGV) browserhttps://usegalaxy.org/), and Nanopolish, a package specific for signal-level analysis of nanopore sequencing data (https://github.com/jts/nanopolish).Data analysis from reads mapping to variant calling was performed in parallel using Galaxy, a web-based platform for processing NGS data (https://github.com/lh3/bwa/blob/master/NEWS.md/#release-079-19-may-2014); the leftalign utility from FreeBayes package was applied to homogenize the positional distribution of insertions and deletions. The BAM files obtained were visualized. Single nucleotide variants (SNV) and insertions/deletions (indels) detection was separately performed with the Varscan software47 , and the VCF files obtained were annotated with ANNOVAR16. The pipeline implemented in Galaxy is reported in Supplementary File In Galaxy, reads were aligned on GRCh37 human reference genome with the BWA-MEM method using specific Nanopore platform parameters .48 and BSgenome.Hsapiens.UCSC.hg19 R packages were used to retrieve the sequences flanking the variants detected, in order to evaluate the presence of homopolymer contexts and their potential association with MinION sequencing errors.The ChIPpeakAnnohttp://shiny.bat.infspire.org/igcllglass/) and GeneScreen49.The filtered variants were then compared with the results obtained using SS; electropherograms were then analyzed by visual inspection, glass free software for SS analysis data (http://bioinfo.biotec.or.th/WASP) was used to design the allele-specific primers for detecting the mutated and wild-type site, using the default parameters.For one SNV present at a level below the sensitivity threshold of SS (about 11%), an Allele Specific Oligonucleotide PCR (ASO-PCR) assay was accomplished. The Web-based Allele Specific Primer (WASP) designing tool (TM Taq DNA Polymerase (Invitrogen), using 100 ng of genomic DNA, in a final volume of 50 uL. Thermal cycling conditions were 95 °C for 30 seconds, 95 °C for 30 seconds, 65 °C for 30 seconds, 72 °C for 30 seconds (35 cycles) and 4 °C hold.ASO-PCR was performed with Platinumhttp://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession n° SRP133465.The sequence data from this study have been submitted to the NCBI Short Read Archive (Supplementary dataSupplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary information