In this paper, we demonstrate plasmonic substrates prepared on demand, using a straightforward technique, based on laser-induced photochemical reduction of silver compounds on a glass substrate. Importantly, the presented technique does not impose any restrictions regarding the shape and length of the metallic pattern. Plasmonic interactions have been probed using both Stokes and anti-Stokes types of emitters that served as photoluminescence probes. For both cases, we observed a pronounced increase of the photoluminescence intensity for emitters deposited on silver patterns. By studying the absorption and emission dynamics, we identified the mechanisms responsible for emission enhancement and the position of the plasmonic resonance.  Metallic nanostructures can modify the spectral properties of quantum emitters localized in their vicinity. When both the distance and the spectral relations between them are properly chosen, absorption and emission rates can be significantly enhanced ,2,3. ModAmong plasmonically active platforms, silver islands films (SIFs) play an important role. The SIF substrate consists of randomly deposited silver islands, formed during a reduction reaction of silver nitrate on glass ,9. The sIn this work, we demonstrate that silver nanostructures fabricated using a laser-induced photochemical reaction ,15 are pThe influence of silver islands, fabricated using the photochemical approach on the optical properties of emitters, was evaluated for both Stokes and anti-Stokes nanostructures, i.e., photoactive protein, peridinin-chlorophyll-protein, and rare-earth doped nanocrystals, respectively. Peridinin-chlorophyll-protein is a well-known light-harvesting complex. It consists of eight optically active peridinin molecules and two chlorophylls . Its absThe sample was prepared by spin-coating (3000 rpm for 1 min) the aqueous solution of PCP (2 μg/mL) on a glass substrate with previously deposited silver stripes. Experiments were carried out using wide-field fluorescence microscopy and confocal fluorescence microscopy for determining the intensities and decay curves of PCP emission, respectively. The wide-field microscope  was equipped with an oil immersion objective , a set of LED illuminators, and an EMCCD camera  mounted in the detection channel. The confocal microscope used the same objective, picosecond excitation lasers , and fast detection based on time correlated single photon counting system  coupled with single photon counting module .Emission intensity maps obtained for four excitation wavelengths of 405 nm, 480 nm, 535 nm, and 630 nm, are displayed in In addition to enhancing the absorption, metallic nanostructures may also—under appropriate geometrical and spectral conditions—influence the radiative properties of the emitters. In order to probe this process, we compared fluorescence transients measured of PCP complexes deposited on silver stripes and on glass. For the excitation, we used picosecond lasers operating at 405 nm, 488 nm, and 630 nm. The emission was extracted using a bandpass filter (670/10 nm), matching the emission of PCP chlorophylls. The comparison plotted in 4 nanocrystals were synthesized by wet chemistry, as described elsewhere [3+ plays the role of a donor, whereas Er3+ is the acceptor of the energy. The mechanism of the energy conversion involves several electronic states of erbium and is presented in 2H11/2+4S3/2→4I15/2 and 4F9/2→4I15/2 electric-dipole transitions in Er3+ ions, respectively  was spin-coated (3000 rpm for 1 min) on a glass substrate with previously fabricated silver stripes, as presented in In analogy to the experiments carried out for the photoactive protein, PCP, in the case of the up-converting NCs, we have also performed time-resolved luminescence measurements in order to demonstrate the influence of metallic nanostructures on the radiative processes. For the excitation, an electrically modulated laser operating at 980 nm generating pulses of about 1 µs in width was used. A typical transient collected from NCs deposited onto the silver stripes is presented in One of the key advantages of the photochemical approach described in this work concerns the ability of printing an arbitrarily shaped plasmonically active pattern. In 3, >99%, Sigma-Aldrich, St. Louis, MO, USA) mixed in 1:1 proportion with 2 mmol of trisodium citrate  was placed on top of the coverslip , equipped with a continuous-wave laser operating at 405 nm. A high numerical aperture oil immersion objective  ensured a small, diffraction-limited diameter of the laser spot (~200 nm) for precise spatial localization of the deposited material. Subsequently, a glass coverslip , previously cleaned in a Hellmanex II  solution, was mounted on an XY piezoelectric stage of the microscope. Then, 10 µL of a freshly prepared 2 mmol solution of silver nitrate . In On the other hand, the areas were obtained with a different delay time following the movement of the stage. This parameter, defined as delay time, was equal to 10 ms for the first area, 50 ms for the second, and 100 and 150 ms for the remaining two areas. The optical and SEM images (taken using a FEI Nova NanoSEM 450 scanning electron microscope) reveal the influence of both parameters on the morphology of the stripes: for the shortest time and the lowest excitation power of the laser, we found no formation of a distinguished metallic stripe. In contrast, for all other parameter pairings, a clear line emerged, which we associated with photochemical deposition of silver nanostructures. In order to gain insight into the morphology of the stripes, the same sample was analyzed using SEM, as shown in In this work, we demonstrate that silver islands deposited photochemically on glass substrates exhibit a strong plasmonic activity for both Stokes and anti-Stokes emitters. From the fabrication point of view, using this approach, we can precisely define positions of silver stripes, which are characterized with widths of about 1 micrometer. Importantly, in the case of photoactive proteins and rare-earth doped nanocrystals we find a strong increase of the emission intensity. This effect is attributed to enhanced absorption and emission rates (Purcell effect) of these emitters, as a result of the interaction with silver stripes. By correlating the laser illumination with the position of the piezoelectric stage, a pattern of any arbitrary shape can be obtained from the photochemically deposited silver islands. The strong plasmonic activity, together with the ability to control positioning, renders these unique structures potentially applicable for optoelectronic, photovoltaic, and sensory devices.


The findings suggested that DPFP is a good dietary supplement in the aquaculture, which can positively influence fish growth, intestinal histomorphology, hepatic antioxidant activity phagocytic percent and index, lysozyme activity, nitric oxide (NO) production, and disease resistance against A. hydrophila challenge.In aquaculture, medicinal plants and their by-products can be effectively used as natural immune-stimulants, growth promoters, anti-stress agents, and antimicrobials. However, very few studies have been conducted to evaluate the possible use of doum palm fruit powder (DPFP) in diets for African catfish. In this study, four experimental diets with four levels of DPFP  is known to be a beneficial medicinal plant. The objective of this study was to investigate the effects of the dietary addition of doum palm fruit powder (DPFP) on growth performance, non-specific immune response, and antioxidant parameters of African catfish, Clarias gariepinus (B.). A total of 120 fish  were randomly allocated to four groups ; a basal diet without DPFP supplementation was used as a control, and three other diets were prepared by supplementing 5, 10, or 15 g kg−1 DPFP for a ten-week feeding period. Following ten weeks of feeding, the fish were challenged with Aeromonas hydrophila , and mortalities were recorded. In comparison to the control diet, dietary DPFP significantly improved growth parameters, including final body weight, body weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), and protein efficiency ratio (PER), along with an increase in the content of dry matter of the whole body, in a concentration-dependent manner. Moreover, the heights of intestinal villi, numbers of goblet cells, and intraepithelial lymphocytes (IEL) exhibited marked escalation in all parts of the intestine by increasing the level of DPFP, except for numbers of IEL in the proximal part. The decline in serum glucose, cholesterol, and triglyceride levels was prominent in DPFP10 and DPFP15 groups respective to the DPFP0 group. Furthermore, DPFP boosted the hepatic level of catalase (CAT) in the fish, in a dose-dependent manner; meanwhile, the activity of superoxide dismutase (SOD) and reduced glutathione (GSH) content were also augmented in DPFP10 and DPFP15 groups respective to the DPFP0 group. Dietary DPFP (DPFP15 followed by DPFP10 then DPFP5) led to a pronounced enhancement in the innate immune response  production, and sialoglycans, namely α 2,3-sialyltransferase and α 2,6-sialyltransferase content); however, the myeloperoxidase (MPO) activity was reduced. Significantly higher relative percentage survival  of the fish, following the A. hydrophila challenge, was observed for the DPFP15 group. We can suggest that DPFP can beneficially influence fish growth, intestinal histomorphology, hepatic levels of catalase (CAT), superoxide dismutase (SOD) activity and glutathione (GSH) content, immune response, and disease resistance against A. hydrophila challenge.Application of herbal immune-stimulants for modulation of fish growth and immune response has received great interest during the past decades. With several pharmacological properties, Doum palm,  Clarias gariepinus (B.), which belongs to the family Clariidae, is a widely cultured species in tropical and subtropical countries. Owing to its hardy nature, high cultivability, fast growth rate, reproductive ability in captivity, high survivorship, the capability to accept formulated feed, and somewhat cheaper in comparison with other fish species, African catfish is one of the most preferred fish species for culture in Africa [As compared to the other food production sectors, the aquaculture industry has flourished significantly in recent times to meet the requirements of the fish market. Fish are considered to be the primary source of nutrition in most poor countries, thereby creating a huge demand . The Afrn Africa ,3. They n Africa ,5. Beingn Africa .Aeromonas hydrophila, a bacterium, can infect numerous fish species inducing hemorrhagic septicemia [Aeromonas infection is usually treated with antibiotics, often developing drug-resistant bacteria and posing a threat to human health [The fundamental objectives of the fish production industry are to improve fish growth, nutrient digestibility, immunity, and decrease feed cost ,7,8. Thepticemia . Aeromonn health ,13. TherOwing to their capability of improving the growth and immunity of fish, herbal feed additives can strengthen the host defense to combat the infectious agents. Henceforth, these herbal additives have grasped the interest of many researchers around the world. However, the roles of different herbal plants in aquaculture need to be elucidated. Various parts of medicinal plants, including leaves, fruits, and roots, have found effective application as functional feed additives in aquaculture . As natuHyphaene thebaica (Mart.) which belongs to the Arecaceae family. Doum palm fruit (DPF) has several applications in the food industry, such as in the manufacture of sweetmeats, cakes, and nutritious drinks [Phoenix dactylifera, a plant in the same family of doum palm, in augmenting growth rate, immune parameters, and antioxidant enzymes activity of common carp, Cyprinus carpio [Sparus aurata and European sea bass, Dicentrarchus labrax, respectively.With its palatable oval fruits, s drinks . The enes drinks ,22. Fatts drinks . Scientis drinks . To avois drinks . Previous carpio ,26. Furts carpio  and Guars carpio  reportedThe available information regarding the effect of dietary DPFP on African catfish is very scarce. Therefore, the present study is attempted to evaluate the potential effect of using DPFP as a feed additive on growth, intestinal histology, hematological and biochemical blood parameters, hepatic antioxidant capacity, and immunity augmentation in African catfish.The DPF was bought from a local market at Zagazig city in Egypt. After cleaning the fruits with water to remove any debris, the pulp and seed were separated with a stainless-steel knife. The pulp was dried, crushed, ground into a fine powder with an electric mixer, strained through a 0.25 mm sieve, and finally stored in sealed polyethylene bottles. According to the Association of Analytical Communities, AOAC , chemicaThe experimental protocol was approved by the Ethics of the Institutional Animal Care and Use Committee of Zagazig University, Egypt (ZUIACUC-2-F-152-2019).−1, pH = 7.2 ± 0.5; ammonia = 0.02 ± 0.001 mg L−1; nitrite = 0.017 ± 0.001 mg L−1; water temperature = 24 ± 2 °C; photoperiod 12:12 light:dark).One hundred and twenty healthy African catfish with an average initial weight of 60.50 ± 0.04 g were obtained from a private fish farm at Abbassa, Sharkia Province, Egypt. No history of outbreaks and no clinical abnormalities were recorded for these fish. According to the norms of CCoA , a routi−1 diet , for a ten-week feeding period . Fish were fed 3% of body weight twice daily (8:00 a.m. and 2:00 p.m.). The feed was adjusted every two weeks based on the weight of the fish.The fish were randomly allocated into four groups with three replicates for each . The experimental groups comprised of four basal diets with the addition of four doses of DPFP 0, 5, 10, and 15 g kgThe initial weight of the fish was recorded at the start of the experiment; then, the body weights and feed intake were recorded every two weeks, as well as at the end of the experiment. The following growth performance parameters and survival rate were calculated according to Reference  as folloAt the end of the feeding trial, three fish from each group were randomly selected for the chemical analysis of the fish body, according to AOAC . Fish sa−1 clove oil  within 3 min [g for 20 min for serum separation for measuring the biochemical and immunological indices. Furthermore, for the evaluation of the antioxidant enzyme activity, liver samples were isolated and frozen at −20 °C until analysis. Samples of the intestine were also removed and preserved in 4% buffered formalin for histological examination.At the end of the experiment, three fish from each aquarium (9 fish/group) were randomly chosen and anesthetized using 95 mg Lin 3 min . Thereaf®, Wetzlar, Germany), tissue sections of 5 µm thicknesses were prepared and stained with hematoxylin and eosin (H&E). Slides were visualized for morphometric analysis and photographed using the AmScope digital camera-attached Ceti England microscope for histopathological examination [At the end of the experiment, approximately 1 cm-long piece of the three intestinal parts  was fixed in 4% buffered formalin. The specimens were dehydrated using ascending grades of ethyl alcohol (70–100%), cleared in xylene, and embedded in paraffin. With the help of microtome  was used to count the total erythrocyte (RBC) and leukocyte (WBC), following the method described by Feldman et al. . AccordiWith the help of the colorimetric diagnostic kits of spectrum-bioscience , total cholesterol, triglycerides, and glucose were estimated following the protocols of Allain et al. , McGowanThe activities of catalase (CAT) and superoxide dismutase (SOD) enzymes and the reduced glutathione content (GSH) of the liver samples from three fish from each replicate  were assessed using the kits from Biodiagnostic Co.  according to the methods described by Sinha , McCord The phagocytic activity (%) of leucocytes was assayed, and phagocytic percent and phagocytic index were calculated according to the formula of Siwicki et al. :PhagocytAccording to the method of Grinde , the lysThe α 2,3-sialyltransferase  and α 2,6-sialyltransferase  activities to galactose were estimated by lectins in a sandwich solid phase assay Kletter et al. .A. hydrophila at a 0.1 mL dose of suspension cell containing 1.5 × 107 mL−1 cells using standard McFarland tubes through intraperitoneal injection. A. hydrophila was previously isolated from the dying fish and confirmed to be pathogenic for African catfish by the Department of Fish Diseases and Management of the Faculty of Veterinary Medicine at Zagazig University. A. hydrophila was identified by conventional biochemical tests and an automated VITEK 2-C15 system for bacterial identification  according to the manufacturer’s instructions at the Department of Microbiology and Immunology, National Research Center (NRC), Dokki, Giza, Egypt as explained by Reference [At the end of the experiment, all experimental groups were inoculated with the pathogenic bacterium eference ,52. Accoeference , fish moeference .p < 0.05. Post-hoc Tukey’s test was applied to determine differences among means based on linear regression. The statistical significance was based on p < 0.05 unless otherwise stated.All data were expressed as means ± standard error (SE). All data were verified for normality after transformation (ASIN). ANOVA  test was used based on polynomial orthogonal contrasts. Linear and quadratic regression equations were performed using SPSS  Version 17 for Windows  on growth, intestinal morphometric, biochemical, hematological, antioxidant, and immune parameters to evaluate the relationship between DPFP levels and these parameters at a significance value of p = 0.00, p = 0.002), total WG , total feed intake , SGR , and PER , and decreased the FCR , respectively, of fish as compared to that of the control group (DPFP0). However, the effect of DPFP on African catfish SR was found to be statistically insignificant.The growth performance and SR of African catfish fed on diets enriched with DPFP is shown in p = 0.001), respectively. Moreover, the crude protein content increased linearly and quadratically , whereas the ash content increased linearly  in DPFP10 and DPFP15 groups, respectively, compared to the control group. A linear elevation in crude lipids  was also perceived in the DPFP15 group relative to the control group.The proximate whole body composition analysis is presented in p = 0.00, p = 0.01) in the proximal and  middle parts, whereas only linearly  in the distal part of the intestine in DPFP5, DPFP10, and DPFP15-fed groups, respectively, relative to the control group.As shown in p = 0.001, p = 0.01) and distal parts , respectively   (p = 0.001) of the intestine also reflected a linear increase in the count of the goblet cells for the group fed on DPFP10 (5-fold) and DPFP15 (6.22-fold) diets compared to the control fish  C and dis = 0.01) E of DPFProl fish D.Histological sections of the posterior part of fish intestine illustrated- moderate goblet cell metaplasia in tall and serrated villi surfaces with mild intraepithelial and lamina propria lymphocytic infiltration in DPFP0 group; thickly branched villi with marked goblet cells metaplasia at several rows and proliferated enterocytes in DPFP5 group; short thick villi with goblet cells metaplasia and lymphocytic infiltrations in DPFP10 group; tall villi with numerous broad tips and goblet cell metaplasia in DPFP15 group .p = 0.00, p = 0.001), WBC count , and Hb  content, and only a linear increase in the HT%  was associated with a concentration-dependent increase in the DPFP level , respectively, as compared to the DPFP0 group.The results, represented in p = 0.002) and triglyceride  and linear and quadratic decreases in the cholesterol level  were noted in DPFP10 and DPFP15 groups, respectively (With regards to the control (DPFP0) group, a linear decline in the levels of glucose  in DPFP10 (1.35-fold) and DPFP15 (1.49-fold) groups with respect to the DPFP0 group. Linear elevation was also obtained in the serum total globulin  and its fraction γ globulin  in DPFP-fed groups , respectively, as compared to the control group. However, DPFP failed to illuminate any significant effect on serum albumin and other globulin fractions.The serum total protein, as demonstrated in p = 0.001) was linearly augmented by the DPFP-supplemented diets  respectively, higher than the DPFP0 group. Compared to the control group, there was a linear increase in the SOD activity  and GSH content  in fish fed DPFP10 and DPFP15, respectively.As shown in p = 0.003), phagocytic index , and lysozyme activity  and decrease in MPO activity , respectively, as compared to the DPFP0 group.p = 0.001, p = 0.01) observed in fish fed DPFP, where NO activity was  in DPFP5, DPFP10, and DPFP15 groups, respectively. DPFP-supplemented diets could linearly augment the α 2, 3-ST  and α 2, 6-ST  activity, respectively, more than the control diet , followed by those fed on DPFP5 (44.45%) and DPFP10 (22.22%), whereas the lowest mortality rate was recorded in those fed on DPFP15 (7.63%). An increase in the concentration of the DPFP was associated with an enhanced survival rate in the fish, where it was 55.55%, 77.78%, and 92.37% in DPFP5, DPFP10, and DPFP15, respectively, as compared with the control group (33.33%). The RPS was found to be highest in the DPFP15 group (88.56%), followed by the DPFP10 group (66.67%), whereas it was lowest in the DPFP5 group (33.33%).The mortality rate, survival (%), and RPS of the African catfish, fed on DPFP-enriched diets for ten weeks and challenged with −1 for eight weeks could induce growth-related gene expression and improve fish weight by 34.6% and feed utilization by 17.53%.Dietary DPFP was responsible for a concentration-dependent increase in the growth performance, feed efficiency, and utilization of African catfish in the present study. Although there is no available data on the impact of doum palm on fish growth and feed intake, the growth improvement offered by dietary doum palm can be returned to better nutritional status. High contents of proteins (2.86–5.01%), fibers 52.26–66.5%), essential fatty acids, vitamins, and minerals in doum palm justify its high nutritional value ,23. More–66.5%, eNelumbo nucifera stamen extract [Ocimum gratissimum leaves extract for 12 weeks.Furthermore, the present study also established the effect of dietary DPFP in augmenting the villous heights and numbers of goblet cells and IEL in all parts of the intestine except count of IEL in the proximal part. Increased surface area for the absorption, improved feed digestion, nutrient utilization, and defense against pathogens by mucin layers on the intestinal mucosa were, therefore, substantiated by these findings, which in turn reflected the effect on the fish growth performance as reported by Reference . The dig extract . Abdel-T extract  also obsTelfera occidentales leaves [The dry matter of African catfish was redundant with increasing the level of DPFP. An increase in the crude protein and ash content was reported in the DPFP10 and DPFP15 groups; however, marked elevation of the crude lipids was witnessed in the DPFP15 group. Enhanced feed efficiency and utilization by DPFP, as well as improved digestibility, might be held responsible for these findings as the carcass composition has been demonstrated to be influenced by the diet . As repos leaves .Vernonia amygdalina, or their combination [The hematological indices are used as biomarkers to evaluate the physiological condition and health status of fish due to the influence of immunostimulants ,66,67. Tbination  have alsThe primary source of energy for fish to withstand unfavorable conditions is blood glucose, which is also an efficient indicator of stress. Cholesterol and triglycerides are the lipids present in the blood in the form of lipoproteins. Among the several factors that influence the concentrations of cholesterol and triglycerides, diet formulation is the most important one . The preZingiber officinale and Roselle, Hibiscus Sabdarifa for 70 days have also documented a parallel increase in the protein profile [Elevated levels of blood proteins, especially globulin, are good indicators of the increased immune response . DPFP in profile . Further profile  also illOxidative stress is detrimental to vital biomolecules, such as DNA and proteins. This oxidative damage of the body tissues is prevented by the antioxidant system that eradicates the reactive oxygen species . DietaryIn contrast to mammals, innate immunity is the major player of defense in the case of fish. Phagocytic cells (neutrophils and macrophages) are important components of the innate immunity, which obliterate the pathogens through a process called phagocytosis . MoreoveActivated neutrophils release the enzyme MPO, a well-known heme-containing peroxidase. The killing of pathogens is a crucial role of this enzyme, and it also acts as a local mediator of tissue damage resulting in oxidative stress and inflammation . PresentA. hydrophila pathogen. The beneficial effects of DPFP on the survival rate of African catfish might be attributed to its active compounds that could enhance the non-specific immune parameters like phagocytic activity, NO, lysozyme, sialoglycans, and antioxidant enzymes and its antimicrobial activity. According to Olusola and Nwokike [A. hydrophila.The challenge test is mostly used as a confirmatory assay to measure the organism’s immune functions as a whole . The eff Nwokike  and Abde Nwokike , herbal A. hydrophila challenge. Further studies are encouraged to elucidate its potential applications on the growth and health of other fishes.The present results established the beneficial effects of DPFP on fish growth, intestinal histomorphology, hepatic antioxidant activity represented by increased catalase, superoxide dismutase activity, and glutathione content, immune response represented by increased phagocytic percent and index, lysozyme activity, nitric oxide production, sialoglycans content, and reduced myeloperoxidase activity, and improving disease resistance of African catfish against


Zinc oxide (ZnO) is one of the main functional materials used to realize chemiresistive gas sensors. In addition, ZnO can be grown through many different methods obtaining the widest family of unique morphologies. However, the relationship between the ZnO morphologies and their gas sensing properties needs more detailed investigations, also with the aim to improve the sensor performances. In this work, seven nanoforms  were prepared through simple wet chemical synthesis. Morphological and structural characterizations were performed to figure out their growth mechanisms. Then, the obtained powders were deposited through screen-printing technique to realize thick film gas sensors. The gas sensing behavior was tested toward some traditional target gases and some volatile organic compounds  and compared with ZnO morphologies. Results showed a direct correlation between the sensors responses and the powders features (morphology and size), which depend on the specific synthesis process. The sensors can be divided in two behavioral classes, following the two main morphology kinds: aggregates of nanocrystals (leaves and bisphenoids), exhibiting best performances versus all tested gases and monocrystal based . Sensors have become of crucial importance in several aspects of our daily life, allowing us to monitor physical, chemical, biological, and environmental parameters ,2,3. SmaMetal oxides (MOX) materials are major players in the field of chemical sensing. Well-established methods, including wet chemistry ,6 and diSeveral MOX semiconductors are known to exhibit gas-sensitive fluorescence and optical responses, permitting their use as optochemical sensors as fluorescent films ,17 or byMOX properties depend significantly on their defect composition and nanocrystals morphology, as discussed in this work. To date, a lot of efforts are addressing to improve sensing, selectivity, and reliability properties of MOX gas sensors ,31. In t2, SnO2, and WO3), ZnO is a n-type semiconductor and certainly a well-performing material for sensor devices the most promising oxide primarily due to its chemical and morphological stability and its high gas sensitivity. In fact, ZnO has been successfully employed to detect various gases, such as H2, NO2, O2, H2S, C2H6O, and NH3 v  > [10–11]v  > [10–10]v .The common and stable zinc oxide phase at ambient conditions is the hexagonal wurtzite structure, which consists of alternating planes composed of tetrahedrally coordinated Otruction . The othtruction . ZnO diftruction , that th2 direct precipitation in the synthesis solution starting from the base addition, while bisphenoids derived from the spontaneous growth of leaves kept in the synthesis solution. Leaves samples are constituted of leaves of rhombic shape, which have the longest diagonal of about 6 μm in size, while the shortest diagonal one is about 3 μm long. ZnO leaves . Leaves and bisphenoids exhibited a highest SSA of about 20 m2/g.Textural characterization gave specific surface areas (SSA) that can be divided into two ranges of values. Stars, needles, long needles, prisms and flowers showed a SSA under 10 m3mc) irrespective both on the synthesis method and the particle morphology, as shown in the XRD patterns  Å, b = 5.1457 (1) Å, c = 8.4780 (2) Å and volume of 214.176 (8) Å3 according to JCPDS Card No. 38–0385. During the calcination process, the solid precursor changes into ZnO nanoparticles through an in situ crystallization [4 that becomes CaSO4 × 2H2O.In the case of stars, needles, long needles, prisms, and flowers, the shape is directly related to the ZnO crystalline phase through a rapid crystallization in slightly different synthesis conditions. On the contrary, as clearly observable in the lization , releasilization . PseudomMorphological analysis of the sensing films after the firing process for 1 h at 650 °C, firstly highlighted two different behaviors, as shown in In metal oxide gas sensors, the electrical properties are firstly dependent on their aptitude to modify the electrical conductivity when they come into contact with the gases of the surrounding atmosphere. On the basis of this peculiarity, a great amount of features influence the sensor characteristics. Of course, the functional material is the key constituent of the sensor, but the morphology and the size of its particles are certainly critical factors to determine its detection properties . In partApart from the aptitude of the specific morphologies to detect the various gases, it is however of the utmost importance to verify for all samples the electrical stability and repeatability. Indeed, they are essential characteristics in order to obtain efficient and reliable sensors able to operate continuously for long time. In Other features of screen-printing technology, such as the high firing temperature or the chance to fabricate in one batch up to 500 sensors, are conditions that contribute to assess screen-printed MOX gas sensors highly reliable among the various kinds of chemical gas sensors.Considering the specific electrical characterizations, an Arrhenius plot, which consists of the measurement of the conductance as a function of temperature in different atmospheres, was carried out. In this work, dry air and a mixture of dry air and acetone (10 ppm) were chosen, for comparison. Such measurement highlighted for all samples a n-type semiconductor behavior, the conductivity exhibiting a trend with temperature divided in more ranges, behavior ascribable to different chemical adsorption phenomena. Such particular trend is typical of MOX gas sensors and justified by the formation of inter-grain Schottky barriers that modulate the film conductivity . Moreove2H4O, acetone C3H6O, ethanol C2H6O, toluene C7H8, isoprene C5H8, and ammonia NH3), the responses toward each tested gas at the concentration of 10 ppm in dry air, for the sensors based on bisphenoids, stars, prisms, and flowers are reported in Passing to dynamical measurements of conductance in presence of the tested gases  that changes into ZnO through an in situ crystallization obtaining aggregates of nanoparticles; (ii) a rapid crystallization in growth solution for monocrystal based morphologies. The corresponding sensing layers were studied with particular interest to their gas sensing properties toward a wide number of reducing gases in relation to the functional material morphology and the respective growing mechanism.The ZnO powders were successfully obtained with optimized routes, starting from the same reagents in water solution by changing the synthesis conditions. Two classes of morphologies were obtained: the nanoparticles’ aggregates (leaves and bisphenoids) and the monocrystals based ones . Their growing mechanisms were systematically analyzed highlighting two main ways: (i) the formation of a solid precursor (ε-Zn(OH)It turned out, similarly to powders, there were two different behaviors. The samples constituted by nanoparticle aggregates exhibited the best performances toward all tested gases with respect to monocrystals based sensors, that, albeit with some difference, all showed worsened performances. Due to the large number of investigated forms, this result confirms that, almost for reducing gases, the three-dimensional shape, like a spherical particle, is the most suitable form in the MOX chemiresistive sensing process.The present work permitted to select sensors with optimal sensing properties , exploiting ZnO aptitude to grow in different forms. At the same time, the synthesis involved is easy to do, not expensive, and further chemical processes are not necessary. Moreover, this strategy opens the way for future applications in other functional material preparation.


Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”. EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy. Extracellular vesicles (EVs) are released by cells into the extracellular space and are classified according to their size and biogenesis , 2. AccoEVs are quite abundant in biofluids as they are continuously released by cells . In someAdditionally, since EVs contain signaling molecules, they are considered be a potential source of diagnostic biomarkers for the prediction of disease, as well as in disease monitoring and treatment decision making , 16. To In this study, we characterized the protein content of small EVs (diameters under 200 nm), most likely exosomes and small microvesicles. These were isolated by the combination of UF and SEC from cell culture supernatant of three distinct cancer cell lines: oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC), and melanoma, all with poor prognosis , 22. We 2 atmosphere incubator.Small EVs were isolated from the culture media of human oral squamous cell carcinoma (OSCC) , pancreatic ductal adenocarcinoma (PDAC) , and human melanoma brain metastasis . The cells were cultured as previously described . In ordeCell culture supernatants were collected once a week. After collection of the cell culture supernatant, the CELLine AD1000 flasks were washed with warm PBS. Cell culture supernatant and PBS were then pooled. Samples were centrifuged at 4000g for 5 min at room temperature, to remove cell debris and the supernatant was then centrifuged for another 45 min at 15000g, 20°C. Next, 15 ml of the 15000g supernatant was concentrated by UF using Amicon-Ultra 15 Centrifugal Filter Units (Merck Milipore) with a 50 kDa molecular weight cut-off (MWCO), to a final volume of 4 ml. Concentrates were loaded into 30 ml sepharose CL-2B  size-exclusion chromatography (SEC) columns. The concentrated cell culture supernatants were eluted through the columns with the continuous addition of filtered PBS. Eluates were collected by gravity in 20 fractions of 1 ml. For each fraction, protein quantification was determined by spectrophotometry (Absorbance 280nm) in a Nanodrop spectrophotometer (Thermofisher).®, Thermo-Fisher Scientific)), western blot (WB) ) and transmission electron microscopy (TEM), as described elsewhere [Following protein quantification, the EV enriched fractions were identified and pooled. Samples were characterized by nanoparticle tracking analysis (NTA), immunoaffinity capture , as described in detail in . BrieflyValidation of liquid chromatography-mass spectrometry (LC-MS)-based peptide and protein identifications was carried out using Scaffold_4.9.0 . Here protein threshold was set at 99.0% to ensure that only proteins with 99% of probability of being present were produced, and the minimum number of peptides was set to 2. Furthermore, the peptide threshold was set at 95%, the minimum probability to determine if a given spectrum identifies a peptide.Data from the vesicles isolated from E10, BxPC3, and H3 cell culture supernatants were analyzed and extracted individually from Scaffold. All datasets were screened and proteins that were not present in all 3 replicates within a cell line were not included for further analysis.http://www.funrich.org). In addition, the proteins identified in the preparations from E10, BxPC3, and H3 were compared with the Vesiclepedia database (http://microvesicles.org), a web-based open source compendium of biomolecules  found across all classes of EVs from published and unpublished studies [Comparison of the proteins from the different cell lines was carried out using the Functional Enrichment Analysis Tool (FunRich)  Bioinformatics Resources 6.8. The proteins were classified according to their gene ontology (GO) term and categorized into functional groups under the term “Biological processes” (GOTERM_BP_FAT). Next, the GO terms were grouped into “Functional annotation clusters”, where the terms are organized into groups based on the premise that genes with similar annotation are functionally related to each other . Functio10, 3.14x1010, and 2.22x1010 particles/ml for E10, BxPC3, and H3, respectively  analysis for identification and characterization of their protein content.Proteomic analysis of the vesicles isolated from cell culture supernatant from the three cell lines identified 678 different proteins from the E10 derived EVs, 618 proteins from the BxPC3 derived EVs, and 762 proteins from H3 derived EVs . InteresThe top 20 most highly expressed proteins in the EVs from each of the cell lines are listed in Thus, despite the presence of common proteins, the EVs isolated from the different cell lines showed an individual profile.When the proteins in our datasets were analyzed in view of the Hallmarks of cancer, we identified proteins with roles in the processes of angiogenesis, inflammation, cell proliferation, migration, immunity, and cell adhesion .The EVs from each of the cell lines demonstrated varied protein profiles related to these processes. For example, the panels of proteins associated with cell proliferation, migration, and adhesion were fairly extensive for both E10 and BxPC3. Of note is the presence of several members of the integrin family (IT), associated with cell proliferation, migration, and adhesion, such as ITGA2, ITGA3, ITGA6, ITGA5, ITGB1, and ITGB4 in the EVs derived from E10 and BxPC3. In contrast, only half of the cell proliferation-related proteins were found in EVs from the H3 cell line, most of them poorly expressed (with SC below 10). The number of proteins related to adhesion were also reduced in the H3 derived EVs. Here ITGB1, ITGA3, and ITGA2 were found in the vesicles from H3, the latter with fairly low SC.Overall, the protein content of the EVs isolated from the different cell lines carry signals that promote cancer traits. However, the EVs from the H3 melanoma cell line have a more distinct protein signature than the other two, with respect to both the type of proteins as well as their amount present.To better understand the roles and relationships of the proteins in the isolated EVs, Gene Ontology (GO) analysis was performed as described in the Materials and Methods section.In the dataset of proteins identified in the E10 vesicles, the 8 most enriched biological processes in the functional annotation cluster analysis  includedExtracellular vesicles carry complex biomolecular cues and thus, play an important role in the communication between cells. In this study we analyzed and categorized the protein content from EVs derived from oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC), and melanoma brain metastasis cell lines , all cancers with poor prognosis for the patient. Isolation of the EVs from the cell culture supernatants, prior to proteomic analysis, was carried out combining UF and SEC. The EVs were characterized by NTA, WB, immunoaffinity capture, and TEM.et al., indicates that SC of less than five is unrealiable in studies with a small number of replicates [Characterization of the protein content of the isolated vesicles by LC-MS identified a similar number of proteins for all preparations: 678 proteins for E10, 618 for BxPC3, and 762 for H3 . When coplicates , 38, 39.plicates . Neverthplicates . The graplicates .The proteins identified in the EVs of each cell line were ranked according to their average SC for further analysis. Due to the large datasets for each of the cell lines, the top 20 most abundant proteins were extracted . A charaIn summary, our results highlight that the EVs isolated from each of the different cancer cell lines have distinct protein profiles. Cell line-specific profiles already begins to emerge when comparing the 20 most adundant proteins. Here, proteins that seem to provide tumors with signals that promote its progression are found among EV-related proteins. These EV related proteins are present in the datasets from all three cell lines.The panel of proteins detected in the EVs from OSCC, PDAC, and melanoma cell lines  indicates that these vesicles can play roles in angiogenesis, inflammation, proliferation, migration, as well as in modulation of immunity, all representing Hallmarks of cancer. Within this selection of proteins , there wIn contrast, Muc5AC was found to be highly enriched in the BxPC3 derived EVs, while present in much lower amounts in the EVs from the other two cell lines. Muc5AC is a protein that stimulates angiogenesis in PDAC and might also suppress the immune response towards the tumor . In patiLastly, FN1 was abundant in the H3 derived EVs but detected in a smaller amount in the E10 derived EVs. Furthermore, a dramatic drop in the FN1 content was found in the BxPC3 derived EVs. FN1 plays a role in cell adhesion, migration, and differentiation . Furtheret al the levels of integrin expression in exosomes derived from melanoma brain metastasis are down regulated [et al in saliva from OSCC patients  [Differences among the cell lines were also noticeable regarding members of the integrin family. The members of this family of cell adhesion receptors are relevant in cancer processes such as cell migration and invasion , 83. In egulated . Moreovement C3) . Similarment C3) , 87. ThiOverall, the EV content reflects the characteristics of each cell line. Additionally, the cell line specific EV protein combinations point to the cellular phenotypical characteristics described as Hallmarks of cancer. Furthermore, the proteins identified have been detected in clinical cancer tissues in other studies, indicating that these may be relevant as diagnostic biomarkers for these cancers.Functional enrichment analysis was performed using DAVID annotation clustering. Here, we identified the most enriched biological processes regulated by the hundreds of proteins identified in the EVs from each cell line . We founThe EVs derived from the three cancer cell lines also had some biological processes in common. “Interspecies interaction between organisms”, related to viral reproduction is one example. This process reflects the similarity of EVs and viruses even though EV biogenesis is a naturally occurring biological process while viruses hijack the cellular machinery for its propagation . In addiTogether, these findings reveal that the EVs analyzed in our study contain proteins that can regulate biological processes towards a cancer phenotype, in a manner that is characteristic for each of the cell lines.In conclusion, in the present study we used LC-MS to characterize the protein content of small EVs derived from OSCC, PDAC, and melanoma brain metastasis cell lines. More than 600 proteins were identified in the EVs of each cell line. Analysis of the datasets revealed cell-specific protein panels. In short, EVs derived from E10 had a high expression of EGFR, ITGB4, and NT5E, the EVs isolated from BxPC3 were enriched in mucins , and the most abundant proteins in the EVs from the H3 cell line included HMCN1, HSP8A, and APOE. Furthermore, screening of the protein datasets for proteins involved in the Hallmarks of cancer also exposed cell-specific protein panels. Here, proteins associated with the promotion of processes such as angiogenesis, proliferation, migration, or adhesion were found to be present in the EVs derived from all three cell lines. This is relevant due to the role of EVs in the modulation of the cells in the tumor microenvironment  into a more permissive setting for tumor progression. The list of proteins involved, for example, in cell proliferation was quite extensive in E10 and BxPC3 in comparison to H3 where only half of the proteins were found and most of them poorly expressed. Functional enrichment analysis by gene ontology (GO) was carried out to extract a broader biological significance. Here, processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”, all associated with aspects of cancer, were found. However, the processes identified from the proteins from the H3 derived EVs were distinct from the ones in the datasets from E10 and BxPC3, revealing an underlying difference between the cell lines. This study focuses on the protein content of small EVs, and does not explore other classes of biomolecules such as RNAs. Nevertheless, the protein lists generated and analyzed here may provide important information regarding the complex biomolecular cues EVs carry. In a clinical setting, biofluids from cancer patients will include a higher number of cancer-derived EVs in addition to non-cancer-related EVs. This will complicate the collection and examination of spacific caner-derived EVs. Hence, we believe it is important to start with a broad analytical approach that may assist in pinpointing the cancer-derived EVs in biofluids.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


ED3 and ED5 nets were coated with SiO2 nanoparticles of different diameter, while in the case of ED3-P and ED5-P, paraffin was added to increase the mass of the deposited particles on the net’s surface. In the first series of bioassays, the knockdown and mortality rates of these species were evaluated after exposure to the aforementioned net formulations for 5, 10, 15, 20, 25, 30, 60, 90, and 180 min. In the second series of bioassays, knockdown and mortality of these species were recorded after 1, 7, and 10 days of post-exposure to the nets for different time intervals . Based on our results, all nets significantly affected A. fabae, since all insects were dead at the 1-day post-exposure period to the silica-treated nets. Conversely, at the same interval, no effect on either S. oryzae adults or T. confusum larvae was observed. However, in the case of S. oryzae, the efficacy of all nets reached 100% 7 days after the exposure, even for adults that had been initially exposed for 15 min to the treated nets. Among the species tested, T. confusum larvae exhibited the lowest mortality rate, which did not exceed 34% at the 10 days of post-exposure interval. Our work underlines the efficacy of treated nets in pest management programs, under different application scenarios, at the pre- and post-harvest stages of agricultural commodities.Insect proof nets are widely used in agriculture as mechanical and physical barriers to regulate pest populations in a greenhouse. However, their integration in the greenhouse ventilation openings is highly associated with the decrease of air flow and the adequate ventilation. Thus, there is need for alternative pest management tools that do not impair adequate ventilation. In the present study, we tested four net formulations of relatively large mesh size coated with SiO Despite the association of many insecticidal compounds with mammalian toxicity and increased environmental footprint, conventional pesticides are widely used as a means of pest management. Over the years, many insects species have developed resistance mechanisms to the vast majority of active ingredients that are used as synthetic insecticides ,2,3. AccNets are widely used in agriculture as mechanical or physical barriers in order to protect crops from either biotic or abiotic stresses. In the Mediterranean region, protection against insects is considered significantly important even compared to the management of excessive heat during the summer . Insect Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodoidae) and the western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), could lead to a ventilation reduction of approximately 88%. As a result, higher temperature regimes are recorded in the interior of the protected greenhouse plants leading to non-uniform production, increased yield losses, as well as quality deterioration [According to Kittas et al. , the envioration ,14. Smalioration . Moreoveioration , showed Myzus persicae (Sulzer) (Hemipetra: Aphidedae) and Aphis gossypii (Glover) (Hemipetra: Aphididae) immigration without affecting the parasitoid Aphidius colemani Viereck (Hymenoptrea: Aphididae). However, during field trials, LLINs effectiveness was degraded after sun exposure whereas, the efficacy of either bifenthrin or deltamethrin was suggested as insufficient to control B. tabaci culture due to its small size. Notwithstanding, according to Arthurs et al. [F. occidentalis invasion. Hence, both the effect of LLINs on beneficial enemies and their effectiveness in small sized insects should be further evaluated [The establishment of insect proof nets for the stored product pest management is directly linked to environmental protection, as their use does not lead to residues or to contamination in storage facilities and stored commodities . Many sts et al. , nettingvaluated .®, a polyester net coated with alpha-cypermethrin, against two stored tobacco insect pests, the cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: Anobiidae) and the tobacco moth, Ephestia elutella (Hübner) . Moreover, Paloukas et al. [® could be effectively used to control different major stored-product insects such as the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), since 98% mortality was recorded two weeks after the exposure to the treated net.Recently, Rumbos et al.  successfs et al.  suggeste2) begins with the adherence of the nanoparticles on the cuticle of the insect, inactivating epicuticular lipids, which leads to death through desiccation [The use of nanoparticles as a novel biological insect control agent has been well established in the recent years. Silicon is known as the most frequent metalloid and the most copious element on earth after oxygen ,22,23. Ticcation ,25. FurtTribolium confusum Jacquelin DuVal (Coleoptera: Tenebrionidae) is considerably difficult compared to soft-bodied greenhouse pests such as aphids. On the other hand, aphids are among the most common pests in horticultural crops and are related to the transmission of many economically hazardous viruses even after injecting their sucking mouthparts on the leaves for a very short time interval [Rhopalosiphum padi (L.) (Homoptera: Aphididae), when applied on wheat plants. However, in that study, the authors stood eventually against these DE applications, because of negative photosynthetic rates [Diatomaceous earth (DE) that consists mainly of amorphous hydrated silica has been extensively used in stored product insect management. Vayias and Athanassiou  stated tinterval ,27. Unliinterval  found thic rates .Plutella xylostella (L.) (Lepidoptera: Plutellidae) to 1 mg × cm−2 of a siliceous dust formulation resulted in 85% mortality after 72 h. The authors suggested that the mortality rate is improved as dose and exposure interval are increased. Moreover, silica nanoparticles, concerning their physical and chemical properties, differ significantly from their bulk formation [S. oryzae. At the post-harvest stages of agricultural commodities, stored product protection could be enhanced by the use of silicon-based nanoparticles as non-conventional pesticide agents [Recently, Shoaib et al. , indicatormation . Accordiormation , nanopare agents .Aphis fabae Scopoli (Hemipetra: Aphididae), and the stored product pests T. confusum and S. oryzae. The aforementioned species were exposed to insect proof nets coated with two different silicon dioxide dust formulations, Syloid®ED3 and Syloid®ED5 with and without the addition of paraffin in order to achieve better particle adherence to the net, respectively.To date, little attention has been given to the incorporation of physical inert dusts to the yarns of insect proof nets. The application of non-toxic and environmental-friendly nets could replace the insecticidal treated nets leading to insecticide-free products. Thus, the objective of our study was to assess the effectiveness of silicon dioxide-treated nets under laboratory conditions for the control of the black bean aphid, A. fabae individuals took place at the Laboratory of Agricultural Entomology, Benaki Phytopathological Institute, Attica, Greece. Aphids were reared on Vicia faba L. plants at 20 ± 1 °C, relative humidity (r.h.) of 65 ± 2% and a photoperiod of 16 h in light: 8 h in dark [The rearing of the  in dark . Aphid cS. oryzae and T. confusum were maintained in LEAZ under controlled conditions in a growth chamber as suggested by Faliagka et al. [Established laboratory colonies of a et al. .® ED3 or Syloid® ED5 which are amorphous silica dust formulations . The former silica dust consists of 99–100% SiO2 with particle size of 5.8 μm, while the latter consists of 95–100% SiO2 with particle size of 9.0 μm. Paraffin was added as an organic primer in two of the four netting samples (ED3-P and ED5-P) consisting of different silica formulation in order to enhance the adhesion properties of each dust to the net. ED3 and ED5 samples were used without the addition of paraffin. The purpose of the primer is the integration of up to 40% more SiO2 particles on the net. The mass of the deposited silica particles on the net’s surface was fluctuated from 0.4 to 0.9 g × m−2 depending on the dust formulation as well as the addition of paraffin. However, paraffin may reduce the mesh size and therefore, the air permeability properties of the tested nets. In the present study, the insecticidal properties of SiO2 nanoparticles were evaluated. In the case where adequate results are provided, it is suggested to incorporate silica on nets of larger pores in order to alleviate insufficient ventilation. Experiments were carried out with four samples of the same insect proof net, the textiles of which were performed by the Institut für Textiltechnik (ITA) of RWTH Aachen University. The weaving and the evaluation of the nets was provided from Thrace Nonwovens & Geosynthetics S.A. . All textiles were coated either with Syloid2 free-nettings with the same mesh size as well as clear Petri dishes without the addition of nets were used as a control treatment. The silica coating procedure was repeated two times for each tested net.All samples were produced by the same 50 mesh net that was constructed by using high density polyethylene (HDPE) monofilament yarns. SiO2 in surface), using a thin layer of silicon. The lids of plastic dishes are usually loose-fitting making it easy for insects to escape, so, the “neck” of each Petri dish was covered with polytetrafluoroethylene dispersion, . A single fine brush was used to insert the species into the Petri dishes to avoid wounding.Nets were fine cut and adjusted at the bottom of plastic Petri dishes , and untreated nets for the intervals of 5, 10, 15, 20, 25, 30, 60, 90, and 180 min. For each exposure interval, mortality and knockdown effect of all species in each Petri dish were recorded. For each of the four nets, the bioassay was replicated eight times per exposure interval. Thus, in this series of bioassays 144 Petri dishes were used in total.In the short-term effect bioassay series, adults of S. oryzae and T. confusum, cracked wheat (0.5 ± 0.1 gr) and wheat flour (1.0 ± 0.1 gr) were added in all petri dishes, respectively. In the case of A. fabae no food was supplied, since the bioassay lasted one day, unlike that of the stored product insects which was completed after 10 days. All bioassays were carried out at 25 °C and 65% r.h. Mortality and knockdown effect of S. oryzae and T. confusum were assessed after 1, 7, and 10 days, whereas in the case of A. fabae after 1 day. For each of the four nets, the bioassays were replicated eight times. A total of 624 Petri dishes were used to perform this series of bioassays.The purpose of this series of bioassays was to evaluate the effect of the nets on the knockdown and mortality rates of the exposed individuals at certain post-exposure intervals. Thus, as above, ten individuals of each species were transferred to Petri dishes, with different plates per species, and exposed to the nets for 15, 30, and 60 min. Knockdown and mortality were recorded immediately after the completion of the exposure periods and then all insects were carefully transferred to new dishes consisting of untreated nets or clear Petri dishes (without nets). When transferring the insects, a clean single fine brush was used to avoid possible contamination of the dust in the treated nets. Within each exposure interval of 15, 30, and 60 min no food was added in the Petri dishes. However, during post-exposure period for t50, KDt95, and KDt99 for each species and net formulation. The intervals tested yielded a variety of knockdown results only for A. fabae, which allowed the calculation of KDt50, KDt95, and KDt99 values. For the second series of bioassays, separately of each species, the data  were converted to percentages and then were submitted to a two-way ANOVA, to determine the differences among the exposure intervals  and among the net formulations (including controls as well), while means of knockdown and mortality were separated using the Tukey-Kramer HSD test at the 5% level. Moreover, the same approach was conducted for the long-term effect for each species tested. The data were analyzed using SPSS 25.0 .The results of the first series of bioassay (short-term effect) were analyzed using Probit Analysis to estimate the knockdown time, i.e., KDt values were not found to fit the data well, since P values were <0.01 .The mortality rate of all tested species was relatively low. In the first series of tests, KDre <0.01 . In mostt99 values for A. fabae at ED3-P resulted in the highest values among treatments, corresponding to 1590.9 min (approximately 26 h)   and 1281.4 min (approximately 21 h), respectively. For KDt50 the lowest value (51.3 min) for A. fabae was recorded at ED5, whereas the highest value (279.7 min) was noted at ED3-P  . Contrarely 6 h) . Moreoveat ED3-P .A. fabae was low and did not exceed 28.7% , while at the 1-day post-exposure period, no significant differences were noted among the exposure intervals . Larval knockdown of T. confusum was also negligible (data not presented). After 7 d of exposure in all formulations, mortality did not exceed 30%, whereas after 10 d the highest mortality level was 34% at ED5-P  no immediate mortality was achieved for either of the stored product species tested. However, for Particle size is highly associated with the insecticidal efficacy of inert dusts. Many authors have emphasized on the importance of the particle size, a significant physical property of dust formulations, underlining that formulations of smaller particles may enhance the insecticidal efficacy on a variety of stored-product beetles ,37,38,392 nanoparticles are enhanced when added to liquid paraffin. In the present study, it was concluded that the susceptibility of A. fabae and S. oryzae was high, regardless of the addition of the primer or the size of silica nanoparticles, since no significant differences were recorded concerning the long-term effect. Notwithstanding, our results suggested that the use of paraffin on the net’s surface contributes to the reduction of fabrics’ shrinkage whereas it increases the rate of the particle deposition. Thus, the knockdown effect of A. fabae was increased when insects were exposed to ED5-P nets since in this case, the deposition of silica particles was the highest among all tested samples due to the addition of paraffin. In particular, it was shown that 99% of the adults were knocked down after six hours when exposed to the net coated with the ED5-P dust formulation, which consisted of the largest silica particles at the highest deposition rate.Peng et al.  stated t2-free nets were also affected by the presence of the net as opposed to the insects exposed to the dishes without net. Concerning the above, silica-treated nets could be applied on the vent openings of a greenhouse limiting the number of infestations due to greenhouse pests’ invasions, which occurs mechanically, i.e., without the use of the dust. A similar approach has been investigated in the case of stored product insects, with similar results regarding the detrimental effect of the net to the insect bodies [Silica particles should not be considered as the only lethal factor in our study, as mortality can be also attributed to the presence of the net itself. As shown in the results corresponding to the control treatments, insects exposed to the SiOt bodies ,20.S. oryzae and the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptrea: Bostrychidae) when exposed to a mixture of DE and abamectin. Our data show that, despite low initial effect, delayed mortality for the two beetle species was high, which clearly suggests that the exposed individuals were heavily affected by the presence of the dusts in their cuticles. As inert materials are slow-acting, it has been observed that mortality may take several days or even weeks to occur, even if the insects are in continuous contact with the dust particles [Although no immediate mortality was recorded for all the insect species tested, delayed mortality was high, reaching 100% even in the shortest exposure time (15 min). Similarly, Kavallieratos et al.  observedarticles ,44. NeveTo conclude, this study indicates that all of the nets tested here were effective for the control of aphids and less for beetles , despite variations in efficacy among treatments. Practically, the nets that are coated with inert materials can serve as a good alternatives to traditional pesticides in greenhouses but also in food and processing facilities, significantly moderating insect immigration. This technology can be adopted in a wide range of facilities, and also in food packaging, where the larval stage can penetrate with ease. At the same time, in a “real world” greenhouse scenario, immigration of aphids from the outside toward the plantation will force the insects to pass through the treated net, which is expected to be lethal, even if some aphids eventually pass successfully.


The immune dysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome is a rare disorder that most often manifests in the early stages of life. IPEX syndrome with a late onset, presenting with severe gastritis has rarely been reported.Two male adolescents presented with recurrent vomiting, severe malnutrition, and growth retardation due to severe gastritis.FOXP3 in the patients who were diagnosed with the IPEX syndrome.Esophagogastroduodenoscopy of the 2 patients revealed rare presentations of severe gastritis with multiple ulcers and stenosis of the pylorus. Next-generation sequencing revealed 2 novel variants in gene Both patients were treated with a high calorie formular enteral nutritional therapy. In addition, the pylorus of patient 1 was enlarged by balloon dilation, while patient 2 was treated with mercaptopurine and low dose prednisone.Symptoms and nutritional status of the patients improved after treatment.Chronic severe gastritis with stenosis of the pylorus could be an atypical manifestation of the IPEX syndrome. The use of next-generation sequencing is highly suitable for the diagnosis of atypical IPEX syndromes. The syndrome is characterized by severe enteropathy, chronic dermatitis, early-onset type I diabetes mellitus (T1DM), autoimmune thyroiditis, antibody-mediated cytopenia, and other autoimmune diseases. IPEX syndrome is caused by mutations in the forkhead box protein 3 (FOXP3) gene, which is located in the centromeric region of the X chromosome and encodes a key factor in human CD4+CD25+ regulatory T (Treg) cell development.,5The IPEX syndrome  is a rare immune disorder that most commonly presents in the early stages of life but can occur antenatallyFOXP3 gene sequencing, indicating various manifestations of IPEX syndrome.,7 IPEX syndrome patients who present with atypical symptoms have often been misdiagnosed before the correct diagnosis, or received the information late in life. An early diagnosis of IPEX syndrome could provide better treatment and improve patients’ quality of life. Here, we report 2 rare IPEX syndrome patients who presented with late-onset severe gastritis and pyloric stenosis, which has not been reported previously.Clinical features of the IPEX syndrome have previously been described, and most IPEX syndrome patients suffer from severe diarrhea during the first days of life. However, an increasing number of atypical cases have been diagnosed by 2This study was approved by the Children's Hospital of Zhejiang University, School of Medicine. Informed written consents were obtained from the patients and their parents for publication.33.1Helicobacter pylori (HP) infection.Patient 1, a 14-year 1-month-old, was the second child in his family, born at term with a birth weight of 3350 g after an uneventful pregnancy. The baseline information is shown in the Table FOXP3 gene, chrX:49107810, exon11, c.1280dupC, insertion, p.P427fs , and Treg/CD3+ expression was 1.98% (reference range: 2.49–6.61%). This indicated a decrease in expression of the FOXP3 protein on T cells. The patient was then diagnosed with the IPEX syndrome and prescribed mercaptopurine because of persistent gastritis. However, the patient experienced recurrent respiratory bacterial infections and therefore stopped the treatment of mercaptopurine. Due to the economic situation of the patient's family, HSCT was not an option. Despite the discontinuation of the medication, during a period of more than 3 years, the patient did not experience any symptoms. The Z score of height for age and body mass index (BMI) for age were both between -3 SD and -2 SD.He received anti-HP therapy, and a repeated HP test subsequently returned negative. However, the ulcers of the stomach did not recover. Because of the stenosis of pylorus, he received balloon dilation for four times in prior years. The patient's vomiting decreased after this treatment, and he began to gain weight with enteral nutrition therapy; however, the gastritis and ulcers of the stomach did not show any significant improvement. The latest balloon dilation was performed approximately 2 years ago. Because of the persistent gastritis and low immunoglobin levels, an immunodeficiency was suspected and he had whole exon sequencing (WES) examination. WES revealed a hemizygous mutation of the 3.2Patient 2, a 13-year 3-month-old boy, was born as the second child of his mother. He had a healthy 17-year-old sister. Patient 2 was diagnosed with an autoimmune liver disease 3.5 years before presentation to the authors and was treated with prednisone at a dose of 1.5 mg/kg per day, which was gradually decreased to 2 mg per day. The patient had taken prednisone 2 mg per day for more than 2 years with no detectable effect on liver function. However, the patient continued to suffer from watery diarrhea and vomiting for already 7 months and was diagnosed multiple ulcers of the stomach and chronic diarrhea in another hospital. In this facility, he was treated with omeprazole and supportive treatment; however, his condition did not improve.Z score of height for age was -2.69, and the Z score of BMI for age was -6.20. The cervical and inguinal lymph nodes were enlarged (with the largest being 1.5 cm × 1 cm in size), and he did not have hepatosplenomegaly or edema. The laboratory investigations showed a normal white blood count and mild anemia, as well as a slight reduction in albumin, while the liver function was normal. The lymphocyte subsets and serum immunoglobulin levels of the patient were all within normal range. The expression of autoimmune diseases related antibodies, described in the first case, were all negative. His levels of serum total triiodothyronine and free triiodothyronine were decreased due to malnutrition. EGD showed severe chronic inflammation, multiple ulcers in the stomach, and pyloric stenosis, which resemble with the first case , while also continuing to take prednisone (2 mg per day). The patient's vomiting drastically improved, while the gastritis did not get much better, as indicated by the EGD repeated 1 month later. Despite this, 8 months after the beginning the above-described treatment, the patient could tolerate a normal diet, and his 4FOXP3 gene, which encodes a member of the forkhead transcription factor family expressed primarily in CD4+CD25+ regulatory T cells, and leads to multiorgan autoimmunity in IPEX patients. Furthermore, other mutations in genes such as IL2RA, LRBA, CTLA4, STAT1 GOF, STAT3 GOF, STAT5b, TTC7A, TTC37, and DOCK8 are related to IPEX-like syndrome. The clinical presentations of IPEX syndrome are variable and typically involve severe enteropathy, T1DM, and eczema, while the symptoms often overlap with other distinct clinical manifestations, including those of hematologic disease, renal disease, hepatic disease, lymphadenopathy, and severe infectious disease. Diarrhea usually begins within the first few months of life in the majority of IPEX patients, and some patients have histories of food allergies. Histologically, nearly half of the patients have villous atrophy on small bowel biopsy. The immunoglobulin levels are generally normal, except for elevated Ig E levels in most IPEX syndrome patients. CD4+ and CD8+ lymphocyte subset counts are usually normal.IPEX is a primary immunodeficiency disease caused by mutations of the  However, the severity of symptoms is unpredictable and is not related to the mutation type. In addition, patients with the same mutations could have completely different phenotypes, which could reflect the complex intracellular interactions of FOXP3.More than 70 mutations have been revealed in IPEX syndrome.FOXP3 gene mutations, an increasing number of atypical IPEX syndrome patients have been identified. Some patients with IPEX syndrome have mild and recurrent courses, which can delay the diagnosis. Notably, some patients even have gastritis along with other immune diseases.,13 Here, we reported 2 patients with rare late-onset atypical IPEX syndrome who mainly presented similar symptoms of severe gastritis with pyloric stenosis. We previously reported an IPEX syndrome patient who had a similar presentation as these 2 patients. Villus atrophy was observed in the descending part of the duodenum, and pathological eosinophil infiltration was reported. The 2 patients had disease onset during adolescence without the typical enteropathy observed in most IPEX patients, and their EGD had similar findings with multiple ulcers throughout the stomach, especially around the sinus and the body of the stomach, along with pylorus stenosis. Patient 1 had HP infection at the first biopsy; however, after anti-HP treatment, the inflammation and ulcers did not show improvement. Moreover, the appearance of the EGD did not show a chronic infection caused by HP. Histology revealed an infiltration of multiple inflammatory cells, which did not resemble a HP infection. The serum IgG, IgA, and IgM levels in patient 1 were lower than the normal range, indicating immunodeficiency, which is not commonly observed in IPEX patients. Recently, several patients with hypogammaglobulinemia were reported; here, a functional B cell class switching defect was suggested as a possible explanation. Patient 2 was first diagnosed with autoimmune hepatitis in another hospital and was treated with prednisone; however, the histological investigation showed rapid deterioration, severe fibrosis, and atrophy of the liver. According to this presentation, autoimmune hepatitis could be one of the symptoms of IPEX syndrome. The mutation of patient 2 was not previously reported and was not validated by a study addressing the protein function. The level of Treg cells of this patient was normal. It has been reported that patients with missense and splicing mutations exhibit varying expression levels of the FOXP3 protein, and 2 patients with missense mutations even had markedly elevated percentages of FOXP3 expressing CD4+ cells. [10] According to the previously experienced symptoms and his comorbidity of other autoimmune disease, he was considered to be suffering from IPEX. To fully confirm this diagnosis, it would be good to perform a study on the FOXP3 protein function. Table FOXP3 gene in 3 of the reported IPEX syndrome patients with gastritis. Except for 1 patient we reported previously, the other 2 patients both had early disease onset and underwent gastrectomy. The patients reported here experienced a late disease onset. Supporting our diagnosis and intervention, the patients achieved remission under the medication therapy and supportive treatment. The stenosis of pylorus may be caused by the chronic ulcers in these 2 patients.With awareness of IPEX syndrome and recognition of ,17 Normally, the effect of T cell-targeted immunosuppressant (IS) is better than that of other forms of IS. The only cure for the disease is HSCT, and it is better to perform HSCT before more organs are affected. According to the latest multicenter study, the survival rate after IS treatment was comparable to that after HSCT. Although most of the symptoms of the 2 patients were drastically reduced in the 2 cases described in this article, their gastritis did not recover. The previously reported patient by our team underwent HSCT, his symptoms got dramatically better, and his gastritis fully remitted. Therefore, we suggest that HSCT may be the best choice for patients with severe gastritis.Regarding the treatment of the IPEX syndrome, there are no standard therapeutic strategies, but the main methods include immune suppression and HSCT.POXP3. However, the impact of these novel mutations was not confirmed by a functional study. Moreover, the mechanism that causes the gastritis in these patients remains unclear and requires further research.In summary, we have diagnosed 3 IPEX syndrome patients, 2 patients in the present report and 1 previously reported. The EGD of the 3 patients revealed similar extents of gastritis, which were not reported in the literature. In all 3 patients, next-generation sequencing was performed, which revealed 2 novel mutations of Altogether, according to our experience, chronic severe gastritis with stenosis of pylorus could be an atypical presentation of the IPEX syndrome and they may be a late-onset of the disease. Next-generation sequencing could help in the diagnosis of such atypical patients.YF participated in the coordination and interpretation of data and wrote the manuscript. JC participated in the study design and helped draft the manuscript. YL and JL collected the clinical data of the patients and interpreted the results of molecular analysis. All authors read and approved the final manuscript.Conceptualization: Jie Chen.Data curation: Youyou Luo, Jingan Lou.Project administration: Youhong Fang.Resources: Youyou Luo, Jingan Lou.Supervision: Jie Chen.Writing – original draft: Youhong Fang.Writing – review & editing: Jie Chen.The funding number appeared incorrectly as LQ19H030005 and has been corrected to LQ19H030010.


Biological therapies are widely used for the treatment of ulcerative colitis. However, only a low proportion of patients achieve clinical remission and even less mucosal healing. There is currently scarce knowledge about the early markers of therapeutic response, with particular regard to mucosal healing. The aim of this prospective study was to evaluate the role of fecal calprotectin (FC) as early predictor of mucosal healing.A prospective observational study was conducted on patients with ulcerative colitis, who started biological therapy with infliximab, adalimumab, golimumab, or vedolizumab at our center. All patients underwent colonoscopy, performed by 2 blinded operators, at baseline and week 54 or in case of therapy discontinuation because of loss of response. FC was assessed at baseline and week 8 and evaluated as putative predictor of mucosal healing at week 54.P < 0.0001). Sensitivity, specificity, positive predictive value, and negative predictive value were estimated to be 75%, 88.9%, 86.6%, and 75.5%, respectively, based on a cutoff of 157.5 mg/kg.We enrolled 109 patients, and 97 were included in the analysis. Twenty-six patients (27%) experienced loss of response. Over 71 patients (73%) with clinical response at week 54, clinical remission was obtained in 60 patients (61.9%) and mucosal healing in 45 patients (46.4%). After 8 weeks of treatment, FC predicted mucosal healing at week 54 (The present study suggests that FC assessment after 8 weeks of treatment with all the biological drugs could represent a promising early marker of response to therapy in terms of mucosal healing. Ulcerative colitis (UC) is a chronic relapsing disease that involves the colorectal mucosa. Over the years, the therapeutic target has been upgraded from the resolution of symptoms to deep remission to prevent relapses and complications. With this goal, the STRIDE consensus suggested that the primary therapeutic target to be achieved in patients with UC is both clinical/symptomatic  and endoscopic remission . In this4β7 integrin expressed in a subset of T lymphocytes, preventing their endothelial adherence and migration toward the bowel mucosa (The anti–tumor necrosis factor (TNF) monoclonal antibodies infliximab (IFX), adalimumab (ADA), and golimumab (GOL) have greatly improved treatment expectations in patients with UC refractory or intolerant to standard treatments ,6, allowl mucosa . It is al mucosa –13. Howel mucosa ,15.The early prediction of response to biological therapies is one of the most important challenges for the clinicians. In this respect, the identification of a reliable biomarker would allow to optimize the management of patients with UC, improving the cost-effectiveness of biological therapies .Calprotectin is a 36-kDa calcium- and zinc-binding protein, which represents approximately 60% of soluble proteins of granulocyte cytoplasm . Fecal c5 mg/kg i.v. at weeks 0, 2, and 6 and then every 8 weeks for IFX;160 mg at week 0, 80 mg at week 2, and 40 mg subcutaneously starting from week 4 for ADA;200 mg at week 0, 100 mg at week 2, and then 50 mg or 100 mg (if patient's weight was >80 kg) subcutaneously every 4 weeks for GOL; and300 mg at weeks 0, 2, and 6 and then every 8 weeks for VDZ.Consecutive adult patients with UC, who underwent biological treatment with IFX (biosimilar CT-P13), ADA, GOL, or VDZ at the Pisa University Hospital from October 2016 to March 2018, were enrolled prospectively. Patients had to meet the indications for treatment with TNF antagonists or VDZ and agreed to participate in the study signing informed consent. Patients enrolled in this prospective study the day of the first dose of the biological treatment and were evaluated every 8 weeks for 54 weeks. We excluded from the analysis patients who were primary nonresponders to anti-TNF or VDZ therapy, defined as a decrease in Full Mayo Score ≤2 or a lack of improvement of rectal bleeding at week 8. Patients treated concomitantly with immunosuppressants were excluded as well. Treatment regimen was as follows:Treatments with IFX and VDZ could be escalated after week 6 to a monthly regimen, whereas ADA treatment could be escalated to a weekly regimen, in case of a mild worsening of UC after a careful medical evaluation, which included testing for antidrug antibodies. We recorded general patient features, such as age, sex, age at diagnosis, previous immunosuppressant, and previous anti-TNF treatment; moreover, baseline disease extension, concomitant corticosteroid treatment, FC, Partial Mayo Score (PMS), and MES were recorded.A team of clinicians performed clinical evaluations, defining clinical status by PMS. At each time point, FC and C-reactive protein (CRP) were assessed. FC was determined using the ELISA Bühlmann fCAL Turbo , known to perform with high sensitivity and specificity .Colonoscopy was performed at baseline and after 54 weeks of treatment to evaluate the therapeutic outcome in terms of mucosal healing. In case of loss of response (after at least 14 weeks of treatment) with a subsequent discontinuation of therapy, the therapeutic outcome was assessed on the basis of endoscopy and clinical examination at discontinuation. All colonoscopies were performed by 2 operators expert in the evaluation of MES, blinded to the results of FC and CRP. Mucosal healing was obtained in case of MES ≤1. Clinical remission was defined with a PMS ≤1. A value of CRP <0.5 mg/dL was considered normal.The primary endpoint was to evaluate whether FC after 8 weeks of treatment could be used as a possible predictive marker of mucosal healing or clinical remission after 54 weeks of treatment in patients with UC treated with anti-TNF or VDZ in monotherapy. The secondary endpoint was to evaluate the role of CRP after 8 weeks as a possible marker of mucosal healing or clinical remission after 54 weeks of treatment.To evaluate the primary endpoint, we set power, α error, and effect size at 95%, 5%, and 60 mg/kg as delta FC between the responders and nonresponders group in terms of mucosal healing, respectively. At least 40 patients per group were needed in both the groups. According to real-life studies, we expected a rate of primary nonresponders of 20% ,25 and aCategorical data were described by frequency (%) and quantitative data by mean (SD) or median (interquartile range). To compare FC and CRP with mucosal healing and clinical remission, the Mann–Whitney test (2 tailed) was applied. The receiver operating characteristic (ROC) curve analysis (using a nonparametric test) was performed to calculate the best cutoff in predicting either mucosal healing or clinical remission. Sensitivity, specificity, positive predictive value, and negative predictive value were also calculated. A comparison between drugs in terms of FC levels was performed by the Kruskal–Wallis test. Significance was fixed at 0.05. All analyses were performed using SPSS technology (v.25).This study was conducted in full compliance with the 1975 Declaration of Helsinki and was approved by the Ethical Committee of the Pisa University Hospital (CEAVNO). All of the included patients signed informed consent forms, approving the use of their anonymized data for research purposes.P = 0.332).During the enrollment period, 109 patients with UC started a biological treatment. We excluded 9 primary nonresponder patients and 3 patients treated concomitantly with azathioprine, thus including 97 patients in the analysis. Twenty-seven patients were treated with IFX, 20 with ADA, 18 with GOL, and 32 with VDZ. Their general characteristics are displayed in Table During the follow-up, 26 patients (27%) experienced loss of response : 10 at week 16, 8 at week 24, 3 at week 32, 2 at week 40, and 3 at week 48. Over 71 patients (73%) with clinical response at week 54, clinical remission was achieved in 60 patients (61.9%) and mucosal healing in 45 patients (46.4%). Clinical remission rate was 44.4% in patients treated with IFX, 65% with ADA, 61.1% with GOL, and 75% with VDZ, whereas mucosal healing rates were 29.6%, 60%, 38.9%, and 56.2%, respectively.P = 0.850 and P = 0.665, respectively). However, after 8 weeks of treatment, both FC and CRP correlated with clinical remission , whereas only FC correlated with mucosal healing (P < 0.0001), as shown in Table FC at baseline did not correlate with either mucosal healing or clinical remission at week 54 (http://links.lww.com/CTG/A285). FC levels were similar in patients treated with the 4 different drugs, without significant differences evaluated by means of the Kruskal–Wallis test (P = 0.332 at baseline and P = 0.147 at week 8).To calculate the best cutoff of FC at week 8 related to mucosal healing, a ROC analysis was performed Figure , and senThis study was conceived to identify an early marker of mucosal healing in a prospective cohort of patients with UC treated with biological therapies, and our results support the view that FC could be regarded as a reliable tool in this perspective. To predict mucosal healing during biological treatment with an accurate, rapid, and noninvasive biomarker is currently one of the main goals of the clinicians in the UC setting. Biomarkers of inflammation will attain growing importance in the clinic because we strive for more effective and cost-effective strategies to treat patients with IBD .In our cohort, anti-TNF and VDZ were confirmed as good options for patients with UC in terms of efficacy: in our cohort, 62% of patients achieved clinical remission at 54 weeks, and even 46% displayed mucosal healing. IFX is the most studied drug in the UC setting, and its mucosal healing rate has been reported to range from 30% to 45% . ADA resThe main issue in the evaluation of mucosal healing is the time over which the clinician expects its achievement. In our opinion, an appropriate timing to evaluate the mucosal effect of a biologic drug in patients with UC should be at least 1 year, provided that patients display a clinical response to treatment: in the perspective of a treat-to-target strategy, we should allow enough time to drug therapy to express its full potential because if the target is not reached, the treatment should be optimized or changed. In this respect, because we have waited for 54 weeks to perform the follow-up endoscopy, we could obtain high rates of mucosal healing, even with subcutaneous drugs.Loss of response was observed in 27% patients. These findings are in line with the current literature, where loss of response was reported to range from 23% to 46% 12 months after anti-TNF initiation , althougIn our series, FC levels at week 8 showed a strong correlation with mucosal healing and clinical remission rates after 54 weeks of treatment. Various studies on patients with UC treated with anti-TNF, in particular IFX and ADA, have shown that a rapid decrease in FC levels over the initial weeks is positively associated with the rates of clinical remission and mucosal healing. Molander et al.  observedWith regard to mucosal healing, a prospective multicenter study conducted in Belgium showed that FC <50 mg/kg at week 10 has a very good correlation with mucosal healing  in patients with UC treated with IFX . This fipost hoc analysis of the GEMINI I trial ; moreover, all endoscopies were performed by 2 operators blinded to FC and CRP values. Of note, other important data are provided by our study because this is the first prospective real-life investigation where FC levels have been proven to have a role in the prediction of mucosal healing at 54 weeks in VDZ-treated patients. Moreover, the predictive role of FC is currently proposed only in a few studies conducted during IFX and ADA treatments, mainly with retrospective design, whereas the present prospective study has shown that its use is reliable for all anti-TNF drugs. Furthermore, the statistical strength of the correlation between FC at 8 weeks and mucosal healing, regardless of the mechanism of action of the drug, could allow to conclude that this fecal biomarker is useful as early predictor of response to all the biological therapies available in the UC setting, and therefore, its wider use should be encouraged.The main limitation of this study is the lack of a multiple evaluation of FC: its levels can vary day-to-day, by the time of day, and even within the same bowel movement . MoreoveIn conclusion, we have documented an early predictive value of FC in patients treated with all the biological therapies currently available for the treatment of moderate/severe UC. This finding encourages the use of FC to obtain an early evaluation of treatment outcome. At present, this study suggests that patients with higher levels of FC at week 8 should be tightly monitored, regardless of clinical activity, likely anticipating colonoscopy, to optimize their treatment or even switching to another biological drug. Moreover, the present data pave the way to future investigations aimed at assessing more conclusively the value of FC levels in monitoring the therapeutic management of biological therapies in patients with UC.Guarantor of the article: Lorenzo Bertani, MD.Specific author contributions: L.B.: study concept and design, data collection, writing of the manuscript, and approving final version; C.B.: writing of the manuscript and approving the final version; M.G.M., L.C., E.A., G.T., G.B.S., F.Z., and F.C.: data collection and approving the final version; N.d.B. and M.B.: writing of the manuscript and approving the final version; R.M.: statistical analysis and approving the final version; S.M.: writing of the manuscript and approving the final version; and F.C.: study concept and design, writing of the manuscript, and approving the final version.Financial support: This paper has not required funding in terms of grants, equipment, and drugs. No supportive foundations have funded this article.Potential competing interests: F.C. received board membership honoraria from Takeda, Janssen, and Amgen and lecture fees from Abbvie, Takeda, Zambon, Ferring, Diasorin, Otsuka, and MSD; none of these honoraria had influence on this paper. All other authors have no potential conflict of interest in presenting this paper.✓ FC is widely used as a surrogate biomarker of endoscopic activity in UC.✓ A reliable biomarker able to predict the therapeutic outcome to biological therapies is still missing.✓ An early assessment of FC is able to predict mucosal healing after 54 weeks of treatment.✓ This is the first prospective study where FC showed a potential role as a prospective biomarker in predicting the therapeutic outcome in all biological therapies in UC.✓ A simple ELISA test could be inserted in clinical practice to have an early prediction of treatment outcome and, possibly, guide treatment decisions.