Escherichia coli culture or HeLa cell culture. Results indicated that the piezoelectric biosensor with an annular microelectrode meets the requirements for the real-time detection of E. coli or HeLa cells in culture. Moreover, this kind of piezoelectric biosensor is more sensitive than the sensor with an interdigital microelectrode. Thus, the piezoelectric biosensor acts as an effective analysis tool for acquiring online cell or microbial culture information.Biological information is obtained from the interaction between the series detection electrode and the organism or the physical field of biological cultures in the non-mass responsive piezoelectric biosensor. Therefore, electric parameter of the electrode will affect the biosensor signal. The electric field distribution of the microelectrode used in this study was simulated using the COMSOL Multiphysics analytical tool. This process showed that the electric field spatial distribution is affected by the width of the electrode finger or the space between the electrodes. In addition, the characteristic response of the piezoelectric sensor constructed serially with an annular microelectrode was tested and applied for the continuous detection of  In recent years, piezoelectric biosensors ,2 have bE. coli culture or HeLa cell culture. The new piezoelectric biosensor acted as an effective analysis tool for acquiring online cell or microbial culture information.However, the IDME structure is composed of two parallel groups of tiny finger electrodes inserted with each other. Two main electrodes, which connect the fingers of the IDME, exist at the outer edge. Therefore, the electric field spatial distribution on the surface of the IDME is asymmetric upon operation ,8,9,10. The experimental instruments used in this work include an AT-cut 9 MHz piezoelectric quartz crystal , a piezoelectric biosensor connected serially with a microelectrode (self-developed), whose structure is shown in Escherichia coli O157:H7, and HeLa cells were taken from the College of Biology, Hunan University, China. LB medium, DMEM medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin, and EDTA were purchased from Gibco, USA. The PBS buffer solution was composed of 8.1 mM Na2HPO4 + 136.7 mM NaCl + 2.7 mM KCl + 1.5 mM KH2PO4 (pH = 7.4). All other chemicals were of reagent grade. Ultrapure water (RN 18.2 MΩ/cm) was used throughout the experiment.Yeast extract culture detection of microorganism culture medium used with an series piezoelectric quartz crystal (SPQC) instrument was proposed in our laboratory. BP 212-37 positive UV Photoresist , potassium chloride . 2 glass  was used as substrate of the annular microelectrode. We optically polished the glass surface and cleaned it with a piranha solution (1:3 (v/v) 30% H2O2/H2SO4) for 5 min. Then, the glass surface was sonicated in acetone and double-distilled water (each for 3–5 min) individually and dried under a nitrogen flow [The process of the annular microelectrode is shown in gen flow ,14,15. Agen flow . After e(water) = F0. While replacing the water with KCl standard solution at different concentrations, the piezoelectric resonance frequency was recorded as F(KCl) = Fi . Thus, the sensor’s response frequency shift could be obtained as follows: ΔF The annular microelectrode was connected to the piezoelectric quartz crystal sensor in series. The frequency shift of the sensor was measured at different concentrations of a standard KCl solution. The detailed procedure is as follows: When the sensor’s microelectrode was immerged in purified water , the piezoelectric resonance frequency was recorded as FE. coli culture, 5 mL of LB medium was added to the culture-detection cell, and 1 mL of suspension containing 105 cfu/mL of E. coli was mixed gently with the medium. Then, the annular microelectrode was inserted into the culture-detection cell. After connecting serially with the piezoelectric sensor detecting system, the E. coli suspension was incubated at 37 ± 0.2 °C. The response frequency shift was recorded automatically by the piezoelectric biosensor.For the detection of 4 cell/mL was seeded in the culture-detection cell and incubated in a carbon dioxide incubator under 5% CO2 at 37 °C and saturated humidity. The response frequency shifts, which indicated HeLa cell growth in real time, were recorded automatically by the sensor.For the monitoring of HeLa cell growth, the annular microelectrode was assembled at the bottom of the culture-detection cell, which was used as the culture substrate. An amount of 1.5 mL of HeLa cell suspension at a concentration of 1 × 10−12 F/m), and εr is the relative dielectric constant of the electrolyte solution.The electric field spatial distributions of microelectrodes with different finger or gap widths in the medium were separately simulated under static conditions in three-dimensional (3D) space using COMSOL Multiphysics coupling analysis software . The terThe strength of the electric field spatial distribution in the simulation domain was described using the electric potential (E):Equation (2) was used to calculate the charge conservation equation for the electric potential using the given spatial distribution of the electric charge. The equation is used primarily to model charge conservation in dielectrics under static conditions. The 3D simulation of the microelectrode was performed using these equations and the COMSOL program. Simulation results are showed in In the simulation of the microelectrode, on the upper spatial domain of the annular microelectrode, a closer distance between the microelectrode and the electrode surface, represents a greater change in the electric potential. Near the surface of the microelectrode, the electric line of force field intensity was very dense and displayed an annular shape, while the electric field weakened with increasing height of the electrode. Based on the simulation of the IDME, the electric field concentrated under the upper spatial domain of the interdigital electrode. However, the electric field intensity of the main electrode was stronger on the two sides and two terminals than those of the interdigital electrode, as influenced by the interdigital geometric structure. It is possible that, in the static field, the skin effect of the charge distribution resulted in the uneven distribution pattern of the electric field on the entire electrode surface. The mass transfer of electrolytes in the solution was then carried out in a large range. This phenomenon resulted in the poor transfer efficiency and electrochemical reaction sensitivity of the IDME sensor compared with the annular microelectrode sensor.The geometric parameters of the annular microelectrode are the band width, the gap size, and the number of annuluses of the electrode. We employed the simulation software to simulate the variation in the equivalent capacitance and equivalent resistance with those of the geometric parameters of the microelectrode.The annular microelectrode shown in A was choIn addition, the real capacitance and impedance of the annular microelectrode were measured using a 4192 impedance Analyzer at 9 MHz. As shown in In order to investigate the influence of the number of annuluses on the electric parameters, we designed the annular microelectrode with two, three, four, and five pairs of annuluses (every electrode pair with a 50 µm of electrode band width and 50 µm gap). The capacitance and resistance of the annular microelectrode were measured using the 4192A at 9 MHz. Results are shown in Annular microelectrode piezoelectric biosensors were constructed using two, three, four, and five pairs of annulus microelectrodes. Their piezoelectric frequency characteristics were studied at different concentrations of KCl solution. Results are shown in C0, Lq, Cq, and Rq represent the piezoelectric crystal static capacitance, the motional inductance, the motional capacitance, and the motional resistance, respectively. Cs is the equivalent capacitance of the annular microelectrode. Rs is the equivalent resistance of the solution. The equivalence circuit model showed the total complex impedance, Z, using the following equation:ј is the imaginary unit; ω is the angular frequency.R is the real part of Z; X is the imaginary part of Z; Ј is the imaginary unit. According to the oscillation theory of the piezoelectric resonant phase shift condition, the phase shift of the feedback must be –θ and The response frequency shift of the annular microelectrode piezoelectric biosensor was theoretically deduced as follows. For the convenient calculation, the equivalent circuit of the microelectrode and the piezoelectric crystal in series were simplified as shown in A is defined by the equation By substituting the real and the imaginary parts of Equation (3) in Equation (5), Equation (5) can be transformed into Equation (6), as follows:Because For the annular microelectrode piezoelectric biosensor under the oscillating condition, Equation (7) must be differentiated as follows:F0 is the fundamental frequency of the piezoelectric crystal, C0 is the static capacitance of the crystal in Equations (9) and (10). They were constants. RS and CS are the initial resistance and the initial capacitance of the annular microelectrode, respectively. According to Equations (9)–(11), the frequency shift of the annular microelectrode piezoelectric biosensor was not only affected by the initial resistance and the initial capacitance of the annular microelectrode, but also by the changes in resistance and capacitance. The equivalent electric parameters of the sensor were detected using an HP-4192A Impedance Analyzer, with Therefore, as is evident from E. coli and HeLa cell in real time. Results are shown in E. coli or HeLa cells. However, the maximal frequency shift signal of the annular microelectrode sensor was larger than that of the IDME sensor. This finding suggests that the piezoelectric biosensor series with the annular microelectrode possesses more response sensitivities. The microelectrode piezoelectric biosensor was employed to monitor the growth of In this study, an annular microelectrode was designed and a novel piezoelectric biosensor in series was also constructed. Multi-physics field simulation software was employed to simulate and to calculate the electric field strength of the annular microelectrode. The simulated and calculated results suggested that the annular microelectrode has a very strong intensity electric field. Moreover, the electric field of the annular microelectrode exhibited an annular shape on the surface. The mass transfer of the electrolyte conducted within a large scope and the mass transfer efficiency between the microelectrodes were high because of the electric field shape distribution. Moreover, the influence of the geometric size of the annular microelectrode on the electrical parameters and the response frequency shift characteristics were investigated. The annular microelectrode piezoelectric biosensor, compared to the IDME piezoelectric biosensor, was more sensitive to the changes in the electrical parameters and more effectively monitors the growth of microorganism or mammalian cells in real time. Therefore, the piezoelectric biosensor series with an annular microelectrode is a promising detection method for detecting both microorganism with excellent sensitivity.


The article presents a proposal of a model of fatigue destruction of hydrophobised ceramic brick, i.e., a basic masonry material. The brick surface was hydrophobised with two inorganic polymers: a nanopolymer preparation based on dialkyl siloxanes (series 1–5) and an aqueous silicon solution (series 6–10). Nanosilica was added to the polymers to enhance the stability of the film formed on the brick surface. To achieve an appropriate blend of the polymer liquid phase and the nano silica solid phase, the mixture was disintegrated by sonication. The effect of the addition of nano silica and sonication on changes in the rheological parameters, i.e., viscosity and surface tension, was determined. Material fatigue was induced by cyclic immersion of the samples in water and drying at a temperature of 100 °C, which caused rapid and relatively dynamic movement of water. The moisture and temperature effect was determined by measurement of changes in surface hardness performed with the Vickers method and assessment of sample absorbability. The results provided an approximate picture of fatigue destruction of brick and hydrophobic coatings in relation to changes in their temporal stability. Additionally, SEM images of hydrophobic coatings in are shown. The process of destruction of porous building materials caused by changes in temperature and moisture is a major problem in modern materials science reported in numerous works ,2,3. TheIn line with other studies ,13, placPlastic strains manifest themselves as slip lines and slip bands, as shown in The slip line can be defined as a slip trace on the free surface in one slip plane. In turn, slip bands are made up of slip lines formed along the planes through dislocation.As specified by Fic et al., slip bands can be defined as extrusions and intrusions . ExtrusiAccording to several authors, micro-cracks are initiated and develop in slip bands as a result of concentrated strains (stress) and accumulation of energy in the planes between them ,25,26. TIn order to mitigate the occurrence of destructive processes in construction materials such as bricks, stone, or concrete, various impregnates and hydrophobisation are commonly employed solutions ,31. As rIn a nanopolymer-hydrophobised ceramic brick, the surface layer is reinforced by the formation of a surface film , in whicIn the present study, we analysed samples of ceramic brick hydrophobised by immersion in a nanopolymer for 15 s, which resulted in formation of a thin film with a rough surface on the brick, as shown in According to numerous studies performed by, among others by Hazlett, Baldan, Kuczmaszewski, Rudawska, incorporation of a nanopolymer into the brick surface primarily occurs through mechanical adhesion, which is affected by the rheological properties of the impregnate, roughness and structure of the hydrophobised surface, and the thickness of the formed layer ,37,38,39Other factors determining the structure of the film and its effectiveness in prevention of water penetration include chemical (in particular hydrogen) bonds and Van der Waals forces between the molecules of the polymer and the hydrophobised material, as described by Fic et al. and Van Oss et al. ,43.The glazed film-porous system is influenced by cyclic fatigue loads. Consequently, deformations arise on the surface; they initially cause damage to the surface layer of the film and induce formation of micro-cracks and scratches, thereby promoting water penetration into the material . SecondaThe article presents the destruction processes arising from low-cycle fatigue on the example of a ceramic brick hydrophobised with nanopolymers. The study carries on with the topic described in several papers ,31,35, pThe present research consisted of three stages of examinations: characteristics of ceramic bricks and formulations, method for sample preparation and test methods, and finally determination of the parameters of hydrophobised bricks.The fatigue analyses were performed in 10 series, each comprising 6 samples excised from the ceramic brick.ρ = 2.55 g/cm3, apparent density aρ = 1.42 g/cm3, open porosity oP = 22.12%, and total porosity P = 31.51%.Determination of the physical parameters was conducted according to PN-EN 1936:2010 and PN-EN 1389:2005 ,49. Dens-−3 Pa·s;viscosity η = 1.08 × 10-−3 N/m;surface tension σ = 23.51 × 10-surface tension-to-viscosity ratio η/γ = 21.77;-ρ = 0.80 g/cm3.density at 20 °C—Oligomeric dialkyl siloxanes characterised by the following physical parameters:-preparation-water ratio 1:8;-−3 Pa·s;viscosity η = 0.98 × 10-−3 N/m;surface tension γ = 77.24 × 10-surface tension-to-viscosity ratio η/γ = 78.82;-ρ = 1.03 g/cm3 [density at 20 °C—03 g/cm3 .Aqueous methyl silicone resin in potassium hydroxide characterised by:-−3 Pa·s;viscosity η = 0.89 × 10-−3 N/m;surface tension γ = 72 × 10-surface tension-to-viscosity ratio η/γ = 80.90;-ρ = 0.99 g/cm3.density at 20 °C—Parameters of water for comparison:The samples were dried to constant weight, weighed, and hydrophobised with preparations based on the following:The excised brick samples were hydrophobised by full immersion in appropriately prepared polymer solutions based on small-molecule dialkyl siloxanes and/or silicones.Series of samples used in the investigations and the mode of preparation thereof (with the formula of the hydrophobising preparation) are shown in The disintegration of the solution was carried out with the use of a stationary laboratory homogeniser with a 400-W ultrasonic processor UP400 . The device has been certified in accordance with DIN-EN ISO 9001.All samples were stored for 14 days in laboratory conditions at a temperature of 20–22 °C and 50%–55% air humidity.Sonication was applied as one of the effective methods for changing polymer viscosity and surface tension. It was also an initiator of sonochemical reactions between the molecules of the polymer and the filler . ChangesThe fatigue of the ceramic bricks was achieved via a cyclic alternating process of drying of the samples at a temperature of 100 °C until maximum saturation. Surface hardness was examined after 10, 20, 25, 50, and 60 cycles. Simultaneously, the samples were weighed after each cycle to determine changes in their absorbability induced by the modelled low-cycle fatigue.The investigations consisted in measurements of the hardness of the surface of the hydrophobised samples from all the series with the Vickers method using the Zwick–Roell device at a load range of 0–200 N . The performed measurements were automatically read based on the indentation of a right square-based pyramid applied to the material surface. Hardness was defined as the ratio of the force applied to the sample surface to the surface of the indentation. For each sample, 25 measurements of hardness were performed at a base load of 10 N.Analysis of the distribution of the hydrophobic coating in the pores of the ceramic brick in SEM was carried out. The observations were compared with the images of the standard brick.The method for preparation of the samples for scanning microscopy analyses excludes the possibility of formation of defects related to the sample surface and is considered non-destructive to the microstructure of the material or its coatings. Sometimes defects appearing on the sample surface are generated by the beam of energy applied to the sample. However, this is a clear optical effect, which can be observed on a computer screen. Therefore, low-energy systems generating a low vacuum and a low energy beam should be used. Such research methodology was adopted during the analyses of the ceramic bricks presented in the articleThe surface hardness of the analysed samples was assessed using a fragment of a non-hydrophobised ceramic brick as a standard. Measurement of the surface hardness of samples containing a sonicated and silica-supplemented nanopolymer solution revealed significant changes in the physico-mechanical properties of the analysed surfaces. The results of the measurements of the surface hardness  are presented in The results of the changes in the absorbability of the series induced by hydrophobisation and sonication are shown in There were correlations between the hardness and absorbability of the standard bricks (Series 0) and those with the modified nanopolymer (Series 1–5). A linear regression model with a single input variable was used and the results are presented in A more complex experimental model with two variables is proposed below . The secMicroscopic images showing the structure of the standard brick before hydrophobisation are shown in The investigation results indicate that the adopted model of fatigue tests in the hydrophobising coatings of ceramic materials can be successfully used for determination of the effectiveness of the coatings. Cyclic application of high temperature (in the range of 0–100 °C) and saturation of brick pores with water efficiently models the work of these elements exposed to prevailing weather conditions. The two parameters, i.e., surface hardness and absorbability, adopted for comparison as the basic factors characterising physico-mechanical changes in the analysed materials, are appropriate indicators of the effectiveness of the hydrophobising preparation applied, which are easy to examine and assess. The fatigue tests have demonstrated a positive influence of the addition of silica nanoparticles on these parameters in terms of the capacity of coating reorganisation and reconstruction the during the fatigue cycles. Obviously, ceramic materials are destroyed over time by temperature and humidity and the process is largely related to their porous structure. However, a certain degree of resistance of these materials to deformations leading to formation of extrusions and intrusions and, consequently, scratches and cracks resulting in degradation of the material can be achieved. In the sonochemical reaction initiated by ultrasonic energy, large silicone molecules are not easily incorporated into nanosilica molecules, which do not penetrate deep into the silicone internal structure but remain in its upper layer. This is reflected in reinforcement of the layer and higher susceptibility to damage caused by formation of intrusions and extrusions, which is associated with its more fragile structure. The incorporation of nanosilica into silicone chains is also influenced by attachment of alkyl groups to silicon atoms, which impair the formation of hydrogen bonds.Nanosilica is incorporated as an element of the siloxane chain, and penetration of the new polymer into the pores of ceramic bricks occurs as well as cross-linking of the hydrophobising coating in the material structure. Consequently, the film is more flexible, which is reflected in reduction of its mechanical strength due to the plasticity of the deformations.The investigation results indicate that surface hardness and changes therein are closely associated with the type and method for preparation of the hydrophobising preparation. In samples hydrophobised with the dialkyl siloxane preparation, the hardness value declined along the number of the fatigue cycles. In turn, samples hydrophobised with the methyl silicone resin exhibited an initial increase in the hardness (with the exception of the Series 10 samples) and a decline in its value after 20 cycles. The highest initial hardness value (13.33 HV) was noted in the Series 5 samples; in comparison, the highest surface hardness value in samples hydrophobised with the aqueous silicone solution was noted in Series 8 (11.73 HV). The rate of the changes occurring in the hardness, i.e., the mechanical strength of the samples, was varied. There was a steady 50% decrease in the hardness of the ceramic bricks after 60 cycles; however, this relationship was not obvious in the case of the hydrophobised samples . In eachNeither the siloxane coating nor the silicone coating lose their properties from the beginning of fatigue tests. In turn, one can observe their capability of cyclic reinforcement and sealing of the structure, damage to the coating delaying degradation, and cross-linking of polymer chains on the brick surface, which is enhanced up to a certain moment. In extreme cases in Series 5, the ability of the coating to protect the material against water penetration was stabilised. Consequently, sample absorbability after 60 cycles was only 1.88%; furthermore, the samples exhibited relatively high surface hardness, compared with the final hardness of the non-hydrophobised brick. The mean absorbability of the non-hydrophobised bricks was ca. 14% at the beginning of the investigations. It gradually increased during the consecutive cycles and reached 17.76% after 60 cycles . The lowMaterials with no hydrophobic coating and those hydrophobised with the non-modified polymer were characterised by a steady absorbability increase at prolonged impregnation, which was confirmed in other studies reported by Barnat-Hunek ,32. TherR2 = 0.855.There were correlations between the hardness of the polymer coating and the brick absorbability before, during, and after the fatigue cycles in the case of the bricks hydrophobised with the nanopolymer (Series 1–5) . CorrelaThe simple experimental models with one input variable have confirmed that surface hardness can be a measure of brick absorbability after hydrophobisation without and with application of silica modification. No similar correlations were found for Series 8–10 hydrophobised with soluble silicon. Modification of the polymer with nanosilica by sonication resulted in an absorbability decline in the initial 10 or 20 fatigue cycles followed by damage to the polymer coating caused by material fatigue, which led to increased absorbability, while the material hardness decreased linearly from the beginning of the investigations . This deNext, we presented models  showing The structure of the standard brick exhibited irregularities, fissures, and numerous pores with varied diameters . This inMacromolecular siliconates  Series 6After sonication and mixing with 0.5% silica, the water-dilutable preparation formed a tighter coating, which completely covered the entire brick structure  Series 8All polymers based on small-molecule dialkyl siloxanes with 0.5%, 1%, and 1.5% nanosilica addition coated on the brick samples produced a continuous and evenly distributed film with no discernible scratches or cracks. The non-modified preparation in Series 1 formed a vapour permeable silicon coating composed of tiny, sharp polysiloxane gel plates. These properties of the silicone resin should ensure good water resistance and hydrophobisation efficiency, which was confirmed by the analysis of absorbability, which exhibited a very low value of 0.84%. Similar observations were reported in the article by Barnat-Hunek et al. .The coating containing dialkyl siloxanes with nanosilica is characterised by a fine-pore microstructure and is composed of very small spheres forming an excessively dense gel, which fully covers the mineral components and pores in the brick. The distribution of nanosilica-modified preparations reduces their effectiveness in comparison with the standard preparation (Series 1). The absorbability of the Series 1 brick was by 89%–91% lower than that of brick with the modified coating (Series 3–5). However, as in the case of the methyl silicone resin in Series 8, the absorbability value in Series 5 after 60 fatigue cycles was by 60.2% lower than that in Series 1. No such absorbability decrease was noted in the Series with 0.5% and 1% nanosilica. The excessive density of the polysiloxane gel formed in the structure of the material prevents its precise adhesion to all minerals contained in the brick. It can be assumed that voids that are not fully covered by the thin layer of the hydrophobic coating are formed. This is reflected in the high absorbability, compared with that of the non-modified hydrophobic coating (Series 1). In turn, compared with the standard non-hydrophobised brick, the absorbability of these bricks is by 40% lower.The ineffectiveness of the dense, tight hydrophobic coating was reported in other articles ,32. The (a)Increased mechanical and fatigue strength, e.g., resistance to damage induced by the tests, was achieved by the coating based on the aqueous silicon solution, which is associated with the chemical traits of silicones and the physical properties of the film.(b)The sonochemical reaction proceeds more rapidly and is more effective in the case of incorporation of silica molecules into the internal structure of dialkyl siloxane-based nanopolymers. This is closely related to the size of siloxane molecules and the type of substituents in the main chain .(c)To compare conclusions (a) and (b), the fragility of the silicone coating makes it more susceptible to cracking, especially under the effect of the temperature gradient. The siloxane coating, which is more susceptible to deformation, simultaneously exhibits greater flexibility and higher susceptibility to roughness changes and formation of intrusions and extrusions; hence, it does not crack. Therefore, depending on the preparation applied, the image and course of the coating destruction vary.(d)A very interesting phenomenon of secondary reorganisation of the hydrophobising coating structure caused by water movement in brick pores and capillaries caused by temperature changes was noticed. This phenomenon is highly advantageous from the point of view of the effectiveness of the hydrophobising agent formula.(e)There is no close relationship between the amount of the silica filler added and the efficiency of hydrophobisation in terms of changes in the surface hardness and absorbability of the tested samples.(f)Siliconates and small-molecule dialkyl siloxanes, which do not seal the structure and do not impair vapour and gas diffusion, form a relatively well-distributed coating in the brick structure. Dialkyl siloxane-based coatings modified with nanosilica form an excessive-density layer, which negatively affects brick absorbability. Modified methyl silicone-resin coatings exhibit an appropriate structure of polysiloxane gel, which was confirmed by its high effectiveness observed in the study.Based on the analysis of the research results, the following conclusions are proposed:


The tolerant parents, HKI1105 and CML425, and their hybrid, ADWLH2, were physiologically active under drought stress, since vital parameters viz., chlorophyll, root length and relative water content, were on par with the respective well-watered control. All the genes were up-regulated in ADWLH2, many were down-regulated in HM8 and HM9, and most were down-regulated in PMH1 and PMH3 in the shoots and roots. The nature of the gene action was controlled by the parental combination rather than the parent per se. The differentially expressed genes in all five hybrids explained a mostly non-additive gene action over additivity, which was skewed toward any of the parental lines. Tissue-specific gene action was also noticed in many of the genes. The non-additive gene action is driven by genetic diversity, allele polymorphism, events during gene regulation, and small RNAs under the stress condition. Differential regulation and cross-talk of genes controlling various biological functions explained the basis of drought tolerance in subtropical maize hybrids. The nature of the gene action and the direction of the expression play crucial roles in designing introgression and hybrid breeding programmes to breed drought tolerant maize hybrids.Breeding maize for drought tolerance necessitates the knowledge on tolerant genotypes, molecular basis of drought tolerance mechanism, action, and expression pattern of genes. Studying the expression pattern and gene action of candidate genes during drought stress in the hybrids will help in choosing target genes for drought tolerance breeding. In the present investigation, a set of five hybrids and their seven parents with a variable level of tolerance to drought stress was selected to study the magnitude and the direction of 52 drought-responsive candidate genes distributed across various biological functions,  Zea mays L.) is one of the major crops in the world, with the highest genetic yield potential among cereals. Maize leads the world cereal production, with an annual production of 1,021 million tons from 183 million hectares and a global productivity of 5.5 tons per hectare. The crop has well established in global agriculture owing to its multi-faceted uses  with relative humidity of 50–55%. All the genotypes were sown in plastic cups (6”) filled with sandy loam soil in a randomized complete block design and were replicated three times with three plants per replication . For each gene, the gene ontology, under different categories, such as molecular, cellular and biological functions, was identified using Blast2Go .Selected genes were categorized into different drought-related functional groups Table , includiRoot length (RL), chlorophyll content (CC) and relative water content (RWC) were measured in the stress and the control plants. Three plants per replication were used to measure the root length and other phenotypic traits. Utmost care was taken to avoid the damage to root system while removing the soil and measuring of root length. The RL was measured 5 days after the stress treatment in stressed and well-watered control seedlings and is expressed in centimeters. The CC (%) was recorded daily from day 1 to day 5 of the stress treatment period using a portable SPAD chlorophyll meter . The RWC was measured 5 days after the stress period using the following formula:where FW is the fresh weight, DW is the dry weight, and TW is the turgid weight . From the isolated RNA, 2.5 μg RNA was treated with DNase I  as per manufacturer's protocol to remove any genomic DNA contamination. The concentration of the isolated total RNA was determined using a Thermo Scientific NanoDrop 1000 spectrophotometer . One microgram of RNA was used to synthesize cDNA using an Affymetrix Kit .http://www.idtdna.com/scitools/applications/primerquest/default.aspx), and the 18s RNA coding gene was selected as the internal control  using a SYBR Green-based PCR assay . qPCR was performed using a total reaction volume of 25 μl, which consisted of 12.5 μl of the SYBR green RT-PCR master mix , 1 μl of reverse transcriptase, 0.5 μl of ROX dye, 1 μl of cDNA, 2 μl of each primer, and 8 μl of nuclease free water. Reverse transcription was performed at 50°C for 30 min and was then terminated at 95°C for 10 min; PCR was then performed with 40 cycles of 94°C for 3 s, 58–60°C for 1 min, and 72°C for 30 s with three technical replicates.−ΔΔCT, where ΔΔCT is the difference between the ΔCT of the condition of stress treatment and the ΔCT of the control.The expression data for the genes in the different treatments, tissues, and genotypes were normalized by subtracting the mean reference gene CT value from the individual CT values of the corresponding target genes (ΔCT). The relative abundance of the transcripts was derived using the expression 21 hybrid expression level of each gene related to the average of the two parents  was determined using a d/a ratio method , the dominant (d) is measured by the difference between the F1 (hybrid) and the average of the parents (μ), and the additive (a) is measured by the difference between the parent  and the average of the parents (μ) i.e.,1  allele, F1 = P1, then d/a = 1. Similarly, d/a = −1 explains the complete dominant gene action of the P2  allele. In case of an additive gene action, if F1 = μ, then d/a = 0.The quantitative measurement of the F1 hybrid expression was measured relative to the parental expression /2].Based on the above-mentioned concept, the transcript expression level is considered a phenotype of each gene, and the F1 hybrid from the average of the two parents was calculated as d = F1 – μ. Then, the deviation of the maternal parent from the average was calculated as a = Parent maternal – μ. The d/a ratio was then used to measure the gene action in the hybrids. If d/a = 0, this indicates additive gene action; if d/a = 1, this indicates the dominant gene action where the hybrid expression is skewed toward a maternal parent; if d/a = −1, this indicates a dominant gene action where the hybrid expression is skewed toward the paternal parent; if d/a > 1, this indicates an over-dominant gene action where the hybrid expression is skewed toward the maternal parent; and if d/a > − 1, this indicates over-dominant gene action where the hybrid expression is skewed toward the paternal parent, considering that the maternal parental expression is higher than the paternal parent expression. When the paternal parent expression is higher than the maternal parental expression, then the sign of the d/a ratio is reversed in over-dominant situations.From the actual expression data of the parents and the respective hybrids, the deviation of the Fd/a ratio is between −0.5 and +0.5, this is considered an additive gene action, where both parental lines contribute equally to the gene expression, or the F1 hybrid expression is equal to that of the parental mean. If the d/a ratio > ±0.5, this is considered a non-additive gene action that is skewed toward any one of the parental lines.If the The reduction in the CC was a mere 2.5% in the parents HKI1105 and CML425 and was 3.2% in ADWLH2 on day 5 of stress compared to day 1 Figure . An apprFive hybrids and their respective parental lines were exposed to stress, and the expression level of 52 candidate genes was measured under the stress condition. These 52 genes represent six important functional categories, including stomatal regulation, root development, ROS scavenging, hormone signaling, photosynthesis, and sugar metabolism.u–box protein and ereb155 in the maternal parent HKI1105 were expressed three times than in the paternal parent CML 425 in the roots. Many genes in HM8 and HM9 and in their parents  were positively up-regulated, and the level of expression was lower than that of the tolerant lines HKI1105 and CML425. In all the negatively expressed gene scenarios of the LM13 and LM17 parental combination, the maternal parent LM13 showed a higher level of down regulation than the maternal parent LM17, whereas in the LM17 and LM14 parental combination, neither of the parents showed a higher level of negative expression over the other parent.The expressions of all 10 genes in HKI1105 and CML 425 and in their hybrid (ADWLH2) were up-regulated both in the root and shoot tissues but to a greater extent in the shoots Figure . u–box pzhd 15, PRC protein, u–box, and ereb155) showed additive gene action except in one case where zhd15, in the roots of PMH3, showed a slightly higher activity toward the paternal parent  were negatively expressed in the paternal parent HKI161 and reduced the transcript level in its hybrid HM8. Of the seven genes that were down-regulated in the shoots of HKI1128, four were reduced at the transcript level in the hybrid HM9. The paternal parents LM13 and LM17 of PMH1 and PMH3, respectively, expressed higher levels of down-regulation in both the tissues for nac1 and nac3 and V–type PPase H+ pump over the common paternal parent LM14. Although the rest of the genes in both the tissues were up-regulated, the level of up-regulation was several folds less when compared to the tolerant lines HKI1105 and CML425.All the selected genes for studying root development were highly up-regulated in the roots and shoots of ADWLH2 and its parents but to a greater extent in the roots. e Figure . Two gennac2 and nactf38) in all five hybrids in both shoots and roots were close to the midpoint level, which explained the additive nature of those genes, but PMH3 showed a dominant gene action in the shoots for nac2. The remaining eight genes deviated from the midpoint at varying levels in all the five hybrids . All the hybrids expect for PMH1, showed maternal influence for the NBS–IRR–partial expression in both the shoots and roots. Three hybrids (HM8 and HM9 and PMH1) showed all paternal influence for nac68, whereas the expression of this gene in the rest of the hybrids was largely contributed by the maternal parents.The expression of two genes  showed root-specific expression in the parental lines of ADWLH2 since, the level of expression was 4 to 10 times higher than that of the shoot tissue under the drought condition. Most of the up-regulated genes in HKI161 and HKI1128 were less expressed compared to the tolerant maternal parent HKI1105. The down-regulation of two genes, pld1 and aldh3, in the shoots of HKI161 reduced the transcripts level in its hybrid HM8. The expression level of ROS scavenging genes in the parental lines PMH1 and PMH3 were very low in both the shoot and root tissues. sod2, pld1, mkkk11, aldh3, GPx, and mkk6 showed shoot-specific down-regulation in PMH1 and PMH3 and in their parents  showed additive gene action in all the hybrids. However, the gene action of one of the genes, apx2, in the shoots and roots of PMH3 was not uniform. Phospholipase D (pld1) in the shoots of HM9 explained the highest level dominant deviation (4.58) toward the maternal parent, followed by GPx in the shoots of HM8 (4.16). Several genes showed over-dominance in the tissues of one or another hybrid, but mkk6 showed over-dominance in the seedlings of all the hybrids except in the roots of HM8. ereb101, in all hybrids except in the shoots and roots of PMH1, and mkkk11, in all hybrids except in the roots of HM8 and the shoots of PMH3, showed maternal deviation in both root and shoot. The remaining genes did not show any clear-cut parental preference in the tissues of all hybrids  reduced the level of expression in the hybrid HM9, and this could be the reason for the moderate level of tolerance of the hybrid HM9 over ADWLH2 despite of having a common tolerant maternal parent HKI1105 , which indicated the over-dominant action of the gene. phyC1 in the shoots and MAPKK3 in the roots of the hybrid of ADWLH2 showed the highest degree of dominance toward maternal and paternal parents . artf11 and MAPKK3 showed maternal dominance in all the hybrids except in PMH3 where it was paternal. However, in other cases, the degree and the direction of dominance were distributed across the hybrids and the tissues . Three genes  were negatively expressed in the parental inbreds HKI161 and HKI1128 and hybrids HM8 and HM9. Two root-specific down-regulations (psa6 and rca1) in the paternal parent HKI1128 were observed but did not affect the positive expression of its hybrid HM9 owing to effect of maternal parent HKI1105. All the photosynthetic genes were down-regulated in the hybrids PMH1 and PMH3 as well as their parental lines , whereas the roots showed a higher level of paternal dominance (d/a = −5.46) in the hybrid PMH1  were skewed toward the paternal parent HKI161, and in HM9, three genes  were dominated by the paternal parent HKI1128.1 Figure . In all Sbe2A, sus1, and sodh1) showed shoot-specific positive expression in HKI1105, CML425, and the hybrid ADWLH2. The hybrid HM8 and its sensitive paternal parent HKI161 showed a negative regulation in the shoots compared with the roots for amyb5 and sodh1. Four genes  expressed a root-specific down-regulation in HKI1128, of which, two (ss5 and sus1) were also reduced at the transcript level in the root tissues of its hybrid HM9. All the genes were down regulated in both root and shoot tissues of PMH1 and its parents, and the magnitude of the down-regulation was higher in amyb5 as compared to other genes of sugar metabolism. PMH3 also showed a total down-regulation for all genes, and except for sodh1, the other genes showed a shoot-specific down-regulation in the hybrid as well as in both parents  of the same hybrid for ss5. In contrast, a significant dominant deviation toward the paternal parent was observed in the shoots of ADWLH2 (−4.35), followed by in the roots of PMH3 for ss5. Out of 50 possible cases across the five genes, two tissues, and five hybrids, 28 cases showed paternal dominance for sugar metabolism. Invertase 1 (ss5) showed a dominant deviation toward the paternal parent CML425, whereas sus1 showed a deviation toward the maternal parent HKI1105, and the rest of the genes skewed toward either of the parents.All five genes showed a dominant gene action similar to that of hormone signaling genes Figure . The degThe CC, RL and RWC of the drought tolerant parents (HKI1105 and CML425) were on par or slightly lower under stress than their WW controls. The performance of the hybrid ADWLH2 was also better under stress since its parents (HKI1105 and CML425) were highly tolerant to drought stress. There was a gradual reduction in all three parameters in the parents HKI161 and HKI1128. Despite the sensitive paternal parents, the growth performance of the hybrids HM8 and HM9 was moderate under stress since the maternal parent (HKI1105) in both hybrids was stress tolerant.A robust root system is very important to absorb water, to maintain a higher level of water potential in the aerial systems and for the ability to transpire for a longer duration under stress conditions , one that was similar to that of the parental line (dominant) and one that fell outside of the parental expression (over-dominant)  over the paternal (207) for all the genes, hybrids and tissues combinations.The ) Figure . The d/aIn this experiment, the direction of influence by the parental lines is of great significance in all five hybrids. The direction of skewness did not affect the ADWLH2 performance under stress since the alleles from both parents contributed to the drought tolerance. However, the direction is very important for the hybrids HM8 and HM9, since one of the parents, HKI1105, was stress tolerant and the paternal parents, HKI161 and HKI1128, respectively, were not tolerant. Nearly 40% of the genes in HM8 and HM9 in the different functional categories showed dominance or over-dominance toward their respective sensitive parent HKI161 and HKI1128 in the shoots and roots. The influence of the sensitive parents in the hybrids reduced the transcript level in the respective hybrids, thereby affecting the crucial biological function under the stress condition. However, other genes showed either an additivity or non-additivity toward the tolerant parent HKI1105, which reduced the level of damage caused by stress and provided the necessary support for maintaining biological activities. PMH1 and PMH3 were sensitive to stress, and since their respective parents were stress sensitive, the transcript level in the hybrids also either followed the mid-parental level or was dominant toward the parents.per se, whereas it is characterized by the combination of the parental lines involved. For example, me2 and nced2 in the stomatal regulation category in AWDHL2 showed dominance toward the parent HKI1105 in the shoots and roots, whereas the genes in HM8 and HM9 showed dominance toward the other parent, HKI161 and HKI1128. A hormone signaling gene, camta5, explained the over-dominance toward LM14 in PMH1, but the same gene showed over-dominance toward the parent LM17 in PMH3. The variation in the direction and the magnitude of the same genes in the different hybrids, especially in the hybrids involving one common parent, suggested that the extent of the genetic diversity between the parents also decides the gene action , and the specific growth conditions in which the genotypes were exposed (seedling or flowering and control or stressed). A non-additive gene action, especially over-dominance of the crucial genes under the stress condition, is also the result of the linked-loci of those genes. Since, drought tolerance is a complex trait, and various component traits are involved in stress tolerance, these traits are supported by a cascade of pathways inter-linking with each other. Further, these pathways are triggered in response to stress and results in the abundance of specific transcripts that are needed for the survival of the plant and to maintain various biological functions. Linked loci also play a critical role in the overexpression of genes . Many of the genes that were part of the signaling cascade were activated in the tolerant lines under stress, which explained that these genes were actively involved in sensing the stress and were able to trigger other genes. One of the key hormones is ABA, which plays a variety of roles under stress conditions, including stomatal regulation. An increased accumulation of ABA during stress stimulates the closure of stomata and decreases the transpiration rate under stress conditions  (Furihata et al., The ABA signaling pathway comprises three components, which are a group of ABA receptors, including V–type PPase pump enhances ion homeostasis, which regulates the osmotic balance and, hence, copes with drought (Li et al., ascorbate peroxidase functions as a H2O2 reductant in ROS detoxification (Foyer and Halliwell, ascorbate peroxidase reduces H2O2 to water and mono-dehydroascorbate. Under drought stress, glutathione reductase activity is increased in the roots and leaves, which might facilitate the defense against ROS (Gallé et al., SOD and glutathione peroxidase are also involved in ROS scavenging (Dietz et al., MAPK under drought stress in ROS detoxification (Ning et al., Root development is strongly influenced by abiotic stress conditions. A subfamily of NAC transcription factors is involved in various abiotic stresses such as drought, cold, salinity (Mao et al., Rubisco activase precursor under the drought stress condition reveals that a higher activation state could also have affected the Rubisco activity (Ramachandra Reddy et al., CAMTA, is identified as a regulator of the photosynthetic machinery where the T-DNA insertion line of AtCAMTAs is observed at a low photosystem II efficiency under drought stress (Pandey et al., invertases (Zinselmeier et al., Sucrose synthase enzyme is a key enzyme involved in the synthesis and cleavage of sucrose. Under water deficit conditions, the activity of sucrose synthase is enhanced in the cleavage direction and increases the sucrose synthesis (Castrillo, In response to drought stress, photosynthetic activity is reduced because of a decline in stomatal conductance as well as Rubisco activities, resulting in lower carbon fixation followed by an over reduction in the components of ETS system. The up-regulation of the The tolerant genotypes HKI1105 and CML425 and their hybrid ADWLH2 performed better under drought stress due to the activation of various genes controlling different molecular functions. The genes played individual as well as cumulative roles in maintaining crucial biological functions under the stress condition. The cross-talk of these pathways also played vital role in controlling a cascade of molecular activities under stress conditions. The absence of such gene regulation in the sensitive inbreds and the hybrids is the reason for the susceptibility under drought stress. The role of these candidate genes is very important for understanding the drought tolerance in hybrid genotypes and designing strategies to breed drought-tolerant maize genotypes.The gene action of the differentially expressed genes was either additive or non-additive across all the hybrids and tissues. The non-additive cases switched to any of the parents, depending upon the parental combination and showed tissue specificity in some of the hybrids. The genes mostly show non-additive gene action in the hybrids, and some of them showed over-dominance toward any one of the parental lines. The non-additive gene action could be driven by genetic diversity, allele polymorphism, events during gene regulation. The magnitude and the direction of the gene action of the candidate genes are very important to select the genes for drought tolerance. The magnitude of the gene expression and the expression pattern in the tissues will aid in selecting target candidate genes for drought tolerance breeding. The nature of the gene action and the direction of the expression pattern could play crucial roles in designing introgression and hybrid breeding programmes to breed drought tolerant maize hybrids.TN, HV, and MGM: conceived and designed the experiments; HV, TN, MGM, MS, BP, SJ, PD, AB, ARR, and RG: performed the experiments and analyzed the data; All authors contributed to manuscript preparation. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to evaluate the effect of adding propylene glycol (PG) to mineral trioxide aggregate (MTA) liquid with volume ratio of 20% on the compressive strength (CS) of MTA in two time periods (4 and 21 days) after mixing. n=15) with an internal diameter of 3 and a height of 6 mm were prepared and MTA (groups 1 and 2) and MTA+PG (80% MTA liquid+20% PG) (groups 3 and 4) were placed in to the cylinders. In groups 1 and 3 the CS was evaluated after 4 days and in groups 2 and 4 after 21 days. Data were calculated using the two-ways ANOVA. The level of significance was set at 0.05. Four groups of steel cylinders (P<0.05). Mixing MTA with PG significantly reduced the CS; but passing the time from 4 to 21 days significantly increased the CS. The highest (52.22±18.92 MPa) and lowest (4.5±0.67 MPa) of CS was obtained in 21-day MTA samples and 4-day MTA+PG specimen, respectively. The effect of time and PG were significant on the CS (Considering the limitations of this study, PG had a negative effect on CS of MTA. Mineral trioxide aggregate (MTA) is a calcium silicate-based hydrophilic cement consisting of powdered particles that are composed of Portland cement, gypsum and bismuth oxide which sets in contact with the liquid , 2. Due Several strategies have been proposed to improve the properties of the MTA, one of which is to add propylene glycol (PG) to MTA liquid -6. PG isConsidering the clinical applications of MTA, compressive strength (CS) of this material, is important in some cases; for example when MTA is used to repair furcal perforations where it must tolerate occlusal forces and the placement of restorative material on it , 15. CS Studies conducted on the CS of MTA, indicate that the hydration catalysts such as calcium chloride, etching, the presence of blood contamination, fetal bovine serum (FBS), increased ratio of liquid to powder, all can decrease the CS , 19-22. in vitro study aimed to evaluate the effect of adding PG into MTA liquid (with volume ratio of 20%) on the CS of MTA in two time periods of 4 and 21 days after mixing. The null hypothesis in this study was that PG has no effect on the CS.So far, there have been no published studies on the effect of PG on the CS of MTA. This n=15). Fifteen steel cylinders with an internal diameter of 3 mm and height of 6 mm were prepared for each group. In groups 1 and 2 the cylinders were filled with Angelus MTA  and in groups 3 and 4 they were filled with MTA mixed with 0.2 mL PG  and 0.8 mL MTA liquid. In the other words, 4 volumes of MTA liquid were mixed with one volume of PG. The volumes were measured using insulin syringe.Based on the used material and the assessment time of CS, 60 samples were assigned into 4 groups in this study  4 (groups 1 and 3) and 21 (groups 2 and 4) days after mixing and placement of the materials in to the cylinders. Cross head of the device applied force at a speed of 1 mm/min in the direction parallel to the longitudinal axis of the molds until the materials were crushed. This force was recorded based on Newton and was converted into MPa using the following formula: CS=4P=0.3) and equality of the variances (P=0.06), the two-way ANOVA was used to evaluate the significant effect of the material type and time period and also for the two-by-two comparison of the groups. SPSS software  was used for the analysis of data. The level of statistical significance was defined at 0.05.Data were analyzed using the descriptive statistics (mean±SD). The Kolmogorov-Smirnov test was used to evaluate normal distribution of data and the Levene's test was applied for equality of error variances. Considering the normal distribution of data (The highest (52.22±18.92 MPa) and lowest (4.5±0.67 MPa) values of CS was obtained in the second (MTA/21 days) and third (MTA+PG/4 days) groups, respectively. P=0.02 and P=0.001, respectively). The presence of the PG significantly reduced the strength (P<0.05). Regardless of the presence or absence of PG, the passage of time from 4 to 21 days, significantly increased the CS (P=0.03 for MTA groups and P=0.0001 for MTA+PG groups). Pairwise comparison of the studied groups showed significant differences between all groups (P<0.05). The ANOVA analysis showed a significant effect of the presence or absence of PG and time span on the CS (This study aimed to assess the effect of PG on the CS of MTA. In addition to the presence or absence of PG which was added with volume ratio of 20% to MTA liquid, another variable of the study was to evaluate the effect of time on the CS which was considered 4 and 21 days after mixing. The null hypothesis was rejected and PG significantly reduced the CS. Over time, the CS of all groups in the study was increased.MTA is a calcium silicate material and with regard to its applications in the field of root canal treatment , the CS In this study, we chose two time periods to assess the CS. The shorter time span of 4 days was selected due to the fact that the primary strength is important in clinical applications because initially, the material is exposed to occlusal forces in the patient's mouth. Also in this period, the material will reach to optimum setting and also in the previous studies, the same time period was taken into consideration for the same purpose , 15, 17.In this study, attempts were made to homogenize factors affecting the physical properties of the MTA , 15, 17.In our study, by adding PG to MTA liquid, the CS significantly decreased. PG is a hygroscopic compound and reduces the amount of water available for hydration and MTA setting and affects the nucleation of calcium silicate hydrate, which is the possible reason for the result obtained in this study. The effect of PG on the strength of MTA is similar to the effect of hydration accelerators such as calcium chloride since those materials reduce the CS, as well . IncreasAccording to our results and the results of previous conducted studies, properties on which PG has a negative effect are CS, microhardness and hardness of MTA ; which setc.Although, we have tried to minimize the effect of confounding variables in the study; however, we should still consider the clinical application of this material in interpreting and generalizing the results of the study too. Because a series of factors with which the material is faced in the clinical application, are not included in the study, such as the presence of blood contamination in the area, the presence of an acidic environment due to inflammation, bacterial products, interstitial fluid elements, Under the limitations of this study, the use of PG for mixing MTA is not recommended where the CS is important, such as repair of furcal perforation; because the material must be resistant against being crushed as a result of occlusal forces and the placement of restorative material on it. However, given that PG significantly improves other properties of MTA, it is recommended to use it as apical plug and retro filling material in surgery in cases where the CS is not important.


Sunitinib is an inhibitor of platelet-derived growth factors receptor (PDGFR) and vascular endothelial growth factor receptor-2 (VEGFR2) tyrosine-kinase activities, and it is registered for the treatment of metastatic renal cell carcinoma (mRCC). Indeed, sunitinib 50 mg/day for 28 days every 6 weeks is effective against mRCC and induces significant improvements in progression-free and overall survival  with respect to interferon-α and interleukin-2, even in cytokine-resistant tumors  instead of the standard 4/2 schedule (4 weeks on treatment and 2 weeks of rest). The new schedule has the same dose intensity with respect the classical one, but tolerability has significantly increased  in preclinical model was dependent on the dose , time to progression (TTP) and overall survival (OS) in mRCC patients  and disease stabilization (>95%). Those effective concentrations were obtained with sunitinib 50 mg/day in 7–14 days of a 2/1 schedule . Furthermore, the antiproliferative effect of sunitinib still continues during the rest period of 7 days, because (1) the drug is still circulating within the blood flow at the beginning of the next cycle and (2) the final effect on tumor microvessel density does last for days. At this point, the improved tolerability of the 2/1 schedule helps to support its therapeutic advantages with respect to the standard schedule.On the basis of the available pharmacokinetic and pharmacodynamics data one may argue that the 2/1 schedule is an active treatment because sunitinib achieves effective target plasma concentrations within the range 50–100 ng/mL (Figure Several studies have compared the tolerability of the standard 4/2 schedule with that of the alternative one. Overall, the conclusion of those studies is the same, with a highly significant reduction of moderate-to-severe toxic effects, namely diarrhea, fatigue, mucositis, thrombocytopenia, and hypertension in the alternative schedule (Bracarda et al., At this point, the second question is why the 2/1 schedule is better tolerated than the 4/2 one. Both treatments have the same dose intensity in a 6-week period, and both of them have a rest period that allows patients to recover from toxicities. However, during a 4/2 schedule, patients start to experience sunitinib-induced toxicities at the second week of treatment, and the severity usually increases over the next 2 weeks (Najjar et al., The last point that should be briefly discussed is the duration of the rest period with respect to both treatment tolerability and drug pharmacokinetics. As anticipated above, sunitinib concentrations are measurable at the end of the 7-day rest period (Britten et al., The recent studies on the alternative schedule 2/1 have demonstrated an identical efficacy in comparison with the standard 4/2 treatment because both schedules achieve the same plasma levels and share the same dose intensity, but the first one is characterized by a reduced incidence of moderate-to-severe adverse events (Bracarda et al., In conclusion, it is really impressive how changing the schedule of drug administration by splitting the treatment into two periods has increased tolerability beyond expectancies, hence demonstrating the possibility to optimize tolerability and subsequently efficacy by changing the standard regimens in a rational way.ADP, SB, EA, and RD contributed to the analysis of pertinent bibliography and critically revised the manuscript. ADP is accountable for all aspects of the work to ensure accuracy and truthfulness. All of the authors approved the manuscript.ADP served as a board member for Novartis Farma SpA. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Non-alcoholic fatty liver disease (NAFLD) is characterized by accumulation of intrahepatic lipid. The use of live microorganisms promotes beneficial effects; however, the use of symbiotic and its role in NAFLD is not yet fully understood. Verify if the symbiotic administration influences the occurrence and progression of NAFLD in rats, after induction of hepatic steatosis by high calorie diet.  Forty-five rats were divided into four groups: G1 (control); G2 (control+symbiotic); G3  and G4 , and euthanized after 60 days of diet. Liver disease was evaluated by biochemical analysis, IL6 measurement and histological assessment.  Symbiotic had influence neither on weight gain, nor on coefficient dietary intake in G3 and G4. G2 had the greatest weight gain, while G1 had the highest coefficient dietary intake between groups. G1 showed higher expression of aspartate aminotransferase than those from G2  while G4 showed higher expression of the enzyme compared to G3 (141±9.7 mg/dl to 78±4 mg/dl). Liver histology showed different stages of NAFLD between groups. G4 animals showed increased serum interleukin-6 when compared to G3 (240.58±53.68 mg/dl and 104.0±15.31 mg/dl).  Symbiotic can reduce hepatic aminotransferases and interleukin-6 expression. However, the histology showed that the symbiotic was not able to prevent the severity of NAFLD in rats. Non-alcoholic fatty liver disease (NAFLD) is characterized by accumulation of lipids in hepatocytes, which accounts for at least 5% of the weight of this tissue. It includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Population studies indicate that 10-50% of the population may have relatively benign hepatic steatosis. About 40% of patients with NAFLD develop into non-alcoholic hepatic steatosis, and 5% develop fibrosis after 4-13 yearsIt is believed that the development of DHGNA involves two phases. In the first one occurs the inadequate storage of lipids or ectopic accumulation of this nutrient. The second is marked by oxidative stress that causes hepatocyte injury and leads to the development of inflammationWhen normal physiology of the liver is altered, and inflammatory cells are activated, changes in the mechanisms of control and the activation of factors derived from the intestine occur. The imbalance in the intestinal microbiota can generate lipopolysaccharides that are components of the cell wall of gram-negative bacteria that reach the liver through the portal vein, leading to endotoxemiaThe production of endotoxin by the intestinal microbiota can cause inflammation in the liver of patients with obesity, diabetes, metabolic disease, hepatic steatosis and NASH,Studies using live microorganisms (probiotics) or soluble (prebiotic) fibers in the treatment of various diseases, including NAFLD, are well established in the literatureThe objective of the present study was to verify if the administration of symbiotics influences the occurrence of DHGNA in rats and to verify if the symbiotic conditions reduce the levels of liver enzymes and pro-inflammatory markers after induction of hepatic steatosis by hypercaloric diet.The animal experiments were carried out under institutional terms according to the norms established in Federal Law No. 11,794, of October 8, 2008, with the norms established by the National Council for the Control of Animal Experimentation (CONCEA) and after approval by the Ethics Committee Of Animals (CEUA) from Universidade Positivo, protocol number 181-2013.A total of 45 adult male Wistar rats weighing approximately 200 g were collected from the Positivo University laboratory. The animals were kept in controlled environment conditions, with temperature, luminosity and humidity control of the air. They were divided into four groups as symbiotic supply and diet provided.,,,Before the experiment itself, a pilot study of steatosis induction was carried out in five rats over a period of 30 days, according to the methodology used by Cintra et al.Lactobacillus paracasei LPC-37, Bifidobacterium lactis HN0019, Lactobacillus rhamnosus HN001, Lactobacillus acidophilus NCFM®and fructooligosaccharide (5.5 g) at the daily dose of 200 mg/kg/day were used as the symbiotic agent with strains (doses 1x109 UFC/g) day/animal, orally, corresponding to the approximate dose of 200,000 to 206,109 CFU (colony forming units) administered seven times a week for 30 days orally with the aid of a spatula adapted for this research ; hyperlipid diet developed according to studies by Cintra et alresearch  . This dDuring the experiment, we performed a weighing of the remaining diet every two days. The animals’ body weight was recorded every 30 days to evaluate the weight gain. These data were used to determine the coefficient of food efficiency (CEA)Serum levels of triglycerides, total cholesterol and aspartate aminotransferase (AST or TGO) and alanine aminotransferase (ALT or TGP) levels were evaluated at the end of the experiment. The amount of IL-6 in the blood was also dosed with a kit  at the end of the experiment. The animals were euthanized with isoflurane inhalation followed by carbon dioxide intoxication in a CO2 chamber . Afterwards, the liver and intestine were collected and placed in formaldehyde 10% for fixation and after 48 h they were embedded in paraffin, cut into a microtome, 5 μm thick, stained with H&E and analyzed for the development of steatosis in histopathology. Histological analyzes of the intestine were performed in order to evaluate changes in the architecture, lymphoid follicle and the presence or absence of acute or chronic inflammation. In the liver, the degree of inflammation and its intensity  and the percentage of fat cells were measured, and the classification system was adapted from Brunt et al.Quantitative variables were described in mean and standard error. For the comparison of the groups, the variance analysis model was considered. When, at some point, the hypothesis of equality of the averages of the four groups was rejected, they were compared two by two, considering the LSD test. For the comparisons of each evaluation moment with the first evaluated moment the Student t test for paired samples was considered. For the evaluation of the interaction between evaluation time and group, for the feed intake variable, the Split-Plot model was considered. The non-parametric Kruskal-Wallis test was used to compare the groups in relation to the steatosis classification. For the evaluation of the normality condition of the variables, the Jarque-Bera test was considered. P values less than 0.05 indicated statistical significance.All groups presented weight gain at the end of the experiment and Feed, water and fructose consumption were evaluated and all groups presented similar consumption in the first weeks. After the third week, the G3 and G4 feed consumption decreased by 15% in relation to the other groups. The consumption of water and fructose remained similar throughout all time in all groups.When comparing the groups with control diet (G1 and G2) the G1 group had higher CEA (p=0.012). When comparing the groups with hypercaloric diet consumption was similar between groups, G3=G4 .Cholesterol values did not differ between groups and did not change from reference values for the speciesWhen G1 and G4 were compared, in G1 40% of the animals did not present steatosis, 40% had mild and 20% moderate steatosis. In G4, moderate (60%) and mild (40%) steatosis predominated. Both groups did not show a similar course for severe steatosis.All animals of all groups presented preserved cellular architecture and without chronic or acute inflammation in the intestine. In the G2 and G4 groups, 10% of the animals presented alterations in the lymphoid follicle; in G3, 20%.The role of the symbiotic in controlling the progression of hepatic steatosis is not fully understood. The central objective of this research, after eight weeks of experimental protocol, was to evaluate if the consumption of a symbiotic can impact the development of NAFLD in rats.Bifidobacterium, Lactobacillus and oligosaccharides,It was observed that the symbiotic reduced levels of aminotransferases but was unable to alter the histological progression of NAFLD in an experimental model used. The difference in steatosis evolution may be related to diet composition. However, steatosis does not normally progress to the advanced stage of fibrosis in experimental modelsThe most important finding of this study was the reduction of IL-6 with the use of symbiotics. IL-6 is involved not only in the response of acute liver injury, but also in adipose tissue metabolism, lipoprotein lipase activity, and hepatic triglyceride secretion,Bifidobacteria and Lactobacillus in the gut of patients with colon cancer and irritable bowel syndrome. Future studies should specifically investigate the role of specific bacteria in NAFLDOther human studies reported similar results to the present study. Probiotic therapies reduced hepatic transaminases, TNF-a and IL-6 expression and improved insulin resistance in patients with DGNHA,,,7, 22.Other conclusions of this study deserve special attention. The first is related to the experimental model. Induction of steatosis in rodents usually uses a hyperlipidic or hyperglycemic diet, under sedation and through a feeding tube, which goes from 21 to 30 daysFinally, the symbiotic had no impact on cholesterol levels. A recent meta-analysis has shown controversial results of cholesterol reduction with the use of symbioticsThe present study had some limitations. The first is the short period of follow-up of the animals. The second refers to the fact that we chose to evaluate a single inflammatory marker (IL-6). Finally, common bacteria strains were used in dosage that mimics human use. It is possible that different combinations of different strains and dosages, as well as the composition of the intestinal microbiota of the animals, may interfere in the results. In the future, as metagenomic advances progress, more precise knowledge of all the mechanisms involved in the gut-liver relationship may possibly be predicted, possibly predicting the severity of NAFLD.In summary, administration of symbiotics was associated with reduced levels of aminotransferases and IL-6 expression. However, the symbiotic was unable to prevent the progression of hepatic steatosis in rats.Further studies should be performed, expanding the use of other markers and confirming the chosen model of oral symbiotic administration.Symbiotic can be an alternative in the treatment of NAFLD, because according to the results found it was able to reduce hepatic aminotransferases and to reduce the expression of IL6. However, contrary to the biochemical analysis, histology showed that the symbiotic was not able to prevent the severity of the disease.


Any system applied to the control of parenteral nutrition (PN) ought to prove that the process meets the established requirements and include a repository of records to allow evaluation of the information about PN processes at any time.The goal of the research was to evaluate the mobile health  app and validate its effectiveness in monitoring the management of the PN process.We studied the evaluation and validation of the general process of PN using an mHealth app. The units of analysis were the PN bags prepared and administered at the Son Espases University Hospital, Palma, Spain, from June 1 to September 6, 2016. For the evaluation of the app, we used the Poststudy System Usability Questionnaire and subsequent analysis with the Cronbach alpha coefficient. Validation was performed by checking the compliance of control for all operations on each of the stages  and by monitoring the operative control points and critical control points.The results obtained from 387 bags were analyzed, with 30 interruptions of administration. The fulfillment of stages was 100%, including noncritical nonconformities in the storage control. The average deviation in the weight of the bags was less than 5%, and the infusion time did not present deviations greater than 1 hour.The developed app successfully passed the evaluation and validation tests and was implemented to perform the monitoring procedures for the overall PN process. A new mobile solution to manage the quality and traceability of sensitive medicines such as blood-derivative drugs and hazardous drugs derived from this project is currently being deployed. The use of information and communication technologies (ICTs) in the health field opens a long list of possibilities aimed at achieving benefits at many levels in the health environment. A wide spectrum of research trends emerges. The use of ICTs plays a paramount role in the development of innovative clinical nutrition projects, especially in collaborative environments that go far beyond Web apps. A new area emerges, eHealth, in which great advances, new benefits, and possibilities arise when applying ICT to the health care domain, especially with the use of mobile apps, mHealth.Systems supported by telematic apps enable registries that allow linking and evaluation of information on parenteral nutrition (PN) processes at any time . The actThe good practices guide for the preparation of medicines in hospital pharmacy services establishes that all procedures related to the preparation of medicines, including PN, should be approved and reviewed. This guide establishes the need to evaluate and verify all procedures related to the preparation of medicines under the premise of “applying the principles of risk management for quality and quality by design” . The EurThe Spanish Society of Hospital Pharmacy , in its standards of practice for specialized nutritional support established by its Nutrition Working Group , recommeThe objective of this study was to evaluate the mHealth app and validate its effectiveness in monitoring the management of the PN process.A follow-up study was planned to evaluate a previously developed mHealth app and validate that it covers the real needs of the general process of PN.The units were the PN bags prepared by the pharmacy service of the Son Espases University Hospital (HUSE) of Palma, Spain.For the management of the stages of validation and transcription of the prescription, HospiWin 2000 version 8 (Baxter SL) software was used. This program provided the label that was attached to each PN bag. In addition, the entities to be monitored (PN bags) were identified with a unique code (a QR label was attached).The tracking of the PN bags was carried out from June 1 to September 6, 2016.The flow diagram, including all the stages of traceability and management of the PN, was generated and is published elsewhere . Using tUsing a methodology based on hazard analysis and taking advantage of semantic technologies, a holistic service platform for traceability and process control was designed. Upon the reading of a QR tag attached to an element to be monitored (in this case a PN bag), a data exchange occurs between the mobile app and the analysis server (steps 1 and 2). This server accesses its semantic knowledge base to infer the potential control operations associated with the PN bag. Using this data and other contextual information , a personalized list of ranked operations is sent to the mobile app (steps 2 and 3). The user selects the most convenient operation (step 3), probably the first one on the list, and the app generates a form to capture the values required for the task under consideration (step 4). At this point, the human user completes the form and captures photos when required, and the mobile app acquires any other required pieces of data, such as GPS location or time. These data are sent to the server to update the knowledge base (step 5). Finally, analysts and auditors can use a Web dashboard to visualize the data and trace the operations. A more detailed description of the platform is published elsewhere ,9.The verification of this app was previously published . The appEach one of the requests performed by this app was linked to a certain control operation in a particular stage. In case an operation was invoked more than once, by PN bag, the system only validated the first request, storing the remaining ones as “null requests.”An assessment of the utility and ease of use was performed. For this purpose, the Poststudy System Usability Questionnaire (PSSUQ)  was usedValidation was performed by checking the compliance of all the operations in each of the stages and corroborating that they were adequately monitored, particularly the operative control points (OCPs) and, more importantly, the CCPs. In order to validate the process, the results obtained from 387 PN bags at HUSE were analyzed.In the preparation stage for the gravimetric control, according to HUSE protocol for PN bags with a volume greater than 100 mL, the real weight should not exceed a margin of ±5% of its theoretical weight.For control of the storage temperature of the PN bags according to the procedure for the preservation of thermolabile medications at HUSE , the opeIn the administration stage, in the absence of preestablished limits for time and speed infusion, recommendations for each specific nutrition were observed according to the patient's clinical and social situation and the device used for its administration.The stages of the process were prescription, preparation, preservation, and administration. All control operations at each stage of the process were recorded in a dichotomous way (yes/no), according to whether they were finished or not.The operations characterized as CCPs were gravimetric control, temperature of the refrigerator, and volume and time of infusion (administration). These operations were recorded in a continuous and quantitative manner.The operations characterized as OCPs were microbiological control (which is recorded a posteriori), final check of the PN , and existence of the filter in the infusion pump (according to the PN type). These operations were recorded by means of dichotomous observations (yes/no). In all cases, it was possible to capture an image to prove compliance with a certain operation.The parameters monitored through the mobile device (phone or tablet) were sent to the Web server. This server processed these data and stored them in the knowledge base in the form of facts. The information model used was designed using Semantic Web–based techniques that allowed representation of concepts and relationships and offered advanced query services and analysis capabilities .To access the information managed by the system, the Web service offered a dashboard accessible through a common Web browser. In this dashboard, analysts and auditors were able to study and evaluate the history of records generated in any phase of the process, filter them based on various aspects of the context , download the information in a standardized format , and visualize usage statistics through easily interpretable graphics.A descriptive analysis was performed on the control operations assigned to each stage. The quantitative variables were characterized by their sample mean, standard deviation, median, mode, maximum, minimum, and interquartile interval (IQI). To avoid problems caused by outliers in the mean, the robust average was also calculated. The qualitative variables were characterized by their absolute and relative frequencies. Figures and tables were used to represent the most relevant qualitative variables.t test was used. The significance level used in all hypothesis tests was alpha≤.05.For analysis of the internal consistency of the answers obtained from the PSSUQ , a Cronbach alpha coefficient was used. To evaluate the significance in the difference of means for independent samples, a Student Quality control of the data was performed through double tables and active search for errors. When errors were found, they were corrected by consulting the original source. SPSS Statistics version 22.0 (IBM Corp) was used for the analysis.This work is part of the PI13/00464 project and was approved by the project evaluation body of the Miguel Hernández University with registration number 2013.95.E.OEP.During the implementation of the mHealth system for the traceability and management of PN at HUSE, 50 professionals took part and a total of 387 PN bags were managed.The usability test was performed at HUSE, with a total of 21 hospital professionals collaborating (42% of the staff was invited to participate).The overall result of the responses to the questionnaire presented the following values: mean 96.0 (SD 6.6), median 102, minimum 29, maximum 133, and IQI 72.5 to 118.5 (maximum score 133 = 19 items ×7 maximum score). The statistics obtained according to the different usability dimensions and study of the reliability of the measurement scale using the Cronbach alpha coefficient can be seen in In the validation study, 387 PN bags were managed; 91.2% (353/387) of the bags were from adult patients and 8.8% (34/387) from pediatric patients, with a total of 3860 control operations see . A totalAll operations related to OCP were recorded and audited in the knowledge base, including the type of operation, date, and person who carried out the operation. Compliance with these controls was 100%.The statistics obtained from the control of the operations labeled as CCPs for PN bags intended for adults and children can be checked in As shown on In the administration stage, it was found that for infusion time (measured in hours), the deviation from the mean was 0.3 hours for adults and 0 hours for children see .The results obtained in the period under consideration from daily recording of the temperature by 2 probes in the refrigeration room  can be seen in A nonconformity was detected from June 24 to July 7 that did not influence the validity of the PN bags since although the operative limit was exceeded (8ºC), the critical limit was not reached 12ºC). Likewise, there was a 10-day period when the temperature was lower than 2ºC, although in no case was it below 1.1ºC, so the temperature never fell below the critical limit (0.1ºC). In both situations, measures described in the established protocol had to be adopted [ºC. LikewThe statistics for data collected from the temperature sensors in the refrigeration room were:Maximum temperature (in ºC):Probe 1 (P1): mean 7.2 (SD 0.1) (CI 95% 6.9-7.5); robust average 7.1; median 6.9; maximum 11.8; minimum 5.5; and IQI (6.1-7.5)Probe 2 (P2): mean 7.0 (SD 0.1) (CI 95% 6.7-7.3); robust average 6.9; median 6.7; maximum 11.0; minimum 5.2; and IQI (5.9-7.2)t190=1.0; P=.31. There were no statistical differences in the measure of the maximum temperature between the 2 probes.Comparison of means between P1 and P2 was Minimum temperature (in ºC):Probe 1 (P1): mean 4.1 (SD 0.1) (CI 95% 3.8-4.4); robust average 4.1; median 3.8; maximum 7.4; minimum 1.4; and IQI (3.3-4.8)Probe 2 (P2): mean 3.8 (SD 0.2) (CI 95% 3.5-4.2); robust average 3.7; median 3.4; maximum 7.7; minimum 1.1, and IQI (3.0-4.6)t190=1.3; P=.19. There were no statistical differences in the measure of the minimum temperature between the 2 probes.Comparison of means between P1 and P2 was The geolocation function provided by the app made it possible to know the location, day, and hour of administration of every PN bag see .The evaluation and validation performed verified the ability of the mHealth app to register all designed processes and record nonconformities that occurred. Therefore, it was found that holistic monitoring of the traceability and management system of the PN could be implemented.The results of the evaluation of the mHealth app through a survey revealed that, in general, users assessed its usability positively. The 4 dimensions measured , on average, tended to the highest values of the Likert scale used. This means that the professionals considered the mHealth app useful in their work, and they found it easy to manage even without technological experience. The calculated Cronbach alpha  allowed us to be sure about the consistency of the evaluation test . This result showed that the positive perception of the app is common to all the users who took part in the process.The validation of the system allowed us to observe the monitoring and registration of all stages of the PN system. As already stated in the scientific literature, compliance with protocols minimizes unjustified clinical variability, which directly affects the reduction of risks associated with PN  and evenThe characterization and quantification of CCPs was essential to know to what extent the process designed met the quality standards (scope or degree of compliance with the standards) . The empAll bags prepared were of volumes greater than 100 mL, so the margin of error of actual weight compared to theoretical weight should not exceed 5%. These results are in line with previous studies on gravimetric control in the PN process -20, whicIn the administration stage, characterized mainly by the speed or rate of infusion of PN (volume to be infused per time), there are no specific regulations. Usually, only recommendations based on the clinical situation of the patient and device used for administration are established.A margin of ±1 hour of the scheduled infusion time is a sensible recommendation, and the reasons for larger deviations should be evaluated . In thisOther data on administration, such as location, date, and time, made it possible to collect very important data on control of the processes and holistic traceability of the PN. A large number of errors associated with the use of PN occur at the administration stage. Therefore, every stage of the process must be properly followed and managed. It is appropriate to establish procedures, especially in home care, that provide alerts about unwanted deviations . The sofThe use of a geographic information system made it possible to be certain about the disposition of the PN and get additional information about administration of the PN. In home care, the app would allow us to be aware of the actual arrival date of the PN at the patient’s home . The benBut mobile apps not only help the adherence to treatment; the traceability also contributes to minimizing security issues related to custody of the data and controlling the integrity of the medicine itself .In any case, in a context of progressive penetration of mobile apps for the management and traceability of medicines, it is desirable for health professionals to contribute by increasing health literacy (necessary to adequately recognize risks) and proper decision-making to take advantage of the opportunities inherent in Medicine 2.0 .Furthermore, as has been demonstrated in previous projects, the use of mobile apps designed to guide the medication-related processes significantly reduces, compared to conventional preparation methods, the medicine preparation time, administration time, and rate of medication errors .From the point of view of the authors, it is clear that the use of mobile apps for the management of medication, including parenteral nutrition, is a hot research topic with a bright future ,26,28.To control the volume being administered, it is difficult to establish a margin. The PN has a particular volume. Therefore, the potential hazard is linked to infusions done in less time than established, which would generally be due to a failure in the infusion pump . To avoiIn its recommendations , the AmeFrom the results obtained, we have concluded that the developed app successfully passed the evaluation and validation tests and, more importantly, properly completed the procedures for monitoring the overall PN process. In addition, the repository of records allowed us to see the deviations at all times. Therefore, a system for the management and traceability of PN that quantifies the main control points at a low cost has been successfully implemented and verified at HUSE in Palma, Spain.A new mobile solution to manage the quality and traceability of sensitive medicines such as blood-derivative drugs and hazardous drugs derived from this project is currently being deployed.


The formed Pt catalyst showed the average particle size of 4.1 nm. This Pt catalyst was applied to a polymer electrolyte membrane fuel cell (PEMFC). The PEMFC showed an open voltage of 0.85 V and a maximum output power density of 216 mW/cm The fun−1 . This hi−1 ,4,5.The low operating temperature of PEMFCs also causes a disadvantage in that there is a slow electrochemical reaction between hydrogen and oxygen molecules. Therefore, high loading of platinum (Pt) and/or Pt-based alloy catalyst is required for both the anode and the cathode electrodes in order to enhance the electrochemical reaction and increase the output power of PEMFCs. Because of material costs related to Pt, the catalyst needs to be formed at small sizes. 1–10 nm, on a suitable carbon support in order to increase the electrochemically active surface area (EASA) of Pt and to achieve high mass activity ,7,8,9.The carbon support materials for Pt catalysts are widely manufactured by the pyrolysis of hydrocarbons, such as natural gas or oil fractions from petroleum processes. Valcan XC-72 is one of the most popular commercial carbon support materials for Pt catalysts in fuel cells because of its low cost and high activity . Pt and/Physical syntheses, such as the spattering method, are also a promising way to fabricate small and active Pt catalysts ,14,15. SIn this study, Pt particles were formed on carbon black  using the in-liquid plasma method. The formed Pt particles were applied to a fuel cell as a catalyst. The fuel cells are also characterized.Carbon black (Vulcan XC-72) was used as a support material for the Pt catalyst. The carbon black was dispersed in water at a concentration of 0.0378% using a rotary mixer without the addition of a dispersion assistant. The Pt catalyst was formed on the carbon black surface using the in-liquid plasma method . A glass2 was used for the anode and cathode electrodes. The carbon black dispersion, which was applied with the in-liquid plasma, was painted on the carbon paper at a volume of 10 mL in order to load the Pt particles on the electrodes. Nafion 117 sheet was used as the polymer electrolyte for the PEMFC. A Nafion sheet and two sheets of carbon paper were sandwiched. Thermocompression bonding was carried out at a temperature of 215 °C and a pressure of 19 kg/cm2 for 2 min, in order to assemble the membrane electrode assembly (MEA). This process bonds the carbon papers to the Nafion sheet and enhances the conduction of protons in the MEA. Humidified hydrogen and oxygen gases were flowed into the anode and cathode electrodes of the fuel cell, respectively, at the flow rate of 1000 sccm. The fuel cell was operated at a temperature of 70 °C.Polymer electrolyte membrane fuel cells (PEMFC) were fabricated with the Pt particles, which were formed on the carbon black surface using the in-liquid plasma method. Carbon paper with a size of 3 × 3 cmdesQ the charge of hydrogen desorption on the unit area of the cathode electrode.Cyclic voltammetry measurements were conducted on the fuel cell in order to estimate the electrochemically active surface area (EASA) of the Pt catalyst. The measurements involved humidified hydrogen and nitrogen gases at the anode and cathode electrodes of the fuel cell, with an applied voltage between 0 and 1.6 V at a sweep rate of 100 mV/s. The EASA of the cathode electrode was observed from the charge of hydrogen desorption from the Pt catalyst. The EASA per unit area of the cathode electrode was estimated by using the following equation :(1)EASA2) of the MEA was 174 mC/cm2. Therefore, the EASA of the Pt per unit area (1 × 1 cm2) of the cathode electrode was estimated to be 830 cm2/cm2. The EASA per area of the MEA was available to be converted into EASA per weight of Pt particles. The converted EASA was 79 m2/g, which was similar to previously-reported values for the catalysts of fuel cells [The fuel cell was fabricated with the carbon black after the in-liquid plasma process was carried out for 20 min. el cells ,22,23,242 was observed for the fuel cell with Pt catalyst formed at a processing time of 5 min. The maximum output power increased with an increase in processing time.p), and a series resistor (Rs), as shown in The equivalent circuit for the inner impedance of the fuel cells consists of a capacitor (C), a parallel resistor (Rs), the capacitance (C), and the parallel resistance (Rp) were estimated from the cole–cole plots, using Equation (2), using the least squares method.The series resistance (Rs) is mainly originated from the ohmic resistance of the electrolyte; here, the proton exchange membrane. On the other hand, the parallel resistance (Rp) originated from the charge transfer resistance, which was provided mainly by the catalytic activity of the Pt particles [The equivalent circuit model, as shown in articles ,27,28,292) of the MEA using an in-liquid plasma processing time of 5 min. The weight of the loaded platinum linearly increased with an increase in processing time of the in-liquid plasma.2/cm2 was observed for the fuel cell fabricated with carbon black, which was treated with the in-liquid plasma process for 5 min. The EASA increased to 830 cm2/cm2 with an increase in the in-liquid plasma processing time to 20 min.No EASA was observed for the fuel cell when the in-liquid plasma process was not carried out for active carbon, because the Pt catalyst did not exist on the surface of the carbon black. An EASA of 380 cms) and the parallel resistance (Rp) were 47 and 172 mΩ, respectively, for an in-liquid plasma processing time of 5 min. Although the processing time increased, the series resistance (Rs) of fuel cells was not varied. On the other hand, parallel resistance (Rp) decreased to 75 mΩ with an increase in the in-liquid plasma processing time to 20 min.The series resistance  of the fuel cell. Therefore, the fuel cell, which is assembled using carbon black treated for longer in-liquid plasma processing times, shows a higher output power, because the internal resistance of the fuel cell is decreased, mainly by the reduction of parallel resistance (Rp).The weight of Pt sputtered by the in-liquid plasma increased with an increase in the processing time . Moreove2.The Pt catalyst was successfully formed on the carbon black surface using the in-liquid plasma method. The particle size of the Pt catalyst was in the range of 2–4 nm. Polymer electrolyte membrane fuel cells (PEMFC) were assembled with the formed Pt catalyst. An increase in the processing time of the in-liquid plasma increased the weight of the Pt catalyst loading in the MEA of the fuel cell, and decreased the internal resistance of the fuel cell. The fuel cell, assembled with the Pt catalyst, formed by the in-liquid plasma method, generated a high electrical power density of 216 mW/cm