Natural chloride diffusion test values indicate a sharp decrease in apparent diffusion coefficient (Dapp) equal to about 75% when concrete is protected by CI. Mechanism of action of CI in slowing down the chloride penetration inside the cement matrix is basically due to the water repellent effect as confirmed by data of concrete bulk electrical resistivity.One of the most important parameters concerning durability is undoubtedly represented by cement matrix resistance to chloride diffusion in environments where reinforced concrete structures are exposed to the corrosion risk induced by marine environment or de-icing salts. This paper deals with protection from chloride ingress by a silane-based surface-applied corrosion inhibitor. Results indicated that the corrosion inhibitor (CI) allows to reduce the penetration of chloride significantly compared to untreated specimens, independently of w/c, cement type, and dosage. Reduction of chloride diffusion coefficient (D Concrete alkalinity promotes the formation of a passive protective oxide layer able to prevent corrosion of steel rebars and guarantees an adequate service life of reinforced concrete structures . HoweverTwo different types of corrosion inhibitors are available on the market: the admixed inhibitors, added to fresh concrete, and migrating corrosion inhibitors—also called penetrating inhibitors or surface-applied corrosion inhibitors—applied on the hardened concrete surface . In partnssm also called DRCM) was calculated to quantify the reduction in the penetration of chloride into the cement matrix in accordance with Spiesz and Brouwers [The purpose of this paper is to evaluate the performances of a silane-based corrosion inhibitor applied on the surface of concrete element in order to slow down chloride diffusion in cement matrix and, consequently, to delay the onset of the corrosion process. The experimental program was carried out both in the form of accelerated and natural diffusion tests in different concrete mixtures manufactured in order to evaluate—other than the efficiency of CI treatment—the influence of w/c, cement type, and cement factor on the penetration mechanism. For each concrete, the chloride diffusion coefficient (DBrouwers  and Li eBrouwers .3). Finally, natural siliceous sand and gravel (three different gradings) with a maximum size equal to 22 mm were combined in order to meet the Bolomey curve  was calculated by the following equation [d is the average value of the penetration depths, t is the test duration, erf−1 is the inverse of error function, Cd is the chloride concentration at which the color changes, and C0 is the chloride concentration in the catholyte solution. Accelerated chloride migration tests were carried out according to NT BUILD 492 . A cylinequation :(1)DnssmFor the natural chloride diffusion test, 150 mm cubic specimens were stored in a 3 wt.% NaCl solution at 20 °C for six months. The solution was replaced monthly and, at fixed intervals (1–2–3–6 months), the samples were split into two halves by means of a compression testing machine , Italy) and the penetration of chlorides was measured using the previously described colorimetric method based on silver nitrate ,36.The standard method reported in ASTM C1760 was used to evaluate the bulk electrical resistivity of concrete with and without corrosion inhibitor. The water-saturated concrete sample (100 mm diameter and 100 mm height) was positioned between the test cells used for accelerated chloride migration test containing 3 wt.% NaCl solution and an electrical potential of 60 V DC was applied across the specimen. The bulk electrical resistivity was calculated using the following equation: In Compressive strength at 210 days is 26–30% higher than the corresponding value achieved at 28 days for concretes manufactured with pozzolanic (IV) and blastfurnace (III) cements. The 210-day strength value of limestone Portland cement concrete (II), on the contrary, is only 18% higher than the 28-day compressive strength. Data confirm that when a pozzolanic or blastfurnace cement is used, a higher increase of compressive strength with time is achieved as a consequence of the pozzolanic reaction .The average bulk electrical resistivity of water-saturated concretes is reported in Data highlight the positive role of w/c since the electrical resistivity increases with concrete compressive strength independently of whether the specimen is treated or not, confirming results available in literature ,41,42. Mnssn) resulting from the accelerated diffusion test basically depends on the depth of chloride penetration in concrete  when the accelerated diffusion test is carried out. The reduction of Dnssm is close to 30–40% if measured on samples water cured for 7 days and it slightly decreases at 21–39% when concrete is cured 28 days. The chloride diffusion coefficient (Dconcrete . As expenssn) of pozzolanic cement-based concretes (IV) as a function of w/c ratio. Data are in good agreement with electrical resistivity results confirming the effectiveness of CI treatment in preventing chloride ingress inside the matrix. Moreover, according to electrical resistivity data, the efficiency of the CI treatment seems to be higher than the lower the w/c. nssn) vs. cement factor for concrete manufactured with CEM IV/A-P 42.5 R at the same w/c (0.50). Results confirm the positive role of the CI treatment, independently of the cement dosage. Similar to the bulk electrical resistivity, the chloride diffusion coefficient is not strongly influenced by the cement factor. Experimental results are in agreement with Bertolini et al. [i et al. , affirminssn) vs. cement type for concrete manufactured with the same w/c (0.50). Data confirm the efficiency of CI treatment independently of the cement type. Results also indicate the positive role of pozzolanic and blastfurnace cement in reducing the chloride penetration inside the matrix as a consequence of the binding capacity of pozzolanic reaction products [products ,48,49.On the basis of the experimental results of the accelerated chloride diffusion test, it is possible to affirm that the surface-applied corrosion inhibitor performs better if applied on concrete intrinsically resistant to chloride penetration manufactured with a pozzolanic or blastfurnace cement and with a low w/c ratio.Finally, from the analysis of parameters resulting from the accelerated chloride migration tests and the bulk electrical resistivity tests, it was possible, in accordance with Layssi et al. , to corrAfter three months of exposure, a reduction of 65–90% could be noticed in chloride penetration as a consequence of the CI treatment . After 6app) according to the following equation [SLS is the duration of the exposure to the chloride-rich solution expressed in years, and dx is the thickness of the convection layer depending on the concrete compressive strength. At equal w/c (0.50), Dapp values are in the range of 0.20–0.30 × 10−12 m2/s for untreated concretes manufactured with blastfurnace (III) or pozzolanic (IV) cements while limestone Portland cement-based mixtures (II) evidenced higher apparent diffusion coefficients, close to 1.15 × 10−12 m2/s. The reduction in w/c promotes the formation of denser cementitious matrix with low Dapp in accordance with the study of Neville [Chloride penetration values (X) measured from the natural diffusion test over six months are used for the calculation of the average value  of apparequation :(5)0.66X Neville .app; values of treated concrete are in the range of 0.03–0.21 × 10−12 m2/s, about 75% lower than those detected for concretes without treatment. The efficiency of CI treatment seems to be independent of w/c, type, and dosage of cement. However, the lowest values for Dapp were obtained for those concretes intrinsically resistant to chloride penetration (low w/c and pozzolanic or blastfurnace cement), confirming the same results obtained for the accelerated chloride diffusion test.Treatment by the corrosion inhibitor determines a sharp decrease of Dapp and 28-day Dnssn; the correspondence between the two coefficients is linear and the proportionality factor is consistent, as reported in study of Spiesz and Brouwers [nssm determines a slower growth of Dapp in treated specimens.Brouwers . In partnssn) measured by an accelerated test in treated concrete was in the range 30–60%. Natural chloride diffusion test values indicate a sharp decrease in Dapp equal to about 75% when concrete is protected by the surface-applied CI.The surface-applied corrosion inhibitor allows to reduce significantly the penetration of chloride in concrete, independently of w/c, cement type, and dosage. Reduction of the chloride diffusion coefficient  should be avoided in environments rich in chlorides, preferring pozzolanic (IV) or blast furnace (III) cements.The following conclusions could be drawn from the present study:Further experimental data are in progress to evaluate the effectiveness of the surface-applied corrosion inhibitor at very long ages. Moreover, further studies should be focused on understanding if the migrating corrosion inhibitor is capable to stop corrosion in chloride contaminated concrete where the critical chloride concentration is reached on the steel rebars.


In rice, the conversion of tryptamine to serotonin is catalyzed by SL (sekiguchi lesion), a member of cytochrome P450 monooxygenase family. The SL in an indica rice restore line Minghui 86 (MH86), named as sl-MH-1, − 2 and − 3, all of which present the typical lesions under normal growth condition. Compared with those in MH86, the serotonin content in sl-MH-1 is dramatically decreased, whereas the levels of tryptamine and L-trytophan are significantly increased. The sl-MH-1 mutant accumulates high H2O2 level at its lesion sites and is more sensitive to exogenous H2O2 treatment than the wild type. When treated with the reductant vitamin C (Vc), the lesion formation on sl-MH-1 leaves could be efficiently suppressed. In addition, sl-MH-1 displayed more resistant to both the blast fungus and blight bacteria, Pyricularia oryzae  and Xanthomonas oryzae pv. Oryzae (Xoo), respectively. The pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses, like reactive oxygen species (ROS) burst and callose deposition, were enhanced in sl-MH-1. Moreover, loss function of SL resulted in higher resting levels of the defense hormones, salicylic acid and jasmonic acid. The RNA-seq analysis indicated that after P. oryzae infection, transcription of the genes involved in reduction-oxidation regulation was the most markedly changed in sl-MH-1, compared with MH86.Here we identified three allelic mutants of Our results indicate that SL, involving in the final step of serotonin biosynthesis, negatively regulates rice resistance against (hemi)biotrophic pathogens via compromising the PTI responses and defense hormones accumulation.The online version contains supplementary material available at 10.1186/s12870-020-02724-6. In nature, plants are constantly exposed to a wide range of pathogenic microorganisms, thus they have developed a sophisticated innate immune system to protect themselves from infection . PAMP-trMucuna pruriens, serotonin has been found widely distributed in the plant kingdom and involved in regulation of diverse physiological processes [Serotonin (5-hydroxytryptamine) is originally known as a neurotransmitter controlling fundamental physiological processes, such as mood, sleep and anxiety, in mammals . Since irocesses , 9. Serorocesses . Serotonrocesses . And therocesses .Oryza sativa) is the staple food for more than half of the world’s population. Rice blast and bacterial blight, caused by the hemibiotrophic fungal pathogen Pyricularia oryzae (P. oryzae) and the biotrophic bacterial pathogen Xanthomonas oryzae pv. Oryzae (Xoo), respectively, are the most devastating rice diseases [sekiguchi lesion (sl), originated from the rice cultivar Sekiguchi-asahi. The SL gene, cloned by Fujiwara et. al in 2010, encodes CYP71P1, belonging to the cytochrome P450 monooxygenase family [Bipolaris oryzae  infection was abolished in sl mutant, and sl displayed increased susceptibility to B. oryzae [sl mutant to biotrophic pathogens infection and the mechanisms of SL involved in plant immunity are still ambiguous.Rice (diseases . Increase family . SL poss. oryzae . Howeversl mutants in MH86 background (sl-MH) and found that the lesions of sl-MH-1 is caused by excessive accumulation of ROS, which could be suppressed by the reductant treatment. The absence of SL results in the enhanced PTI responses and high resting levels of defense hormones, and thus the broad-spectrum resistance against P. oryzae and Xoo.In this study, we identified the rcd1–1, in an indica rice restore line Minghui 86 (MH86). This mutant spontaneously exhibits orange-colored lesions on its leaves when grown in field or greenhouse  to detect cell death and H2O2 accumulation, respectively. As shown in Fig. sl-MH-1 leaves (shown by the dark trypan blue staining) displayed more H2O2 accumulation as indicated by DAB staining (dark brown color). To assess the defects of sl-MH-1 responding to exogenous ROS stress, sl-MH-1 and MH86 seeds were germinated on Murashige and Skoog (MS) medium with or without H2O2. After kept in a 28 °C growth chamber with 12-h light for 1 week, we found that there was no significant difference between sl-MH-1 and MH86 seedlings on MS medium without H2O2  than the wild type to H2O2 treatment , which is one of the major redox buffers in plant cell and functions as an antioxidant to conduct ROS detoxification [sl-MH-1 seedlings grown in green house, whose leaves did not exhibit visible lesions, were treated with 0.1% VC or water (as control) by spraying for 10 days. Then the lesion numbers on the top second and third leaves were counted. As shown in Fig. sl-MH-1 leaves was significantly decreased by VC treatment, indicating that exogenous antioxidant application could suppress sl-MH-1 lesion formation. Taken together, our data suggest that the cell death in absence of SL is caused by high endogenous ROS accumulation.In order to further determine whether lesion of fication . And it fication . 3-week-sl-MH-1 mutant was more resistant to rice pathogens. Firstly, we challenged 3-week-old sl-MH-1 and MH86 seedlings with conidial spores of the compatible blast fungal isolate, FJ-1, by spraying. As shown in Fig. sl-MH-1 mutant exhibited sekiguchi lesions at the FJ-1 infection sites. To quantify the resistance of sl-MH-1 to the blast fungus, we carried out punch inoculation on sl-MH-1 and MH86 leaves and investigated the relative fungal biomass within the infected region, which showed that the sl-MH-1 mutant supported significantly less blast fungus growth than MH86  in MH86 could extend to the neighboring cells of the first infected cell at 48 h post inoculation (hpi) and further extended to the adjacent cells at 72 hpi  in 6 weeks-old sl-MH-1 and MH86 plants, when the lesions were fully appeared on sl-MH-1 leaves. As shown in Fig. sl-MH-1 was about twice as high as that in MH86; both JA and JA-Ile contents in sl-MH-1 were burst to approximately 30 times higher than those in MH86. Meanwhile, levels of SA, JA and JA-Ile were also measured in sl-MH-3 to confirm that the high accumulation of the defense hormones was resulted from SL mutation. It was found that sl-MH-3 accumulated similar level of free SA with sl-MH-1 and even higher levels of JA and JA-Ile than those in sl-MH-1  and jasmonic acid (JA) are the major phytohormones involved in defense against pathogens. To assess whether sl mutant and wild type in response to pathogen infection, we carried out transcritome analysis in sl-MH-1 and MH86 plants with and without P. oryzae inoculation. The leaf tissues of sl-MH-1 and MH86 were harvested at 48 hpi with FJ-1 to conduct RNA-seq analysis, water treatment was used as control (mock). The number of significant differentially expressed loci (SDEL) in sl-MH-1-mock compared to MH86-mock (sl-MH-1-mock/MH86-mock) and sl-MH-1-48hpi compared to MH86-48hpi (sl-MH-1-48hpi/MH86-48hpi) with fold change ≥2 or ≤ 0.5 were 95 and 149, respectively  encodes a ω-3 fatty acid desaturase and a OPR isozyme, respectively, which are involved in JA biosynthesis [sl-MH-3 before and at 48 h after P. oryzae inoculation. As shown in Fig. sl-MH-3 are similar with those in sl-MH-1 transcritome data.Among the other eighteen redox-related DEGs in nditions ; Os08g01rocesses ; Os03g02ynthesis ; Os04g04ynthesis . To confSL transcription in the japonica rice cultivar Nipponbare (NPB) and Piz-t-transgenic NPB (NPB-Pizt) plants before and after inoculation with the blast fungal isolate KJ201, which is avirulent to Piz-t [SL transcriptional levels in NPB and NPB-Pizt was detected before or at 48 h after inoculation. With disease development, SL transcription in NPB was significantly up-regulated at 72 hpi, when it was not obviously altered in NPB-Pizt -12-oxophytodienoic acid (OPDA), is derived from oxidatively modified polyunsaturated fatty acids [sl-MH-1 may also be caused by the raised oxidative level, as high levels of JA and its derivant were detected in Arabidopsis when more ROS was accumulated by deletion of Fd2, the major ferredoxin in chloroplasts [sl-MH mutants, which could contribute to JA and JA-Ile production as well.Our results indicated that SA constantly accumulates in nous ROS , we specty acids . Thus throplasts . On the sl mutants in MH86 background (sl-MH) and revealed the roles of SL in rice innate immunity. Our results suggest the following conclusions: (i) the sl-MH mutant is more sensitive to exogenous ROS stress, its lesions formation is mainly resulted from excessive accumulation of internal ROS; (ii) SL negatively regulates rice defense against blast fungus and blight bacteria via compromising the PTI responses and suppressing the defense hormones accumulation; (iii) loss function of SL dramatically alters transcription of the genes involved in redox pathway after inoculation with P. oryzae; (iv) success P. oryzae infection up-regulates the transcriptional level of SL in rice. Taken together, our study decipher the negative roles of SL in rice defense against (hemi)biotrophic pathogens.In this study, we identified indica rice restore line Minghui 86 was originally bred at Institute of Rice, Fujian Academy of Agricultural Sciences, Fuzhou, China. The sl-MH-1 mutant was generated from the tissue culture-induced mutation of MH86 as described previously [sl-MH-2 and sl-MH-3 mutants in this study were generated from 60Co ~ γ-ray irradiation of MH86 by us. The rice cultivar Nipponbare, its transgenic line carrying Piz-t (NPB-Pizt) and the P. oryzae isolate KJ201 were originally obtained from Dr. Guo-Liang Wang’s laboratory . The P. oryzae isolate FJ-1 (virulent to Minghui86) was provided by Dewei Yang . The Xoo strain race 6 was provided by Dr. Dingzhong Tang . For the H2O2 treatment, P. oryzae inoculation, PTI responses detection and defense hormones measurement assays, the plants were germinated and grown in a growth chamber at 28 °C under a 12-h light (600–800 μmol/m2.s) /12-h dark cycle. For the metabolites measurement, lesion staining, VC treatment and Xoo inoculation assays, the plants were grown in a humidity-controlled greenhouse under natural conditions during summer season.The eviously . The sl-P. oryzae and Xoo inoculations were carried out in growth chamber and greenhouse, respectively. P. oryzae isolate FJ-1 was cultured on CMII medium for 2 weeks under light for sporulation. Then a conidial suspension with 3 × 105 spores/ml was sprayed on 3-week-old rice leaves. After inoculation, the seedlings were maintained in the dark for 24 h at 28 °C with high humidity, then transferred into the growth chamber for 5 ~ 7 days to evaluate their disease symptom. Four-week-old rice plants were used to perform punch inoculation as previously described [5 spores/ml) was applied. Investigation of the fungal biomass in infected rice leaf tissue was carried out as the method described previously [Xoo was conducted during the tilling stage by the leaf clipping method [600) = 0.5 was used to inoculate rice leaves as follows: scissor was dipped in the bacterial suspension and then used to cut the tip of a rice leaf. The inoculated plants were kept in the greenhouse for 14 days before disease symptom analysis. The blight lesion length and bacterial population accumulated in the infected leaves were evaluated as reported previously [escribed , and a 1eviously . Two pung method . The baceviously .VC  was sprayed onto the 3-week-old rice seedlings three times per day  for 10 days. Water spraying was used as control.1000 μl of precooled extract solution  was added to each 50 mg ground leave samples of MH86 and sl-MH-1. Metabolites extraction was performed as described previously  with minsl-MH-1 or sl-MH-3 plants were harvested as one biological replicate; the leaves from eight 3-week-old MH86 or sl-MH-1 plants were harvested as one biological replicate. The method of measurement is as previously described [For measuring the defense hormones contents, the leaves from three 6-week-old MH86, escribed .sl-MH-1 plants were soaked in 2 mg/mL 3,3′-diaminobenzidine (DAB)  for 4 ~ 5 h then transferred to the destaining solution (ethanol:lactic acid: Glycerol = 3:1:1). After heating to 90–95 °C for 15 min, the leaves were transferred to a fresh destaining solution and shaked gently overnight. Trypan blue staining was carried out as previously described [The leaf samples of 8-week-old MH86 and escribed .For observing callose deposition, the rice leaves from 7-day-old seedling were treat with chitin (hexa-N-acetylchitohexaose). The assay was carried out as previously described . Finally2O2. The plates were put in a 28 °C growth chamber with a 12-h light/12-h dark cycle for 7 days. Then the seedlings were imaged for analyzing sensitivity to H2O2 treatment.The dehusking rice seeds were sterilized with 75% ethanol for 2 min and 3% sodium hypochlorite for 30 min, then germinated on 1/2 MS medium with or without 0.02% HLeaf discs from 7-day-old rice seedlings were floated on sterilized water overnight, then put into 100 ul reaction solution  containing 500 nM flg22 or 400 nM chitin to immediately test the ROS burst. The luminescence was measured by Berthold Mithras luminometer every 1–2 min for 1 h. Each data point represented eight replicates.sl-MH-1 leaves before and after blast fungus spraying inoculation was extracted using the RNAeasy kit  and treated with an RNase-Free DNAse Set , according to the manufacturer’s instructions. The RNA quality, library construction and size were assayed using a 2100 Bioanalyzer system . The libraries were synthesized using the TruSeq RNA Sample Preparation v2 kit . Total RNA from each treatment was pooled and then two libraries were constructed and used for sequencing. The samples were run in the NovaSeq system and raw sequences of paired 150-bp were obtained.The total RNA from MH86 and https://www.i-sanger.com). Raw reads were evaluated using fastx_toolkit_0.0.14. SeqPrep and Sickle software were used for clean data. TopHat2 was used to map reads individually for each biological replicate in MH86 or sl-MH-1 against O. sativa japonica cultivar Nipponbare sequence according to MSU 7.0. Cufflinks was used for assembly into transcripts with reference annotation to guide assembly [sl-MH-1 mutant compared to MH86 were identified after applying multiple corrections (FDR adjusted p ≤ 0.05). The SDEL with fold change ≥2 or ≤ 0.5 were used for further analysis. Functional enrichment analysis (FEA) was performed on SDEL in sl-MH-1 compared to MH86. Functional annotation and expression correlation analysis were conducted on the gene set related to oxidation-reduction biological process. Heat map was generated using cluster analysis.The analysis below was run on Majorbio .Additional file 4: Fig. S4. Resistance of sl-MH-3 to P. oryzae and Xoo. a. 3-week-old MH86 and sl-MH-3 seedlings were inoculated with P. oryzae conidia by spraying and the diseased leaves were imaged at 7 dpi. b. Punch inoculation with P. oryzae conidia was performed on the leaves of 4-week-old MH86 and sl-MH-3 plants. The diseased leaves were photographed at 9 dpi (left), and the fungal biomass was measured (right). Bars represent mean values ± SD from three biological replicates. Statistically significant difference was indicated by ** . c. MH86 and sl-MH-3 plants were inoculated with Xoo. The infected leaves from three independent MH86 or sl-MH-3 plants were imaged at 14 dpi. d. Blight lesion length on sl-MH-3 and MH86 leaves were measured (left), bars represent mean values ± SD . Blight bacterial populations were counted 14 dpi with bars representing mean values ± SD from three independent MH86 or sl-MH-3 plants (right), ** means p < 0.01 by Student’s t-test. e. Callose deposition on MH86 and sl-MH-3 leaves after chitin treatment was imaged with a microscope under UV light (left). Number of the callose deposition per view was counted (right). Bars represent mean values and SD (n = 5). Statistically significant difference was indicated by ** .Additional file 5: Fig. S5. Contents of defense hormones in 3-week-old MH86 and sl-MH-1 plants. The resting levels of SA, JA and JA-Ile in 3-week-old MH86 and sl-MH-1 leaves were measured by UPLC. Bars represent mean values ± SD from three biological replicates. Statistically significant difference was indicated by ** .Additional file 6: Fig. S6. qRT-PCR analysis of the representative genes of redox pathway in MH86 and sl-MH-3. The transcriptional levels of Os01g0963000, Os08g0104600 and Os03g0290300 were determined in MH86 and sl-MH-3 at 48 hpi with P. oryzae (water spraying was employed as mock treatment). UBQ was used as the internal control. Bars represent mean values ± SD (n = 3). Statistically significant difference was indicated by ** .Additional file 7: Table S1. Significant differentially expressed genes in sl-MH-1-mock/MH86-mock.Additional file 8: Table S2. Significant differentially expressed genes in sl-MH-1-48hpi/MH86-48hpi.Additional file 9: Table S3. Quantification of serotonin, tryptamine, L-trytophan and L-glutamine by UHPLC-MS.Additional file 10: Table S4. Sequences of the qRT-PCR primers in this study.


RBV) of the calcium salt of the hydroxy analog of dl-methionine  to dl-methionine  as Met sources fed to pigs. In experiment 1, 42 crossbred barrows  were allotted to 7 treatments in an N-balance study. The basal diet (BD) was formulated to contain 15.4% CP and 0.22% Met (70% of requirement). Diets included (1) BD, (2) BD + 0.025% dl-Met, (3) BD + 0.050% dl-Met, (4) BD + 0.075% dl-Met, (5) BD + 0.038% MHA-Ca, (6) BD + 0.077% MHA-Ca, and (7) BD + 0.115% MHA-Ca. An increase in dietary inclusion rates of both Met sources linearly increased (P < 0.01) N retained (g/d) and N retention (% of intake). Using linear slope-ratio regression, the RBV value of MHA-Ca to dl-Met for N retained (g/d) was 63.0% on a product-to-product basis (75.0% on an equimolar basis). In experiment 2, 40 crossbred barrows  were allotted to 5 treatments in another N-balance study. The BD was formulated to contain 17.0% CP and 0.22% Met (70% of requirement). Diets included (1) BD, (2) BD + 0.030% dl-Met, (3) BD + 0.060% dl-Met, (4) BD + 0.046% MHA-Ca, and (5) BD + 0.092% MHA-Ca. Increasing levels of dl-Met or MHA-Ca increased N retained (g/d) and N retention (% of intake) linearly (P < 0.001) and quadratically (P < 0.05). Using linear slope-ratio regression, a product-to-product RBV value of MHA-Ca to dl-Met was 68.4% (81.4% on an equimolar basis) for N retained (g/d). In experiment 3, 276 pigs  were used in 3 diet preference studies. Pigs were randomly allotted to 1 of 3 treatment comparisons of feed choice: (1) BD (0.23% Met) or BD + 0.07% dl-Met; (2) BD or BD + 0.0825% MHA-Ca, and (3) BD + 0.07% dl-Met or BD + 0.0825% MHA-Ca. Pigs consumed a higher percentage  of their total feed intake from the diet supplemented with 0.07% dl-Met in Comparison 1, but a lower percentage  of their total feed intake from the diet supplemented with 0.0825% MHA-Ca in Comparison 2. There was no diet preference for dl-Met or MHA-Ca in Comparison 3. The observed Met source preference differences occurred in the barrow replicates but not in the gilt replicates. These results demonstrated the mean RBV of MHA-Ca to dl-Met of 65.7% on a product-to-product (wt/wt) basis or 78.2% on an equimolar basis and that a preference for Met sources was observed in barrows but not in gilts.Experiments were conducted to determine the relative bioavailability ( Alternatively, the calcium salt of the hydroxy analog of dl-Met  and liquid dl-Met hydroxy analog-free acid  are utilized as alternative Met sources in swine diets to improve N retention by pigs.Methionine is a limiting sulfur-containing amino acid (AA) in conventional corn–soybean meal-based diets fed to pigs . CrystalRBV) of Met sources in pigs have been focused on liquid MHA-FA. For instance, dl-Met of 65.2% on a product-to-product basis (74.1% on an equimolar basis) for N retained per day (g) in growing pigs weighing from 16 to 21 kg. Also, dl-Met in growing pigs was 65.7% on a product-to-product basis (74.4% on an equimolar basis) for protein deposition using the indicator AA oxidation method in a slope-ratio comparison.Most of the published studies dealing with the relative bioavailability (dl-Met of 71.2% on a product-to-product basis (84.8% on an equimolar basis) for N retention rate expressed as % of intake in growing pigs weighing from 18 to 22 kg, more information about the RBV of MHA-Ca to dl-Met in pigs is needed to provide a more robust estimate of the RBV of MHA-Ca compared with dl-Met in pigs. We hypothesized that the RBV of MHA-Ca would be lower than that of dl-Met for N retention of starter pigs.While dl-Met than the MHA-FA-supplemented diets regardless of the inclusion level of MHA-FA. We, thus, further hypothesized that if a preference for Met-sources existed, that the preference observed would similarly be for greater consumption of dl-Met compared with MHA-Ca. Therefore, the objective of the experiments presented was to determine the RBV of MHA-Ca compared with dl-Met to support N-retention in 15- to 21-kg starter pigs and to determine whether pigs demonstrated a preference for different sources of Met.Regarding dietary preference of nursery pigs for Met levels and sources, dl-Met and MHA-Ca, were provided by Evonik Operations GmbH, and results of analyses confirmed the concentrations of 99.7 and 84% active substance in dl-Met and MHA-Ca, respectively.Three experiments were conducted using nursery pigs. The first experiment was conducted at the Institute of Agricultural Sciences for Southern Vietnam under experimental protocols approved by the Animal Care Committee of the Institute. The second and third experiments were conducted at the University of Kentucky under experimental protocols approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Prior to diet formulation, ingredients contributing AA were analyzed for AA composition and the analyzed AA contents and the standardized ileal digestibility coefficients according to AminoDat (3.0) were used in diet formulation in all experiments. Both test Met sources, BW) of 15.0 ± 0.7 kg were used in an N-balance trial with 2 groups of 21 pigs each. Each group of pigs provided 3 complete replicates (blocks) of the experimental diets. The basal diet  or MHA-Ca  at the expense of corn starch. The corresponding supplementation levels of MHA-Ca were based on an assumed average RBV of MHA-Ca to dl-Met of 65% (product-to-product basis) to provide similar animal performance. Supplemental AA other than Met were added to the BD to exceed the requirement for AA other than Met and Met + Cys ] barrows with an initial body weight  BD + 0.060% dl-Met, (4) BD + 0.046% MHA-Ca, and (5) BD + 0.092% MHA-Ca. The supplemental levels of MHA-Ca were based on an assumed average RBV of MHA-Ca to dl-Met of 65% (product-to-product basis) to provide similar animal performance. To prevent unintended variations from potential weighing or mixing errors, experimental diets were prepared by adding both Met products to a single common batch of the BD.A total of 40 barrows [Hampshire × (Landrace × Yorkshire)], with an initial BW of 15.5 ± 1.5 kg, obtained from the University of Kentucky Swine Research herd, were used in an N-balance trial with 2 groups of 20 pigs each. Each group of pigs included 4 complete replicates (blocks) of the experimental diets. The Met-deficient BD was formulated to contain 17.0% CP and 0.22% Met  based onIn both experiments 1 and 2, pigs were placed in metabolism crates for a 7-d adaptation to their diet and crate. Feed allowance was equalized within block at ~3.5% of the average BW. One-third of the daily feed allowance (in mash form) was provided at 0800, 1200, and 1600 hours in experiment 1 and at 0600, 1300, and 2000 hours in experiment 2 for the 7-d adaptation period and the 5-d collection period, mixed with a sufficient quantity of water to create a gruel. Feed allowance for the collection period was adjusted based on the final BW for the adaptation period. In experiment 1, after a 7-d adaptation period to the diets and metabolism crate, feces and urine were collected quantitatively for 5 d in a manner similar to During the collection periods, the total quantities of feces excreted were collected daily, stored in plastic bags, and frozen at −20 °C until the end of the collection period. The total feces collected for 5 d were dried in a forced-air drying oven for 72 hr at 55 °C. The dried fecal samples were ground to pass a 1-mm screen in a Wiley Mill  for analysis of DM and nitrogen (N) content. The collection of urine was initiated 14 hr after feeding of the first marked meal and was completed 14 hr after feeding of the second marked meal at the end of the collection period. A total of 10 mL of 6 N HCl (experiment 1) and 150 mL of 3 N HCl (experiment 2), respectively, were added to the collection container at the beginning of each collection to prevent volatilization of urinary N. Urine was collected every 24 hr and stored at −20 °C. At the end of the collection phase, the total quantity of urine collected from each pig was allowed to thaw, then measured and pooled. Two aliquots (~200 mL) of urine from each pig were subsampled for N analysis. The average of the analyzed dietary values in experiment 2 was used for diet N content calculation because of the mixing of a common BD for all dietary treatments.dl-Met; (2) choice of BD or BD plus 0.0825% MHA-Ca, and (3) choice of BD plus either 0.07% dl-Met or 0.0825% MHA-Ca. The pigs were housed 4 pigs per pen for a total of 23 replicates .A total of 276 barrows and gilts [Hampshire × (Landrace × Yorkshire)], with an initial BW of 7.09 ± 1.1 kg, were weaned at an average of 21 d of age (range of 18 to 25 d) and used in 3 4-wk diet preference studies to determine whether pigs preferred diets based on the diet Met content and the different Met sources. The BD  was formTwo feeders, each with one of the two diets in the treatment comparison, were placed in each pen and the location of the feeders was rotated each Monday, Wednesday, and Friday to avoid the potential of feeder location being confounded with potential feed preference exhibited. Pigs were given ad libitum access to feed and water  as a response of supplemental Met level (%) using the following equation: The experimental data were analyzed as a randomized complete block design using GLM procedures of SAS  with individual pig considered the experimental unit. The statistical model included treatment, group, and block (group). Orthogonal polynomial contrasts were conducted to determine linear and quadratic effects of increasing the graded levels of y = response criterion (N retained); a = intercept; b1, b2 = the slope of dl-Met and MHA-Ca, respectively; x1, x2 = dietary inclusion level (%) of dl-Met and MHA-Ca, respectively. The RBV of MHA-Ca when compared with dl-Met was calculated as the ratio of their linear slopes  as described by P < 0.05 and tendencies if 0.05 ≤ P < 0.10.in which t-test using Graph Pad Prism  with the pen considered the experimental unit. Differences were considered significant if P < 0.05 and tendencies if 0.05 ≤ P < 0.10.Feed disappearance values were converted to percentage consumption of each diet. The percentages for each period and for the cumulative time periods were subjected to unpaired dl-Met and MHA-Ca were very close to the formulated levels. The analyzed contents of AA including the supplemented dl-Met and MHA-Ca were slightly lower than the calculated values across the experimental diets. However, the corresponding dl-Met to MHA-Ca ratio of ~65% (wt/wt) was maintained in the Met-supplemented diets  fecal N output per day (g). Urinary N excretion per day (g) was linearly decreased (P = 0.001) with graded levels of dietary dl-Met from 0% to 0.075%. Increasing dietary inclusion rate of both Met sources resulted in a linear increase (P < 0.01) in retained N per day (g) and N retention rate (% of intake). Quadratic effects of including dietary Met sources on retained N or N retention rate were not observed.The BD contained 0.22% Met, and the added levels of d values . The effdl-Met of 63.0% was observed based on N retained expressed as gram per day  in urinary N excretion per day (g), whereas retained N per day (g) linearly increased (P < 0.001) with graded levels of both Met sources. Increasing graded levels of dl-Met and MHA-Ca from 0% to 0.060% and 0% to 0.092%, respectively, linearly increased (P < 0.001) N retention rate (% of intake). In addition, quadratic effects of supplemental Met sources on retained N or N retention rate (P < 0.05) were observed.The BD contained 0.22% Met, and the added levels of expected . The effdl-Met in the current experiment was 68.4% based on N retained expressed in gram per day  than the BD for days 14 to 21 and 21 to 28 periods. Based on the percentage of feed consumption during the periods (from days 14 to 21 and 21 to 28), there was a pronounced preference (P < 0.01) in cumulative consumption for the diet containing 0.07% dl-Met. At the termination of the study on day 28, the pigs chose to consume more of the dl-Met-supplemented diet, with the ratio of 0.07% dl-Met to Met-deficient BD being ~55:45.The diet preferences of pigs for the comparison of the Met-deficient BD relative to P < 0.05). By the conclusion of the study on day 28, pigs consumed less of the MHA-Ca-fortified diet (P < 0.05) with a ratio of ~55:45. The diet preferences of pigs for the comparison of 0.07% dl-Met relative to 0.0825% MHA-Ca are shown in dl-Met and 0.0825% MHA-Ca existed during any of the study periods.The diet preferences of pigs for the comparison of the Met-deficient BD compared with 0.0825% MHA-Ca are provided in dl-Met or 0.0825% MHA-Ca were further divided into evaluations of the different gender response and are provided in dl-Met-supplemented diet (P < 0.05) than the Met-deficient BD, whereas no diet preference was exhibited by gilts. Barrows in 8 of 12 pens consumed more of the dl-Met-supplemented diet, with the ratio of dl-Met to Met-deficient BD being ~57:43. For the total 28-d study for comparison 2, barrows consumed less of the MHA-Ca-supplemented diet than the BD (P < 0.05), whereas gilts exhibited no diet preference. Barrows in only 2 of 12 pens consumed more of the MHA-Ca-fortified diet, with the ratio of Met-deficient BD to MHA-Ca being ~60:40. No preference differences were observed for either the barrow or gilt replicates for the comparison of 0.07% dl-Met relative to 0.0825% MHA-Ca in comparison 3.The feed intake preferences of pigs for diets supplemented with either 0.07% dl-Met using the slope-ratio procedure  based on the N retention per day (g), resulting in the lower biological efficacy of MHA-Ca than that of dl-Met of 78.2% on an equimolar basis. The lower RBV of MHA-Ca may have been due, as postulated by l-Met compared with that of dl-Met to l-Met by pigs. To be utilized for protein synthesis, both the d- and l-isomers of MHA-Ca must be converted by l-hydroxy acid oxidase, d-hydroxy acid dehydrase, and d-amino acid oxidase, respectively, into keto-Met via oxidation, followed by transamination into the bioactive l-Met , which equates to 70.5% on an equimolar basis. In the study of dl-Met in growing pigs weighing from 16 to 21 kg was 65.3% on a product-to-product basis, which equates to 74.1% on an equimolar basis for N retention. Furthermore, dl-Met was 73.2% on a product-to-product basis in growing pigs, which equates to 83.2% on an equimolar basis for N retention.The mean value of the RBV of MHA-Ca to ve l-Met . dl-Met sorption . The lowdl-Met-supplemented diet at 0.07% than the BD. The diet preference of pigs for the Met-fortified diets over the Met-deficient diets could be attributed to the distinctive ability of pigs to identify AA deficiency in diets. However, the mechanisms by which pigs exhibit a preference for Met-adequate diets over Met-deficient diets and the main reason for this response by pigs are unknown. The results of the current study are in agreement with the results reported in the study of dl-Met and MHA-Ca diets were compared directly, where the results of the first two comparisons would suggest extreme discrimination against MHA-CA supplemented diets, there was no preference exhibited. The differences in preference for Met sources were exhibited strictly in barrows but not in gilts. To our knowledge, this is the first report that demonstrates a gender-specific preference for Met sources. Thus, further research is warranted to investigate preference of nursery pigs for various Met sources by gender.In the current study, pigs preferred to consume more of the dl-Met resulted in significant improvements in N retention by pigs as evidenced by reductions in urinary N excretion. The results of the current studies demonstrated that the mean RBV of MHA-Ca to dl-Met for N retention was 65.7% on a product-to-product basis, which equates to 78.2% on an equimolar basis. When given a choice, pigs preferred the diet supplemented with dl-Met more than the diet with no supplemental Met. In contrast, pigs showed a preference for the diet without supplemental Met over the diet supplemented with MHA-Ca. Barrows exhibited a stronger preference than gilts for the source of supplemental Met in the diets.In conclusion, supplementation of Met-deficient diets with MHA-Ca or


This paper describes the testing of effectiveness and behavior of dowels placed in transversal joints of concrete pavements, while focusing on dimensions and quality of commonly used materials. The analysis uses experimental tests in laboratory conditions which were performed independently in the Czech Republic and Slovak Republic. The comparison of quality as well as potential use of alternative materials of dowels is made with the use of developed tests focusing on main requirements, such as longitudinal displacement in cement concrete, resistance of coating to damage, and reduced potential to concrete damage. Furthermore, the paper describes and compares loading results of the relative concrete deformations around dowels by strain gauges that were analyzed. Results of deformations on beams with an inserted dowel and the findings that were observed during the measurement are presented.  This article is an outcome of research in the Czech and Slovak Republics that aimed perform measurements of stress on beams with an inserted dowel, to assess their effectiveness and to test the concentration of tensile stress in the concrete around dowels intended for jointed plain concrete pavements (JPCP).Together with the aggregate interlock, dowels are a solution to improve interaction between slabs at transversal joints (LTE—load transfer efficiency) of concrete pavements. However, this technological complication may not have a long-term effect in cases when concrete is damaged along the dowels and their function may be lost. An analysis of the current JPCP loading showed another potential complication in the form of existence of high concentrations of tensile stress of concrete at faces of transversal joints around dowels and potential occurrence of microcracks in this area. Theoretical modelling results by the finite element method (FEM) method show that stress in the concrete slab increases under static and thermal loading, particularly in the vicinity of dowels . The FEMMany models deal with the dowel−concrete interaction through theoretical modelling and applied nonlinear calculation along the whole length of the dowel based on an analysis of stress distribution ,11,12,13The issue of the real behavior of dowels in transversal joints of JPCPs and its long-term monitoring is described by several foreign authors ,16,17. AAlthough the real behavior of dowels by laboratory testing, which verifies results of FEM modelling, dowels effectiveness, behavior during loading and alternative dowel materials, was not satisfactory, it was studied in detail. The cooperation of Transport Research Centre and the Faculty of Civil Engineering, STU Bratislava, was based on analyses of dowel effectiveness, since the effect of dowel dimensions on cement concrete slabs behavior is still not completely known and clear in the field of road pavements design. In its first phase, the experimental research in the Czech Republic and Slovakia focused on overall durability, deformation properties, and the damage type of beams with inserted dowels. Furthermore, cooperation was established with the aim of focused research and the development of alternative materials suitable for installations in motorway and highway structures as well as airport pavements. Nowadays, a single dowel type is used in the Czech Republic ; in Slovakia and foreign countries (apart from Germany and Austria) dowel dimensions are prescribed based on the concrete pavement thickness . In additilt of the dowels relative to the length of 500 mm  may be up to 25 mm, but this must be met by a minimum of 75% of dowels in the joint and the remaining 25% of dowels in the joint may be up to 40 mm;tolerance in vertical translation (depth under the pavement surface) may be up to 30 mm, but this value must be met by a minimum of 75% of dowels in the joint and the remaining 25% of dowels in the joint may be up to 50 mm;tolerance in longitudinal translation  may be up to 75 mm, but this value must be met by a minimum of 75% of the dowels in the joint and the remaining 25% of dowels in the joint may be up to 120 mm.The material parameters of common dowels comply with Czech standard ČSN 736123–1 and European standards EN 13877–1 and EN 13877–3 ,21,22. Tm (MPa), in the range of 1 to 2 500 dowels. The requirement of steel quality to achieve class S 235, which is commonly used, and the quality of coating is not monitored.Durability of dowels must be ensured by protective factory-applied coating or applied in a construction site in compliance with national standards applicable in the country of use. Anticorrosion measures must meet the requirements of national standards or regulations in the country of use. The standards define dowels used for class CB I cement concrete road pavements, which must be of the minimum diameter of 25 mm and minimum length of 500 mm. The whole length of the dowel must be covered with a thin plastic film of 0.3 mm thickness, which must guarantee corrosion protection and at the same time allow the dowel slip in concrete. The coating must meet special guideline (TP 136) requirements. Different coatings can be used for class CB II. The coating for CB I must be factory-applied. However, the manufacturer must document thickness measurement of the dowel plastic coating as well as the dowel tensile strength RDowels must have tensile strength Rm at least 250 MPa in compliance with EN ISO 15630–1 . DiameteThe above mentioned sources and information make it obvious that the issue of using dowels in JPCPs may significantly affect their durability. Some of the main aims of the study include verifying behavior of different types of dowels  in laboratory conditions and show shortcomings when used. Another aim of the research activities is to formulate research conclusions and results while taking into account foreign authors’ experience, which would subsequently be integrated in effective regulations and use in practice in Slovakia and the Czech Republic.A concrete mixture was prepared in laboratory. The concrete used in this test was prepared in accordance with the design of mixtures for constructions of highways by the Road and Motorway Directorate of the Czech Republic. The selected water to cement ratio was 0.40, and the used cement was CEM I 42.5 R, which was manufactured by Heidelberg Cement Group. The coarse aggregate was granite, gradation category Gc 90/15, with nominal maximum aggregate size of 22 mm. Fine aggregate was river sand complying with ČSN EN 933–1 and ČSN 736123–1 specification ,25. The The specified expanded measurement uncertainty is a product of standard measurement uncertainty and the coverage factor k = 2, which corresponds with the coverage probability of approximately 95% for normal distribution. The standard measurement uncertainty was determined in compliance with the document EA 4/16 .basic position (in the middle of the beam);vertical translation (downwards—20 mm);vertical tilt (towards the force—20 mm).In order to verify theoretical modelling results of tensile stress of concrete around dowels, static and cyclic tests were performed on concrete beams with dimensions of 150 × 150 × 350 mm with selected cases of defective positions of dowels:The aim of the tests was to find whether the dowel position in the slab has an impact on tensile stress in the vicinity of a dowel (particularly the existence of high tensile stress) and whether there is a risk of concrete damage. Findings from American tests were extended and beams with defective positions of dowels were tested. The selected variants were those that showed the highest stress in concrete by the theoretical calculation of the finite element method by ANSYS software and whose built-in dowel position allowed the installation of strain gauges ,34.The scheme of the loading test is based on an American study and the tests performed at West Virginia University, USA. The study confirmed occurrence of high concentrations of stress in concrete in the vicinity of dowels located in the central line plane of the concrete beam. The loading scheme for tests created by CDV’s researchers is shown in The measured relative concrete deformations were determined with the use of glued strain gauges. The results were converted to stress according to Hooke’s Law and compared. Tensile and compressive stress σ is considered linearly proportional to its fractional extension or strain ε by the modulus of elasticity E:Adhesive strain gauges were used for these tests. They are applied onto clean, dry and flat base of the concrete beam face in areas with the highest stress concentration, theoretically determined by the finite element method  50 and 2The principle of the static test was gradual loading of the beam by force applied by the head of the press machine and located above the dowel. The load increment was selected to be 50 N/s, the deflection of the top of concrete beam were monitored by displacement sensors and values of measured relative deformations by strain gauges. This procedure allowed to accurately determine relative deformations and deflections in relation to the applied force.Beams “B”, so-called “superbeams” with dimensions of 270 × 270 × 300 mm were already tested with the aim of simulating loading in the scale 1:1 to the real road pavement thickness . After aCoating of dowels used nowadays in constructions of highway pavements and airports is produced from thermoplastic powder on the basis of modified polyolefins (marked PE plastic), as mentioned above. Dowels with these coatings have been used in the Czech Republic since the beginning of cement concrete pavement construction in 1996. Therefore, laboratory tests also focused on the quality of this epoxy coating. A detailed analysis of possible causes for low values of interaction found the potential main cause to be the low hardness of dowel coatings, which may cause its squeeze and damage at the interface with the cement concrete. Low resistance of the existing coating was clearly proven by the following loading tests of beams with an inserted dowel . ConsideAdjacent concrete slabs expand and shrink due to temperature loading. Dowels are required to allow horizontal displacement of concrete slabs. The aim of the tests was to assess the longitudinal displacement of dowels in the cement concrete.The tests were performed by gradual extracting and pushing of the dowel in a modified tensile testing machine. This test is not a standardised method and is used for a relative comparison of the force necessary to pull out and push back dowels to the beam under different temperatures and use of different dowel types. The maximum of ten cycles of gradual extracting and pushing of dowels into the beam were performed .Regarding solutions to theoretical issues in the field of concrete road pavement behavior and reinforcement of transversal joints, an experiment in cooperation with Slovakia and Poland was performed with an aim to determine concrete slab behavior in relation to different designed diameters of dowels. The experiment was applied to currently ongoing construction of highway S8 Wroclaw–Warsaw.Six testing specimens were designed and produced directly in the construction site of the highway for testing. Their dimensions were as follows : beam heConcrete B40 (C35/40) was used for the beam production. The concrete was produced directly in the concrete plant in the construction site with the original recipe that was designed and used for highway S8 Wroclaw–Warsaw. In the experiment, the used dowels were those with the PE coating. They were produced from smooth steel S 235 JR with the diameters of 20, 25, 30, 40 mm and length of 500 mm, PE coating thickness of 0.3 mm in compliance with standard EN 13877-3.The experiment focused on properties and behavior of a steel dowel in a concrete beam in terms of overall durability, deformation properties and potential beam damage type. Forces applied to a single dowel were determined according to an analytical model derived from the solution by Timoshenko and Lessels, for a differential equation of the deflection of a beam on an elastic foundation .A loading system a was preThe results in The results in Dowel coatings with PE plastic, new plastic TUR, and new fiberglass with special surface treatment that should ensure dowel longitudinal displacement in the beam were tested. The comparison of results in Static loading test results for individual beams are shown in more detail in During gradual loading up to the state when the beam with the lowest diameter (Ø 20 mm) was damaged, its deformation occurred already at the force of 10.3 kN and loading could not continue. The concrete beam remained undamaged a. In theThis paper presents the main findings of laboratory testing of dowels used in the Czech Republic and Slovakia. The results show high tensile stress in concrete around dowels and confirm the results of theoretical modelling by FEM. Laboratory test results showed high stress around the dowel, which is higher for the traditional diameter of 25 mm than for the diameters of 30 and 40mm. Tensile stress values were experimentally determined in concrete for loading of 10 kN, which is the maximum force that traffic loading may be applied to a dowel. Since the tensile strength of concrete is lower  than theThe effects of defective positions of dowels in critical variants were analyzed. The downward vertical translation of the dowels by 20 mm has no major effect on tensile stress results. The highest tensile stress values were examined in the variant with the vertical tilt (20 mm) of the dowel. Therefore, it is essential to maintain the specified position with tolerances given in standard ČSN 736123-1, with particular attention paid to the vertical and horizontal tilt of the dowel. The number of defectively installed dowels in every transversal joint must be taken into account and their effect on road pavement durability must be assessed, e.g., by pavement modelling with the use of the finite element method.New coating types with higher hardness and resistance to mechanical damage were tested. Higher quality of monitored parameters, such as longitudinal displacement of dowels in concrete and resistance against loading, was reached and verified with the use of newly developed nonstandard testing. Fiberglass based coatings failed to prove to be effective in terms of longitudinal displacement in concrete and they would probably work in the structure as anchors. Therefore, damage may occur during the JPCP expansion. A new type of coating of dowels, TUR, has lower values of tensile stress around the dowels and is currently fully used in the constructions and repairs of JPCPs on highways and in airports.


Infections caused by antibiotic resistant pathogens are increasing, with antibiotic overuse a key contributing factor.The CareTrack Kids (CTK) team assessed the care of children in Australia aged 0–15 years in 2012 and 2013 to determine the proportion of care in line with clinical practice guidelines (CPGs) for 17 common conditions. This study analyses indicators relating to paediatric antibiotic overuse to identify those which should be prioritised by antimicrobial stewardship and clinical improvement programs.A systematic search was undertaken for national and international CPGs relevant to 17 target conditions for Australian paediatric care in 2012–2013. Recommendations were screened and ratified by reviewers. The sampling frame comprised three states containing 60% of the Australian paediatric population . Multi-stage cluster sampling was used to select general practices, specialist paediatric practices, emergency departments and hospital inpatient services, and medical records within these. Medical records were reviewed by experienced paediatric nurses, trained to assess eligibility for indicator assessment and compliance with indicators. Adherence rates were estimated.Ten antibiotic overuse indicators were identified; three for tonsillitis and one each for seven other conditions. A total of 2621 children were assessed. Estimated adherence for indicators ranged from 13.8 to 99.5% while the overall estimate of compliance was 61.9% (95% CI: 47.8–74.7). Conditions with high levels of appropriate avoidance of antibiotics were gastroenteritis and atopic eczema without signs of infection, bronchiolitis and croup. Indicators with less than 50% adherence were asthma exacerbation in children aged > 2 years , sore throat with no other signs of tonsillitis , acute otitis media in children aged > 12 months who were mildly unwell , and sore throat and associated cough in children aged < 4 years .The results of this study identify four candidate indicators  for monitoring by antibiotic stewardship and clinical improvement programs in ambulatory and hospital paediatric care, and intervention if needed. There have been substantial concerns relating to antibiotic overuse in recent decades. Antibiotic overuse can accelerate the rate of development of antibiotic resistance , and conThe extent of overuse drives the concern about emerging resistance. In Australia, General Practitioners (GPs) are estimated to be prescribing between four and nine times as many antibiotics as would be expected for acute respiratory tract infections (RTIs) if they were following published guidelines . For paeA variety of drivers of antibiotic overuse have been proposed, including: that parents have expectations for prescribed antibiotics for mild infections ; that heTo identify intervention targets for unnecessary antibiotic use it is important to first identify conditions where antibiotics are used regularly but without known benefit. The CareTrack Kids (CTK) team assessed care of Australian children aged 0–15 years in 2012 and 2013 to determine the proportion who received care in line with clinical practice guidelines (CPGs) for 17 common conditions . Across The CTK methods have been described in detail, elsewhere , 22. We We modified and applied the RAND-UCLA method to develop indicators . A cliniA systematic search for Australian and international CPGs relevant for the years 2012–2013 yielded 99 CPGs for clinical conditions under consideration, from which 1266 recommendations were extracted. We screened recommendations for eligibility and excluded based on four criteria: (1) their strength of wording (“may” and “could” statements were excluded); (2) a low likelihood of the information being documented; (3) guiding statements provided without recommended actions; and (4) aspects of care deemed out of scope of the CTK study such as “structure-level” recommendations; 322 recommendations were excluded, with the remaining 944 recommendations used to draft initial indicators. The recommendations were converted into a structured and standardised indicator format [n = 55) and external review (n = 91) [Proposed indicators were ratified by experts over a two-stage multi-round modified Delphi process, which comprised an email-based three-round internal review and a collaborative, online, wiki-based two-round external review, custom-designed for the study . In tota(n = 91) . A clini(n = 91) , using a(n = 91) , and wor(n = 91) .In the internal review, experts scored each indicator against three criteria; acceptability, feasibility and impact ; recommeCTK identified 6689 medical records for 17 conditions. If any of the 6689 medical records we sampled contained a visit for any of the 17 conditions, a separate assessment of appropriateness was made for each visit. Detail on the general sampling methods are provided elsewhere ; additioAt indicator level, estimates of compliance were measured as the percentage of eligible indicators  which were scored as ‘Yes’. Weights were constructed to reflect the estimated incidence of presentation of each condition in each sampling unit separately for each setting, as briefly outlined in the Additional file We received primary ethics approval from relevant bodies including the Royal Australian College of General Practitioners (NREEC 14–008) and state hospital networks , and site-specific approvals from 34 hospitals. Australian human research ethics committees can waive requirements for patient consent for external access to medical records if the study entails minimal risk to HCPs and patients ; all relDetails of the 2621 children with one or more eligible assessments of CPG compliance for antibiotic overuse are provided in Table Of 12,562 possible antibiotic overuse indicator assessments, 3237 (25.8%) were automatically filtered out by age or setting restrictions, and surveyors designated a further 5344 (42.5%) as not applicable or otherwise ineligible. Surveyors conducted 3981 indicator assessments during 3935 visits. Eligible antibiotic overuse assessments were conducted in 81 GP and 18 paediatric practices, 34 hospital EDs and 29 hospital inpatient services.Children with acute otitis media aged > 12 months who were mildly unwell were not prescribed antibiotics.”) to 99.5% for ECZE07 (“Children with atopic eczema and no signs of infection were not prescribed antibiotics.”). Estimated adherence across the ten indicators was 61.9% (95% CI: 47.8–74.7).The estimated adherence for each of the 10 overuse indicators is shown in Table The overall estimate for compliance with antibiotic overuse indicators masks substantial heterogeneity. The two overuse indicators with near perfect compliance were for children with gastroenteritis or eczema without signs of infection . Overuse indicators for mild to moderate bronchiolitis , croup  and fever with no clinical focus aged > 3 years  all had moderately high levels of compliance.Overuse indicators for asthma exacerbation  and sore throat  had poor compliance, under 50%. The vast majority of children with acute otitis media who were mildly unwell , and children with sore throat and associated cough  received antibiotics contrary to recommendations. As there were three indicators of antibiotic overuse for tonsillitis, we calculated an overall estimate across the three indicators of 24.3% (95% CI: 16.1–34.2).all overuse indicators in the broader CTK study , which included overuse of other tests and treatments [Our overall estimate of compliance with antibiotic overuse indicators was 61.9% (95% CI: 47.8–74.7) across 10 indicators. This estimated rate of adherence to guidelines advocating against antibiotic overuse was markedly lower than that found for eatments . There wFirst, we consider indicators with higher compliance. In relation to antibiotic use for eczema without signs of infection (99% adherence) or fever in well infants aged > 3 years (79%) we are not aware of other studies reporting adherence rates to which we can compare. For gastroenteritis, a study of Welsh GPs found only 2.4% rate of antibiotic use for children , reflectFor indicators with lower adherence, published research provides some insights without directly assessing the indicators measured in our study. For asthma, an assessment of the management of acute exacerbations in children and adolescents found that antibiotic use was only half as frequent in specialist paediatric EDs as it was in general EDs , suggestThe CTK study found an 86% rate of antibiotic prescription for children with acute otitits media (AOM) who were mildly unwell , across ambulatory and non-ambulatory patients. This result may reflect that there is no consensus surrounding the use of antibiotics vs expectant management in the management of mild AOM. Importantly, the CTK study did not distinguish between antibiotics prescribed for immediate use and those prescribed as a ‘back-up’ to allow treatment to commence promptly, if required; both would have been classified as non-compliant. Australia’s move to nationally funded universal coverage with the pneumococcal vaccine for infants in 2005 , followiOur study found a 59% rate of antibiotic prescription for sore throat on its own, and an 86% antibiotic prescription rate for sore throat and cough in children aged < 4 years. The results for sore throat and cough broadly agree with Australian GP survey data for ‘tonsillitis’ in children aged < 5 years (89% prescribed antibiotics); data for diagnosis of ‘throat symptoms/complaint’ was not examined in this study .Given the many decades of concern about antibiotic overuse, our findings for asthma, sore throat and otitis media were disappointing. A number of potential reasons for antibiotic overprescribing have been suggested, including clinical time constraints, diagnostic uncertainty, risk aversion associated with fear of litigation, patient health beliefs and literacy and the knowledge, skill and attitude of clinicians –38. It hSoundly executed interventions can improve compliance with antibiotic guidelines. A review of clinician-targeted interventions to reduce antibiotic prescribing for acute respiratory infections in primary care found benefits – particularly, those interventions targeting point of care testing for C-reactive protein (22% reduction), shared decision-making (56% reduction) and procalcitonin-guided management (90% reduction). The quality of evidence for interventions focused on clinician educational materials and decision support was too poor to confidently assess . TraininThe strengths and weaknesses of the CTK study are described in additional detail elsewhere , and areThere are also strengths and weaknesses which are specific to our assessment of antibiotic overuse in the present study. The study was not designed to assess compliance with CPGs which address antibiotic overuse overall; rather, we assessed compliance with CPG indicators associated with 17 clinical conditions, ten of which were about antibiotic overuse. The overall estimates of compliance with overuse indicators can only therefore be generalised to these conditions. As a strength, however, it should be noted that most studies examining antibiotic use are restricted to reporting prescribing rates, without an assessment of compliance with a specific CPG; our study, by contrast, trained experienced paediatric nurses to assess compliance with specific CPGs.Our results help to identify conditions with high levels of antibiotic overuse in paediatric settings. The methods we used can be adapted to measure non-adherent antibiotic prescribing for a broader range of clinical conditions, to prioritise targets for intervention and increase guideline compliance. Such studies should consider distinguishing between prescriptions provided for immediate filling and those provided in case of deterioration. This information can be used by existing antimicrobial stewardship programs and clinical improvement programs in primary care, to prioritise targets for intervention.There is a need to achieve substantial and sustained reductions in over prescription of antibiotics. Our study identified four presentations with > 50% antibiotic prescription, contrary to guidelines: AOM in mildly unwell children aged > 12 months; children with sore throat and cough in children aged < 4 years; children with sore throat and no other signs of tonsillitis; and children aged > 2 years presenting with an acute exacerbation of asthma.Additional file 1. Additional details relating to study methods.


Antibiotics are not indicated for treating acute bronchitis cases, yet up to 70% of adult acute bronchitis medical visits in the USA result in an antibiotic prescription. Reducing unnecessary antibiotic prescribing for acute bronchitis is a key antibiotic stewardship goal set forth by the Centers for Disease Control and Prevention. Understanding what factors influence prescribing for bronchitis cases can inform antimicrobial stewardship initiatives. The goal of this study was to identify factors associated with antibiotic prescribing at a high-volume student health center at a large US university. The Pennsylvania State University Health Services offers on-campus medical care to a population of over 40,000 students and receives over 50,000 visits every year.We conducted a retrospective chart review of acute bronchitis visits for the 2015–2016 academic year and used a multivariate logistic regression analysis to identify variables associated with antibiotic prescribing.Findings during lung exams increased the likelihood of an antibiotic prescription . Individual clinicians had dramatically different rates of prescribing (OR range 0.03–12.3). Male patients were more likely than female patients to be prescribed antibiotics . Patients who reported longer duration since the onset of symptoms were slightly more likely to receive prescriptions , as were patients who reported worsening symptoms . Visits with diagnoses or symptoms associated with viral infections or allergies were less likely to result in prescriptions (upper respiratory tract infection (URI) diagnosis OR 0.33, 95% CI 0.18–0.58; sneezing OR 0.39, 95% CI 0.17–0.86; vomiting OR 0.31, 95% CI 0.10–0.83). An exam finding of anterior cervical lymphadenopathy was associated with antibiotic prescribing .Suspicious findings during lung examinations  and individual healthcare providers were important factors influencing antibiotic prescribing rates for acute bronchitis visits. Patient gender, worsening symptoms, duration of illness, symptoms associated with viral infections or allergies, and anterior cervical lymphadenopathy also influenced prescribing rates. In the United States, 30% of outpatient antibiotic prescribing is estimated to be unnecessary, resulting in almost 47 million unnecessary antibiotic prescriptions each year , 2. ExceA major source of unnecessary outpatient antibiotic prescriptions is acute bronchitis cases –10. AcutAlthough acute bronchitis presents an opportunity to improve antibiotic stewardship, there is little consensus regarding effective stewardship interventions for ambulatory care –24. A diIdentifying drivers of prescribing for acute bronchitis could suggest potential interventions, but relatively few studies have focused on identifying these predictors. Prior studies of upper respiratory tract infection prescribing (including for acute bronchitis) in the USA have shown higher rates of antibiotic prescribing in rural (vs. urban) practices , 10, wheUniversity student health clinics provide an opportunity to study antibiotic prescribing in young adult patient populations. In the US, college students comprise a sizeable cohort of the population, with 20.1 million students enrolled in higher education, including 13.8 million students enrolled at 4-year degree-granting institutions . At thesThe goal of this study was to identify patient and visit factors associated with antibiotic prescribing for young adults diagnosed with acute bronchitis at a high-volume student health center at a large US university. We conducted a retrospective chart review of all visits with an acute bronchitis diagnosis for the 2015–2016 academic year at the Pennsylvania State University’s Student Health Center. This work is part of a multi-study interdisciplinary effort to improve antibiotic stewardship in emerging adult populations, with an initial focus on students at residential colleges.The Pennsylvania State University Health Services (UHS) offers on-campus medical care to PSU students and their dependents, serving over 40,000 students in more than 50,000 visits yearly. At the time of the study, 28 clinicians saw patients at UHS. During the study period, 21 of these clinicians (9 doctors of medicine (MDs), 2 doctors of osteopathic medicine (DOs), 8 physician assistants (PAs), and 2 nurse practitioners (NPs)) diagnosed at least one patient with acute bronchitis. The remaining clinicians did not diagnose acute bronchitis in the period studied, and therefore they do not appear in the data set.N = 69; ~ 5%) of the visits. This check indicated adequate data quality  for the intended analyses; identified discrepancies were corrected [UHS staff identified 1451 visits with acute bronchitis diagnoses during the 2015–2016 academic year (August–May). Honest brokers were then employed and trained to access the electronic medical records for these visits, extract deidentified data , and enter it in the secure database manager REDCap for use by the researchers. Data extracted included patient characteristics, visit characteristics, symptoms recorded, exam findings, secondary diagnoses, tests ordered, and antibiotic prescriptions . One thousand thirty-one visits were included in the final analysis  were uncommon in acute bronchitis visits. To narrow the list to variables that might be important in acute bronchitis visits, as well as to eliminate variables with zero frequency cells in univariate contingency tables, we only considered symptoms and exam findings recorded for > 20 patients for subsequent analysis Table .Four visits had onset durations that were extreme outliers (> 100 days since onset), and we substituted missing values for these onset durations. Models excluded visits with missing values in predictor variables. This strategy resulted in 33 visits being excluded from analysis in the final multivariate model due to missing values in predictor variables. It is important to note that for two patient-reported variables, severity and progression, “not recorded” was coded as a factor level, and these entries were not considered missing values.p-values < 0.05. Analyses were carried out using R (version 3.4.3).In all analyses, the response variable was whether an antibiotic was prescribed at a visit. All variables listed in Table The data set included 1031 visits with an acute bronchitis diagnosis Table a. FigureFactors independently associated with antibiotic prescribing in a multivariate regression model are summarized in Table The model showed that prescribing rates decreased slightly over the course of the academic year . Male patients were more likely than female patients to be prescribed antibiotics . Patients who reported longer duration since the onset of symptoms were slightly more likely to receive prescriptions , as were patients who reported their symptoms were worsening . Visits with additional diagnoses or symptoms associated with viral infections or allergies were less likely to result in prescriptions . An exam finding of anterior cervical lymphadenopathy was associated with antibiotic prescribing .N = 149) for previously diagnosed conditions , despite treating the same patient population at the same clinic. These results suggest that a subset of providers can drive a disproportionate amount of unnecessary antibiotic prescribing for acute bronchitis. In the current study, provider traits  were not included in the logistic regression analysis due to the small number of providers in the data set . Previous studies have identified provider specialty, provider age, and perceived patient demand for antibiotics as factors influencing provider prescribing rates for upper respiratory tract infections , 36, 37.A second important driver may be diagnostic uncertainty. In the present study, prescriptions were much more likely when findings of rales, rhonchi, or percussion abnormalities were recorded during lung examination, and somewhat more likely when external anterior cervical lymphadenopathy was reported. Rales and percussion abnormalities increased prescribing 13-fold, and rhonchi increased prescribing 5-fold. This increase in prescribing may reflect suspicion of pneumonia. Orders of chest x-rays, which also indicate suspicion of pneumonia, were a significant predictor of prescribing in a bivariate analysis, but were not significant in a multivariate model due to high correlation with other lung exam findings. Providers may prescribe antibiotics when there is suspicion of a condition that would respond to antibiotics or general diagnostic uncertainty , and thiOther predictors of prescribing in this study included symptoms of sneezing and vomiting, reported worsening of symptoms, diagnosis of an upper respiratory tract infection, duration of illness, and patient gender. Duration of illness has previously been associated with prescribing for upper respiratory tract infections . PatientStreptococcus and influenza [The identification of provider variation and diagnostic uncertainty as drivers of prescribing suggests possible interventions for this clinic and similar settings. Provider variation points to a need for provider-targeted interventions such as audit and feedback, communication training, provider education, or clinical decision support tools , 23. In nfluenza . There influenza .While unnecessary prescribing for acute bronchitis was common in our data, the rate of prescribing was substantially lower than the nationwide average. In the 2015–2016 academic year, antibiotics were prescribed at less than a third of acute bronchitis visits, compared to national rates near 70% , 16. TheOur study is unique in its focus on antibiotic prescribing practices at a university health center. University health services are important centers for antibiotic prescribing serving millions of patients, yet they have largely been overlooked as sites for antibiotic stewardship. To our knowledge, the Pennsylvania State University is the first university with a student antibiotic stewardship program. This study is the first to identify drivers of antibiotic prescribing in a university health center, and one of the few to focus on young adults or consider exam findings and symptoms from patient charts as possible predictors of prescribing. We hope that these findings can be used to inform antibiotic stewardship initiatives at university health centers and similar clinical contexts. Our results suggest that unnecessary antibiotic prescribing is disproportionately driven by a subset of clinicians, and interventions targeting providers may be effective at reducing unnecessary prescribing.Reducing unnecessary antibiotic prescribing for acute bronchitis cases is a national antibiotic stewardship goal, yet rates of unnecessary antibiotic prescribing remain stubbornly high nationwide. Here we identified factors that influence antibiotic prescribing for acute bronchitis cases at a large university health center. Suspicious findings during lung examinations  and individual healthcare providers were the most influential factors affecting antibiotic prescribing rates for acute bronchitis visits. Patient gender, worsening symptoms, duration of illness, symptoms associated with viral infections or allergies, and anterior cervical lymphadenopathy also influenced prescribing rates.


Medulloblastoma is an embryonal tumor that shows a predilection for distant metastatic spread and leptomeningeal seeding. For most patients, optimal management of medulloblastoma includes maximum safe resection followed by adjuvant craniospinal irradiation (CSI) and chemotherapy. Although CSI is crucial in treating medulloblastoma, the realization that medulloblastoma is a heterogeneous disease comprising four distinct molecular subgroups  with distinct clinical characteristics and prognoses has refocused efforts to better define the optimal role of CSI within and across disease subgroups. The ability to deliver clinically relevant CSI to preclinical models of medulloblastoma offers the potential to study radiation dose and volume effects on tumor control and toxicity in these subgroups and to identify subgroup-specific combination adjuvant therapies. Recent efforts have employed commercial image-guided small animal irradiation systems as well as custom approaches to deliver accurate and reproducible fractionated CSI in various preclinical models of medulloblastoma. Here, we provide an overview of the current clinical indications for, and technical aspects of, irradiation of pediatric medulloblastoma. We then review the current literature on preclinical modeling of and treatment interventions for medulloblastoma and conclude with a summary of challenges in the field of preclinical modeling of CSI for the treatment of leptomeningeal seeding tumors. Medulloblastoma is a posterior fossa embryonal tumor of neuroectodermal origin that is thought to arise from progenitor cells present during early brain development ,2,3. It Like many embryonal tumors, medulloblastoma shows a predilection for distant metastatic spread and leptomeningeal seeding, with subarachnoid dissemination present in approximately 20% to 35% of cases at diagnosis and in most cases at relapse ,8. CSI tThe evolution of RT for medulloblastoma has principally involved delaying or omitting RT in very young patients and reducing RT dose and target volumes in older patients. In the past, patients with medulloblastoma, irrespective of age or tumor characteristics, were treated to high doses to the craniospinal axis, with doses ranging from 36 to 40 Gy, followed by focal boost to the posterior fossa for a cumulative dose of 54 to 60 Gy ,21. DevaNotably, these studies did not directly compare reduced-dose CSI in combination with chemotherapy to high-dose CSI and boost RT alone in standard-risk patients specifically. The only randomized trial in which this was done, in fact, showed that adding chemotherapy to RT had a negative impact on survival in standard-risk patients . That saReductions in the primary-site boost volume have also been evaluated. Initial retrospective pattern-of-failure studies suggested a low incidence of isolated posterior fossa recurrence outside the primary tumor bed. Subsequent pilot studies demonstrated the feasibility of reducing boost fields from the entire posterior fossa to the more limited postoperative surgical bed ,42,43. MBased primarily on DNA methylation analysis, medulloblastoma is known to be a heterogeneous disease comprising four distinct molecular subgroups  with significant differences in their clinical and pathologic characteristics, genetic drivers of disease development and progression patterns, and clinical prognosis ,46,47,48Irradiation techniques and delivery modalities for CSI have also evolved. Multiple studies have demonstrated the critical importance of ensuring adequate coverage of the entire target volume, encompassing the meninges covering the brain and spinal cord, including extensions along the nerve roots, with more frequent relapses and inferior survival being observed in patients treated with CSI with targeting deviations ,59,60. TDeveloping new and more effective therapeutic approaches to treating pediatric medulloblastoma requires the use of accurate preclinical models to inform clinical trial design. Several groups have developed murine models of medulloblastoma, primarily in an effort to create preclinical testing tools. These models range from genetically engineered murine models (GEMMs) to patient-derived orthotopic xenografts (PDOXs) . Next-geMedulloblastoma presents unique challenges with regard to preclinical modeling and therapeutic evaluation. The genetic and molecular heterogeneity of the disease dictates the use of multiple in vitro and in vivo models to evaluate response variability. Interrogation of novel and optimized therapies has been conducted in cell lines or xenograft models derived from naïve patient biopsies or in mouse models of primary disease. These experimental therapies are, thus, delivered “at diagnosis” and not in a recurrent setting after standard therapy. Although upfront treatment approaches are certainly under evaluation, most clinical trials enroll patients who have already undergone surgery, chemotherapy, and/or RT. Another assumption often made in preclinical testing is that the biology of recurrent disease is largely similar to that of the tumor at primary diagnosis. Work by Taylor and colleagues revealed that medulloblastoma recurrence is driven largely by clonal selection and that dominant clones present at recurrence may contain molecular targets that differ considerably from those in clones that predominate at diagnosis . Medullo®) micro-irradiator  and the X-Rad 225Cx image-guided small animal irradiator  [Analysis of in vitro and in vivo preclinical studies of radiation-related interventions suggests that improving their efficacy will require the incorporation of standard-of-care modalities that use clinically relevant and carefully delivered RT regimens . Optimizy, Inc.) ,77. BothThe first study to evaluate image-guided delivery of radiation in a preclinical setting for medulloblastoma was conducted by Taylor and colleagues . Using tMYCN amplified, TP53 mutation) and G3 medulloblastoma (MYC amplified) [6 photons/s, which corresponds with histologically identifiable tumor burden but no overt clinical symptoms or detection by MRI in vivo. The intent was to model minimal disease burden, as in a patient who had undergone tumor resection. CSI proved superior to whole-brain irradiation with regard to tumor coverage and recurrence patterns, highlighting the necessity of encompassing the entire neuraxis and the importance of image guidance in facilitating this   labeled ing this . IteratiMore recent work by Huang et al. incorporated CSI and boost irradiation in a preclinical setting to explore potential combination therapy in G3 and G4 medulloblastoma PDOX models . Using tIncorporating RT into the preclinical pipeline of medulloblastoma models will be critical to translational efforts to define novel therapeutic approaches for this disease, particularly for the highest-risk subsets, for which overall survival rates remain disappointingly low. Although limited in their scope, the studies performed to date have provided valuable insights into study design for these therapies and have raised important questions .The strong response to CSI in murine PDOX models is consistent with the positive responses in patients after upfront therapy. However, in several preclinical medulloblastoma models, clinically comparable CSI doses appear to be curative, with no tumor burden or disease progression being detected for hundreds of days after the completion of treatment. One important consideration is that the low-energy photons characteristic of small animal irradiators may exert a more substantial biological effect than the particles from clinic-based high-energy sources. In addition, there may be significantly fewer tumor clonogens, or tumor stem cells, within any orthotopic xenograft than in human patients; if this is the case, a “murine-equivalent” CSI dosing regimen will be much different from that currently employed clinically. This also raises critical questions for translational brain tumor research in general: What is the optimal tumor burden at the start of preclinical survival or tumor growth-delay studies, and what is the best modality for determining enrollment threshold  (discussed below) ,80,81? AOutstanding questions also remain regarding boost treatment. Preclinical studies employing CSI in medulloblastoma models have not routinely incorporated a boost phase of radiation after the CSI. However, patients aged 3 years or older most commonly receive wide-field radiation encompassing the brain and spine followed by a local boost to the tumor bed, whereas tumor-bed irradiation alone has been evaluated in younger children. Therefore, it is important to evaluate preclinically the role played by the additional boost RT or focal boost therapy alone. Our group has initiated studies to determine the value of an acute boost of irradiation after chronic treatment, but the spatial and temporal control of the delivery is not yet optimized, in part because of the enhanced RT response observed in our PDOX models. With regard to the RT modality, a recent advance in preclinical RT studies that may hold particular promise for medulloblastoma translational research is the development of image-guided proton therapy for use in small animals ,84.Another important consideration illustrated by the studies discussed above that employed image guided CSI is the optimal enrollment threshold. Whether imaging modalities or clinical symptoms are assessed, a consensus must be reached on the most appropriate time for study initiation. It has been challenging to determine the murine parallel to intracranial tumor burden while ensuring reasonable disease progression and study timelines. Similarly, the incorporation and timing of surgical resection in the preclinical pipeline has yet to be optimized. Whereas Taylor and colleagues were able to model resection and CSI in vivo to molecularly characterize disease recurrence , Smith aFindings from our laboratories suggest that additional dose titration is needed to accomplish clinically relevant exposure while facilitating a therapeutic window for adjuvant therapy. A short-term survival advantage with subsequent recurrence of disease was achieved using 10 Gy of CSI, but the administration time (5 days) was limited when compared to the RT treatment window of approximately 6 weeks. Unpublished work by our group has evaluated the feasibility of protracted CSI dosing in several murine and PDOX models of medulloblastoma, in which the daily dose of RT is reduced to between 1 and 0.5 Gy per fraction but the cumulative CSI dosing is similar to that delivered with 2 Gy fractions. By using these lower daily doses of radiation (0.5 to 1 Gy fractions) over 2 to 4 weeks for a cumulative dose of 10 to 20 Gy, we were able to establish a therapeutic window through lifespan extension, albeit with eventual disease progression . This protracted study design will facilitate the evaluation of concurrent systemic therapy  with CSI in the preclinical setting.TP53 status has a significant impact on radiation response, whereas WNT activation may overcome this effect [Finally, although the use of adjuvant chemotherapy has confounded the issue, it is tempting to speculate that there is a subgroup- and/or subtype-specific response to irradiation, particularly given the data from several laboratories demonstrating enhanced radiation sensitivity in medulloblastoma cells ,86. An es effect . With thMB encompasses a heterogenous group of embryonal tumors, consisting of four genetically defined subgroups with distinct molecular and clinical features and varied prognoses with current therapies. RT, targeting the entire neuraxis, represents a critically effective adjuvant therapy, yet carries the risk of significant long-term toxicities. Through the development of accurate and reproducible preclinical radiotherapy platforms modeling the clinical delivery of CSI, researchers are now poised to experimentally define optimal radiation dose-volume parameters, delineate molecular determinants of radiation response, identify radiotherapy adjuncts, and investigate normal tissue toxicity in emerging animal models systems that represent the distinct subgroups of MB. While important questions remain as to the optimal approaches of preclinical CSI, integration of this modality into translational studies offers the potential to better inform modifications to what has been the most studied treatment modality of clinical trials of MB.