Microbial reactor sensors  or microbial membrane sensors  can be used as convenient instruments for studying processes that cause the response of a biosensor, such as the properties of enzymes or the characteristics of metabolism. However, the mechanisms of the formation of biosensors responses have not yet been fully understood to study only one of these processes. In this work, the results of studies on the formation of a response to juglone for intact and immobilized bacterial cells used as receptors are presented. It was shown that the contribution of reactive oxygen species (ROS) to the formation of the biosensor response depends on the culture receptor and the form of juglone, quinone, or phenolate used. The response to the quinone form of juglone both for intact and immobilized cells of catalase-positive actinobacterium is formed regardless of the presence of ROS. The response of freshly harvested intact actinobacterial cells was caused by the rate of the enzymatic conversion of juglone. The rate of the response of immobilized actinobacterial cells was influenced by the activity of transport systems and metabolism. The response of immobilized pseudomonad cells was caused by the transport of juglone into cells, the inhibitory effect of juglone-induced ROS, and juglone metabolism. Juglone was obtained for the first time in 1851 while processing walnut fruits. Today, natural sources of juglone are plants such as black walnut (Juglans nigra) and white walnut (Juglans cinerea) and microorganisms; juglone is also obtained synthetically [Juglone is a natural compound of the naphthoquinone group, 5-hydroxy-1,4-naphthoquinone, Cetically .It is known that naphthoquinones are slightly soluble in water. A small amount of juglone (a quinone form) dissolves in water . When juJuglone is widely used in industry, agriculture, and medicine. This reddish-yellow crystalline substance is a natural pigment that is used in the textile industry as a natural dye for clothing and fabrics and in the cosmetics industry . JugloneIt was found that when naphthoquinones penetrate into a cell, they can induce oxidative stress  as a resThe enzyme that initiates the decomposition of juglone under aerobic conditions is juglone 3-monooxygenase  ,21,22. T2 + 1/2 O2 = 3,5-dihydroxy-1,4- naphthoquinone + A + H2O.5-hydroxy-1,4-naphthoquinone + AHA rapid method for the determination of juglone is based on application of sensor analyzers. A microbial sensor can be used for these purposes, for which the response to juglone is evaluated by the change in oxygen consumption by microbial cells in response to substrate addition, since the metabolism of juglone is initiated by an enzyme–substrate interaction that occurs with oxygen consumption. The development of a laboratory model of such a microbial sensor was started at IBPM RAS . MoreoveThe aim of this research was to study the formation of a response to juglone  for intact and immobilized bacterial cells used as receptors for microbial reactor sensors or microbial membrane sensors.Rhodococcus sp. 3 and bacterial cells of Pseudomonas sp. 4(c4), stored in a culture collection created by the authors of this study, were used in the present study. The cultures were maintained on the agarized Luria–Bertani (LB) medium at +4 °C and passaged every six months.Non-spore-forming actinobacterial cells of 2PO4 ; NaOH ; CuSO4 × 5H2O ; acetone ; catalase from bovine liver ; medium L.B. ; malt extract agar .In this work, the following reagents were used: KH−1 M stock solution of juglone in acetone. Using the stock solution, 1 × 10−2 M, 1 × 10−3 M, and 1 × 10−4 M solutions of juglone in acetone were prepared by the method of successive dilution. During the preparation of the dilutions and measurements, the solutions were kept in hermetically sealed vessels without access to light. Similarly, 1 × 10−1 M, 1 × 10−2 M, 1 × 10−3 M, and 1 × 10−4 M solutions of juglone were prepared using 5% NaOH solution and distilled water as solvents. The resulting alkaline solutions contained alkali in an amount of 0.008 g/mL.Crystalline substance, juglone, was used to prepare a 1 × 10Rhodococcus sp. 3 and Pseudomonas sp. 4 (c4) were grown on malt agar and on the agarized LB medium, respectively. Bacterial biomass was harvested and suspended in a 50 mM K-Na-phosphate buffer (pH 7.0) to give a final concentration of 100 mg wet weight per mL. Suspension of freshly harvested bacterial cells (intact cells) was immediately used for formation of the microbial reactor sensor [2 piece of paper. The obtained receptor elements were air-dried within 30 minutes at room temperature. Receptor elements were kept in a refrigerator at +4 °C for 24 h and then used for measurements or stored in the refrigerator.r sensor . The rem2 Amplifier, Switzerland-USA) was used to amplify this signal, and a two-coordinate recorder  was used to record electrical signals. The recorded rate of signal change was proportional to the rate of alteration in oxygen consumption by microbial cells in response to the juglone injected. Based on this, the first derivative of the electrode current change in the response to juglone injection  was calculated. The obtained parameter characterized the rate of change in oxygen consumption by bacterial cells in response to juglone (cells’ response to juglone).Laboratory models of microbial sensors were formed with the use of intact or immobilized cells of bacteria. The designs of recognizing parts of the microbial reactor sensor and microbial membrane sensor were given in our article . Figure In order to plot the “response-concentration” dependence, sets of standard solutions of juglone in acetone or in NaOH solution with different concentrations of juglone were used. The volume of juglone solution added into the measuring cell was no more than 20 μL.t-test (p < 0.05). It was thought that the differences between the values were statistically significant if the confidence intervals failed to cross.The presented results were averaged values of the measurements which were taken in triplicate in two independent series of experiments. Statistical analysis of the data was carried out using a Student’s The response of microbial cells to juglone has been studied in water medium. Only a small amount of the quinone form of juglone dissolves when it is added to water. Bearing this in mind, before the addition of juglone into the measuring cell, it was dissolved in acetone and, after that, in order to minimize the effect of acetone on the cell response, small quantities of acetone solution (the quinone form of juglone) were injected into the aqueous solution. A water-soluble alkaline solution of juglone (the phenolate form) without any treatment was added into the measuring cell. The addition of a small amount of alkali or acetone did not affect the cell respiration (data not shown).Rhodococcus sp. 3 . The addition of a water-insoluble substance—for example, acid in the form of water-soluble salt—is the conventional microbiological technique. However, in our case the curves plotted for the phenolate form and quinone form of juglone differed significantly. The rate of response to the quinone form of juglone was an order of magnitude higher than that of the phenolate form, and it attained its maximum when the substrate concentration was an order of magnitude lower. It was recorded both for intact (Rhodococcus sp. 3. A decrease in the response to the quinone form of juglone (the substrate) was observed when the juglone concentration was above 4×10−6 M and 1×10−4 M for intact and immobilized Rhodococcus sp. 3 cells, respectively, as a result of substrate inhibition.The phenolate form and quinone form of juglone were used to plot the curves of the dependence of the rate of the response to juglone on the initial juglone concentration for intact and immobilized cells of us sp. 3 . The mear intact a and immr intact b cells oThe detected difference in responses was obviously due to the presence of a hydroxyl group in the aromatic ring of the quinone form of juglone, which was involved in the induction of oxidative stress. However, there was no phenolic hydroxyl in the phenolate form of juglone. Therefore, both forms of juglone were used in the study of the cells’ response to juglone.Rhodococcus opacus 1CP [The rate of response to the substrate for intact cells was caused by the rate of enzyme–substrate interaction in the presence of the enzyme, which initiates substrate metabolism. It was shown in our previous studies with the laboratory model of the microbial reaction sensor formed with the use of intact cells of acus 1CP .Rhodococcus opacus 1CP to oxidative stress during cultivation in the presence of juglone [Rhodococcus sp. intact cells grown on the medium in the absence of juglone (without enzyme induction by substrate). There is no inducible enzyme in non-substrate-grown cells. The rate of intact cells’ response to substrate is 0 pA/s or does not exceed 1–1.5 pA/s in the presence of the base concentration of an inducible enzyme in non-substrate-grown cells. It should be noted that in the case of Rhodococcus sp. 3 (Rhodococcus sp. 3) is a constitutive enzyme.Juglone 3-monooxygenase is the enzyme that initiates the metabolism of juglone. We previously reported on the presence of a constitutive enzyme in rhodococci ; this wa juglone . “Responus sp. 3 a, the raPseudomonas putida bacterium, Rettenmaier and Lingens reported [4 can inhibit the enzyme activity completely. When copper sulfate inhibits the activity of juglone 3-monooxygenase of Rhodococcus sp. 3, the rate of interaction between the enzyme and juglone will be reduced. To estimate a contribution of the enzyme-substrate interaction to the formation of Rhodococcus sp. 3 cells response to juglone, intact and immobilized cells were incubated in the presence of CuSO4 before the addition of juglone  production occurs under the effect of the quinone form of juglone on microorganisms. Catalase, glutathione reductase, superoxide dismutase, peroxidase, and other enzymes take part in protecting microbial cells against oxidative stress.In aerobic conditions, juglone is a Hcompound ,30. Obvi2O2 and protected cells against the harmful effects of this compound. All the strains of rhodococci (Rhodococcus sp. 3 is one of the rhodococci) under study, unlike pseudomonades, were catalase-positive . Pseudomonades require externally introduced catalase to protect them from the action of H2O2. Thus, besides intact cells of Rhodococcus sp. 3, the cells of Pseudomonas sp. 4(c4) were applied for the formation of the laboratory model of the microbial reaction sensor.Seemingly, ROS formed under the action of the quinone form of juglone could take part in the formation of the response to juglone for microbial cells. With the aim of checking this supposition and estimating susceptibility of the cells of the culture-receptor to ROS, cell responses to the quinone form or the phenolate form of juglone were measured in the presence of catalase. Since catalase degrades hydrogen peroxide, this enzyme should have inactivated HRhodococcus sp. 3 and Pseudomonas sp. 4 (c4) display different levels of resistance to oxidative stress. H2O2 formed in the presence of the quinone form of juglone probably had an inhibitory effect on the cells of pseudomonade, leading to a decrease in the intensity of the cells’ response to juglone. In the presence of catalase, the effect of H2O2 on Pseudomonas sp. 4(c4) was inactivated, thereby confirming the findings of Zhang et al. [2O2 should not have a killing effect on intact cells of Pseudomonas sp. 4(c4), and juglone uptake into the cells should not have an inhibitory effect on the culture that can lead to an increase in the culture’s response to the substrate. This is what we observed. The increase in the response to the quinone form of juglone for Pseudomonas sp. 4(c4) intact cells was registered when the quinone form of juglone was used in the presence of catalase   cells did not contain their own catalase and could not inactivate H2O2 themselves.As expected, H. 4 (c4) b. UnlikeRhodococcus sp. 3 (Pseudomonas sp. 4(c4) (Rhodococcus sp. 3 possessed their own (intracellular) catalase, which protected them against ROS, without the need for the extra addition of extracellular catalase.Intact cells of us sp. 3 a, unlikep. 4(c4) b, did noPseudomonas sp. 4(c4) immobilized cells was similar to that of intact cells. As expected, no changes in the rate of the response to alkaline solution of juglone was observed for immobilized cells of Pseudomonas sp. 4(c4) (Pseudomonas sp. 4 (c4) contained an enzyme that initiates the metabolism of juglone. This was evidenced by the presence of the response to juglone for intact culture cells (Pseudomonas sp. 4 (c4). Therefore, enzymatic transformation of the substrate took place in these cells. The process of metabolism of the substrate in cells could occur only after substrate transport into the immobilized bacterial cells. The presence of the response to the quinone form of juglone indicated the activity of both processes: the process of metabolism of juglone in cells and the process of transport of juglone into cells. When ROS formed in the presence of the quinone form of juglone was inactivated by the addition of catalase, the transport of juglone into the immobilized cells of the culture receptor did not inhibit the immobilized cells and the transport could be activated. Furthermore, an increase in the rate of substrate transport into the cells of the culture–receptor led to an increase in the rate of enzymatic conversion of substrate. Therefore, the addition of catalase to immobilized cells of Pseudomonas sp. 4(c4) was accompanied by activation of both processes, which caused the response of immobilized cells of the microbial membrane sensor—namely, substrate transport into the cells of the culture–receptor and enzyme–substrate interaction. If ROS influenced both these processes, then ROS had an effect on the formation of the response to the quinone form of juglone in immobilized bacterial cells. Thus, the response to the quinone form of juglone for immobilized cells of Pseudomonas sp. 4(c4) was caused by processes such as juglone transport into the cells of the culture–receptor, the inhibitory effect of juglone-induced ROS, and juglone metabolism catalyzed by cell’s enzymes.p. 4(c4) b similarp. 4(c4) b in the Rhodococcus sp. 3 in the presence of catalase. ROS had no effect on the formation of the response of catalase-positive Rhodococcus sp. 3 immobilized cells.No changes in the cell response to the quinone form of juglone were observed for immobilized cells of Thus, the results of this study demonstrated that models of the microbial reactor sensor and the microbial membrane sensor formed on the basis of cells of various cultures can be used to explore the activity of an enzyme, which initiates the metabolism of juglone, the process of transport of juglone into cells of a microorganism, and the effect of ROS on microbial cells.


To explore and compare the age, source and future risk of COVID-19 infection in Hong Kong SAR China and Singapore as of March 5, 2020.We find significant difference in age patterns of confirmed cases in these 2 localities early in the pandemic.We highlight the potential importance of population age structure in confirmed cases, which should be considered in evaluation of the effectiveness of control effort in different localities. The coronavirus disease 2019 (COVID-19) pandemic first broke out in Wuhan, China in December 2019, and was declared as a global pandemic by the World Health Organization (WHO) on 11 March 2020 . SingapoWe obtained the data of the COVID-19 cases of Singapore and Hong Kong from Singapore and Hong Kong’s governmental websites , 6. The From the onset of COVID-19 to March 5, there have been 23 imported cases in Singapore and Hong Kong, respectively. The age distribution of imported and local cases can be seen in Additional file Since the elderly are more vulnerable to get infected after exposure and develop into severe cases, thus the population age structure, among other factors, should be considered when comparing the effectiveness of control measure in different localities.The situations of COVID-19 change fast worldwide. The COVID-19 data of Singapore with age information has been unavailable for public after we first submitted this work. Hence, our work performed on merely the data of Singapore and Hong Kong as of March 5. Although the patterns of infected cases may stay similar to some extent, the age structures of infected cases in both locations may be different now.Additional file 1: Table S1 The cases of Hong Kong and Singapore by March 5. Figure S1 Histogram of local/imported case numbers by age distribution in HK and Singapore by March 5.


We found that the associations between the average concentrations of NO Both th2, SO2, and CO on the transmission of coronavirus diseases 2019 (COVID-19) in China. Many studies have been done on the impact of meteorological factors and air pollutants on the spread and COVID-19 and the patient outcome, for example, focusing in China  growth rate, denoted by r, of the epidemic curve over the a 16-day period starting from the confirmation of the first case in each city , where C0 denoted the number of seed cases at the start of the outbreak. Using the formula R0 = 1/M(–r) that is derived from the Lotka-Euler equation , of the probability distribution of the COVID-19 serial interval. The distribution of serial interval is approximated by a Gamma distribution with mean at 5.5 days and standard deviation at 3.3 days  univariable, and (ii) multivariable linear regression, using R0 = 2.5 was detected in Wuhan City. The average concentrations of NO2, SO2, and CO were 31.0, 11.9 μg/m3, and 1.0 mg/m3, respectively. The spatial distributions of NO2, SO2, and CO are shown in The descriptive statistics of the basic productive numbers, three pollutants, temperature, and relative humidity for 154 cities are given in 2, SO2, and CO on the initial transmissibility of COVID-19 across Chinese cities. We performed sensitive tests by using spline regression (three degrees of freedom) and restricting the cities outside Hubei province only and found no significant association.In R0 varied in provinces with different population flow  and the initial transmissibility of COVID-19 in Chinese cities. Since we focus on the initial 16 days after the confirmation of the first case, the lockdown of cities should not have reduced the pollutant level in each city, as normally it will take time for pollutants to drop. Our averaged concentration should be a fair indicator of the level of air quality in each city.In this large-scale ecological study, we find no significant association between the three gaseous air pollutants (NOOther factors for the insignificant results include that the outbreak period covered the spring festival when factories were closed. And the closure of factories was extended by the government. Many cities adopted different levels of lockdown, and emission of vehicles were reduced. Thus, the concentration of air pollutants may be low in general. Strict and effective mitigation measures stopped the transmission timely.All datasets presented in this study are included in the article/JR and SZ conceived the study and carried out the analysis. LH collected the data. JR, SZ, and DH drafted the letter and discussed the results. All authors critically read and revised the letter and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rolling contact fatigue (RCF) damages often occur, sometimes even leading to shelling on locomotive wheel treads. In this work, the RCF damage behaviors of two locomotive wheel materials with different molybdenum (Mo) contents were studied, and the influence of depth of wheel material was explored as well. The result indicates that with the increase in the Mo content from 0.01 wt.%  to 0.04 wt.% , the proeutectoid ferrite content and the interlamellar spacing of pearlite decreased, the depth and length of the RCF cracks increased and the average RCF live of locomotive wheel steel improved by 34.06%. With the increase in the depth of material, the proeutectoid ferrite content and the interlamellar spacing of pearlite increased, the depth of RCF cracks increased, the length of RCF cracks of wheel 1 increased and then decreased whereas that of wheel 2 decreased, the RCF live showed a decrease trend for wheel 1, while the RCF life increased and then decreased for wheel 2. The processes of shelling can be divided into three patterns: cracks propagating back to the surface, crack connection and fragments of surface materials. As is whakedown ,3. When chetting ,3. The ef wheels ,5,6. Basf wheels ,9,10,11.As one of the typical RCF failures, shelling often occurred on the locomotive wheel treads on the heavy haul railways in north China, as shown in A change of alloying elements could alter the mechanical properties of wheel materials significantly. Various improved wheel materials with a good combination of strength and toughness have been developed ,19,20. ZIn the industrial field, tensile and impact tests were the general methods of evaluating the mechanical properties of railway wheel materials, during which the microstructures were observed, and the stress-strain curves and impact absorbed energy were analyzed ,20,24. HIn the present study, the RCF processes of two locomotive wheels with different Mo contents were explored via the twin-disc tests. The influences of Mo content and the depth of material on the RCF lives were studied. Besides, the processes of shelling were discussed in detail.eH) referred to the upper yield point and the ultimate tensile strength (Rm) referred to the highest point in the stress-strain curve. The yield strength and the ultimate tensile strength of wheel 2 were larger than those of wheel 1, but the impact toughness of the two wheels was similar. The matrix hardness of the two wheels was measured at 25 mm below the tread. The matrix hardness of wheel 1 was larger than that of wheel 2. In this study, two kinds of locomotive wheels  with different alloying elements were examined. The two wheels were produced through the following processes: smelting of steel, casting, stamping, rolling, dimension and heat treatment. The heat treatment process included: heating the wheels to 830~850 °C for 1.5 h, followed by water quenching to 350 °C, then tempering at 520 °C for 1 h and then cooling to room temperature. The steel grade of wheel 1 was J1, which was a kind of locomotive wheel material according to the Chinese railway industry standard TB/T 3469-2016. The main difference was that wheel 1 contained 0.01 wt.% Mo and wheel 2 contained 0.04 wt.% Mo. In the production process, composition analysis was carried out two times during the smelting progress. Meanwhile, composition analysis was also conducted for wheels after production. Therefore, the error of Mo content in wheels was controlled within 0.01 wt.%. The chemical compositions of the wheel materials are listed in y5) of about 2 μm. On each wheel, three rollers were cut from different depths . The sampling position and sizes of the wheel and rail rollers are shown in The wheel rollers were cut from the two types of wheels and the rail roller was from a U75V railhead, with a diameter of 55 mm and a contact width of 8 mm. The contact surfaces were ground to reach a roughness (RA twin-discs test machine (MJP-30A rolling-sliding test rig) was used to conduct the RCF experiments in this study. Details of the test rig have been described in the previous work . Two disIn all experiments, the rotational speed of the rail roller was 1500 rmp and the creep ratio was 0.3%. According to the Hertzian simulation rule , the nor3 in ethanol). The hardness was measured via the Victorinox hardness tester . The subsurface morphologies were inspected via optical microscope (OM)  and scanning electron microscope (SEM) . The surfaces of wheel and rail rollers were observed via optical microscope (OM) . Then, longitudinal sections of the wheel rollers were cut, mounted in resin, ground to 2000 grit silicon carbide paper, polished and etched with natal  were too thin, and the wheels were re-profiled before application to remove the top layers. Thus the “complex layers” were not further explored via rolling-sliding tests. The wheel rollers for rolling-sliding tests were cut from the 1st, 2nd and 3rd layers.0.5. The surface hardness of wheel rollers before and after tests is shown in In order to study the hardening phenomenon, the hardness was measured on the cross sections after tests, as shown in Specifically, the RCF lives of wheel 1 were around 836,000, 815,000 and 514,000 cycles at different depths, and the RCF lives of wheel 2 were around 946,000, 1,157,000 and 800,000 cycles. Thus, the addition of Mo could improve the RCF life of the wheel; the increase rates were 13.23%, 41.90% and 55.46% at different depths, respectively. When RCF damage such as shelling occurs on locomotive wheels, the wheels need to be re-profiled, leading to a reduction in wheel diameter. When the diameter of the wheel is reduced to 1150 mm, the wheel will be replaced. In order to compare the RCF lives of two wheels in the whole service process, the average RCF lives of the wheel rollers with different depths were calculated. The average RCF life of wheel 1 (standard wheel) was 722,000 cycles, and the average RCF life of wheel 2 (improved wheel) was around 968,000 cycles. The increase rate was 34.06%.2 without significant regularity. There was no significant difference in the damage forms of rollers taken from different depths of the same wheel. The key issue of this study was to clarify the effect of Mo addition on RCF. Therefore, damage analysis was carried out on the second layer tests , as shown in After the test, shelling occurred at one or more places on the wheel roller surface. The area of shelling ranged from 2 to 12 mmThe wheel rollers were cut along the rolling direction. The cutting line passed through the bottom of the shelling. Then the cross sections were observed, as shown in As shown in By comparing the subsurface damage around the shelling of two kinds of wheels, we found that the main differences between wheel 1 (standard wheel) and wheel 2 (improved wheel) were: (i) the size and quantity of cracks in wheel 2 were smaller than those in wheel 1; (ii) there was milder fragment damage of materials near the surface in wheel 2 than in wheel 1.There were also a large number of cracks at the surface far away from the shelling, as shown in The depth, length and angle of cracks on the wheel rollers were measured. The maximum lengths of the RCF cracks of the two wheels are similar at the first layer. At the second and third layers, the crack length of wheel 1 is longer than that of wheel 2, as shown in The distribution of RCF cracks angles is shown in tw and Ftr). In this study, the direction of the cracks on the wheel roller was consistent with the rolling direction, while the direction of the cracks on the rail roller was opposite. When cracks entered the wheel/rail contact zone, the openings of cracks were closed. The liquid in the crack on the wheel roller was sealed and trapped, but the liquid in the cracks on the rail roller was squeezed out. Then, under the cyclic normal load, the trapped liquid on the wheel roller accelerated the crack propagation due to high hydro-pressure [The objective of this work was to investigate the RCF behaviors of two kinds of wheel materials, thus the water was added into the wheel/rail interface during the rolling-sliding tests to enhance the RCF damages. As shown in pressure ,11. TherIn previous studies, the shelling processes were described as the propagation and union of adjacent cracks, or a result of branched cracks which propagated back to the surface ,30. But Concerning pattern (i): when branch cracks or parallel cracks propagated back to the surface, the material above cracks was removed from the matrix a,b, leadIt was found in this study that RCF life was greatly improved by adding only a small amount of Mo into the wheel material. With the increase in Mo content from 0.01 to 0.04 wt.%, the RCF lives of the wheel rollers were increased by 13.23%, 41.90% and 55.46% at different depths of the wheel, and the average RCF life was increased by 34.06%. RCF life was mainly related to initiation and propagation of RCF cracks. With the increase in Mo content, the yield strength of the wheel was increased to 578 MPa . Therefore, the initiation of cracks was delayed. Moreover, the volume fraction of proeutectoid ferrite in wheel 2 (improved wheel) was smaller than in wheel 1 (standard wheel). The reduction of proeutectoid ferrite content was also helpful to alleviate the RCF cracks because the crack mainly initiated and developed along the ferrite ,31. BesiThe RCF performance of wheel rollers at different sampling depths was also different. On the whole, the RCF life of wheel 1 decreased with the increase in sampling depth . This haIt was also found that the form of RCF damage changed due to the increase in Mo content. As can be seen from In summary, improving the Mo content from 0.01% to 0.04% could increase the anti-fatigue properties of the wheel material, and the average RCF life was improved by 34.06%. The following study will focus on the RCF properties of wheel materials with a series of Mo contents to find the optimal wheel material.With the increase in Mo content from 0.01 wt.%  to 0.04 wt.% , the proeutectoid ferrite content and the pearlite interlamellar spacing were reduced. With the increase in depth, the proeutectoid ferrite content and the pearlite interlamellar spacing increased.With the increase in Mo content from 0.01 to 0.04 wt.%, the RCF lives improved by 13.23%, 41.90% and 55.46%, at the first, second and third layers. With the increase in depth, the RCF lives increased and then decreased for wheel 2 (improved wheel), while the RCF lives showed a decreasing trend for wheel 1 (standard wheel). Shelling and RCF cracks were generated on the wheel materials after the tests. The crack depth and length of wheel 2 (improved wheel) were smaller than those of wheel 1 (standard wheel). With the increase in the depth of the sampling position, the crack depth increased, whereas the crack length of wheel 1 first increased and then decreased and the crack length of wheel 2 decreased.The processes of shelling could be divided into three patterns: cracks propagating back to the surface, crack connection and fragments of surface materials.In this work, the RCF damage behaviors and lives of two locomotive wheel materials with different Mo contents were studied via the rolling-sliding tests. Meanwhile, the influence of the depth of the wheel was also explored. The following conclusions can be drawn:


A theoretical model was established to calculate the EEC with optimization. Subsequently, an exposure experiment was carried out to test the performance of this detector, and we compared the chamber experiment and the theoretical model by estimating and measuring various parameters. The deposition flux of progeny derived from the prediction agreed well with the value measured in the exposure chamber. The energy-weighted net track density (NTD) measured by this detector is much more reliable to reflect the linear relation between NTD and time-integrated EEC. Since the detector is sensitive to the exposure environmental condition, it is recommended to apply the detector to measure the EEC after its calibration in a typical indoor environment.An improved passive CR-39-based direct  Several designs of measuring devices using SSNTD have been used for indoor 222Rn and 220Rn surveys, such as RADUET [220Rn based on its quite short half-life, the low diffusion rate chamber detects mainly 222Rn and the high diffusion rate chamber detects both 222Rn and 220Rn. However, evaluation of the internal exposure to 222Rn and 220Rn does not directly use the activity concentration of 222Rn and 220Rn gas instead of the equilibrium equivalent concentration (EEC). It should be noted that the equilibrium factor depends on various parameters, such as the radioactive decay, ventilation, and reactions with the structure and the surface of furnishing. Because of the short half-life of 220Rn (55.6 s), the indoor 220Rn concentration distributes heterogeneously, decreasing with distance from the source [220Rn concentration directly measured by the integrating detectors to evaluate its internal exposure. Otherwise, study of the behavior of 222Rn and 220Rn decay products indoors is important for assessing the natural background radiation exposures on the public through the inhalation route. Therefore, the direct measurement technique of 222Rn and 220Rn progeny is desirable and necessary for the evaluation of internal indoor 222Rn and 220Rn exposure.In the conventional integrating measurements for s RADUET , Pin-hols RADUET ,4, and sof 220Rn 5.6 s, th220Rn progeny measurement technique based on CR-39 detector for EEC of 220Rn was developed and used by Zhuo and Iida [222Rn and 220Rn progeny measurement techniques using LR-115 detector. CR-39 is the most sensitive and popular detector for recording α-particles [222Rn and 220Rn progeny detector using CR-39 was developed and applied because its energy windows for recording α-particles is quite wide, and it is unable to distinguish alpha energy directly, although some researchers make great efforts to develop dedicated tools based on the track geometrical characteristics to measure alpha particles. Moreover, the application of the direct measurement technique for determining their progeny concentrations depends on estimation of the effective deposition velocities of combinations of 222Rn and 220Rn progenies in a typical indoor environment. Some researchers [222Rn/220Rn progeny detector. The 222Rn and 220Rn behavior model and particle deposition model were used to estimate the concentration of each 222Rn and 220Rn progeny as well as the EEC.In the recent 20 years, a direct and Iida , Tokonamand Iida , Zhuo anand Iida , Sorimacand Iida , and Hu and Iida . Mishra and Iida  were thearticles  and perf222Rn and 220Rn progeny. Each detection channel was mounted with a different thickness of aluminum-vaporized polyethylene films (Mylar films), which can only detect the alpha particles emitted from corresponding progeny radionuclides and ensure that lower energy alpha emissions  do not pass through the absorber. In the conventional passive direct 222Rn/220Rn progeny detector, there are only 2 detecting channels: the 220Rn progeny (TnP) channel and the 222Rn progeny (RnPII) channel. The TnP channel can only detect the 8.78 MeV alpha particles emitted from 212Po atoms, which are formed from the decay of 212Pb and 212Bi atoms deposited on the film surface without any interference from other alpha emissions of natural radioactivity decay series. The RnPII channel selectively detects 212Po and 214Po (7.69 MeV) from the decay of surface deposited 222Rn and 220Rn progeny. In the improved passive detector, we added one more detecting channel RnPI to detect the alpha emission energy higher than that of 218Po (6.0 MeV). The design information is present in 222Rn and 220Rn progeny environment, there are interferences from the high energy alpha particles, which could be subtracted using the tracks on the TnP channel. Therefore, 4 slices of CR-39 detector are used on the TnP channel instead of 2 slices on the other channels to decrease the error.The structure of the detector is shown in 222Rn and 220Rn, apart from the decay losses, deposition and ventilation are two major mechanisms to remove the decay products from indoor air. Unlike the removal process by ventilation which depends essentially on the air exchange rate in a given room environment, the removal by deposition depends on the activity size distribution and the structure of turbulence at the air–surface interface. Since this detector is designed using the deposition mode, it is necessary to avoid uncontrolled static charges from affecting the deposition rates. Hence, the aluminized side of the Mylar is chosen to act as the deposition surface. Among the several aspects of indoor behaviors of 222Rn and 220Rn progeny, the relationship between the attached 222Rn and 220Rn progeny deposition flux, J (atom cm−2 s−1), and track density, N (tracks cm−2) can be expressed as follows:η is the track registration efficiency, which is the multiplier of the branching ratio of its progeny, the geometric efficiency, and etching efficiency. For each channel, the geometric efficiency depends on the energies of incident α-particle, incident angles against the absorbers, and the thickness of the absorbers. The track registration efficiency measured in this study is present in t is the exposure period (s).To describe the behavior of eV (cm s−1), is defined as follows:C is the atom concentration of the progeny (atom cm−3).The effective deposition velocity of the progeny, 222Rn progeny, a fraction of all the alpha particles emitted from the atoms of 218Po, 214Pb, and 214Bi deposited on the absorber can form tracks on the CR-39. Therefore, the tracks counted at the end can be directly proportional to the sum of the 218Po, 214Pb, and 214Bi atoms deposited. Similarly, the total tracks registered by the 220Rn progeny will be proportional to the sum of the 212Pb and 212Bi atoms deposited. Accordingly, we can define the following:Tn, Rn1, and Rn2 mean the parameters of the TnP channel, RnPI channel, and RnPII channel, respectively, and the subscript of each radionuclide means the corresponding parameter. Some of the parameters used in this study are shown in e,TnV is the effective deposition velocity of 220Rn progeny, which is a combination of deposition velocities of 212Pb and 212Bi. e,RnV is a combination of deposition velocities of 218Po, 214Pb, and 214Bi. e,Rn,Po218V is the only effective deposition velocity of 218Po.To determine the deposited progeny atoms from the alpha tracks registered in CR-39, we need to check the progeny atoms’ ultimate decay on the surface of the detector, which causes the alpha track to the CR-39. In the case of 222Rn and 220Rn environment, we can easily count the registered tracks on the TnP channel from 212Po decay, which come from the decay of the deposition flux of 212Pb and 212Bi, as shown in Equation (6). Accordingly, from Equations (3)–(5), we can also calculate the counts of registered tracks from 214Po on the RnPII channel and 218Po on the RnPI channel as in Equations (7) and (8).In the mixed 218Po and 212Pb can react with the ions in the air and form clusters in a short time by the neutralization process. Due to the larger diffusion coefficient of these cluster particles, 222Rn and 220Rn progenies can attach themselves to various surfaces, such as the surfaces of aerosol particles and droplets in the atmosphere, thereby giving rise to a consecutive activity size distribution. This distribution is broadly classified into two groups: the unattached fraction and the attached fraction. The effective deposition velocities combine the contribution from both the unattached and attached fractions of each progeny species. Considering the contributions of these two fractions, the deposition velocity of each radionuclide can be demonstrated as follows:ip denotes the unattached fraction of radionuclide i, and In the atmosphere, a large fraction of 212Po decay come from the decay of the deposition flux of 212Pb and 212Bi, according to the concept of the effective deposition velocity, the deposition velocity of TnP channel could be written as follows:Since the registered tracks from Similarly, the deposition velocity of the RnPII and RnPI channels could be written as follows:iA, Bq m−3) by using the decay constant iλ, s−1. Subsequently, Equations (6)–(8) could be modified as follows:r3 = ABi212/APb212.r1 = APb214/APo218 and r2 = ABi214/APo218.The atom concentrations in these equations could also be reexpressed in terms of the activity concentrations ; the rate constant for deposition of unattached progeny, λdu (s−1); the rate constant for deposition of attached progeny, λda (s−1); and the ventilation rate constant, λv (s−1). The details of the equations of the Jacobi room model for both 222Rn and 220Rn behaviors could be found elsewhere [APb212 can be solved by the data of the TnP channel and APo218 can be solved by both the RnPI channel and RnPII channel in the equations.To solve these equations above, we introduced the Jacobi room model  for bothlsewhere . FinallyIn the theoretical model, Lai and Nazaroff’s  three-lai, with the allowed values a and u, allows us to distinguish the two important states of 222Rn and 220Rn progeny: attached and unattached. uS, vS, and dS are the areas of upward-facing surfaces, vertical surfaces, and downward-facing surfaces, respectively; and cn is the correction coefficient of the surface area [In practice, the indoor rooms can be regarded as a rectangular cavity. The rate constant for deposition in the Jacobi room model is estimated by using the deposition velocity calculated by Lai and Nazaroff’s three-layer model . For theace area , which cace area .222Rn and 220Rn progeny to the ambient aerosol. The charging process depends on the electric charge distribution of an aerosol in steady state. In most used theories, attachment is diffusion-controlled as a result of electrostatic attraction and follows the gas kinetic laws [oN is the aerosol concentration, reflecting the concentration of condensation nuclei and where Z(pd) is the number size distribution of aerosol for unit aerosols concentration and can be described as a frequency function of the lognormal distribution with the count median diameter (CMD) and geometric standard deviation . β(pd) is the attachment coefficient. The rate constant for attachment was estimated by the compound trapezoid formula with the Latin-hypercube sampling method.The rate constant for attachment in the Jacobi room model reflects the attachment velocity of the unattached tic laws ,22. The 222Rn and 220Rn exposure chambers located in Hirosaki University, Japan. The chamber verification system includes 4 function cells, as shown in 222Rn gas and 220Rn gas generation system, which employs the natural uranium rock and commercially available lantern mantles as the 222Rn and 220Rn sources, respectively, to generate and import 222Rn and 220Rn to the mix chamber. The humidifier in this cell is responsible for maintaining the stability of the inlet gas concentration. (2) The aerosol generation cell contains 3 components, a compressor, a constant output atomizer , and a diffusion dryer filled with silica gel. The 5000 μg mL−1 NaCl solution is employed to generate droplets with the median diameter of 0.3 μm (GSD < 2.0) to mix with 222Rn gas or 220Rn gas in the mix chamber. (3) The exposure cell includes two chambers: a mix chamber and an exposure chamber. The volumes of the mix chamber and exposure chamber are 150 L and 3.24 m3 , respectively. The mix chamber provides a buffer to create more chances for the aerosol particles and gas to form a stable progeny flux to the exposure chamber. To maintain stable concentration in the exposure chamber, the experiment adopted a circulating connection that applies the outlet of the exposure chamber as the inlet of the source generation cell with an airflow rate of 3 L min−1. (4) The measurement cell is responsible for measuring progeny concentrations and environmental parameters in the verification experiment. The portable Si-photodiode detector with a sampling flow rate of 0.5 L min−1 and an EMD Millipore MF-Millipore AAWG02500 Mixed Cellulose Ester Filter Membrane were used to measure the EEC of 222Rn in real-time. The details of the Si-photodiode detector can be found elsewhere [220Rn. The Model 3034 SMPSTM Scanning Mobility Particle Sizer produced by TSI® was adopted to measure the concentration of condensation nuclei, CMD, and its GSD (σg). The concentration of condensation nuclei in the exposure chamber ranged from 2.05 × 104 to 4.32 × 104 particles per cm3. The CMD of the aerosol in the exposure chamber ranged from 0.187 to 0.218 μm. The friction velocity was calculated using a fan-turbulence model [sN is the rotation speed of the fan (2300 rotation min−1), td is the blade length for rotation (5 cm), and V is the volume of the exposure chamber. In this exposure chamber, we set two fans in opposite directions; as a result, we obtained u* as 23.53 cm s−1. In the verification experiment, each channel had 4 slices of CR-39 in the vertical and upward orientations as one group. For the total verification experiment, each exposure period had 3 groups of progeny detectors in the front, middle, and back of the bottom of the exposure chamber. In the theoretical model, the genetic algorithm is adopted to optimize the parameters in the calculation process. The measured values of the concentration of nuclei condensation number, CMD and its GSD, estimated friction velocity, and ventilation rate are the input parameters. Since the friction velocity, ventilation rate, and particle distribution fluctuate and change all the time, the equilibrium value of each parameter during the exposure period should be optimized depending on the different circumstances for each survey. To verify the feasibility of measuring the EEC, a verification experiment was conducted in lsewhere ,24. The ce model ,25, as ff(x) as follows:n is the number of objects of the model; 1,i,x and 2,ix are the measured data from actual measurement of EEC; and iω is the weight. The measured ranges of the concentration of nuclei condensation number, CMD, and its GSD are the optimized parameters, and the goodness-of-fit function is carried out to decrease the error of the average EEC of 222Rn calculated by the calibrated portable Si-photodiode detector and the CR-39-based progeny detector. The average track density of detectors deployed in the upward and vertical orientations was used to calculate the EEC of 222Rn by the progeny detector, which is in the same orientation as that placed in use. The same verification experiment was also conducted for 220Rn progeny except for the measurement method for the EEC of 220Rn, which used the periodic grab sampling method to replace the portable Si-photodiode detector. The comparison results of the progeny detector and experiment are shown in 220Rn progeny with an exposure time of 36.9 h, the average experimentally estimated deposition velocities in the upward and vertical orientations were 0.193 ± 0.067 and 0.217 ± 0.045 m h−1, respectively. Additionally, the model-simulated deposition velocities were 0.243 and 0.238 m h−1, respectively, which were located in the ranges of the experimental data. In the experiment, the deposition velocity in the upward orientation presented insignificant variation with that of the vertical orientation. The exposure time also presented an insignificant influence on the deposition velocity between 25.5 and 36.9 h. For deposition flux, the variation of deposition flux on the TnP channel estimated by the model showed excellent agreement with experimental values. As shown in 222Rn progeny, among distinct exposure times of 30, 70, and 102 h, the calibrated deposition velocities varied narrowly in the upward and vertical orientations, with the ranges of 0.123–0.136 and 0.117–0.131 m h−1, respectively. The average deposition fluxes in the theoretical model of 222Rn progeny on the RnPII channel and of 218Po on the RnPI channel were located in the ranges of the experimental values in most cases. The deposition flux was calculated by the net track density (NTD). The calculation process of NTD itself has uncertainty, especially caused by the sensitivity of the CR-39 detector, read-out of the tracks, counting statistics, and assumed background of the CR-39 detectors [A genetic algorithm with an improved goodness-of-fit objective function was adopted to optimize the results of the theoretical method . The imp222Rn and 220Rn progeny of the other researchers [−1 and 0.072 to 19.08 m h−1, respectively. To analyze the impact factors of the deposition velocity of 222Rn and 220Rn progenies, except the deposition velocities measured by Bigu [222Rn and 220Rn progenies.Compared with the deposition velocities of the total earchers ,11,15,27 by Bigu , other d by Bigu . When th222Rn and 220Rn progeny exposure in test rooms and dwellings.The design of TnP detectors from Zhuo and Iida  and Sori222Rn and 220Rn is shown in 222Rn and 220Rn progeny, the NTD caused by 212Po on the TnP channel can be used to estimate the total potential alpha energy of 212Po emitted by the deposited 212Pb and 212Bi. The detectors were exposed to a total of 220Rn progeny concentration of 83 ± 20 Bq m−3 during the exposure period from 0.2 to 2.5 d. The time-integrated EEC of 220Rn ranged from approximately 16 to 168 Bq m−3 d, which corresponded to the time-integrated EEC of 220Rn in an exposure period between 1 week to 3 months with the average EEC of ca. 2 Bq m−3 per day indoors [220Rn. Accordingly, the NTD caused by 218Po on channel RnPI can be used to estimate the atom number of the deposited 218Po on the detector firstly decaying to 214Pb. Furthermore, the NTD of 214Po can be used to calculate the total potential alpha energy of 214Po emitted by the deposited 218Po, 214Pb, and 214Bi during the decay. Therefore, in ε (i) is the alpha energy of the radionuclide i emitted during the decay. ε(218Po) is 6.0 MeV, and ε (214Po) is 7.7 MeV. The progeny detectors were exposed to an average EEC of 222Rn of 685 ± 6 Bq m−3 during the exposure period. The time-integrated EEC of 222Rn ranged from approximately 863 to 2839 Bq m−3 d, which corresponds to the time-integrated EEC of 222Rn in an exposure period from 1 month to 3 months with an average EEC of 222Rn of ca. 30 Bq m−3 per day. As a result, the values of the three NTDs increased linearly with the increase of the time-integrated EEC of 222Rn. Thus, it is possible to estimate the EEC of 222Rn indoors during a certain period. To compare the three NTDs as a function of the time-integrated EEC of 222Rn, the R2 of NTD of 218Po, energy-weighted NTD, and NTD of 214Pb were successively decreased from 0.9861 to 0.5625. Therefore, compared to the other deposition-based passive 222Rn progeny detectors which only measure the NTD contributed by 214Po, the detector with two channels is much more reliable. Besides, the progeny detector with 4 slices of CR-39 for the TnP channel designed rather than the same slices of CR-39 for each channel is recommended to decrease the error in calculation.The relationship between the NTD and the time-integrated EEC of 222Rn/220Rn progeny detector was developed and tested to measure and calculate the EEC of both 222Rn and 220Rn without the equilibrium factor. In the verification chamber experiment, the genetic algorithm was implemented to optimize the estimation result of the theoretical model. The optimized deposition fluxes of 220Rn progeny on the TnP channel, the 222Rn progeny on the RnPII channel, and 218Po on the RnPI channel agreed well with the experimental measurements. The EEC of 220Rn and 222Rn measured by the calibrated devices are comparable to the estimated value of the detector. Compared with the two channels’ detectors, the energy-weighted NTD is much more reliable to estimate the EEC of 222Rn rather than only measuring the NTD contributed by 214Po. Moreover, because the exposure conditions, especially the airflow and turbulence in the room, are smaller than that in the small-size chamber, the deposition velocity measured in this study is close to the value measured in the room. Therefore, a detector calibrated in a similar size to the chamber test as in this study is more reliable for assessment of 220Rn and 222Rn progeny exposure. At last, since the detector is sensitive to the exposure environmental condition, in the site survey, it is recommended to use the improved passive CR-39-based direct progeny detector after its calibration in a typical indoor environment.In this study, a 3-channel passive CR-39-based direct


N) of Zircaloy-4 corroded in lithiated water with 0.01 M LiOH at 633 K/18.6 MPa. The results showed that the Li ions segregated in the grain boundaries and subgrain boundaries in the oxide film, but nearly no Li ions were found in the oxide around the interface between the oxide and matrix. Finally, we discussed the mechanism of the LiOH influence on the corrosion resistance of Zircaloy-4.Transmission electron microscopy (TEM), second ion mass spectrum (SIMS) and atom probe tomography (APT) techniques are used to study the Li ion distribution in the oxide formed on the rolling surface (S Therefore, a lot of research work has been performed in order to provide a better explanation of the mechanism concerning the degradation of corrosion resistance of zirconium alloys corroded in lithiated water.In pressurized water reactors (PWRs), zirconium alloys are widely used as fuel cladding materials. During working, LiOH is added in the coolant to adjust the pH value for reducing the corrosion rate of structure materials due to the addition of boric acid, in which the 2 or t-ZrO2 were present prior to dissolution in LiOH solution and where many pores formed in the oxide film, then the corrosion rate of zirconium alloys in lithiated water was accelerated. However, Liu et al. [2 to the solution came from. Jeong et al. [+ substituted to Zr4+ so that it could enhance the corrosion rate. However, it is well known that the energy for the process of Li+ to replace Zr4+ in the oxide film is high; there is no way of knowing where the energy comes from. Pêcheur et al. [2 transformation to m-ZrO2 since the Li+ can substitute the Zr4+ in the oxide lattice; however, it supports the view proposed by Jeong [2 to m-ZrO2 is limited. Ramasubramanian et al. [Up to now, several mechanisms regarding the effect of lithiated water on the corrosion resistance of zirconium alloys have been proposed. For example, Cox et al. ,2,3 suggu et al.  raised ag et al. ,6 suggesr et al. ,8 considr et al.  suggesteby Jeong ,6. In 20n et al.  believedn et al. ,12 held Based on the discussion above, there are many possible mechanisms for the Li ions to influence the corrosion resistance of zirconium alloys, but further work is required. However, the mechanism of the effect of Li ions on the surface energy of oxide holds potential for future research. Although many articles about how LiOH affects the corrosion resistance of zirconium alloys have been reported, none have shown the distribution of Li ions in the oxide film in detail. Therefore, this was studied by transmission electron microscopy (TEM), atom probe tomography (APT) and second ion mass spectrum (SIMS) in the present work. We hope our results will be helpful for understanding the effect of Li ions distributing in oxide film on the corrosion behavior of zirconium alloys corroded in lithiated water.3 in size. The specimens were annealed at 853 K for 10 h. Prior to the corrosion tests, these specimens were pickled in a mixed solution of s 10% HF +45% HNO3 + 45% H2O (in volume fraction), and rinsed in cold tap water and boiling water. Then, these specimens were exposed to lithiated water with 0.01 M LiOH at 633 K/18.6 MPa in a static autoclave.We used plates of Zircaloy-4  specimens about 24 × 15 × 2 mmN) of the cladding tube reacts with coolant, so we only focused our attention on the oxide that formed on the SN surface. The thickness of the oxide film was near 2 µm after corroding for 70 d. Then, TEM, SIMS and APT methods were used for analyses. The SIMS analysis was conducted by Physical Electronics (PHI) Adept 1010 SIMS ; the O2+ voltage was 3 kV, and the initial current was 200 nA. The diameter of the analysis spot was 300 μm. Needle-shaped atom probe specimens perpendicular to the SN surface of the specimen were made using a Helios NanoLabTM 600i focus ion beam (FIB) workstation . Some TEM thin foil specimens of the cross-section of oxide film were also fabricated by the FIB method to observe the morphology of the oxide grains by JEM-2100 transmission electron microscope . The APT analysis was conducted by IMAGO LEAP 4000XHR with a laser pulsing mode at a frequency 125 kHz and 300 pJ laser energy . During testing, the temperature of the specimens was maintained at 80 K, and the pressure in the analysis chamber was approximately 1.3 × 10−10 mbar. About 8.6 million data points were collected during analysis. Then, IVAS 3.6.8 software  was used to analyze the experimental data. During analysis, we used the default k-factor of 3.3 and the evaporation field of 20 V/nm to reconstitute the maps of atomic distribution for different elements.In the reactor, only the rolling surface (S2 [2 ). Geng e2  evaluateN surface of the specimen corroded in lithiated water after 70 d exposure. From N surface of the Zircaloy-4 specimen, which was corroded in lithiated water with 0.01 M LiOH at 633 K/18.6 MPa for 70 d exposure, detected by SIMS. In +, and the abscissa represents the depth of oxide film. The depth at 0 means the outer surface of the oxide film, and the line marked on the abscissa at the depth near 2 μm identifies the O/M interface. In + is about 1014 in the O/M interface and matrix. However, the results reported by Jeong et al. [14 as shown in the ordinate. Therefore, we know the characteristics of the Li-ion distribution in the oxide film from g et al.  and Liu g et al.  showed t2 (red), Fe (blue), Li (black) and Sn (green) in the needle-shaped specimen prepared in the oxide film were about 1 μm apart from the O/M interface, as shown in 3. It is clearly shown that the Fe atoms segregated in some regions of the oxide rather than in a uniform distribution. The distribution of Li-ions was also not uniform, and the Li-ions segregated in the positions similar to that of the Fe atoms. The Sn atoms were uniformly distributed in the oxide. The Cr atoms were not detected in the present test. When comparing the morphology shown in The 3D reconstruction maps for ZrO2 (red), ZrO (yellow), Zr (purple), Fe (blue) and Sn (green) obtained by another needle-shaped specimen fabricated around the O/M interface. ZrO2 was mainly located in the upper-right part of the map, and Zr was mainly located in the lower-left part of the map. The distributions of Fe and Sn were nonuniform, and there was Fe and Sn segregation in localized areas. 2 enrichment region. From the concentration curves of Zr, O, Fe and Sn in the analysis cylinder shown in 2 occurs at the O/M interface. Therefore, the oxide grain boundaries provide an important path for the corrosion medium’s diffusion into the oxide film during the growth of the oxide [Based on the discussion in the introduction, we know that the mechanism of the Li ions’ effect on the surface energies of the grain boundaries of the oxide is more reasonable than others. So, we will discuss the mechanism of the LiOH influence on the corrosion resistance of Zircaloy-4 from this angle. The oxidation reaction between oxygen and zirconium to form ZrOhe oxide ,18. If t2 formed on the Zircaloy-4 specimen corroded in lithiated water would be lower than that corroded in deionized water since the Li ions segregated in the grain boundaries. So, the gathering of defects in the grain boundaries of the ZrO2 formed on the Zircaloy-4 specimen corroded in lithiated water could have been easier than that corroded in deionized water. Scanning probe microscopy (SPM) was employed to examine the surface morphology of the oxide covered on the specimens corroded in lithiated water in the early stage of pretransition [TEM and scanning electric microscope (SEM) techniques were employed to examine the microstructure and the fracture surface morphology of the oxide film covered on the Zircaloy-4 specimens corroded in lithiated water and deionized water at 633 K/18.6 MPa by Zhou et al. ,12. Theiansition . Based oansition ,12. Howeansition . Then, mansition ,12. It iThe findings of this study help to better understand the promotion of the enlargement or extension of the pores in the oxide film formed on the specimens corroded in lithiated water, as suggested by Cox et al. ,2,3, andThe distribution of Li ions in the oxide film formed on the Zircaloy-4 specimen, which was corroded in lithiated water with 0.01 M LiOH at 633 K/18.6 MPa for 70 d exposure was investigated by TEM, SIMS and APT methods. The results showed that the Li ions segregated in the grain boundaries and subgrain boundaries of the oxide rather than as a uniform distribution in the oxide film, but nearly no Li ions were detected at or around the O/M interface of the specimen. It was suggested that the degradation mechanism for the corrosion resistance of the Zircaloy-4 specimens corroded in lithiated water was attributed to the promotion of the formation of pores and microcracks along the oxide grain boundaries and subgrain boundaries, due to the segregation of Li ions there.


In the published article, there was an error regarding the affiliation for Minseo Kim and Jong-Min Lee. Instead of affiliations 3 and 2, respectively, Minseo Kim and Jong-Min Lee should both have affiliation 1.The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Introduction: The curricular implementation of events (or programs) for science-related training in human medicine has been on the agenda of the medical faculties since the publication of the Federal-State Working Group [ng Group . The MedProject description: The program “Research and Medical Studies” (RaMS) consists of several components: these elements of the mandatory curricular  and optional components , Research Track (RT), Research Fair Cologne (RFC)) are described here. Results were recorded at various levels: Likert Scale evaluation of the event’s elements were collected as satisfaction parameters from the studentsProcess data on participation in the voluntary events were collected and evaluated as absolute and relational figures (WS 12/13-SS 17). Data on the outcome of the RaMS program were collected: Type of scientific projects in the academic years 2011/12-2014/15), number and type of available projects offered at the RFC (in the years 2011-18) and number of student research funding applications in a comparison of the periods 2010-13 vs. 2014-17). Results: The students’ acceptance of mandatory and paracurricular courses of the RaMS program is pleasingly high, which is not surprising, at least in the case of the voluntary courses. The participation of students in RiMS, RT and RFC is satisfactory for voluntary courses. In the case of the RT, with certified participation of approximately 47% of all registrations , this is comparable to similar programs. It can be shown that the number of experimental science projects has more than doubled over time in parallel with the development of RaMS. The average number of provided projects according to the RFC is 42 (which corresponds to a placement rate of approx. 1:4). The number of successful student applications for a research support grant during the period the measures were implemented has doubled.Discussion and conclusion: The RaMS program shows a route for the implementation of the SP required by the next licensing regulations in medical education, which was initially supported and expanded solitarily, later by further elements (RiMS), also in the sense of a science-based career development . The student acceptance and the measured success, in the form of successful participation in the Research Track, increased choice of experimental projects, significant increase of submitted as well as approved research grants and the high project placement rate of the Research Fair, encourage the further development of the program, which is indicated in the conclusion.  The teaching of scientific competence in medical studies is the subject of an intensive, current academic debate , 3], , . The Fedhttp://www.nklm.de], adopted by the Medical Faculty Association (MFT) in 2015, following the CanMEDs model  as  as http:Ds model . In contDs model . FurtherDs model . The German Science Council has published “Recommendations for the Further Development of Medical Education”, which, among other things, requires the strengthening of the scientific competence of students . For theBased on these recommendations, the question arises for the medical faculties, how it is possible to establish curricular and paracurricular courses in order to integrate the teaching of scientific competences into the already time-consuming medical studies. This thesis aims to answer two research questions in the context of local (para-) curricular implementation: are the described (para-) curricular elements well accepted by students and lecturers and how are they evaluated by students in particular? is it possible to describe a qualitative change in the scientific activities of students on the basis of the selected parameters ?Mainly curricular elements were designed, implemented and evaluated as a methodological basis. The elements  was used to assess the individual parts of the course, the overall evaluation of the course by the students and additional characteristics  (see attachment 1 ). The data was collected continuously for the courses from the winter semester 2012/13 to the summer semester 2018.The RT is an optional program that is intended to give students interested in research the opportunity to carry out experimental research projects at an early stage . The core learning objective is to understand the scientific method of developing hypotheses based on a research question and to test these hypotheses experimentally. Practical teaching elements include visits to research laboratories, the presentation and explanation of large-scale research equipment and techniques, and visits to research facilities of cooperating institutions such as the “German Aerospace Center” or the “German Sport University Cologne”. RT participants must complete a final experimental internship of at least 60 hours (this can be accredited as Scientific Project I). The successful participation in the RT will be certified. The continuation of the RT In the second stage of study is offered as a “lunch seminar”. Here, the main focus is finding a suitable doctoral project, not obtaining a second certificate. The time course of the RT curriculum is shown in figure 2 st stage of studies Wednesdays 6-8 p.m.; for 2nd stage of studies lunch break 1-2 p.m.), the agreement to English-language lectures and other items (see attachment 2 ) and the overall assessment of the event by the participants. The proportion of successful RT participants was expressed as a percentage of the number of students who completed the RT completely  in relation to the number of students who registered for the RT by e-mail after the first lecture of the respective semester.In both parts of the RT, a student evaluation was carried out after each course, in which student satisfaction was assessed on a 6-level Likert scale (liked it – disliked it). At the end of the semester, an evaluation sheet was distributed, on which items were assessed according to the time of the event  to “disliked it” (6); mean±SD; 12 semesters from WS 12/13 to SS 18 were considered, see figure 3 Of the all the students in the second pre-clinical semester, an average of 21% of each cohort (approx. 40 persons) registered for participation in the program through e-mail; the number of successful participants averaged 47% of all those registered  and in the clinical stage as an average of 1.73±0.14  to “disliked it” (6); mean±SD; pooled evaluation data from SS 14-SS 18; evaluation sheet, see attachment 3 ). In the pre-clinical stage, English as the language of instruction was rated positively by the participants 1.7±0.4  to “disliked it” (6); mean±SD, pooled evaluation data from WS 12/13-WS 17/18, see table 1 Every year, an estimated 150 - 200 students and 100 scientists from an average of 30 different clinics and institutes take part in the Research Fair with approximately 50-70 posters. The evaluation of student satisfaction of the Research Fair resulted in a rating of 2.25±0.21  to “disliked it” (6); mean±SD; pooled evaluation data from 2013-2018, see figure 5 The outcome evaluation showed that on average 42 projects (41.8±8.4) were placed on the Research Fair. This corresponds to an average placement rate of approximately 1:4 to 1:5 per student participant. On average, 67.3% of these were experimental, 20.7% clinical-statistical and 11.9% literature work . The number of experimental projects as a proportion of all scientific projects increased from 8% in the academic year 2011/12 to 19% in the academic year 2014/15  (response rate 54%).The students rated the basic idea of conducting SPs as slightly positive with an average of 6.0 points , better with regard to the project they had conducted themselves . There is a moderate mathematical correlation between the ratings . The students work in median 60 h (25%-quartile 32 h and 75%-quartile 120 h). Only 6 out of 411 (1.5%) evaluations reported that the students felt “exploited”.With the introduction of the model curriculum in human medicine in Cologne, competence-oriented scientific education was integrated into the curriculum as early as 2003 with the implementation of the Scientific Projects . In addiIt should not be forgotten that the Cologne model is merely a possible implementation of the requirements of WR and MFT, which is now more longitudinally structured at the Medical Faculty in Cologne under the label “Research and Medical Studies (RaMS)”. The model focuses on the support of top-level and broad-based scientific research. SP and RiMS ensure the basic qualification of all students, while RT and RFC particularly target the top group of students. Similarly complex, albeit differently structured programs  also shThe acceptance of the individual optional curricular elements by the students is very positive. The Scientific Projects as a mandatory curricular component still perform positively overall (at least with regard to their own projects). With a median of 60 hours, the time spent on the projects is on average within the time frame provided for this in the study regulations (60-120 hours), although it is at the lower end of the scale. This fits in with the students' statements that only very few students (1.5%) report to have been exploited.http://www.nklm.de]) are already taught in the SP, students who attend the voluntary preparative and advanced courses  have the opportunity to knowingly plan their careers in a scientifically guided manner (or to promote young scientists in the sense of the faculty). Only some of the students take advantage of the voluntary elements. The number of participants who complete the Research Track amounts to approx. 10% of the students in a year and is thus comparable to similar programs ). In our opinion, the performance of experimental work represents the entire research process from the formulation of the research question to the conduction of experiments and the evaluation and interpretation of the data. Literature work in the context of Scientific Projects (which are not systematic reviews) does not fulfill this. In this context, the increase in experimental work seems to represent a qualitative improvement in terms of mapping the entire research process. Whether the demonstrated effects on the scientific activities  are causally related to the RaMS program or not, cannot be proven. However, the close temporal relationship to the newly introduced teaching elements and effects studied suggests that this is the case.Since the competencies required by the NKLM . In addAlthough or perhaps because of the positive experience for the past 15 years with the model curriculum at the Medical Faculty in Cologne in conveying science in medical studies in accordance with the recommendations of the WR and MFT, the next consecutive steps in the further development of the concept are already becoming apparent:The recently founded Graduate School of Human and Dental Medicine (GSHZ Cologne) guides the qualified paracurricular science program in the first own scientific achievement, the doctorate.med) was founded, whose task is the methodical hands-on preparation of students for training in scientific projects (and doctorates).With the faculty's decision to change the academic regulations for the Model Study Program 2017, the Cologne Science Skills Lab (KWiSThe reorganization of teaching in the first study stage, which is expected to start in 2021, will be challenging. This will also involve an even better combination of traditional and new elements in the scientifically-oriented curriculum of the model study program.Taken together, it becomes clear, particularly through the description of the complex intervention to improve the teaching of science in the study program, that a simple strategy, for example one oriented towards research-based learning , cannot The authors declare that they have no competing interests.


Outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported in Brazil since 1999 when the vaccinia virus strain Cantagalo was first isolated in the State of Rio de Janeiro. However, the genomic diversity of the viral isolates associated with these outbreaks is not well known, particularly in the southeastern states that represent the focal point of virus spread to other regions. Here, we report the genomic sequences and an analysis of the polymorphic site profiles and genotypic diversity of four clinical isolates of vaccinia virus strain Cantagalo collected from 1999 to 2006 in southeastern Brazil. Poxviridae.,,Vaccinia virus (VACV) is a large and complex double-stranded DNA-containing virus that belongs to the family ,In Brazil, outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported since 1999.,,,,,,,,,,,After the first outbreaks in 1999, the number of reported cases increased with the rapid spread of the infection to the other southeastern states, i.e., Minas Gerais (MG) and Espírito Santo (ES).de novo assembly using Velvet vs 1.2.10 with a kmer of 149.,Outbreaks of vesiculopustular disease affecting dairy cattle were previously reported by the state secretariats for animal health surveillance and their affiliated agencies in the municipalities of Miracema (RJ), Vieiras (MG), Campos de Goytacazes (RJ), and Alegre (ES) in 1999, 2001, 2003, and 2006, respectively . Scab sa,The Supplementary data (Table).The median-joining network explores the relatedness of the field isolates of VACV in greater detail and zooms in on the SNP-based distances among them . We can The most striking difference observed in the base-by-base profile is an insertion of 3,753 bp in the left end of the genomes of CTGV-ALEH2 and Serro2 viruses  from MI233 (RJ) and ALEH2 (ES). However, ALEH2 is more distant genetically from CG04 than MI233 is. Additionally, the deletion in the region of the CPXV 77 kDa and C9L genes present in the ALEH2 genome is not shared with the 3 RJ and VI04 isolates, but is shared with Serro2 virus, which was collected in the previous year but approximately 500 km apart. However, although sharing this structural feature, ALEH2 and Serro2 genomes have accumulated 81 SNPs. Taken together, our data suggest a different event of virus spread into ES, not originated from the Northwestern region of RJ and not directly originated from Serro2, reflecting the complex genomic diversity of CTGV isolates related to the first years of the outbreaks that emerged in the Southeastern region of Brazil in 1999. These studies will lead to further investigation of the focal point of VACV dissemination in Brazil, and the biological and virulence diversity of the isolates.


This Ca2+ influx triggers the activation of a cascade of phosphorylation-dephosphorylation reactions that eventually affects the expression of C-repeat-binding factors , which were shown in many plants to control resistance to cold stress by regulating the expression of cold-regulated (COR) genes. Based on experimental observations mostly made on Arabidopsis thaliana, we build a computational model for the cold response pathway in plants, from the transduction of the cold signal via the transient influx of Ca2+ to the activation of the phosphorylation cascade leading to CBF3 expression. We explore the dynamics of this regulatory network by means of numerical simulations and compare the results with experimental observations on the dynamics of the cold response, both for the wild type and for mutants. The simulations show how, in response to cold stress, a brief Ca2+ influx, which is over in minutes, is transduced along the successive steps of the network to trigger the expression of cold response genes such as CBF3 within hours. Sometimes, instead of a single Ca2+ spike the decrease in temperature brings about a train of high-frequency Ca2+ oscillations. The model is applied to both types of Ca2+ signaling. We determine the dynamics of the network in response to a series of identical cold stresses, to account for the observation of desensitization and resensitization. The analysis of the model predicts the possibility of an oscillatory expression of CBF3 originating from the negative feedback exerted by ZAT12, a factor itself controlled by CBF3. Finally, we extend the model to incorporate the circadian control of CBF3 expression, to account for the gating of the response to cold stress by the plant circadian clock.Understanding the mechanism by which plants respond to cold stress and strengthen their tolerance to low temperatures is an important and challenging task in plant sciences. Experiments have established that the first step in the perception and transduction of the cold stress signal consists of a transient influx of Ca Low temperature has adverse effects on the survival, growth, and development of plants . In ordeArabidopsis have led to the identification of key factors in the transcriptional network of the cold acclimation pathway, where three C-repeat (CRT)-binding factors (CBFs), the most commonly studied being CBF3, also known as dehydration-responsive element (DRE)-binding proteins (DREBs), play vital roles in cold acclimation , a widely studied plant second messenger, is involved in nearly every aspect of cell physiology and development  cycle is indeed circadian and peaks at about ZT8, i.e., 8 h after light onset, just after the early morning peak of the proteins CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) involved in the circadian clock mechanism  in plant cells shows a sharp rise before returning to a basal level. The steady-state cytoplasmic calcium concentration in plants is in the range of 80~250 nM , which is activated through the binding of Ca2+ in a cooperative manner, and ultimately regulates, via a signaling cascade, the expression of target genes , the autophosphorylation of this CRLK —particularly CRLK1—is activated. The activation of CRLK1 leads to the phosphorylation of downstream targets such as the mitogen-activated protein kinases 6 (MPK6) and 4 (MPK4), which elicits the cold response  and the 14-3-3 protein kinases play negative roles to prevent excessive cold responses . Cold-int growth , prolong2+ influx to the rise in CBF3 that controls the expression of genes which confer tolerance to low temperatures. The model based on the experimental observations summarized above in “The Cold Response Pathway: Brief Overview of Experimental Aspects” section (where relevant references are indicated) is represented schematically in Our goal is to develop a detailed computational model for the plant response to cold stress involving the succession of biochemical events, from the initial Ca2+. This initial signal leads to the activation of CaM upon cooperative binding of Ca2+ (with a Hill coefficient of 4) to the protein. The activation of CaM brings about the activation of two protein kinases, CRLK1 (left branch) and CRPK1 (right branch). We first describe the series of reactions initiated by the activation of CRLK1. We will return afterwards to the right branch initiated by CRPK1, for which available experimental observations are not as detailed as for CRLK1.Starting from the top left, we can follow the reactions that are successively triggered by a cold stress. First, the decrease in temperature elicits a brief transient influx in CaThe kinetic equations (4)–(17) governing the time evolution of the 14 state variables of the model are listed in 2+ in plant cells. The rise in [Ca2+] takes the form of a single brief spike, or of a train of spikes. A discrete quantitative description of [Ca2+] dynamics for the generation of a single spike after a drop in temperature was proposed by 2+. Parameters were selected so as to produce a single peak in [Ca2+] of appropriate magnitude and duration  indicates that this steady state is stable for the parameter values listed in 2+ steady-state level is relatively low at this temperature, so that CRLK1 is predominantly in its inactive form while MPK6, MPK4, ICE1, and MYB15 are mostly unphosphorylated. The high level of active, unphosphorylated MYB15 prevents the transcription of CBF3 so that before the cold stress, CBF3 remains at a low stable steady-state level.We first examine the behavior of the model at a constant temperature of 20°C. At such temperature, the concentration of CaCBF3 mRNA expression as the output measuring the response to cold stress. The model allows us to follow in a detailed manner how all the elements of the cold response pathway, i.e., the state variables of the model, change in time after a drop in temperature, from the initial Ca2+ influx to the expression of CBF3 and ZAT12. The advantage of the model is that it permits us to follow the evolution of every state variable, as shown in CBF3 mRNA and ZAT12 mRNA. The accumulation and subsequence disappearance of these two mRNA species have been measured over the hours that follow the drop in temperature  triggered by the cold stress, for activated CRLK1 (B), phosphorylated MPK6 , phosphorylated ICE1 , MYB15 (E), phosphorylated MPK4 , nuclear HOS1 , CBF3 mRNA (H), and ZAT12 mRNA (I). In CBF3 mRNA and ZAT12 mRNA (solid curves) with experimental data (red dots for CBF3 mRNA and blue dots for ZAT12 mRNA) available for these state variables  and 4 h (B), after the cold stress. The Ca2+ pulse, triggered by the shift to cold temperatures, starts at t = 2 h. Here, plotting the different curves on the same graph allows us to clarify the sequence of activation steps that leads to CBF3 expression in the pathway. The state variables start from their steady state levels reached before the cold stress and, after the cold signal is given, increase or decrease in response to the Ca2+ pulse. This pulse is followed, successively, by the peaks in CRLK1a, ICE1P, HOS1n, MPK6P, MPK4P, then by the trough in MYB15, and the peaks in CBF3 mRNA and ZAT12 mRNA. Some of the variables, such as Ca2+, CRLK1a, MYB15, ICE1P, MPK6P, and MPK4P, return more rapidly to their steady state than other variables which take more time to do so  takes more time, because the degradation of CBF3 mRNA proceeds at a relatively slower rate at cold temperatures.To better grasp how the dynamics of the cold response pathway unfolds in the course of time, we plot in o so see . The retvs1 in eq. (10), and its conversion, activated by MPK6P, into the phosphorylated form ICE1P. This slow rise continues until ICE1P reaches its steady state. We included a term for the synthesis of ICE1 to avoid the depletion of the protein and to ensure the existence of a steady state, because we take into account the degradation of ICE1P, which is enhanced by HOS1n.The predicted evolution of ICE1P appears to be biphasic. First ICE1P rises and peaks in advance of its upstream variables MPK6P and MPK4P, and later decreases below steady state, due to the rise in HOS1n which promotes the degradation of ICE1P. After MPK6P, MPK4P and HOS1n reach their steady state, ICE1P begins to rise again after t = 6 h see , because2+ pulse to the synthesis of CBF3 mRNA.The results of 2+] transients, with an amplitude of about 125 nM and a period of about 150 s, associated with stomatal closure , while we increase 5-fold the total WT concentration for HOS1 overexpression (OX) mutants. Moreover, the rate of synthesis of ICE1 is reduced to 20% to represent the effect of mutations, while the rate of synthesis of ZAT12 is increased 5-fold to represent the effect of overexpression. We compare the predictions of the model  with experimental data (red dots) in The model of the cold response pathway allows us to predict the effect of mutations in the various elements of the network and to compare the results with available experimental data. We determine the expression of ice1 mutation blocks the CBF3 transcripts, in agreement with experimental observations . The situation in regard to CBF3 mRNA is, therefore, similar to that in ZAT12 mutant.Finally, we turn to ZAT12, which dampens the expression of the CBF cold response pathway . The ove2+ or a train of Ca2+ spikes. However, the temperature fluctuates seasonally, daily, as well as on a short time scale due to stochastic fluctuations. The time scale over which Ca2+ changes occur varies greatly, from rapid increases within seconds –(19);  display a peak at 24 h  characterized experimentally. For example, kinetic studies have yet to be performed to yield values for the various rates of phosphorylation or dephosphorylation, for the Michaelis constants that characterize these enzyme reactions, for the activation or inhibition constants that measure the regulatory interactions, and for the total concentrations of the different molecular species involved in the pathway. For all these parameters, we made a semi-arbitrary choice, in a physiologically reasonable range, as explained in Section 4.1 of As explained in detail in Section 4 of CBF3 expression, which is the major response measured in the experiments, in regard to its amplitude, timing, and half-width. This was achieved by trial and error, i.e., by performing simulations for a large set of parameter values and selecting a set that produced (1) a CBF3 mRNA peak in a range extending from 1 to 6 h after the cold stress, as observed in different experiments –(2d) as described in Section 1.2 of CBF3 mRNA has been determined experimentally. Thus, the CBF3 transcript has a half-life of 7.5 min at warm temperatures, which gives an approximately degradation rate of CBF3 mRNA at 20°C of the order of 5.5 nM/h, while at low temperature this degradation rate is 10 times smaller, of the order of 0.55 nM/h  an eriments ; and (3)eriments . For Ca2.55 nM/h . This quCBF3 expression elicited by a cold stress: the amplitude of the peak in CBF3 mRNA, the timing of this peak, and the half-width, measuring the duration of the response. This analysis allows us to determine which parameters might be targeted experimentally to modify most effectively the amplitude, timing, or half-width of the peak in CBF3 expression elicited by the cold stress.We also performed a sensitivity analysis, as detailed in Section 4.2 in CBF3 expression, which, so far, have not been observed experimentally, and to investigate the effect of coupling the plant cold response pathway to the circadian clock. In a certain range of parameter values, the model indeed predicts the possible occurrence of sustained oscillations in the expression of CBF3. The phenomenon is accompanied by oscillations in ZAT12 but not in the other state variables of the network. The mechanism of CBF3 oscillations involves the negative feedback exerted by ZAT12 on the expression of CBF3. Numerical simulations indicate, as shown in “Putative CBF3 Oscillations Resulting From Negative Feedback Exerted by ZAT12” section and CBF3. Besides augmenting the stability of CBF3 mRNA, enhancing the level of phosphorylated ICE1 might, therefore, be a promising approach for investigating the possibility of an oscillatory expression of the genes that control the plant response to cold stress. The possible physiological significance of such oscillations in CBF3 expression and their very occurrence nevertheless remain an open question, given that they only occur for appropriate parameter values and that so far they have not been observed experimentally. Besides these ZAT12-controlled oscillations, the cold response pathway is subjected to periodic alternations between cold and warm periods and to control by the endogenous plant circadian clock, which appears to gate the plant response to cold stress, as shown in “Circadian Gating” section and An additional interest of the computational model is to reveal the possibility of new modes of behavior, such as oscillations in CBF3 expression into the model allowed us to account for circadian gating of the response to a drop in temperature. We represented the circadian input phenomenologically by means of a sine function multiplying the rate of CBF3 mRNA synthesis. It would be possible to replace this description by using a compact model for the plant circadian clock (CBF3 occurs during the day, several hours before the L to D transition. This timing does not correspond to the time at which the cold stress is expected to occur in physiological conditions, which should be in late evening or at night. The same remark holds for the results of 2+ pulse is negligible. From a physiological point of view, we would not expect the cold response pathway to operate optimally if the cold stress arrives at such phases. In discussing the relative timing of the system’s response with respect to the cold stress, we should keep in mind that a delay likely exists between the rise in CBF3 expression and the build-up of the cellular response to cold stress. A large number of COR genes must indeed be induced by CBF3, and their translation into the corresponding proteins takes time, while additional delays should arise from the time taken by these proteins to organize cold acclimation and freezing tolerance at the cellular level.Incorporation of the effect of the circadian clock on an clock  in whichan clock , the resBesides the pathway responsible for resistance to a cold stress, plants display a separate cold-dependent response, which occurs on a time scale of weeks or months. This major cold-dependent physiological process, known as vernalization, is the phenomenon by which prolonged cold exposure leads to the silencing of a floral repressor gene , therebyThe original contributions presented in the study are included in the article/XY, XH, and AG designed the study. RZ, AG, and DG built the computational model. RZ and DG performed numerical simulations. RZ and AG wrote the manuscript. RZ, DG, XH, XY, and AG contributed to discussing the results and to finalizing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.