However, less inclusions such as Al2O3, MnS and Ca-Mg-Al-Si composite oxides were formed at the interface after pickling treatment. For the vacuum degrees of 10−2 Pa, 1 Pa and 105 Pa, the oxidation reaction became more intense with the decrease in vacuum degree. The interface inclusions were gradually changed from Al2O3 and Si-Mn complex oxides to oxide scale and MnCr2O4 spinel oxide. The interfacial bonding strength of stainless steel clad plate was improved with the increase in degree of vacuum. The bonding strength was 55 MPa at vacuum of 105 Pa, but it was 484 MPa at vacuum of 10−2 Pa, which is far greater than that of the national standard, and an excellent performance was obtained.In this study, the impact of different surface treatment and degree of vacuum on the interface and mechanical properties of 304/Q345 stainless steel clad plate was investigated. The study indicated that more continuous or aggregated Al The stainless steel clad plate is a new means of processing dissimilar materials. The clad plate consists of thicker carbon steel as a substrate and thinner stainless steel as a cladding material. Stainless steel clad plates combine the excellent corrosion resistance of stainless steel and the good mechanical properties and low cost of carbon steel. This has led to the stainless steel clad plates being widely used in the chemical, petroleum, and shipbuilding industry, etc. ,2.At present, there are many methods for producing stainless steel clad plate, among which, hot roll-cladding is the most economical and efficient manufacturing process ,4. HowevThe usual method to solve the problem of bonding interface oxidation is to isolate the outside atmosphere during the preparation process by, for example, welding around the base cladding plates and vacuum extraction , vacuum −2 Pa, 1 Pa and 105 Pa. The formation mechanism of oxides and the influence on mechanical properties are analyzed, thus providing a reference for controlling the oxidation products of hot-rolled stainless steel clad plates.In this study, stainless steel clad plates were prepared by Vacuum Rolling Cladding (VRC) technology, which ensured that the plates had extremely excellent interface bonding performance with rare oxidation due to the use of vacuum electron beam welding (VEBW) technology. We studied the influence of two different surface treatment methods, brush grinding and pickling treatment, and three different vacuum degrees, which were 10The base and clad materials of the clad plates were Q345 carbon steel and 304 austenitic stainless steel, respectively, and the chemical composition of both these materials are shown in A schematic of vacuum roll-cladding (VRC) is shown in The nature, composition, morphology and formation mechanism of oxide inclusions at composite interface, as well as the impact on mechanical properties for different methods of surface treatment and different degrees of vacuum are discussed based on the high temperature oxidation experiments and hot rolling experiments.−2 Pa. One set was used for high temperature oxidation experiments and the other set was used for hot rolling experiments. Similarly, to study the effect of vacuum on the oxidation products, two sets of billets were combined by pickling, and the stacked billets were welded under vacuum of 1 Pa and 105 Pa for high temperature oxidation and hot rolling experiments (the billets at 10−2 Pa high vacuum were prepared as described above). The above-obtained stacked billets were placed in an electric resistance furnace and heated to 1200 °C for 2 h. The billets for the high-temperature oxidation test were directly cooled to room temperature after heating while the hot rolling experiment were carried out on a 450 Two-High Hot Rolling Mill made by NEU. The rolling speed was 1.0 m/s, and the total reduction rate was 50%.First, the surface of the billets was cleaned by means of brush grinding and pickling, respectively, to remove contaminants and the oxide layer. Two sets of billets were prepared in each method. The billets were then electron beam welded at vacuum of 10The specimens for microstructural examination were obtained from the central part of the clad plates in a plane parallel to the rolling direction, and the specimen was etched with 4% nitric acid alcohol solution when picking was adopted as the surface treatment. The interfacial microstructure was observed using optical microscope , scanning electron microscopy , energy dispersive spectroscopy  and transmission electron microscope . The composition and type of interfacial inclusions were determined by electron probe microanalyses  in conjunction with wavelength dispersive spectroscopy .For clad plates, the bond strength of the composite interface is the most important performance, and the shear strength of the interface bond strength will determine the main properties of the clad plates. In this paper, the bonding strength of the interface of stainless steel clad plate was tested by shear testing. According to the standard of GB/T 6396-2008 , the cla2O3 passive film at atmosphere or in the presence of oxidizing medium. During the process of brush grinding, the Cr2O3 passive film on the surface of the stainless steel was lifted by the grinding force, and was subsequently covered irregularly on the surface of stainless steel. Three different positions for chemical composition analysis were used, as shown in The SEM images of the interface for different surface treatments are shown in The SEM images of the surface of 304 stainless steel and carbon steel after high-temperature oxidation experiments for the two kinds of surface treatment are shown in 0) depending on metal oxide is formed, which indicates the affinity between the metal and oxygen. The lower the value, the higher the stability of oxide and easier the metal is oxidized under lower free energy [0–T diagram of the metal oxidation reaction [0 of Al, Si, Mn, Cr, and Fe oxides are different, and the increasing sequence is ΔGAl2O3 < ΔGSiO2 < ΔGMnO < ΔGCr2O3 < ΔGFeO < ΔGFe3O4 < ΔGFe2O3, that is, the affinities of Al, Si, Mn, Cr, Fe, with O decrease. Thus, Al was preferentially oxidized to Al2O3. Meanwhile, Al diffused to the surface due to concentration difference. The content of oxygen in the valley or depressed surface region is high, and Al below the surface did not diffuse to the interface to react. While Al on the surface was consumed, the oxygen continued to react with Si and Mn. However, the oxygen content was limited, which resulted in the formation of composite inclusions dominated by Al2O3 and Si-Mn oxides.When the metal is oxidized, the free energy , and the atomic ratio of Al to O was 2:3. Thus, the inclusions were Al2O3. The cross section of stainless steel was clean and clear without oxide. 2O3 and Si-Mn composite oxides were detected.5 Pa. With the decrease in vacuum, the oxide content clearly increased, and an oxide layer and larger oxides were formed on the carbon steel side near the interface. At high-temperature and high-oxygen content, Fe was oxidized rapidly to form an oxide layer. Because sufficient oxygen was enriched on the surface, Al2O3 and Si-Mn composite oxides were sequentially generated based on the preferential selective oxidation mechanism. The cross section of stainless steel was relatively clean, but there were a certain number of holes in the localized area, which were formed on the stainless steel surface after pickling and could not be completely eliminated during heating at a high temperature. Thus, a large amount of oxygen is trapped here, which easily reacted with the active metals in the stainless steel to form oxides. The microstructure of high-temperature oxidation of stainless steel and carbon steel at different vacuum degree are shown in −2 Pa are shown in 2O3 after pickling in the high-temperature oxidation experiments. A small amount of Ca-Mg-Al-Si composite oxides were formed on the surface of stainless steel according to high temperature oxidation experiments. The forming mechanism of surface inclusions has been discussed in Micrographs of carbon steel and stainless steel corresponding to the vacuum of 102O3 and Si-Mn composite oxides were detected and distributed on the surface because of selective oxidation. With the decrease in vacuum, the content of oxides was increased on the surface of the depressions for stainless steel, and larger Al-Si-Mn oxides were formed. When the vacuum was 1 Pa, the interface was in a low-vacuum sealed state. Although the content of oxygen at the lower vacuum was higher than that under the higher vacuum, and the initial oxygen partial pressure was also high, the total oxygen content was limited. With the oxidation reaction, oxygen was gradually consumed until the partial pressure of oxygen was reduced to a level such that oxide was not formed. Thus, the composite interface did not undergo severe oxidation at lower vacuum, but was similar to the degree of reaction at high vacuum and a had a slightly higher degree of oxidation. The result was an increase in oxide production compared to the high vacuum [Morphologies of the samples with a vacuum of 1 Pa are shown in h vacuum .5 Pa are shown in 2O4. The composite interface was in contact with the outside atmosphere at vacuum of 105 Pa. During subsequent heating, the stainless steel was severely oxidized. Due to the high content of Cr in stainless steel, a layer of Cr2O3 protective film was first formed on the surface of stainless steel at high-temperature and high-oxygen conditions according to the selective oxidation mechanism. With the progression of the oxidation, Cr2O3 was continuously generated and covered the entire stainless steel surface, which isolated the stainless steel matrix from the atmosphere. If Cr continues to oxidize on the composite surface, alloying elements must diffuse through the Cr2O3 layer to the surface and react with oxygen. Meanwhile, the lattice diffusion coefficient of Mn ions in Cr2O3 was two orders of magnitude higher than that of Cr ions, and its diffusion rate was faster than Si, Fe and Ni in stainless steel [2O3 and reacts with oxygen to form MnO. When Cr2O3 and MnO are present on the surface of stainless steel, MnCr2O4 spinel oxides are formed [Morphologies of samples with the vacuum of 10ss steel . Therefoe formed ,28.−2 Pa, or less.Thus, when the vacuum was reduced, the degree of interfacial oxidation became more serious, which was unfavorable to obtaining the excellent interface of the hot-rolled clad plate. Therefore, the degree of vacuum at the composite interface should be controlled between 1 Pa and10The shear strength-displacement plot of the interface of stainless steel clad plate at different degrees of vacuum after pickling is shown in Thus, with a decrease in the degree of vacuum, the bond strength of the composite interface became less. The interfacial shear strength of stainless steel clad plates prepared at high vacuum, low vacuum and atmospheric pressure was 484 MPa, 397 MPa and 55 MPa, respectively. In addition, with the decrease in vacuum degree, the deformation of the specimen in the shear test became smaller, which indicated that the deformation resistance of the composite interface also deteriorated when the degree of vacuum decreases. In summary, inclusions at the composite interface at different vacuum degrees was the key factor affecting the strength and toughness of the composite interface. Therefore, welding pickled stainless steel clad plates in high vacuum and hot rolling can provide excellent bonding performance.2O3, Si-Mn composite oxides. A small amount of dispersed Al2O3, MnS and Ca-Mg-Al-Si composite oxides were formed after pickling, indicating that a cleaner interface can be obtained by pickling. Additionally, the type of oxide inclusions at the interface after hot rolling was unchanged, the inclusions were formed at the stage of high-temperature oxidation.At high temperature, the oxides that formed at the interface after brush grinding were continuous or aggregated Al−2 Pa, the content of oxide inclusions was very small, and were mainly Al2O3 and Si-Mn composite oxides. When the vacuum was 105 Pa, the composite interface was almost completely covered with an iron oxide skin and MnCr2O4 spinel oxides. Thus, during vacuum hot rolling cladding to prepare stainless steel clad plate, the vacuum at the composite interface should be controlled between 1 Pa–10−2 Pa, or less.The lower the vacuum level, the more intense the oxidation of the composite interface. When the vacuum was 1 Pa and 10−2 Pa, 397 MPa at 1 Pa and 55 MPa at 105 Pa. The mechanical property of the stainless steel clad plate was mainly affected by the type and morphology of interface inclusions.The interfacial shear strength of stainless steel clad plate after pickling at different vacuum degrees decreased with the decrease in vacuum degree, and was 484 MPa at 10


In 1983–1984, interventions were implemented focusing on person-to-person transmission  and regulations to control irrigation waters containing fecal contaminates. In 1991, a second intervention was quickly implemented to avoid the cholera epidemic affecting neighboring countries . We explored the potential impact of these interventions on the epidemic. We created a yearly database of the MR TF cases, population, and contextual factors of TF from 1969 to 2012. We first analyzed the epidemic (Joinpoint regression), identified predictors of TF (Poisson multiple regression), and then analyzed the effect of the interventions (interrupted time series model). The main predictor of the TF epidemic was the rate of unemployment. In relation to the 1983–1984 person-to-person interventions, TF came down by 51%  and continued to decrease at a rate of 10.4% (95% CI: 5.8–15.6%) per year until 1991. In 1991, with the strong environmental control of the sewage-irrigated crops, TF further decreased by 77% (95% CI: 69.0–83.1%) and continued decreasing thereafter at 13% (95% CI: 11.3–15.6%) per year until the end of the study period. Today, 40 years after the epidemic, TF is a rare disease in the MR of Chile.From 1975 to 1983, a large epidemic of typhoid fever (TF) affected the metropolitan region (MR) of Chile (incidence rate [IR] of 219.6 per 10 Supplemental Figure 1).1 This has been the largest TF epidemic reported in Latin America, occurring in a country with the best health indicators in the region.3 The highest incidence rates (IRs) were seen in the metropolitan region (MR), one of Chile’s 16 geographical regions, located in the center of the country. The MR includes Santiago, the country’s capital city and seat of government, home to 40% of the national population; this region had better sanitary conditions than the rest of the country.5 The TF epidemic was attributed to various causes, including person-to-person transmission of Salmonella enterica serovar Typhi (ST), the short cycle associated with poor personal hygiene,4 and deterioration of safe food handling practices,2 deterioration of drinking water chlorination,7 or the use of untreated sewage in irrigation systems.9 Important public health interventions directed to the control of TF were first put in place in 1983–1984.11 In 1991, the government implemented a second public health intervention to avoid the cholera epidemic affecting neighboring countries.13 Our aim was to evaluate the association of these interventions with changes in the IR of TF. We collected all available data and used sound statistical analysis, to evaluate this association, which could inform policy makers in areas of high TF incidence.From 1950 to 1976, typhoid fever (TF) was highly endemic in Chile and did not seem to respond to improvements in household coverage of safe drinking water or sewage collection; in 1977, a large epidemic cycle began , environmental data (sanitation), and contextual data (socioeconomic metrics). The unit of analysis for this study was the MR, and the unit of observation was each year between 1969 and 2012 (44 time points). We collected the age–gender distribution of the population and of the TF cases reported and TF determinants (sanitation and socioeconomic).14 The study was approved by the Pontificia Universidad Católica de Chile Ethical Committee.We used interrupted time series (ITS) analysis to model the two interventions implemented for the control of TF in the MR.15 and the number of new TF cases , by age, place of residence, month and year were obtained from the compulsory transmissible diseases notification system of the Ministry of Health (MoH)16. We analyzed seasonality , age distribution , and S. enterica serotype distribution . We analyzed the following environmental risk factors of enteric infections: drinking water, sewage, final wastewater treatment, sanitary inspections of food handling, inspections of irrigation waters, measurements of residual chlorine in drinking water, and vegetable production .21 We also included the following socioeconomic markers: poverty, schooling, gross domestic product, Human Development Index, public health expenditure, and unemployment .25We obtained the MR population data by age and year between 1950 and 2012 from the national census and intercensal projections,TF cases  and deat11 the second was implemented over the course of a few months with the objective of protecting Chile from the cholera epidemic affecting neighboring countries.13We collected data on two interventions: the first was implemented over 2 years and was directed specifically toward controlling TF,26 This result prompted the prohibition of cultivating or marketing vegetables generally consumed raw, that grew at ground level , and initiation of mass communication and education campaigns about hygienic handling of crops. The clinical trials of a new oral typhoid vaccine, with an estimated efficacy of 70%, immunized 486,319 schoolchildren aged 5–19 years, covering a large proportion (60%) of the target population, with at least one dose of the oral vaccine.10 The food handler management included sanitary control and education in restaurants and school kitchens; in addition, small studies of chronic carrier detection with Vi serology and treatment with ciprofloxacin were carried out.10At the beginning of 1980, the MoH, in collaboration with the World Health Organization and the University of Maryland, designed the TF Control Program; its transference to public health occurred mainly during 1983 and 1984. This TF Control Program included a series of epidemiological studies to clarify the main transmission mechanisms of TF and included an evaluation of a new oral vaccine, educating food handlers, new methods for detection of chronic carriers, and a clinical trial of a new drug for chronic carriage treatment. They found ST cultivable in 10% of the irrigation water samples studied.13In early 1991, the government implemented emergency measures to avoid the propagation of the cholera epidemic affecting neighboring countries into Chile. It instituted an emergency action commission to implement and enforce control measures to prevent transmission of enteric infections in the MR and the rest of Chile. All measures were directed toward immediately stopping the long transmission cycle of TF from crop irrigation with sewage-contaminated water to crop consumption by the population. Through the joint action of the ministries of health, education, agriculture, and internal affairs, this commission intervened in improvement of water quality and irrigation practices, and further regulated the commercialization of crops. Specific measures included increasing the number of prohibited crops; banning restaurants from serving raw vegetables, raw fish, and seafood; construction of new irrigation channels separated from sewage discharges; chlorination of water channels; prohibition of any human activities near water sources with sewage discharges; and establishment of sanitary barriers in those areas.Supplemental Table 2). Significant factors were included in a Poisson multiple regression model. We then conducted a Joinpoint regression of TF over the study period with complete TF data (1950–2012) to identify significant breaks in the trends of the incidence of the disease.27 The second step was to analyze the effect of the interventions using ITS analysis.14 Records of the interventions performed in Santiago between 1969 and 2015, to control enteric transmission, demonstrated that there were two distinct governmental interventions, the first mass intervention extended over a 2-year period—1983 to 1984—and the second was a more focused and intense intervention in the summer of 1991. Based on the timing of these two , we divided the study period into four segments: 1969–1973 or 1950–1973 (pre-epidemic), 1974–1983 (epidemic), 1984–1991 (post-intervention 1), and 1992–2012 (post-intervention 2). We used the annual IR over the study period to estimate the change in disease level (intercept terms) and in disease trend (slope terms) for each intervention period. Variables significant in the Poisson multiple regression were entered in the ITS analysis as possible confounders. For the ITS analysis, our first approach was modeling TF incidence data with a Poisson generalized linear model.28 Nevertheless, with Cameron–Trivedi test, we found data overdispersion29; therefore, we used the negative binomial regression30 presented here:Yt: represents the number of cases of TF in year t,T: represents the calendar times beginning with 1969 = 1; T1 is the time of the initiation of the epidemic, T2 and T3 represent the initiation of each intervention. These time points were selected based on the date of the interventions initiation,X: represents the presence of the epidemic (X0) and of each intervention (X1 and X2), andt: Represents the population of Santiago in the year t.PopThe first step was to identify the main risk factors of the TF epidemic as a means to identify and control possible confounders of the interventions. We evaluated the association of individual risk factors with the annual IR of TF by bivariate correlations, using r-Pearson correlation  to select the best model. We analyzed the residuals of the selected model to detect autocorrelation, R’s autocorrelation, and partial autocorrelation functions. We estimated confidence intervals (CIs) for the change in the slope and the slope in the segment with the open statistical package R (application multcomp and function confint). We used the delta method to estimate the CIs for the TF IR per 105. Statistical analyses were performed with SPSS version 23.0  and R Core Team (2017).31 We compared the ITS analysis for two time intervals 1969–1973, when we had complete information on contextual factors, and 1950–1973, when we only had TF data. Finally, we analyzed the decrease of TF IR in four age groups  and three study periods , using a Poisson regression analysis, we included interaction terms for age and periods. Based on this model, we estimated the reduction of TF IR for each age group from the epidemic to the end of the study periods (1983 versus 2005–2012) and from the endemic to the end of the study periods (1969 versus 2005–2012).We ran the full model, including the previously selected confounders and evaluated the results of each β parameter using the Supplemental Figure 1). Annual cases of TF in the MR in 1969 were 3,463, in 1983 peaked to 9,825, then in 1984 fell to 5,819, in 1992 the cases fell further to 447, and by 2012 there were only 65 TF cases reported . The IR of TF dropped in all ages, but the size of the reduction varied significantly among the age groups; by the end of the study, the TF IR, compared with the pre-epidemic level, dropped 25 times (95% CI: 19–34) among 0–4 year olds, 116 times (95% CI: 100–134) among 5–19 year olds, 121 times (95% CI: 102–143) among 20–34 year olds, and 51 times (95% CI: 41–63) among 35 year olds and older .Typhoid fever in the MR was highly endemic, without a clear trend in incidence, from 1950 to 1976; in 1977, TF increased abruptly reaching its worst epidemic ever reported, which lasted until 1985. Typhoid fever incidence had a strong downward trend until 1991 and in 1992, its decline accelerated reaching, by the end of the study period, the lowest TF IR reported in the MR . Typhoidreported . The marS. enterica serotypes suggest that during the epidemic, ST was predominant until 1993 (nearly 80% of cases) when it began to decrease, reaching 40% at the end of the study period. At this time, the proportion of Salmonella Paratyphi B increased reaching up to 60% of cases, whereas Salmonella Paratyphi A ran from 0% to 2% throughout the period . Environmental sanitization variables had not deteriorated before nor during the epidemic. Coverage of drinking water and sewage collection were improving during the epidemic; final sewage treatment was not implemented until 2002 in the MR, when the disease had been controlled . Contrarily, most socioeconomic indicators deteriorated during the epidemic period ; in the Poisson multiple regression, only unemployment was selected .The available data on Without including external a priori information, the Joinpoint regression analysis from 1950 to 2012 showed four statistically significant changes in trend: a stable period from 1950 to 1975, a rapid increase from 1975 to 1978, a short stable period from 1978 to 1983, and then a rapid and significant drop from 1983 to 2012 .Supplemental Table 2).The TF IR pre- and post-intervention periods are presented in Our aim was to analyze the trend of TF in the MR from 1969 to 2012 and to evaluate its association with the public health interventions of 1983–1984 and 1991. We collected a large body of data to understand the trend of TF, including the causes of the 1977–1984 epidemic. We concluded that the main explanatory factors were the long cycle of transmission of TF through the contamination of irrigation waters by sewage, worsened by the intensification of person-to-person transmission during an economic crisis, with unemployment being its single strongest marker.32In Santiago, sewage collection coverage was increasing rapidly before, during, and after the epidemic years. Nonetheless, all sewage collected from the households was discharged untreated into the water courses used for irrigation of the city agricultural lands, thus increasing the enteric contamination of the crops eaten by the citizens. Because of this return of the contamination through the crops, the sewage collection did not benefit the Santiago population. Between 1969 and 1976, the coverage of sewage collection from homes increased by 75% in Chile. By 1982, the MR reached 82% and Chile 70% of sewage coverage. Since sewage sanitation in Santiago was implemented in 2010, the increase in sewage collection from 1969 to 1976 augmented the microbiologic burden in the irrigation waters of Santiago by at least 75% immediately before the epidemic years. This recirculation of sewage had been alerted a century before: “The present water closet system, with all its boasted advantages, is the worst that can generally be adopted, briefly because it is a highly extravagant method of converting a mole-hill into a mountain. It merely removes the bulk of our excreta from our houses to choke our rivers with foul deposits and rot at our neighbors’ door. It introduces into our houses a most deadly enemy…”—chemist quoted in the Scientific American, 1869.36 suggesting a broad enteric contamination in the MR of Santiago.9 Unemployment is a complex variable representing a summary of the underlying socioeconomic and political crisis; it was clearly associated with the initiation of the epidemic as other authors also noted.7 The abrupt increase in the unemployment in Santiago (reaching 19% of the population), a marker of an important socioeconomic crisis, resulted in an increase in person-to-person transmission of enteric infections, in turn rapidly increasing the force of infection in the city causing the huge epidemic.The TF epidemic coincided with increases of other enteric infections, in particular hepatitis A,The 1983–1984 TF Control Program was associated with the break of the TF epidemic, which returned to the previous endemic level and the 1991 cholera prevention measures were associated with the definitive control of TF which became a sporadic disease in the MR.35 In association with the 1983–1984 interventions, TF came down by 51% (95% CI: 30.2–65.0%) and kept decreasing by 10.4% (95% CI: 5.8–15.6%) per year until 1991. In 1991, TF further decreased by 77% (95% CI: 69.0–83.1%) and continued decreasing thereafter at 13% (95% CI: 11.3–15.6%) per year until the end of the study period. Today, 40 years after the epidemic, TF is a rare disease in the MR of Chile. This presents strong evidence that the environmental contamination with ST was the main cause of the high endemicity of TF in the MR. The socioeconomic crisis which preceded the epidemic or hyper-endemic years, as many authors called the 1977–1983 period, offered the conditions to aggravate person-to-person transmission of ST, increasing the burden of infection in the irrigation waters and crops, thus canceling out the benefits of households sanitation. The sewage contamination of irrigation waters explains the epidemiology of the disease in the MR, with high IRs in all socioeconomic sectors, independently of the availability of drinking water or connection to sewage systems.1The 1983–1984 intervention placed a higher emphasis on interrupting person-to-person transmission of ST, by vaccination of schoolchildren and food handlers, increase of food handling controls, and chronic carrier identification. They also conducted actions to prevent the environmental contamination and the long cycle of transmission. The 1983–1984 intervention required community participation (in vaccination programs) and changes in behavior of the public and providers. Contrarily, the emphasis of the 1991 intervention was in the interruption of the environmental transmission of enteric infections by strict control of irrigation waters and crops cultivation, transport, and marketing, forcefully interrupting the contact of sewage-contaminated items with the public; its success did not depend on the public’s willingness to follow the health recommendations.37 Borgoño, in 1958, reviewed the epidemiology of TF in the MR; he described the decreasing trend in case-fatality rate between 1940 and 1949 (19.3% and 13.9%), later markedly accelerated by the introduction of chloramphenicol from 1950 to 1956 (6.5–1.5%). Regarding causality, he pointed out that 50% of the population was connected to sewer lines whose waters were used for crop irrigation in the MR; he concluded that TF endemicity was largely attributable to environmental transmission, urging the authorities to develop an integral sanitary program to definitively control enteric infections in the region.38 In 2007, Laval, in a historical review of TF in Chile, concluded that “although the hypothesis of environment contamination as the cornerstone in typhoid persistence was present since the recognition of the disease in 1894, it was faced efficiently and perhaps in a definite manner only almost 100 years later.”1 Cabello gave most of the causal weight of the epidemic to the lack of chlorination of drinking water.7 Drinking water chlorination cannot explain the cause nor the control of the TF epidemic because the MR reached the highest national coverage of chlorinated drinking water and yet presented the highest rates TF since 1950 ; the statistical model selected, which appropriately fit the data; and Chile’s very unique situation regarding TF, a quasi-experimental design in which we could analyze the factors associated with the increase and the decline of the disease in the population.The main limitation is the observational nature of the study, in which we could not rule out that unmeasured confounders were causing the changes in the disease. Also, the 1983–1984 intervention is not an ideal clear-cut intervention; instead it extended over a broad period and consisted of various measures of different efficacy, being impossible to individualize the contribution of each factor to the control of the disease.41 Thus, we could not estimate how much of the changes could have been explained by exhaustion of the susceptibility in the community. The latter would have come into effect in the years immediately after a large outbreak, but by itself it cannot explain the maintenance of the low TF transmission 40 years after the peak of the epidemic. The near disappearance of TF in the MR is better explained by the definitive interruption of its main transmission route.Another important limitation is related to our models, which did not include the dynamics of the disease.Intervention 1 was complex, consisting of vaccination, intensive mass communication, and increases in environmental controls. It is not possible to disentangle the effects of these various components; the vaccine effect in the community may last longer than the duration of the immunological protection reached by the vaccines. Not having a dynamic model, it was not possible to estimate the duration of the effect of the particular interventions. Thus, we assumed that the first intervention changed the dynamics of the disease from then on, maintaining its effect until the end of the study period; this may represent an overestimation of its effect.Our findings can be applied to many areas in which sewage is discharged into irrigation waters. Our results suggest that ST could be transmitted through this mechanism and this transmission route can be effectively interrupted by short-term public health measures which do not require very large investments; these should be maintained and enforced until a structural solution, such as sewage treatment plants, is in place. We showed that the definitive control of ST transmission was achieved immediately after 1991, in concordance with a powerful government decision to completely stop the irrigation with sewage-contaminated waters.Supplemental figures and tables


This study aims to describe the spatial and temporal characteristics of human infections with H7N9 virus in China using data from 19 February 2013 to 30 September 2017 extracted from Centre for Health Protection of the Department of Health (CHP) and electronic databases managed by China’s Center for Disease Control (CDC) and provincial CDCs synthetically using the Geographic Information System (GIS) software ArcMap™ 10.2 and SaTScan. Based on the multiple analyses of the A(H7N9) epidemics, there was a strong seasonal pattern in A(H7N9) virus infection, with high activity in the first quarter of the year, especially in January, February, and April, and a gradual dying out in the third quarter. Spatial distribution analysis indicated that Eastern China contained the most severely affected areas, such as Zhejiang Province, and the distribution shifted from coastline areas to more inland areas over time. In addition, the cases exhibited local spatial aggregation, with high-risk areas most found in the southeast coastal regions of China. Shanghai, Jiangsu, Zhejiang, and Guangdong were the high-risk epidemic areas, which should arouse the attention of local governments. A strong cluster from 9 April 2017 to 24 June 2017 was also identified in Northern China, and there were many secondary clusters in Eastern and Southern China, especially in Zhejiang, Fujian, Jiangsu, and Guangdong Provinces. Our results suggested that the spatial-temporal clustering of H7N9 in China is fundamentally different, and is expected to contribute to accumulating knowledge on the changing temporal patterns and spatial dissemination during the fifth epidemic and provide data to enable adequate preparation against the next epidemic. The first human infection with avian influenza AH7N9) was observed in Shanghai in March 2013 . As a reN9 was obThere are many challenges to face in responding to this threat, including the frequent occurrence of cases, the high fatality rate, the unknown and potentially intractable genetic variation of the virus, the limitations to medical technology, the potential for pandemics, and the unpredictability of the virus. These issues have become increasingly prominent . The A In the early stage of transmission, the A(H7N9) virus is strongly concealed. Scientists are rarely recruited to detect typical symptoms in poultry until they are transported to other cities, even those carrying the virus . (2) DueThe number of cases peaked in 2014 and generally decreased in subsequent epidemics up to a strong rebound in A(H7N9) cases in December 2016. Some researchers found that the fifth epidemic started earlier in the year spread to more regions and counties in affected provinces and had more confirmed cases than previous epidemics before 1 October 2016 . The strChina is the original site of the virus outbreak and the most severely affected area in the world. Chinese people have suffered severely from the avian influenza A(H7N9) virus over the years. The Chinese government has been working to establish an effective system of disease surveillance and to monitor the epidemic. In addition, the statistical department of the Chinese government is collecting detailed and up-to-date data on infections around the world. This study provides the most comprehensive and up-to-date detailed data on human infections with A(H7N9) virus over five outbreaks in China. This study aims to conduct multiple analyses of the H7N9 epidemics to increase understanding of the current features of the epidemiological distribution of the virus and to explore its temporal and geographical patterns, especially its spatial-temporal clustering. The findings are expected to contribute to accumulating knowledge on the changing temporal patterns and spatial dissemination during the fifth epidemic and provide data to enable adequate preparation against the next epidemic.We established a multi-source database on laboratory-confirmed human cases of A(H7N9) virus infections. It included basic information such as the event ID, patient age, patient sex, date, clinical symptom severity, affected area, latitude and longitude, and activation record of every case. We collected this information mainly from avian influenza weekly reports produced by the Centre for Health Protection of the Department of Health (CHP) and electronic databases managed by China’s Center for Disease Control (CDC) and provincial CDCs. Local CDSs are required to conduct a field investigation for each A(H7N9) virus infection and report aggregated data to a national surveillance system. China’s CDC is responsible for publishing the data in standard form. CHP is responsible for extracting data on both humans and birds and for highlighting global avian influenza activity. Uniform-format data contribute to analyses and exploration of characteristics. To eliminate the inconsistency between the onset date and the report date, we selected the time at which the patient was identified as infected with the A(H7N9) virus and was reported to the local CDS by the hospital as the date in this study. The diversity of multiple-source data increases the complexity of processing spatial attributes and increases the margin of error. Here, the administrative levels defining prefecture-level cities were used as the default values. Province-controlled divisions were regarded as administrative cities. The total number of patients in prefecture-level cities were compiled, including all counties and districts. Sometimes it was difficult for officials to identify the specific site of the infection, such as when the infected person frequented different cities before seeking medical advice or before infection was confirmed. Under such circumstances, after referring to the literature and consulting experts, the hospital location was temporarily assigned as the outbreak location . We extrGIS analysis of the human infection database was conducted for the entire study area. The Spatial Autocorrelation and Getis-Ord Gi* (Getis-Ord Gi* statistic (pronounced G-i-star) calculated in Hot Spot Analysis) tool of the software ArcGIS10.2  was used in the study. This tool provided intuitive visual presentations of results regarding the distribution of infections and spatial clusters at various stages of historical outbreaks in Mainland China. Using this tool, Global Moran’s I, Getis-Ord Gi* statistic, and Moran’s I index value were calculated, and z-scores and p-values were calculated to examine the null hypothesis that the attribute under analysis was randomly distributed among the regions . These rSpatial-temporal analysis, as an epidemiological method, can extract and mine information from epidemic data. The SaTScan model adapts the spatial-temporal interactive scan and statistics method to detect the cluster characteristics of cases. Relevant date and location information is the only required inputs in the space-time permutation version of the model . When orIn the model, a cylindrical window corresponding to space at the base and to time in the vertical direction is moved in space and time. The cylinder is centered at a county with various spatial radii to search for clusters and expands in height with different temporal values . The cylThe circulation period of the avian influenza A(H7N9) virus starts from 1 October of each year and ends on 30 September of the following year. Taking the novel strains of the A(H7N9) virus into consideration, the first epidemic period continues from February 2013 to 30 September 2014. The following four cycles follow the standard pattern. By the end of 30 September 2017, the database contained a total of 1558 cases of clinically confirmed infections reported by the government. The percentages of male and female patients in this infection group were 69.9% and 30.1%, respectively. Young people accounted for 67.7% of cases. The underage and older groups had lower incidences of infection with percentages of 3.6% and 5.5%. Elderly females represented only 24 cases. For the entire studied period, the weekly number of H7N9-infected individuals was also calculated, as shown in The avian influenza A(H7N9) virus caused various levels of damage in the 28 provinces and 183 prefecture-level cities in Mainland China, as shown in As shown in p < 0.0001). The value of the third epidemic phase had the maximum index value (0.4208), indicating the highest level of clustering. Although most cases occurred in the fifth epidemic phase, the distribution was scattered; thus, the Moran’s I index value was not the highest observed. Across all five epidemics, the Moran’s I index value was 0.0820, the Z-score was 5.6341, and the p-value was 0.0000, indicating significant clustering of the cases.Here, we conducted a GIS analysis using the A(H7N9) database and spatial autocorrelation. Moran’s I index was calculated to identify any clustering in the spatial locations of infections in the past five epidemics. As shown in the As shown in p-values less than 0.05) in Mainland China, with one primary cluster and 10 secondary clusters. The single primary cluster was located in Northern China and covered the Beijing-Tianjin-Hebei region, Shanxi Province, Shaanxi Province, Henan Province, and Shandong Province. The clustering time spanned from 9 April to 24 June 2017, and 79 cases were involved. The secondary clusters were used to detect the most likely aggregation patterns in local regions or time periods after the primary cluster scanning. It was therefore expected that they might overlap areas of other secondary clusters or the primary cluster. The secondary clusters had extremely wide geographical distributions and scattered time periods, accounting for 511 cases. The 10 secondary aggregation areas were mainly distributed in the southern coastal areas, especially in Eastern China and a small part of Southern China. Most of them were distributed in the 2013 and 2017 time frames. The model did not consider data from Jilin Province, Liaoning Province, Gansu Province, or Tibet, because these regions failed to meet the requirement of only sporadic infections in some restricted periods. Furthermore, Tibet and Gansu Provinces exhibited a long geographical span in an east–west direction. This pattern was different from that in Midwestern Jiangsu Province and Eastern Guangdong Province. There was a large group of A(H7N9) infections in each epidemic. The highly decentralized incidence in different cities was a concern in this analysis, although they accumulated in a continuous time span. Evaluation of the historical infected areas along with the spatial information of the clusters revealed that they represented 61.5% of the number of cities in the whole study area. As shown in As shown in Our study provides a comprehensive description of the epidemiology of laboratory-confirmed cases of A(H7N9) virus infection during the last five epidemics, presents the temporal and spatial dissemination pattern of A(H7N9) epidemics in Mainland China, and tests the cluster characteristics of patients infected with A(H7N9) in time and geography.A previous study indicated a higher risk in the cool months, especially when the minimum temperature is between 5 and 9 °C and the maximum temperature is between 13 and 18 °C . AlthougGeographic data normally show autocorrelation, rather than being independent. It is the same with avian influenza A(H7N9) data. Spatial autocorrelation methods are effective ways to measure the spatial interdependence of geographic data . HoweverSome studies have shown that the spatial density of A(H7N9) virus infections in waterfowl and poultry is positively correlated with the incidence of A(H7N9) infections in humans ,31,32,33(1)The fifth epidemic started earlier in the year and affected more individuals and more districts than did the previous four epidemics based on analyses of the timeline and geographical distribution. Cases shifted from coastline areas to more inland areas over time.(2)The cases exhibited local spatial aggregation, with high-risk areas most found in the southeast coastal regions of China. Shanghai, Jiangsu, Zhejiang, and Guangdong were the high-risk epidemic areas, which should arouse the attention of the local governments.(3)Combining GIS and SaTScan methods increased our understanding of the development of A(H7N9) virus epidemics. A strong cluster from 9 April to 24 June 2017 was identified in Northern China, and there were many secondary clusters in Eastern and Southern China, especially in Zhejiang, Fujian, Jiangsu, and Guangdong Provinces.Our study constructed a comprehensive, up-to-date geographic database of the avian influenza A(H7N9) infections and conducted spatial-temporal analyses of five consecutive epidemics. The following primary conclusions were attained:The up-to-date database of avian influenza A(H7N9) cases and the temporal-spatial assessments of the epidemiological characteristics can help prevention in the next pandemic outbreak. However, the problem is complex, and further analysis, especially from the perspectives of space and time expansion, is warranted.


Following publication of this article , the jouAnalytical Chemistry article was not cited in the PLOS ONE article; these figures were included in the PLOS ONE article without attribution to or permission of the original publication.Figs 3A and 4A were published previously as Figure 3 in . The AnaIn Fig 5, the ‘tear’ stamp pattern shown vertically on the left side of the image for CA-Cdc42, Pattern B, 18 hours, is vertically inverted and in a different position as compared to the placement of the tear stamp detectable on the original micrograph.Concerns were raised about whether Golgi staining in Fig 2 accurately represents the primary data.The University of Cincinnati has completed an investigation of this work and recommended retraction based on the above concerns.PLOS ONE Editors retract this article, as the concerns about these figures call into question the validity of the results and conclusions reported in the article.In line with the University of Cincinnati’s recommendation, the GK, C-CH, and CCC agree with the retraction.


Upon the growth of clusters, the structures tend to become well-knit gradually. As for the evolution of electronic structures of AlN clusters through the HSE06 calculations, the density of states curves become more and more nondiscrete with the atomic structures evolving from small to large size and tend to resemble that of the Wurtzite AlN. The chemisorption characteristics of the large Al60N60 cluster towards different elements such as Al, N, Fe and Cu are also theoretically unveiled, in which it is interestingly found that the N and Cu atoms are likely to be adsorbed similarly at the growth edge position of the Al60N60 cluster and the density of states curves of these two chemisorption systems near the Fermi level also show some interesting similarities.A theoretical insight into the structural evolution of AlN atomic clusters and the chemisorption of several common alloying elements on a large cluster has been performed in the framework of state-of-the-art density functional theory calculations. We report the findings that the longitudinal growth takes precedence during the early stage of structural evolution of small AlN clusters, when the longitudinal dimension becomes stable, the AlN cluster proceeds with cross-growth and blossoms into the large-size Al It is worthwhile to perform fundamental research on clusters to reveal their structures and properties at the atomic and electronic levels. Li et al. systematically investigated the structural evolution of gold–germanium bimetallic clusters and the nonlinear optical properties, chemical properties of a series of alkali-metals-adsorbed gold–germanium bimetallic clusters, in which it was found that the atomic structure of gold–germanium bimetallic clusters with adsorbed alkali metals did not change significantly and alkali metals tended to adhere to the surface or edge of clusters . Yan et observed . Meanwhiobserved ,14,15,16ronments . BelBrunclusters . Furtherclusters ,20. Howeclusters ,22,23,2460N60 large clusters to unveil their chemisorption characteristics. In the previous experimental study, we found that copper can be observed on the AlNp/Al interface by adding copper powder to AlNp-reinforced Al composites. In addition, when the Fe power is added to the AlNp-reinforced Al composites, it is not aggregated at the interface but dispersedly distributed in the composite material [In this article, we have theoretically investigated the structural evolution concerning the growth of AlN clusters from the perspective of atomic and electronic structures using the HSE06 hybrid functional, along with the characteristics of the bond lengths and energetics of AlN clusters. In order to understand the chemisorption of common alloying elements such as Fe, Cu, Al, N on AlN clusters, we also simulated the interaction between these elements and the Almaterial . This wo6N6 cluster). In order to avoid interaction between clusters in the x, y and z directions, all the models are separated by the vacuum space of 20 Å. The K-points mesh of 1×1×1 has been used within the Monkhorst–Pack scheme in the stage of geometry optimizations and electronic structure calculations. The plane-wave energy cut-off of 520 eV has been employed for all the structure optimizations. We used a conjugate gradient algorithm to perform the structure optimizations and relax all ionic positions until the force on each ion is lower than 0.02 eV/Å. In order to calculate the electronic structure of the clusters and their chemisorption systems more accurately, we have considered the effects of nonlocal exchange and used the screened hybrid functional of Heyd, Scuseria, and Ernzerhof (HSE06) [In this study, the calculations of total energies, atomic forces, and structure optimizations have been performed using the generalized gradient approximation (GGA)  in the f (0001¯) , we have (HSE06) . In HSE06N6. When the Al6N6 cluster first grows longitudinally into a stable two-layer hexagonal prism structure, the representative cluster is Al9N9. For the Al15N15 cluster, it is formed when the longitudinal dimension of the cluster becomes stable. The Al15N15 cluster then evolves to Al30N30 in the transverse direction after the cluster size is stabilized in the longitudinal direction. The Al60N60 is a large cluster structure evolved from Al30N30 with continued transverse growth. The structural evolution process of AlN clusters in the atomic scale is shown in 15N15 cluster, each aluminum or nitrogen atom of the hexatomic ring connects with a nitrogen or aluminum atom to lead to the formation of the Al30N30 cluster. The dangling aluminum and nitrogen atoms in the Al30N30 cluster would further absorb atoms, forming complete hexatomic rings and growing into a large aluminum nitride cluster (Al60N60 cluster).During the structural evolution and growth of AlN clusters, the five most representative clusters are selected. The smallest cluster unit is Al6N6, Al9N9, Al15N15, Al30N30 and Al60N60 clusters are compared with the Wurtzite AlN crystal, which can be shown in 6N6, Al9N9 and Al15N15, have the discrete density of states curves. With the evolution and growth of AlN clusters, the density of states curves tend to be continuous and become more similar to the DOS curve of the Wurtzite AlN crystal gradually. For the larger Al30N30 and Al60N60 atomic clusters, the density of states curves show four and two deep levels, respectively, in the band gap through the HSE06 calculations. Near the Fermi level, in accordance with the increase of AlN clusters, the HOMO–LUMO gap is also gradually increased.In addition to the evolution of AlN clusters in atomic structures, we have also investigated the evolution of AlN clusters in electronic structures. In order to obtain the more accurate electronic structure, we have employed the HSE06 hybrid functional in calculations. The electronic structures of the Aln clusters: with the growth and evolution of clusters, the average bond length gradually increases; when the cluster reaches a certain size, the bond length attains the maximum value. After that, the average bond length begins to decrease and the large cluster tends to shrink. n clusters, which are calculated using the previous method [n is the total energy of the investigated (AlN)n  cluster system, while EAl and EN stand for the total energies of a single aluminum atom and nitrogen atom, respectively. When the AlN clusters grow in the early stage, the cohesive energies tend to increase. While, the cohesive energy decreases rapidly when n = 30, which is related to the structure of the Al30N30 cluster. Each atom in the hexatomic ring of the Al30N30 cluster is connected with a hetero-atom, which dangles outside and results in a sudden decrease in cohesive energy. After those dangling bonds are saturated by growth, the cohesive energy of the cluster would increase again. In order to further investigate the properties of AlN clusters, the average bond length, cohesive energy and total energy changes during the structural evolution of atomic clusters have been analyzed, which can be seen in s method ,34 based60N60 large cluster are investigated considering different chemisorption sites. There are three different positions on the Al60N60 large cluster: top, edge and side. Due to the existence of equivalent sites, two different sites are studied for the top position: one above the center of the six rings and the other above the Al-N bond. There are four different sites for the edge position: near the N atom with four bonds, near the Al atom with three bonds, near the N atom with three bonds and near the Al atom with two bonds. There are two different sites for the side position: one is near the recessed Al atom, and the other is near the Al-N bond. The adsorption energy Ead is calculated as follows:Al60N60+M is the total energy of the system with M element  adsorbed on the Al60N60 cluster, while EAl60N60 and EM stand for the total energies of the Al60N60 cluster and the isolated M  atom, respectively. We considered the chemisorption of Al, N, Fe and Cu at different positions  of the Al60N60 large cluster. A total of eight chemisorption sites for every elemental sorption are studied. After calculations, we have chosen the respective site with the largest adsorption energy for each position  in our research.As is known, the large clusters especially, in short- or medium-range order could play important roles in nucleation of the corresponding solid phase in materials science. Take the in-situ AlN-reinforced Al alloys for example, Cu is a common strengthening element in aluminum alloys, which can significantly increase strength by precipitation hardening; Fe is also an important element in rapidly cooled aluminum alloys. For chemisorptions of Al and N atoms, it is useful to understand the evolution and growth of AlN clusters. It is essential to study the interactions between the AlN cluster and the alloying elements, which can help to understand the distribution of those alloying elements in solidified materials. In this work, the fundamental interactions between several common alloying elements  and the Al60N60 large cluster, the Ead is the maximum of 10.4775 eV, almost three times those at the top or edge positions. Thus, the aluminum is likely to adsorb and accumulate at the side of the Al60N60 cluster. ad is the maximum of 3.0128 eV. Thus, the nitrogen is more likely to be adsorbed at the edge of the growth frontier of AlN. In the Al melt, the growth atmosphere of AlN is rich in aluminum atoms and poor in nitrogen atoms, so the growth of AlN mainly depends on the deposition/adsorption of nitrogen atoms on AlN. As shown in The chemisorption characteristics towards the Al atom are shown in 60N60 large cluster at different positions is shown in 60N60 cluster, compared with the top and side sites, the copper atom has the highest adsorption energy at the edge site. It can be seen that copper is more likely to be adsorbed at the edge site of the cluster, which is similar to the stacking mode of nitrogen atoms. The chemisorption of iron atoms at different positions of the Al60N60 cluster is shown in ad of Fe element at the side is the largest. It is shown that the Fe atom is easier to accumulate at the side of the Al60N60 cluster.The chemisorption of Cu atoms on the Al60N60 cluster, we have also investigated the electronic structures based on the configurations corresponding to the respective chemisorption systems with the largest adsorption energies of Al, N, Fe and Cu atoms on the cluster through the HSE06 calculations (shown in 60N60 large cluster is sensitive to the atomic chemisorption. What is more, the electronic density of states curves of the N-chemisorption and Cu-chemisorption systems near the Fermi level show some interesting similarities. This corresponds with the above absorption site similarity of the two systems. After adsorbing N or Cu atoms, a small number of deep levels are produced between the HOMO and LUMO.In order to discuss the effects of Al, N, Fe and Cu atoms on the electronic properties of the Alshown in . After A60N60 large cluster. It is also found that the electronic density of states curves of the N-chemisorption and Cu-chemisorption systems near the Fermi level show some interesting similarities through HSE06 calculations. This work is proposed to provide valuable theoretical clues and ab initio analyses for the experimentalists in the field and help to deepen understanding of the broader aluminum–pnictogen system at the basic atomic and electronic levels.In summary, the AlN clusters prefer to grow longitudinally in the early stage and then evolve to the large cluster transversely by using a first principles method. During the structural evolution process, the cohesive energy generally increases and the large-size AlN clusters tend to shrink. The elemental chemisorption studies show that the copper and nitrogen atoms have similar chemisorption characteristics on the Al


Scientific Reports 10.1038/srep02827, published online 3 October 2013Correction to: The following information was omitted from the Methods section.The images in Figures 2, 3B, and 4A were captured through a coverslip. To achieve this, we cut the boundary of tissue culture dishes and mounted with fluoromount-G. The contrast/brightness of the Golgi stain in Figure 5 was adjusted using Image J software which can alter certain details of the Golgi structure. However, the key point was to show the brief position of the Golgi apparatus during the release period. They are not relative to the certain details of the Golgi structure.


Recuperative techniques have been used to anticipate and potentiate recovery. The massage is one of the most widely used in sports. Among the ways to demonstrate the recovery of the organism is the resumption of autonomic modulation of heart rate, which can be analyzed in situations that cause disturbances in the behavior of the cardiovascular system with the objective of verifying the responsiveness of the autonomic nervous system (ANS). Recovery can be assessed through heart rate variability (HRV) which analyzes the oscillations in consecutive heartbeats, thus allowing an important non-invasive alternative for the study of modulation of the ANS. The objective of the study will be to measure the effects of massage as a recuperative technique on the autonomic modulation of heart rate and cardiorespiratory parameters at different moments of application.This is a randomized, cross-over clinical trial. Forty men aged 18 to 30 years, healthy and physically active according to the International Physical Activity Questionnaire will participate in the study. Participants will be randomized into groups, which will perform the five interventions of the study at randomized moments, one intervention per session: Intervention 1: control; Intervention 2: participants will receive the massage protocol; Intervention 3: performance of the stress protocol; Intervention 4: participants will perform the stress protocol and immediately after receive the massage; Intervention 5: participants will perform the stress protocol and 1 h after conclusion of the protocol will receive the massage. The sessions will occur with an interval of 1 week between them and, due to the technique used, blinding participants and therapists is not possible. The primary outcome measure is HRV that will be measured 2 h after the conclusion of each intervention, and secondary outcome measures, which include heart rate, respiratory rate, blood pressure, oxygen saturation, and individual touch perception, will be measured at specific moments in the course of each intervention.The implementation and use of this standardized protocol should provide important and reliable information regarding the use of massage in post-exercise recovery, with the identification of its effects on the ANS and the best timing and form of massage application. The data obtained in the present study will provide subsidies for the best management of application of the technique in sports clinical practice, considering periods of training and, mainly, of competitions.NCT03094676. Pre-results. 12 March 2018.ClinicalTrials.gov, ID:The online version of this article (10.1186/s13063-018-2830-1) contains supplementary material, which is available to authorized users. Training in its simplest form represents acute challenges to the body intended to optimize chronic improvements in physiological capabilities . The sucPhysical exercise can be a stressor mechanism to body systems, promoting several organic modifications, and recovery processes are commonly employed in the sense of restoring the systems to their basal condition . To acceClinical, functional, and metabolic modifications are already described in the relevant literature regarding the effectiveness of massage –9. It isHowever, the physiological adaptations promoted by massage can also be monitored through autonomic nervous system (ANS) and cardiovascular responses . StudiesIn relation to the markers of autonomic recovery, massage has also demonstrated positive results in the recovery capacity. Arroyo-Morales et al.  performeHowever, in the study of Arroyo-Morales et al.  some gapThe recovery of autonomic modulation of heart rate has been considered an important global marker of athlete recovery. Ihsan et al.  reportedThe present study is a randomized clinical trial aimed at investigating the effects of massage as a recuperative technique on the recovery of autonomic modulation of heart rate and cardiorespiratory parameters in physically active young people comparing two distinct scenarios: (1) immediately after stress in order to investigate if the technique anticipates the recovery of the ANS, and (2) immediately after the recovery moment of the ANS in order to investigate whether the technique potentiates the recovery of the ANS.A randomized, cross-over, controlled clinical trial will be conducted at the Center for Studies and Care in Physiotherapy and Rehabilitation of the Universidade Estadual Paulista (FCT/UNESP), Presidente Prudente, São Paulo, Brazil. The study design is illustrated in Fig. 2) according to the World Health Organization [A total of 40 participants will be recruited at the local university  through online advertising and approaching participants by telephone, SMS, and social networks, from January 2018 to April 2018. Participants will be considered eligible if they are apparently healthy, male, aged between 18 and 30 years, and have a Body Mass Index (BMI) classified as normal . This researcher will be instructed not to previously inform the participants or other researchers about the procedures that will be performed in each session. The following figure presents the schematization of the aforementioned randomization processes Fig. .Fig. 2ScThe study will be conducted in five interventions (Intervention 1 (I1); Intervention 2 (I2); Intervention 3 (I3); Intervention 4 (I4); Intervention 5 (I5)), and the groups will be randomized to perform these interventions at several moments, with a 1-week interval between interventions. Data collection will always be performed at the same period of day and in air-conditioned environments.Intervention 1 (I1): the HRV behavior of the participant will be evaluated without exposure to any recovery technique or any type of stress. The follow-up period will be 2 hIntervention 2 (I2): the HRV behavior of the participant will be evaluated before the application of the massage technique. Evaluations will be performed 2 h after massage applicationIntervention 3 (I3): the HRV behavior of the participant will be evaluated before the implementation of the stress protocol. The autonomic modulation of heart rate will be evaluated 2 h after the end of the stress protocolIntervention 4 (I4): the HRV behavior of the participant will be evaluated before the stress protocol, followed immediately by the massage technique, as well as 2 h after the completion of the massage in order to observe if HRV is anticipatedIntervention 5 (I5): the HRV behavior of the participant will be evaluated before the stress protocol, and the massage technique will be applied at the exact moment of ANS recovery, determined by the study of Almeida et al. [a et al.  who usedThe description of the interventions is as follows:The first three interventions of the study  will be performed to establish the basal behavior of the study variables and/or before the two different stimuli (massage and stress), and for possible explanations and discussion of the study.In Interventions 4 and 5, analysis of the effects of massage on post-exercise recovery, and the comparison of the effects of massage when performed immediately after stress or 1 h after stress, will be performed.It is emphasized that at I5 the massage will be applied 60 min after the end of the stress protocol, as this was the exact time identified in the study by Almeida et al.  for the -1 of the participant’s body mass. During the sprints and test, verbal stimuli will be provided to stimulate maximum performance.Participants will be submitted to a stress protocol consisting of a jumping program and a maximum short-term cycling test . The jumThis stress protocol was chosen due to the study of Almeida et al.  which inIt is worth mentioning that after the stress protocol all participants will be required to drink 180 ml of water in order to standardize hydration and avoid the influence of blood volume on the results of the present clinical trial.According to a recent systematic review with meta-analysis , the effFor massage on the anterior thigh (quadriceps femoris muscle) the participant will be placed in the dorsal decubitus and the massage initiated on the dominant limb and then the non-dominant limb, and posteriorly, in the ventral decubitus position, massage will be performed on the trunk . The techniques of surface and deep slip will be used, the latter being presented at two intensities, moderate or intense, being gradually increased. The massage will be performed towards the muscle fibers, from distal to proximal and following the lymphatic flow.The massage will be performed by three physiotherapists specializing in sports physiotherapy, previously trained by a sports physiotherapist with 10 years’ experience in sports massage.A pilot study will be conducted for the training of the physiotherapists who will apply the massage in a standardized manner regarding the intensity, rhythm, form of application of massage, and application dynamics through data collection. The physiotherapists will be required to present excellent correlations between them in order to be eligible to perform the technique.The massage will have the rhythm: 1 s of slip and 1 s for return of the hand to the initial position, totaling 2 s of slip  and individual touch perception. Evaluations will be performed by the same independent trained assessors throughout the study.software Kubios HRV – version 2.0 [HRV will be used as the primary outcome as it is an important overall marker of post-exercise recovery . The HRVsion 2.0 .software Kubios HRV – version 2.0, supplemented by manual filtering, to eliminate premature ectopic beats and artifacts, and only series with more than 95% of sinus beats will be included in the study [For this analysis, the time series of RR intervals will initially be subjected to digital filtering using the he study . ThroughDuring the recovery period, the evaluated moments will be: R1 , R2 , R3 (fifth to 10th minutes of recovery), R4 (10th to 15th minutes of recovery), R5 (20th to 25th minutes of recovery), R6 (30th to 35th minutes of recovery), R7 (40th to 45th minutes of recovery), R8 (50th to 55th minutes of recovery), R9 (60th to 65th minutes of recovery), R10 (70th to 75th minutes of recovery), R11 (90th to 95th minutes of recovery), and R12 (115th to 120th minutes of recovery). It is worth mentioning that 256 consecutive RR intervals will be obtained at these moments .HRV indices in the time domain (rMSSD and SDNN), frequency domain  and high-frequency spectral component (HF)), and the Poincaré plot (SD1 and SD2) will be evaluated .In the time domain, the rMSSD Index corresponds to the square root of the square mean of the differences between the adjacent normal RR intervals in a time interval expressed in milliseconds and evaluates the behavior of the parasympathetic autonomic modulation , while tIn the frequency domain the low-frequency spectral component (LF – frequency between 0.04 and 0.15 Hz) is considered an index that evaluates sympathetic and parasympathetic behavior , 23, whiThe Poincaré plot is a map of points in Cartesian coordinates constructed from the values of the RR intervals obtained . For quaBlood Pressure○ Blood pressure will be verified by an indirect method using a stethoscope  and an aneroid sphygmomanometer  fixed on the left arm of the volunteers. To avoid errors in determining blood pressure, these measurements will be performed by a single evaluator. The measurements will be performed according to the criteria established by the VII Brazilian Hypertension Guidelines ► Heart rate○ To verify the heart rate, a cardio frequency meter  will be used ► Respiratory rate○ Respiratory rate measurements will be performed by counting the respiratory incursions for 1 min without the participant being aware of the process, so that the usual breathing characteristics are not modified ► Oxygen saturation○ Oxygen saturation will be verified by means of a pulse oximeter . The pulse oximeter is a device that provides blood saturation readings by evaluating the absorption behavior of oxyhemoglobin and deoxyhemoglobin in relation to red and infrared light lengths ► Questionnaire on individual touch perceptionTouch Avoidance Questionnaire [○ The ionnaire  is a que► The study will include five measures of secondary outcomes: individual touch perception, which will be collected at baseline of the first randomized intervention, and blood pressure, heart rate, respiratory rate, and oxygen saturation, which will always be collected at baseline and during the 2 h of recovery at minutes 1, 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120. In addition, in I5 these variables will be collected 1 h prior to the application of the massage at minutes 1, 3, 5, 7, 10, 20, 30, 40, 50, and 60.n is added to statistically supply the analyses in the case of withdrawals during data collection. The value obtained for the sample size was 33 participants, adding the 10% gave a sample of 36 volunteers. However, there are other indices besides the SDNN Index within the HRV, and this situation generates an environment where the sample size could be underestimated. In the same sense, due to the new proposal presented by the study, there are no trials in the literature that have the same proposal dynamics, and thus, for safety we will add four volunteers to ensure the statistical analysis of all HRV indices, thus totaling a sample of 40 participants.As described above the primary outcome will be the HRV. The sample size calculation was carried out based on the study of Arroyo-Morales et al.  in whichFor analysis of the data of the population profile, the descriptive statistical method will be used and the results presented as values of means, standard deviations, median, minimum and maximum numbers, and confidence interval. The normality of the data will be evaluated through the Shapiro-Wilk test.The comparisons of cardiorespiratory parameters and HRV indices between interventions  and moments (rest (R1) vs recovery moments (R2–R12)), the data are analyzed from the prism of the covariance analysis (ANCOVA), which will be adjusted for the baseline value of the evaluated variable and generated estimated means after such adjustment (extracting the variance explained by the confounding variables). The Levene test will be used to prove the homogeneity of the variances at the created moments, and the Bonferroni post hoc test will be used for comparison. To guarantee homogeneity of data the variable can will be transformed into logarithm.For analysis of the moments (rest vs recovery moments), repeated measures ANOVA with the Tukey post test for parametric distribution or Friedman test with Dunnett’s post test for non-parametric distribution will be used and the analysis of the different groups will be performed by means of one-way ANOVA with the Tukey post test or the Kruskal-Wallis test with Dunnett’s post test.p < 0.05 for all tests. The SPSS statistical program (version 13.0)  will be used for the analyses. Data integrity will be monitored by regularly scrutinizing data files for omissions and errors. Participants will be given an anonymous study ID to protect confidentiality, and only study investigators will have access to the final trial data set.The level of significance will be This massage protocol was created from recent findings in the literature and its standardization was performed according to the union of the best results found. The implementation and use of this standardized protocol will provide important and reliable information regarding the use of massage in post-exercise recovery, with the identification of its effects on the ANS and the best moment and form of massage application, representing an important advance in the sports field and in clinical practice.A strong point of this study is that the protocol created was based on the latest in the literature on the theme. Another strong point of the study is the comparison of the effects of massage when applied at different moments on post-exercise recovery. In addition, its design of a randomized clinical trial of the cross-over type, the easy reproducibility of the protocol from the description of the same, the fact of the massage being performed by three physiotherapists specialized in sports physiotherapy also enhances the study. However, one limitation of this study is the impossibility of blinding the physiotherapists and participants regarding the application of the technique, and although standardized there is no control of the pressure exerted during the massage, only a subjective control through the level of comfort of the volunteer with the specific scale application.With the accomplishment of this study we will verify which HRV indices demonstrate a significant influence of the massage in post-exercise recovery, and which moment of application presents better recovery after exercise. The data obtained in the present study will provide subsidies for the best management of application of the technique in sports clinical practice, considering periods of training and, mainly, of competitions.Number Protocol: NCT03094676Patient recruitment is currently underway.Study start date: 9 April 2018Primary completion date: 14 May 2018Study Completion date: 30 July 2018Additional file 1:Recommendations for Interventional Trials (SPIRIT). (DOC 120 kb)


The purpose of this research was to evaluate the effect of immediate placement of different restorative materials in comparison with a temporary restoration on the surface microhardness of mineral trioxide aggregate (MTA).Access cavities were prepared in 40 extracted human molars, and a 3-mm layer of MTA was placed in the pulp chamber. The samples were divided into eight groups (n=5). Ten minutes after the MTA placement, two groups were restored with Zonalin temporary restoration, while the other six groups were restored with glass-ionomer cement (GIC), resin-modified glass-ionomer (RMGI), or resin-based composite. In each group, the Vickers microhardness (VMH) of MTA was determined after 7 and 21 days. Data were entered into SPSS 17 software program and were analyzed by two-way analysis of variance (ANOVA). The significance level was set at 5%.The type of restorative materials had a statistically significant effect on the microhardness of MTA (P=0.002). However, the microhardness of MTA was neither significantly influenced by the timing of final restoration (P=0.246) nor by the time-material interaction (P=0.116).Based on the results of the present study and by considering the limitations of laboratory studies, it is recommended to postpone the placement of final restorations until the underlying MTA is completely set. Otherwise, in the clinical conditions in which early covering of MTA is recommended, sufficient moist-curing and hydration should be guaranteed by selecting a restorative material with the lowest hydrophilic interaction energy. Mineral trioxide aggregate (MTA) was first introduced in 1993 as a root-end filling material . NowadayIn this in-vitro experimental study, 40 extracted human molars with mature apices and without any root resorption, canal calcification, endodontic treatment, or coronal restoration were selected. After extraction, the teeth were cleaned and stored in 0.5% Chloramine-T solution at 4°C before the preparation of the samples. An access cavity was prepared in each tooth, and the pulp chamber was rinsed with 5.25% sodium hypochlorite (NaClO) solution followed by a rinse with normal saline. The root canals were filled with normal saline to the level of canal orifice.Control groups (groups 1 and 2): A 2-mm layer of a temporary filling material  was placed on the wet cotton pellet. Next, the teeth were incubated at 37°C and 100% relative humidity for 7 (group 1) and 21 days (group 2).TM Plus, Kerr Dental Co., California, USA) with the intensity of 900 mW/cm2. The samples were then incubated at 37°C and 100% relative humidity for 7 (group 3) and 21 days (group 4).RMGI groups (groups 3 and 4): The cotton pellet was removed after 10 minutes. The powder and liquid of the RMGI  were mixed with the standard ratio according to the manufacturer’s instruction, and a 2-mm layer of the mixture was placed over the MTA. The RMGI was cured for 20 seconds by using a light-emitting diode (LED) curing light (DemiGIC groups (groups 5 and 6): The cotton pellet was removed after 10 minutes. The powder and liquid of the GIC  were mixed with the standard ratio according to the manufacturer’s instruction, and a 2-mm layer of the mixture was placed over the MTA. After the completion of the initial setting reaction of the GIC (10 minutes), the samples were incubated at 37°C and 100% relative humidity for 7 (group 5) and 21 days (group 6).Resin composite groups (groups 7 and 8): The cotton pellet was removed after 10 minutes. The self-etch primer and bonding agent of the Clearfil™ SE Bond  were applied according to the manufacturer’s instruction. Afterwards, a 2-mm thickness of the flowable composite resin  was placed over the MTA and was cured for 40 seconds by using the LED curing light. The teeth were then incubated at 37°C and 100% relative humidity for 7 (group 7) and 21 days (group 8).According to the manufacturer’s instruction, the powder and liquid of the MTA  were mixed with the standard ratio, and a 3-mm layer of the mixture was placed in the pulp chamber. A moistened cotton pellet was placed on the MTA surface, and the teeth were randomly divided into eight groups (n=5) according to the type of coronal restoration:The samples were mounted in a custom-made mold by using a self-curing acrylic resin . The teeth were sectioned longitudinally by using a low-speed saw and were polished with silicon carbide papers (300 to 1200 grit).2.In each sample, VMH testing was performed by using a VMH tester  at three points and at a 200-μm distance from the MTA-filling material interface by applying a 500 gram-force (gf) load with the dwell time of 10 seconds. The angle between the opposite faces of the diamond indenter was 136 degrees. The diameter of the indentation was measured at each point, and the mean of three points was calculated as the Vickers hardness number (VHN) of each sample in Kg/mmData were analyzed by using SPSS version 17 software program . The effects of the type and time of placement of the filling material on the microhardness of MTA were measured by two-way analysis of variance (ANOVA). The significance level was set at 5%.The results of the present study are summarized in The VHN also showed significant statistical differences between the RMGI and resin composite groups (P=0.029).The MTA powder is a refined Portland cement that is extensively used in endodontics. This bioceramic has two main characteristics that differentiate it from other available restorative materials: biocompatibility and a superior sealing ability .® (2-ethoxybenzoic acid), IRM® , and amalgam [Based on the results of a meta-analysis, MTA is the most biocompatible substance when compared with Super-EBA amalgam . During  amalgam . However amalgam .The sealing ability of MTA depends on physical and chemical properties of the material after the finalization of the setting process . StudiesIn the clinical settings, especially when MTA is applied to the coronal part of the tooth, the physical properties of the material such as surface microhardness also play an important role in achieving an ideal seal . The occAlthough the long setting time of MTA may result in a two-session application of this material, immediate placement of a final restoration could promote the coronal seal . MoreoveIn case of immediate coronal restoration, clinical manipulations including the condensation pressure, etching, rinsing, and priming could affect the setting of MTA . Since tBased on the results of the present study, the null hypothesis was rejected, and there were significant differences between the groups in terms of the microhardness of MTA after the use of different restorative materials; however, the time of final restoration and the time-material interaction did not significantly change the microhardness of MTA. In the groups evaluated after 7 days, the highest and lowest mean VHNs were recorded for Zonalin/RMGI and resin composite, respectively. In the groups evaluated after 21 days, the Zonalin groups showed the highest, while the resin composite groups showed the lowest mean VHNs. During the examination time, the VHN increased in the Zonalin and GIC groups, while it decreased in the resin composite and RMGI groups. The ultimate microhardness of MTA was similar in the RMGI and GIC groups.Yesilyurt et al  measuredTsujimoto et al  investigPatil et al  evaluateBased on the outcomes of the current study, the application of the self-etching resin composite significantly reduced the microhardness of MTA. The initial setting of MTA is not completed 10 minutes after the MTA placement, and a 10-minute moist-curing of MTA may not be sufficient for the completion of the setting process. Therefore, the early covering of MTA with a resin composite is not recommended.Many factors could influence the setting reaction and microhardness of a bioactive material like MTA ,19. The More condensation, less material thickness, the presence of a chelating agent, and acid-etching might adversely affect the microhardness of MTA . An evalAccording to Lee et al , the phySince the moisture cannot effectively diffuse through thick layers of MTA, a bilateral moisture exposure has been recommended for the completion of the setting process . GancedoThere are contradictory results with regard to the impact of the moist-curing time on the final microhardness of MTA ,8,24,25.In the clinical conditions in which early covering of MTA is recommended, sufficient moist-curing and hydration should be guaranteed by selecting a restorative material with the lowest hydrophilic interaction energy. Otherwise, it is recommended to postpone the placement of the final restoration to after the completion of the MTA setting process.