Several advantages have been established regarding the efficiency of self-ligating brackets (SL). In spite of some controversy surrounding this question in the literature, clinical results confirm that “arch development” requires fewer extractions.  The objective of this study was to compare changes in the transverse and sagittal planes in patients treated with conventional ligating brackets (CL)as well as in patients treated with SL brackets and oversized arches. 0) to conclusion (T1) of treatment. Lateral cephalograms were analyzed for changes in the lower incisor (IMPA) and the first lower molar distal angulation (MAng). Intraoperator reliability was tested with linear regression analysis. To assure all groups were comparable at T0, an ANOVA test with a 95%confidence interval (CI) was performed for all values. To assess changes from T0 to T1 in all groups, a Student’s t-test with 95% CI was used. Finally, results from the three groups were compared using an ANOVA-test (95% CI) and a post-hoc test. A sample was selected from a pool of 300 consecutive cases treated by a single clinician: 51 patients with SL brackets and oversized wires, and 55 with CL brackets. These two groups were compared with a control group of 20 patients. All plaster models were scanned and dental landmarks were measured to identify changes from commencement  took place in the SL group.  Increases in all the transverse variables were recorded in the two groups treated (SL and CL), except for the lower intercanine distance in the SL group. IMPA difference from TSelf-ligating brackets with oversized arches and conventional ligating brackets showed increases in all variables in the transverse plane, except for the SL group at the mandibular intercanine distance. In comparison with the CL group, fewer different IMPA values were observed in the SL group, in which distal molar angulation occurred. To identify these benefits, it is necessary to compare passive SL brackets with conventional brackets to ascertain their true effect in the sagittal and transverse planes, and then match these findings with a control group. A combination of transverse and sagittal variables was studied to determine the existence of the “lip bumper effect”When orthodontists plan a treatment, they can only resort to expansion, protrusion, distalization, extraction and/or IPR . However, patients began to demand shorter treatments without the need for extractions. In this context, self-ligating (SL) brackets have gradually seen a relaunch over the last 15 years. Although the self-ligating system was first described in 1935, brackets and different shapes of wires made of the latest alloys continue to be developed. Indeed, these systems have grown exponentially from 8.7% in 2002 to 42% in 2008.Universitat Internacional de Catalunya. The sample was selected from a pool of 300 consecutive cases treated by a single clinician in his dental offices in Lleida and Tarragona, Spain, from 2005 to 2010. To detect a difference of 1 or more units, taking into account a standard deviation of 2, a sample of 50 patients per treated group was needed to achieve a statistical power of 80% with a significance level of 0.05. A group of 20 patients was necessary in order to identify differences and achieve a statistical power close to 100% in a control group. The sample size calculation was performed using “Sample size and power calculator GRANMO” . The orthodontist investigator was highly experienced and has worked with conventional and SL brackets indistinctly. In accordance with this technique, the patients treated with passive SL brackets  wore oversized wires during the first phase of the treatment. Those patients treated with conventional CL brackets  wore a different sequence of wires, which were customized for each patient  and repeated at 24 months (T1).Three groups were studied: 1) SL group, consisting of 51 patients (36 women/15 men), with a mean age of 19.9 ± 11.3 years, treated with self-ligating brackets and oversized arches; 2) CL group, consisting of 55 patients (34 women/21 men), with a mean age of 16.38 ± 9.86 years, treated with conventional brackets; 3) CT group, consisting of 20 individuals (12 women/8 men), with an approximate age of 24.05 ± 2.15 years. The control group was made up of students of Dentistry  with no previous orthodontic treatment, who met all the inclusion criteria. Orthodontic records of this group were taken (T0) and upon completion of treatment (T1).Two casts, taken without any appliances on the teeth, were scanned in 1:1 proportion using an HP 1315  and calibrated with a millimeter ruler as a reference. All measurements were performed by a single operator blinded to the group. Calibrations and measurements of the images, obtained with Adobe Photoshop CS  showed different values in maxilla and mandible. Data were collected and stored in an Excel® 2013 file . Sagittal values were measured with lateral cephalograms taken at T0 and T1. In order to avoid any radiological distortion, only angular values of the lower incisor to mandibular plane (IMPA) and the molar distal angulation to the mandibular plane (MAng) were measured. Mandibular arch depth (ArchD) was determined using the same procedure as that used for all variables in the transverse plane  Intraoperator analysis. Five variables were randomly measured again at two weeks, by the same operator, followed by a linear analysis of regression.0, an ANOVA test with a 95% CI and comparative post-hoc tests (LSD)were performed, and Pearson’s chi-square test was used to analyze dichotomous variables.2) Validation of the sample. To assure that the three groups were similar and comparable at T0 to T1. A Student’s t-test with a 95% CI was used to observe changes after treatment in each group.3) Changes in SL, CL and CT from Tpost-hoc tests (LSD), to compare changes in the three groups.4) Comparison of changes among SL, CL and CT. An ANOVA test with a 95% CI was carried out, together with comparative All statistical tests were performed, in conjunction with the statistical service of the Universitat Internacional de Catalunya, using Statgraphics Plus Centurion XVI .A regression line with five variables of the first measurements and results was repeated after two weeks, by the same operator. A value of 0.985 was obtained in the correlation coefficient, with a standard error of ± 0.2. p = 0.0134). The transverse and sagittal variables studied among the three groups at the beginning of treatment showed no statistically significant differences  and CL (16.38 ± 9.86 years) groups. However, CT showed a higher mean age .The sagittal plane showed a significant increase in the angulation of both the lower incisors (IMPA T0- T1 = 3.15 ± 6.78o) and molars (MAng T0- T1 = 7 ± 4.41o). A significant increase in CL was noted in all the transverse plane variables, the largest of them in the second premolar area . In the sagittal plane, only the variable of the lower incisor angulation increased (IMPA T0- T1 = 5.53 ± 7.13o). However, in CT, a significant reduction occurred in the transverse variables in both the mandible and the premolar region of the maxilla. No changes were observed in the sagittal plane variables.0 -T1). In this variable, changes in SL and CL were the same. The most significant changes in the rest of the variables were observed in SL. The same differences were noted in both the mandible and the maxilla, except for the intercanine distance (DCMd T0-T1), where no differences in SL were seen when comparing CL and CT. 0-T1 = 7.13 ± 5.53o) followed by SL (IMPA T0-T1 = 3.15 ± 6.78o). The angulation of the mandibular molar also exhibited differences in relation to SL, CL and CT: SL showed the greatest increase in molar distal angulation (MAng T0-T1 = 4.41 ± 7.00o),while CL and CT presented no difference between them. The arch depth (ArchD T0-T1) had an ANOVA p-value > 0.05, suggesting there were no changes in this variable among the three groups.In the sagittal plane, IMPA revealed differences among the three groups. The largest increase occurred in CL . Therefo6 In the second study, measurements in both the conventional and self-ligating type were also performed with CBCT; however, the sample in that study was smaller than the sample used in the present paper, and the measurements were only taken at the beginning of the treatment and at seven months post-treatment.All the variables studied were dental. To observe real changes in the transverse dimension, skeletal measurements should be taken. In two papers citing measurements taken with CBCT, only one compared changes between the two kinds of self-ligating brackets, but not with conventional brackets.7-9 in which SL brackets without oversized arches were used showed fewer increases in the transverse plane than increases achieved with oversized wires, possibly suggesting the effect of the shape of the wires used in the technique. In this regard, other authors6,10 have claimed that the changes are due to the arch shape, and not to the type of bracket. Hence, further studies may confirm the results obtained in the present study. Other authors consider that besides the bracket, patient’s individual characteristics may also be responsible.11 All variables were observed to decrease in the control group (CT), but, when smaller than 1 mm, this decrease was not clinically perceptible. These results are in accordance with those of another study in which the arch shape was observed from 6 months to 25 and 45 years. Once the whole dentition has erupted, a minimal decrease can be expected.The self-ligating brackets and oversized arches used in the present study produced a significant expansion of all variables in the transverse plane, except for the mandibular intercanine distance. In contrast, a significant increase in all variables was observed in the conventional appliances, including the mandibular intercanine distance. It is important to note that in the case of the conventional appliance group, the orthodontist took into account the initial intercanine mandibular distance of each patient, and the torque prescription in the lower canine showed higher negative values in CL. Some studies  have inv,,-8,13,17,19 highlight the direct relation between the initial crowding and the increase in IMPA. The present study enrolled only patients with low to moderate crowding (0-5 mm). Molar angulation (MAng) increased significantly in SL (7.00 ± 4.41o), while no variations were observed in CL. To our knowledge, no studies have evaluated this variable. The combination of arch expansion and distal angulation of the molar in SL may explain the smaller inclination of the lower incisor, as well as the lower values in the arch depth. The present results showed that arch depth values from T0 to T1 are very similar to those of a recent study in which no differences were found between groups.,,The increase in the lower incisor angulation was statistically significant in both SL (3.15 ± 6.78) and CL (7.13 ± 5.53) groups, and higher in CL. A number of studies2,10,14,21 Described as the liberation of the dentition from the perioral muscles, this effect facilitates the development of the dental arches, by means of the effect of low tongue forces. In SL, in which the super-elastic oversized arches were used, there was less inclination of the lower incisor, distal angulation of the mandibular molar, and expansion of the dental arches. These effects are observed in classical lip-bumper therapy.22 In a recent RCT on CBCT scans of active and passive brackets, the author was unable to confirm apposition and bone growth in the transverse plane.6 Both self-ligating brackets and oversized arches in the present study produced dentoalveolar expansion with distal molar angulation, which may be attributed to the “lip-bumper effect”, although this has not been demonstrated to date.There is little mention in the literature on the “lip bumper effect”.,,,The results obtained in the present study show that in patients with low to moderate crowding, the use of self-ligating brackets and oversized arches increases the transversal measurements, except for the lower intercanine distance, with less protrusion of the lower incisor. However, given the direct relation found in the literature» Self-ligating brackets, oversized arches, and conventional ligating brackets showed an increase in all variables in the transverse plane, except for SL in the mandibular intercanine distance. The most substantial expansion was located in the first premolar in SL and in the second premolar in CL. A significant reduction in the control group was observed in all mandibular variables, as well as first and second premolars of the maxilla.» Distal molar angulation occurred only in SL.» No changes in arch depth were found among groups.» The lower IMPA observed in SL might be caused by the distal angulation of the molar and arch expansion due to oversized wires.


Salvia miltiorrhiza Bge and decreases the yield and quality of crude drug, thus resulting in serious economic losses in China. Here, we selected four replanted field soils with 1, 2, 3, and 4 years of monoculture history to investigate the influence of continuous monocropping soil on the property of medicinal materials by pot experiments. Results showed that the commodity appearance and active ingredient contents of Salvia miltiorrhiza were significantly affected by soil with different continuous monocropping years. Along the time series of plantation soil, the diameter of main roots, weight of fresh roots, and total contents of hydrophilic and lipophilic components demonstrated a decline tendency. With the method of PCA, the property of medicinal materials affected by continuous monocropping soil was evaluated by the following formula: F = (0.3762 × F1 + 0.2320 × F2 + 0.1913 × F3 + 0.0994 × F4)/0.8989. Eventually, crude drug properties ranked according to comprehensive scores were as follows: CK (0.380) > 1 year (0.360) > 2 years (0.348) > 3 years (0.337) > 4 years (0.245). For the medicinal plant of Salvia miltiorrhiza Bge, continuous monocropping soil had significant effects on the property of Salvia miltiorrhiza and should be ameliorated by some measures. The results provide support for the optimal continuous cropping year selection and continuous cropping obstacle abatement of Salvia miltiorrhiza Bge.High-efficiency monoculture severely inhibits the growth of  Salvia miltiorrhiza Bge, recorded in the Chinese Pharmacopoeia with the name of Danshen, are commonly used to treat a wide variety of ailments including cardiovascular and cerebrovascular diseases, liver diseases, and diabetes mellitus in traditional Chinese medicine [Salvia miltiorrhiza Bge on the same area with or without short rotation increased dramatically. However, high-efficiency monoculture often leads to continuous cropping obstacle, which eventually causes poor growth, low yield, and serious insect disease. In China, this problem has resulted in severe economic losses and hindered the sustainable development of Salvia miltiorrhiza industry.The roots and rhizome of medicine . At presmedicine , it has medicine . With suPseudostellaria heterophylla [Consecutive monoculture problem, popularly known as replant disease or soil sickness, usually leads to a series of problems such as inhibition of plant growth, reduction in the yield and quality, and exhaustion of soil fertility . As to trophylla , potato rophylla , and somSalvia miltiorrhiza Bge roots to the soil contribute to the inhibition of its self-growth [Salvia miltiorrhiza Bge and then results in the change of the biomass, yield, and quality of crude drug. Therefore, in this study, a pot experiment with soil from the field that had undergone continuous cropping for Salvia miltiorrhiza Bge was designed to evaluate the influence of continuous cropping on the quality of Salvia miltiorrhiza. In traditional cognition, the quality of Salvia miltiorrhiza mainly consisted of commodity appearance and active ingredient contents or biomass, so these kinds of indices were determined in this study.Our previous studies have also confirmed that some phenolic acids released from two-year consecutive planting of f-growth . Hence, Salvia miltiorrhiza Bge had been cultivated for a period of 4 years, 3 years, 2 years, and 1 year in the Germplasms Nursery of Shandong University of TCM (SDCM), in March 2016. The second type is the control and collected from where Salvia miltiorrhiza Bge had never been planted in the same field. The soil was then further sieved through a 2 mm mesh to remove debris and root tissues. Samples of rhizosphere soils of Salvia miltiorrhiza Bge were collected by pulling plants from the soil and shaking soil off from plant roots [Two types of soil were used in this experiment. The first type was, respectively, collected from where Ø = 12.5 cm; 25pots, each containing 750 g soil). The robust and uniform semiannual Salvia miltiorrhiza seedlings were selected and planted in each pot and allowed to grow for eight months under the same management conditions. All of the pots were kept in bright sunlight, allowing the medicinal plants to grow naturally with normal watering and farmyard manure just like those planted in the field. Twenty pots were maintained per treatment as replicates.The collected soil was immediately put into polyethylene pots .During the pot experiment, plants were carefully uprooted in the root harvest period of late October, keeping the root system intact, and the roots were collected. Firstly, the biomass of fresh Eight reference standards, including rosemary acid (20120809), salvianolic acid B (PA0418RA13), lithospermic acid (PF225SA14), dihydrotanshinone I (KN1113GU14), tanshinone II (20130111), danshensu sodium (YY90062), tanshinone I (110867-200406), and cryptotanshinone (110852-200806) with purity not less than 98%, were obtained from Yuanye Biological Technology Company . All chemical reagents were analytical grade and purchased from Tianjin Fu-Yu Chemical Reagent Factory.Accurate weight of the reference compound was dissolved in 70% methanol or methanol, respectively, to yield eight individual standard stock solutions.The hydrophilic working standard references including danshensu sodium, salvianolic acid B, rosemary acid, and lithospermic acid were prepared by diluting mixed standard stock solutions with 70% methanol to get six different concentrations for calibration curves. The lipophilic standard references including dihydrotanshinone I, tanshinone IIA, cryptotanshinone, and tanshinone I were also prepared by diluting mixed standard stock solutions with methanol to obtain six different concentrations for calibration curves.All the standard stock and working solutions were prepared in dark-brown calibrated flasks and stored at 4°C.μm membrane for component analysis.The air-dried material was powdered to a homogeneous size using an electrical blender and sieved through a 40-mesh sieve. For quantitative analysis, the accurately weighed sample (0.25 g) was suspended in 25 mL of 75% methanol in a conical flask with a cover. The weight of the flask along with the contents was recorded, and the mixture was ultrasonically extracted at 50°C for 50 min. After ultrasonic treatment, the original solvent weight was restored by adding 75% methanol. Subsequently, the extract was filtered through a 0.45 The hydrophilic component solution was prepared following the above method with methanol as the solvent.18 column  at 30°C. The mobile phase consisting of acetonitrile (A) and 0.1% aqueous phosphoric acid (B) in a gradient elution mode for hydrophilic component determination was as follows: 0~20 min, 3%-23% A; 20~35 min, 23%-25% A; 35~40 min, 25% A; 40~50 min, 25%-90% A; and 50~60 min, 90% A. The flow rate was 1.0 mL·min−1, the sample injection volume was 10 μL, and the detection wavelength was set at 286 nm.Chromatography was performed on Agilent 1260 Series HPLC equipment  with a ZORBAXSB-C−1, the sample injection volume was 10 μL, and the detection wavelength was set at 270 nm.The mobile phase in a gradient elution mode for lipophilic component determination was as follows: 0~20 min, 20%-60% A; 20~50 min, 60%-80% A; and 50~55 min, 80% A. The flow rate was 1.0 mL·minY) versus concentration (x) of each analyte. Under optimal chromatographic conditions, the diluted six appropriate concentrations of working standard solutions were injected in triplicate, and their regression equations were calculated in the form of Y = Ax + B.The calibration curve for each compound was established by plotting the peak area , and the comprehensive scores were calculated to evaluate the continuous cropping effects.In the study of many indexes, the phenomenon of information overlap of observational data exists because of too many variables and possible correlations between them. So we used principal component analysis to search for several factors which have no correlation to represent all the variables. In this study, the values of the biomass including the diameter of main roots, length of main roots, and fresh weight and eight analyte contents of the roots of Salvia miltiorrhiza Bge. All the data were represented as means ± standard deviation, and means were compared using Duncan's multiple range test at a 5% level.One-way analysis of variance (ANOVA) procedures were performed in the statistical software SPSS 19.0  to assess the effects of different cropping years of plantation soil on biomass and chemical contents of the roots of S. miltiorrhiza roots. Along the time series of plantation soil and compared to the control, the diameter of main roots decreased by 12.63%, 18.99%, 32.95%, and 36.33%, the length of main roots decreased by 31.70%, 39.60%, 33.76%, and 37.25%, and the fresh weight or the yield decreased by 24.9%, 38.6%, 48.9%, and 53.8%, respectively. The facts indicate that continuous planting would cause yield and quality decline of Salvia miltiorrhiza. In addition, the treated plantation soil showed no significant effect on the length of main roots.R2 > 0.9991) in a relatively wide concentration range. The overall stability and repeatability variations were less than 3%. The HPLC chromatogram of the eight reference compounds is shown in Figures The regression equations of the eight analytes are shown in Salvia miltiorrhiza compared with the control. With the exception of danshensu sodium, the contents of rosemary acid, lithospermic acid, and salvianolic acid B in S. miltiorrhiza roots showed significant difference among treated groups, while the contents of dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA varied significantly among different treated groups (P < 0.05). For hydrophilic components or lipophilic ingredients, the total contents of the 8 determined ingredients reduced with the increase in continuous cropping years of soil.Figures The data including biomass and component contents from all of the treatments were analyzed using PCA to identify the primary factors. The PCA results demonstrated that the four principal components  contained 89.88% information of the original indexes from the results of the eigenvalue and variance contribution rate of the principal component  and factThe first principal component F1 (37.62%) reflected the comprehensive variation information of danshensu sodium, rosemary acid, lithospermic acid, salvianolic acid B, and dihydrotanshinone I. The second principal component F2 (23.20%) reflected the comprehensive variation information of cryptotanshinone, tanshinone I, tanshinone IIA, diameter of main roots, and fresh root weight. The third principal component F3 (19.13%) revealed comprehensive variation information of dihydrotanshinone I, tanshinone IIA, and length of main roots. The forth component F4 (9.94%) reflected that the diameter of main roots contributed the maximum value.The column elements of F1, F2, F3, and F4 from the factor load matrix  are diviThe scores of the tested groups on the four principal components, which were the weight for the linear combination equation, were calculated by using the input standardized raw data into the above linear equations, and the comprehensive model was established by the following formula:S. miltiorrhiza roots were the control > 1 − year continuous cropping soil planted > 2 − year continuous cropping soil planted > 3 − year continuous cropping soil planted > 4 − year continuous cropping soil planted.Finally, with the aid of the eigenvector of eleven variance, the comprehensive scores of property assessments of crude drug for the control, 1-year continuous monocropping soil, 2-year continuous monocropping soil, 3-year continuous monocropping soil, and 4-year continuous monocropping soil groups were 0.380, 0.360, 0.348, 0.337, and 0.245, respectively. So the magnitude sequence of the comprehensive score for Salvia miltiorrhiza was closely associated with its commodity appearance, active ingredient contents, and yield. So the high yield and quality are the primary goal of medicinal plant growers. In addition, the fresh weight index of Salvia miltiorrhiza can represent its yield, and the hydrophilic and lipophilic component contents which were stipulated in the Chinese Pharmacopoeia can represent its quality to some extent, while the main root length and diameter indicators could reflect commodity appearance to a certain extent. Till now, the active ingredients of Salvia miltiorrhiza mainly consisted of two categories of lipophilic and hydrophilic compounds. Among the former, tanshinone and its derivatives display a range of biological activities including antibacterial and antioxidant effects and prevention of angina pectoris and myocardial infarction [Salvia miltiorrhiza. Thus, those indicators of the main root length, diameter, component content, and fresh weight could comprehensively reflect the overall quality of Salvia miltiorrhiza together.In commercial trade, the profit of farction , 11, Whifarction , 11. ThuSalvia miltiorrhiza Bge was one of about 70% of medicinal plant species with tuber roots suffering from consecutive monoculture problems and its quality was not always very palatable [Salvia miltiorrhiza Bge roots and no influence on the length of main roots. When it comes to the content change of the eight lipophilic and hydrophilic compounds, there was no regularity with increasing years of monoculture. Because there was no regularity to explore for those indicators and a single indicator could not reflect the comprehensive effects induced by continuous cropping soil on Salvia miltiorrhiza Bge, a strategy must be adopted to evaluate those regularly changed or irregularly changed indicators from the whole. So a linear feature extraction was designed by using the principal component analysis method to calculate the comprehensive score of all those indicators in each test group. The PCA statistical method, with the merits of simplifying the complexity in high-dimensional data while retaining trends and patterns, was an appropriate strategy to evaluate those indicators including the diameter, length, and fresh weight of roots and contents of danshensu sodium, rosemary acid, lithospermic acid, salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone IIA comprehensively. The results of the final comprehensive scores showed that the overall quality of Salvia miltiorrhiza declined along the time series of 1, 2, 3, and 4 years of continuous planting.However, in China, Salvia miltiorrhiza Bge, the aerial parts and taproots were removed, while some fibrous root residuals were left behind in the planting soil, which will be decomposed, releasing allelochemicals and changing soil microecological balance. These chemicals may contain some water-soluble substances, which are regarded to contribute to replant failure at the same time. With the increase in the period of continuous cultivation, more and more allelochemicals accumulate in the soil and soil microecological imbalance becomes more and more serious. Then, autotoxic substance mediation, along with soil microecological balance, would disturb the successive season planting. So the yield and quality of Salvia miltiorrhiza in our study demonstrated downward trends with the period of continuous cultivation of soil increased. The same findings also concurred with a previous report of Angelica sinensis (Oliv.) Diels. Pot experiments showed that the root yield, content of essential oils, and ethanol extract of Angelica sinensis (Oliv.) Diels planted in continuous cropping soil declined significantly compared with the control [Angelica sinensis (Oliv.) Diels cropping significantly decreased the plant height, main root length, and biomass of Angelica Sinensis (Oliv.) Diels plants during the vegetative period and then led to lower yields [Besides structural imbalance of microorganisms, autotoxin accumulation mainly induced by plant residues and root exudates was also popularly accepted as an important factor contributing to continuous cropping obstacle . In actu control . Anotherr yields .Salvia miltiorrhiza cultivation in our further work.Recently, with the advance of modern agriculture, serious continuous cropping obstacles occurred in more and more medicinal plant cultivation. Seeking effective measures to confront this problem has attracted the attention of many scholars. Till now, crop rotation, tillage, and fertilization are proven to be the effective strategies to overcome continuous cultivation obstacle in agricultural production through altering soil properties . These kSalvia miltiorrhiza Bge but also affected its active ingredient content obviously. Because a single indicator could not evaluate the influence wholly, the PCA method was applied to establish linear equations to analyze those multiple indicators. The results provide support for the optimal continuous cropping year selection and continuous cropping obstacle abatement of Salvia miltiorrhiza Bge.The continuous cropping obstacles can decrease the yield and quality of many medicinal plants. Although the reason was complex, the soil microbes and autotoxic substances released into the soil were popularly proven to be the major determinants. With the continuous cropping years increased, the soil physiochemical property changed differently and then would have a different effect on the quality and yield of medicinal materials. Pot experiments proved that 1-, 2-, 3-, and 4-year continuous cropping soil not only reduced the quality and yield of


Smart structures have attracted significant research attention in the last decade, mainly due to the capabilities of advanced concrete in electrical resistance-enabled self-sensing. In this study, we present a type of environment-friendly, self-sensing concrete enabled by electrical resistance. Environment-friendly, self-sensing concrete was casted with the additions of byproduct wastes , blast furnace slag (BOF) and red mud (RM)) at various volume fractions and cured using the conditions of 3, 7 and 28 days. The self-sensing concrete samples were experimentally tested to investigate the effects of the byproduct wastes on the mechanical and electrical properties . In the end, parametric studies were experimentally conducted to investigate the influences of the byproduct wastes on the mechanical and electrical properties of the reported environment-friendly, self-sensing concrete.  Approximately over 30 million tons of concrete are consumed worldwide each year, making concrete the second most used resource after water . The desStudies in the literature have demonstrated that properties of Portland cement such as electrical conductivity can be affected applying certain fillers to its matrix ,9,10,11.Here, we study a type of environment-friendly, self-sensing concrete enabled by electrical resistance. The reported concrete is self-sensing by using the fractional change in resistivity (FCR) to monitor and indicate the status of the concrete. We first fabricated the self-sensing concrete with the additions of byproduct wastes, including coal fly ash, blast furnace slag and red mud. Three curing conditions of 3, 7 and 28 days were particularly investigated. Cement mortars prepared with Portland cement type I were examined with the byproduct wastes at different volume fractions. The compressive strength and electrical resistance of the environment-friendly, self-sensing concrete samples were experimentally investigated with respect to the byproduct wastes. To accurately reveal the status of the concrete, it is necessary to investigate the relationships between the byproduct wastes and the performance. Therefore, we experimentally calibrated the mechanical and electrical characteristics of the self-sensing concrete. Finally, parametric studies were experimentally conducted to investigate the influences of the byproduct wastes on the mechanical and electrical properties of the self-sensing concrete. 2/Al2O3 equal to 1.53, 4 and a weight ratio of SiO2/CaO of 3.3 and 0.35, respectively , blast furnace slag (BOF) and red mud (RM), were used to partially replace the Portland cement in the environment-friendly, self-sensing concrete samples. The chemical compositions composition of the byproduct wastes  were obtained via x-ray fluorescent spectrometry (XRF); the loss on ignition results are presented in The particle size distribution of the by-product wastes and Portland cement type I was measured via a 3071A Analyzer; the observations are presented in The weight ratios of the solid raw materials used to produce the environment-friendly, self-sensing concrete were 5%, 10%, 15%, 20% and 25% of the total weight of Portland cement type I, as shown Production of the environment-friendly, self-sensing concrete samples was completed by following the steps shown in 3). The blast furnace slag had a bulk density of 2027 kg/m3 and was supplied by Phoenix-Services. The red mud had a specific gravity of 1.9 and was collected from Pavlodar alumina plant in Pavlodar, Kazakhstan that has a 1.5-million ton annual production of alumina [Specimens were casted in 50 mm cubic molds following ASTM C109 ; and two alumina .In the experiments, we first evaluated the microstructural characteristics of the environment-friendly, self-sensing concrete using x-ray diffraction (XRD) analysis and pH approaches. The performance of the self-sensing concrete was evaluated with respect to the electrical resistivity, setting time and compressive strength. Ordinary Portland cement (OPC) was used as a reference for comparing electrical conductivity. The mineralogy of the developed environment-friendly, self-sensing concrete was assessed using the x-ray diffraction (XRD) technique. A Bruker D8 x-ray diffractometer equipped with Cu x-ray radiation operating at 40 kV and 30 mA was used for performance of the XRD tests; the covering a reflection angle range 2θ was between 5–60°. A continuous PSD fast was adopted in the scan mode and the counting time for each step was set to be 0.2 s. pH was used to measure the 1% aqueous solution of the solid material using Fisher Scientific Accumet AB15 instrument. In this test, 1 g of the concrete powder was dissolved in 100 g of distilled water. The solution was placed on a shaker for 30 min at 200 rpm. The solution was left to settle for 30 min and then the pH value was measured. It can be seen from BOF contained belite and alite phases in crystal form, and ferrite, mayanite and wuestite in amorphous form. The main components of FA were quartz, lime, magnetite and hematite, which were in the amorphous form as well. RM was in the amorphous phases of hematite, lime, rutile and aluminosilicate.The compressive strength of 50-mm cubic mortar specimens was measured based on ASTM C109 at 3, 7 and 28 days of curing  using thTwo embedded copper electrodes were used to assess the voltage parameters; AC current flows was measured between the two external foil electrodes. Due to contact resistance issues associated with the two-probe approach, a four-probe approach was adopted to measure the electrical resistance of the concrete samples, as shown in ρ, can be calculated as: V, I, A and L are the voltage, current readings, the cross-sectional area and distance between the inner electrodes, respectively.The electrical resistivity of the mortar specimens, 2O3) found in these byproducts, as shown in The electrical resistivity of the mortars was measured at the early and late ages without applying any mechanical load. Note that the results in In this study, the maximum fractional change in resistivity (FCR) is calculated asNext, the stress sensitivity coefficient of the mortar was calculated asIn this study, we reported the environment-friendly, self-sensing concrete. The self-sensing concrete was fabricated with the byproduct wastes  and cured using the conditions of 3, 7 and 28 days. The self-sensing concrete samples were experimentally tested to investigate the effects of the byproduct wastes on the mechanical and electrical properties . In the end, parametric studies were experimentally conducted to investigate the influences of the byproduct wastes on the FCR the reported concrete. The main findings can be drawn as: (1) The addition of byproducts wastes was observed to significantly affect the electrical properties  of the reported concrete samples, (2) FCR can be used to indicate the working conditions of the reported concrete for the purpose of self-sensing and (3) FA might be the most suitable addition for the self-sensing concrete since the obtained FCR were more stable and reliable comparing with other additions.


To the Editor:C. difficile and other adverse events, with consideration of healthcare value” (The American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) recently published updated guidelines for the diagnosis and treatment of adults with community-acquired pneumonia (CAP) . In the e value” . The Wore value” , 5.We are therefore concerned that the 2019 version of the ATS/IDSA CAP guidelines seems to give disappointingly little weight to such antibiotic stewardship principles while continuing to recommend WHO Watch and Reserve antibiotics as first-line options for CAP in most of the target populations. We suggest that Access antibiotics would be sufficient for many patients and would be preferable from an antibiotic stewardship perspective. Amoxicillin, which is the first-choice treatment for CAP based on the 2019 WHO Model List of Essential Medicines, and is also listed as a first-choice option in many guidelines outside the United States, is only recommended in the ATS/IDSA guidelines, together with doxycycline and macrolides as equivalent options, for patients without comorbidities  , 7.Clostridioides difficile infections, as well as an increased risk of adverse events   , 10.This will hamper the CDC’s efforts to reduce overall antibiotic consumption and fluoroquinolone use in the United States . The HeaStaphylococcus aureus and Pseudomonas aeruginosa. Efficacy data from phase III trials suggesting that ceftaroline is superior to ceftriaxone with regard to clinical cure require further scrutiny, and its current listing as a first-choice option violates basic antibiotic stewardship considerations  in hospitalized adults without risk factors for methicillin-resistant erations , 13.Given that respiratory tract infections are one of the most frequent reasons for antibiotic prescriptions worldwide, and that in many countries the U.S. treatment recommendations are still considered an important reference, it seems to us, as members of the WHO EML Antibiotics Working Group, that this represents a lost opportunity for antibiotic stewardship. We believe there is a clear need to better align all treatment guidelines to the same guiding principles, and to establish a global set of evidence-based recommendations with a focus on enhancing the use of Access group antibiotics.


Case Presentation. Here, we present the clinical course of a patient with IgG4 ophthalmic disease who achieved a favorable response from radiotherapy. Our patient initially presented with a history of recurrent painful flares of orbital inflammation, a pathologic diagnosis follicular lymphoid hyperplasia from a right lacrimal gland biopsy, and MRI imaging noting expansion of the lateral rectus muscle of the right eye. Initial treatment with dacryoadenectomy and multiple courses of corticosteroids failed to keep his symptoms at bay. Further evaluation revealed florid IgG4 staining. In this context, he was evaluated for image-guided intensity-modulated radiotherapy (IG-IMRT) to the orbit to 20 Gy in 10 fractions. His ophthalmic symptoms resolved.  IgG4-related ophthalmic disease is a rare, newly recognized entity with high failure rates on first-line therapy of systemic corticosteroids and no other proven management options.  This treatment experience suggests radiotherapy may be a favorable option for symptom relief in patients with IgG4-related ophthalmic disease not controlled by corticosteroids. IgG4-related ophthalmic disease (IgG4-ROD) is a relatively recently recognized ocular inflammatory entity characterized by the infiltration of IgG4-positive plasma cells into tissues, typically ocular muscles. The etiology and pathogenesis of this process is currently unknown , 2. IgG4Current treatments for IgG4-ROD limited to the orbit are guided by systemic agents used to address extraorbital multifocal disease. First-line therapy is often systemic corticosteroids, but retrospective data suggests progression/relapse rates as high as 72% . Other sHere, we describe the clinical course of a patient found to have IgG4-ROD, treated with image-guided intensity-modulated radiation therapy (IG-IMRT) who had an excellent and rapid clinical response at 3-month follow-up.The 39-year old patient initially presented in 2017 for recurrent episodes of dry, painful, swelling of the right eye . ComputeThe patient received dozens of courses of oral steroids over the course of the two years prior to presenting to radiation oncology. The steroid courses were of varying doses and duration and were not well tolerated due to medical comorbidities, mainly malignant hypertension. In 2019, he began to experience increased severity of symptoms during flares, rating pain 10/10 with no response to systemic steroids. Symptoms included new onset diplopia, severe ocular pain, and worsening upper and lower palpebral edema described by the patient as intermittently “so bad I can't open my eye at all.” His physical exam was significant for mild right proptosis, right-ptosis, evoked diplopia with inability to fully medialize the right globe, and incomplete conjugate gaze at the far medial and lateral fields without appreciable impairment of visual acuity. The patient was referred to radiation oncology, where his constellation of findings prompted consideration of the possibility of IgG4-ROD and a request for IgG4 staining of tissue from his 2017 orbitotomy was sent and shown to be 100 per high-powered field . A numbeTreatment options were reviewed. In the context of his comorbidities and polypharmacy, radiotherapy was selected as the preferred treatment approach by the patient over other second-line immunomodulators that have been described for use in IgG4-ROD to avoid the addition of another systemic therapy. We discussed that IgG4-ROD is one of a number of benign inflammatory conditions of the orbit including graves ophthalmopathy, orbital pseudotumor, and precursor lymphoproliferative process  lymphomas). We discussed that low-dose radiotherapy can be given to address the symptoms in the context of these other orbital processes and discussed the risks and benefits of a course of IG-IMRT with the goal of symptom control. After informed consent was obtained, the patient underwent IG-IMRT of 20 gray (Gy) in 10 fractions to the right orbit with attention to sparing the brain and lens.Although he experienced an inflammatory flare during the first week of radiotherapy, he subsequently had an impressive clinical response . In the The patient was immobilized with a short aquaplast mask and underwent a CT simulation without IV contrast and an orbital MRI with gadolinium. The treatment field was defined as the bony orbit. An intensity-modulated radiotherapy plan with volumetric modulated arc radiotherapy (V-MAT) using 6 MV photos was created with two arcs delivered with the couch at 0 degrees and one arc delivered with the couch at 280 degrees. The quality of the plan was assessed for conformality and homogeneity. Daily image-guidance with cone beam CT was undertaken with a match to the bone. The dose was 20 Gy in 10 fractions.IgG4-ROD typically presents with fibro-inflammation of the orbit and symptoms of unilateral or bilateral proptosis, eyelid swelling, ocular pain, and impaired ocular movement , 10. OthTherapeutic options are important for this cohort of patients as recurrent symptoms are common and long-term steroids do not always produce the desired clinical relief. Additionally, inadequate steroid dose can lead to insufficient immunosuppression, and as was seen in this case, steroids could not be tolerated due to a history of malignant hyperthermia treated with multiple antihypertensives. Even when steroids do provide relief, long-term corticosteroid use is associated with many negative side effects including osteoporosis, hyperlipidemia, hyperglycemia, and adrenal insufficiency . When stGiven the variety of clinical scenarios in which radiotherapy has been used, as well as variable reported efficacy, this case report provides an important and unique perspective , 12. ThiWhile there have been case reports of the use of radiotherapy in IgG4-ROD, the toxicities are often not described in the context of clinical decision-making in these cases. One of the most common side effects of radiotherapy to the orbit is dry eye syndrome. In this case, the patient had previously had a dacryoadenectomy in the affected eye that was to be treated with radiotherapy, which helped guide decision-making as the patient already had iatrogenically induced dry eye syndrome due to previous surgery. Radiation is often reserved as a second line therapy due to the increased risk of secondary malignancy following radiation.Additionally, this case demonstrates a diagnosis of IgG4-ROD two years after initial presentation. In the time between initial surgery in 2017 and definitive diagnosis, the patient continued to receive many courses of oral steroids and experienced adverse effects until steroids were no longer tolerable. As a relatively new pathology, it is expected that more cases of IgG4-ROD will be diagnosed after several therapies have been initiated. Understanding the role that radiotherapy can play in these cases provides a valuable addition to the literature and provides treatment experience that may apply to patients diagnosed under similar circumstances.Without available prospective data, case reports are an important way to prompt our colleagues to consider IgG4-ROD when evaluating patients with inflammatory orbital disease. As with all newly described entities, given a tool to assess for IgG4 staining, the number of patients who can be assessed as having IgG4-ROD will rise and, with it, a desire for data related to treatment options. This case report provides an important perspective in that the diagnosis of IgG4-ROD was made several years after initial evaluation and attempted disease management with steroids that were not well tolerated. Consideration of low-dose radiotherapy may be reasonable in the right clinical circumstance.


In wireless sensor networks (WSNs), detection and report of continuous object, such as forest fire and toxic gas leakage, is one of the major applications. In large-scale continuous object tracking in WSNs, there might be many source nodes simultaneously, detecting the continuous object. Each nodes reports its data to both a base station and mobile workers in the industry field. For communication between the source nodes and a mobile worker, sink location service is needed to continuously notify the location of the mobile worker. But, as the application has a large number of sources, it causes a waste of energy consumption. To address this issue, in this paper, we propose a two-phase sink location service scheme. In the first phase, the proposed scheme constructs a virtual grid structure for merging the source nodes. Then, the proposed scheme aggregates the merging points from an originated merging point as the second phase. Simulation results show that the proposed scheme is superior to other schemes in terms of energy consumption. The amount of damages from either disasters or accidents is influenced by the level of the prevention and countermeasures system. Especially, in case of the distant areas from the social infrastructures, such as a mountainous region and a forest area, a prevention system exerts a stronger influence then the case of a downtown area because of their low accessibility. As a promising solution for a prevention system in inhospitable areas, Wireless Sensor Networks (WSNs) have been receiving significant attention. A WSN consists of low-cost and small sensor nodes and is able to provide sensing information to remote users by self-organizing manner. These advantages enable construction of high-level prevention systems using WSNs with low-price. This paper is the part of an effort to facilitate advanced disaster prevention and environment monitoring system using WSNs. Concretely, we focus on increasing the usefulness by reducing energy consumption in communication parts. After sensor nodes are deployed in interested field, they organize a network by self-organizing manner. The information about the base station, called a sink, is embedded into sensor nodes before deployment or advertised on the network after deployment. Once an event is occurred, the sensor nodes exchange messages with each other to elect a reporting node, called a source. The source generates a data packet and sends it to the sink by multi-hop transmission. These features leverage the applications of sensor networks ranging from military surveillance to habitat monitoring ,2.Object tracking is one of the important applications in WSNs where sensor nodes detect where and when the object exists. There have been many researches on object tracking with sensor networks. Target objects are divided into two categories: individual objects and continuous objects. The individual objects usually have very small size comparing with the large area with sensor network deployed. An individual object is sensed by one or few sensor nodes. For example, an individual object might be an animal in habitat monitoring or an intruder in military surveillance.In contrast with the individual object, a continuous object is a large-scale phenomenon which is originated from a specific point  and spreads continuously. Then, the continuous object occupies large area and it is detected by many sensors simultaneously. The continuous object might be fire in forest fire monitoring system and harmful gas in leakage alarm system. Therefore, continuous object tracking is suitable for prevention of disasters and environment monitoring. Sink mobility support: In case of a static sink, it is possible that providing sensor nodes with its location information as a priori knowledge because its location is fixed. On the other hand, a mobile sink has to notify its location to the network periodically because it moves continuously.Reducing data report cost: Because a continuous object is a very large phenomenon and continuously diffuses, the detecting information generated by a number of sources brings considerable communication overhead for reporting data. The overhead might shorten network life time. To prolong network lifetime, the data report cost have to be reduced.In the scenario described above, the applications should satisfy the following requirements:The existing continuous object tracking studies ,9,10,11 Intuitively, the most simple approach is the flooding. A mobile sink could notify its current location by flooding the location to whole network like To avoid the problems in flooding-based or intermediary-based approach, the adoption of existing studies for supporting sink mobility is a reasonable alternative. One of the most important issues in sink mobility support studies is how to notify the current location of a mobile sink. However, the studies are not suitable in continuous object tracking because they have dealt with not continuous objects but individual objects. In case of tracking individual objects like We intensively investigated the energy efficiency in continuous object tracking with mobile sink by analyzing the location update and data report to prevent hot spot problem and to prolonging network lifetime.The proposed scheme provides the integrated location update by mobile sink and distributed data report for reducing overall energy consumption and mitigating hot spot problem.Our simulation results in various environment provides the analytical insights for revealing the radical limitation of the existing schemes and the key principles of the proposed scheme thereby promoting the future developments.Our aim is to design an energy efficient sink mobility support scheme for continuous object tracking. The previous continuous object tracking researches focus on reducing communication cost of reporting data from a number of sources without considering of sink mobility. Unfortunately, the existing studies for supporting sink mobility are inappropriate in continuous object tracking because they have dealt with individual objects. To facilitate advanced continuous object tracking, the sink mobility support scheme being suitable for continuous object tracking is required. The main problem in existing sink mobility support studies is the individual location updates to the sources detecting a continuous object. The location updates are essential for supporting sink mobility, but lead to wasting energy. Our previous work focuses on the connection the origin point to each sinks . We exteThe rest of the paper is organized as follows. We present the related work in WSN is used for various applications such as forest fire monitoring, gas leakage monitoring, and radiation monitoring because it is easy to use and provides cost-efficiency and flexibility compared to wired networks . For exaIn wireless sensor networks, several protocols to detect and track continuous object have been developed. They focus on how to efficiently detect the continuous object and reduce the amount of reports about continuous object toward a static sink. On the other hands, many protocols to support sink mobility have been proposed with the aim of disseminating data to mobile sinks. In this chapter, we briefly introduce continuous object tracking schemes and evaluate the protocols for sink mobility support with features of continuous object tracking.According to ,18, sinkA number of WSN researchers have interested in supporting sink mobility . They assource-based structure and independent structure.In such a context, the difficulty for sensor nodes is to efficiently update the mobile sink location to report sensor data. In many researches, overlaying a virtual infrastructure over the physical network has been investigated as an efficient strategy for an effective data dissemination to mobile sinks ,22. TheySome researches on the sink mobility exploit source-based structure. In the approach, a source detecting an event initiates construction of a structure. The structure provides the route tending to the source on whole network. A mobile sink might loads location update message thereby delivering the message to the source. The source reports detecting data to the sink through the reverse route. The most representative is Two-Tier Data Dissemination (TTDD) model for large-scale WSNs .In TTDD, once an event is detected, the source pro-actively builds a virtual grid structure, as shown in On the other hands, in continuous object tracking, TTDD might causes a serious dissipation of energy. As mentioned above, in TTDD, the grid structure is constructed by a source as the initiator. In continuous object tracking, a number of sources are generated because a continuous object is large and diffusing continuously. Therefore, the sources initiate the construction of grid structures respectively like in Meanwhile, several researches exploit independent structure for supporting sink mobility ,24,25,26w wide and consists of h sized groups. The line is placed in the center of the sensor field so that sensor nodes and a mobile sink might easily access it. The nodes within the line are In LBDD, the independent structure is vertical virtual node① in  left. Thnode① in  left. Thnode① in  left. EvHowever, in continuous object tracking, LBDD might lead to a serious concentration of messages. Inherently, all messages generated in the sensor field, including data from sources and queries from sinks, pass through the line area like in Existing continuous object tracking studies focus on reducing reporting cost considering a static sink. To support sink mobility, the adoption of existing sink mobility support researches is a reasonable solution. The researches provide sink mobility efficiently in individual object tracking by overlaying virtual structures. However, in continuous object tracking, they cause critical problems shown as following A mobile sink should notify its current location to sources to collect data from the interested data. Generally, the location update message is directly delivered by unicasting. Namely, the location update cost increases proportional to the number of sources. So, in continuous object tracking, the location update consumes an amount of energy because a continuous object is detected by a number of sources. In energy constrained WSNs, reducing the cost of sink location update should be considered.R for presenting the efficiency of the integrated location updated compared to the individual location update asx is the x-coordinate of the location agent, n is the number of boundary points  when the location agent is within the continuous object. The cost of the integrated location update tends to decrease as the agent is closer to the center of the continuous object whereas the value increases sharply as the agent far from the continuous object. Second, the location of the agent should be fixed and unique. In R is approximately 0.1 . It means the most efficient location of the agent is not deterministic but vary according to the shape of the continuous object and the location of the sink. However, calculating the most efficient location is extremely difficult because it has to consider both the location of a mobile sink and the locations of all sources in real time. Moreover, the location update process for the agent is needed if the agent’s location can be changed. To summarize, we derive the following requirements about an agent from above observation.An agent should be within targeted continuous object.An agent should be at fixed and unique location.We select the origin point of a continuous object as an agent. Generally, a continuous object tends to be originated at specific point and to diffuse slowly. With the features, designating the origin point as the location of an agent completely satisfies above requirements. First, the origin point is located within a continuous object because the continuous object diffuses toward its surroundings. Second, the origin point is fixed although the continuous object changes its shape and size. Additionally, there is no need to calculate the location of the origin point because it is surely detected by a specific source. With the origin point, we exploit overlaying virtual structure for supporting sink mobility. The structure should satisfy the following design principles: construction of structure regardless of the number of sources and decentralization of messages for avoiding hot-spot problem.We exploit the origin-based structure for supporting sink mobility. The brief introduction of our design is as followings. The origin-based structure delivery location update messages from mobile sinks to the origin point. The node on the origin point is responsible for forwarding the messages to sources. The sources receiving the location update message report data to the sink’s location in the messages directly. The origin-based structure is constructed regardless of the number of sources. Moreover, the direct data reporting prevents concentration of messages of the structure. With this, our design satisfies above principles. Additionally, we exploit clustering to reduce data report overhead.A two-dimensional sensor field is covered by a large number of homogeneous sensor nodes which communicate with each other through short-range radios. So, long distance message delivery is achieved by multi-hop transmission. All sensor nodes in the sensor field are aware of their own locations through either the GPS  or otherThe sensor nodes are periodically awoken and observe the targeted object. Once a stimulus appears or the message is forwarded at specific geographical location, the sensors surrounding it collectively process the signal and one of them becomes the source of the stimulus or the destination of the location.The scheme uses simple greedy forwarding  as underWe assume for the design of OCOT as followings:grid construction messages to the four neighboring grid points. When the location of the origin node is When a continuous object occurs at the origin point, the first source node becomes the origin node that starts the construction of grid structure. The messages spread the whole network and the node closest to each grid point becomes the grid node. A grid node keeps the information about the grid point O indicated with a black square, and its virtual grid structure. The black dots indicate grid points and the white triangles around each grid point are the grid nodes. The gray area indicates a cluster. After the grid structure and clusters are completely built in the sensor field, all sensor nodes become to know their cluster heads, to whom they should deliver their detection information. The cluster head receiving data from ordinary nodes becomes boundary cluster head and has responsibility for data reporting to sinks.A in A is activate node because its all neighbors detect the continuous object, so it does not report data to the cluster head. The number of boundary nodes K can be estimated by calculating boundary area. For example, the continuous object has circular shape,R is the radius of the continuous object, and r is the communication range of sensors. If R is 100 m and r is 15 m, the A continuous object gets a great number of sensor nodes activated so it is more efficient to gather detection information only from boundary nodes rather than all activated sensor nodes. As an integrated location update scheme, in OCOT, the location update message from the mobile sink is delivered to the origin node through the grid structure as shown in We evaluate our Origin-based Continuous Object Tracking (OCOT) through computational simulation with existing studies: TTDD  and LBDDWe choose two metrics to analyze the performance of OCOT. The total energy consumption is defined as the communication  energy the network consumes during a simulation round. The total energy consumption means efficiency of message delivery. The idle energy is not counted since it depends largely on the message generation interval and does not indicate the efficiency of massage delivery. Thedistribution of energy consumption is the map including the location of each node and used energy of the node. We might deduce the possibility of the hot-spot problem in each scheme from the distribution of energy consumption. We divide the network into 20 × 20 cells and observe the average used energy of the nodes in each cell. We implement OCOT in MATLAB . The defUnfortunately, comparison with existing studies is not reasonable because they focus on sink mobility support in individual object tracking. So, we assume that TTDD and LBDD have exploited DCS  for tracThe distribution of energy consumption indicates the operational efficiency and existence of hot spot problem. Therefore, we can tangibly estimate overall energy efficiency and network lifetime. We divide the 1000 m × 1000 m network into 20 × 20 cells and observe the average used energy of the nodes in each cell. In this paper, we propose a novel continuous object tracking scheme, Origin-based Continuous Object Tracking (OCOT), which provides energy-efficient sink mobility support in wireless sensor network. OCOT integrates location updates from a mobile sink using regional locality of sources detecting a continuous object. Also, OCOT exploits distributed data report to avoid the hot-spot problem. Additionally, the reactive and static clustering in OCOT increases the efficiency of location update and data report. Simulation result proves that OCOT is greatly efficient in terms of the total energy consumption and distributional energy consumption. Especially, OCOT reduces at least 49% of energy compared to the existing scheme during 100 s of continuous object tracking process. Under the same simulation setting, in addition, the over 7.25% and 1.25% of sensors consumed over 25% of the energy budget in the existing schemes and OCOT respectively, which suggests that the enhanced network lifetime of OCOT.


Escherichia coli as an example.The genes encoding DNA adenine MTase and cytosine MTase, dam and dcm, respectively, were separately knockedout using the λRed system in E. coli MG1655. MG1655 and the two knockout strains were separately exposedin 96-well plates to 20 antibiotics from five classes. The EC50 values of almost all of the tested antibiotics were lowerin the dam and dcm knockout linesthan that of the control. Our statistical analysis showed that thevariations observed in EC50 values were independent ofthe mechanism underlying each antibiotic’s mechanistic action.Antibiotic-resistantbacteria are causing more antibiotic treatmentfailures. Developing new antibiotics and identifying bacterial targetswill help to mitigate the emergence and reduce the spread of antibioticresistance in the environment. We investigated whether DNA methyltransferase(MTase) can be an adjunct target for improving antibiotic toxicity.We used  In Escherichia coli, DNA adenine methyltransferase  mediates the generationof N6-methyladenine (6mA), and DNA cytosine methyltransferase mediates the generation of C5-methylcytosine(5mC) at the second C in the 5′-CC(A/T)GG-3′ motif.32 DNA methylation, one of the most important and widespread epigeneticmodifications, was shown to be involved in bacterial survival duringantibiotic exposure.34 Moreover, DNA methylation (6mA or 5mC) plays an importantrole in regulating many biological processes including restriction–modificationsystems, replication initiation, mismatch repair, virulence persistence,and global gene regulation.41To mitigatethe emergence and spread of antibiotic resistance,reduction in antibiotic use and improvement in antibiotic efficacyis crucial, which requires investigation of adjunct targets for antibiotics.dam and dcm, respectively)were knocked out in E. coli MG1655using the λRed system. The microdilution method was then usedto assess the exposure of 20 antibiotics from five classes  using96-well plates. The inhibition rates of different antibiotics againsteach E. coli strain were then determined.The concentrations required for the 50% maximal effect (EC50) were determined to compare and assess the toxicity of each antibioticagainst three different E. coli strains.The overlap of 95% confidence intervals was used to assess significantdifferences in the EC50 values. This investigation providesa new insight to reduce the entry of antibiotics into the environmentfrom the source and to extend the service life of existing antibiotics.In view of the above, this paper aims to (1) investigate whetherDNA methyltransferase (MTase) can be an adjunct target and (2) whetherthe antibiotic toxicity can be improved. Here, the genes encodingDam and Dcm . This confirms that dam or dcm knockouts in MG1655 do not affect the growth rate under normal cultureconditions, though 6mA and 5mC were demonstrated to be functionalin regulating many biological processes. Therefore, we consider thatDam and Dcm are not proper targets of the antibacterial agents perse but are potential adjunct targets in the presence of antibiotics.Followingthe construction of the 2.2dam, and Δdcm. As shown in Figure S3a, the growth of MG1655 is a continuous process,which means that the growth rate will not decrease or increase sharplyand suddenly under stable culture conditions. Therefore, we assessedthe effects by comparing the OD600 value of the negativecontrol with those of the exposure groups after 12 h of incubation.As a result, MG1655, Δdam, and Δdcm did not show any obvious differences in the OD600 values under the exposure of 1% DMSO or 100 μM NaOH .Before conducting the exposure experiments, we assessed the effectsof the solvents on the growth of MG1655, Δ2.3dam and dcm) were both confirmed to not influence the growth rate of MG1655,Δdam, and Δdcm . Therefore, we performed antibioticexposure experiments wherein each strain was exposed to 11 concentrationsof each antibiotic, and the OD600 values of the exposedsamples were measured after 12 h of incubation when the bacteria werein the log phase.As discussed above, the varioussolvents used to dissolve the antibioticsand the gene knockouts , CTE,and procaine penicillin (PG) promoted the growth of Δdam at concentrations <0.5, <0.1, and <30 μg/mL,respectively, inducing the hormesis effect. This phenomenon does notappear in the exposure experiment against Δdcm and MG1655. However, it can be concluded that knocking out dam or dcm improves the inhibition propertiesof almost all the antibiotics tested against E. coli MG1655.This enabled us to curve fit the inhibitionrate of each antibiotic against the different strains using the software.As a result, the curve for the inhibition rate of a single exposurewas well fitted by the logistic function and the Levenberg–Marquardt(LM) iterative algorithm 1. All 20and Δdcm 1. Furthe2.4dam, and Δdcm was characterized bythe EC50 values obtained in this work. The EC50 for each antibiotic was calculated at the same time as the curvefitting was performed. Contrasting with the minimum inhibitory concentration(MIC) determination method, this method can avoid the potential errorscaused by visual inspection. We also applied the overlap of the 95%confidence interval (CI) to evaluate significant differences in theEC50 values for each antibiotic against the three strains.45The toxicity of the antibiotics tested against MG1655, Δdam and the exposure of clarithromycin (CLR) and spectinomycin(SC) against Δdcm , azithromycin(AZM), SC, doxycycline (DOX), ERY, CLR, roxithromycin (ROX), ticarcillinsodium (TC), and PG) against Δdam did not significantlydiffer from those against Δdcm (dam and Δdcm simultaneously (50 of each antibiotic againstthe three strains in ascending order (Table S6). The EC50 values for CIP, enrofloxacin (ENR), CT, andofloxacin (OFX) are the minimal four of all of the antibiotics againstMG1655, Δdam, and Δdcm. Furthermore, there are only slight variations in the EC50 ranking of each antibiotic against the different strains. In otherwords, the EC50 variation of a tested antibiotic is relatedto the absence of Dam or Dcm, but the effect is limited. Even so,according to the above discussion, we consider that Dam or Dcm deficiencyimproves the toxicity of antibiotics against E. coli.Our results showed that the 95% CI overlap for MG1655 wasonly observed for the exposure of streptomycin (SM) against Δnst Δdcm 2. Thus, nst Δdcm 2. Notablaneously 2. We the2.550 values of the tested antibiotics and classified it according totheir mechanisms of action. Quinolone antibiotics block DNA synthesis;aminoglycosides, tetracyclines, and macrolides inhibit bacterial proteinsynthesis; and β-lactams inhibit bacterial cell wall biosynthesis.48 We found that the EC50 values of the antibiotics withdifferent modes of action against Δdam or Δdcm were lower than those against MG1655 .We performed a statisticalanalysis to investigate the observed variation in the EC50 valuesof almost all of the antibiotics that inhibit bacterial protein synthesiswere lower for the Δdcm strain than for theΔdam strain . Thus, Dcm may be more important than Dam for bacterial survivalduring the exposure of antibiotics that inhibit bacterial proteinsynthesis. The reduction of EC50 induced by the deficiencyof Dam or Dcm indicates the universality of DNA MTase as an adjuncttarget for improving the toxicity of antibiotics, and this is independentof the mode of action of the antibiotic. We then calculated the relativereduction in EC50 as a percentage, as shown in 50 reduction for AZM against Δdam reached 67.4% was purchasedfrom Tiandz Gene Technology . Plasmids used for geneknockout were stored in our laboratory, including pKD46 (GenBank: AY048746.1)and pKD13 (GenBank: AY048744.1). l-Arabinose  was purchased from Solarbio . The Q5 high-fidelity polymerase for polymerase chain reaction(PCR) was purchased from New England Biolabs . Primersfor the PCR were synthesized by Sangon Biotech .Lysogeny broth (LB)  was applied to bacteriacultures. The LB solid medium (LB plate) was prepared by adding 2%agar (w/v) into the LB medium. Ampicillin  and kanamycin  usedfor bacteria screening were purchased from Sangon Biotech, and theworking concentrations were 100 and 20 μg/mL, respectively.Antibiotics used for exposure including β-lactams, tetracyclines,and quinolones were also purchased from Sangon Biotech. Other antibioticsincluding aminoglycosides and macrolides were purchased from Macklin. Dimethyl sulfoxide  was purchased from Beyotime . NaOH  and NaCl were purchased from Sinopharm Chemical Reagent .4.2dam and dcm genes as described previously.53 However, in this work,the lengths of the homologous arms of the substrate DNA were extendedto 500 base pairs (bps) using the overlapping extension PCR (overlappingPCR).56 Briefly, the kan cassette, whichencompasses a 500 bp DNA fragment upstream and downstream the targetgene (dam or dcm) was PCR-generatedusing pKD13 and MG1655, respectively, as templates. After agarosegel purification, the three products were mixed at a mole ratio of1:1:1 and used together as the template for the overlap PCR generatingthe substrate DNA. The gene knockout was further verified by the bacteria-brothPCR as well as Sanger sequencing. The elimination of pKD46 plasmid,which was temperature-sensitive, was accomplished in MG1655 by furtherbacterial culturing at 37 °C. The genotypes of the dam and dcm knockout strains were MG1655 Δdam::kan (Δdam)and MG1655 Δdcm::kan (Δdcm), respectively. All of the PCR primers were designedas shown in Figure S1, and their DNA sequencesare listed in Tables S1–S3.The λRed knockout systemwas used to knock out 4.3600 measurements were determined on aVarioskan Flash microplate reader . Bacteria cells (200μL/well) were cultured in flat-bottomed 96-well plates  at 37 °C and 200 rpm. OD600 was measuredin triplicate every 2 h from the initial bacteria culture. Thus, thegrowth curves of the three strains were determined from the OD600 measurements over a 24 h period.58OD4.4Table S4). Antibiotic dilutions were performedin 1 mL centrifuge tubes, and each antibiotic was successively dilutedwith the corresponding solvent to achieve 11 concentrations by themultiple dilution method.60 The initial dilutionconcentrations and ratios of the various antibiotics were pre-experimentallydetermined (Table S5). All of the dilutionswere protected from light at 4 °C.Theexposure experiments included five antibiotic classes: β-lactams, tetracyclines , quinolones  aminoglycosides , andmacrolides , which were classified by their chemicalstructures. According to the standard from the Clinical and LaboratoryStandards Institute (CLSI), 20 antibiotics were separately dissolvedin the correct solvents at the proper concentrations  were separately inoculated into10 mL of fresh LB broth and incubated at 37 °C. When the OD600 reached ∼0.6, the bacteria broth was placed in icefor 15 min and then diluted with LB (1:10 000) to an OD600 of ∼0.01. The broth dilutions of MG1655, Δdam, and Δdcm strains were storedat 4 °C and then used as bacterial inoculums. All bacterial inoculumsand antibiotic dilutions were prepared on a clean bench.Newlygrown clones from the three strains , and 10 mM NaOH. The final concentrationof the solvent in the antibiotic exposure tests was 1% for DMSO (v/v)or up to 100 μM for NaOH. MG1655, Δ4.7Figure S2). Altogether, 60 mL of each bacterial inoculum  was prepared for the antibiotic exposure experiments. The VarioskanFlash microplate reader was used to measure the initial OD600 at 0 h of culturing the 96-well plate to which 200 μL/wellof the samples was previously added. The plates were placed at 4 °Cuntil all samples of the three strains had been added to the wellsof 12 96-well plates. Every three 96-well plates containing the samestrain were stacked together, fixed with rubber bands, and then carefullyand smoothly placed in a constant temperature shaker. The three strainswere then incubated at 37 °C and 200 rpm. OD600 wasdetermined for each sample every 2 h over a 24 h period.Dilutionsof the 11 different antibiotic concentrations were added to wellsin the same row of a 96-well plate. A 2 μL aliquot of the correspondingsolvent was added to the first well of each row as a control. A 2μL aliquot of antibiotic dilution was added to the wells fromlow to high concentration in the same row. Thus, 20 different antibioticswere added to three 96-well plates. Next, a 198 μL inoculumof a bacteria strain was carefully added to a well and mixed withthe antibiotic dilution using a pipette (4.8https://www.originlab.com/index.aspx?go=Products/Origin). First, the inhibition rates were calculated by taking the OD600 values at 12 h of culture as parameters  iterative algorithm of the various antibiotic concentrations against MG1655, Δdam, and Δdcm was calculated at thesame time by curve fitting.64 A statisticalanalysis of the EC50 values was accomplished using thesame software (R50EC refers to the EC50 reduction rate of the test antibiotic, as induced by the dam or dcm knockout. ECW50 is the EC50 value of an antibiotic targeting MG1655,whereas ECΔ50 is the EC50 valueof an antibiotic targeting Δdam or Δdcm.The curve fittingof the inhibition rates was performed by OriginProsoftware (rameters 1.61,6212lgorithm2.63 Thirsoftware 3. REC50


Logic gates are important components in integrated photonic circuitry. Here, a series of logic gates to achieve fundamental logic operations based on linear interference in spoof surface plasmon polariton waveguides are demonstrated at terahertz frequencies. A metasurface-based plasmonic source is adopted to couple free-space terahertz radiation into surface waves, followed by a funnel-shaped metasurface to efficiently couple the surface waves to the waveguides built on a domino structure. A single Mach-Zehnder waveguide interferometer can work as logic gates for four logic functions: AND, NOT, OR, and XOR. By cascading two such interferometers, NAND and NOR operations can also be achieved. Experimental investigations are supported by numerical simulations, and good agreement is obtained. The logic gates have compact sizes and high intensity contrasts for the output “1” and “0” states. More complicated functions can be envisioned and will be of great value for future terahertz integrated computing.  •Six major plasmonic logic gates are designed by the linear interference approach•The logic gates are fabricated with other waveguide components on the same platform•The logic gates are characterized using scanning near-field terahertz microscopy•The logic gates are compact and have high-intensity contrasts for the output states Circuit Systems; Photonics; Metamaterials Terahertz (THz) technology is of great potential in developing next-generation, ultrahigh-speed communications, owing to its broad frequency band and capability in carrying ultralarge amounts of information . In partTHz surface plasmonic waves allow chip-scale THz manipulation, propagation, and processing simultaneously. The development of THz on-chip system based on surface plasmons is considered an important avenue for THz systems to be compact and multifunctional. This scheme is also the most promising one to achieve the simultaneous transmission of electrical and optical signals, considering the vital role THz waves might play in future communications. At the same time, THz surface plasmonic waves share similar wave properties with their optical counterparts, and thus the excitation and propagation control methods can be well transformed between the two regimes . With thLogic gates are the basic components of an optical signal processing system, and they are the bridges between electrical and optical calculations. Therefore, logic gates have great potential applications in the field of optical calculation and ultra-high-speed information processing. As is well known, transistor-based Boolean logic gates are the rudimentary units of electronic circuits. In photonic circuits, logic functions can be realized by linear interference effects  and non-In this work, we report on the design, simulation, and experimental characterization of a whole set of fundamental logic gates formed by THz spoof SPP waveguide structures. These waveguides can support transverse-magnetic (TM)-like SPP modes, and these SPP modes propagate in the direction parallel to the gold film and are suitable for on-chip integration applications. A single Mach-Zehnder interferometer can work as AND, OR, NOT, and XOR logic gates. NAND and NOR operations can be achieved by cascading two Mach-Zehnder interferometers with one arm used for the control beam. The working principle is discussed in detail and verified by experiment. These compact logic devices are stable and robust, meeting the requirements for future on-chip integration applications.w = 120 μm, length l = 50 μm, and height h = 80 μm arranged on the top of a metallic surface, also known as a domino structure , the input intensity is divided into two paths by a beam splitter and connected to the logic gates through S-bend waveguides based on the cosine function. For the  and  input singles, the input intensity is also divided into two paths, one of which is connected to the logic gate as logic 1, and the other is not connected to the logic gate as logic 0. The input signal of logic 0 is realized by guiding the SPPs to the outside of the structure with an inclined straight waveguide. This design is to realize the presence or absence of signals in the integration of actual logic gates. As explained earlier, such a beam-splitting design is not necessary but is used to obtain more easily two or more signal sources with specific amplitude or initial phase in the experiment. In the practical application of the proposed logic gate, only the second part is connected to the system. For logic gates in Part 2, the phases of the two arms are controlled by changing the length of the metallic pillars. Destructive interference can be reached in the output waveguide when the phase difference of the two arms is an odd integer multiple of π, whereas constructive interference can be reached when the phase difference is an even integer multiple of π. The remainder port denoted by O is used as the output of the logic gate.The proposed two-port logic gates with detailed structural parameters are provided in k1 after Part 1 and finally combines by the Y-shaped waveguide to form the output. The distances from the input ports of the two waveguides to the interaction point have exactly the same value, which will ensure constructive interference in the output waveguide.I1 and I2 are E1 and E2, respectively. The constructive or destructive interference between E1 and E2 is determined by the phase difference of the input signals, resulting in a different output Eo. The decision mechanism of the logic gates is based on binary on-off-keying, that is, the amplitude of the signal is used as the criterion of the logic , |E|2 for inputs  and , and 4|E|2 for input . When It is chosen to be below |E|2, the output state is logic 1 for input , , and , realizing the OR logic function. When It is chosen to be above |E|2 and below 4|E|2, the output is logic 1 only for  input, corresponding to the AND logic operation. The normalized output power for different situations are obtained and presented in Ez|2 near the same position of the output port of the waveguides. The results are normalized by the input power measured in the initial position of the interferometer arm to eliminate the influence of propagation loss. As can be seen, the two logic operations can be implemented by correctly defining the threshold intensity. The experimental intensity contrast is about 5.46 dB for the AND gate. The experimentally measured total loss is about 3.78 dB for the AND/OR gates. The loss is calculated as the ratio of the power coupled to the fundamental mode of the input waveguide to the output power: Loss = 10log(Pin/Pout). Since in our analysis the actual metallic loss and radiative loss inside the waveguides are the only source of loss. The insertion loss of the AND/OR gates is about 1.87 dB, which is extracted by comparing the total loss of the structure with that of a straight waveguide of the same length. The corresponding propagation loss of the straight waveguide is about 1.06 dB/mm.To verify the functions of the logic gates, simulations based on the time domain solver of CST Microwave Studio are performed. As the source, the SPPs are excited by a plane wave irradiated vertically on the excitation region of the sample from the bottom side. In order to obtain a maximized and optimized SPP field, we calculated the transmission spectra of the logic gates in the paper. The results show that the center frequency excited and supported by the structure is 0.58 THz. In order to obtain the maximum output energy, the devices are designed at an operating frequency of 0.58 THz. O, Io , as a function of frequency is shown in Io  and Io  are shown as the solid pink line and dotted green line, respectively. The value of the power is obtained by integrating the longitudinal component of the Poynting vector on vertical planes near the input and output of the waveguide. The integrating regions have the same dimension of 600 μm × 600 μm to ensure the correct calculation of energy distribution. The power is then normalized by that of I1 or I2 output from Part 1. The high output power of Io  due to complete interference is attributed to the single-mode characteristics and structural symmetry of the waveguide, as shown by the black line. By defining the corresponding threshold intensity It, the logic functions can be realized. For a threshold value of It = 0.7I1, the AND operation can be realized. For a threshold value of It = 0.2I1, the device functions as an OR gate  required to accumulate a phase difference of π is 2,100 μm at 0.58 THz.E1 and E2 are –E and E, respectively. Because of the symmetry of the gate structure, complete destructive interference can be realized at port O, resulting in large interference visibility. Ez|2 distributions for different input signals of the NOT/XOR gates with a scanning area of 8 × 3 mm2 at 0.58 THz. For destructive interference, the complex amplitudes of the outputs for the individual inputs Io is 0 for inputs  and  and |E|2 for inputs  and . When It is chosen to be below |E|2, the output state is logic 1 for inputs  and , realizing the XOR logic function. If the signal –E at port I1 is used as the control signal, for the inputs 0 and –E at port I2, i.e.,  and , the output states are just opposite to the input states, which leads to the function of a NOT gate. The experimental intensity contrast is about 16.60 dB for the XOR and NOT gates. The experimentally measured total loss is about 3.82 dB for the NOT/XOR gates, and the insertion loss is about 1.91 dB.The truth table of the NOT/XOR gates is shown in O, Io , as a function of frequency is shown in Io  and Io  as indicated by the solid pink line and dotted green line, respectively, remains unchanged, whereas the output for Io  decreases sharply owing to the destructive interference. It should be noted that there is a slight difference in the amplitudes of the transmitted powers owing to the structural difference between the two arms, so complete destructive interference cannot be fully realized. For a threshold value of It = 0.2I1, the XOR and NOT operations can be realized. The dotted red line in The normalized output power at port I1 and I2 can be first realized. To realize the 2 × 2 cascaded logic gates, a control signal IC is added to coherently interfere with the output of the first-stage operation to determine the final output at port O. The value of IC is controlled by the coupling length of the parallel waveguides in the first part. The gap between the two parallel waveguides is 80 μm and the length of the parallel section is L. When two identical waveguides are close to each other, there are two supermodes supported by the entire structure. The difference in the propagation constants kSPP between these two supermodes at the same frequency will lead to a phase difference. After the SPPs have propagated a certain distance, the mode power will be shifted from one waveguide to the other in the case of identical waveguide geometries. According to the coupled-mode theory, the output powers from the two parallel waveguides can be obtained as a function of L  with a phase difference of π, the output intensity Io is 0 for input , |E|2 for inputs  and , and 4|E|2 for input . When the threshold value It is chosen between |E|2 and 4|E|2, the output state is logic 1 for input  and the NOR operation can be realized. For a threshold value It below |E|2, the output state is logic 1 for inputs ,  and , and the function of a NAND gate can be realized. The experimental intensity contrast is about 5.48 dB for the NOR gate and 3.14 dB for the NAND gate. The experimentally measured total loss is about 7.75 dB for the NOR/NAND gates, and the insertion loss is about 3.72 dB.The truth table of the NOR/NAND gates is shown in in phase A and 3D.O as a function of frequency for different inputs. The red crosses mark the corresponding area and power threshold for the three logic functions. For |E|2 < It < 4|E|2, the NOR operation can be realized. For It < |E|2, the device functions as a NAND gate. For a threshold value of It = 0.3I1, the NOR operation can be realized. For a threshold value of It = 0.15I1, the device functions as a NAND gate. The dotted red and blue lines in Since the coupling between waveguides is the main limitation to be considered to minimize the device dimensions, according to the coupled mode theory, the coupling between the waveguides decreases as the distance between the waveguides increases and reduces almost to zero when the distance is about 280 μm . At thisThe operation speed of the logic gates proposed in this paper is mainly determined by the time required for the surface waves to propagate on the waveguide. Here, the propagation length of the logic gates is defined as the output position of the beam splitter to the interference position of the logic gates. For the AND/OR gates, the required operation time is about 15 ps, and for the NOT, XOR, NAND, and NOR gates, the operation time is about 35 ps.2 by high precision lithography, while integrating several cascaded SPP waveguides and logic elements on the same substrate. Assembles of these gates can lead to complex functionalities and open up an avenue toward developing THz plasmonic computing chips.In summary, a series of novel THz basic logic operations based on spoof SPP waveguides are designed and experimentally demonstrated based on the linear interference effect. A single Mach-Zehnder interferometer can function as an AND, OR, NOT, or XOR logic gate. By using two cascaded Mach-Zehnder interferometers with one arm as the control input, NAND and NOR operations can be accomplished. The SPP waveguide systems have been fabricated on an area of 10 × 5 mmExperiments to investigate XNOR gate as well as assembles of the gates were not included in this work and require further investigation.yanfengli@tju.edu.cn).Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Associate Professor Yanfeng Li (This study did not generate new unique reagents.This study did not generate/analyze datasets/code.All methods can be found in the accompanying