China has committed to reaching carbon neutrality by 2060, which will require a drastic cut in greenhouse gas (GHG) emissions from all sectors, including those from agricultural activities. A comprehensive, long-term, and spatially-precise profile of agricultural GHG emissions can help to accurately understand drivers of historical emissions and their implications for future mitigation. This study constructs province-level agricultural GHG emissions in China from 1978 to 2016. It considers primary and secondary emissions from a full range of agricultural activities related to crop farming, including crop residue open burning, rice cultivation, cropland change, cropland emissions, machinery use, nitrogen fertilizer production, and pesticide production. Annual or interpolated activity data from official sources and the latest emission factors available for China were adopted in this study. The data can be used in spatial and temporal analysis of emissions from cropping systems as well as the design of mitigation strategy in China. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14753940 In particular, agricultural activities were the key sources for emissions of the non-CO2 GHG including CH4 and N2O, which have global warming potentials (GWP), respectively, of 28 and 265 times the value of CO2 on a 100 year time scale2.According to the Synthesis Report (SYR) of the IPCC Fifth Assessment Report (AR5)3, China had more than 367 million people working in agriculture in 20194. Changes in agricultural activities caused by urbanization in turn pose challenges for GHG emission mitigation in agriculture. Urbanization in China has developed rapidly with a continuous decrease in the rural population. The percentage of agricultural labor decreased from 83.5% in 1952 to 25.1% in 20195. The Reform and Opening policy adopted in 1978 accelerated this process by encouraging agricultural labor to move to cities for jobs with higher salaries. To ensure crop production, agricultural activities have become more and more reliant on fertilizers and machinery inputs rather than labor and land. The use of fertilizers, pesticides, and machinery power has increased rapidly since 19785. The total power of agricultural machinery reached 1027 GW in 20195, over 8 times the value in 19785. China peaked in fertilizer and pesticide use in 20176. Associated with urbanization and agricultural modernization, the use of machinery is expected to continue increasing7. The GHG emissions associated with this increasing use of machinery raise challenges in agricultural mitigation in China. In 2012, although crop farming and animal husbandry only contributed 7.8% of total GHG emissions in China, they contributed over 50% of total non-CO2 emissions8. To realize carbon neutrality in China by 20609, GHG emission mitigation in agriculture will be an important target10.With 18% of the global population13. Based on Tier 1 IPCC Guidelines14, FAOSTAT calculated 1961–2017 GHG emissions from agriculture, land use and forestry for 234 countries, which included emissions from enteric fermentation, rice cultivation, agricultural soil, crop residues and savanna burning, and energy use in agricultural activities11. Burney et al.12 calculated global agricultural GHG emissions from 1961 to 2005, which included emissions from agricultural soils, rice cultivation, cropland expansion, and production and use of fertilizers. Parton et al.13 used ecosystem models to estimate GHG emissions from agriculture from 1870 to 2000 in the U.S. Great Plains, including emissions from soil cultivation, N mineralization, N fertilizer application, livestock, fossil fuels used in fertilizer production, and burning of fossil fuels by farm equipment.A number of studies have investigated long-term GHG emissions in the U.S. and globally14, China published official GHG inventories for 199415, 200516, and 20128, in which enteric fermentation, manure management, rice cultivation, cropland soil, agricultural wastes burning, and land-use change were included for agricultural and land-use emissions16. Zuo et al.17 calculated GHG emissions from changes in crop mix, multiple cropping, fertilizer application, irrigation, and paddy rice water management in China, but only presented emissions for 1987, 2000, and 2010 rather than a continuous result. However, those studies selected different agricultural activities and parameters, which made it difficult for comparison. In addition, agricultural machinery uses and emissions from fertilizer and pesticide production were excluded from those studies. Most importantly, the existing datasets could not offer the GHG emissions profiles for agriculture in China either for the long-term perspective or on the provincial level and thus had limited application in designing mitigation strategies.A holistic and integrated database for provincial agricultural GHG emissions is still lacking in China. Based on IPCC GuidelinesTo fill those gaps, this study builds a database for provincial agricultural GHG emissions in China from 1978–2016. It covers crop residue open burning, rice cultivation, cropland change, cropland emissions, machinery use, nitrogen fertilizer production, and pesticide production. Up-to-date annual activity data was used for most agricultural activities and numerical interpolation was adopted for cropland change. Our analysis selected the latest emission factors available for China. Providing a complete and continuous profile of agricultural GHG emissions in China at the provincial level, this study can aid further analysis of contributions from different activities as well as spatial and temporal emission patterns, which are the foundation for designing mitigation strategies.18. Primary emissions are defined as GHG emissions coming directly from tillage, seeding, harvesting and transportation18. Secondary emissions refer to GHG emissions from the production, packaging, and storage of fertilizers and pesticides18. Tertiary emissions denote GHG emissions from the acquisition of raw materials, manufacture of agriculture equipment like agriculture machinery, and construction of agricultural buildings18. According to the IPCC definition, primary emissions are direct emissions, and secondary and tertiary emissions are defined as embodied emissions and indirect emissions19. Primary emissions gain wide attention in GHG inventory compilation and carbon management. Agricultural activities mainly emit CO2, CH4, and N2O in primary emissions through livestock enteric fermentation, manure management, rice cultivation, cropland, crop residue burning, energy use, and land cultivation20. Secondary emissions are often accounted for in the life cycle analysis (LCA) of agricultural products. Since the boundary of tertiary emissions is broad, most studies concentrated on the primary and secondary emissions to guide the practice of agricultural GHG control17.Agricultural GHG emissions can be categorized into three types of sources: primary, secondary, and tertiary8. For secondary emissions, we considered machinery use, N fertilizer production, and pesticide production based on FAO201420. Crop residue open burning can emit CO2, CH4, and N2O14, however only CH4 and N2O emissions are taken into account as CO2 released from burning is countered by carbon uptake from the air during the growth of crops. During rice cultivation, the anaerobic decomposition of organic materials in inundated rice fields emits CH414. Emissions from cropland change can be positive or negative, accounting for CO2 emission or absorption due to the release or absorption of carbon by vegetation and soil during land-use changes between cropland and other land-use types like wood, grassland, and residential land14. Application of synthetic nitrogen fertilizers, manure, and crop residue on land directly emits N2O through nitrification and denitrification, and indirectly emits N2O through volatilization and leaching22. Agricultural machinery use consumes energy like diesel, gasoline, and electricity, which emits CO2, CH4, and N2O emissions20. Production of nitrogen fertilizers and pesticides produces CO2, CH4, and N2O emissions during manufacture and transportation.The study mainly focuses on crop farming and covers primary and secondary emissions. For primary emissions, the study included crop residue open burning, rice cultivation, cropland change, and cropland emissions based on China’s official inventory in 20122 equivalent, and Global Warming Potential (GWP) values for the 100-year time horizon published by IPCC Fifth Assessment Report (AR5) in 2013 were adopted to transform CH4 (GWP100 = 28) and N2O (GWP100 = 265) emissions2. Total emissions calculated with the GWP values for the 20-year time horizon were included in the Supplementary Information . Since Hainan province was founded in 1987 and Chongqing city was founded in 1997, their emissions before establishment were calculated under Guangdong province and Sichuan province respectively. The following GHG emissions were counted as COn Figure . Missinget al.23, CH4 and N2O emissions from crop residue open burning were calculated through Eq. 30 published by the Ministry of Ecology and Environment of the People’s Republic of China in 2015. CH4 emission factor was developed from the mean value of EPA1992 Open Burning table31, Andreae et al.32, IPCC Guidelines14, and the three most recent studies in China33. N2O emission factor was collected from the mean value of Andreae et al.32, IPCC 2006 Guidelines14, and one study in China33.The study considered 13 types of crops, including rice, wheat, corn, millet, jowar, other cereal, beans, tubers, cotton, oil-bearing crops, fiber crops, sugar crops, and tobacco. Annual crop production data for each province was collected from the National Bureau of Statistics of China (NBSC)et al.34, CH4 emissions from rice cultivation were calculated as in Eqs. ::et al.345. The length of the rice-growing period and emission factors of each rice type and province were obtained from Yan et al.34. For Shanghai, Jiangsu, Anhui, Henan, and Hubei , we adopted the mean values of emission factors of middle rice and single late rice in Yan et al.34 for middle rice and single late rice in our study. For Chongqing, Sichuan, Guizhou, and Yunnan (AEZ 6B regions), we adopted the emission factors of middle rice in Yan et al.34 for early rice and double late rice in our study.The present study considered 3 rice types, including early rice, middle rice and single late rice, and double late rice. The annual rice cultivation area for different provinces was collected from annual data from the NBSC20, CO2 emissions from cropland change were calculated as in Eqs. ::20, CO2 35. The annual area of cropland change was calculated as the 10-year or 5-year average of the area of cropland change. The annual area of cropland change of 1978 and 1979 was assumed to be the same as that in 1980, and the annual area of cropland change of 2015 and 2016 was assumed to be the same as that in 2014.The present study considered 8 types of cropland use change, which are land-use changes between cropland and wood, grassland, water and residential areas, and others. The land-use remote sensing data in 1980, 1990, 2000, 2010, and 2015 were acquired from Data Center for Resources and Environmental Sciences, Chinese Academy of Sciences (RESDC)et al.12, which assumed consistent values from 1978 to 2016. According to Burney et al.12, this study assumed 70% of cropland was for crops, 15% was for oil crop plantations, and 15% was for fruits. The carbon content in biomass of cropland was selected as 10.075 t C/ha, which is 50% of the weighted average of global peak values of crops, oil crop plantations, and fruits. For wood, biomass carbon was 116.2 t C/ha, and soil organic carbon was 97.6 t C/ha, which were calculated from the mean value of the tropical evergreen forest, tropical deciduous forest, temperate broadleaf evergreen forest, temperate needle leaf evergreen forest, temperate deciduous forest, boreal evergreen forest, boreal deciduous forest, evergreen/deciduous mixed forest, dense shrubland, and open shrubland in Burney et al.12. Carbon content in water and residential areas was 0 t C/ha. For others, biomass carbon was taken as 11.5 t C/ha, and soil organic carbon as 14.5 t C/ha, the mean value of savanna, tundra, desert and polar desert/rock/ice in Burney et al.12. For soil organic carbon in cropland, the study obtained soil organic carbon in cropland in 1980 and its annual change rate during 1980–2009 in China from Yu et al.36. The annual change rates in 1978 and 1979 were assumed to be the same as that in 1980, and the annual change rates during 2010–2016 were assumed to be the same as that in 2009. Then the soil organic carbon in cropland for each year was calculated based on the above values.Carbon content values for biomass of cropland, biomass and soil of wood, grassland, and others were obtained from Burney 16, the study adopted IAP-N model22 to calculate direct N2O emissions from cropland, and calculated indirect N2O emissions from cropland based on IPCC Guidelines14.According to the People’s Republic of China Second National Communication on Climate Changei in year j; Nitri,j is the nitrogen input in the corresponding province and year, including the application of synthetic nitrogen fertilizer , application of manure , and crop residue on cropland , which were calculated through Eqs.   11) were5. Application of synthetic nitrogen fertilizers (effective component) during 1979–1986 was collected from the Fifty Year Agricultural Statistics37, and the data during 1987–2016 were collected from annual data from the NBSC5. For poultry population data, the data during 1978–2008 were obtained from the Fifty Year Agricultural Statistics37, and data during 2009–2016 were obtained from the China Agriculture Statistical Report38. Parameters used for calculating nitrogen input were obtained from Zheng et al.21. Emission factors were obtained from Zheng et al.39 and this study adopted the mean value of all types of cropland in each region.The rural population, livestock population, and crop production were collected from annual data from the NBSCi in year j; Atmosi,j is the amount of N2O-N produced from atmospheric deposition of N volatilized from managed soils in corresponding province and year; Leachi,j is the amount of N2O-N produced from leaching and runoff of N additions to managed soils in regions where leaching or runoff occurs in the corresponding province and year. In Eq.   14) were20, CO2 emissions from agricultural machinery were calculated as in Eqs.  from generalized agriculture  41. The emission factors of electricity for different years were acquired from the Energy Research Institute (ERI) of the National Development and Reform Commission (NDRC).The present study considered 9 energy types, which are coal, coke, gasoline, kerosene, diesel, fuel oil, LPG, natural gas, and electricity. Energy consumption from generalized agriculture and average low calorific value were collected from the China Energy Statistical Yearbook (CESY) see Eq. . GDP ratThe study applied Eq.  to calcui in year j; NFerti,j is the application of synthetic nitrogen fertilizers, which is the same as cropland emissions; EFFERT is the emission factor of nitrogen production. The emission factor was calculated from total emissions from nitrogen fertilizer production in 2005 calculated by Zhang et al.42 and nitrogen fertilizer production in 2005 published in the 12th Five-Year Plan of the Fertilizer Industry43.where i in year j; Pesti,j); EFPEST is the emission factor of pesticide production.Eqs.  and 20)20) were 5. We calculated the proportion of effective components in pesticides during 2010–2014 with the application of effective components during 2010–2014 from the National Agriculture Technology Extension Service Center44 and the total amount of pesticide application during this period from annual data from the NBSC5. Then we regressed the proportion of effective components on pesticides on year to get the proportion for 1990–2009 and 2015–2016. The emission factor of pesticide production was calculated using the mean value of emission factors of all types of pesticides in Zhang et al.45.This study only calculated emissions from pesticide production after 1990, when the Chinese government started to publish data on pesticide use. The total amount of pesticide application was collected from annual data from the NBSC46. It contains one national inventory by agricultural activity and inventories by province. For the national inventory by agricultural activity, one matrix shows point estimates with 39 rows representing years and 8 columns representing agricultural activities and total emissions, while the other matrix shows 95% confidence intervals. For the inventories by province, one matrix shows total agricultural emissions with 39 rows representing years and 31 columns representing provinces, and the other seven matrixes show point estimates for emissions in 7 agricultural activities. Figure The dataset is available on figshare2-eq; contributing 41% of GHG emissions in agriculture (938 Mt CO2-eq) and 3.4% of total GHG emissions (11320 Mt CO2-eq) in China8.In 2012, the total emissions from crop residue open burning, rice cultivation, and cropland emissions in the crop system were 382.59 Mt COet al.34, CVs for cropland change were from Burney et al.12, and CVs for machinery use were from Shan et al.47. CVs for emission factors of electricity in machinery use and emission factors in nitrogen fertilizer production and pesticide production were assumed to be 50% due to high uncertainty, according to Zhao et al.48. For cropland emissions, normal distributions and triangular distributions were assumed, where CVs and parameters for distribution were derived from Zheng et al.39 and IPCC Guidelines14. CVs and parameters for distribution are shown in Table Uncertainty mainly comes from measurement errors of activity data, emission factors, and other parameters (see Table We generated 50,000 random samples of those parameters and gained 95% confidence interval for national GHG emissions from each agricultural activity. The 95% confidence intervals of total emissions and emissions from each agricultural activity are shown in Fig. Our analysis may have the following limitations: First, activity level data on cropland change adopted in this study are only available for 1980, 1990, 2000, 2010, and 2015, and we used numerical interpolation for other years. Incorporation with multi-dimensional data in interpolation in future studies may enhance continuity. Second, although the study considered the spatial heterogeneity of emission factors in crop residue open burning, rice cultivation, and cropland emissions and temporal heterogeneity of emission factors in cropland change, machinery use, and pesticide production, it applied national emission factors for cropland change, machinery use, nitrogen fertilizer production, and pesticide production, and uniform emission factors for 39 years in crop residue open burning, rice cultivation, cropland emission, nitrogen fertilizer production, and pesticide production. Further research using emission factors specific for province and year can narrow down spatial and temporal uncertainty. Lastly, this study used energy consumption in crop farming and effective components in pesticide applications derived from the GDP ratio rather than actual statistics. Uncertainty can be further reduced with more specific energy consumption data from agricultural machinery use and the number of effective components in pesticides applied to the field.Supplementary information


Prader–Willi syndrome (PWS) is a rare disease determined by the loss of the paternal copy of the 15q11-q13 region, and it is characterized by hypotonia, hyperphagia, obesity, short stature, hypogonadism, craniofacial dysmorphisms, and cognitive and behavioral disturbances. The aims of this retrospective study were to analyze interictal EEG findings in a group of PWS patients and to correlate them with genetic, clinical, and neuroimaging data. The demographic, clinical, genetic, EEG, and neuroimaging data of seventy-four patients were collected. Associations among the presence of paroxysmal EEG abnormalities, genotype, and clinical and neuroimaging features were investigated. Four patients (5.4%) presented drug-sensitive epilepsy. Interictal paroxysmal EEG abnormalities—focal or multifocal—were present in 25.7% of the cases, and the normalization of the EEG occurred in about 25% of the cases. In 63.2% of the cases, the paroxysmal abnormalities were bilaterally localized over the middle–posterior regions. Brain magnetic resonance imaging (MRI) was performed on 39 patients . No relevant associations were found between paroxysmal EEG abnormalities and all of the other variables considered. Interictal paroxysmal EEG abnormalities—in particular, with a bilateral middle–posterior localization—could represent an important neurological feature of PWS that is not associated with genotype, cognitive or behavioral endophenotypes, MRI anomalies, or prognosis. Prader–Willi syndrome (PWS) is a rare genetic condition with an estimated prevalence of about 1:15,000 that is characterized by multisystemic features, such as severe early hypotonia, hyperphagia, childhood obesity, short stature, small hands and feet, hypogonadism, growth hormone or other endocrine deficiencies, craniofacial dysmorphisms, developmental delay, intellectual disability (ID), behavioral disturbances, and autism spectrum disorder [PWS is a genomic imprinting disorder, as it occurs due to the loss of a paternal copy of the 15q11-q13 region. Most frequently (in approximately 60% of cases), this is the result of a de novo paternal deletion of the 15q11-q13 region, leading to a lack of expression of paternally derived genes; in about 35% of cases, maternal uniparental disomy (UPD)—with both instances of chromosome 15 inherited from the mother—is present. Imprinting center (IC) defects  in the 15q11-q13 region or other chromosomal abnormalities are found in the remaining 5% of PWS patients .Until now, no peculiar interictal electroencephalographic patterns have been identified in PWS, as they have in other chromosome abnormalities, and heterogeneous EEG pictures have been reported, such as focal, multifocal, or generalized epileptiform abnormalities ,6,7,8,9.Although epilepsy does not represent a typical neurological feature, recently, a prevalence ranging from 4% to 26% was estimated in several retrospective PWS series worldwide. Seizures usually start before two years of age, and epilepsy is mostly of the focal type, but generalized epilepsy can also occur, with generalized tonic–clonic seizures and, more rarely, atypical absences and atonic seizures ,10,11,12Specific EEG patterns were previously described in a series of genetic disorders , but notWe retrospectively recruited subjects with PWS referred to 4 Italian Referral Centers, i.e., Oasi Research Center , the Pediatric Unit of Casa Sollievo della Sofferenza , the Pediatric Unit of University of Modena e Reggio , and the Department of Human Pathology of the Adult and of the Developmental Age “Gaetano Barresi” .All of our patients were representative of all PWS patients because they were initially diagnosed and then clinically managed at centers that specialized in pediatric endocrinology and clinical genetics. The necessary inclusion criteria of this study were an established genetic alteration that was typical of PWS and the availability of EEG recordings. In the above-mentioned centers, the EEG was a common diagnostic tool included in the neuropediatric workup of developmental disorders and rare comorbid diseases, such as PWS, even in the absence of seizures.This study was approved by all of the local ethics committees of the centers in which the study was performed. Written informed consent was obtained from the patients’ parents or from the patients, as appropriate. All investigations were conducted according to the principles expressed in the Declaration of Helsinki.The clinical diagnosis of PWS was made according to the consensus clinical diagnostic criteria  and was For all of the subjects, we evaluated their gender, age (range and mean) at the first and last EEG, duration (range and mean) of the EEG follow-up, BMI (range and mean) at the first and last EEG, genotype, degree of intellectual disability, behavioral disturbances, presence of seizures, age (range and mean) at seizure onset, seizure type, EEG and MRI findings, and drug therapy. MRI was carried out in order to detect possible pituitary or brain anomalies. When clinically suspected, the presence of sleep apnea was assessed by using polysomnography.Intellectual disability (ID) and borderline intellectual functioning (BIF) were classified according to the DSM 5 criteria . SeizureWe investigated the possible correlations between the presence of paroxysmal EEG abnormalities and other clinical and neuroimaging features, such as the degree of cognitive dysfunction (subjects with BIF or mild ID versus subjects with moderate or severe ID), behavioral disturbances (presence versus absence), genotype (deletion 15q11-q13 versus UPD), and brain MRI abnormalities (presence versus absence).p-values < 0.05 were considered statistically significant.A statistical analysis was performed by using SPSS version 20.0 for Mac OS . The age did not follow a normal distribution (Kolgomorov–Smirnov test), and thus, a non-parametrical Mann–Whitney U test was used for comparisons between groups. The Yates-corrected chi-square test was used to assess any differences between categorical variables. The data are presented as the range and mean ± SD or rate. In addition, we checked for possible simultaneous associations of age, presence of behavioral disorders, pathological findings in MRI, and genetic conditions, which were used as independent factors/predictors; abnormal or normal EEG or the level of intellectual disability was considered as the dependent variable. This was achieved by means of the General Regression Models module offered by the commercially available STATISTICA v.6, StatSoft Inc . This module allows one to build models for designs with categorical predictor variables, as well as with continuous predictor variables. For the dependent variable, the statistical significance of the association of each independent factor was obtained by considering the effects of the other independent factors.We studied 74 subjects with PWS who were aged 2–42 years; 39 were males (52.7%) and 35 were females (47.3%). Thirty-two patients (43.2%) had a 15q11-q13 paternal deletion, thirty-eight (51.4%) had a maternal UPD, and four (5.4%) had an imprinting center defect (ICD). ID was diagnosed in 67 subjects. Four patients (5.4%) presented epilepsy with various types of seizures, such as absences, focal seizures with impaired awareness, or generalized tonic–clonic seizures. One patient had febrile convulsions. Seizure onset ranged from 1 year and 3 months to 7 years, and in all cases, seizures were controlled with antiepileptic drugs in monotherapy or polytherapy. Sleep apnea was diagnosed in 17 patients out of the 28 who underwent polysomnography. The following drugs were given to treat seizures and/or behavioral disturbances: topiramate in five cases, valproate in three cases, levetiracetam in two cases, and oxcarbazepine in one case. The complete demographic and clinical findings are reported in A total of 59 subjects (79.7%) took drugs during the follow-up period, i.e., growth hormone or other hormones (49 cases), hypoglycemic medications (6), antiepileptic drugs (11), and psychopharmacologic treatments (11).2, with a mean of 24.2 ± 12.1 kg/m2; and the BMI range at the last EEG was 13.5–67.4 kg/m2, with a mean of 25.6 ± 11.1 kg/m2.A total of 198 EEGs were recorded : 92 during wakefulness and 106 during sleep . The ageIn all cases, the background activity was normal for the age. EEG abnormalities were found in 19 patients , and they were classified as multifocal and middle–anterior (five subjects)  when locIn two cases, focal slow waves were present. In five out the nineteen patients (26%), the EEG picture became normal at the end of the follow-up at 9.01 (range: 4.7–16.3) years of age and 3.6 (range 1–11.3) years after the first abnormal EEG pattern . Only onBrain MRI was performed in 39 subjects . In 23 on = 19) and without paroxysmal EEG abnormalities ; behavioral disturbances were present in five PWS EEG+ patients and in 12 PWS EEG- patients. The genotypes were deletion 15q11-q13 in 10 PWS EEG+ patients and 22 in PWS EEG- patients, and UPD in nine PWS EEG+ patients and 29 in PWS EEG- patients; ICDs were evident only in four PWS EEG- patients. Brain abnormalities were found in 6 out of 12 PWS EEG+ patients who performed MRI ; they were evident in 17 out of 27 PWS EEG- patients . The main characteristics of the PWS patients with paroxysmal EEG abnormalities and PWS patients without paroxysmal EEG abnormalities are summarized in The mean ages were, respectively, 11.2 years (range: 3–33 years) and 10.3 years (range: 0.1–33 years) in the subgroups of PWS subjects with paroxysmal EEG abnormalities , behavioral disturbances (p = 0.93), genotype (p = 0.66), or brain MRI abnormalities (p = 0.77). Finally, as a further confirmation of the results reported above, none of the factors considered  as independent predictors were significantly associated with abnormal EEG or with the level of intellectual disability as a result of the analysis by means of the General Regression Models module.In summary, no differences were found between patients with and without paroxysmal EEG abnormalities in terms of age  carried out an EEG study in 26 out of 50 patients with PWS, and 10 of them 38.5%) showed abnormalities. In particular, five subjects (with or without seizures) presented high-voltage EEG activities at 4–6 Hz, four  showed focal paroxysmal discharges, and one  showed short generalized discharges of polyspike and wave complexes [8.5% show carried Three subsequent retrospective studies revealed focal, multifocal, or generalized paroxysmal EEG abnormalities, respectively, in 13 of 23 56.5%), 13 of 22 (59%), and 38 of 38 (100%) patients with PWS and epilepsy .5%, 13 o,8. FurthMultifocal or focal EEG abnormalities were found in 19 25.7%) of our 74 patients, and epilepsy was found in 4 (5.4%). In only two cases, psychopharmacological treatment with risperidone could facilitate the appearance of interictal EEG abnormalities. These prevalence rates are at the lower end of the prevalence ranges reported in the previous literature: 12.8–100% for EEG abnormalities and 4–26% for epilepsy; however, this apparent discrepancy in the prevalence rate could be due to the different settings, as all of our patients were recruited in centers that specialized in pediatric and endocrinological management of PWS .7% of ou,12. In aWe did not find statistically significant associations between the presence of interictal paroxysmal EEG abnormalities and any other clinical, genetic, or neuroimaging features of the PWS phenotype. However, the interictal EEG abnormalities—and, in particular, their middle–posterior localization—could represent an important and supportive neurological feature of PWS, even though they do not contribute by providing information about the genotype, cognitive or behavioral endophenotypes, possible structural anomalies in MRI, or prognosis.Although our study seems to confirm that a peculiar interictal EEG pattern does not exist in PWS, the paroxysmal abnormalities are mostly focal or multifocal, and in approximately two-thirds of our patients, spikes were localized over the middle–posterior regions. Thus, this localization could represent a rather common EEG trait that could be considered as a potential marker of PWS.Both epilepsy and interictal EEG abnormalities are more frequently found in PWS patients than in the general population (3.54%) , but theBrain MRI abnormalities were relatively frequent (59%) and heterogeneous in our cohort of patients with PWS. Most frequently, we found enlargements of the lateral ventricles (39.1%) and pituitary hypoplasia (39.1%). Dilation of lateral ventricles, a non-specific finding, was previously observed in patients with PWS in up to approximately 45% of cases, and it can represent the consequence of lost or reduced growth of white or gray matter, or both ,8,11,12.Our study has an important strength. The recruitment of a rather large series of patients with PWS, who were all managed for a long period of time in highly specialized centers, allowed us to establish the prevalence of interictal EEG abnormalities with a particular accuracy considering the great number of EEG recordings carried out. On the other hand, some methodological limitations should be taken into account. In fact, this study was retrospective; all patients were managed in pediatric and endocrinological settings, and the sample under study was limited by the rarity of the syndrome, and was not representative of PWS as a whole, with 43% presenting a deletion of the 15q11-q13 region and 51% presenting a UPD, which had an impact on the correlations. Brain MRI was available for approximately 52% of the sample studied; a machine-/deep-learning approach to predicting the subtypes or outcomes in PWS patients based on the EEG, genotype, and clinical and neuroimaging data was not used because these capabilities were not available.In conclusion, we showed that about 25% of PWS patients presented EEG abnormalities, irrespective of their genetic defects, gender, and intellectual disabilities, and that about 5% presented epilepsy. The EEG normalized in about 25% of the patients with EEG abnormalities. Our study showed that paroxysmal abnormalities are mostly focal or multifocal and are localized over the middle–posterior regions in two-thirds of the patients. This localization might be considered a rather common EEG trait and a potential marker of PWS in about 25% of patients during follow-up. A peculiar interictal EEG pattern does not exist in PWS. Further large prospective studies are needed to clarify the pathogenesis of electroclinical findings in PWS, and they should also address more strict correlations with molecular genetics and high-definition neuroimaging.


Innovative approaches to the dissemination of evidence-based COVID-19 health messages are urgently needed to counter social media misinformation about the pandemic. To this end, we designed a short, wordless, animated global health communication video (the CoVideo), which was rapidly distributed through social media channels to an international audience.The objectives of this study were to (1) establish the CoVideo’s effectiveness in improving COVID-19 prevention knowledge, and (2) establish the CoVideo’s effectiveness in increasing behavioral intent toward COVID-19 prevention.In May and June 2020, we enrolled 15,163 online participants from the United States, Mexico, the United Kingdom, Germany, and Spain. We randomized participants to (1) the CoVideo arm, (2) an attention placebo control (APC) arm, and (3) a do-nothing arm, and presented 18 knowledge questions about preventive COVID-19 behaviors, which was our first primary endpoint. To measure behavioral intent, our second primary endpoint, we randomized participants in each arm to five list experiments.P<.001) arm. We observed high baseline levels of behavioral intent to perform many of the preventive behaviors featured in the video intervention. We were only able to detect a statistically significant impact of the CoVideo on one of the five preventive behaviors.Globally, the video intervention was viewed 1.2 million times within the first 10 days of its release and more than 15 million times within the first 4 months. Knowledge in the CoVideo arm was significantly higher  than in the do-nothing (mean 16.86, 95% CI 16.83-16.90; Despite high baseline levels, the intervention was effective at boosting knowledge of COVID-19 prevention. We were only able to capture a measurable change in behavioral intent toward one of the five COVID-19 preventive behaviors examined in this study. The global reach of this health communication intervention and the high voluntary engagement of trial participants highlight several innovative features that could inform the design and dissemination of public health messages. Short, wordless, animated videos, distributed by health authorities via social media, may be an effective pathway for rapid global health communication during health crises.German Clinical Trials Register DRKS00021582; https://tinyurl.com/6r4zkbbnRR2-10.1186/s13063-020-04942-7 Soon after the outbreak of the COVID-19 pandemic, health-related misinformation flooded the social media space . CompellThere is a critical need to rapidly disseminate evidence-based informational videos on social media channels to counteract the epidemic of COVID-19 misinformation. To date, public health efforts have focused on correcting misinformation and debunking myths . As suchIn March 2020, we designed a short, wordless, and animated video to disseminate information about preventing the spread of COVID-19 ,8. The iIn this study, we evaluate the effectiveness of the CoVideo to improve knowledge and behavioral intent toward COVID-19 prevention. According to the Theory of Planned Behavior, the intention to act is considered to be the immediate determinant of action . Here, wThis is a multisite, parallel-group randomized controlled trial (RCT) comparing the effectiveness of a short informational video on COVID-19 prevention. To evaluate the effectiveness of the CoVideo, we enrolled participants from five countries into a large, online RCT. We randomly assigned participants to the CoVideo , an atteWe used an online platform called Prolific  to enrolParticipants began by answering basic demographic questions about their age, sex, primary language, country of residence, and highest education completed.The Gorilla algorithm then randomly assigned participants 1:1:1 to the CoVideo, APC video, or do-nothing groups. Participants were required to watch the CoVideo or the APC video once from start to finish. The CoVideo is animated with sound effects but does not include any words, speech, or text. It explains how the novel coronavirus is spread  and recommends best practices to prevent onward transmission . It also covers the mass media coverage of the outbreak and the public’s response to this media coverage, which includes a subplot on the stockpiling of essential goods, and the impact thereof on health care services and resources . The total duration of the CoVideo is 2 minutes, 30 seconds.The APC is also a wordless, animated video with the same duration as the CoVideo. Its content describes how small choices become actions, which become habits, which become a way of life. We included an APC to account for possible attention effects elicited by the video format. APC conditions should mimic the “inactive” components of an intervention—the effect of watching the video—while not containing any of the “active” intervention components—the content delivered by the video . We did After completing the intervention , participants answered 18 knowledge questions on preventive COVID-19 behaviors. All items required true or false responses, and all participants received the knowledge items. After completing the knowledge questions, participants then completed five list experiments. For each list experiment, we randomized participants 1:1 to a control list or a control list plus a sensitive item about behavioral intent toward social distancing, washing hands, cleaning dishes, cleaning kitchen surfaces, and the stockpiling of essential goods. The control group received a list of 5 items that were unrelated to COVID-19. For example, in the first list experiment, we asked: “How many of the five statements do you agree with? We don’t want to know which ones, just answer how many: 1. Spend time watching TV, 2. Do the vacuuming, 3. Pick a fight with my partner, 4. Eat a low sugar diet, 5. Rinse my nose with salt water daily.” The treatment group received the same 5 items and 1 additional “sensitive” item, “Go out with my friends,” which indicates behavioral intent to social distance (or not) during lockdown restrictions. We used the list experiments to reduce social desirability bias ,27 and dWe summarized the participant characteristics by obtaining mean (SD) values for age, gender, primary language, country of residence, and education status. Using the Gorilla platform, we identified and excluded participants from the analysis who were lost, defined as those who did not complete the survey from start to finish. Because we could not determine if participants watched some or all of the CoVideo or APC video, we used an intention-to-treat analysis.y=b1VideoArm, where y is the number of knowledge statements that the participant correctly answered and VideoArm represents the treatment arm.For the first endpoint, we calculated a knowledge score for each participant by adding the correct responses . Participants had a time limit of 30 seconds to answer each knowledge item, preventing them from searching for answers on the internet. If the participant timed out, they received a missing value of 9. This missing value was recoded as an incorrect answer to the knowledge item, since the participant could not correctly answer the question in the allotted time. We used an analysis of variance (ANOVA) model with and the Tukey honestly significant difference to test for statistically significant differences (with α=.05) in mean knowledge between the CoVideo, APC, and do-nothing arms. The ANOVA model is jC denote the number of items that the jth participant selected from the control list , and let jT be the number of items that the jth participant selected from the treatment list . We calculated the mean score for the control list, denoted by  and treatment list, denoted by , for the ith list experiment . Let the superscripts cov denote the CoVideo, apc denote the APC, and no denote the do-nothing arms, and let k denote the kth trial arm . For list experiment i and trial arm k, we then estimated the prevalence of behavioral intent, denoted by ikP, as the difference between the treatment and control, such that ikP= (ik – CikT) × 100. From these estimates, we calculated the total, content, and attention effect of the CoVideo. Let iTotD denote the total effect, which is estimated by icovP – inoP; and let iAttD denote the attention effect, which is estimated by iapcP – inoP. These analyses are analogous to difference-in-difference analyses, which we implemented by specifying the main and interaction terms in an ordinary least squares (OLS) regression model. The OLS equation for the ith list experiment is given as:For the second endpoint, we calculated the prevalence of behavioral intent to perform COVID-19 preventive behaviors for each list experiment. Let y is the number of statements in the list that the participant agreed with, VideoArm indicates the kth arm, and TreatList indicates assignment to the treatment or control list. We calculated standard errors, 95% CIs, and P values (with α=.05) for linear combinations of coefficients from the OLS model.where All participants underwent a process of informed consent on the Prolific platform. The consent form explained the purpose of the study, the risks and benefits of the research, and how to contact the study investigators (or the Stanford University ethics review board). By clicking the link, participants agreed to participate in our study, and were redirected to the Gorilla platform, where additional information was given. Participants could withdraw from the study at recruitment or at any point during the experiment.Each participant was assigned a unique, anonymized ID on Prolific and had no identifying information associated with it. We informed participants that their names could be revealed to us if they emailed the study investigators. The study investigators kept this information confidential.Because Prolific handled the interaction between the study investigators and participants, the participants were completely anonymous to the study investigators. Participants self-responded to the survey questions and self-submitted their responses anonymously on the Gorilla platform. Only the participant’s unique, anonymized ID was used to manage the linking between the Prolific and Gorilla platforms. The study investigators were blinded to the group allocation .No adverse events or harms were observed given the online format of the trial.The data that support the findings of this study are available from the corresponding author upon request.Between May 13, 2020, and June 23, 2020, 15,163 participants from the United States, Mexico, the United Kingdom, Germany, and Spain were enrolled in our RCT. Between recruitment and randomization, 171 participants were lost and 14,992 participants were randomly assigned to the CoVideo (n=4940), APC (n=4954), and do-nothing (n=5081) arms . After rThe majority of participants reported their residence in the United Kingdom  or the United States , and 84.9%  of participants reported English as their first language. The sample was relatively well educated, with 81.6%  having some college education or higher . P=.002), which represents an increase of 7.6% (0.09/1.14) (P=.06), which represents an increase of 5.3% (0.06/1.11). The knowledge questionnaire had an acceptable reliability correlation coefficient of 0.65 . Overall, there was extraordinarily high attainment of COVID-19 knowledge. In the do-nothing arm, participants correctly answered 16.86 (95% CI 16.83-16.90) out of 18 items, which is a 93.7% correct response rate . With th09/1.14) . The aveP values  × 100 = 17.2, as shown in  × 100 = (2.33–2.03) × 100 = 29.4. For our secondary outcome, we report that participants in the CoVideo arm had lower behavioral intent to go out with friends when compared with the APC  and do-nothing  arms.Figure S1 in P values . Scores In this study, we tested an intervention with several innovations in global health communication that catalyzed a broad, organic global reach on social media ,8-10. ThTo evaluate the effectiveness of the CoVideo on knowledge and behavioral intent toward COVID-19 prevention, we used a large, online RCT to enroll 15,163 participants from the United States, Spain, Germany, the United Kingdom, and Mexico. The results for our first endpoint showed high knowledge of COVID-19 prevention behaviors across the five countries. For the three trial arms, the average number of correct answers was nearly 17 out of 18 items, a correct response rate of approximately 94%. Moderate to high knowledge levels about COVID-19 prevention measures among the general public were also observed earlier in Ecuador  and the An important study finding was that the CoVideo improved already high levels of COVID-19 prevention knowledge. In the do-nothing and APC arms, only 1.14 and 1.11 additional correct items were needed to reach a perfect (100%) score, respectively. Our results showed that the CoVideo boosted COVID-19 prevention knowledge by another 7.6% relative to the do-nothing ceiling and by 5.3% relative to the APC ceiling. It seems plausible, therefore, that the CoVideo could significantly improve COVID-19 prevention knowledge in countries where baseline knowledge levels are currently low or moderate.For our second endpoint, we nested a list experiment in each trial arm to evaluate the effect of the CoVideo on self-reported behavioral intent toward COVID-19 prevention. We used this experimental approach because it is likely that participants (at the time of enrollment) were already primed to give socially desirable responses to questions about COVID-19 prevention. The indirect questions  provide protection to participants who have no behavioral intent toward COVID-19 prevention, without revealing this intention directly . Our resOur study is innovative in its use of both a list experiment and an APC video. Our APC video was selected to account for the possible attention effects elicited by the CoVideo intervention. The APC was designed to mimic the inactive components of the CoVideo intervention (the effect of watching a video of the same length), while not containing the active intervention component  . The APCOur study had several limitations. At the time of our study, no validated scale on COVID-19 knowledge prevention existed. Nevertheless, we used best practices from the survey methods field to inform the design and development of the knowledge questions . AnotherTogether, the findings of this study present innovative propositions for content design, dissemination, and evaluation of rapid global health communication interventions. Content designs that emphasize cultural accessibility, convey a compelling narrative, and elicit high-arousal emotions could fuel rapid dissemination across the 3.8 billion global citizens currently using social media. The wordless, animated approach also minimizes barriers traditionally associated with underlying differences in language and literacy levels. Given the massive global penetration of social media, short, animated, wordless video messages, designed to spread organically, may help public health authorities reach people where they are . Evaluating these interventions using online trials, APCs and list experiments can help expedite results and strengthen our efficacy evaluations. The value of such an approach becomes especially apparent during global crises in which lost weeks translate into lost lives. Accessible and compelling video health messages that lean on the shared characteristics of our global community could facilitate the spread of time-sensitive health messages. Public health authorities poised to implement these innovative health communication solutions could better support a global community facing unprecedented, shared challenges.AV, MA, and VH wrote the paper. AV and CF undertook the statistical analysis. MA designed, produced, and created the CoVideo. TB, AV, and MA designed the trial. AV, TB, MA, and MG contributed to the questionnaire development. All authors provided comments and feedback.


Bone metastases induce pain, risk of fracture, and neural compression, and reduced mobility and quality of life. Electrochemotherapy (ECT) is a minimally invasive local treatment based on a high-voltage electric pulse combined with an anticancer drug. Preclinical and clinical studies have supported the use of ECT in patients with metastatic bone disease, demonstrating that it does not damage the mineral structure of the bone and its regenerative capacity, and that is feasible and efficient for the treatment of bone metastases. Since 2009, 88 patients with bone metastasis have received ECT at the Rizzoli Institute. 2014 saw the start of a registry of patients with bone metastases treated with ECT, whose data are recorded in a shared database. We share the Rizzoli Institute experience of 38 patients treated with ECT for a bone metastasis, excluding patients not included in the registry (before 2014) and those treated with bone fixation. Mean follow-up was 2 months (1–52). Response to treatment using RECIST criteria was 29% objective responses, 59% stable disease, and 16% progressive disease. Using PERCIST, the response was 36% OR, 14% SD, and 50% PD with no significant differences between the two criteria. A significant decrease in pain and better quality of life was observed at FU. According to a Global Cancer Observatory (GLOBOCAN) 2020 publication, cancer is one of the leading death causes worldwide and the most significant disease burden globally . In 2019Although this scenario could be perceived as discouraging, since 1970, with the implementation of chemotherapy as adjuvant treatment along with radiotherapy and surgical resection, significant steps towards disease control have occurred. However, because of survival times prolonging and cancer rates increasing due to demographic changes, accumulative cancer cases are rising . This coMany complications that patients with cancer might experience and compromise their life quality arise from bone metastasis . SymptomStandard local treatments include radiation therapy, surgery, embolization, focused ultrasound treatment, or cryotherapy ,8,9,10. http://reinbone.wng.it accessed on 15 February 2022) protected by security passwords.Since 2009, 88 patients with bone metastasis have received ECT at the Rizzoli Institute. In 2014, a registry of patients with bone metastases treated with ECT was started. Since then, all patient data are recorded in a shared database  Protocollo Generale 0028598, 25 August 2014). Inclusion criteria for ECT treatment were as follows: age >18 years, and histologically proven involvement of appendicular or axial skeleton by metastatic carcinoma or melanoma. Exclusion criteria were as follows: coagulation disorders, severe pulmonary edema or fibrosis, pregnancy or lactation, known allergy to Bleomycin or cumulative dose exceeding 400,000 IU, and chronic renal dysfunction. For this paper, 18 patients undergoing fixation after ECT were excluded; also, one patient died prior to achieving the intended length of follow-up.Prior to data gathering, ethics committee and data protection authority approval was requested. Then, for the remaining group of 38 patients, data concerning age, diagnosis, previous treatments, metastases localization, ECT parameters, and clinical outcome were recorded. Imaging evaluation to assess tumoral morphology included conventional X-ray and MRI, CT with contrast enhancement, or FDG-PET scans.® VITAE . Hardware included trocar-type electrodes of diameter 1.8 mm. Length selection was according to localization, morphology, and dept of the metastasis. Available electrodes had a total length to choose between 12 to 20 cm composed of two parts, one insulated for soft tissue protection and the other considered the “active part” non-insulated, which might be 30 or 40 mm. Electrode diameter, length, and positioning were selected according to careful pre-operative planning to accomplish configuration requirements for effective electroporation.The electroporation system used was the CliniporatorUsing diagnostic radiological images, the lesion to be treated was modeled using specific PULSAR planning software . Treatment planning was done considering the size and location of individual tumors, with respect to major blood vessels and preferred electrode insertion geometry, so that the number and the geometric distribution of the electrodes, their distances, pairs of electrodes for the pulse the delivery and the pulses for each pair of electrodes were calculated accurately verified. This procedure has made it possible to determine the optimal positioning of the electrodes to ensure complete and homogeneous electroporation of the tumor mass.ECT procedure was carried out following ESOPE guidelines . Eight pElectrochemotherapy requires covering the entire tumor with a pulsed electric field, the distribution of which depends on the biology of the treated tissues.The Cliniporator calculates the most appropriate electric field to achieve electroporation in all of the treated tissues; after the delivery of the current, the Cliniporator communicates if electric field between the pairs of electrodes was effective in inducing the electroporation of the tissue crossed by the current; if not, the machine recalculates the parameters of the current, and the electrical supply with the new parameters is then repeated. In the case of particularly complex or highly uneven lesions, pre-treatment planning can be a good solution to optimize the procedure.2 of body surface .Bleomycin was the selected chemotherapeutic with a dosage of 15 mg/mAfter patients underwent peripheral blocking or general anesthesia, electrodes were positioned with imaging assistance, using either fluoroscopy or CT scan. Then, intravenous Bleomycin bolus was administrated, and 8 min were waited to allow drug distribution. Next, electroporation was immediately performed to meet the 30-min time-frame limit after Bleomycin injection to be efficacious.Evaluation of clinical and radiological response was performed at two timeframes, the first one within 60 to 90 days after ECT and the second one, among surviving patients, at 6 months. For clinical evaluation, quantifying tests were submitted to analyze data. The Visual Numeric Scale was used to evaluate pain , while tp value have been reported. Data were statistically analyzed using the Mann–Whitney U test used for nonparametric analyses. Statistical significance was defined as p\0.05. All analyses were performed with IBM SPSS version 21.0 .Continuous variables were described by median value and range, mean and standard deviation. Categorical variables were described by absolute number and percentage. The relationship between each criterion of response was assessed using a Chi-square test or, where appropriate, Fischer exact test. Logistic univariate analysis was performed to identify clinical or instrumental variables that could influence the objective response rate among those registered in the database; relative risk (RR) and A total of 38 patients met our inclusion criteria. The median age of the 38 patients was 59 years (range 41–91 years) and the median time since diagnosis of the primary tumor was 49 months (range 0–226 months). Thirty-seven patients underwent a single ECT session, while one underwent two ECT sessions, for a total amount of 39 ECT sessions. According to tumor size and geometry, the number of needles used for each session varied from 3 to 11. For correct electrode positioning, CT scan guidance was used in 16 cases and fluoroscopy in 22. Detailed information regarding ECT sessions and treated lesions are reported in The most treated sites were the long bones of the limbs and the pelvis. Treatment with variable-geometry electrodes requires keeping the active tips of the electrodes parallel to each other, and this limits the use of ECT in the vertebral sites, for which it is necessary to insert the electrodes trans-peduncularly. However, in 2019, Cornelis et al. [The total procedure lasted from 20 to 80 min (median 40 min) depending on the size of the target lesion. The median follow-up time was 2.2 months (range 1–52 months) with a mean value of 6.6 ± 4.9 months. One out of 38 patients died before the first follow-up due to the rapid worsening of the systemic conditions, and was excluded. Therefore, follow-up was available for 37 patients, and 15 of them (39%) achieved a follow-up longer than three months with an average of 12.3 ± 14.5 months.One patient presented early skin necrosis with bone exposure after the treatment, requiring amputation. The patient was a 91-year-old suffering from squamous cell carcinoma of the forearm. He underwent mass excision and local flap coverage. He was referred due to a further recurrence with tumor invasion of the proximal 2/3 of the ulna. Given the extent of the lesion, arm amputation was proposed. The patient refused the amputation, so ECT was performed with custom geometry electrodes to treat the skin tumor and its extension into the bone. However, treatment-induced necrosis caused bone exposure, eventually requiring amputation.The response to treatment is summarized in Overall, the response to treatment by RECIST criteria comprised nine objective responses (25%), three complete responses (9%), and six partial (16%). Twenty-two patients had stable disease (59%) and six a progressive disease (16%).p = 0.44). The univariate logistic model revealed that gender, age, histology, number of metastases, and fractures did not influence the outcome. Only ECOG 0–1 is a predictor of objective response in this cohort of patients (p < 0.0001). Analyzing the radiological result based on the type of lesion , of the 29 lytic lesions, three had a CR, five a PR, 16 SD and four PD . The sclerotic lesion remained radiologically stable; of the eight mixed lesions, one showed a partial radiological response, five remained stable and two progressed. Twenty-three of the 29 patients with osteolytic lesion were taking anti-resorption drugs. The radiological outcome of these patients was not considered to be affected by this therapy, because it has been ongoing for many months prior to ECT treatment. Regarding pain intensity before ECT, four patients (11%) reported no pain, three patients (8%) had mild pain, 18 (47%) moderate, and 13 (34%) had severe pain, with a median value of 6 ± 2.7. All four patients in whom ECT treatment was performed in the absence of pain had a lytic lesion at the femoral neck, a site where, in the event of growth of the lesion, the risk of pathological fracture increases with subsequent indication for surgical hip replacement. Treatment in these cases was therefore performed to prevent major complications with minimally invasive treatment. Additionally, the analysis of pain management in these patients revealed that before ECT, four patients (11%) did not require any painkillers because they had no pain. Six patients (16%) sometimes used analgesics, another six (16%) continuously used non-opioids drugs, 18 (47%) required opioids, and three (8%) had uncontrolled pain (one unknown).According to PERCIST criteria, 13 objective responses (36%) were recorded, of which five were complete (14%) and eight partial (22%). Five patients had stable disease (14%) and 19 a progressive disease (50%). No significant differences were observed between the two criteria . Ten patients did not experience any change in their pain status (including the four patients with no pain before ECT), and two experienced worsening pain.p = 0.0049).To summarize pain response, 17 out of 37 patients (45%) reduced painkillers after the ECT with a significant improvement of pharmacologic pain management .A significant decrease in pain intensity was observed at the mean Visual Numeric Scale (VNS) after ECT at early (2 ± 2.7) and late (2 ± 2.6) follow-up visits concerning pre-ECT values , bone quality improved in two, and the other two remain without changes. Additionally, among the eight patients with stable or worsened pain, three had no X-ray changes, and five displayed disease progression .Twelve patients performed local radiation therapy one year before ECT at least (1 ± 0.8 years). One patient resulted in CR, three PR, seven SD and one PD. Eight out of 12 patients improve their performance status.p = 0.0450). However, although not significantly, it is clear that the quality-of-life condition improved at follow-up compared with the pre-ECT status. The univariate logistic model revealed that ECOG values 0–1 were significantly associated with a higher objective response rate (p < 0.0001).Finally, there are no significant trends in EQ-5D items, probably due to the low number of cases, except for the pain item .Surgery provides structural stability, but may be technically demanding and associated with prolonged recovery.Radiotherapy (RT) is the most commonly used therapy to locally deal with bone metastases, with estimated rates of pain relief reported in the literature between 50% to 80%. Conventional external body RT is considered a non-invasive treatment but has complications inherent to tissue toxicity. Fibrosis, bone necrosis, or vascular insufficiency are somehow present to a certain extent in areas subjected to RT. This toxicity can potentially limit repetitive RT, often for 20% of patients under single-fraction and 8% of multi-fraction regimens. Additionally, it could compromise wound healing when future surgical procedures are required ,25. To mTo avoid RT side effects, other more recent technologies have been used. Regarding cryoablation, Callstrom et al.  reportedDupuy et al.  suggesteThe common endpoint to all ablation techniques is the induction of the largest possible thermal necrosis of the target lesion to destroy periosteal nociceptors and reduce cancer size. There is no current evidence favoring the use of one ablation technique over another. In other hands, those techniques lead to a physical necrosis of the tumor cells, while ECT provides a chemical necrosis: in 2010, a study conducted by Fini et al.  demonstrECT does not induce bone necrosis, and therefore, in the case of fracture of the ECT-treated bone, healing is possible with usual fracture callus quality and healing time . BetweenPain is one of the most relevant quality-of-life indicators in patients who undergo palliative treatments. Pain relief was achieved in 68% of patients, and painkiller usage decreased in 45% of them. Our results show a significant pain reduction after ECT in more than half of patients at early follow-up. Moreover, we observed a reduction in pain even in the absence of an objective radiological response.However, if we consider the radiological response (RECIST and PERCIST) by itself, it seems that the objective response to ECT is not very favorable. Nevertheless, among patients with pain improvement after ECT and those without pain before treatment (29 patients), the real radiological progression of disease was present in only one patient. Furthermore, objective changes in control imaging showed stable metastases behavior in 19 cases and improvement  in nine.Complications related to ECT are usually few, predictable, and avoidable. In our patient cohort, complications associated with ECT were reported in only one patient, who developed tissue necrosis. The risk of skin necrosis and ulceration with further bone exposure in patients with poor soft tissue quality or previously irradiated areas was already described ,32. ThisTo conclude, ECT offers the possibility to use chemotherapy, a systemic treatment, in a localized fashion. Although it is not a complication-free treatment, drawbacks can be substantially decreased with meticulous pre-operative planning and careful case selection. In addition, conversely from other local treatments for bone metastases, ECT spares mineralized bone structure from irreversible damage, enabling bone healing. Finally, ECT is a valuable tool to alleviate pain and improve the quality of life for those patients facing the bone metastases burden.


T1 was a control without any filter bedding material, whereas rice straw, rice husk, coconut husk, and charcoal were used as filter bedding materials in the T2, T3, T4, and T5 treatment systems, respectively. Each treatment showed significant treatment efficiency wherein T3 had the highest removal efficiency of 76.21% followed by T4 (67.57%), T5 (65.18%), T2 (46.46%), and T1 (45.5%). T3 and T4 also showed higher salt accumulation, such as sodium (Na) and potassium (K). Further, the pollution load in terms of TDS and chemical and biological oxygen demand significantly reduced from leachate in the T3 and T4 treatments in comparison with other treatments. Parameters of the soil, such as electrical conductivity, exchangeable sodium percentage, and cation exchange capacity did not show values corresponding to high salinity or sodic soils, and therefore, no adverse impact on soil was observed in the present study. Also, Eucalyptus camaldulensis plant species showed good response to wastewater treatment in terms of growth parameters, such as root/shoot weight and nitrogen, phosphorus, and potassium (NPK) uptake, plant height, biomass, and chlorophyll content. Root and shoot dry weight were in the order T3 (51.2 and 44.6 g)>T4 (49.3 and 43.5 g) > T5 (47.6 and 40.5 g) > T2 (46.9 and 38.2 g) > T1 (45.6 and 37.1 g). Likewise, the total chlorophyll content was highest in T3 (12.6 μg/g) followed by T4 (12.3 μg/g), T5 (11.9 μg/g), T2 (11.5 μg/g), and the control, that is, T1 (11.0 μg/g). However, the most promising results were obtained for T3 and T4 treatments in comparison with the control (T1), which implies that, among all organic raw materials, coconut and rice husks showed the highest potential for salt accumulation and thereby wastewater treatment. Conclusively, the findings of the study suggest that organic raw material–based amendments are useful in managing the high salts levels in both plants and leachates.The present study focuses on determining the phyto-treatment efficiency for treatment of moderately saline wastewater using organic raw materials, such as rice husk, coconut husk, rice straw, and charcoal. The moderately saline wastewater with total dissolved solids (TDS) concentration up to 6143.33 ± 5.77 mg/L was applied to the lysimeters at the rate of 200 m Rapid industrialization and population growth has intensified the generation of a huge amount of waste and other pollutants in the environment . The preTo manage the enormous volumes of wastewater, several innovative technologies have been deployed globally for its recycling, treatment, and reuse. However, the amount generated is far more than the amount that is treated. For instance, India generates approximately 38,000 million liters per day (MLD) of wastewater, whereas the sewage treatment capacity of the whole country is limited to only 12,000 MLD . In factBrassica campestris. The regulatory authorities in India, for instance, the Central Pollution Control Board, also issue guidelines for disposal of wastewater under land-based systems for achieving zero discharge , and directly impacts soil fertility, seed germination, and crop growth and productivity . Furtherischarge . In the ischarge . A revieLand-based treatments are employed using plant species having a high salt tolerance capacity. Eucalyptus genus is one such potential plant species, which has been widely employed for different activities such as afforestation, social forestry, wastewater treatment, and carbon sequestration . NumerouMoreover, Eucalyptus camaldulensis, and examining the changes in physicochemical and nutrient loads in plants and soil irrigated with saline wastewater.The hypothesis of the study included the treatment strategy of saline wastewater having high TDS (up to 6000 mg/L) as proposed in the Food and Agriculture Organization (FAO) guidelines. According to the FAO, water having TDS up to 2000 mg/L are put under severe restriction use as per the guidelines for water quality for irrigation as the salt accumulates in the crop root zone to a concentration that causes a loss in yield. As a result, it was a mandate for all the industries to treat and discharge the wastewater for irrigation purposes only after meeting the desired criteria defined. Industries mostly release wastewater through reverse osmosis reject having TDS in the range up to 2500–3500 mg/L, which is higher than the prescribed limits and, therefore, face problems in treatment and disposal. Treatment of wastewater to get the desired value using expensive and sophisticated treatment systems was itself a difficult task considering the economic viability. In a similar context, not much attention has been paid globally to the role of natural treatment systems involving forestry plant species to study the impact of saline wastewater irrigation (having higher TDS than prescribed guidelines) other than agricultural crops. Therefore, the present study focused on studying the impact of industrial wastewater with TDS up to 6000 mg/L on forestry species, Eucalyptus camaldulensis plant species. In this study, the effect of wastewater on plant growth and the role of FBM in TDS management were continuously monitored. The novelty lies in the treatment of wastewater having very high TDS levels by using a plant combination of FBM, which is a low-cost simple treatment system, and such a system is desirable in developing countries, not only from the point of view of cost, but also in acknowledgment of the difficulty of the reliability of operating potentials of the present complex treatment systems. Most studies  collected from a common effluent treatment plant (CETP) has been treated using lysimeters aided with low-cost organic raw material, such as rice husk, rice straw, coconut husk, and charcoal, as filter bedding materials (FBM) planted with  studies  report t sensors . The Euc sensors . FurtherThe study was conducted at CSIR-NEERI, Nagpur, Maharashtra, under field conditions from August 2020 to February 2021. The study area belongs to the agro-ecoregion of Maharashtra, where the climate is characterized by hot summers and mild winters. The mean annual precipitation ranges between 600 and 1000 mm, which covers about 40% of the annual potential evapotranspiration in the range of 1600 to 1800 mm. The moisture availability in the growing period ranges from 90 to 150 days. The common soilscapes in the region are ustorthents, ustropepts, and chromusterts.2), magnesium chloride (MgCl2), and potassium dihydrogen phosphate (KH2PO4). The salts were dried at 100°C for 2 h and mixed in a homogenizer to get a salt mixture. The salt-amended treated wastewater  was stored at 4°C as per the standard method prescribed in Treated effluent was collected from the CETP, Butibori, Nagpur, Maharashtra. The wastewater was amended with a mixture of anhydrous salts, such as potassium chloride (KCl), sodium chloride (NaCl), calcium chloride , marble chips (10–15 mm), black cotton soil, and plant-derived FBMs, which included coconut husk, rice husk, rice straw, and charcoal.The experimental setup included five treatments (four plus one control) as shown in Eucalyptus camaldulensis, was planted in each lysimeter  was placed, followed by sand. All lysimeters were rinsed with distilled water and filled with a layer of a mixture of sand (10–15 mm) and marble chips (<2 mm). Black cotton soil (up to 55 cm) filled each of the columns above the sand/marble chip layer. A layer of different FBMs (30 cm) followed by a topsoil layer (10 cm) was made in each of the lysimeters. A salt-tolerant plant species, ysimeter  and alloEucalyptus camaldulensis + soil (T1); soil + rice straw + Eucalyptus camaldulensis (T2); soil + rice husk+ Eucalyptus camaldulensis (T3); soil+ coconut husk+ Eucalyptus camaldulensis (T4), and soil + charcoal + Eucalyptus camaldulensis (T5). Each treatment has triplicate replications with a constant depth of 30 cm for FBM. Results obtained for physical characteristics of FBM are shown in The five treatments designed in this study were as follows; control without FBM + Different physicochemical characteristics, such as color, electrical conductivity (EC), pH, total hardness, TDS, BOD, COD, alkalinity, sulfate, sodium, potassium, calcium, magnesium, nitrates, ammonia, and heavy metals were analyzed according to standard protocol . pH and EC of wastewater was determined by a pH and EC meter (Hach Model – VSI = 06) as described by +, K+, Ca2+, and Mg2+ ion concentrations were determined by flame photometry, and the heavy metals were analyzed by ICP-OES  as described by Jackson. (1973). Na ICP-OES . The met ICP-OES . Organic ICP-OES  and the  ICP-OES . Total p ICP-OES .Eucalyptus camaldulensis of height 30 and 8 cm root length were planted in the lysimeters. During the period of acclimatization, each lysimeter was initially fed with tap water for 15 days. After that, moderately saline wastewater was fed to each lysimeter at a rate of 200 m3 day–1 ha–1 . The hydraulic loading rate can be calculated using the following equation , root length, number of shoots, fresh and dry weight, chlorophyll content, total Kjeldahl nitrogen, heavy metals, and uptake of salts in terms of calcium, magnesium, sodium, potassium, and phosphorus, respectively. Height measurements were taken manually. Dry weight was determined by oven drying at 70°C for 48 h .The data was analyzed statistically using one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test. The test was performed for the parameters of soil and wastewater quality before and after treatment to assess the efficiency of FBM in salt removal in between the different treatments and the treatment efficiency and the associated plant growth. The significant differences were tested at 95% confidence level (+) ions and bicarbonates (HCO3–) were found to be 1170 ± 1.04 and 433.43 ± 4.62 mg/L, respectively. Concentration of the potassium (K+) divalent cations, calcium (Ca2+), magnesium (Mg2+), and SAR was found to be 95.14 ± 2.41, 2305.74 ± 15.51, and 1237.12 ± 10.24 mg/L and 2.21, respectively. Heavy metal concentrations were negligible. The characterization of amended wastewater implied that it belonged to the “severe restriction for use” category in irrigation as it had higher values of parameters such as EC, TDS, and SAR . Heavy metals were not detected in the leachate because the initial concentrations were very low. The changes in various physicochemical properties of leachate during the period of study are shown in The physicochemical parameters of the leachate are given in –3, but after the treatments, it was found to be in the range of 1.21–1.36 g cm–3. The reduction of bulk density was observed to be in the range of 3.50–15.38%. Similarly, maximum water-holding capacity and porosity increased in soil after treatments as compared with controls and were in the range of 56.29–63.29 and 65.25–68.07%, respectively. Similar trends were also reported by The physicochemical properties of the soil after treatment are shown in +, K+, Ca2+, and Mg2+ were observed to be 0.19, 0.99, 4.31, and 6.36 Cmol+/kg, respectively. It was observed that there was a decrease in the concentration of the exchangeable cations in soil after the application of saline wastewater as shown in The initial values of exchangeable cations of soil Na–1) in all treatments after wastewater application increased, the saline conditions were not developed. EC in the present study was in the range of 0–2 dSm–1 and show no impact of saline wastewater to the soil and, therefore, is classified as nonsaline  > (T4) > (T5) > (T2) > Control (T1).Though the EC  was the least, and therefore, there was the highest leaching of salts in percolate, which was collected from the control treatment. The other treatments, which had FBM, showed significant salt reduction and thereby lower TDS levels in the percolate (leachate) as shown in Various mechanisms are involved in the adsorption of various pollutants using natural adsorbents. Coconut husk and coconut coir consist of hollow internal conduits, xylem and phloem, where xylem facilitates the uptake of water and phloem serves the purpose of transport of dissolved solids into the plant vacuoles, pits, voids, and lacunae or lumina . SimilarThe larger specific surface area and higher water-holding capacity in coconut husk, rice husk, charcoal, and rice straw facilitate a higher percentage absorption of inorganic salts and other pollutants from the wastewater. Hence, the adsorption capacity of the pollutants is determined by the surface area and porous structure of the adsorbents along the chemical interactions between the functional groups of adsorbents and inorganic ions present in the wastewater. Therefore, the higher removal of TDS in this study may be due to the adsorption–desorption mechanism due to physical interactions at a dynamic equilibrium . The varSignificant variations were observed in pH and other parameters among all treatments. Consecutively, negligible concentrations of phosphate, sulfate, nitrate, ammonia, BOD, and COD were observed in the treatments containing rice and coconut husk leachate, making the systems’ efficiency range between 65 and 76% and justifies the role of FBM in management of TDS as compared with control and other treatments. FBMs such as rice straw and charcoal also show significant results but had lower efficiencies as compared with rice and coconut husk.In the context of degradability of the FBM, coconut husk, rice husk, and rice straw contain high lignocellulose biomass, which consists of a complex structure of cellulose, hemicellulose, and lignin. These complex compounds are not easily degradable and, therefore, take longer for degradation by microorganisms . SimilarEucalyptus camaldulensis showed good response to wastewater treatment having high TDS in terms of growth, chlorophyll content, root/shoot weight, and uptake of sodium and potassium and other nutrients. In addition, the increase in plant height and significant nutrient uptake showed the salt-tolerance capacity of Eucalyptus camaldulensis. Changes in the growth parameter of the plant during the period of study are shown in p > 0.05) except for T1, which has the least value. The total chlorophyll content was highest in T3 (12.6 mg/L) followed by T4 (12.3 mg/L), T5 (11.9 mg/L), T2 (11.5 mg/L), and control, that is, T1 (11.0 mg/L). Our results were compared with the study of The height and the root length of the plant were 30 and 8 cm at the start of the experiment. Enhanced heights were obtained at the end and were highest in T3 and T4 treatments. The average percentage increase in plant height was 194; 39%, where the highest increase was in T4 (213.6%) followed by T3 (212.09%), T5 (182.5%), T2 (197.6%), and T1 (166%). Likewise, a similar trend of growth rate was observed in root length. Percentage increase in root length was as follows: 342% (T4), 328% (T3), 266% (T2), 247% (T5), and 241% (T1). Similar results are also reported by Eucalyptus camaldulensis is also shown in the study of Eucalyptus camaldulensis. The plant irrigated with wastewater showed increased stem height, biomass, leaf count, and dry weights. Nutrient accumulation was also higher in wastewater-irrigated plants. Similar results are also found in the present study. Another study concludes that Eucalyptus shows better performance in terms of survival, biomass, and growth among different plant species when irrigated with saline wastewater -based treatment of wastewater. One such study by  species . Such en+/EC/TDS. However, further studies are needed that can emphasize the use of a proper combination of FBM for different wastewater treatment by extending the knowledge generated in this study. In this study, Eucalyptus plants accumulated Na, Ca, Mg, and K and higher biomass with no adverse impact on the growth as a function of wastewater, which indicates that sal- tolerant plant species can be utilized in the treatment of wastewater. The absolute combination of forestry plant species and organic raw material showed the feasibility of treatment of wastewater with very high TDS (up to 600 mg/L). The study can be further extended to other salt-tolerant plant species, such as Prosopis, Acacia, Casuarina, etc. in the field conditions in combination with other organic raw materials to further enhance the treatment and TDS management efficiency of the lysimeters or land-based treatment systems. Such approach can also be applied to restore the productivity of degraded lands by saline wastewater irrigation. Furthermore, utilization of organic raw materials such as coconut husk, rice husk, and rice straw as FBMs for saline wastewater management might offer socioeconomic solutions and sustainable management options of these organic residues, which are generated in huge quantities and have various environmental implications.Based upon outcomes of the study, it can be concluded that the relative capacity of rice and coconut husk as bedding material for TDS management is highest among all other treatments. However, other bio-adsorbents, such as rice straw and charcoal, can also be effectively utilized for treatment purposes based on their local availability. The salt reduction from wastewater was also very high (60–70%) because of the adsorption of salts on FBM. Consequently, about 80–88% of total leachate volume reduction was obtained in the treatments. Therefore, this approach can efficiently be modified to a large scale, such as constructed wetland, to fulfill the regulatory need of zero discharge from industries. The soil after application showed no saline or sodic conditions because of the presence of FBM in treatment systems, thereby maintaining the physicochemical and biological integrity of the soil. Therefore, it was inferred that FBM not only plays a significant role in management of TDS but also favors plant growth. The plant growth data confirms the role of FBM asa buffer not allowing the roots to experience toxicity due to NaThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.DM has prepared the first draft of the manuscript, performed the experiments, and compiled the results, and also performed data interpretation. KR helped in the finalization of data and data interpretation and final draft preparation. AS reviewed the manuscript for grammatical and language errors and helped in preparation of graphs and performed the statistical interpretations. KK and PT designed the experiments, performed data analysis, and provided editorial advice in the manuscript and supervised the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Phenylpropanoids are specialized metabolites playing crucial roles in plant developmental processes and in plant defense towards pathogens. The attachment of sugar moieties to these small hydrophobic molecules renders them more hydrophilic and increases their solubility. The UDP-glycosyltransferase 72 family (UGT72) of plants has been shown to glycosylate mainly two classes of phenylpropanoids, (i) the monolignols that are the building blocks of lignin, the second most abundant polymer after cellulose, and (ii) the flavonoids, which play determinant roles in plant interactions with other organisms and in response to stress. The purpose of this review is to bring an overview of the current knowledge of the UGT72 family and to highlight its role in the homeostasis of these molecules. Potential applications in pharmacology and in wood, paper pulp, and bioethanol production are given within the perspectives.Plants have developed the capacity to produce a diversified range of specialized metabolites. The glycosylation of those metabolites potentially decreases their toxicity while increasing their stability and their solubility, modifying their transport and their storage. The UGT, forming the largest glycosyltransferase superfamily in plants, combine enzymes that glycosylate mainly hormones and phenylpropanoids by using UDP-sugar as a sugar donor. Particularly, members of the UGT72 family have been shown to glycosylate the monolignols and the flavonoids, thereby being involved in their homeostasis. First, we explore primitive UGTs in algae and liverworts that are related to the angiosperm UGT72 family and their role in flavonoid homeostasis. Second, we describe the role of several UGT72s glycosylating monolignols, some of which have been associated with lignification. In addition, the role of other UGT72 members that glycosylate flavonoids and are involved in the development and/or stress response is depicted. Finally, the importance to explore the subcellular localization of UGTs to study their roles in planta is discussed. In order to adapt to terrestrial environments and biotic interactions, plants have developed the capacity to produce a wide range of specialized compounds. These compounds can undergo various chemical modifications, such as hydroxylation, methylation, or glycosylation. This capacity has been allowed by the diversification of enzymes able to add new functional groups to molecules. Among these modifications, the glycosylation reaction can occur on –OH, –COOH, –NH2, –SH, and C–C groups in diverse molecules such as proteins, carbohydrates, primary and specialized metabolites, as well as xenobiotics . GlycosyGT genes in each GT superfamily differs among organisms in function of the peculiarities of their metabolism . Be. Be11]. ric acid . Alternatyrosine . During royl-CoA ,17,18, wroyl-CoA . In addiroyl-CoA . All theroyl-CoA . In thatUGTs are classified into families in which members share more than 45% of amino acid sequence identity . HoweverPlant UGTs display a typical GT-B fold which consists of two Rossman fold domains connected by a linker and forming a cleft . The C-tAbout 450 million years ago, embryophytes, deriving from green algae emerged from the aquatic habitat and colonized the terrestrial environment thanks to the acquisition of novel biochemical pathways, phytohormones, and signaling pathways ,42. EspeUGTs increased during evolution, consisting of five in Chlamydomonas reinhardtii to more than 200 in trees such as Pinus taeda (gymnosperm) and Populus trichocarpa (angiosperm). This divergence suggests an early and continuous expansion of the UGT family in plants which may be related to their constant adaptation to the terrestrial environment and biotic interactions by producing and regulating specialized metabolites . Th. ThUGT72P. tremula × P. alba), the overexpression of UGT72AZ1 and UGT72AZ2 triggers the accumulation of coniferin, and the overexpression of UGT72AZ1 causes also the accumulation of syringin (In poplar (syringin . Howeverβ-glucosidase) and nuclear magnetic resonance (NMR) spectroscopy analysis of cell wall and lignin fractions of wood from gymnosperms and angiosperms . Th. ThpUGT7ectively . Anotherand pith . Finallyfication ,89, are ectively .ugt72e1, ugt72e2 or ugt72e3 Arabidopsis mutants do not show any difference in lignin quantity in the floral stem when using the acetyl bromide method, a higher proportion of lignin in the xylem and interfascicular fiber cell walls of the ugt72e3 mutant was evidenced by Raman microspectroscopy and safranin O ratiometric imaging technique, as compared to the wild type [ugt72e3 young stem as compared to the wild type. This phenotype was related to an increased expression of lignin-specific PRX71 and LAC17 [ugt72b1 mutant exhibits a higher lignin content in the whole floral stem, as well as an ectopic lignification in the interfascicular fibers and the pith as compared to the wild type and the rescued line (insertion of the UGT72B1 cDNA under the control of the native gene promoter) [PRX34, PRX37, PRX71, PER64, LAC5, LAC12, LAC15, ABCG29, ABCG40, RBOHA and RBOHD) [UGT72B1 mutation, which depletes the content of monolignol glucosides, may trigger a signal upregulating the genes involved in monolignol biosynthesis, transport, and polymerization, generating an overproduction of monolignols, hence an increased and ectopic lignification. Consequently, the increase in coniferin in the mutant may be a secondary effect of this monolignol overproduction [ugt72b1 mutant [Functional characterization of mutants has evidenced phenotypes to clarify this question. Although single ild type . This dind LAC17 . The ugtromoter) . In addid RBOHD) . The autoduction . The alt1 mutant .UGT72s encoding proteins glycosylating monolignols and/or their precursors were expressed in vascular tissues of stem or roots , when ectopically expressed in tobacco, induces a 60% increase in Klason lignin content in the stem of a 2-month-old plant  was detected in the cytosol in uncatula . The firuncatula . In popluncatula ,102,103.uncatula ,105, andPolygonum tinctorium indoxyl-β-D-glucoside synthase (IGS), a member of the UGT72B sub-family, was localized in both cytosolic and microsomal fractions, suggesting a reversible binding of the protein to the membranes. The ER localization was confirmed using ultracentrifugation with a sucrose density gradient [Pyrus bretschneideri PbUGT72AJ2 fused to GFP was also located mainly in the cytosol and cytomembrane compartment [The gradient . The Pyrpartment . The enzpartment ,109. Asspartment . The chlpartment ,111,112.partment . The subE. coli as an expression host, the significance of several post-translational modifications occurring in plants is not adequately addressed. For instance, various patterns of protein N-glycosylation modified the affinity of human UGT2B7 towards zidovudine and morphine [UGT72E2 and UGT72B1 in Saccharomyces cerevisiae or Schizosaccharomyces pombe engineered with the vanillin pathway, thereby allowing to produce vanillin β-D-glucoside which is less toxic than vanillin [Our knowledge of the glycosyltransferase activity of UGT72s remains limited because the enzymatic assays are performed in vitro and do not reflect the availability of the appropriate substrates in planta. In addition, since most of the recombinant proteins are produced using morphine . Such anvanillin . Otherwivanillin . Chen anvanillin .UGT72E3/2 gene is overexpressed in Arabidopsis plants, there is 145% more syringin in leaves than in the transgenic Arabidopsis overexpressing UGT72E3 [UGT72AM1 is 4.2-fold more expressed in this variety than in the wild type (the ‘Longjing43′ tea variety). As the protein coded by this gene can glycosylate in vitro kaempferol, quercetin, myricetin, naringenin, eriodictyol, and cyanidin, its higher expression may be linked to the higher content of glycosylated flavonoids in the ‘Zijuan’ variety [The glycosyltransferase activity of UGT72s against monolignols and flavonoids opens avenues across numerous applications. The glycosylation of these compounds is extensively studied in the food and pharmacology sectors, because the biological functionalities of small molecules may be enhanced by increasing their hydrophilicity and stability through glycosylation . For ins UGT72E3 . Flavono UGT72E3 . The pur UGT72E3 . Molecul variety ,76.The monolignol biosynthesis pathway and the impact of its modification on biomass recalcitrance is extensively studied to improve cellulose extraction for the production of paper pulp and bioethanol. Actually, lignin is the main limiting factor in these sectors because it restricts polysaccharides’ accessibility and inhibits enzymatic activity. Reducing the lignin content in the cell wall and/or altering its composition are considered as two strategies to improve pulping and saccharification . The invSubstrates of UGT72 enzymes, using mainly UDP-glucose as sugar donor, cover a wide range of specialized metabolites and xenobiotics.