In the 1920s, Lewis Stadler initiated the introduction of permanent improvements to the genetic makeup of irradiated plants. Since then, studies related to breeding mutations have grown, as efforts have been made to expand and improve crop productivity and quality. Stadler’s discovery began with x-rays on corn and barley and later extended to the use of gamma-rays, thermal, and fast neutrons in crops. Radiation has since been shown to be an effective and unique method for increasing the genetic variability of species, including rice. Numerous systematic reviews have been conducted on the impact of physical mutagens on the production and grain quality of rice in Southeast Asia. However, the existing literature still lacks information on the type of radiation used, the rice planting materials used, the dosage of physical mutagens, and the differences in mutated characteristics. Therefore, this article aims to review existing literature on the use of physical mutagens in rice crops in Southeast Asian countries. Guided by the PRISMA Statement review method, 28 primary studies were identified through a systematic review of the Scopus, Science Direct, Emerald Insight, Multidisciplinary Digital Publishing, and MDPI journal databases published between 2016 and 2020. The results show that 96% of the articles used seeds as planting materials, and 80% of the articles focused on gamma-rays as a source of physical mutagens. The optimal dosage of gamma-rays applied was around 100 to 250 Gy to improve plant development, abiotic stress, biochemical properties, and nutritional and industrial quality of rice. Climate change poses a significant threat to national agricultural production, with climate change fingerprints causing 76 droughts, floods, temperature anomalies, and storms in the last 5 years . Rice, bvia physical and chemical mutagenesis or somaclonal variation. Total of 81.3% (693) of the total rice mutant develop using physical mutagens. The number of rice mutant varieties in the countries of Southeast Asia is shown in To mitigate this issue, it is necessary to develop new rice varieties that can adapt to climate change. One such breeding technique is mutation breeding, which uses physical or chemical mutagens to generate new forms of valuable agricultural production. There are approximately 852 varieties of rice mutants publicly released to farmers  contribuMutation breeding was introduced to obtain the highest mutagenesis efficiency in terms of a maximum number of targeted mutations while conserving plant variety and a low background mutation rate. Few studies have been clarified that several factors influence by the effect of irradiation including the radiation aspect in terms of mutagen dosage, source of mutagen, and duration of exposure , and theth century which was introduced by Stadler  develop tolerance to biotic and abiotic stress after being exposed to Cobalt-60. After a while, PSB RC82 with improved grain quality and tolerance to abiotic stress, as well as a mutant variety of PSB Rc72 was released in 2000. This variety was widely used after it was released because it was high yielding and had good grain quality, similar to IR64  as a guide to retrieve relevant articles on physical mutagens in rice cultivation. Relevant articles were assessed from a specific perspective before being selected as the primary references for exploring the physical mutagens used in rice production in Southeast Asian countries.Oryza sativa”, “rice cultivation” alone or in combination with “OR” and/or “AND”.To conduct the review, publicly available databases such as Scopus, Science Direct, Emerald Insight, and MDPI journal databases were searched. A systematic review was performed using Mesh terms including “induced mutation”, “physical mutagen”, “physical agent”, “mutagen”, “ionizing radiation”, “radiation”, “mutation breeding”, “rice”, “paddy”, “The PRISMA Statement, which comprises a 27-item checklist and a four-flow diagram, was utilized as a guide to retrieve relevant articles on physical mutagens in rice cultivation . The revThe systematic review process was carried out in a progressive manner, starting with the identification stage, followed by screening, eligibility, and inclusion  and ionizing radiations . In Southeast Asia, physical mutagens have been used in rice production to create new cultivars, improve genetic traits, and increase yield potential. The following section provides more details on the specific physical reagents that have been used. Gamma rays are electromagnetic waves with the highest energy and shorter wavelengths that can penetrate deep into biological matter. Analysis of the studies reviewed revealed that gamma rays were used as a source of physical mutagens in 21 out of 28 studies to enhance crop traits. Of these, 13 studies used Cobalt-60, and seven studies used Cesium-137 as the gamma-ray source. The radiation source was not specified in eight of the studies. Cobalt-60, which has a half-life of 5.27 years, is produced by irradiating the stable isotope Cobalt-59 with neutrons in a nuclear reactor . On the Indonesia has developed the most varieties of rice over the last five (5) years, utilizing 28 varieties of rice, namely Inpago Unsoed 1, Rojolele, Inpari 13, Cirata, Cempo Ireng, Cempo Melik, Melik, Sigupai, Cantek Manis, Rom Mokot, Ramos Mirah, Sanbei, Ciherang, Mentik Wangi, G10 Line, G16 Line, Baas Selem, Inpago Unram-1, Situpatenggang, Batutegi, Sintanur, Sigah, Mira-1, Inpari 10, IR64, Unsyiah-1 Simeulu, Unsyiah-3 Sanberasi, and Unsyiah-5 Sibahak. In contrast to genetic improvement, a physical mutagen is also used in food production for example in Malaysia, a study by An ion beam is a charged particle beam that contains an ion. Along with gamma-rays, ion beams have been widely exploited as novel mutagens in mutation breeding programs for the production of new genetic resources in plants, with the increasing number of mutants in the database . In Taka+), atomic nitrogen ions (N2+), argon, and carbon under-voltage potential of 30 and 50 kV to an ion fluence of around 1 × 1016 to 4 ×1016 ions/cm2 (the bombarding time was about 2 min for the fluence of 1016 ions/cm2). The results showed that in the preliminary test, the optimum conditions to achieve the survival rates of ion bombarded rice seeds from 50% to 70% was the combination of 30 kV and 1,2 and 4 × 1016 ions/cm2 fluences or 50 kV and 1 × 1016 ions/cm2.Recent inquire shows there are five (5) studies that utilized ion beams in improving rice traits and consists of four (4) from Thailand and one (1) from Indonesia. In Thailand, the rice varieties KDML-105 and Sangyod were utilized in addition to RD6, whilst, in Indonesia, Pare Ambok, and Pare Lea were used. The seeds were blasted with various ion beams including mixed molecular , electron beams, gamma-rays, and X-rays are classified as low-LET radiation . Low-LETOnly one study from Thailand, conducted by Neutrons have the strongest ionization power among the physical mutagen agents. Neutrons are high-speed nuclear particles with a remarkable penetration power on materials. Neutrons can travel great distances in air and require very thick hydrogen-containing materials (such as concrete or water) to stop them. Fortunately, neutron radiation occurs predominantly within a nuclear reactor, where feet of water provide sufficient protection. The neutron, stable within the perimeters of the atom nucleus, once released will decays with a mean lifetime of approximately 15 min, releasing various kinetic energy. In Thailand, Several physical mutagens, including gamma-rays, ion beams, and fast neutrons have been explored in this study in terms of their efficient research for rice production enhancement. Gamma rays revealed the most significant mutagens utilized to increase rice plant growth, biochemical characteristics, industrial quality, and abiotic stress. Even though neutrons are potent mutagens, fast neutrons were less popular in rice development techniques due to the significant cost involved.In mutation treatment, the plant material used should be relatively selective to ensure consistency and success in its genetic modification, particularly its mutation characteristics. This section explores the plant materials utilized in the great invention of rice mutant breeding in more detail.in vitro such as leaf and stem explants, calli, cell culture, ovules, protoplasts, etc., has been employed in irradiation research  of the reviewed articles in this study involve seeds (containing seed embryos) as the material of choice, which is most typically used as an explant for mutation induction. In general, every part of the rice plant, including dried seeds, dormant seeds (whole plants), pollen (vegetative organs), cuttings, tissue or cells research . However minutes .50), while exposure to 40 krad caused changes in the rice plant’s color from green to brown , whereas the equivalent and effective doses are measured in millisieverts (mSv).The optimal dosage used in rice breeding with different sources of physical mutagen with encouraging results is assessed and discussed in this article. Mutagen dose in irradiation studies may result in different outcomes depending on the purposes, radiation safety standards, and issues involving the radiation protection of humans. In general, the higher the irradiation dose applied, the greater the possibility of mutant formation, as well as the greater the degree of destruction will arise.etc. On the other hand, lower doses induced less chromosomal damage and fewer negative side effects that are related to small and large insertions, deletions, and genomic rearrangements. As an example, the effect of overdose application of ion beam (high LET) may lead to the creation of aneuploids has been discussed in detail by The dosage applied in gamma-ray treatment ranges from 20 to 500 Gy, whereas, ion beams are around 30 to 50 kV, electron beam (440 Gy), and neutron (33 Gy). The same trend can be seen in countries other than Southeast Asia, such as Sri Lanka and India, where the optimum dosage range for gamma-ray (200–350 Gy), electron beam (290–330 Gy), X-ray (200–250 Gy), and proton beam (150–200 Gy) was discovered . Gamma rIn this respect, data from etc.), yield variation and including industrial quality . Overall, irradiation treatment applied to rice varieties has a positive influence at the appropriate dose. However, it is important to exercise caution when selecting mutant genotypes obtained from plant material to ensure their reliability. This precautionary is necessary due to the substantial genetic variability observed in higher generations, for example resulting from the irradiation of mature pollen with gamma-ray, as noted by 15.The genetic heterogeneity caused by induced mutation has enhanced current plant breeding. During the 2016–2020 study period, researchers concentrated on improving many useful traits affecting plant size, flowering and harvesting period, abiotic tolerance, nutritional quality  compared to non-irradiate (0.15 µMg−1) at the same concentration PEG. In Indonesia, rice seeds  were irradiated with 100 or 150 Gy gamma rays were immersed in sodium azide (SA) for two (2) or six (6) h before being treated with 5% PEG at a pressure of −0.03 MPa. Under the drought stress level of −0.03 MPa, the genetic variants in the M1 generation show diversity, and the best combination of mutagen and cultivar is Inpago Unsoed 1, which was irradiated with gamma-ray 100 Gy and then soaked in SA for 2 h  and seeded in a field nursery, with M1145 emerging as the salinity tolerant line. Even though the lines were similar under salt stress to Pokkali and FL530 , the researcher proposed that M1145 should be evaluated in the field to confirm the potential for salt stress. Furthermore, in the flood tolerance study, approximately 3,000 M3 lines of irradiated electron-ion beam 0.44 KGy were screened for submergence tolerance and 317 tolerant mutant lines were survived and selected for planting for the following season (M4 and M5).A combination of the above study results reported to the impact of irradiation on abiotic stress is expected to be used as a benchmark, particularly in drought, flood, and salinity, in designing the upcoming abiotic resistant/tolerant rice genetic line.Radiation may also alter the gene that regulates biochemical compounds in plants. Anthocyanin, the bioactive substance with antioxidant properties in rice that may give anti-inflammatory and anti-cancer benefits, is one of the primary target qualities of the next generation of genetically modified crops particularly in black rice . Accordietc.), post-harvest handling , cultivar varieties and manipulations, followed by selection through breeding and genetic mechanisms, all have an impact on the nutrient content of rice. It is necessary to carefully manage the entire process to avoid nutritional losses including in the selection of grains for seeds to be used in the following cropping. The current study provides an update on the effect of physical mutagens on the rice’s nutritional properties.Higher nutritional value food is constantly required for human health. Agricultural practices ) found that OSRD6-4 and OSRD6-20 are 9.1/100 and 8.7/100 g, respectively, and are higher than the wild RD6 (8.3/100 g). Similarly, OSSY-23 (8.9/100 g) and OSSY-17 (9.3/100 g) were mutants selected from Sangyod rice mutants in M2 generation irradiated with an ion beam (4 × 1016 ions/cm2 (50 kV)) also showed high protein content compared to wild Sangyod (8.6/100 g). The findings demonstrated that when physical agents are used in the induced mutation breeding technique, rice characteristics have a high possibility and chance of improving rice quality.The protein content is one of the nutrients that represents the nutritional quality of the rice yield. According to 16 ions/cm2 (50 kV)). The amylose content of the M2 generation of RD6 rice which is OSRD6-4 (16.4/100 g) was higher than that of the RD6 (wild type) with amylose content of 7.0/100 g and OSRD6-20 (6.8/100 g). Meanwhile, M2 generation from Sangyod rice revealed no significant difference in amylose content between rice mutants OSSY-14 (19.0/100 g), OSSY-17 (15.5/100 g), and OSSY-23 (16.9/100 g) compared to wild Sangyod (17.7/100 g) when exposed to ion beam (4 × 1016 ions/cm2 (50 kV)). This implies that wild rice Sangyod, Sangyod mutants , and OSRD6-4 belong to the low amylose content group, whereas wild RD6 and RD6 mutants (OSRD6-20) belong to the group very low amylose content. When cooked, rice with a very low amylose content becomes softer, moister, and stickier in texture than rice with low amylose content. Furthermore, according to Amylose content was the next characteristic that represents nutritional quality in rice. The amylose content of the milled rice is a major determinant of rice texture, specifically rice hardness, and this contributes to amylose content playing an important part in decision making in rice breeding programs. Amylose content in rice is divided into five (5) categories: waxy (0–2%), very low (3–9%), low (10–19%) intermediate (20–25%), and high (>25%) . TecharaThere is currently no common definition of “industrial rice quality,” and there is even less agreement on how it should be quantified. Rice quality was difficult to define precisely because “rice quality” is subjective and context-specific compared to agronomic traits which include the ability of rice to boost production and/or alleviate abiotic or biotic stresses in crops . ConsumeIn The use of physical mutagens in rice breeding is a promising tool for improving rice production and addressing rice cultivation challenges. Previously, this approach may have aided in the selection and evaluation of desirable traits such as improved yield, drought stress tolerance, and disease resistance by exposing rice seeds to ionizing radiation to induce genetic mutations.To accelerate the development and deployment of improved rice varieties in the future, it is advisable to combine radiation-induced mutation breeding with modern molecular and genetic techniques, such as gene editing and marker-assisted selection. Furthermore, as demonstrated in the current study, gamma rays were the most widely used as a mutagen agent, owing to the high cost and the need for specialized facilities of using neutrons as a mutagen. It is crucial to develop new irradiation methods that are more cost-effective and efficient such as using low-dose or targeted radiation exposure. In addition, current research is primarily focused on agronomic characteristics, but there is a gap in the nutritional quality of rice through radiation-induced mutation breeding, which involves selection of mutations that increase the levels of essential nutrients such as iron and vitamin A. To address the scarcity of arable land, it is crucial to increase the sustainability of rice production by developing new rice varieties that require less water, fertilizer, and pesticides and that are more resistant to pests and diseases. Rice remains an important food crop in many countries and thus continuing to advance the understanding of induced mutation and its application will help in making the supply enough for all.Handful integrated research on the impacts of chemical mutagens on rice has been conducted in recent years, with only a few related to physical mutagens. As a result, the current works emphasize the need for additional research on the use of physical mutagens in rice breeding, particularly in Southeast Asia. Overall, the database shows that gamma rays exhibit physical mutagens that are commonly utilized in rice variety enhancement from 2016 to 2020.To achieve better rice production traits , gamma-rays have played a significant role in plant genetics studies with Cesium-137 and Cobalt-60 as a radiation source. While the ideal dosage rate is between 100 to 250 Gy to produce a more stable plant character and lower the chromosomal damage and less negative side effects. In addition, it was found that seeds were widely used as a source of plant materials in physical mutagen research. The findings from this review are useful as a guideline to determine the correct irradiation dosage and the benefits from this usage of physical mutagens that might contribute to the enhancement of global food security.


In conclusion, the seedling quality in the straw incorporation practice was improved through rotary after deep ploughing and compaction after sowing for lime concretion black soil in Huaibei Plain, China or a similar soil type.The straw incorporation in lime concretion black soil compromises the emergence and quality of winter wheat seedlings in Huaibei Plain, China, lowering the potential of wheat productivity. To overcome the disadvantage, a two-year field experiment was conducted in 2017–18 and 2018–19 to investigate the effects of different tillage modes on seedling emergence and subsequent seedling growth, and final grain yield (GY) in winter wheat. The modes are rotary tillage with compaction after sowing (RCT), rotary tillage after deep ploughing (PT) and rotary tillage after deep ploughing with compaction after sowing (PCT), with the traditional rotary tillage (RT) method as the control. Compared to RT, greater soil moisture content (SMC) at the seedling stage was observed in deep ploughing or compaction treatment, and the highest SMC was achieved in PCT; the time of reaching the maximum number of seedlings was 1 d sooner in RCT or PT, and 3 d in PCT; the seedling number in RCT, PT and PCT was significantly increased by 32.6%, 34.5% and 61.5% respectively. The population size, shoot and root growth of winter wheat in ploughing mode was significantly enhanced than that of rotary treatment at the over-wintering stage; compared to no compaction after sowing, plant growth in compaction treatments was significantly promoted with greater plant population size and height of seedlings. At harvest, GY in RCT, PT and PCT was significantly improved by 5.87%, 10.8% and 16.4%, respectively, compared to RT and the highest GY was achieved in PCT by up to 8, 350.1 kg ha Triticum aestivum L.) belongs to the genus Triticum of the family Gramineae, which is the first food crop cultivated by human beings [Wheat , located in the Huaibei Plain region, Anhui Province, China in 2017–18 and 2018–19 winter wheat growing seasons. It has a typical semi-humid, monsoon-prone climate with an average annual precipitation of 821.5 mm. During winter wheat growing seasons, the total precipitation was 284.7 mm in 2017–18 and 474.4 mm in 2018–19, respectively . The soi2 (5.0 m×6.8 m). Under the maize straw retention, four tillage methods were applied prior to wheat growing seasons, namely, the traditional rotary tillage (RT), the traditional rotary tillage + compaction after sowing (RCT), deep ploughing + rotary tillage (PT), deep ploughing + rotary tillage + compaction after sowing (PCT). The compacting stone with a weight of 150 kg, was used to crush the soil. And the location of each experimental plot in the two years were kept consistent and the wheat straw was evenly returned to soil surface prior to following maize season. The winter wheat cultivar i.e. Yannong 19, as one of the most widely planted varieties in Huaibei Plain, was used in the experiment. Prior to sowing, 135.0 kg N ha−1, 75 P2O5 kg ha−1, and 75 kg K2O ha−1 were applied as the base fertilizer. Total nitrogen was 225 kg ha−1, with 135 kg ha−1 as the base fertilizer and 90 kg ha−1 as the top dressing at the jointing stage in winter wheat. Wheat seeds were sown on October 28, 2017 and October 16, 2018, with 25 cm row spacing, and harvested on June 5, 2018 and June 10, 2019.A randomized complete block design was used to arrange the treatments of different tillage methods, each treatment with three replications. The area of each individual experimental plot was 34.0 mIn this experiment, precipitation varied greatly with the growing seasons, which reflects the typical rainfall characteristic at sowing or seedling growth period of winter wheat in Huaibei Plain, and there were some fluctuations in annual air temperature . As show−1 dry soil) was assayed by oven-drying samples at 105°C for 48 h to a constant weight [Soil moisture content (SMC) was measured prior to sowing and at two weeks after sowing (seedling stage), and the samples were collected from 0−20 cm soil layer with a soil core sampler in each plot. The soil gravimetric water content (g water gt weight .th day after sowing (DAS), and ended at the fifteenth DAS with 1 m2 of area, composed of 1 m length and 4 lines adjacent wheat plants, was randomly selected from each experimental plot and then marked. The number of seedlings at each treatment in the marked investigation area was counted daily in this study.The emergence of seedlings was investigated under different cultivation treatments daily, starting from 72.The number of wheat seedlings (NS), number of the germinated seeds in soil (NGS), number of ungerminated seeds but in soil (NUG), number of ungerminated seeds exposed on the soil surface (NUGS), number of dead wheat seedlings (ND), number of wheat seedlings missing  and average distance of wheat seedling deficiency (AD) were investigated at three weeks after sowing, and the survey area of each plot is 1 m2.Seedling population and tiller number were investigated at 20 DAS, and the seedling population and tillers of plant were investigated at 50 DAS and 80 DAS in this study. The sampled area of each experimental plot is 0.5 mFive plants per plot were destructively sampled to determine leaf length (L), maximum width (W) at 20 DAS, 50 DAS and 80 DAS. LAI was calculated as 0.70×L×W .The SPAD-502 chlorophyll meter was used to conduct nondestructive measurements for leaf relative chlorophyll content . SPAD va2) were sampled in each plot at 80 DAS (over-wintering period). Root extraction was performed using the soil core method according to Böhm (1979) [Wheat plants in adjacent two lines with one meter (0.5 mm (1979) , and sam2 prior to harvest. The grain number per spike was determined by counting the grains in each spike from 50 randomly selected plants in each plot prior to harvesting. Wheat plants from a 4 m2 area in each plot were harvested at maturity and threshed to determine grain yield. Actual grain yield was reported on a 13% moisture basis. The 1000-grain weight (TGW) was calculated by weighing 1000 grains from each sample and averaging three replicates.To determine grain yield, spikes were counted in 2 mThe effects of different tillage methods on grain yield, yield components, tillage depth, soil moisture content, seedling quality, seedling population size, plant dry and root etc. Data were analyzed by analysis of variance using the GLM procedure in SPSS ver. 19.0 . Differences were judged by the least significant differences test using a 0.05 level of significance. All figures were created using OriginPro 2016 Version 9.3 .Prior to winter wheat sowing, the tillage depth of different treatments was presented, based on 5 points investigated in each plot . From thAs shown in -2; NS in RCT and PT was significantly greater compared to RT, and there was no great difference between RCT and PT, and they were all significantly lowered than that of PCT. However, compared to RT, NS was increased by 31.1%, 33.0% and 60.0% in RC, PT and PCT, respectively. Across the two years investigated, NGS in RCT and PT was significantly reduced than those in RT, and there was no significant difference between RCT and PT, but they were greatly higher than PCT. No significant difference was observed in NUG and NUGS among different treatments in this trial. There was little gap in ND and AD among RT, RCT and PT, but they significantly higher than PCT. It was significantly increased in NSM of RT than RCT, RCT significantly higher than PT, and PCT was the lowest. From the results of analysis of variance, compared with rotary tillage, deep ploughing tillage significantly increased NS, but decreased the NGS, NUGS, ND, NSM and AD. The NS in compaction after sowing was significantly increased than those of no compaction (NC), and the NGS, ND, NSM and AD were significantly decreased in NC. In brief, the deep ploughing before sowing combined with compaction after sowing (PTC) achieved the highest NS at the seedling stage at three weeks after sowing, mainly due to the least NGS, ND, NSM and AD.As shown in th and 10th day after sowing in 2017–18 and 2018–19 under compaction treatment (CT), while the time was delayed more than 3 days for seedlings under RT. The seedling density at 15th day after sowing in PCT was the highest among all the treatments of the two years’ study, and there was no significant difference between RCT and PT, RT was the lowest. Overall, deep ploughing with compaction after sowing was more beneficial in accelerating the emergence of wheat seed and form a high seedling density of emergence stage of wheat.Different tillage methods had a significant effect on the dynamic characteristics of seedling emergence in winter wheat . From th4 ha-1, which increased by 57.2%, 21.6% and 20.2% than RT, RC and PT, respectively. Overall, PCT treatment was more conducive to the formation of large population at seedling over-wintering stage of wheat.The seedling population increased as the time went by after sowing . In thisnd leaf was not significant different for RCT, PT and PCT, but they were significantly higher than RT. However, in 2018–19, the chlorophyll content of the 2nd leaf in PCT was significantly higher than those of all other treatments while the content for RT was significantly lower than those in RCT and PT. The chlorophyll results for the 3rd to 6th leaf, the different treatments were consistent for the two years. In the 3rd leaf, RT was significantly lower compared to RCT and PT, and there was no significant difference between RCT and PT, but they greatly lower than PCT. In 4th to 6th leaf, the chlorophyll content in RT was significantly lower compared to RCT, and RCT lower than PT, while PT lower than PCT. In brief, the highest chlorophyll content of different leaves was achieved in PCT treatment.The results of the two-year experiment showed that the chlorophyll content in leaf first increased and then had little difference with the increase of seedling leaf position in all treatments . In 2017The leaf area of the wheat plant showed weak difference between the two growing seasons . HoweverAlthough there were some differences in plant height, dry weight, root number, root length and dry weight under different tillage methods during the two years, the changes were essentially consistent . The pla-1 and 7579.8 kg ha-1 in 2017–18 and 2018–19 growing season, respectively. Compared with RT, GY in RC, PT and PCT was increased by 5.53%, 9.95% and 16.7%, respectively in 2017–18, and increased by 6.21%, 10.8% and 16.0%, respectively in 2018–19. Across the two years results, compared to RT, the SN of RCT was significantly higher, while the SN of PT was higher compared to RCT, and PCT was the highest; the GN was significantly higher under RT than under RCT, while RCT was higher than those of PT and PCT, and there was no significant difference between PT and PCT. In 2017–18, the TGW in RT was significantly increased than RCT, there was little difference between RT and PT, and TGW in RCT was significantly higher than that in PCT. In 2018–19, there was no difference between RT and PT, RCT was similar to PCT, but the former was significantly higher than the later. However, compared with rotary method (R), the percentage of GY in deep ploughing method increased by 10.3%, which mainly was due to the significant increase in SN. The percentage of GY of compaction after sowing increased by 5.74% compared to no compaction, and it significantly increased SN, while decreased GN. Across the two years, PCT had the highest GY, the increase percentage in production was 16.4% compared to the traditional tillage method, and the increase in grain yield mainly owing to the raise in SN, which was up to 31.1%, while it significantly decreased the GN and TGW under PCT.GY, grain number per spike (GN) and thousand grain weight (TGW) were significantly affected by year type, and GY was significantly higher in 2018–19 than that in 2017–18 growing season , due to Different tillage methods caused great variations in the rate of seed germination and the quality of wheat seedling at over-wintering after the maize straw incorporation. In this present study, PCT could improve the seed emergence rate, the quality of seedling in the lime concretion black soil area mainly via the greater soil water moisture, and cause accelerated seed emergence and seedling leaf growth, the stronger roots of seedling, thus alleviating the adverse effects of drought on seed germination and growth.The soil was too loose to contact the seed closely with soil in RT due to large amount of maize residue, causing reduced soil bulk density, increased soil porosity, and accelerated soil moisture evaporate ion , which hThe seedling quality of winter wheat is critical for its resistance to adverse environments during the subsequent growth stage. The air temperature for wheat seedling growth decreased after sowing, so the early emergence is favorable to prolong the time from seedling to over-wintering producing stronger seedlings and greater number of tillers. In our study, PCT and RCT significantly increased the seeding emergence, possibly by maintaining optimal soil moisture for seed germination and thus accelerated to reach the maximum seedling density (SD). The leaf chlorophyll content and leaf area of seedlings were significantly increased by deep ploughing, and the compaction after sowing can increased soil water content (soil water availability) at 10 and 30 cm soil depths compared to the non-compacted treatment  and furtSeedling number, tillering number and strong seedlings had great relationship to SN, GN and TGW of winter wheat , 33. ComThe different tillage techniques have great influence on the seedling emergence and growth of wheat . In thisRCT, PT, PCT treatments could improve seed emergence rate and seedling quality in winter wheat. Of which, PCT achieved the fastest emergence and highest germination rate after sowing, which produces strongest seedlings whilst over-wintering. In addition, the uniformity of seedling emergence and the population of seedlings were increased in deep ploughing or compaction after sowing treatment, and PCT was the optimized treatment in this study. In the end, maximum grain yield over the two years has been achieved in PCT. The findings indicated that PCT could improve the rate of wheat seed emergence, and obtain stronger individuals, which lays the foundation for stable and high yield under the condition of straw incorporation for lime concretion black soil in Huaibei Plain, China or a similar soil type.S1 FileANOVA for tillage depth and soil moisture content (SMC) among different tillage methods at seedling stage in winter wheat is based on this dataset.(XLSX)Click here for additional data file.S2 FileANOVA for the seedling quantity among different tillage methods in winter wheat is based on this dataset.(XLSX)Click here for additional data file.S3 FileComparisons of leaf area of seedlings under different tillage methods in 2017–18 and 2018–19 growing seasons are based on this dataset.(XLSX)Click here for additional data file.S4 FileANOVA for the population size under different tillage treatments at the seedling stage in winter wheat is based on this dataset.(XLSX)Click here for additional data file.S5 FileComparisons of leaf chlorophyll content  under different tillage methods at seedling stage in 2017–18 and 2018–19 growing seasons are based on this dataset.(XLSX)Click here for additional data file.S6 FileComparisons of leaf area of seedlings under different tillage methods in 2017–18 and 2018–19 growing seasons are based on this dataset.(XLSX)Click here for additional data file.S7 FileANOVA for shoot and root growth among different tillage treatments at over-wintering stage in winter wheat is based on this dataset.(XLSX)Click here for additional data file.S8 FileANOVA for grain yield and yield components among different tillage treatments in winter wheat is based on this dataset.(XLSX)Click here for additional data file.


The Russia-Ukraine war has undeniably impacted global science and healthcare in Ukraine. Many Ukrainian researchers have had their projects disrupted by this war, either due to loss of life, displacement, or destruction of resources. Despite these challenges, these researchers have sought to make their voices heard. This scoping review highlights the trend of healthcare-related publications on the current Russia-Ukraine war and characterizes the contribution of Ukrainian authors to these publications.A comprehensive literature search was performed using two databases (Scopus and Pubmed) for publications related to the ongoing Russia-Ukraine war. We included articles only related to healthcare. We then extracted and analyzed bibliometric data.One hundred and eighty-three articles were identified, including 12 (6.6%) original articles, 26 (14.2%) cross-sectional studies, 19 (10.4%) letters to the editor, 10 (5.5%) commentaries, 5 (2.7%) perspectives, 35 (19.1%) editorials, 2 (1.1%) randomized controlled trials, 11(6.0%) correspondences, 13 (7.1%) opinions, 8 (4.4%) reviews and 42 (23.0%) are identified as others. 180 (98.4%) studies were in English, and 3 (1.7%) were in German. 54 (29.5%) papers on the war had at least one author affiliated with a Ukrainian institution, and 29 (15.9%) studies had authors with Ukrainian affiliation as first authors.our study shows that there has been a significant number of publications on the Russia-Ukraine war and only a small portion of first authors, co-authors, and last authors of these publications are affiliated to an institution in Ukraine. Therefore, despite the relatively high number of publications, most publications do not arise from the perspective of Ukrainian authors.The online version contains supplementary material available at 10.1186/s12939-023-02070-7. It has been over a year since the Russia-Ukraine war began, and this war has led to high civilian casualties in the country, resulting in 9,369 deaths and 16,646 injured individuals .Apart from the various setbacks the country has faced, the war has also significantly impacted Ukraine’s scientific contributions in healthcare , 4. ManyUnderrepresentation of authors from Low- and middle-income countries (LMICs) as first and last authors of publications about LMICs have been reported in the literature . This unA comprehensive literature search of two databases (PubMed and Scopus) was performed for articles published on the current Russia-Ukraine war with a focus on the ones related to healthcare, from the start of the war in February 2022 up till February 2023 inclusively. Major keywords such as “war”, “conflict”, “invasion”, “Ukraine”, “Russia” etc. (see appendix 1 for search strategy) were used to identify articles relevant to our research. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for scoping reviews (PRISMAScR) were applied .All articles were screened by two independent reviewers at the abstract and full-text screening stages and any conflict was resolved by a third independent reviewer. The inclusion criteria were (1) articles published from February 2022 inclusive, (2) articles on the ongoing Russia-Ukraine war, (3) articles related to healthcare, (4) articles which full-text is available, and (5) no restriction on language. The exclusion criteria were (1) articles published before February 2022, (2) not related to the ongoing Russia-Ukraine war, (3) not related to healthcare, (4) with no authors’ affiliation, (5) not peer-reviewed, and (6) with full text is not available.Bibliometric data such as date of publication (month and year), journal, article type, affiliation of the authors (whether any author and the proportion of authors affiliated with Ukraine), language of publication, and article specialty  were extracted and analyzed using Excel  to identify the trend in publication and authorship distribution in the first 12 months of the war . The firWe considered authors unique for each included manuscript and analyzed, and for articles with a single author, the author was considered both as first and last author for that manuscript in our analysis.A total of 419 publications were identified in the literature search, with a total of 122 duplicates removed , The Lancet , BMJ  , and Asian Journal of Psychiatry  were the journals with the most publications. The specialty of the articles published was mostly public health , followed by Psychiatry , and nephrology , with specialties categorized as others coming in 4th .Regarding authorship, 54 (29.5%) of the publications had at least one Ukrainian author, while 129 (70.5%) did not have any author affiliated with Ukraine (Fig. Our study indicates that while there is a significant amount of publication within one year of the beginning of the Russia-Ukraine war, Ukrainian authors are underrepresented both as first authors, last authors, and co-authors. These findings are particularly concerning given that the war directly impacts Ukraine, and the voices of its researchers are crucial for a comprehensive understanding of the war’s effects on healthcare.Our findings further support findings from previous studies published on output of local researchers during conflicts or political instabilities. For example, Rema A. et al. showed that 59.3% of articles published during the period of conflict in Syria had no author affiliated to a Syrian institution , which iThe underrepresentation of Ukrainian authors in the scientific literature on the Russia-Ukraine war is a multifaceted issue. One of the primary challenges is the death and migration of researchers and medical personnel due to the ongoing conflict, which has resulted in a shortage of healthcare manpower and disrupted research activities . It is iThe underrepresentation of Ukrainian authors not only limits the diversity of perspectives but also risks skewing the narrative and understanding of the war’s impact on healthcare. Local researchers can provide unique insights into the challenges and needs of the healthcare system, which are essential for effective interventions and policymaking . TherefoTo address this underrepresentation, several steps can be taken. First, international organizations and journals could offer translation services to Ukrainian researchers to help them publish their work. Second, special issues or sections could be dedicated to research conducted by Ukrainian authors on the impact of the war on healthcare. Third, collaboration between Ukrainian researchers and international teams could be encouraged to ensure a more balanced representation . Lastly,Our study has some limitations to be considered despite our efforts to conduct a comprehensive scoping review. Firstly, the literature search was restricted to only two databases, therefore the possibility of excluding relevant studies from other databases, as well as non-English studies, exists. The findings of this study may not be generalizable to other fields as only articles related to healthcare were included. Lastly, the study does not explore the quality or impact of the publications, which could provide further insights into the contribution of Ukrainian authors.Since February 2022, the Russia-Ukraine War has influenced the lives of millions both within and outside Ukraine. As the events of the war and its profound effects continue to unfold, it is essential that these effects, especially on healthcare, are documented by those most directly involved; that is to say, by those affiliated with Ukraine. However, there is underrepresentation of Ukrainian authors and affiliates either as first authors, last authors, or co-authors. We advocate that greater effort be put into ensuring that Ukrainian voices are heard.Below is the link to the electronic supplementary material.Supplementary Material 1


Their lifestyle was assessed using the Fantastic Lifestyle questionnaire. Moreover, physical activity quantification was assessed using the International Physical Activity questionnaire, and musculoskeletal discomfort was quantified using the Corlett diagram. The mean total time of physical activity was 546 ± 276 min per week. No differences (p = 0.0832) were found between the Adm (454 ± 217 min) and TF (623 ± 301 min) groups. Concerning lifestyle, in general the sample presented very good (42%) and good (42%) style classification. For this parameter, no significant differences were found, but only a tendency was discovered ; indeed, the TF presented a better classification (63%) of very good, compared to the Adm (53%) of good. No differences (p > 0.05) were found in musculoskeletal perception of discomfort between the right and left sides (p > 0.05) for all police officers and between the Adm and FT groups (p > 0.05). Military police officers showed high and moderate risk for waist circumference and waist-to-hip ratio, respectively; however, lifestyle and total time of physical activity were considered adequate without differences between military administrative and tactical force sectors.Our aim was to evaluate musculoskeletal discomfort and the lifestyle of military police officers of administrative and tactical force departments. Military police officers were distributed into two groups: administrative (Adm,  The military profession provides to its operating agents tasks of different physical demands on a daily basis, from long periods with less demanding activities , to critical situations, with short periods of physically demanding tasks such as running, crawling, jumping, lifting, pushing, pulling, and transporting objects or people, and controlling uncooperative suspects . Araújo Some military personnel, due to the characteristics of the work, may spend time carrying large amounts of weight (work equipment), but at other times may also remain frequently seated for a long time. This combination of mandatory equipment, such as the ballistic vest, tactical belt, leg holster, weapons with their main chargers and loaded spares, boots, handcuffs, radio communicator, and flashlight, and the large amount of time that military police remain seated, especially for motorized patrols, for example, can impact musculoskeletal health and physical fitness indicators that contribute to performance in operational tasks ,5,6,7,8.According to Calheiros et al., military police officers in the field, responsible for ostensive patrolling, are more predisposed to problems related to the spine, especially in the thoracic and lumbar regions, due to the maintenance of orthostatic postures for prolonged periods, and the aggravation of the ballistic vest . TherefoBecause of these well-known conditions, assessing the risk of incurring musculoskeletal disorders and injuries for these particular workers is essential to provide tailored interventions and occupational health protection measures. However, to the best of our knowledge, although the analysis of musculoskeletal discomfort is an intervention that has already been consolidated in clinical practice and ergonomics, information on military police is still inconclusive. Considering that operational military police officers are more likely to remain inactive due to motorized patrolling, in this study we hypothesized that skeletal muscle discomfort as well as lifestyle would have a greater impact in military from the tactical force. Additionally, the anthropometric, working time, and lifestyle parameters could be associated with skeletal muscle discomfort. Thus, the objective of this cross-sectional correlational study was to evaluate the musculoskeletal discomfort and the lifestyle of military police officers in the administrative and operational tactical force sectors.After approval by the Research Ethics Committee of the Federal University of Espírito Santo (no. 6.275.609/2023), military police officers working in the 17th Independent Company of the Military Police of the municipality of Vila Velha, Espírito Santo, were invited to participate in the study. The invitation to participate in the study was carried out through direct contact between the researchers and the military and through verbal and digital dissemination strategies. Being active in employment was adopted as an inclusion criterion and recent return to work (3 months) as an exclusion criterion. Individuals who answered the questionnaire incorrectly and who did not present the signed informed consent form were excluded.n = 15) and military from the operational tactical force .The company is composed of 120 military personnel: 20 from the administrative sector and 21 from the operational tactical force, with the rest having other functions. Of the 41 personnel from the administrative or operational tactical forces, only 10 subjects did not agree to participate in the study. Thus, the study sample comprised 31 military police officers, distributed into two groups: military from the administrative sector  was calculated according to the following equation: BMI = weight/height2. The circumferences of the abdomen, waist, and hips were evaluated using an anthropometric tape  to estimate abdominal adiposity and cardiovascular risk. The abdomen circumference was measured at the level of the umbilicus for men and women; the waist was measured at the midpoint between the last lower rib and the iliac crest for men and at the greatest protrusion of the buttocks for women; and, finally, the hips were measured at the largest circumference around the buttocks. Anthropometric variables were classified according to the recommendations of the World Health Organization [Height was measured using a Stadiometer, model WCS  with a precision of 0.1 cm. Body mass was measured using a Scale, Personal Line Model 150  with an accuracy of 0.1 kg. The body mass index , short version ,16,17. T2 test and the unpaired t-test were used to compare the data of the qualitative and quantitative variables, respectively. In addition, Pearson’s correlation was used to identify the correlation between length of service and perception of general pain with anthropometric parameters, level of physical activity, and lifestyle. The software GraphPad Prism version 6.00 for Windows  was used, adopting a significance level of p < 0.05.Data are presented as absolute frequency (F), relative (%) for qualitative variables, and mean and standard deviation for quantitative variables. The xp = 0.0043).Of 31 participants, 4 (13%) were women working in the administrative sector, and 27 (87%) were men, of which 11 were from the Adm group and 16 were from the TF group. The general mean age of the participants was 32 ± 6 years, with 35 ± 6 years in the Adm group and 29 ± 3 years in the FT group, which was a significative difference .Regarding operational function, in general the sample consisted of 20 (65%) soldiers, 5 (16%) corporals, 3 (10%) sergeants, 1 (3%) lieutenant, 1 (3%) captain and 1 (3%) major. The TF group was composed of 13 (81%) soldiers and 3 (19%) corporals. The Adm group comprised 7 (47%) soldiers, 2 (13.5%) corporals, 3 (20%) sergeants, 1 (6.5%) captain, 1 (6.5%) lieutenant, and 1 (6.5%) major. No differences were found between groups regarding operational function (xp = 0.0014) were found between the service time of military personnel in the Adm (12.63 ± 5.65 years) and TF (6.38 ± 3.70 years) groups. Regarding the level of physical activity, military police officers in general were classified as active, with a mean total time of physical activity of 541 ± 272 (coefficient of variation 50.40%) minutes per week. No significant differences (p = 0.0832) were found in the time of weekly physical activity practice between the active military in the Adm  and TF  groups. The sample characteristics are shown in The general average service length corresponded to 9.04 ± 5.64 years of activity. Significant differences  and TF  groups, no significant differences were found . However, these results may be related to more lean mass instead of fat mass.Although the general classification of BMI of 26.28 ± 4.07 kg/m2: 1.060; p = 0.7868) between the Adm group  and the TF group . Concerning waist circumference (WC), the military personnel in general were classified as low risk, with 35% presenting high risk and 3% very high risk. No significant differences  were revealed between soldiers in the Adm group  and those in the TF group (56% for high risk). Regarding the risk classification according to the waist-to-hip ratio, in general, the military personnel were considered low risk; however, 29% presented low risk, 49% moderate risk, 16% high risk, and 6% very high risk. When comparing the ratings of the military, no differences were found in risk  between the Adm  and TF  groups.Regarding the risk classification for abdominal circumference (AC), the general average of the military personnel was normal, with 49% of the personnel having normal AC, 13% having medium risk, 32% having high risk, and 6% having very high risk. No significant difference was found ; indeed, the TF group presented a better classification (63%) of very good compared to the Adm (53%) group of good.As regards the assessment of lifestyle , in genep > 0.05) were found in the right and left sides between administrative and tactical force groups. Similarly, no differences (p > 0.05) were revealed in the perception of discomfort between the Adm and TF police officers among anatomical regions.As shown in The aim of this study was to assess the lifestyle and musculoskeletal discomfort of military police officers in the administrative and operational sector of Espírito Santo district. No difference between administrative and operational officers was found.Sample characteristics of administrative and tactical force showed no significant difference, except for age. Moreover, participants were classified as overweight but low risk for cardiovascular diseases, with no differences between military police officers in the administrative sector and ordinary troops. The high prevalence of overweight military police officers is not a new or recent finding ,20,21,22Regarding lifestyle, the military police were classified as good or very good, and better results were detected among operational troops compared to those in the administrative sector; however, no differences were revealed between the groups. The poorer lifestyle classification of police officers in the administrative sector may be explained by older age, longer service, greater body mass, and less time practicing physical activity ,28. UnpuRegarding musculoskeletal discomfort, we found that the perception of pain in military police officers can be considered low. In addition, no differences in pain perception were found between military police officers in the administrative sector and those in operational troops. However, the latter reported greater pain intensity in the dorsal region, particularly in the lumbar spine, which is still the most reported site of pain in both groups. Previous studies have also shown that low back pain is the main pain complaint in military police officers ,33,34. TAdditionally, some important limitations are present in this study, including the very small sample, which was limited to administrative and tactical force military police officers; low quantitative distribution of men and women in the sample; assessments of skeletal discomfort without identifying the primary cause; lack of objective parameters of skeletal muscle strength associated with skeletal discomfort; and evaluation of only the body composition and physical activity behavior, which limits the generalization of the results.In conclusion, although the evaluated military police officers presented high and moderate risk considering waist circumference and waist-to-hip ratio, lifestyle and total time of physical activity were considered adequate and did not show indications of musculoskeletal discomfort. Nor were differences found between military personnel working in the administrative sector and tactical strength troops. New studies need to be carried out in order to identify the influence of personal, environmental, and socioeconomic factors on the lifestyle of military police officers from Espírito Santo, as well as the relationships between work characteristics, use of protective equipment, and length of service in the occurrence of musculoskeletal discomfort.


Racial and ethnic inequalities in all-cause mortality exist, and individual-level lifestyle factors have been proposed to contribute to these inequalities. In this study, we evaluate the extent to which the association between race and ethnicity and all-cause mortality can be explained by differences in the exposure and vulnerability to harmful effects of different lifestyle factors.The 1997–2014 cross-sectional, annual US National Health Interview Survey (NHIS) linked to the 2015 National Death Index was used. NHIS reported on race and ethnicity , lifestyle factors , and covariates . Causal mediation using an additive hazard and marginal structural approach was used.additional deaths per 10,000 person-years whereas Hispanic/Latinx women experienced 9.3  fewer deaths per 10,000 person-years; no statistically significant differences were identified between White and Hispanic/Latinx men. Notably, these differences in mortality were partially explained by both differential exposure and differential vulnerability to the lifestyle factors among Black women, while different effects of individual lifestyle factors canceled each other out among Black men and Hispanic/Latinx women.465,073 adults (18–85 years) were followed 8.9 years (SD: 5.3); 49,804 deaths were observed. Relative to White adults, Black adults experienced 21.7  and 11.5  Lifestyle factors provide some explanation for racial and ethnic inequalities in all-cause mortality. Greater attention to structural, life course, healthcare, and other factors is needed to understand determinants of inequalities in mortality and to advance health equity.The online version contains supplementary material available at 10.1186/s12889-023-16178-6. Long-standing and stark racial and ethnic inequalities in health and mortality are widespread in the United States (US) , 2. It iLifestyle factors  are an important driver of inequalities in health, explaining, for example, more than two thirds of the association between SES and all-cause mortality , 15. Altdifferential exposure to lifestyle factors, and (ii) differential vulnerability to the harmful effects of each lifestyle factors across different race and ethnicity groups. The lifestyle factors considered were smoking, alcohol use, physical activity, and body mass index (BMI).Overall, the extent and means by which lifestyle factors might explain racial and ethnic disparities is largely unknown. Using a comprehensive model Fig.  and a laData came from the National Health Interview Survey (NHIS) linked to the National Death Index (NDI) using probabilistic record matching . NHIS isThe outcome was time to all-cause mortality, operationalized as the time from the NHIS survey to death or last presumed alive. Race and ethnicity, the independent variable of interest, was self-reported and categorized as non-Hispanic White , non-Hispanic Black/African American (henceforth Black), and Hispanic/Latinx. We further distinguished all other non-Hispanic racial and ethnic groups  for descriptive analyses, though sample size was too small for inclusion in the main analyses.2), normal weight , pre-obesity (25-29.99 kg/m2) or obese (≥ 30 kg/m2) [Participants’ report of the frequency and quantity of alcoholic beverage consumed in the past 12 months was converted to grams of pure alcohol consumed per day, assuming 14 g of pure alcohol per drink. Alcohol use was categorized according to the standards of the World Health Organization  and incl0 kg/m2) .The covariates used in all models were age (continuous), sex, educational attainment, marital status, and survey year (continuous). Educational attainment was categorized as low (high school diploma or less), medium (some college but no bachelor’s degree), or high (bachelor’s degree or more), and was treated as a proxy for socioeconomic status; given its ubiquity in the extant literature, stability over time, and completeness of data  in the NHIS. Marital status was a binary variable indicating whether the individual was married or living with partner.Causal mediation analysis using the marginal structural approach with Aalen’s additive hazard models was used, as described by Lange et al. –29. Brietimereg package (version 2.0.2) [timereg package does not allow for complex sampling designs and survey weights were not utilized given the analytical and computational complexity of the analyses.All analyses were completed in R 4.1.3, using the n 2.0.2) ; the staIn a sensitivity analysis, causal mediation models were repeated without education included as a covariate, recognizing that race and ethnicity are deeply tied to SES in the US , and priParticipants were 465,073 adults , of whom 63% were non-Hispanic White, 15% non-Hispanic Black, 17% Hispanic/Latinx, and 5% non-Hispanic Other , of additional deaths per 10,000 person-years, whereas Hispanic/Latinx women experienced 9.3  fewer deaths per 10,000 person-years after adjusting for covariates. Mortality was similar among White and Hispanic/Latinx men, after adjusting for covariates.Table fewer deaths per 10,000 person years in Black and Hispanic/Latinx adults, relative to White adults (depending on the subgroup). However, Black men and Hispanic/Latinx adults were also more vulnerable to the adverse effects of smoking, which resulted in 2.2 to 11.7 additional deaths per 10,000 person years. The opposite pattern was observed for physical activity, finding that greater exposure to a sedentary lifestyle was associated with 4.6 to 7.8 additional deaths per 10,000 person years among Black and Hispanic/Latinx adults, relative to White adults, and that Black women and Hispanic/Latinx adults were less vulnerable to the adverse effects of physical inactivity, resulting in 1.7 to 5.0 fewer deaths per 10,000 person years. With respect to alcohol use, Hispanic/Latinx men were similar to White men. Among Black adults and Hispanic/Latinx women, exposure to alcohol use was associated with 1.4 to 5.1 additional deaths per 10,000 person-years, relative to White men and women. But this effect was partially offset by a greater resilience  to the adverse effects of alcohol use in these groups. Lastly, with respect to BMI, differential exposure and vulnerability effects were relatively small and offset each other.Table additional deaths, whereas a lower prevalence of smoking was associated with fewer deaths. The results of the sensitivity analysis excluding education as covariate were consistent with our main analysis and did not change our conclusions (Supplementary Table Notably, the net indirect effect through lifestyle factors was not significant among Black men and Hispanic/Latinx women, and did not contribute overall to racial and ethnic inequalities in these racial and ethnic groups; different levels of physical activity were associated with The current study sought to evaluate the mechanism and extent to which lifestyle factors contribute to racial and ethnic inequalities in mortality among US adults. Specifically, we examined whether these inequalities can be explained by indirect effects through differential exposure and differential vulnerability to harmful effects of different lifestyle factors.additional deaths among Black men and Hispanic/Latinx women were attributed to a higher exposure to sedentary physical activity, while a lower prevalence of smoking resulted in fewer deaths, relative to White men and women. This finding that particularly highlights the differential exposure to different lifestyle factors across racial and ethnic groups is consistent with past studies [First, and consistent with the extant literature –4, we fo studies , 33, 34. studies  and for  studies . Such fa studies , will noOur findings for the somewhat limited role of lifestyle factors in explaining racial and ethnic inequalities in mortality stand in contrast to research on socioeconomic disparities in mortality, which report that the latter inequalities are largely attributed to the net indirect effect of lifestyle factors , 15. ThiIn interpreting the results presented above, limitations should be considered. First, the choice of covariates is important given that causal mediation models assume no unmeasured confounders for the exposure-outcome, exposure-mediator, and mediator-outcome relationships, and no mediator-outcome confounders caused by the exposure. Mediators are also assumed to have no causal effect on each other. Residual confounding by unmeasured risk factors in our analyses is possible. In particular, chronic health conditions and diet quality were not taken into account and might vary across racial and ethnic groups. Second, because the data arose from participants’ self-report from a single time point, reporting bias and changes in lifestyle factors over time may have introduced misclassification and underestimated the association between lifestyle factors and mortality. Third, the exclusion of the institutionalized population in the NHIS may have led to an underestimation of racial and ethnic inequalities in mortality, as well as the indirect effects of lifestyle factors, as racial and ethnic minorities are likely to be overrepresented in some of these populations . MoreoveOverall, our study of multiple lifestyle factors demonstrates that their net effect helps to explain some portion of the observed racial and ethnic inequalities in all-cause mortality. While differential exposure and vulnerability to multiple lifestyle factors contributed to the disparity in Black women’s all-cause mortality, in other groups, indirect effects of individual lifestyle factors canceled each other out. Importantly, lifestyle factors do not develop in isolation but are a product of more fundamental forces associated with structural and social determinants of health . Future Below is the link to the electronic supplementary material.Supplementary Material 1


Background: The brain is an extraordinarily complex organ with multiple anatomical structures involved in highly specialized functions related with behavior and physiological homeostasis. Our goal was to build an atlas of protein-coding gene expression in the goat brain by sequencing the transcriptomes of 12 brain regions in seven female Murciano-Granadina goats, from which three of them were 1-month pregnant.Results: Between 14,889 (cerebellar hemisphere) and 15,592  protein-coding genes were expressed in goat brain regions, and most of them displayed ubiquitous or broad patterns of expression across tissues. Principal component analysis and hierarchical clustering based on the patterns of mRNA expression revealed that samples from certain brain regions tend to group according to their position in the anterior-posterior axis of the neural tube, i.e., hindbrain (pons and medulla oblongata), midbrain  and forebrain . Exceptions to this observation were cerebellum and glandular tissues , which showed highly divergent mRNA expression profiles. Differential expression analysis between pregnant and non-pregnant goats revealed moderate changes of mRNA expression in the frontal neocortex, hippocampus, adenohypophysis and pons, and very dramatic changes in the olfactory bulb. Many genes showing differential expression in this organ are related to olfactory function and behavior in humans.Conclusion: With the exception of cerebellum and glandular tissues, there is a relationship between the cellular origin of sampled regions along the anterior-posterior axis of the neural tube and their mRNA expression patterns in the goat adult brain. Gestation induces substantial changes in the mRNA expression of the olfactory bulb, a finding consistent with the key role of this anatomical structure on the development of maternal behavior. The mammalian brain is an extraordinarily complex organ integrating multiple highly specialized structures involved in the regulation of memory, behavior, learning, sensory function, motor skills and body homeostasis, amongst others. During embryo development, three vesicles emerge from the neural tube , which rTranscriptomic analyses may hold the key to significantly advance our knowledge about the biological functions of brain regions. In mice, patterns of gene expression have been used to establish a molecular atlas of the adult brain , and as Very few atlases of brain gene expression have been generated in domestic animals. Recently, Seven adult non-lactating Murciano-Granadina goats raised in the experimental farm of the Faculty of Veterinary Sciences at the Universitat Autònoma de Barcelona (UAB) were slaughtered because of reasons unrelated with this project. Sampled goats were kept under the same management and environmental conditions and had similar ages (6.28 ± 1.38 years). Besides, three of these goats were 1-month pregnant at the time of slaughtering. To minimize pain, goats were administered pentobarbital (150 mg/kg) in the jugular vein. A lethal intravenous dose of sodium pentobarbital causes a mammal to lose consciousness within seconds and results in clinical death within just minutes, but we cannot completely rule out the possibility that the euthanasia procedure used in the current work might have altered the brain mRNA expression of certain genes. Since animals were killed due to the routine culling process implemented in this experimental farm, no permission from the Ethics Committee on Animal and Human Experimentation at UAB was required.After slaughtering, goats were transported to the Necropsy room of the Faculty of Veterinary Sciences at UAB. The cranial vault of each goat was opened with a bone saw and twelve brain anatomical structures were carefully dissected and biopsied by an expert anatomist. Samples were drawn from the adenohypophysis, cerebellar hemisphere, cerebellar trunk or peduncle, frontal neocortex, hippocampus, hypothalamus, medulla oblongata, neurohypophysis, pineal gland, pons, olfactory bulb and rostral colliculus. Biopsies were immediately submerged in RNAlater  to be stored at -80°C until processing.To purify total RNA, tissue samples were mixed with 1 ml QIAzol  and homogenized using the Lysing Matrix D reagent  in a Precellys 24 tissue homogenizer . The extraction of total RNA was performed using the RNeasy lipid tissue mini kit  following the protocol described by the manufacturer. The concentration and purity of extracted RNA molecules were analyzed using the Nanodrop ND-1000 spectrophotometer  and RNA integrity was assessed with a Bioanalyzer-2100 equipment  using the RNA 6000 Nano Kit 4.2 .Paired-end sequencing (2 x 50 bp) of total RNA was carried out at the Centre Nacional de Anàlisi Genòmica (CNAG). Sequencing methods have been reported by http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapters were trimmed using the TrimGalore v.0.6.6 tool . In addition, reads with more than five ambiguous bases (Ns) were removed. Filtered sequences were aligned to the goat ARS1 reference genome (https://github.com/gpertea/stringtie/blob/master/prepDE.py):The quality of the sequences was assessed with the FastQC software v.0.11.9 , count data from protein-coding genes were normalized to CPM with the edgeR package . Finally2(CPM + 1)) considering all protein-coding genes  to identify outlier samples. The distribution of distances between samples from the same tissue was analyzed using the boxplot.stats function from the grDevices package in R , and samples deviating more than 1.5 times from the interquartile range were considered as outliers and, in consequence, eliminated. Additionally, we built a heatmap based on the gene expression level of 61 genes selected by their high tissue-specificity in a set of 12 human tissues  equivalent to those investigated in goats herewith. This analysis was performed to make sure that the expression patterns of the goat samples match what is expected based on human transcriptomic data.In order to detect errors during the sampling procedure, which might alter our results , the quality of the regional sampling of the brain was evaluated in two ways. First, Euclidean distances were estimated in a pairwise manner based on the corrected level of gene expression (logprcomp function from the stats R package (hclust function from the R stats package (c) was estimated from the output of a traditional Fisher exact test and a Z-score was estimated from expected ranks and variances of the set of genes. In accordance with c is the combined score, p corresponds to p-values estimated with the Fisher exact test, and z is the Z-score from the expected rank.A PCA was constructed by using the  package . Additio package . For eac package . The KEG package  was usedn is the number of brain regions, and xi corresponds to the expression profile component normalized by the maximal component value  defined by nt value . This cant value . A τ-valCV) of the expressed genes and genes differentially expressed between conditions:Before doing the differential expression analysis, the level of variability across replicates from the same condition (pregnant or non-pregnant) was assessed by calculating the coefficient of variation  Statistical significance threshold: q-value < 0.05. Differentially expressed genes were used to perform pathway enrichment analyses using the Enrichr R package  of the 84 RNA extractions was 7.53 ± 0.62, ranging from 6.2 to 9 . These vPHOX2A and SLC6A2, which are considered markers for the pons based on human data. In congruence with our results, the expression of these genes is reported to be high in the medulla oblongata of mice  in each tissue ranged from 14,889 genes for the cerebellar hemisphere to 15,592 genes for the pineal gland. Considering all brain regions, 17,054 protein-coding genes passed the quality control (QC) filters and were expressed in at least two samples. One pineal gland sample deviated more than 1.5 times from the interquartile range, so it was identified as an outlier and excluded from any further analysis. As a complementary measure to detect sampling errors, we built a heatmap based on the expression levels of 61 marker genes which in humans show high tissue specificity for each one of the brain regions considered in this research. As shown in  of mice . Furtherand mice . We alsoand mice . This reand mice .In close agreement with data reported above, the PCAs, with  and withτ = 0.15–0.85) and 1,363 protein-coding genes (7.99%) had a highly region-specific profile (τ > 0.85) of mRNA expression (τ ≥ 0.85) identified for each tissue is shown in The distribution of the tissue-specificity index τ-values was highly skewed to the left . Indeed,pression . A heatmτ = 0–0.15) revealed that they are mainly involved in general biological processes such as RNA transport , autophagy , oxidative phosphorylation , spliceosome , proteasome  and ubiquitin mediated proteolysis , as well as in the development of neurological diseases such as amyotrophic lateral sclerosis , Parkinson disease , and Huntington disease , to mention a few.As shown in τ = 0.85–1, listed in τ = 0.85–1 in several of the analyzed regions, i.e., frontal neocortex, hippocampus, hypothalamus, and olfactory bulb. Other pathways associated with neuronal transduction were also enriched in genes with a tissue-restricted expression, e.g., serotonergic synapse  and GABAergic synapse . Besides, the cholinergic synapse pathway showed enrichment in the rostral colliculus . Several brain regions showed an enrichment pattern that agreed well with their biological functions .As indicated in unctions . For insq-value < 0.05), e.g., synaptic vesicle cycle, endocrine and other factor-regulated calcium reabsorption, endocytosis, phagosome, long-term potentiation, dopaminergic synapse, glutamatergic synapse, circadian entrainment, gap junction, gastric acid secretion and adrenergic signaling in cardiomyocytes. We also detected many pathways related to neurological conditions that attained high statistical significance  and were represented in a broad array of encephalic regions, e.g., Alzheimer disease, Huntington disease, Parkinson disease, prion disease, amyotrophic lateral sclerosis, and, to a much lesser extent, spinocerebellar ataxia. Pathways related with Vibrio cholerae and Salmonella infection were also commonly found. Protein processing in endoplasmic reticulum , RNA transport  and ribosome  were amongst the most significant pathways in the adenohypophysis  while in the remaining brain regions it did not reach statistical significance.The results of the Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis for the 1,000 mRNA genes with the highest expression in each region  revealedpophysis  and neurpophysis , but notpophysis  phototrac = 89.246; q-value = 7.29 E-07), dopaminergic synapse , neuroactive ligand-receptor interaction , cholinergic synapse  and phenylalanine, tyrosine and tryptophan biosynthesis . In contrast, downregulated DEGs in the olfactory bulb of pregnant goats were enriched in pathways related to nervous development and control of cellular activities, including migration, differentiation, and proliferation , affective behavior, sociability and exploration , anxiety and depression , autism , aggression , cognition, memory and learning , response to stress , feeding behavior  and diverse neuropsychiatric disorders .By making a literature search for each one of the genes showing differential expression in the olfactory bulb of pregnant vs. non-pregnant goats, we observed that many of them are related to human behavioral traits , includiPROKR2 and SALL3), olfactory bulb angiogenesis , olfactory bulb morphogenesis and establishment of a functional olfactory neural circuitry , axonal growth and guidance  and sensitivity of olfactory sensory neurons to food cues . A heatmap displaying the gene expression patterns of 207 genes related to behavior (As shown in behavior  and 53 gbehavior  that shoτ = 1). Results obtained in the Genotype-Tissue Expression (GTEx) project were consistent with this finding , implying that they have a ubiquitous pattern of expression. Indeed, only 469 genes (2.75%) were expressed in just one of the 12 sampled brain regions  and non-pregnant (N = 4) category is low, sensitivity to detect DEGs is expected to be quite limited . To circIn comparisons involving cerebellar hemisphere, cerebellar trunk, hypothalamus, medulla oblongata, neurohypophysis and rostral colliculus, the total number of DEGs when comparing pregnant vs. non-pregnant goats was very low or inexistent . In contBrain gene expression during pregnancy is dynamic and it might change depending on the time point under consideration. By using magnetic resonance imaging, it has been shown that the brain of primiparous women experiences substantial morphological changes during gestation which mostly affect the right middle temporal gyrus, inferior frontal gyrus and posterior cingulate cortex . A longiNFATC3 and CCL2, and apoptosis (CTSH) were upregulated in these tissues in pregnant goats  and 70 DEGs (55 upregulated and 15 downregulated in pregnant goats) were detected, respectively . The hipnt goats . Interesnt goats . Besidesnt goats .RYR2, ROCK2, GNAQ, NFATC3 and ADCY7) and cortisol synthesis and secretion  were upregulated in pregnant goats. The anterior pituitary is strongly enlarged during gestation in response to hormones produced by the placenta and ovaries, and the secretion of prolactin increases gradually to prepare the female for lactation (In the adenohypophysis 201 DEGs (188 upregulated and 13 downregulated in pregnant goats) were identified . Adenohyactation .Differential mRNA expression in the pons, with 190 DEGs (133 upregulated and 57 downregulated in pregnant goats) is harder to interpret . This brWe have detected 94 genes that are differentially expressed in more than one tissue when comparing pregnant and non-pregnant goats . The most significant pathways detected in this list of 94 genes are Wnt signalling pathway, adherens junctions signalling pathways, and FoxO signalling pathway . These pDDC) mRNA, which was consistently downregulated in the pons and olfactory bulb . This gene is responsible for the synthesis of dopamine, essential for a healthy pregnancy  gene was significantly upregulated in the olfactory bulb of pregnant goats (log2FC = 1.15), while in the frontal neocortex was downregulated in pregnant goats (log2FC = -0.67). The VAMP1 gene encodes a protein that plays an important role in synapsis and synapsis plasticity and forms part of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex  or of the prokineticin receptor 2  genes results in olfactory bulb hypoplasia  inhibits the formation of the glomerular layer in the olfactory bulb  gene is fundamental for the proper development of mouse olfactory system  or olfactomedin 2  impairs olfactory function  , which is essential for blood vessel formation in the olfactory bulb  , and anoory bulb . So, modDBH, |log2FC| 0.93, q-value < 0.05), which encodes an oxidoreductase that catalyzes the conversion of dopamine to norepinephrine. In mice, the inactivation of the DBH gene causes a profound impairment of maternal behavior characterized by the abandonment of pups which die a few days after birth  and the 5-hydroxytryptamine receptor 2A  show an increased expression in the olfactory bulb of pregnant goats. The DRD1 gene is involved in the regulation of mothering in rats  and the prion protein  mRNAs. The inactivation of these genes leads to increased aggression  mRNA, which in zebrafish has the opposite effect  mRNA is upregulated in the olfactory bulb of pregnant females. Interestingly, the intracerebroventricular injection of neurotensin in mice decreases aggression associated with the maternal protection of the offspring , neuritin  and glycine receptor β , and pro-depressive or pro-anxiety genes , prodynorphin  and histamine receptor H1  in pregnant goats. Similarly, we have found increased mRNA levels of genes which promote feeding, e.g., neuropeptide Y, NPY; C-X-C motif chemokine ligand 14, CXCL14 ; and neural EGFL like 2, NELL2 ; or inhibit it, e.g., G protein-coupled receptor 45, GPR45 ; and acyl-CoA binding domain containing 7, ACBD7 , as reported in previous studies , a mediator of second messenger signaling in the olfactory epithelium which also regulates spatial and associative memory , which is important for olfactory bulb neurogenesis, learning and memory (There is also a subset of genes with increased expression in the olfactory bulb of pregnant goats which is particularly interesting because they display dual functions related with both olfaction and behavior. Examples of this category are genes encoding hippocalcin (e memory , and thed memory .The results of the differential expression analysis should be interpreted with caution because sample size is small. Despite this caveat, we consider that the disproportionate number of DEGs detected in the olfactory bulb when compared to other brain regions is a robust result. Importantly, this anatomical structure is strongly involved in the development of maternal behavior in mice, i.e., its complete removal eliminates such capacity and, very often, implies that litters are cannibalized by their mothers soon after parturition . MoreoveIt has been proposed that in sheep and goats the increase of estrogen levels in blood during prepartum, combined with the vaginocervical stimulation provoked by the expulsion of the fetus, trigger modifications in a neural network including the main olfactory system, the medial preoptic area and the paraventricular nucleus of the hypothalamus, in order to induce maternal behavior . Our tra


High-temperature body-centered cubic (BCC)  As a metallic nuclear fuel, uranium zirconium (UZr) alloy is being considered as a candidate fuel for fast reactors due to its advantages of having high thermal conductivity and evolution under burn-up ,2,3,4. GPure uranium exhibits three different crystal structures: the orthorhombic e 1045 K . Among tadiation ,9. Unforar fuels ,12,13 haar fuels ,15,16. ABCC uctivity ,17, makiZr alloy ,22,23,24Zr alloy  also proIn our present work, molecular dynamics (MD) simulations were performed to calculate the mechanical and dynamical stability of All MD simulations were carried out with LAMMPS , which iTo analyze the thermodynamic stability of MPS code . The PhDN is the number of particles, i at time t, respectively. The current density can be decomposed into longitudinal and transverse parts, which can be expressed as The dynamical structure factor simulations were performed using the Dynasor package from MD trajectories . The dynThe current correlations can be transformed to the frequency domain via Fourier transformation. For the convenience of calculations, the scattering lengths are set to unity. The dynamical structural factor related to the frequency can be expressed as follows: NPT ensemble. After relaxation, we calculated the cohesive energy, an essential parameter for determining the stability of solids. For different Zr concentrations, we found that the crystal structures still maintained the BCC structure.Pure aterials ,29,30. IThe dependence of lattice constants and the cohesive energy of  studies ,32,33,34To understand the structural features of In order to verify the effectiveness of d in γ-U ,36 and od in γ-U  are alsol values , which wAccording to the mechanical stability criteria for the cubic crystal system as in (5)C11>Cof γ-UZr  and γ-U UZr [γ-U  at 0 K dMoreover, the elastic constants afford information on the mechanical strength and hardness. Armed with these outcomes, we further calculated the various moduli, Vicker’s hardness, elastic wave velocities, and Debye temperatures for imations ,39,40: (The Young’s modulus It can be seen that the equation :(9)Hv=0.Our results for the Vickers hardness, velocity ,43:(10)ΘApproximately, given by :(11)vm=[The mechanical properties of an alloy are also manipulated by the concentrations. The mechanical properties of In order to study the dynamical stability, we also calculated the phonon dispersions at T = 1200 K. The initial system was firstly equilibrated at 1200 K with zero external pressure in an NPT ensemble before calculating the dynamical properties. In ed above ,46 and ted above ,48. Due In previous studies ,49,50, tg et al.  measuredSubsequently, we calculated the phonon dispersion curves and phonon density of states (PhDOSs) for As mentioned above, the vibration of atoms can provide critical insights for understanding the structural and thermodynamic properties of materials. In general, the atomic vibrations can be probed using inelastic neutron scattering experiments due to the energy and momentum of thermal/cold neutrons being comparable to those of phonons. Inelastic neutron scattering has been widely used to verify the correct structure and vibrational dynamics . The theThe calculated contour maps of the longitudinal and transverse currents at 1200 K for k) for We calculated the contour maps of the dynamic structure factor on the plane of ,S=12 effects . NCrysta effects ,54.The results of coherent elastic, incoherent elastic, inelastic, absorption, and total cross-section of In summary, we have systematically performed MD simulations on the mechanical and dynamical properties of These theoretical results of mechanical and dynamical properties provide a reference for the design and application of UZr as a nuclear fuel. We hope our work can provide some directions and information for scientists in the fields of nuclear reactors and neutron scattering. Due to approximations in potential, limitations in simulation size, and statistical uncertainty of using the MD method, these results still need to be verified in future experimental measurements, such as by using inelastic neutron scattering and a powder diffractometer.


Mulberry leaves are excellent for health care, confirmed as a ‘drug homologous food’ by the Ministry of Health, China. The bitter taste of mulberry leaves is one of the main problems that hinders the development of the mulberry food industry. The bitter, unique taste of mulberry leaves is difficult to eliminate by post-processing. In this study, the bitter metabolites in mulberry leaves were identified as flavonoids, phenolic acids, alkaloids, coumarins and L-amino acids by a combined analysis of the metabolome and transcriptome of mulberry leaves. The analysis of the differential metabolites showed that the bitter metabolites were diverse and the sugar metabolites were down-regulated, indicating that the bitter taste of mulberry leaves was a comprehensive reflection of various bitter-related metabolites. Multi-omics analysis showed that the main metabolic pathway related to bitter taste in mulberry leaves was galactose metabolism, indicating that soluble sugar was one of the main factors of bitter taste difference in mulberry leaves. Bitter metabolites play a great role in the medicinal and functional food of mulberry leaves, but the saccharides in mulberry leaves have a great influence on the bitter taste of mulberry. Therefore, we propose to retain bitter metabolites with drug activity in mulberry leaves and increase the content of sugars to improve the bitter taste of mulberry leaves as strategies for mulberry leaf food processing and mulberry breeding for vegetable use. Mulberry (Morus spp.) is an important economic crop widely distributed from temperate to subtropical regions [Mulberry leaves are rich in a variety of nutrients and bioactive substances, including sugar, protein, fiber, vitamins, minerals, polyphenols, alkaloids and amino acids required for the human body [ regions . Mulberrman body ,3. For tman body ,5. Howevman body . They haman body ,8,9. Theman body ,11,12. MThe bitter taste of mulberry leaves is a problem when used as food. Bitterness is the least popular of the five tastes, and mammals generally dislike or even reject bitter foods ,14. The The rise of metabolomics provides a new research strategy for the formation of bitter taste in mulberry leaves. MMHub, a database for the mulberry metabolome, annotates 124 metabolites of mulberry leaves, among which 90 metabolites contain detailed information about related chemical structures, including alkaloids, flavonoids, bitter amino acids, etc., related to the bitter taste of mulberry leaves . MetabolIn this study, metabolites related to bitterness in mulberry leaves were analyzed by comparing the metabolites of leaves with different bitterness. The genes and pathways involved in the synthesis of bitter substances in mulberry leaves were analyzed via comparative transcriptome. Combined with metabolome and transcription analysis, the main metabolites, genes and their synthetic pathways affecting the bitter taste of mulberry leaves were analyzed, and the physiological mechanism of the formation of the bitter astringency of mulberry leaves was preliminarily determined. This provided a theoretical basis for mulberry breeding for food mulberry leaves.Mulberry (Morus spp.) plants were grown at the Central China Branch of National Mulberry Germplasm Resources, located in Wuhan, Hubei Province, China. A total of 18 Mulberry resources were tested for sensory evaluation  and M. alba L. var. heyebai (HYB) were collected from the mulberry plants for metabolome and transcriptome analysis.aluation . Three tYoung leaves from different varieties of mulberry were picked, cleaned, and boiled for 1 min without any seasoning. The bitterness level of mulberry leaves was then rated from 1 to 9. We defined significant differences in the bitterness levels of the white bud of Guisang12, Taiwan No. 2 and 3 mulberry resources as 2, 4 and 8. The young leaves of three mulberry resources were presented to a trained sensory panel  in sequence after being treated as described above, and the bitterness level of each mulberry leaf was judged using a triangulation test. The group members who completed mulberry leaf bitterness sensory training rinsed their mouths with purified water before and after sample evaluation. The sample evaluation interval was 5 min, and each sample was evaluated twice. The significance of the bitterness level of mulberry leaves was analyzed using SPSS 22.0 software.The sample extraction and professional analytical processes of metabolite detection were performed at Metware Biotechnology Co., Ltd.  following their standard procedures .www.rproject.org, accessed on 15 September 2021), and the data were scaled via unit variance before PCA analysis. The cor function and ComplexHeatmap packages of R software were used to calculate the Pearson correlation coefficient (PCC) between samples and the hierarchical cluster analysis (HCA) of samples and metabolites, respectively. R-package MetaboAnalystR was used to generate VIP values of OPLS-DA results, which also included score plots and permutation plots. Significantly regulated metabolites between groups were determined using VIP ≥ 1 and |log2 fold change| ≥ 1. The identified metabolites were annotated using the KEGG compound database, and then mapped to the KEGG pathway database. The pathways with significantly regulated metabolites were then input into MSEA , and their significance was determined using the p-values of hypergeometric tests.The qualitative and quantitative mass spectrum data were analyzed using Analyst 1.6.3 software . Unsupervised principal component analysis (PCA) was performed within the statistical function prcomp in R software  was introduced to map reads to the reference genome of Morus notabilis (taxid:981085). The mapped reads of each sample were assembled using StringTie (v1.3.4d) with default parameters and gffcompare software (v2.2.1) to reconstruct a comprehensive transcriptome from all transcriptomes of each sample. The novel genes were predicted by assembling the mapped reads and comparing them with the original genome. To obtain the annotation information, the unigenes were compared with public databases, including KEGG, GO, KOG, NR, Swiss-Prot, Pfam and Trembl. To evaluate gene expression levels, transcript abundances were calculated and normalized to FPKM (fragments per kilobase of transcript per million fragments mapped). featureCounts v1.6.2 was used to calculate the FPKM of each gene based on the gene length. PCA was performed within the statistical function prcomp in R software to obtain the relationships among, and variability between, samples. The differential expressions between the two groups were analyzed using DESeq2 (v1.22.1), and the p value was corrected using the Benjamini and Hochberg method. The corrected p value < 0.05 and |log2 fold change| ≥ 1 were used as the threshold for significant difference expression. Enrichment analysis was conducted based on a hypergeometric test. For KEGG, a hypergeometric distribution test was conducted with the pathway as a unit. For GO, it was based on the GO term.Libraries were constructed from enriched mulberry leaf mRNA and sequenced using the Illumina HiSeqTM 4000 platform  by Metware Biotechnology Co., Ltd. . The We performed integration analyses of differentially accumulated metabolites (DAMS) and differentially expressed genes (DEGs) from metabolic fractions and transcriptomes to determine the enrichment of pathways. The transcription–metabolite network was constructed using the gene metabolite network with a Pearson correlation coefficient (PCC) > 0.8.g for 20 min and the supernatant was collected. All supernatants were diluted 20 times for the determination of soluble sugars. In total, 40 µL of diluted sample solution was added to 40 µL distilled water, 20 µL anthrone-sulfuric acid solution, and 200 µL concentrated sulfuric acid, mixed, and then placed in a 95 °C water bath for 10 min. After cooling to room temperature, 200 µL was taken from a 96-well plate at 620 nm to determine its absorbance value. The 5 mg/mL glucose standard solution was diluted into 0.8, 0.6, 0.5, 0.4, 0.3, 0.2 and 0.1 mg/mL gradient solutions, which were measured at the same time as the sample, and the standard curve was drawn according to the relationship between the concentration and light absorption value. Total soluble sugar (mg/g) = C × V ÷ M × F. C; total soluble sugar content calculated from the standard curve, mg/mL; V, extract liquid volume, 1 mL; M, sample weight, g; F, dilution ratio. The software GraphPad Prism 5 was used to analyze the Pearson correlation coefficients of the soluble sugars and bitter taste levels.To detect the concentration of soluble sugars, 100 mg leaves were sampled and ground into a homogenate with 1 mL distilled water. After being incubated at 80 °C for 20 min, they were centrifuged at 12,000× p ≤ 0.05). The mulberry variety with the highest bitter taste out of all the mulberry leaves was Heyebai, followed by Dabaiya and Ribentiancheng. The two varieties with the lowest bitter taste of mulberry leaves were Bai436 and Bai185. The mildly bitter mulberry variety Bai436 (B436) and the extremely bitter mulberry variety Heyebai (HYB) were selected for wide-target metabolomics and transcriptomics analysis  of DEGs and DAMs showed significant differences between the B436 and HYB groups, explaining 51.3% and 71.8% of the total variation, respectively A,B. Thesp-adjusted value < 0.05 and |log2FC| ≥ 1, p-value < 0.05, DESeq2 v1.22.1 was used to analyze the DEGs between the two groups. B436 compared with the HYB of mulberry leaves was detected in 3809 DEGs, of which there were 2231 and 1578 DEGs raised and lowered , indicating that the soluble sugar content was one of the main factors for the bitter astringency difference in mulberry leaves (According to the multi-omics analysis, the content of sweet-taste metabolites may be closely related to the difference in bitter taste between the two mulberry young leaves. We detected the soluble sugar content in the young leaves of six mulberry resources with different bitterness levels, as shown in y leaves B.Bitter taste is one of the basic tastes, and its most prominent feature is that it is easily perceived by people with a very low threshold . Among wBitter substances in mulberry leaves are very varied. In this study, the changes in metabolites related to bitter taste in the DAMs of the young mulberry leaves of two mulberry varieties were very complex. Compared with HYB, 41 kinds of phenolic acids were up-regulated and 21 were down-regulated in B436. There were nine kinds of L-type amino acids, of which six were up-regulated and three were down-regulated. There were 68 flavonoids, 38 of which were up-regulated and 29 which were down-regulated. There were 16 kinds of alkaloids, 8 of which were up-regulated and 8 of which were down-regulated. These results indicate that the formation mechanism of bitter taste in mulberry leaves is the comprehensive embodiment of a variety of bitter metabolites . The L-tIn the food industry, adding an appropriate amount of sweetness to bitterness can suppress part of the bitterness and make the overall taste mild and delicate ,38. A toTo sum up, bitter substances in mulberry leaves are diverse and complex, and the formation of a bitter taste in mulberry leaves is a comprehensive reflection of a variety of bitter metabolites. In this study, many amino acids, flavonoids, phenolic acids, alkaloids and other metabolites related to the bitter taste of mulberry leaves detected were found to have drug activity, and have medicinal effects such as antibacterial, anti-tumor and anti-aging effects, as well as blood sugar and blood pressure lowering effects ,40,41. A