Background: Hepatitis B virus (HBV) is a considerable public health concern in India, characterized by a significant number of chronically infected individuals. India falls into the category of an intermediate HBV endemic country, with approximately 40 million people chronically infected. Public awareness and knowledge about HBV are crucial for prevention, diagnosis, and treatment. This study aimed to assess the knowledge and vaccination status of the general public regarding hepatitis B in India.Methods: An online cross-sectional survey was conducted with 3,545 participants, excluding healthcare workers and individuals below 18 years old. The survey evaluated knowledge in four domains: cause and spread of the disease (Domain A), organ affected and consequences (Domain B), treatment (Domain C), and vaccination (Domain D). The survey used a scoring scale from -20 to +22 points. Vaccination status was also assessed.Results: The mean knowledge score of the respondents was 6.89 (±5.88). Only 25% of the respondents demonstrated good knowledge (score ≥12), while 31% had fair knowledge (score 6-11), 40% had poor knowledge (score 0-5), and 4% had incorrect knowledge (score <0). Males, individuals aged 18-60 years, respondents with higher education levels, and urban residents had significantly higher mean knowledge scores (p < 0.01). In the domain-specific analysis, 47% of the respondents had good knowledge in Domain B, while 43% of the respondents had good knowledge in Domains C and D. Only 17% of the respondents had good knowledge in Domain A. Overall, 22.7% of the respondents had received all three doses of the HBV vaccine, with higher vaccination rates among males, individuals aged 18-60 years, those with higher education levels, and urban residents.Conclusion: The study revealed limited knowledge about HBV among the general public in India. Vaccination rates were also suboptimal. Public health interventions, including educational campaigns and targeted vaccination programs, are needed to improve awareness and vaccination coverage, ultimately reducing the burden of HBV infection. Hepatitis B virus (HBV) infection remains a significant global public health concern, with an estimated 296 million people living with chronic HBV infection and nearly 887,000 deaths occurring annually due to its complications, such as cirrhosis and hepatocellular carcinoma -4. DespiThere is a lack of large-scale population studies on the prevalence of HBV in India, with most data coming from blood bank screening. India is classified as an intermediate HBV endemic country, with an estimated 40 million chronically infected individuals . EstimatSeveral studies have been conducted globally to assess the knowledge, awareness, and vaccination status of hepatitis B among various populations -22. HoweThis study aimed to assess the knowledge and vaccination status of hepatitis B among the general public in India using an online cross-sectional survey. The findings of this study will provide valuable insights for policymakers and healthcare providers to improve public awareness, increase vaccination coverage, and ultimately reduce the burden of hepatitis B in India.Study design and participantsA cross-sectional survey was conducted to assess the knowledge and vaccination status of hepatitis B among the general public in India. The study population comprised adults aged 18 years and above, excluding healthcare workers.Aims and objectivesThe two specific objectives of the study were (1) to assess the knowledge of the general public regarding hepatitis B in India, specifically focusing on understanding the cause and spread of the disease, the affected organ and its consequences, treatment options, and vaccination, and (2) to evaluate the vaccination status of the general public in relation to hepatitis B in India, including the proportion of respondents who had received all three doses of the HBV vaccine and identifying any demographic factors associated with higher vaccination rates.ParticipantsThe inclusion criteria were all consenting adults aged 18 years and above, while the exclusion criteria were healthcare workers.Data collectionThe survey was conducted using a validated questionnaire, which was administered through the SurveyMonkey platform. The questionnaire was distributed to potential participants via text message, social media, and WhatsApp. The participants provided informed consent before responding to the survey. The institutional ethics committee of Sir Ganga Ram Hospital, New Delhi, India, approved the study on August 9, 2021 (vide EC/07/21/1920). Repeated participation by the same person (using the same IP address) was prevented by the survey platform.The questionnaire was carefully designed to cover various aspects of hepatitis B knowledge and awareness among the general public, providing a comprehensive understanding of the participants' knowledge and vaccination status related to hepatitis B. To ensure the validity of our scoring system, we undertook a thorough process of questionnaire validation and piloting, which was carried out in a test sample of 635 subjects. During this process, the consistency and reliability of the questionnaire (including the scoring criteria) was assessed. The validation phase aimed to confirm that the scoring system appropriately differentiates between participants' knowledge levels and aligns with established standards for evaluating health-related knowledge in public health research. The validation was done by the "Validation Committee" of Sir Ganga Ram Hospital, and the final approval was given on December 14, 2021. The internal consistency of the questionnaire, as measured by Cronbach's alpha, was found to be 0.799, indicating good reliability.Survey questionnaireThe questionnaire used for this survey was designed by the authors to assess the knowledge and awareness of hepatitis B among the general public in India. The design process involved an extensive review of existing literature -22 on HBThe questionnaire was divided into several sections, comprising a total of 22 questions Table .The domains and their respective questions are as follows: (1) Inclusion and exclusion criteria (two questions): These questions were used to determine eligibility for participation in the survey based on the predefined inclusion and exclusion criteria. (2) Demographic profile of subjects (four questions): This section collected demographic information of the participants, including age, gender, education level, and geographic location. (3) Domain A (knowledge about the cause of disease and its method of spread (six questions)): This domain assessed participants' understanding of the causes of hepatitis B and how the virus is transmitted from one person to another. (4) Domain B (knowledge about which organ gets affected by HBV and what HBV can cause (four questions)): This section evaluated the participants' knowledge of the primary organ affected by hepatitis B (the liver) and the potential complications or consequences of the disease, such as cirrhosis, liver failure, and liver cancer. (5) Domain C (knowledge about treatment (three questions)): This domain assessed the participants' awareness of the available treatment options for hepatitis B, including antiviral medications and liver transplantation, and the importance of regular monitoring and follow-up care. (6) Domain D (knowledge about vaccination (three questions)): This section evaluated the participants' understanding of the hepatitis B vaccine, its effectiveness, and the recommended vaccination schedule. (7) Vaccination status of the subject (one question): This question aimed to determine the vaccination status of each participant, specifically whether they had received the hepatitis B vaccine or not.Scoring of points in the questionnaireThe scoring system for the questionnaire was designed to assess the participants' knowledge about hepatitis B based on their responses. The questionnaire had a total of 22 questions, with a maximum possible score of 22 points and a minimum possible score of -20 points. The scoring categories were as follows: (A) Good knowledge (≥12 points): Participants who scored 12 points or higher were considered to have a good knowledge of hepatitis B. This indicates a strong understanding of the disease, its causes, transmission, affected organs, complications, treatment options, and vaccination. (B) Fair knowledge (6-11 points): Participants who scored between 6 and 11 points were considered to have fair knowledge about hepatitis B. This suggests a moderate understanding of the disease and its related aspects, but it also indicates that there might be room for improvement in certain areas. (C) Poor knowledge (0-5 points): Participants who scored between 0 and 5 points were considered to have poor knowledge of hepatitis B. This indicates a limited understanding of the disease, with significant gaps in knowledge about its causes, transmission, complications, treatment, and vaccination. (D) Wrong knowledge (<0 points): Participants who scored less than 0 points were considered to have wrong knowledge about hepatitis B. This suggests that not only do they lack accurate information about the disease, but they also hold misconceptions or incorrect beliefs about it.The scoring system enabled the researchers to categorize the participants' knowledge about hepatitis B effectively, helping to identify areas where educational interventions may be needed to improve public understanding of the disease.Data analysisDescriptive statistics were used to summarize the demographic characteristics of the participants and their knowledge and vaccination status concerning hepatitis B. Frequencies and percentages were calculated for categorical variables, while means and standard deviations were computed for continuous variables. The internal consistency of the questionnaire was assessed using Cronbach's alpha to determine the reliability of the items. Associations between demographic factors, knowledge, and vaccination status were examined using appropriate statistical tests. Statistical analysis was performed using IBM SPSS Statistics version 25 . The significance level was set at p < 0.05.Demographic profile of the respondentsThe survey was rolled out on December 15, 2021, and the last response was recorded on March 11, 2023. During this period, the total number of responses received was 4,158. Of these, healthcare workers were 507 and subjects below 18 years of age were 106, so these responses were excluded. Hence, the remaining 3,545 responses were included for analysis.Table The majority of the participants were between 18 and 60 years old, with 89% falling within this age range, while only 9% were over 60 years old and 2% did not reveal their age. In terms of gender, 61% were males, 22% were females, and 17% did not reveal their gender. Regarding education status, 69% had graduated or completed post-graduate education, 9% had completed education till 12th class, and 5% had education less than 12th class. A proportion (17%) did not reveal their education status. Finally, 73% of the participants lived in urban areas, 11% in rural areas, and 17% did not reveal their place of residence.Overall score of the respondentsThe mean score obtained by all the respondents (n = 3545) was 6.89 (±5.88). The maximum score was 22 and the minimum score was -10. Only 882 (25%) respondents had good knowledge and scored ≥12 points, while 1111 (31%) respondents had fair knowledge and scored between six and 11 points; 1407 (40%) had poor knowledge and scored between zero and five points, and 145 (4%) respondents had wrong knowledge and scored less than zero points.Figure Males scored higher points (mean 8.16 ± 5.54) than females and those who did not specify their gender . The respondents in the age group of 18 to 60 years scored higher points than those who are more than 60 year old or those who did not specify . The respondents who were graduates or attained post-graduate education scored higher points than those who were less educated or those who did not specify . Finally, respondents living in the urban area scored more points than those living in the rural area or those who did not specify .Domain-specific analysisThe survey questionnaire tested the respondents’ knowledge on HBV in four domains: Domain A (knowledge about cause of disease and its method of spread), Domain B (knowledge about which organ gets affected by HBV and what HBV can cause), Domain C (knowledge about treatment), and Domain D (knowledge about vaccination).Figure In Domain A, the maximum possible score was 12 and the minimum was -10. The mean score of all the respondents was 2.5 ± 3.62 points. The mean scores of all the subgroups were below 3, except the subgroups of graduate and post-graduate level respondents and urban respondents, both of which had scores barely above 3. In Domain B, the maximum possible score was 4 and the minimum was -4. The mean score of all the respondents was 2.06 ± 1.77. In the subgroup analysis, the mean scores of the male respondents, 18- to 60-year-old respondents, graduate and post-graduate level respondents, and urban respondents were above 2. In Domain C, the maximum possible score was 3 and the minimum was -3. The mean score of all the respondents was 1.05 ± 0.97. The mean scores of the male respondents, 18- to 60-year-old respondents, graduate and post-graduate level respondents, and urban respondents were above 1. Similarly, in Domain D, the maximum possible score was 3 and the minimum was -3. The mean score of all the respondents was 1.28 ± 1.29. The mean scores of the male respondents, 18- to 60-year-old respondents, more than 60-year-old respondents, graduate and post-graduate level respondents, and urban respondents were above 1.When the scores obtained in each domain were categorized into good knowledge, fair knowledge, poor knowledge, and wrong knowledge, it was observed that 17% respondents had good knowledge in Domain A, 47% had good knowledge in Domain B, and 43% had good knowledge in Domains C and D  and 44% having either poor or incorrect knowledge. Our survey has several strengths that lend credibility to its findings. First, the large sample size of 3,545 respondents increases the statistical power and generalizability of our results. Second, the comprehensive survey design, which assessed knowledge across four domains , allows for a more in-depth understanding of the respondents' knowledge gaps and areas of strength. In addition, the inclusion of demographic variables  enabled us to identify specific subgroups with lower knowledge scores and vaccination rates, providing valuable insights for targeted interventions. Lastly, the extended data collection period, spanning from December 2021 to March 2023, offers a more accurate representation of the general public's knowledge and vaccination status over time, accounting for potential fluctuations due to changes in public health messaging or other external factors.This study sheds light on the public's understanding and hepatitis B vaccination status in India, revealing an overall suboptimal level of knowledge, with just 25% of participants demonstrating a good level of understanding -11. ThisOur findings align with previous studies showcasing similar subpar levels of hepatitis B awareness across different populations and geographical locations -22. ThisOur survey unveiled trends within the dataset, identifying males, younger respondents 18-60 years), those with higher education levels, and urban residents as having relatively higher knowledge scores across all domains. These demographic and socioeconomic factors seem to influence the level of knowledge about hepatitis B, an insight that aligns with previous studies and could guide future targeted educational campaigns . Convers-60 yearsThe domain-specific knowledge analysis exposed distinct areas of understanding among respondents. Only 17% had a good grasp of the disease's cause and its transmission methods, indicating a considerable gap in comprehension that could contribute to ongoing virus transmission . This arRegarding vaccination status, the study found a concerning shortfall with only 22.7% of respondents having received all three doses of the HBV vaccine, falling significantly short of the World Health Organization's target of 90% coverage . These lThe limitations of our study include the reliance on self-reported data, which may be subject to recall bias, and the use of an online survey, which may not be representative of the general population due to potential differences in internet access or digital literacy. Nonetheless, our findings provide valuable insights into the current state of hepatitis B knowledge and vaccination status among the general public and highlight areas for future intervention.The low level of knowledge about hepatitis B and the suboptimal vaccination rate among our survey respondents underscore the need for targeted public health interventions to improve awareness and vaccination coverage. Educational campaigns should focus on the general public, with particular emphasis on females, older individuals, those with lower education levels, and rural residents, who demonstrated lower knowledge scores and vaccination rates in our survey. In addition, efforts should be made to increase awareness about the importance of completing the full vaccination schedule, as partial vaccination was reported by 8.6% of our respondents. Comprehensive strategies addressing both health literacy and vaccination coverage are vital for achieving the national and global targets for HBV control .This study sheds light on the inadequate knowledge and low vaccination rates related to hepatitis B among the general public in India. The findings highlight that only a quarter of respondents demonstrated good knowledge, while a considerable proportion lacked accurate understanding. Similarly, the study identified a concerning shortfall in vaccination rates, with only 22.7% having completed the recommended vaccination schedule.These outcomes underscore the urgency for targeted public health interventions. Tailored educational efforts are crucial, particularly for vulnerable groups, such as women, the elderly, those with lower education levels, and rural residents. Equally important is the need to enhance accessibility to vaccination services and promote adherence to the complete vaccination regimen. The study underscores the need for comprehensive initiatives that bridge knowledge gaps and enhance vaccination coverage. By addressing these issues, policymakers and healthcare providers can work toward reducing the impact of hepatitis B in India.


Different from the transparent PLA filament, 10% PBS/PLA filament presented a milky white appearance. The printed 10% PBS/PLA specimen had a smooth surface, while the surface of the printed PLA was rough. All the results indicated that the printed 10% PBS/PLA specimen had good comprehensive properties, including improved mechanical properties, crystallization performance, and surface quality than PLA, as well as proper wettability and water absorption. The prominent conclusion achieved in this work was that 10% PBS/PLA should be an ideal candidate for biodegradable feedstock among all the PBS/PLA blends for FDM 3D printing.Poly(lactic acid) (PLA) and Poly(butylene succinate) (PBS) were chosen as raw materials and melt blended by a twin screw extruder and pelletized; then, the pellets were extruded into filaments; after that, various PBS/PLA blending samples were prepared by Fused Deposition Molding (FDM) 3D printing technology using the filaments obtained and the effect of the dosage of PBS on technological properties of 3D-printed specimens was investigated. For comparison, the PLA specimen was also prepared by FDM printing. The tensile strength, tensile modulus, thermal stability, and hydrophilicity became poorer with increasing the dosage of PBS, while the flexural strength, flexural modulus, impact strength, and crystallinity increased first and then decreased. The blend containing 10% PBS (10% PBS/PLA) had the greatest flexural strength of 60.12 MPa, tensile modulus of 2360.04 MPa, impact strength of 89.39 kJ/m This teAs one of the most common 3D printing technologies that can be used to make both daily supplies and industrial products, fused deposition modeling (FDM) has attracted engineers and scientists greatly in recent years because of its wide range, non-toxicity of materials, convenient operation, high production efficiency, little maintenance needed, simple structure of equipment, powerfulness to provide durable printing procedures, simple operation, high strength and toughness of materials ,5. It caHowever, PLA by itself has shortcomings; it is relatively hard, its toughness, flexibility, and elasticity are not so good, its printed specimens are easy to be broken, and it also has poor appearance quality. All these limit the widespread application of PLA in the 3D printing area. It is thus of great significance to modify PLA to improve its weak points and meet the requirement for 3D printing; one of the primary methods is by blending PLA with other polymers .w/w) blends and compared them with those of PLA. The impact strength of each blend was greater than that of the PLA when highly flexible PBS was blended with PLA. However, the tensile performances became poorer. Suparanon et al.  = [10/90] was 62.9 °C, and for the blend [PBS/PLA] = [20/80], Tg reduced to 61.7 °C, which were 0.9 °C and 2.1 °C lower than that of pure PLA, respectively. The reason was that the content of PBS in the printed samples was relatively low and thus could disperse well in the blend, leading to partial miscibility of the polymers in the amorphous phase [The glass transition temperature  increased, as evidenced in Many factors would affect the water absorption by the printed samples, such as infill density, the composition of the sample, the relative dosage of different components, and the interfacial compatibility . For theThe MFR was investigated to determine the rheological characteristics of the samples. The results are illustrated in 2, which were increased by 54.65%, 61.04%, and 14.78%, respectively, from those of printed PLA samples. After blending with PBS, the fracture of the sample turned from a brittle manner to a little ductile one. SEM observation showed that the fracture surface of the printed PBS/PLA sample became rough while that of the printed PLA sample was smooth. When more PBS was used, serious defects could also be found in the SEM pictures.The tensile properties became poorer when more PBS was used. However, the samples containing 10% PBS had the best bending and impact performances; the bending strength, the flexural modulus, and the impact strength of the printed 10% PBS/PLA sample were 60.12 MPa, 2360.04 MPa, and 89.39 kJ/mi and Tp were only reduced by 4.0 °C and 3.7 °C, respectively. When 20% or more PBS was used, a shoulder peak appeared on the right of the main peak in each DTG curve.The printed specimens became more thermally unstable with increasing dosage of PBS. When 10% PBS was used, however, the TThe samples containing 10% PBS had the greatest degree of crystallinity, the least water absorption among all the samples, and a much better visual appearance than pure PLA.With the increasing dosage of PBS, both the water contact angles and the MFR of the samples increased monotonically.In this study, PBS was used to modify PLA for FDM 3D printing, and the mechanical properties, fracture surface morphologies, thermal stability, melt and crystallization behavior, wettability, visual appearance, as well as flowability of various samples were investigated. The concluding remarks of this study are as follows:To sum up, blending with the proper amount of PBS contributes to improving the comprehensive printing performance and the quality of the printed samples, among which the best percentage of PBS should be 10%. The excellent comprehensive performances made the 3D-printed PBS/PLA blends have good market prospects in many areas, such as wearable smart devices, bioengineering, and medical devices.


Pruning is an important agronomic measure in tea plantation management. In this study, we analyzed the effect of pruning on gene expression in tea leaves from a transcriptomics perspective and verified the results of a transcriptomic analysis in terms of changes in physiological indicators of tea leaves. The results showed that pruning enhanced the gene expression of nine metabolic pathways in tea leaves, including fatty acid synthesis and carbohydrate metabolism, nitrogen metabolism, protein processing in the endoplasmic reticulum, and plant hormone signal transduction, thereby promoting the growth of tea plants and increasing tea yield. However, pruning reduced the gene expression of nine metabolic pathways, including secondary metabolites biosynthesis, flavonoid biosynthesis, phenylpropanoid biosynthesis, and sesquiterpenoid and triterpenoid biosynthesis, and lowered the content of caffeine, flavonoids, and free amino acids in tea plant leaves. In conclusion, pruning could promote the growth of tea plants and increase the yield of tea, but it was not conducive to the accumulation of some quality indicators in tea leaves, especially caffeine, flavonoids, and free amino acids, which, in turn, reduced the quality of tea. This study provides an important theoretical reference for the management of agronomic measures in tea plantations. Tea plants are an important economic crop, and the number of the world’s tea-producing countries and regions reached more than 60 in 2022, with a tea-drinking population of more than 2 billion. China is the largest tea producer and consumer, accounting for 47% of global tea production. In 2021, total tea production in China reached 3.08 million tons, with the total output value of dried gross tea exceeding CNY 290.9 billion, driving 80 million tea farmers to increase their incomes and become rich [Fujian Province is the main tea-producing region in China, and Wuyi rock tea is produced in Wuyi Mountain, Fujian Province, which is unique because of the particularity of its planting environment. The production process of Wuyi Rock Tea is semi-fermented according to the degree of fermentation, Wuyi Rock Tea belongs to a kind of oolong tea. At present, the main varieties of tea plants in Wuyi Mountain are basically shrubs. It is well known that tea plants require a lot of manual management in the planting process to improve tea yield and quality, including irrigation, fertilization, weeding, pest control, pruning, and other agronomic measures ,3,4,5. ICamellia sinensis cv. Meizhan), the main tea plant variety grown in Wuyi Mountain, and verified the results of the transcriptomics analysis in terms of the changes in leaf physiological indicators, with a view of providing a certain reference for the cultivation and management of tea plants. Pruning is also a form of stress for the tea plant itself. It has been reported that plants under stress stimulate secondary messengers to activate cellular signaling, which, in turn, triggers transcriptomic responses related to plant defense, allowing plants to adopt different strategies to cope with the effects produced by different stresses ,14. Jia 2 values of the three biological replicates of the unpruned tea plant (MC) transcriptome were above 0.991, and the R2 values of the pruned tea plant (MP) transcriptome were above 0.988. It can be seen that the transcriptome data of different samples in this study were repeatable and reliable for in-depth analysis.After Illumina sequencing, a total of 251,514,572 raw reads were obtained from the six libraries. In total, 243,980,532 clean reads were obtained after filtration, and the percentage of clean reads relative to raw reads in each library was over 99.97% , indicatp = 0.92). The results of PCoA analysis showed  and unpruned (MC) tea leaves showed A that ths showed B that ths showed C that cop < 0.005), and the Q2 value for predictability was 0.994 (p < 0.005). It can be seen that both R2Y and Q2 values of the model reached significant levels, indicating that the model had a good degree of fit and high reliability and could be used for further analysis. The results of the OPLS-DA scoring map showed  . Meanwhip showed E that OPp showed F that 71p < 0.05). The gene expression of 19 metabolic pathways was further analyzed, and the results showed . Visible pruning was beneficial to promote the growth of tea plants and thus increased the yield of tea. Interaction analysis showed , with a planting area of about 0.8 ha. The tea plants were 8 years old and had not been pruned before. In August 2021, the test area was evenly divided into 6 areas, three of which had tea plants pruned for treatment (MP), i.e., three replicates, and the other three areas had tea plants not pruned, i.e., three control replicates (MC). Tea plants were pruned by trimming 3–5 cm from the tea plant canopy, and pruning litter was left on the surface of the original tea plant soil. The experimental site was Foguoyan tea plantation located in the Wuyi Mountain Scenic Area  at an altitude of 239 m. The tea plant variety was Meizhan  were applied, respectively. Other tea plantation management measures were treated the same for both pruned and unpruned treatments. The basic conditions of the tea plantation during the experiment are shown in 2 in each area of the tea plantation and picking them according to the traditional tea picking standard (three leaves and one core), taking the average, i.e., one replicate, and converting it to the yield of fresh tea leaves per hectare of the tea plantation. Chlorophyll content was determined by randomly selecting 10 mature leaves from the tea plantations in each area, using a chlorophyll detector , and taking the average value, which was one replicate. During the experiment, there were three independent areas for both pruned and unpruned treatments and three independent replicates for the determination of each indicator.Leaf area, hundred-bud weight, chlorophyll content of tea plants, and tea yield in pruned and unpruned areas were measured . Leaf arThe quality indicators of tea leaves were mainly determined by the content of tea polyphenols, theanine, caffeine, free amino acid, flavone, and soluble sugar, with 3 replicates per treatment. The specific testing methods were briefly described as follows: the tea leaves collected were fixed at 105 °C for 15 min, dried at 80 °C to constant weight, ground, and passed through 60 mesh sieves for the determination of the quality indicator of tea leaves. The determination of tea polyphenol content was referred to the national standard of the People’s Republic of China, “Determination of total polyphenols and catechins content in tea (GBT 8313-2018)”, and was determined via the folinol colorimetry method . TheaninRNA was extracted using the TRIzol method  and treated with RNase-free DNase I . RNA degradation and contamination were monitored on 1% agarose gels. RNA was quantified using Agilent 2100 Bioanalyzer  and assessed for quality and integrity by NanoDrop spectrophotometer .® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations, and indicator codes were added to assign sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations at elevated temperatures in NEBNext First Strand Synthesis Reaction Buffer (5×). The first strand of cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second-strand cDNA synthesis was then performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends through exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, the NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments preferentially 200–250 bp in length, library fragments were purified with the AMPure XP system . Then, 3 μL USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Indicator (X) primers. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system. Library preparations were sequenced on an Illumina Hiseq 4000 platform by Beijing Allwegene Technology Company Limited , and paired-end 150 bp reads were generated.A total amount of 1.5 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNextQuality control: Raw data (raw reads) with fastq format were first processed via internal perl scripts. First, Trimmomatic software (v0.33) was used to remove reads containing adapters, and then reads with N-containing ratios greater than 10% were removed, and reads in which the number of bases with a quality value of Q < 20 accounted for more than 50% of the entire read were removed to obtain lean data (clean reads). At the same time, Q20, Q30, GC content, and sequence repeat levels were calculated for clean data. All downstream analysis was based on clean data with high quality .Mapping analysis: Adaptor sequences and low-quality sequence reads were removed from the dataset. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence via STAR. Only reads with a perfect match or mismatch were further analyzed and annotated based on the reference genome.Quantification of gene expression levels: HTSeq v 0.5.4 p3 was used to calculate the number of reads mapped to each gene. Gene expression levels were estimated by fragments per kilobase of transcript per million mapped fragments (FPKM) .Difference analysis: DESeq2 v1.22.1 was used to analyze the differential expression between the two treatments, and the P value was corrected using the Benjamini & Hochberg method. The corrected P value and |log2foldchange| were used as the threshold for significant difference expression .Key gene screening: The orthogonal partial least squares discrimination analysis (OPLS-DA) model between two treatments was constructed based on the acquisition of differential genes . The varExcel 2017 software was used to calculate the mean value and variance of the data. Rstudio 3.3 software was used to make cloud and rain plots, principal component plots, volcanic plots, OPLS-DA models (Package used in this regard was ropls and mixOmics), box plots, and trend plots. Based on the Pearson correlation coefficients between different indicators, Cytoscape_v3.9.1 software was used to produce a correlation network plot.In this study, we analyzed the effect of pruning on growth indicators, quality indicators, and leaf gene expression of tea plants. The results showed  that pru


Environmental pollutants’ (EPs) amount and diversity have increased in recent years due to anthropogenic activity. Several neurodegenerative diseases (NDs) are theorized to be related to EPs, as their incidence has increased in a similar way to human EPs exposure and they reproduce the main ND hallmarks. EPs induce several neurotoxic effects, including accumulation and gradual deposition of misfolded toxic proteins, producing neuronal malfunction and cell death. Cells possess different mechanisms to eliminate these toxic proteins, including heat shock proteins (HSPs) and the proteasome system. The accumulation and deleterious effects of toxic proteins are induced through HSPs and disruption of proteasome proteins’ homeostatic function by exposure to EPs. A therapeutic approach has been proposed to reduce accumulation of toxic proteins through treatment with recombinant HSPs/proteasome or the use of compounds that increase their expression or activity. Our aim is to review the current literature on NDs related to EP exposure and their relationship with the disruption of the proteasome system and HSPs, as well as to discuss the toxic effects of dysfunction of HSPs and proteasome and the contradictory effects described in the literature. Lastly, we cover the therapeutic use of developed drugs and recombinant proteasome/HSPs to eliminate toxic proteins and prevent/treat EP-induced neurodegeneration. One of the most pressing health-related issues that societies face at present is the increase of non-transmittable diseases such as neurodegenerative diseases (NDs), as their incidence has increased exponentially in the last decades. In parallel, contaminants released into the environment have increased in both quantity and compound classes, leading to pollution and deleterious effects on living beings. Environmental pollutants (EPs) have been pointed out as one of the possible etiological factors of NDs, even though the complete causes that mediate their induction remain unclear. This is supported by the parallel increase in contaminants  released into the environment and the incidence of NDs, as well as by the fact that these compounds have been found to induce the main hallmarks and symptoms of several NDs in both in vitro and animal studies ,2,3,4,5.In this regard, several environmental contaminants, such as metals including cadmium (Cd), manganese (Mn), aluminum (Al), and lead (Pb), among others, produce neurotoxic effects and have been linked to the generation of various NDs ,9,10,11.The brain regions in which EPs accumulate are the ones in which neurodegenerative events leading to NDs occur; this, together with the fact that EPs share several mechanisms hypothesized to induce them, supports the involvement of EPs in ND induction  1,3,4,1,16,17,22New drug research for ND treatment is an arduous task which has led to few favorable discoveries. A common characteristic of most NDs is the induction of misfolded toxic protein that accumulate, leading to neuronal dysfunction and loss ,24. As sThe refolding and degradation of toxic proteins are mediated by the proteasome system and the heat shock protein (HSP) family, among other mechanisms ,29,30,317β7β7α7), presenting catalytic activity in the β1, β2, and β5 subunits  . T. T74]. TEnvironmental pollutants  have been related to proteinopathy generation and detrimental effects on the expression and function of HSPs ,52,53,61Toxic proteins have been described as inducing ROS generation and initially increasing NRF2 levels; however, in later stages they produce downregulation of the NRF2 pathway ,75. ThusDifferent research works have explored the effects of EPs on proteinopathies and HSP disruption. In this regard, exposure to the essential metal manganese, which is used in several industrial applications, leads to the aggregation of α-synuclein protein , increasCadmium, an industrial heavy metal, induces α-synuclein protein aggregation  and incrArsenic (As), an industrial heavy metal, increases the levels of p-Tau and Aβ peptides ,3 as welMercury (Hg), present in both organic and inorganic compounds, has been shown to increase Aβ and p-Tau levels both in vitro and in vivo . InorganAluminum, a heavy metal widely used in industry, has been observed to increase the content of Aβ and p-Tau proteins and induce their aggregation by increasing their synthesis and decreasing their degradation ,118,119.Paraquat, a widely used herbicide, and Maneb, an extensively used fungicide, have been described as increasing p-Tau and α-syn levels, and PQ, has been shown to increase Aβ levels ,54. ManeAtrazine, a widely used herbicide, has been related to the increase of α-synuclein and TDP-43 levels . AtrazinBisphenol A (BPA), a chemical compound with multiple industrial applications, has been reported to induce Aβ and p-Tau accumulation . BPA 24 Perfluorooctanoic acid (PFOA), a perfluorinated compound with wide industrial applications, and was described to induce Aβ and p-Tau accumulation ,128. PFOOchratoxin A (OTA), a fungal toxin, has been reported to increase the levels of α-synuclein . OTA sinExposure to several insecticides has been related to the induction of proteinopathies, increasing the accumulation of toxic proteins including Aβ, p-Tau, α-syn, and HTT, among others ,135,136.Carbamates  and pyrethroids (bifenthrin) has been described as inducing GRP78 while downregulating HSP72/73 in A549 pulmonary cells after 3 days of treatment. Bifenthrin increased the expression of GRP94 and decreased the expression of HSP27, and methomyl downregulated HSP90 expression . A studyIn addition to the NRF2 mediation of the differences reported in HSP expression following EP exposure, they may be explained by the diversity of several factors employed in the research, such as the use of different protocols, biological models, vehicles, or salts, as well as by gender differences  144,145145. ThesThe accumulation of toxic proteins leads to HSP induction with the aim of reducing their harmful effects. It has been reported that HSPs can help to avoid the conglomeration of Aβ proteins, one of the main hallmarks of AD, and induce the UPS, leading to the elimination of misfolded proteins ,35,151. 1–40/Aβ1–42 and p-Tau proteins are induced by several HSPs ,20731,20UPS dysfunction has been shown to be involved in the pathogenesis of AD . In thisUPS dysfunction and proteasome activity decrease have been reported in the substantia nigra of PD patients. This effect was associated with the decrease of P20S α-subunits levels ,228,229.Several studies have linked UPS and proteasome dysfunction with HD. Proteasome inhibition has mainly been described in the striatum in HD patients, as well as in several HD cell line models . HoweverProteasome dysfunction has been reported in ALS, with inhibition of the proteasome in the spinal cord and a decrease in proteasome β5 subunit levels . These eEP exposure has been associated with the induction of proteinopathies through proteasome inhibition ,187,188.Different therapeutic strategies have been developed to treat the neurotoxic effects induced by proteinopathies. In this sense, antibody therapy has been developed against specific toxic proteins  by dissolving and removing their aggregates ,244. AnoNDs are characterized by being mediated through more than one known toxic protein, and possibly others that are unknown, requiring a therapeutic strategy to target the removal of all types of toxic proteins present in the cells. several strategies have been developed to address this issue, including gene expression upregulation, activation, or administration of recombinant HSPs and/or P20S proteins  24,49,5,246,247.EP-induced stress has been shown to be reduced through HSPs as a protective mechanism, and their overexpression has been shown to reduce the deleterious effects of EPs ,35,158. Different natural compounds such as geldanamycin, carbenoxolone, celastrol, curcumin, gambogic acid, withaferin-A, jujuboside-A, paeoniflorin, myricetin, quercetin, glycyrrhizin, geranylgeranylacetone, resveratrol, capsaicin, and ginkgo biloba, among others, have been shown to induce the expression of HSPs such as HSP70, HSP40, HSP27, HSP30, and others through HSF1 upregulation, HSP90 inhibition, or both ,252,253.2O2 or staurosporine-induced apoptosis [However, there have been several cases in which the compounds induced toxicity, such as the case of GA and 17-AAG ,261, or poptosis ,263. Thupoptosis  has beenpoptosis ,265,266.The proteasome is necessary for the clearance of damaged or misfolded toxic proteins; however, in NDs and after environmental pollutant exposure its activity declines ,204,267.Denaturalizing compounds such as sodium dodecyl sulfate or poly-lysine have been reported to increase the activity of the proteasome, which is suggested to be mediated through partial denaturalization of the α-ring subunits . HoweverProteasome activation has been carried out by increasing catalytic processing, inducing kinase phosphorylation in specific sites of P26S. Multiple P26S phosphorylation sites (>455) which modulate its assembly, stability, and activity have been identified . RolipraNRF2 factor has been shown to induce the upregulation of proteasome subunits . In thisSeveral natural compounds have been identified as P20S activators or inductors as well. In this sense, betulinic and ursolic acids, both of which are triterpene compounds, have been described as activating P20S . BetulinRecombinant HSPs and P20S proteins have been studied as possible therapeutic agents for neurodegenerative processes . IntranaHSP90 and HSP70 levels and P20S activity were decreased following Mn treatment on SN56 cells for 24 h and for 14 days . Aβ and Only small lipophilic molecules can cross the BBB freely, while hydrophilic or large molecules are impeded from crossing it . EndogenAnother strategy is to use nano-pharmaceutical formulations, which can allow these molecules to cross the BBB and reach the specific brain locations where the drugs are needed, thereby reducing the dosage frequency and toxicity ,282. SevThere is increasing evidence pointing out a relationship between environmental pollutants and proteinopathy diseases, highlighting the need to develop further studies researching the involvement contaminants in disease induction and development as well as the mechanisms through which HSPs and proteasome dysfunction mediate its induction. Contaminants with highly different structures are able to induce the same proteinopathies and neurodegenerative effects, making it important to understand the mechanisms through which they mediate these effects in order to improve treatment of proteinopathy-neurodegenerative diseases.Contradictory effects on HSPs have been described after contaminant exposure, highlighting the need to perform further studies in order to elucidate the reasons for these opposite effects and the relevance of different facts such as time of exposure, dose, and gender, among others, as well as which specific HSPs are altered in the brain. Moreover, it should be determined whether the reduction in proteasome activity observed in NDs and after environmental contaminant exposure is mediated by the direct action of EPs or through indirect action due to the accumulation of toxic proteins. Proteasome regulation is very complex, and it is necessary to understand its complete regulation in order to aid in the development of new, efficient, and specific drugs for the treatment of proteinopathies and neurodegenerative diseases induced by EPs. Such data would lead to a better management and treatment of EP-induced proteinopathies.However, several of the drugs developed to date have been shown to present toxicity, and others could potentially induce it. One example of this occurs with NRF2 activators, as proteasome inductors may be able to induce cell proliferation, making them possible tumor inductors . ProteasRecombinant HSPs and P20S appear to be promising non-toxic tools for global treatment of proteinopathies. However, combined treatment with recombinant HSPs and P20S seems to not completely compensate for the effects induced by toxic proteins ,52, indiAddressing all these issues will allow for a complete understanding of these contaminants’ effects on HSPs and the proteasome system, as well as the associated factors that could modify these effects. These developments will allow appropriate calculation of risk following contaminant exposure, help to establish protective actions, and a present more efficient therapeutic approach.Several NDs, including AD, ALS, MS, PD, and PRD, among others, have been suggested to have EPs as possible etiological factors. Dysregulation of the HSP family and decreased proteasome activity, along with the resulting inability to eliminate the high levels of accumulated toxic proteins, appear to mediate the induction of neurodegenerative disorders and neurodegeneration produced after EPs exposure. There is a bidirectional relationship between the accumulation of aberrant proteins and the dysfunction of HSPs and the proteasome system, which together lead to cell damage and loss. Increased proteasome activity and HSP overexpression prevent the accumulation of aberrant proteins and the cell damage induced by them. Therefore, several therapeutic approaches have been developed to achieve this effect, although a number of the drugs designed to date induce toxic effects, and insufficient drug safety studies have been carried out. A possible therapeutic strategy which may have better outcomes and avoid toxicity could be the treatment of individual NDs with specific recombinant proteins for the involved downregulated HSPs together with recombinant P20S. However, new recombinant protein formulations which allow for adequate delivery to the brain need to be developed. Further studies are needed to clarify the role of EPs on HSPs and proteasome dysfunction, as well as to develop more efficient drugs to prevent and treat neurodegenerative proteinopathies.


This study found that microbiota, metabolites, and epithelial genes were coenriched in pathways related to lipid metabolism, energy metabolism, and immune metabolism, which may be involved in the regulation of Tibetan sheep adaptation to plateau environmental changes.Plateau adaptation in animals involves genetic mechanisms as well as coevolutionary mechanisms of the microbiota and metabolome of the animal. Therefore, the characteristics of the rumen microbiome and metabolome, transcriptome, and serum metabolome of Tibetan sheep at different altitudes  were analyzed. The results showed that the rumen differential metabolites at 3500 m and 4500 m were mainly enriched in amino acid metabolism, lipid metabolism, and carbohydrate metabolism, and there was a significant correlation with microbiota. The differentially expressed genes and metabolites at middle and high altitudes were coenriched in asthma, arachidonic acid metabolism, and butanoate and propanoate metabolism. In addition, the serum differential metabolites at 3500 m and 4500 m were mainly enriched in amino acid metabolism, lipid metabolism, and metabolism of xenobiotics by cytochrome P450, and they were also related to microbiota. Further analysis revealed that rumen metabolites accounted for 7.65% of serum metabolites. These common metabolites were mainly enriched in metabolic pathways and were significantly correlated with host genes ( Tibetan sheep is a unique dominant breed on the Qinghai–Tibet Plateau, and it evolved from wild sheep after long-term adaptation. The current livestock inventory exceeds 50 million . They arThe rumen evolved approximately 40 million years ago  and is aTherefore, we hypothesized that rumen microbiota and metabolites could affect host metabolism (reflected by the serum metabolome) through the rumen epithelium, thus affecting Tibetan sheep adaptation to the plateau environment. In this study, the rumen microbiome and metabolome, host transcriptome, and serum metabolome of Tibetan sheep at different altitudes were studied, aiming to answer the following basic question: Do the rumen microbiota, metabolites, rumen epithelial-related gene expression, and host metabolites respond to plateau environmental changes? The differences in the rumen fluid metabolome and host metabolome in Tibetan sheep at different altitudes were compared to explore the regulation of rumen epithelium-related gene expression on metabolite transport and to reveal the influence of common metabolites on host adaptation to plateau environments. This study will reveal the mechanism of the effects of rumen microbes and metabolites on the host metabolic activities of Tibetan sheep at different altitudes, which will help improve the plateau adaptation performance of Tibetan sheep.p value < 0.01, and VIP > 1 were used as screening criteria for differential metabolite analysis. There were 194 differential metabolites between LA and MA, 216 differential metabolites between MA and HA, and 227 differential metabolites between LA and HA, among which 5 common differential metabolites were found between the three altitudes  and cluster analysis revealed distinct differences in the metabolites among the three altitudes. FC > 2, ltitudes , Data S1ltitudes , fructosKEGG functional annotation found  that thep < 0.05); 11 modules in MA-HA were significantly correlated with Fibrobacteres and Synergistetes (p < 0.05); and 12 modules in LA-HA were significantly correlated with Kiritimatiellaeota, Fibrobacteres, and Acidobacteria (p < 0.05). In a previous study, 19 phylum-level and 243 genus-level microbiotas were found between the three altitudes , and theAnaeroplasma, Shuttleworthia, Lysobacter, Pedomicrobium, Solirubrobacter, etc. (p < 0.05); megastigmatrienone was strongly related to Phyllobacterium, Lysobacter, and Lachnoclostridium_1 (p < 0.05); leukotriene D4 was strongly correlated with Solirubrobacter and Pedomicrobium (p < 0.05); and distearoyl phosphatidate was strongly correlated with Lachnospiraceae_FE2018_group, Lysobacter, and Solirubrobacter (p < 0.05). Between MA and HA, ricinoleic acid was correlated with Ruminococcaceae_UCG-007, Ruminococcaceae_UCG-004, and Fretibacterium (p < 0.05); ferulic acid was positively correlated with Butyrivibrio_2, Mailhella, and Tyzzerella_3 (p < 0.05); ferulic acid was negatively correlated with Ruminococcaceae_UCG-007, Phyllobacterium, and Ruminococcaceae_UCG-004 (p < 0.05); Fretibacterium and Ruminococcaceae_UCG-007 were positively correlated with alpha-linolenic acid (p < 0.05); and Lachnospiraceae_NK3A20_group and Mailhella were negatively correlated with alpha-linolenic acid (p < 0.05). Between LA-HA, Anaeroplasma was positively correlated with butyryl-CoA (p < 0.05) and negatively correlated with alpha-linolenic acid (p < 0.05).Correlation analysis of differential metabolites and differential microbiota (at the genus level) found that there was a strong correlation between the differential metabolites at different altitudes and the microbiota at the genus level . Screen|A total of 27,916 expressed genes were detected in the rumen epithelia of Tibetan sheep at three altitudes by transcriptome sequencing, and 2184 differentially expressed genes were identified . Based op value < 0.01, and VIP > 1, we identified 290 differential metabolites between LA and MA, 894 between MA and HA, and 543 between LA and HA  and cluster analysis showed that there were certain differences in serum metabolite composition at the three altitudes. By using the screening criteria of FC > 2, A and HA , while 1A and HA . ScreeniA and HA , adenosiKEGG functional annotation analysis of serum metabolites showed that LA-MA differential metabolites were mainly annotated in amino acid metabolism and lipid metabolism . UpregulLysobacter, Pedomicrobium, and Solirubrobacter (p < 0.05), while they were negatively correlated with Lachnoclostridium_1 (p < 0.05); asparaginyl-glutamine was negatively correlated with Lachnoclostridium_1 (p < 0.05) and positively correlated with Phyllobacterium and Solirubrobacter (p < 0.05). Between MA and HA, 20 metabolites were found to be correlated with 11 microbiota. Specifically, cytochalasin B was negatively correlated with Mailhella and positively correlated with Ruminococcaceae_UCG-007 (p < 0.05). 13,14-dihydro-lipoxin A4 was positively correlated with Butyrivibrio_2 and Mailhella (p < 0.05) and negatively correlated with Fretibacterium, Ruminococcaceae_UCG-004, and Ruminococcaceae_UCG-007 (p < 0.05). Aspartic acid was negatively correlated with Fretibacterium, Quinella, Ruminococcaceae_UCG-004, and Ruminococcaceae_UCG-007 (p < 0.05) and positively correlated with Mailhella (p < 0.05). Between LA and HA, 15 metabolites were found to have certain correlations with 11 microbiota, and biochanin A was found to be positively correlated with Lysobacter, Ochrobactrum, Phyllobacterium, and Solirubrobacter (p < 0.05).WGCNA revealed that the serum metabolites of the three comparison groups were divided into 15, 10, and 11 metabolite modules, respectively, and these modules were correlated with microbiota to a certain extent. Further correlation analysis was carried out on the differential serum metabolites and differential microbiota (genus level). From the heatmap, it can be found that the differential serum metabolites at different altitudes were strongly correlated with the microbiota at the genus level . Using tGPCPD1 and GPD1 genes, and alpha-linolenic acid was negatively correlated with the PECR gene and positively correlated with the LOC101107420 gene. In the comparison of LA and HA, 3-(3-hydroxyphenyl) propanoic acid was found to be positively correlated with the MOAO gene.Further comparative analysis of rumen fluid metabolites and serum metabolites showed that 274 rumen fluid metabolites were consistent with serum metabolites, accounting for 7.65% of the total host metabolites. There were 3 common differential metabolites between LA and MA, 10 common differential metabolites between MA and HA, and 6 common differential metabolites between LA and HA . In LA-MRuminococcaceae and Butyrivibrio_2 at medium and high altitudes, and Ruminococcaceae can degrade cellulose and starch [Butyrivibrio is positively correlated with the number of methane bacteria [Anaeroplasma is positively correlated with butyryl-CoA at low and high altitudes. Anaeroplasma is positively correlated with body obesity [Previous studies on plateau animals have mainly focused on the genome level and physiological and biochemical levels of blood, lacking the perspective of microbes, metabolites, and host interactions. In this study, by integrating rumen microbes, rumen fluid metabolomes, serum metabolomes, and host transcriptomes, we studied the mechanism of influence of rumen microbes, metabolites on host genes, and the metabolic activities of Tibetan sheep at different altitudes to reveal the response of Tibetan sheep to plateau environmental changes. This study found certain differences in the rumen fluid metabolites of Tibetan sheep at different altitudes. Fructoselysine in MA increased significantly, while fructoselysine in LA and HA decreased. Fructoselysine can be converted to butyric acid , which id starch ,41. The bacteria . Meanwhibacteria , and ferbacteria . Metabol obesity . In the  obesity . These rMHCII gene is upregulated, thereby affecting immune indices and reducing the content of downstream metabolite leukotriene D4, which is a lipid mediator closely related to inflammation [ACADS, HMGCL, HMGCS2, EHHADH, ECHS1, etc.)  pathway at low altitudes compared with medium and high altitudes. Purines are important factors in cell proliferation and immune regulation [How are rumen metabolites transported to the corresponding organs and tissues for regulation? We further analyzed the interaction between these metabolites and the host and found that there was a certain correlation between the gene module and the metabolite module of the rumen epithelium. The differentially expressed genes and metabolites between low altitude and high altitude were enriched in asthma, arachidonic acid metabolism, biosynthesis of amino acids, and other pathways. The asthma pathway regulates respiratory pathways , in whicammation . It alsoammation . These r1, etc.) , the con1, etc.) . In addi1, etc.) , which pgulation . The adegulation . Some upgulation ,51, imprgulation . HoweverLysobacter, Pedomicrobium, and Solirubrobacter) between low and middle altitudes. 6-hydroxynicotinic acid is involved in regulating respiratory-related diseases and plays a positive role [gene-GPCPD1 and gene-GPD1 in the comparison between middle altitude and high altitude, and gene-GPCPD1 was related to choline metabolism [PECR gene. In the rumen, the increased content of alpha-linolenic acid at high altitude may be involved in the regulation of intestinal flora, as well as anti-inflammatory effects, and studies have also found that the PECR gene regulates lipid metabolism [MOAO gene, and the metabolite was enriched in phenylalanine metabolism (ko00360), which was found to reduce the occurrence of inflammation [What is the relationship between serum metabolites and rumen microbiota? The conjoint analysis revealed that there was a significant positive correlation between the differential metabolite 6-hydroxynicotinic acid and some microbiota from plant soil ,54 , and all the experimental animals were in good health, in the nonpregnant stage, and in the same physiological state. During the same period, they ate natural grass freely without any supplementary feeding in the plateau environment. Forage species and chemical composition were described in the previous article [g, 20 min, 4 °C), and serum was isolated and stored at −20°C for serum metabolome determination. Rumen fluids were collected by a rumen vacuum sampler (pipe from the mouth into the rumen), and rumen fluids were divided into frozen tubes and stored in liquid nitrogen for subsequent 16S rRNA sequencing and metabolome determination. Subsequently, according to the traditional slaughter method, a sterilized knife was used to cut the jugular vein of the neck of the test sheep, and death was caused by instant bloodletting. Then, the abdominal cavity was dissected, the rumen organs were removed, and epithelial tissue samples from the ventral portion of the rumen were collected, divided into freeze-storage tubes, and stored in liquid nitrogen for subsequent RNA sequencing.The animals involved in the research were subject to the Regulations of the Ministry of Science and Technology, PRC on the Administration of Laboratory Animal Affairs . The sample collection program was approved by the Ethics Committee of Animal Husbandry Specialty of Gansu Agricultural University . Plateau-type Tibetan sheep were selected as the study object  (accessed on 5 September 2022). The raw data returned by the Illumina MiSeq platform were subjected to merging of paired-end reads. Using FLASH v 1.2.7 software, the reads of each sample were spliced according to the minimum overlap length of 10 bp and the maximum mismatch ratio allowed in the overlap region of 0.2, and the spliced sequence obtained was the original tag data (raw tags). Trimmomatic v 0.33 software was used to filter the raw tags obtained by splicing, and the parameter was set to a 50 bp window. If the average quality value in the window was lower than 20, the base at the back end was cut off from the window, and the tags whose lengths were less than 75% of the lengths of tags after quality control were filtered to obtain high-quality tag data. Finally, UCHIME v 4.2 software was used to identify and remove the chimeric sequences to obtain the final effective tags. Usearch v 11.0.1 software [https://www.arb-silva.de/) (accessed on 6 Sptember 2022). Based on the OTU analysis results, taxonomic analysis was performed on the samples at various taxonomic levels, and the community structure of the samples at the taxonomic levels of phylum, class, order, family, genus, and species was obtained. Then, a t-test was performed on the species abundance data between groups using Metastats V1 (http://metastats.cbcb.umd.edu/) (accessed on 6 Sptember 2022) software [p value was obtained, and the q value was obtained by correcting the p value. Finally, the species leading to the composition difference between the two groups of samples were screened according to the q value.An MN NucleoSpin 96 Soil kit  was used to extract microbial DNA from the rumen content samples, and the concentration and purity were measured by a NanoPhotometer-N60 . Through PCR amplification of the V3–V4 region of the 16S rRNA gene, the community structure of the rumen microbiota was obtained. The primers were 338F 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′. The library obtained by this PCR amplification process was then sequenced on an Illumina MiSeq platform , and bioinformatics analysis was performed using BMKCloud  (accessed on 3 October 2022), and the differential metabolites were screened by combining the difference multiple, the p value of the t-test, and the VIP value of the OPLS-DA model. The screening standard was fold change (FC) > 1, p value < 0.05, and variable importance in projection (VIP) > 1. Kyoto Encyclopedia of Genes and Genome (KEGG) functional annotation and enrichment analyses were performed for differential metabolites [The metabolites in the rumen contents and serum of 18 Tibetan sheep were determined by liquid chromatography–mass spectrometry. The samples were defrosted at room temperature, pretreated according to the methods of Dunn et al. (2011) and Want et al. (2010) ,69, and abolites .® kit  in strict accordance with the kit procedure. A VAHTSTM DNA Clean Beads Kit  was further used for product purification. The constructed library was sequenced using an Illumina NovaSeq6000 . Bioinformatics analysis was performed on BMKCloud (www.biocloud.net) (accessed on 20 October 2022), and clean data were obtained after data filtering. Sequence alignment between clean data and the specified reference genome, Ovis_aries (Oar_rambouillet_v1.0.Ovis_aries), was performed by HISAT (v 0.1.6) [p value. FC > = 2 and FDR < 0.01 were used as the screening criteria for differentially expressed genes.TRIzol reagent  was used to extract total RNA from rumen epithelial tissue of Tibetan sheep. The Nanodrop2000  was used for concentration detection. Agient2100, LabChip GX , was tested for integrity. The cDNA Library was constructed by a VAHTS Universal V6 RNA-seq Library Prep Kit for an Illuminav 0.1.6) . Mapped v 0.1.6)  was usedv 0.1.6)   < 0.05, and then the occurrence frequency of metabolite clusters/microbiota was counted, and the correlation result table of metabolite clusters/microbiota with top 30 frequency was used to draw a chord diagram (circlize package). Using the R tool, the WGCNA package was used to perform WGCNA dimensionality reduction analysis on all metabolites and genes in the differential groups  [p values with at least one set of correlations to satisfy CCP < 0.05 data through the pheatmap package for correlation map visualization and heatmap drawing. In addition, the pathways involved in genes in the transcriptome and those involved in metabolites in the metabolome were compared to obtain the number of coinvolved pathways. The Venn diagram was drawn using the Venn.diagram function in the Venn Diagram function package through the R language tools [According to McHardy et al.’s method , conjoinapplied) , and genge tools . ThroughThe purpose of this study was to elucidate the response of rumen microbiota and metabolites to host (mRNA and metabolite) interactions in the Tibetan sheep plateau environment. Compared with low altitudes, metabolites at medium and high altitudes were enriched in amino acid metabolism and lipid metabolism, which participate in the regulation of amino acid metabolism and fatty acid absorption, thereby affecting production performance. Especially at an altitude of 3500 m, upregulated metabolites were enriched in vitamin B6 metabolism and participated in the regulation of the body’s immune and energy metabolism processes, suggesting that Tibetan sheep were more suitable for grazing and breeding in an environment of approximately 3500 m. In addition, this study also confirmed the interaction between rumen metabolites, metabolites, and genes; the differentially expressed genes and metabolites in high-altitude areas were coenriched in asthma (ko05310), arachidonic acid metabolism, and biosynthesis of amino acids pathways and participated in the regulation of respiratory and immune-related functions to adapt to the plateau environment. Host metabolome analysis revealed that the same functional pathways as the rumen metabolome were enriched in amino acid metabolism, lipid metabolism, and metabolism of xenobiotics by cytochrome P450. The host metabolome is involved in the regulation of amino acid synthesis, lipid metabolism, and immune metabolism pathways. A total of 7.65% of common metabolites were found in the host serum and rumen, and methyl cinnamate was mainly enriched in the metabolic pathway. Methyl cinnamate increases at the middle altitude of 3500 m to participate in the regulation of adipocyte differentiation and thus energy metabolism of the body. Moreover, these metabolites are associated with some genes related to inflammation and lipid metabolism, suggesting that metabolites interact with host genes to regulate the immune level of the body, thus responding to plateau environmental changes.


BRs are unsuitable for use in the germination stage when Sodium chloride is below 100 mM, and the effect is also weakest in the seedling stage. Exogenous BRs promoted photosynthesis, and antioxidant enzyme activity increased the accumulation of osmoregulatory and antioxidant substances and reduced the content of harmful substances and Na+, thus reducing cell damage and improving plant salt tolerance. BRs induced the most soluble protein, chlorophyll a, stomatal conductance, net photosynthetic rate, Glutathione peroxidase, and root-Ca2+, with BRs causing Ca2+ signals in roots probably constituting the most important reason for improving salt tolerance. BRs first promoted the accumulation of Ca2+ in roots, which increased the content of the above vital substances and enzyme activities through the Ca2+ signaling pathway, improving plant salt tolerance.Brassinosteroids (BRs), the sixth major phytohormone, can regulate plant salt tolerance. Many studies have been conducted to investigate the effects of BRs on plant salt tolerance, generating a large amount of research data. However, a meta-analysis on regulating plant salt tolerance by BRs has not been reported. Therefore, this study conducted a meta-analysis of 132 studies to elucidate the most critical physiological mechanisms by which BRs regulate salt tolerance in plants from a higher dimension and analyze the best ways to apply BRs. The results showed that exogenous BRs significantly increased germination, plant height, root length, and biomass  of plants under salt stress. There was no significant difference between seed soaking and foliar spraying. However, the medium method (germination stage) and stem application (seedling stage) may be more effective in improving plant salt tolerance. BRs only inhibit germination in Solanaceae. BRs (2 μM), seed soaking for 12 h, and simultaneous treatment with salt stress had the highest germination rate. At the seedling stage, the activity of Brassinolide (C In recent years, saline land has been expanding due to climate change, wrong irrigation, indiscriminate cutting, the unreasonable use of chemical fertilizers, and numerous natural factors, including climate, water quality, and terrain reasons ,2,3. To  leakage . In addi leakage ,7. This  leakage ,9.+ exclusion and plant salt tolerance [2+ application also increased the superoxide dismutase activity, catalase activity, chlorophyll content, and root activity in cereal grains, thereby reducing the injury caused by salt stress [2+ plays an important role in plant salt tolerance.Brassinosteroids (BRs) are a class of polyhydroxylated sterols, the sixth major phytohormone critical in plant growth and development . Grove eolerance . In addit stress . In conc2 concentration (Ci), transpiration rate (E), K+, and maximal photochemical efficiency (Fv/Fm) in plants under salt stress have been inconsistent in different studies. To date, the most appropriate application method of BRs in increasing salt stress tolerance in plants and the differences in the response of different classifications of plants to BRs under salt stress are unknown. What role does Ca2+ play in regulating plant salt tolerance by BRs? Meta-analysis is a systematic and comprehensive analysis process that combines data from the multiple experiments of the same type to reach an accurate and comprehensive conclusion, essentially solving the aforementioned problems [−1 and that the effects of soil application were more pronounced than those of foliar spraying. Tahjib et al. [What is the best application strategy for BRs to improve seed germination rate?29H50O6 or C28H48O6), concentrations, and application methods for exogenous BRs in the seedling stage?What are the best donor compounds ?2+, Na+, K+, and Mg2+), and other standard physiological parameters? Which physiological processes are most important in BRs increasing the plant salt tolerance levels?How do exogenous BRs affect plant growth , antioxidant substances (ascorbic acid (ASA), reduced glutathione (GSH), dehydroascorbate reductase (DHAR), superoxide dismutase (SOD), guaiacol peroxidase (POX), POD, glutathione reductase (GR), glutathione peroxidase (GPX), catalase (CAT), and APX), photosynthesis , E, stomatal conductance (Gs), and Ci), ion changes , with a digital object identifier (DOI) of 10.17605/OSF.IO/ZCQY9. The OSF is an open source platform to share the review with others transparently and assist researchers throughout the review process. In this study, research papers were collected from the Web of Science, PubMed, CNKI, China Science and Technology Journal Database, and WanFang Database, and the search deadline was 9 February 2023. An advanced search form was used, i.e., subject terms + free words, and the search form for each database is detailed in Since PROSPERO is limited to registering health-related research, this study was registered in the Open Science Framework (OSF) on 26 September 2023, at 28H48O6) and Homobrassinolide (C29H50O6)) treatments in the experiment, with the donor of BRs excluding its analogs. (3) The experimental data must include at least one predetermined and identifiable physiological parameter. (4) The experimental results must include complete and valid data, including at least the mean, error values, and the number of replications. (5) Must have clearly described experimental methods. (6) Excluding the reviews and apology letters, duplicate published or cited data will be removed. Based on the above guidelines, 4595 research papers were found, and 132 (4740 independent studies)  all studies are from journal papers, and experiments must be conducted in non-field conditions. (2) The experimental material must be wild-type, and there must be separate NaCl and NaCl + BRs(Brassinolide (Cstudies)  were finstudies) .https://automeris.io/WebPlotDigitizer/index.html (accessed on 25 March 2023)). When standard errors (SEs) are given in this paper, the SD value is obtained using the formula SD = SE × An Excel database was created to extract valid data from the paper. The extracted data included mean, standard deviation (SD), number of replicates, and experimental description information . Data ineriments . For anap < 0.05), and a fixed-effects model was used in the opposite direction [Meta-analysis was performed using MetaWin 2.1 software, lnR was selected as the effect value type, and the effect size and combined effect values were calculated. A random-effects model was used in the presence of heterogeneity  Czern, accounting for 8%, 7%, 7%, and 7%, respectively , and the promotion effect was significantly higher than that of dicotyledons  under medium-level salt (100~150 mM) and high-level salt (NaCl > 150 mM) stresses and the application of BRs was not promoted under low-level salt (NaCl < 100 mM) stress  stress C. The re) stress C. Regres) stress F.n = 100) had the largest effect value, while shoot DW  had the smallest effect value. Interestingly, the effect values for dry weight were greater than the fresh weight, except for aboveground biomass.29H50O6 and C28H48O6 significantly increased the plant height, root length, and biomass under salt stress, except for shoot FW. However, the combined effect values of all parameters were higher under the C28H48O6 treatment than C29H50O6, with significant differences in plant height, root length, root FW, and shoot FW.The extent of the increased plant salt tolerance level by BRs varied according to donor type. The effect of BRs on the effect values of growth parameters was related to the period of BR application. Pre-treatment and post-treatment significantly increased the effect values of all growth parameters. In contrast, when BRs were treated simultaneously with NaCl, only the effect values of the total DW, aboveground, and below-ground biomass significantly increased. The combined effect values of plant height, root length, root FW, shoot DW, shoot FW, total DW, and total FW were higher under BRs post-treatment than pretreatment, where the total FW was significantly different. However, the number of studies N was only 5. In addition, the combined effect value of the total FW was significantly higher than the other parameters under post-treatment A.The effect values of each parameter differed depending on the method of application of BRs B. The efExogenous BRs under salt stress enhanced the plant height, root length, and biomass parameters in dicotyledonous and monocotyledonous plants, with significant differences in all parameters in dicotyledonous plants. BRs had no significant effect on the shoot FW of monocotyledons under salt stress C. After n = 14). BRs significantly enhanced the plant height, root length, and biomass parameters at all three salt levels (n = 33) when the BRs were applied under high salt level stress.All three concentration levels of BRs significantly increased the plant height, root length, and biomass except for shoot FW A. Overalt levels B. The efn = 115) and the smallest for Ci , and both Pn and Gs had significantly higher effect values than Ci and E. Chlorophyll a > chlorophyll b > total chlorophyll, where BRs promoted chlorophyll a significantly more than the total chlorophyll under salt stress, suggesting that BRs may have a more vigorous regulatory ability on chlorophyll a.Applying BRs under salt stress significantly increased the plants’ total chlorophyll, chlorophyll a, chlorophyll b content, Pn, Ci, Gs, E, and Fv/Fm A. The mo2O2, O2−·, and ·OH contents of plants under salt stress, with the effect values of H2O2 content being significantly lower than those of ·OH (n = 31). However, the range of its confidence interval was so great that it was not significantly different from the other parameters (n = 28), which was significantly higher than CAT and SOD. In addition, BRs also significantly increased the soluble sugar, soluble protein, proline, and relative water content of plants under salt stress , He of ·OH B. Applyirameters A. Regardt stress A, where 2+, K+, and Ca2+ contents and decreased the Na+ content in each part (the Leaf—Na+ difference was insignificant). There were no significant differences between the different parts of K+ and Mg2+, while the Ca2+ effect values in roots were significantly higher than in the aboveground parts and leaves. Regarding the degree of reduction in plant Na+ content by BRs, the most significant effect was observed in leaves, followed by aboveground, and the least in roots. Also, the maximum effect value of Ca2+ in the roots was 0.2977 (n = 26).As shown in Salt stress, one of the most important abiotic stresses, severely restricts plants’ or crops’ development and yield . TherefoThe germination period is the most sensitive to salt stress, and studies have shown that salt stress severely inhibits seed germination . Therefon = 100) was the most commonly used method. Therefore, we further analyzed the soaking time and found that the BRs played a tremendous role when the soaking time was 12 h. In summary, seed soaking may be the most convenient to use, but it does not exert the highest effectiveness; therefore, the use of culture medium for BRs should continue to be explored in the future. In this study, we analyzed the effect of BRs on the germination rate of seeds under salt stress under different application periods. We found that the highest germination rate was achieved when BRs were applied simultaneously with NaCl, while the pretreatment of seeds with BRs did not significantly enhance germination under salt stress. BRs applied at concentrations greater than 0.02 μM positively affected the germination rate, and further regression analysis revealed the best effect when BRs were used at 2 μM. Interestingly, the study of salt levels revealed that the higher the salinity level, the more vital it is that BRs contribute to the germination rate. When the NaCl concentration was lower than 100 mM, BRs did not have a significant effect on the increase in germination rate, which might be because the seeds themselves have a certain resistance to salt stress.As of the current study, the primary uses of BRs in seeds are spraying , Petri d28H48O6, indicating that the activity of C28H48O6 was higher than that of C29H50O6 in exogenously applied plants, which is consistent with the findings of Wani et al. in salt stress on mustard [Plant height, root length, and biomass are the most accurate and straightforward indicators of plant growth, and salt stress can lead to impaired plant growth, mainly in terms of plant height, root length, dry weight, and fresh weight ,40,41. S mustard . There w mustard ,45,46,47In contrast, foliar spraying and seed soaking are the most widely applied uses today ,48,49. T+ content and disrupt the ionic balance, producing osmotic stress and ion toxicity, and affecting plants’ normal metabolism [2+, as an essential element for plant growth, is involved in regulating plant growth and development, salt stress, and other critical physiological processes [+ content and increased the Ca2+, K+, Mg2+, and osmoregulatory substance contents, with the most tremendous increase in Ca2+ in roots. This indicates that BRs promote the synthesis of osmoregulatory substances and thus regulate the osmotic balance, reduce the uptake of toxic ions by plants, maintain the ionic balance, and thus promote the improvement in salt tolerance. And, BRs likely promote the production of root Ca+ primary signals, which in turn trigger a series of salt tolerance processes  to regulate plant salt tolerance [2+ signaling, and Ca2+ can modulate the interaction between CBL4/CBL8 and CIPK24, which in turn causes changes in the activity of the SOS1 Na+/H+ antitransporter protein, with a consequent enhancement of the Na+ efflux, and ultimately leading to enhanced salt tolerance in the plant [SIBRI1 could enhance BR signaling [bHLH/HLH was also related to BR signaling [2+ in the roots and may be the main reason for the increased salt tolerance  was higher than that of Homobrassinolide (C29H50O6), and BR treatment after salt stress was the best application period. The best application method might be stem application, but further study is needed, and there was no significant difference between seed soaking and foliar spraying. The promotion effect of BRs on dicotyledons was higher than that on monocotyledons, and the optimal BR concentration at the seedling stage was recommended to be controlled below 0.02 μM. Exogenous BRs promoted photosynthesis and antioxidant enzyme activity. They increased the accumulation of osmoregulatory substances  and antioxidant substances (ASA and GSH), thus reducing harmful substances and Na+ levels, mitigating cell damage, balancing ion concentrations, and improving plant salt tolerance. BRs regulated soluble proteins, chlorophyll a, Gs, Pn, ASA, GPX, POX, GR, DHAR, and Ca2+ in roots most strongly, with BRs regulated Ca2+ signals in roots probably being the main reason for improving salt tolerance. BRs first promoted the accumulation of Ca2+ in roots, which increased the content of soluble proteins, chlorophyll a, and enzyme activities through the Ca2+ signaling pathway, thus improving plant salt tolerance. Finally, we suggest that, in the future, we should also focus on different application methods of BRs, such as culture medium usage at the germination stage, stem segment application at the seedling stage, and nutrient solution usage at the seedling stage. In addition, the mechanism by which BRs promote salt tolerance should be focused on Ca2+.In this study, 132 papers were screened for meta-analysis to provide evidence that exogenous BRs can improve salt tolerance in plants, and the usage and physiological functions of BRs were further investigated. During the germination period, exogenous BRs significantly increased the germination rate under salt stress, and exogenous BRs promoted the germination rate of woody plants and monocotyledons significantly more than herbaceous plants and dicotyledons, respectively. BRs had the greatest promotion of germination in the flax family and the Malvaceae and had no significant effect on the Solanaceae. The best application method for BRs is the culture medium method, but further research is needed. Seed soaking is commonly used, and its optimal time is 12 h. The best application period for BRs was simultaneously applied with salt stress, and the best concentration was 2 μM. The promotion of salt tolerance by BRs was stronger at high levels of salt concentration than at other levels. At the seedling stage, exogenous BRs significantly increased the plant height, root length, and biomass under salt stress, with the most effect on total DW. The activity of Brassinolide (C