Solea senegalensis). Our goal was to determine how much of the sole microbiome is inherited from the egg stage, and how much is acquired during the remainder of the sole life cycle in an aquaculture production batch, especially regarding potentially probiotic and pathogenic groups. Our work comprises sole tissue samples from 2 days before hatching and up to 146 days after hatching (−2 to 146 DAH), encompassing the egg, larval, weaning, and pre-ongrowing stages. Total DNA was isolated from the different sole tissues, as well as from live feed introduced in the first stages, and 16S rRNA gene was sequenced (V6-V8 region) using the Illumina MiSeq platform. The output was analysed with the DADA2 pipeline, and taxonomic attribution with SILVAngs version 138.1. Using the Bray–Curtis dissimilarity index, both age and life cycle stage appeared to be drivers of bacterial community dissimilarity. To try to distinguish the inherited (present since the egg stage) from the acquired community (detected at later stages), different tissues were analysed at 49, 119 and 146 DAH . Only a few genera were inherited, but those that were inherited accompany the sole microbiome throughout the life cycle. Two genera of potentially probiotic bacteria (Bacillus and Enterococcus) were already present in the eggs, while others were acquired later, in particularly, forty days after live feed was introduced. The potentially pathogenic genera Tenacibaculum and Vibrio were inherited from the eggs, while Photobacterium and Mycobacterium seemed to be acquired at 49 and 119 DAH, respectively. Significant co-occurrence was found between Tenacibaculum and both Photobacterium and Vibrio. On the other hand, significantly negative correlations were detected between Vibrio and Streptococcus, Bacillus, Limosilactobacillus and Gardnerella. Our work reinforces the importance of life cycle studies, which can contribute to improve production husbandry strategies. However, we still need more information on this topic as repetition of patterns in different settings is essential to confirm our findings.Recirculating aquaculture systems (RAS) pose unique challenges in microbial community management since they rely on a stable community with key target groups, both in the RAS environment and in the host (in this case,  HoweverVibrio , influences the farmed fish that is in constant contact with the water, with its own prokaryotic community  that alsum genus , Vibrio  . After the degasification column, where water trickles down, the water passes through the skimmer before returning to the tanks. The total water volume is 370 m3 in the PO system and 60 m3 in the WE system. In both systems, the water recirculation rate is approximately 400% per hour, the feeding regime is approximately 2% biomass/day, and the fish density varies between 2.5 to 5 kg/m2.This study was performed in partnership with an aquaculture production unit, who provided the samples, a sole hatchery (Safiestela S.A.), located in Estela, Portugal. The pre-ongrowing and weaning tanks operate in a recirculating aquaculture system (RAS), while egg and larval stages are kept in a flow-through water system. The water circulation of the pre-ongrowing (PO) and weaning (WE) systems is displayed in CF) A, for flatfish larvae with no potentially probiotic added, was introduced at 65 DAH and replaced by CF B, for nursery with supplemented potentially probiotic Pediococcus acidilactici, at 100 DAH. The exact amount of P. acidilactici was not disclaimed in the commercial diet formulation, but the BACTOCELL CNCM I-4622 strain was used. For this study, the same production batch was accompanied throughout the development stages and tissue samples were collected in duplicate. Eggs were collected at −2 DAH, larvae at 2 and 14 DAH. For juveniles, the separate tissues were collected for microbiome characterization  at the weaning system (49 DAH) and at the beginning and end of the pre-ongrowing . For each sample type, on each day, duplicate samples were collected, one fish per sample in the case of the juveniles, and approximately 2 mL of dry volume in the case of egg and larvae samples. Live feed samples were also collected in duplicate. Information about temperature, salinity, and pH at the sampling time can be found in A description of the age, system, life cycle stage, and feed of the collected samples is presented in 2.2.® sequencer with the V3 chemistry, according to manufacturer’s instructions  at Genoinseq .Total DNA was isolated from the different matrices , in duplicate, with DNeasy Power Soil kit . Samples were prepared for Illumina Sequencing by 16S rRNA gene amplification of the bacterial community. The DNA was amplified for the hypervariable V6-V8 region with specific primers and further reamplified in a limited-cycle PCR reaction to add sequencing adapters and dual indexes. First PCR reactions were performed for each sample using KAPA HiFi HotStart PCR Kit according to manufacturer suggestions, 0.3 μM of each PCR primer: forward B969F 5′- ACGCGHNRAACCTTACC -3′ and reverse BA1406R 5′- ACGGGCRGTGWGTRCAA −3′  and 50 n2.3.Copyright 2019, the R Foundation for Statistical Computing) with the package dada2 (v1.16.0). Primer removal was performed within the pipeline of DADA2 using the filterAndTrim function. Sequence filtering, trimming, error rates learning, dereplication, chimera removal and amplicon sequence variant (ASV) inference were performed with default settings. For taxonomic attribution, the SILVAngs version 138.1 database was used (To obtain a amplicon sequence variant (ASV) table, the DADA2 pipeline  was implwas used . Taxa clphyloseq (v1.38.0) and ggplot2 (v3.3.5) were used for data handling and visualization. Alpha diversity was calculated using the Observed ASVs metric and the Shannon index with vegan (v2.5–7). Beta-diversity was calculated with Bray–Curtis dissimilarity index and plotted with non-metric multidimensional scaling (NMDS), this was also performed for the target groups subsets . Dissimilarity results were tested by permutational multivariate ANOVA (PERMANOVA) using the Adonis function (vegan) for beta group significance  the parameters age (DAH), sample type , life cycle stage  and system  were tested.For the general bacterial community analysis, the package microbiome R package (v. 1.16.0). Additionally, venn diagrams were performed to analyze the membership of shared taxa across the sole life cycle with tissue samples were separated by life cycle stages. Venn diagrams were obtained using the venn R package (v. 1.10) to display the number of shared and exclusive taxa between whole body samples (egg and larvae) and each sole tissue  at different ages .To be part of the core microbiome, we consider the bacterial genera that are present in at least 75% of all samples of the sole life cycle , with an abundance higher than 0% (detection threshold), using the Bacillus, Roseobacter, Phaeobacter, Paenibacillus, Pseudoalteromonas, Alteromonas, Pseudomonas, Aeromonas, Arthrobacter, Clostridium  using the R packages Hmisc (v4.1.1) and corrplot (v0.84).To explore our target groups, potentially probiotic and potentially pathogenic bacterial organisms, these groups were identified at different taxonomic levels to mitigate the effects of unclassified sequences and (in the case of probiotics) to potentially find new promising genera for further studies. For the potentially probiotic group, we selected all genera from the order Lactobacillales  and Bifistridium , Sacchararomyces , Streptoptomyces , and Sheewanella , due to ibaculum , Vibrio  , according to manufacturer instructions, and following SEA EIGHT’s Veterinary Plan. According to the Portuguese legislation DL N° 113/2013, this work is exempted from the need for ethical approval. All methods are reported in accordance with ARRIVE guidelines.The datasets generated and/or analysed during the current study are available in the European Nucleotide Archive (ENA) repository, accession number PRJEB55703.3.The 16S rRNA gene sequencing dataset used had a minimum and maximum read counts per sample (after trimming) of 7,776 and 84,097, respectively. The mean read counts for all samples was 32,709, the complete list of read counts per sample is presented in 3.1.p-values in the Adonis test, with the system having the highest percent variability explained (The most abundant phyla were Proteobacteria (42–91%), Bacteroidetes  and Firmicutes (0–39%). The complete distribution at this taxonomic level can be found in xplained . HoweverAllorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Vibrio, Pseudoalteromonas, Tenacibaculum, Cutibacterium, Methylobacterium-Methylorubrum, Delftia, Pseudomonas, Paracoccus, Peredibacter, Halomonas, and Marinobacter.The core microbiome, at the genus level, can be consulted in Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Cutibacterium, Delftia, Halomonas, Marinobacter, Methylobacterium-Methylorubrum, Pseudoalteromonas, Sulfitobacter, Unclassified Cryomorphaceae, Vibrio). In the gills, there are eight genera (2.2%) present across all samples . A total of eleven genera (2.7%) were present across all intestinal samples  and ten genera (2.9%) in the mucus . There is no apparent trend in the number of exclusive genera per tissue, with numbers varying between ages.Venn diagrams  were use3.2.Bacillus, Enterococcus, Phaeobacter, Pseudoalteromonas, Pseudomonas and Shewanella were already present in the eggs (2 days before hatching). Shewanella disappears at 2 DAH and was only detected again in the WE system (at 49 DAH), after the two sources of live feed (rotifer and brineshrimp) were introduced. Bacillus and Enterococcus also drop below the detection limit (no sequences obtained) at 14 DAH, and only the first re-emerges in the WE system. The remaining three genera  were present throughout the life of the sole in the hatchery. Results showed that more potentially probiotic genera were introduced after hatching, at 2 DAH. At this stage of the life cycle the genera Alteromonas, Streptococcus, Gardnerella, Streptomyces, Pediococcus, Granulicatella, Lactobacilus emerged, but only the first was detected at 14 DAH . However, most of the other (except for Gardnerella) re-emerged in the WE system (at 49 DAH), around forty days after live feed was introduced. At this stage, 19 new genera appeared for the first time: Weissella, Vagococcus, Aerosphaera, Roseobacter, Latilactobacillus, Limosilactobacillus, Aeromonas, Bifidobacterium, Lactococcus, Leuconostoc, Carnobacterium, Ligilactobacillus, Desemzia, Brochothrix, Loigolactobacillus, Lactiplantibacillus, Dellaglioa, Liquorilactobacillus and Facklamia. At 146 DAH only 7 potential potentially probiotic genera were detected: Pseudoalteromonas, Shewanella, Phaeobacter, Pseudomonas, Alteromonas, Roseobacter and Aeromonas. Despite no major changes observed in total relative abundance of potentially probiotic genera, the number of detected genera increased from four at the end of the larval stage (14 DAH) to 22 and 21, in the intestine at day 49 and 119 respectively, and then back to 4 at 146 DAH.Relative abundance of genus distribution of the target groups can be seen in Tenacibaculum and Vibrio accompany the sole microbiome through its development, from egg to 146 DAH. Photobacterium and Mycoplasma were detected, respectively, at 49 DAH and 119 DAH. Photobacterium was also detected in brineshrimp and Mycoplasma was detected in both brineshrimp and rotifer samples.In respect to the potentially pathogenic genera, Alteromonas with Shewanella and Pseudomonas with Phaeobacter). Regarding interactions across the two target groups, there are two positive correlations between Tenacibaculum and potentially probiotic taxa  and one negative with Pseudomonas. There are only negative correlations between Vibrio and six potentially probiotic taxa . Two potentially probiotic taxa have negative correlations with Mycoplasma, Pseudoalteromonas and Streptomyces. Finally, Photobacterium has two negative correlations with Phaeobacter and Alteromonas and 14 positive correlations with potentially probiotic taxa .The spearman correlation matrix between the relative abundances of potentially probiotic and pathogenic genera can be found in 4.Recirculating aquaculture systems have a unique challenge in managing a stable and functional microbial community , with coWe found that alpha-diversity indexes did not change throughout development, although previous studies on a different species (Atlantic cod), refer to a loss of bacterial species diversity when artificial feeding is introduced . BesidesThe most abundant phyla in this dataset, Proteobacteria, Bacteroidetes and Firmicutes are commonly found to be the most abundant in aquaculture systems . At the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Vibrio, Pseudoalteromonas and Tenacibaculum) present in all tissues analysed and in all growth stages. As mentioned before, two of them are potentially pathogenic (Vibrio and Tenacibaculum). One thing to keep in mind is that both the live feed and the border sole tissues collected are in permanent contact with the water, and when studying these frontier environments its complex to disentangle the host from the environment community. Indeed, three of these genera have already been identified in the water, tank biofilm and biofilter carriers in this aquaculture unit, Vibrio, Pseudoalteromonas and Tenacibaculum , all of them included fe cycle . Anotherfe cycle .Pediococcus acidilactici in its formulation, the commercial feed B did not consistently increase the abundance of Pediococcus in the sole intestines fed with this diet (119 and 146 DAH). When considering the increased number of potentially probiotic genera in the fish tissues after day 49, it is worth noticing that most of these genera were not present in the feed itself . An explanation could be that components in these feeds may act as prebiotics, that is, nutrients that are not digested by the fish that may fortify certain components of the intestinal microbiota by stimulating the growth and the activity of particular bacteria  increased their relative abundances when fish were introduced to RAS, during the pre-ongrowing stage  as having the most impact during the sole life cycle in an aquaculture production batch. We found a sharp increase in the number of potentially probiotic genera when sole moved to the WE system (49 DAH), around 40 days after live feed was introduced in the diet. Commercial feed did not appear to substantially increase the number of potentially probiotic genera at 119 or 146 DAH. Despite carrying bacteria . Indeed,galensis . Althouggalensis , a genetgalensis . This shgalensis . Studiesgalensis . Howevergalensis . In our ng stage . Other sng stage . Most prng stage .Tenacibaculum and Vibrio, they appear to be acquired at the egg stage, accompanying the sole microbiome through its development, from egg to 146 DAH. The other two, Photobacterium and Mycoplasma, appear to be detectably colonizing later in the life cycle. In this study they have been identified in the rotifers and brineshrimp and thus the live feed could be a potential vector as has been previously demonstrated , two with Mycoplasma  and two with Photobacterium (Phaeobacter and Alteromonas). For this analysis, we must recognize the potential biases in NGS community correlation studies that may result in misleading positive correlations, derived from the fact that more taxa are detected in deeply sequenced samples and therefore taxa co-vary with sequencing depth  or cases like Bacillus that shows a negative correlation with Vibrio but a positive one with Photobacterium. The genera Streptococcus, Phaeobacter and Limosilactobacillus also have a similar behavior. Four genera had exclusive positive correlations with the potential pathogenic bacteria , therefore these might be the most promising for future empirical studies.In the correlation matrix, we found that six genera with potential probiotic activity were significantly negatively correlated with  species . Only onng depth . Attestihe later . These dibaculum . With th5.Solea senegalensis early development. Two potentially probiotic genera were inherited from the egg stage (Bacillus and Enterococcus), but the main increase in potentially probiotic abundance and diversity occurred around 40 days after live feed was introduced in the diet (at the weaning stage). Notwithstanding this increase, the establishment of this community in the following development stages was not successful. Regarding potentially pathogenic genera, two appear to be inherited (Tenacibaculum and Vibrio), and two are suggested to be acquired during production (Photobacterium and Mycoplasma). These results are relevant, because acquired potentially pathogenic groups may be prophylactically treated with improvement in husbandry conditions, but those that are inherited from the egg stage may be difficult to safely eradicate.This work aimed to describe the sole microbiome development throughout the production cycle in a RAS. Through a description of the inherited and acquired community in the different tissues analysed at different production and life stages, we hope to promote the emergence of life cycle studies in aquaculture and to underscore its applicability. We found that the bacterial community was significantly altered throughout the Solea senegalensis, to our knowledge, the first of its kind. By analyzing the composition of this community, particularly with the definition of key target groups and the definition of the inherited and acquired community in the production cycle, we have highlighted the importance of whole life cycle studies to understand the vulnerability of the stages of fish production with a direct impact in husbandry strategies. The shifts in the composition of key components of Solea senegalensis gut microbiome during its life cycle, open important questions related to the functional significance of the observed taxonomic changes in terms of potentially probiotic activity and pathogenic incidences in the life cycle of this fish that must be explored in future investigations.Our study has conducted a comprehensive description of the bacterial community in different life cycle stages of the The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: European Nucleotide Archive (ENA) repository, accession number PRJEB55703.Ethical review and approval was not required for the animal study because the animals used in this work were not subjected to any experimental protocol and were a part of the routine procedures of a commercial hatchery facility. All animals were handled by the fish farm employees, the euthanasia method used was an anaesthetic overdose of the commercial anaesthetic Aquacen benzocaine 200 mg/ml  following SEA EIGHT’s Veterinary Plan. According to the Portuguese legislation DL N° 113/2013, this work is exempted from the need for ethical approval. All methods are reported in accordance with ARRIVE guidelines.DA, MS, CM, IB, and AM had substantial contributions in the conception and design of the work. DA and IB were responsible for the acquisition of the samples. DA and MS for the analysis. DA, MS, CM, and AM in the interpretation of the data. DA drafted the first manuscript. MS, CM, IB, and AM revised it critically. MS, CM, and AM provided final approval for publication of the content. All authors contributed to the article and approved the submitted version.This work was funded by the project ATLANTIDA (NORTE-01-0145-FEDER-000040), supported by the North Portugal Regional Operational Program (NORTE2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF). DA was supported by the Ph.D. grant with the reference PD/BDE/135542/2018, and Safiestela Sustainable Aquafarming Investments, S.A. (part of the SEA EIGHT group). MS was supported by the project 39948_FeedMi for this work, supported by Portugal and the European Union through FEDER/ERDF, CRESC Algarve 2020 and NORTE 2020, in the framework of Portugal2020. AM and CM were supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT and ERDF.DA and IB were employed by Safiestela S.A., SEA EIGHT group at the time of the study.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bone mineral density (BMD) and balance ability were measured in both groups at two measurement points (at baseline and at the end of Week 12). We found that patients in the DMT intervention group had significant improvements in BMD and balance ability compared with the TAU group by using repeated measures analysis of variance. Treatment with DMT demonstrated a significant improvement in BMD from baseline to week 12 . For the Berg total score and static and dynamic balance, the mean changes in the DMT group were 7.3 (95% CI: 5.6–9.0), 4.0 (95% CI: 0.9–7.1), and 3.7 (95% CI: 2.6–4.8), respectively. Regression analysis showed that baseline BMD was a significant predictor of improvement in BMD from baseline to week 12 in the DMT group . Our results suggest for the first time that DMT intervention may be effective in beneficially regulating BMD and balance ability in SZ patients.Fractures are common accidents for long-term hospitalized patients with schizophrenia (SZ) in psychiatric hospitals, and once they occur, patients usually endure the pain of fractures for a long time. Accumulating evidence has supported the implementation of dance/movement therapy (DMT) as a promising intervention for patients with SZ. However, no research has been conducted to investigate its role in balance ability in SZ. This study was designed to investigate the efficacy of a 12-week DMT intervention in bone mineral density and balance ability in patients with SZ using a randomized, controlled trial design. A total of 58 veterans with SZ were randomly assigned to the DMT intervention group ( The psychiatric symptoms and high rate of relapse of SZ can significantly impair cognitive and social functioning, including problem-solving skills, interpersonal relationships, and work performance3. Moreover, individuals with SZ usually have higher disability and premature mortality rates compared to the general population4.Schizophrenia (SZ) is a chronic and severe psychiatric disorder that affects approximately 1% of the population7. A previous meta-analysis showed that the pooled rate of fractures per 1000 person-years was 5.54 in SZ, compared to 3.48 in the general population8. Between 1% and 3% of falls in hospitals result in a fracture, and even minor injuries can cause distress and delay rehabilitation10. Osteoporosis and its precursors, e.g., osteopenia and low bone mineral density (BMD), have been reported in chronic patients with SZ14, reviewed in Kishimoto et al.12. They were strong predictors for subsequent falls and fractures in psychiatric hospitals15. There is evidence that nearly 52% of patients with SZ have low BMD, 13.2% of patients with osteoporosis, and 40.0% of patients with osteopenia16. Studies have demonstrated that older age, male gender, unhealthy lifestyle factors, increased levels of stress hormones, diabetes, and hypertension were associated with an increased risk of osteoporosis17. Among the risk factors, lower limb muscle strength and balance ability are significant predictors of fractures and falls, which are known to be impaired in patients with SZ19. Considering a deficit in general preventative medical care for osteoporosis in individuals with SZ, specialized exercises provided by specialists such as exercise physiotherapists may play a key role in the management of low BMD in SZ patients20. Therefore, further studies are warranted to investigate and elucidate the role of exercise in improving limb strength and preventing falls in long-term hospitalized patients.Fall is a common concern in hospital settings, with studies reporting a fall rate between three and five per 1000 bed days21, is an emerging therapy for the rehabilitation of patients with SZ in recent years24. DMT therapy uses movement, dance, and interpersonal communication to explore emotional, cognitive, social, and physical integration, enabling patients to enhance self-expression, accept and reconnect with their bodies, and strengthen their fitness25. Patients share their emotions, concerns, and coping strategies with others through dance/movement27. In particular, a few studies have reported a critical role of DMT intervention in negative symptoms in chronic patients with SZ29. However, there is still a lack of evidence on whether DMT is an effective therapy to increase the mean BMD and reduce the risk of fractures due to falls in long-term hospitalized patients with SZ.Dance/movement therapy (DMT), as one of the creative arts therapies36. Although the etiology of low BMD in patients with SZ is complicated, studies have suggested that increased prolactin levels caused by the dopamine D2 receptor-blocking effect of antipsychotics can accelerate bone loss and increase the risk of osteoporosis37. Therefore, the risk of unintentional falls increases with long hospitalizations and long-term antipsychotic use in SZ patients38. Decreased BDM, changes in body composition, and skeletal muscle mass index were reported in elderly chronic SZ patients on long-term antipsychotic medications11. In the present study, we hypothesized that the DMT intervention was effective in increasing BMD and balance ability in long-term hospitalized male veterans with SZ as compared to the control group.Antipsychotic medications are currently effective in treating the symptoms of SZ, but antipsychotics can also cause adverse effects such as weight, muscle stiffness, and low BMD lossEighty long-term hospitalized male veterans with chronic SZ were recruited from Hebei Province Veterans Hospital through advertisements in this study. An experienced psychiatrist performed eligibility screening. The inclusion criteria included: (1) a diagnosis of SZ using the DSM-V; (2) male veterans aged 40–60 years old with a disease duration of 5 years or more and a long-term hospitalization of 3 years or more; (3) able to understand Mandarin Chinese; (4) legally eligible to sign an informed consent form; (5) stability of current antipsychotic medications for more than 2 years; (6) reduced bone mass, osteoporosis, and severe osteopenia after bone density analysis; (7) no comorbid serious physical illness and physical impairments and able to cooperate with nurses to complete the general intensity of activity training; and (8) consent for pre- and post-session interviews. The exclusion included: (1) substance dependence or abuse; (2) hypercalcemia and hyperuricemia; (3) history of kidney stones or renal calculi; and (4) lower limb injury and motor dysfunction, and inability to complete DMT intervention for various reasons. These situations are often associated with the inability to complete the therapy and dropout, and could thus lead to unequal sample sizes and biased results and threaten the validity of the results. The study protocol was approved by the institutional review board of Hebei Province Veterans Hospital. Written informed consent was obtained from each participant.Of the 80 patients, 6 patients were ineligible, 7 patients did not meet the inclusion/exclusion criteria, 7 patients withdrew consent, and finally, 60 patients were randomly assigned. Thirty patients were assigned to the intervention group, and 30 patients were assigned to the treatment-as-usual (TAU) group. However, 1 participant in the TAU group dropped out of this study due to discharge from the hospital in the first week of treatment, and 1 patient in the DMT group was unable to complete DMT therapy due to marked fluctuations in the psychotic symptoms of SZ. Therefore, we included 58 patients (29 patients in each group) in the following analyses , a power of 80%, and α = 0.05. A flowchart of the present trial is displayed in Fig. Sample size power was calculated based on expected changes in BMD, which was the primary outcome of this study. The sample size is considered to achieve significance with a moderate effect size (ES)  to receive either DMT intervention or TAU based on a computer-generated sequence. The independent third party, who was blinded to the subject groups, divided the patients into two groups.The clinical trial used a two-arm parallel group design to compare TAU and DMT plus TAU on outcome measures at two time points.39. In brief, the therapist used a body-based approach that included movement warm-ups followed by an improvisational dance. It starts with simple warm-up exercises such as breathing and muscle relaxation and continues with body awareness exercises. First, meeting, warming up, determining the group rules, explaining the principles, and determining the expectations activities. Then, the greeting is completed with warm-up, initiation, continuation, and closing activities. During the warm-up phase, the whole group comes together to form a circle, generally standing, in order to ensure the group dynamic. The detailed process of DMT intervention was described in the The group consisted of a dance movement therapist and 30 hospitalized veterans with SZ who chose to participate in this study. The DMT intervention was given twice a week for one hour each time. All patients received 24 sessions of DMT over a period of three months. DMT intervention was conducted by an experienced dance/movement therapist with over 10 years of experience in DMT interventions. The DMT intervention protocol was developed based on the therapist’s experience and the theoretical frameworks, and the DMT approach was described in a previous studyThe patients in the control group did not participate in the DMT intervention, but they were treated as usual. The TAU group sessions were conducted by licensed professionals employed at the hospital. The patients received regular daily antipsychotic medication and supportive psychotherapy once a week. Supportive psychotherapy was applied to the treatment of the patients once a week as a total of 12 sessions of 45 min over 3 months. They also participated in daily activities from Monday to Friday for at least 2 h each day, including indoor activities  and outdoor activities .The primary outcome measure was the BMD of the patients recruited, which was assayed by an Erik AKDX-09W-1 bone density device (Shenzhen Ekere Electric Co.). The test was performed using the dual-energy X-ray absorptiometry method. Measurements were made at the distal end of the flexor bone of the patient’s left forearm for fan beam and line scan, which were reviewed by an experienced radiologist who was blinded to the clinical and pharmacological characteristics and randomization of the patients. The measurement time was 2–5 min, and the measurement error in testing the accuracy of the manikin was ≤3%.41, the operational definition of osteoporosis in men was given as a BMD that was greater than 2.5 standard deviations (SD) below the mean value of young, healthy men, ie, a T-score < −2.5 SD. A T-score between 1.0 SD and −1.0 SD is considered normal or healthy, and a T-score between −2.5 SD and −1.0 SD indicates that you have low bone mass, although not low enough to be diagnosed with osteoporosis. The greater the negative number, the more severe the osteoporosis. We have previously measured the BMD of 12,159 normal individuals to obtain mean BMD values for different age and sex groups and obtained a mean peak BMD of 0.5340 g/cm2 for men. We then converted the range of peak BMD at each level into absolute values based on the T-score, i.e., absolute values of BMD greater than 0.467 g/cm2 is considered normal, less than 0.467 g/cm2 and greater than 0.364 g/cm2 is bone loss, and less than 0.364 g/cm2 is osteoporosis.The World Health Organization (WHO) criterion for osteoporosis is based on the measurement of BMD. According to the WHO diagnostic criteria42. It is a quantitative test that combines most components of postural control through 14 functional activities, including reaching, bending, transferring, and standing: sitting and transferring safely between chairs; standing with feet apart, feet together, standing on a single leg, and feet in the tandem; Romberg position with eyes open or closed and reaching and stooping down to pick something off the floor. Each item is scored on a 5-point scale ranging from 0 to 4. Zero indicates the lowest level of function, and 4 is the highest level of function. The total score for all items is 56. The BBS is reliable (both inter- and intratester) and has been validated for validity43. The Cronbach alpha in this study was 0.851. The lower the score on the scale, the worse the balance and the higher the risk of falling. A score of 40 or less indicates the probability of falling.The secondary outcome measures were the balance ability. The balance ability (static and dynamic) of patients with SZ was assessed by three raters using the Berg balance scale (BBS) in the two groups pre- and post-test, to evaluate the changes in balance ability. The BBS scale was first reported by Katherine Berg in 1989 and is now widely recognized as an informative way to evaluate balance ability in clinical practicep-values lower than 0.05.Statistical analysis was performed in SPSS Statistics, version 22.0 (IBM). All statistical analyses were teo-sided, and the significance threshold was set at Considering that patients who dropped out at the beginning of treatment did not receive treatment, the last observation carried forward analysis was not performed for patients who dropped out in the first week of treatment in this study. Demographic characteristics and markers of BMD at baseline between the DMT group and the TAU group were compared using analysis of variance (ANOVA) or chi-square tests.Then, repeated-measure multivariate analysis of variance (RM-MANOVA) was performed to compare the efficacy of the two treatment groups by assessing the main effects (group and time) and the group-by-time interactions on the primary endpoint variables. Using the RM-MANOCA, researchers can control for an increased risk of Type 1 error.After the RM-MANOVA, separate repeated measure ANOVA (RM-ANOVA) tests were followed up with the tests of individual outcome variables if RM-MANOVA yielded significant results. In the RM-ANOVA model, time (baseline and 3-month follow-up) was added as within-effect, group (DMT vs. TAU) was added as between-effect, and BMD and balance ability over time were independent variables, respectively.If a significant difference was observed in the interaction effect of group and time in separate RM-ANOVA, an analysis of covariance with the baseline scores as covariates were conducted to test for the group differences in a 12-week follow-up after treatment. If there was no significant interaction between the group and time, no further statistical analysis was carried out.p > 0.05). The baseline demographic characteristics, BMD, balance ability, and lipid profiles of the two groups were well-matched.Table p < 0.05) (Table p > 0.05).RM-ANOVA analysis showed significant interaction effects of time and group on BMD, Berg total score, and static and dynamic balance  Table , suggestp > 0.05). Treatment with DMT demonstrated a significant improvement in BMD from baseline to week 12 . For the Berg total score and static and dynamic balance, the mean changes in the DMT group were 7.3 (95% CI: 5.6–9.0), 4.0 (95% CI: 0.9–7.1) and 3.7 (95% CI: 2.6–4.8), respectively.Further analysis of the two groups showed that compared to baseline, BMD, the Berg total score, the static and dynamic balance scores increased significantly after treatment in the DMT group, while there was no change in the TAU group  (R2 = 0.35).Multiple linear regression analyses were used to determine potential predictors of treatment response. Considering a strong positive association between age and duration of illness and to avoid including completely collinear covariates in the analysis, we chose age as a covariate in the regression model. The results showed that baseline BMD was a significant predictor of improvement from baseline to week 12 in the DMT group (This study found that after 12 weeks of treatment, BMD, Berg total score, and static and dynamic balance increased significantly in the DMT intervention group compared with the TAU group in hospitalized male veterans with SZ.44. Current studies on the treatment of osteoporosis have mainly focused on menopausal women and older people, and few therapeutic interventions have been reported for high-risk populations of long-term hospitalized SZ patients with osteoporosis. Although there are several studies on falls in the general population, to our best knowledge, this is the first study to investigate the efficacy of a DMT intervention on BMD in long-term veterans with SZ. Moreover, our study also specialized in that, unlike the patients recruited in other studies, we minimized the effect of age on the results by enrolling only hospitalized veterans aged 40–60 years. On the other hand, we also took into account the low incidence of low BMD and osteoporosis in individuals under 40 years of age and the increased risk of falls during DMT intervention in those over 60 years of age.Osteoporosis is a metabolic condition characterized by decreasing bone mass and destruction of the microarchitecture of bone tissue, leading to increased bone fragility and the risk of fracture48. In clinical practice, patients with osteoporosis usually improve significantly with medication and physical activity49. However, there are multiple difficulties in the treatment of osteoporosis comorbidity in veterans with mental disorders who have been hospitalized for a long period. Long-term hospitalization, antipsychotic medications, reduced outdoor activity and exercise, lack of regular bone density testing and calcium supplements in hospitalized patients, and a significant reduction in the duration and frequency of outdoor activity over the last three years due to the COVID-19 epidemic, have contributed to an increased risk of fractures from falls in long-term hospitalized veterans with SZ.In the present clinical study, we showed that the DMT intervention was effective to increase BMD and prevent falls in long-term hospitalized male veterans with SZ. Moreover, we found that the Berg total score, dynamic balance, and static balance scores were significantly increased in the DMT group, but no changes in the TAU group. Numerous studies have found that long-term use of antipsychotic drugs leads to abnormalities in biomarkers of lipid metabolism and bone turnover, which can negatively affect bone metabolism in patients with SZNotably, our findings demonstrated that DMT intervention helped to improve the balance function of long-term hospitalized patients, especially the dynamic balance function. Dynamic balance plays a crucial role before postural changes lead to a fall. Individuals with SZ who do not keep physically active or fit tend to have poorer balance, which increases the likelihood of falling. DMT intervention uses dance and movement to further the emotional, cognitive, physical, spiritual, and social integration of the individual in a non-verbal way. Through simple movement exercises and playful interaction, DMT is particularly applicable for improving the balance ability in long-term hospitalized veterans with SZ. Our findings suggest that early implementation of continuous DMT interventions could reduce or delay the onset of bone loss, increase balance ability, and decrease the risk of fall fractures and the severity of fall injuries of long-term hospitalized veterans with SZ. Thus, this clinical trial provides further evidence for the DMT intervention for patients with SZ.Several limitations should be noted in this study. First, the sample size was small for a clinical trial to examine the efficacy of DMT intervention on BMD and falls in patients with SZ. Thus, we were unable to investigate the effects of sex, age, different types of antipsychotics, duration of antipsychotic exposure, and other fracture risk factors on the outcomes. Second, this study was not a double-blind design. The raters did not know which group the patients had been assigned to during the trial. However, the patients knew which group they were assigned to since they often revealed their treatment groupings when they shared their experiences. Third, it would be better to use the risk of falls, the risk of fractures, the risk ratio for disability, and quality of life as the main outcomes, however, this study only investigated the effect of short-term treatment on the balance ability. Short-term treatment may have little impact on the outcomes such as the risk of falls, the risk ratio for disability, and quality of life. Forth, although the efficacy of DMT on lower limb muscle strength and balance is an important point, which are significant predictors of fractures and falls, the improvements in cognitive functions, social interaction, and depressive symptoms from the intervention also need to be assessed in future studies. Fifth, all participants were male inpatients with SZ and our findings may not generalize to female patients.In conclusion, falling is one of the serious accidents in psychiatric hospitals, and veterans with SZ are at increased risk of falls and fractures due to physical inactivity caused by long-term hospitalization and the use of antipsychotic drugs, combined with decreased BMD and balance ability. DMT interventions can increase bone mass and balance ability in patients with SZ, thereby reducing the risk of fracture in case of falls and further reducing the stress of clinical care. Our study suggests that DMT intervention is a worthwhile therapy for long-term hospitalized veterans with SZ in clinical practice.The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.supplementary file


The effectiveness of place-based policies on carbon emission is controversial, and particularly the mechanism behind its effectiveness is unknown. We treat China’s Old Revolutionary Development Program (ORDP)— a large-scale and novel type of place-based policy targeted at undeveloped regions, as a natural experiment to estimate ORDP’s impact on carbon emission. Employing the panel data of 110 prefecture-level cities in China from 2010 to 2019, we perform a time-varying difference-in-differences (DID) study and discover that ORDP leads to an average of 26.7% increase in carbon emission and this effect takes a period to emerge and is not sustainable in the long term. Three mechanisms that may result in such impact are that ORDP improves economic development, changes industrial structure, and decreases technological progress. Further heterogeneity analysis indicates that ORDP results in a greater increased impact on carbon emission in old revolutionary cities that are located in western China compared to those located in central and eastern China. It is reported that China was responsible for 27% of the world’s total carbon emission in 2019 , exceediNumerous studies have investigated how to reduce carbon emission and have confirmed that governmental policies play a vital role in controlling carbon emission. For example, existing related works of literature have found that China’s Western Development Strategy , China’sIn reality, place-based policies, namely spatially targeted development programs, may serve an important role in controlling carbon emission. On the one hand, place-based policies will decrease carbon emission since the measures implemented in place-based policies can optimize resource allocation, facilitate technological progress, and improve resource utilization efficiency . On the China’s Old Revolutionary Development Program policy (ORDP), a large-scale and novel type of place-based policy introduced by China in 2012, is employed as a quasi-natural experiment of place-based policy to examine the effect of place-based policy on carbon emission. Since 2012, China has launched the ORDP which aims at economic growth and poverty alleviation in five major revolutionary old areas. To obtain the extensive objectives outlined in ORDP, governments have issued a series of preferential policies in finance, land, talent, and industry to support the development of old revolutionary base areas. It is reported that during the “13th Five-Year Plan” period, one out of eight Chinese central budget funds were used in these five major old revolutionary regions, with the investment scale exceeding RMB 300 billion. With these massive preferential investments, the economic growth of the ORDP areas has been considerably boosted. From 2012 to 2019, the average annual growth rate of gross domestic product (GDP) in ORDP areas is 11.7%, while this number is only 9.9% in non-ORDP areas [Our paper makes contributions to four strands of literature. First, we show the first stringent empirical proof of the impacts of place-based policies on the environment. In terms of sustainable economic, social, and environmental development, place-based policies are essential to supporting governments in providing high-quality living and well-being for their people. Most existing studies have examined how place-based policies affect economic growth ,15, goveSecond, in comparison to existing literature that mostly focuses on examining the environmental performance of place-based policies in developed nations, such as America, Australia, and some European countries , we examThird, we contribute to the burgeoning debate about the relationship between economic development and environmental sustainability. A growing literature has been engaged in figuring out the linkage between economic development and environmental sustainability, but there have been opposite views for a long time . TraditiFourth, we formulate a theoretical framework and empirically test the mechanisms through which the effectiveness of place-based policies on carbon emission happens. The majority of current research focuses on evaluating the effect of place-based policy . HoweverThis study is organized structurally as follows. Section 22.1Old Revolutionary Regions are revolutionary base areas founded under the Communist Party of China’s leadership during the Agrarian Revolutionary War and the Anti-Japanese War, which cover more than 1, 300 counties (cities and districts) in 28 provinces around mainland China, occupying one-third of China’s total land area. Compared with other regions in China, old revolutionary regions are in a relatively lagging stage of development. Therefore, in recent years, the Chinese government has given a high priority to supporting the revitalization and development of old revolutionary regions. ORDP is one of the most massive supportive programs.Since 2012, China has introduced strategies for the revitalization and development of five key revolutionary old areas, including Shanxi-Gansu-Ningxia, Jiangxi-Fujian-Guangdong, Dabie Mountains, Left and Right River, and Sichuan-Shanxi old Revolutionary Regions, covering 283 counties (cities and districts), with an area of 845,600 square kilometers and involving 166.7 million people as  shows. WORDP provides a series of financial, fiscal, industrial, and talent policies in the old revolutionary regions, which may have impacts on carbon emission. For example, ORDP proposes to strengthen oil and gas exploration and increases investment in infrastructure construction in the old revolutionary regions. Exploration for oil and gas results in higher carbon emission . While p2.22.2.1Hypothesis 1 that ORDP will lead to an increase in carbon emission. The reasons are as follows.As we mentioned in the introduction section, the impact of place-based policies on carbon emission is unclear, it can either increase or reduce carbon emission. However, based on the specific measures implemented in ORDP areas, we propose our Unlike other place-based policies, such as China’s special economic zones, which always targeted regions that have a stronger economic base or favorable geographic conditions , ORDP spHypothesis 2. The reasons for this Hypothesis are as follows. First, the policies implemented in ORDP need to be given a certain amount of time for the local government and enterprises to respond and thus it takes a period for the effect of ORDP to emerge. Second, ORDP realizes its policy effect primarily through massive infrastructure construction, physical investment, and industrial development, rather than by technological progress or human capital investment. These measures may have a short-term effect on economic development and carbon emission, but this effect may gradually reduce in the long term.However, we also propose that the impact of ORDP on carbon emission will take a period to emerge and is not sustainable in the long term, which is our 2.2.2Hypothesis 3 that the impact of ORDP on carbon emission is mainly caused by scale effect, structural effect, and technological effect. The following are detailed explanations.In the above First, ORDP will promote the economic development of the ORDP areas, leading to an increase in carbon emission. As we stated above, the fundamental target of ORDP is to enhance economic development. ORDP issues many policies to encourage economic development in the old revolutionary regions. Previous studies have verified that economic development will result in environmental pollution, such as carbon emission . Just asSecond, ORDP changes the industrial structure and promotes secondary industry development in ORDP areas, resulting in carbon emission growth . The ORDThird, previous studies have shown that technological progress has a favorable impact on environmental quality, which means a decrease in carbon emission . However33.1This study aims to assess the causal relationship between ORDP and carbon emission. The multiple regression method is often used to catch such effects. However, this method cannot eliminate endogeneity problems, such as missing variables and time trend disturbances. A quasi-experiment based on a DID estimator can well address these endogeneity problems; hence the causal effect can be recognized accurately. A traditional DID method requires that the policy must begin to implement at the same time, but ORDP in this study was introduced and implemented in batches over years 2012–2016, which means that the policies impacted ORDP areas at different times, Therefore, we use the time-varying DID approach to catch the effect of ORDP on carbon emission. Following Beck T. et al. , we adopModel (1) can only measure the average treatment effect of ORDP on carbon emission from a static standpoint, it does not account for the dynamic change of effects over time. Therefore, we adopt an event analysis method to evaluate the dynamic treatment effect of ORDP on carbon emission. The model (2) is as follows:3.23.2.12, 3.101 kg/kg, and 1.302 kg/(kW·h), respectively. We use industrial carbon emission to represent carbon emission in China since the industrial sector contributes to more than 70% of the total emissions in China. Therefore, according to the method proposed by Han F. et al. , we empl2) and carbon emission per 10,000 yuan of industrial value-added (PerCO2) to measure carbon emission.According to Sun P. et al. , we appl3.2.2We treat ORDP as the core explanatory variable. We define ORDP for a city in year t and following years as 1 if the ORDP is applied in the city, and 0 otherwise.3.2.3(1)Economic development. We measured it using the logarithm of real GDP. Economic development is an important factor influencing carbon emission. On the one hand, economic development contributes to massive energy consumption and thus increases carbon emission . On the (2)Urbanization level. Determined by dividing the total population by the fraction of urban residents. Academics are controversial concerning the effect of urbanization on carbon emission. Some consider that urbanization will increase carbon emission in that it will raise people’s income and enhance their consumption capacity . Others (3)Private saving. We express it in the logarithm of year-end resident deposits. The impact of private saving on carbon emission is controversial. On the one hand, private saving boosts carbon emission. Because when society contains more private savings, the investment in the economy is also higher, this is beneficial to economic growth. With the rapid economic development, carbon emission will increase . On the (4)Human capital. Defined by a logarithmic based on the total number of college students. Uncertainty exists on how human capital affects carbon reduction. On the one hand, an improvement in human capital level results in carbon emission reduction, because it upgrades industrial structure, and promotes economic growth . On the To avoid the endogeneity problem that affects the research results due to omitted variables, we control some factors that affect carbon emission. Based on the existing studies, we choose the subsequent four indicators as control variables: economic development, urbanization level, private saving, and human capital. The specifics are as follows.3.3Based on the comprehensive consideration of the rational selection of research samples and the available data, this paper selects 110 prefecture-level cities in the scope of ORDP areas as our research samples. Specifically, 33 prefecture-level cities—15 in 2012, 10 in 2015, and 8 in 2016—are treated as the experimental group, and the rest 77 prefecture-level cities are recognized as control groups. Due to data availability, our research period is from 2010 to 2019.4In this part, firstly, we assess the average treatment effect of ORDP on carbon emission employing the DID approach, and then investigate the dynamic treatment effect of ORDP on carbon emission based on the event study method; in addition, we conduct some robustness tests; and then we test the underlying mechanisms of ORDP on carbon emission. Finally, we examine the heterogeneous impact of ORDP on carbon emission across cities in different regions.4.12. The ORDP coefficient in column (3) is 0.223 and significant at the 5% level, demonstrating that ORDP raises PerCO2. Furthermore, to acquire a more robust result, we control some factors that influence carbon emission. The estimated results including the control variables are shown in columns (2) and (4). The results indicate that the increasing effect of ORDP on carbon emission persists, with an increase in ToTCO2 by 26.7% at the 1% significance level and an increase in PerCO2 by 18.4% at the 5% significance level. Therefore, we conclude that ORDP significantly increases carbon emission, which validates our Hypothesis 1.Based on the model (1), we estimate the average treatment effect of ORDP on carbon emission, and the baseline estimation outcomes are displayed in 4.2The baseline results estimate ORDP’s average treatment effect on carbon emission based on a static perspective only. However, considering that controlling carbon emission is a necessary part of sustainable economic development in the long term, ORDP’s dynamic treatment effect on carbon emission also merits attention.Based on the model (2), we examine the dynamic treatment effect of ORDP on carbon emission, and 4.3To improve the reliability of our results, we conduct seven robustness tests, containing a common trend test, a placebo test, a randomness test, advancing the time of ORDP implementation, changing the definition of ORDP, employing the propensity score matching and the difference-in-differences (PSM-DID) model, and eliminating the interference of other policies.4.3.1The presence of a common trend in carbon emission between ORDP cities and non-ORDP cities before ORDP implementation is an essential prerequisite for estimating the net effect of the ORDP on carbon emission using the DID approach. Therefore, it is necessary to examine the common trends or pre-existing trends.2 and PerCO2, respectively. We find that the coefficients of ORDP for all years are non-significant at the 90% confidence interval before the ORDP implementation, which means the common trend exists and our research design passes the pre-existing trend test. Therefore, we can explore how ORDP influences carbon emission using the DID model.4.3.2To remove any possible effects of observable factors on the baseline results, we perform a placebo test according to Li P. et al. . Specifi2 and PerCO2 are displayed in The probability density distribution of the placebo ORDP variable’s coefficients for ToTCO4.3.3The deployment of ORDP is assumed to be unaffected by initial carbon emissions in different cities in our empirical research. However, local governments may choose when and where to implement ORDP based on each city’s carbon emission status. Thus, the implementation of ORDP may have a selection bias problem. We conduct a randomness test to prove that the cities in ORDP are not chosen based on their initial carbon emission. Referring to Shen K.R. et al. , to eval4.3.42 and PerCO2 are not significant with or without control variables, indicating that the increase in carbon emission is indeed attributable to ORDP and our baseline regression results are robust.To exclude some factors before the ORDP implementation to confound our results, we also conduct a counterfactual test by constructing the false ORDP variables. Specifically, first, we advance the implementation time of ORDP by 5, 4, and 3 years, which are represented by 4.3.5Considering the differences in the specific implementation month of ORDP in the five old revolutions, this paper refers to Lu Y. et al.  to chang4.3.6The baseline results directly use DID method to analyze the distinctions in carbon emission between ORDP and non-ORDP cities. However, systematic differences may exist between ORDP and non-ORDP cities before the implementation of ORDP. Referring to Zhang C. et al. , a combiSpecifically, the first step of PSM-DID is to compute the propensity score for each observation sample based on the logit regression coefficients, where the ORDP dummy is the explanatory variable, and four control variables shown in The outcomes of the PSM-DID method are shown in 4.3.7Other policies that can also influence carbon emission might skew the impact of ORDP. Therefore, for the baseline regression results to be credible and robust, we must eliminate the interference of other policies. By combing existing literature and policies, we identify two policies—the Targeted Poverty Alleviation Policy (TPA) and the National Innovation City Pilot Policy (NICP), which occurred during our research period—that may influence carbon emission and thus distort our evaluation results.Firstly, TPA can promote economic development and may affect carbon emission. To achieve poverty eradication among the rural poor, China has implemented TPA since 2013. Significant funding is distributed to industrial development, transportation improvement, and infrastructure construction in TPA counties, which may affect carbon emission.Secondly, NICP, aiming to foster innovative cities, will also have an impact on carbon emission. To increase innovation abilities and reach sustainable social development, China has implemented NICP since 2008. Up to 2020, 78 cities have implemented NICP . ThroughFollowing Li P. et al. , we incoThe regression results, which are shown in 4.4The investigation mentioned above has established that ORDP causes an increase in carbon emission. The following question is what mechanisms lead to this result? In other words, what are the transmission mechanisms of ORDP on carbon emission? As we stated in section . et al. , the sca. et al. , we use . et al.  did. TheThe following are the specific test procedures. First, we identify the significance of 4.5The ORDP covers five old revolutionary areas, each with different resource endowments and policy implementation efficiency, so the impact of ORDP on carbon emission may differ amongst old revolutionary regions. Therefore, we further test the regional heterogeneity effects of ORDP. Analyzing the heterogeneity effects of ORDP in different old revolutions is helpful to distinguish between old revolutions that facilitate the growth of carbon emission and others that do not, which can inform policy customization for different old revolutions.The influence of ORDP on carbon emission is significantly regionally heterogeneous, as seen in 5Controlling massive carbon emission has become a topic attracting global attention and the target of all governments' endeavors. What is the place-based policy’s role in carbon emission? The answer to this question is crucial to establishing a better understanding of place-based policy’s role in carbon emission in developing countries. In this article, we use 110 prefecture-level cities from 2010 to 2019 to assess the effect of ORDP on carbon emission by considering ORDP as a natural experiment. Our results indicate that: first, ORDP can significantly increase carbon emission, and this effect takes a period to emerge and is not sustainable in the long term. Seven robustness tests demonstrate the reliability of our findings. Second, according to a mechanism analysis, the effect of ORDP on carbon emission is mainly caused by the scale effect, structural effect, and technological effect. Third, heterogeneity analysis suggests that ORDP results in a greater increased impact on carbon emission in old revolutionary cities that are located in western China compared to those located in central and eastern China.Therefore, our paper offers the following policy implications. First, the Chinese central government should put greater emphasis on carbon emission decline and environmental management when designing specific measures in ORDP, not just only focus on promoting economic development in the revolutionary old regions. According to our baseline results, ORDP leads to an increase in carbon emission, which is harmful to the environment. Therefore, the local government in the old revolutionary regions should include environmental protection in their development objectives and pay more attention to carbon emission reduction, thus realizing a “win-win” scenario between carbon emission reduction and economic growth. Measures include establishing laws and regulations about environmental protection, increasing the supervision of polluting enterprises, raising the proportion of resource and ecological indicators in the political performance assessment, improving energy use efficiency, and so on. Second, improve technological innovation in old revolutionary areas. Our mechanism analysis shows that ORDP fails to improve technological progress and decreases the ratio of expenditure on science and technology to total government expenditure, which leads to a significant increase in carbon emission. Therefore, the government in the old revolutionary regions needs strong incentive mechanisms for technological innovation. Measures may include strengthening the investment in science and technology, accelerating the accumulation of human capital, improving the ability of ORDP to absorb and digest advanced technology and knowledge, and so on. Third, promote the industrial structure transformation of old revolutionary areas in the western region. According to the result of the heterogeneity analysis, ORDP leads to a greater increase in carbon emission in old revolutionary cities that are located in western China compared to those located in central and eastern China. Consequently, the Western regions should transform their industrial structure to create sustainable and clean industries instead of continuing to promote the expansion of energy-intensive industries.With much work still to be done. Although the results of our paper, which examines the impacts of ORDP on carbon emission over a relatively brief timeframe, are informative for policymakers, the availability of data prevents us from capturing the effects of ORDP on carbon emission over extended time horizons. Future studies may investigate the ORDP’s long-term effects on carbon emission to further comprehend the ramifications.DAN PAN: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Yiqun Chen: Performed the experiments; Wrote the paper.Fanbin Kong: Contributed reagents, materials, analysis tools or data; Wrote the paper.National Natural Science Foundation of China ; Humanities and Social Sciences Research Project of the Ministry of Education in China (No. 19YJCZH270).This study was supported by the Data will be made available on request.The authors declare no conflict of interest.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


During the first 2 weeks, children wore a mitt over the less-affected hand and engaged in functional and play activities with the affected hand. Starting in week 3, bimanual play and functional activities were added progressively, 1 hour/week. This intervention was compared to two different schedules of block interventions: (1) 3 weeks of mCIMT followed by 3 weeks of BT, and (2) 3 weeks of BT followed by 3 weeks of mCIMT. Hand function was tested before, after, and two months after therapy with the Assisting Hand Assessment (AHA), Pediatric Evaluation of Disability Inventory (PEDI), and Canadian Occupational Performance Measure (COPM). All three groups of children improved in functional independence , goal performance  and satisfaction , which persisted two months post-intervention. All groups showed similar amounts of improvement, indicating that the delivery schedule for mCIMT and BT does not significantly impact the outcomes.Constraint-induced movement therapy (CIMT) and bimanual therapy (BT) are among the most effective hand therapies for children with unilateral cerebral palsy (uCP). Since they train different aspects of hand use, they likely have synergistic effects. The aim of this study was to examine the efficacy of different combinations of mCIMT and BT in an intensive occupational therapy program for children with uCP. Children ( Intensive hand therapy is among the most effective, evidence-based therapies for children with hemiplegia . A key qSince CIMT and BT target different aspects of hand use, they likely have synergistic effects on hand function . By requWhile most therapies have directly compared CIMT and BT, either by comparing one therapy against the other or administering both therapies in a block design, other combinations have not been well-studied. We developed a six-week occupational therapy program that combines CIMT and BT over the course of treatment. The goal was to engage the affected arm and hand using modified constraint-induced movement therapy (mCIMT), then to focus on functional activities that require both hands. We rationalized that an optimal therapy would first increase awareness and engagement of the impaired arm and hand, then train that hand in functional bimanual tasks. We compared this approach to two groups who received blocks of interventions. One of these groups completed three weeks of mCIMT, followed by three weeks of BT, following the same rationale of first focusing on improving the skill of the impaired upper limb and then introducing bimanual training. For a comparison, one other group completed three weeks of BT, followed by three weeks of mCIMT. This group was included to control for the order of the types of training. The goal of this study was to determine if efficacy is impacted by the schedule of delivery of mCIMT versus BT. We hypothesized that children who received mCIMT before BT would show a greater improvement in hand function at the end of the intervention, as focusing on strengthening the more-affected hand first would optimize the efficacy of the subsequent BT.Thirty-five children participated in the study during the summers of 2011–2018. Five to eleven children participated each summer. Demographics and clinical characteristics of participants are shown in Inclusion criteria: (1) unilateral brain injury resulting in impairment of one side of the body, (2) ability to move all joints of affected upper extremity, (3) age 4–12 years, and (4) ability to comply with study protocol. Exclusion criteria: health problems or uncorrected vision that would interfere with study participation. The study was approved by the Institutional Review Board of Blythedale Children’s Hospital. clinicaltrials.gov (NCT02840643) before the first child was randomized to one of the crossover groups.The interventions were offered as part of a clinical program in the Department of Occupational Therapy. IRB approval was obtained to analyze data from the intervention for the group that received the blended intervention. Children in the crossover groups were prospectively enrolled and provided written assent. Their caregiver provided written informed consent. The study was registered on All intervention groups used combinations of modified constraint-induced movement therapy (mCIMT) and bimanual therapy (BT). Therapy was conducted in a large room, such that all children and occupational therapists had the opportunity to interact throughout the program. The program was coordinated by an experienced occupational therapist, who was in the therapy room during the duration of the program. Each intervention took place for 6 h per day, 5 days per week, for 6 weeks (180 h).3 weeks of mCIMT followed by 3 weeks of BT (group CB);3 weeks of BT followed by 3 weeks of mCIMT (group BC);2 weeks of mCIMT followed by stepwise incorporation of BT, increasing the amount of BT by 1 h per day for each of the next 4 weeks (group Step).Three different interventions were tested:Materials: Both mCIMT and BT used toys, board games, art supplies, craft supplies, and sports equipment selected and structured by occupational therapists. Children brought items to the intervention for practicing caregiver/child-identified self-care and functional goals, such as a shirt with buttons or shoes with laces.Participants engaged in age-appropriate training 6 h/day for 30 days (180 h).Providers and Location: Therapy was provided in one room at a pediatric rehabilitation center. Therapy was provided by occupational therapists. The ratio of therapists to children was approximately 1:4. For 60 min daily, each child received 1:1 training with an OT. In addition to the OTs, there were 1–2 volunteers and/or OT interns always present in the therapy room. Duration of therapy and therapy regimen: Therapy was provided 6 h/day for six weeks . The CB group received three weeks of mCIMT, followed by three weeks of BT. The BC group received three weeks of BT, followed by three weeks of mCIMT. The Step group had a stepwise integration of BT after starting the intervention with mCIMT. During the first two weeks of the intervention, children received mCIMT. During the third week of the intervention, children received mCIMT for the first five hours of each day, then received one hour of BT. In each subsequent week of intervention, the duration of mCIMT was reduced by one hour per day, while the duration of BT was increased by one hour per day. Thus, in week four of the intervention, children began each day with four hours of mCIMT, followed by two hours of BT. In week five of the intervention, children began each day with three hours of mCIMT, followed by three hours of BT. In the sixth and final week of the intervention, children began each day with two hours of mCIMT, followed by four hours of BT .During mCIMT, children wore a mitt over their less-affected hand, which restricted use of that hand. Children engaged in intensive therapy to improve the active range of motion, strength, motor control, and sensory awareness of the affected hand. Activities were functional and play-based. Daily structure included: morning gym, fine motor, gross motor, sensory motor, therapeutic feeding, sports, and self-care activities. During training, children performed play-based and functional activities with the affected hand. Example activities included playing card and board games, arts and crafts, and activities that provided sensory stimulation to the affected hand, such as finger painting. Activities also included stretching and strengthening, and reciprocal coordination exercises.During BT, children did not wear a mitt over the less-affected hand. Children were provided individualized activities that facilitated active use of both hands. Therapists adapted and graded activities and guided children to problem-solve for success. Bimanual activities included self-care , sports activities, and manipulation of classroom tools (cutting with scissors). Tailoring of therapy: Activities were selected for each participant based on the child’s preferences, interest, and functional goals. Examples of the children’s preferred interests include sports, arts and crafts, model construction, music, dancing, and computer games. Some examples of functional goals include donning and doffing clothing, using eating utensils, pouring liquid into a cup, carrying a lunch tray, and opening zippered food storage bags. This study began as a clinical program at Blythedale Children’s Hospital, held once annually during the summer. From 2011 to 2015, the Step protocol was used exclusively. In 2016, we decided to add the CB and BC groups. In 2016–2018, only the CB and BC protocols were used. During this time, children were randomized to either of the two groups. Thus, the Step group was not randomized, while the CB and BC groups were randomized. Each cohort was split into two equally sized, age-matched groups. Then, each group was randomized to either the CB or BC interventions. Randomization was done off site by a scientist not otherwise associated with the study.All study participants were evaluated at three time points: before the first day of treatment, within two days of the end of treatment, and two months after treatment. Bimanual performance was tested for in the CB and BC groups after week three of the intervention, when they switched between mCIMT and BT. Three outcome measures were chosen to quantify bimanual performance, motor function of the impaired upper extremity, and functional goal performance. The Assisting Hand Assessment (AHA) was used to measure how children use the two hands together. The AHA quantifies how well children with unimanual upper limb impairments use their impaired hand when performing bimanual activities. The AHA shows excellent validity, reliability (0.97–0.99) and responsiveness to change . The tesThe Canadian Occupational Performance Measure (COPM) was used to measure performance and satisfaction levels in functional goals in self-care, productivity, and leisure domains . The COPThe Pediatric Evaluation of Disability Inventory (PEDI) was used to assess each child’s functional independence with activities of daily living. For this study, only the PEDI self-care domain was used, as evaluated by a caregiver. Scaled performance scores ranging from 0 (poor) to 100 (excellent) were used to assess change over time. The MCID is 11 points . It has The AHA was conducted by staff who were not therapists in the intervention. The AHA was scored by a blinded, trained, certified evaluator who was not involved in any other aspect of this study. The COPM and PEDI were given by one of the therapists, who may or may not have worked with a particular child.p-value of less than 0.05 was considered statistically significant.A group × time repeated-measured analysis of variance (ANOVA) was used to evaluate differences in outcome measures after the intervention and a two-month follow-up, for each intervention group, using SPSS Software . Missing data were interpolated based on average changes in measures from pre- and immediate post-intervention to 2 months follow-up. Two-month AHA follow-up data were missing for two children in the Step group, one child in the BT group, and two children in the CB group. Two-month COPM follow-up data were missing for one child in the Step group, one child in the BC group, and two children in the CB group. Two-month PEDI follow-up data were missing for seven children in the Step group, one child in the BC group, and two children in the CB group. Analyses were done with and without the inclusion of the missing data estimates, and the statistical outcomes were not different between these methods. The findings presented below include the missing data estimates. When main ANOVA effects were found, post-hoc analyses were performed, using Bonferroni corrections to correct for multiple comparisons. The Fisher’s exact test was used to compare baseline categorical variables, sex, and side of lesion, among the groups. A p = 1.0), lesion side , age  = 0.48, p = 0.63), COPM-Performance  = 2.4, p = 0.11), COPM-Satisfaction  = 1.75, p = 0.19), or the PEDI  = 2.6, p = 0.087). There was a difference in baseline AHA among groups  = 3.7, p = 0.037)), which was a limitation of this study. The AHA for the CB group was significantly higher than the AHA for the Step group (p = 0.033). The BC group AHA scores were not significantly different from the other groups (p > 0.45).Thirty-five children with unilateral CP participated in a six-week intensive occupational therapy program that combined unimanual and bimanual training. Participant demographics and baseline clinical measures are presented in p = 0.37). We assessed how many children per group met the functionally meaningful difference for the AHA, which is 4 points. In the Step group, 71.4% children improved by 4 or more points, while 70% of children in the BC group and 36% of children in the CB group reached the functionally meaningful difference.Bimanual hand function was measured before, after, and two months after intervention with the Assisting Hand Assessment AHA; A. There p < 0.031). For COPM Performance, the CB group improved slightly more than the BC group (p = 0.034), while the Step group did not differ from the CB (p = 0.89) or BC (p = 0.24) groups. For COPM Satisfaction, there were no significant differences in improvement among the groups (p > 0.1).Changes in self-care skills performance were measured before, after, and two months after intervention with the Pediatric Evaluation of Disability Inventory (PEDI). Caregiver reports of self-care skills performance were obtained. There was a significant improvement in skill performance B outcomep < 0.0001; Satisfaction: F = 35.2, p < 0.0001) that was retained two months after intervention. These represent clinically meaningful improvements in both functional goal performance and satisfaction.Changes in functional goal performance and satisfaction were measured before, after, and two months after intervention with the Canadian Occupational Performance Measure (COPM). Caregiver reports of goal performance and satisfaction with performance were obtained. There was a significant improvement in both performance C and carp = 0.034). There were no significant differences between groups  = 1.31, p = 0.28), meaning that the order of mCIMT or BT delivery did not impact efficacy.In the BC and CB groups, we measured bimanual function using the AHA after three weeks of the intervention. We examined whether bimanual function changed differently if children received mCIMT or BT in the first block of the intervention . The AHAThis study compared the efficacy of different combinations of mCIMT and BT for improving hand function in children with uCP. The intervention improved self-care skills independence and performance and satisfaction in caregiver-rated functional goals. Improvements were maintained two months after therapy.While many clinical trials have shown that CIMT and bimanual therapy have equivalent efficacy in children with hemiplegia ,7,8,9,10In our study, we did not find clinically meaningful differences in outcomes among the three groups. We had hypothesized that children receiving mCIMT before BT would have the greatest improvements. In our comparison of pre, post, and two-month follow-up time points for all three groups, we found some group differences, but these did not differ by a clinically meaningful amount. Children in the CB group improved in COPM Performance more than the BC group, by a difference on 0.8 points, whereas the clinically meaningful difference is 2 points. Children in the Step group improved in the PEDI more than the BC group, by a difference of 7.3 points, while the clinically meaningful difference is 11 points. Thus, we conclude that the three interventions are not meaningfully different in their efficacy. This could indicate that at the intensity delivered, either type of training was sufficient to drive change. Despite the abundance of research into the optimal therapies for children with uCP, all available therapies remain unable to ameliorate impairments. Children, and their families, invest a massive amount of time, effort, and hope in the best available therapies. Nevertheless, these children spend a lifetime with movement impairments. Much more work is needed to develop effective therapies that enable children to sustain long-term improvements in function.Further study is needed to better understand the optimal combination, schedule, and intensity of therapeutic strategies. There are a wide variety of factors that contribute to movement, and even the best available therapies do not address all factors. For example, many children with uCP have impairments in motor planning and motor imagery ,27,28,29Moreover, a better understanding of individual differences in responsiveness to therapy is needed. There is a high variability in responses among children in the studies cited in this manuscript. Efficacy can depend on how engaged a child is in the intervention , the intThere were several limitations to this study that pertain to how the study was done, and how the findings can be interpreted. First, we will discuss limitations regarding how the study was done. The study is underpowered to be an efficacy study or a non-inferiority study. However, our results are consistent with prior studies comparing unimanual and bimanual training ,10,36,37There are also limitations regarding how our findings can be interpreted. The children in this study were school aged between 4 and 12 years old. We chose this age group because they are capable of remaining engaged in training tasks for 6 h/day, but it is possible that younger children may show greater improvements in a similar intervention, since neuroplasticity is greater in young children . Our finMoreover, our conclusions are limited by the length of follow-up of this study. We included a two-month follow-up evaluation, but ideally the effects would be sustained long-term. In the future, longer follow-ups, such as six months later, would provide important information about the longevity of improvements. It is possible that our three schedules of the intervention led to different rates of long-term retention of improved function.In this study, we hypothesized that children who received unimanual training before bimanual training would improve more than children who did not receive unimanual training first. Our findings indicate, however, that the order of training strategies does not significantly affect outcomes. The optimal schedule of training approaches is likely to be specific to an individual’s impairments and other unknown factors. More work is needed to better understand how to optimize a child’s improvements.This study compared the efficacy of blocks of mCIMT and BT or progressive shift from mCIMT to BT for improving hand function in children ages 4–12 with uCP. All groups improved equally in self-care skills independence and performance and satisfaction in caregiver-rated functional goals. This interpretation is limited by a small sample size, lack of long-term follow up, and differences in baseline bimanual function among the three groups.


Moringa oleifera seeds was studied. For this purpose, a central composite design (CCD) was applied to optimize ultrasound-assisted extraction (UAE) parameters such as amplitude (25–75%), solute-to-solvent ratio (1:10–1:30), and pH (9–13) for obtaining the maximum protein yield. At the optimized conditions of 75% amplitude, 1:20 solute-to-solvent ratio, and 11 pH, a protein yield of 39.12% was obtained in the UAE process. Moreover, the best sonication time at optimized conditions was 20 min, which resulted in about 150% more extraction yield in comparison to conventional extraction (CE). The techno-functional properties, for instance, solubility, water (WHC)- and oil-holding capacity (OHC), and emulsifying and foaming properties of the protein obtained from UAE and CE were also compared. The functional properties revealed high solubility, good WHC and OHC, and improved emulsifying properties for protein obtained from UAE. Although protein from UAE provided higher foam formation, foaming stability was significantly lower.Plant proteins can be an important alternative to animal proteins subject to minor modification to address sustainability issues. The impact of ultrasound application on the yield, techno-functional properties, and molecular characteristics of protein extracted from  Proteins impart some important techno-functional properties such as emulsification, foaming, and/or gelling, owing to which they are considered to be one of the main components of food products . ProteinMoringa oleifera (Lam.), which is a widely cultivated species in native parts of Asia and Africa, belongs to the family Moringaceae , th(38–39%)  and currThe main challenge while extracting the proteins is to choose the appropriate extraction technique. Traditionally, methods such as alkaline, organic solvent, salt, and enzymatic extraction, which are used for protein extraction, are less efficient, require long extraction time, give less protein yield, involve a high amount of solvents, and ultimately lead to an environmental burden . The appM. oleifera seed protein extracted via the sonication technique. Moreover, fluorescence and Fourier-transform infrared spectra will be taken to identify changes in functional groups.Conclusively, the study is mainly focused on the effect of techno-functional properties such as protein solubility, water- and oil-holding capacity, and emulsifying and foaming properties of M. oleifera seed protein (MOSP) was evaluated to observe the maximum recovery of the protein using RSM. For this, a central composite design (CCD) was applied using three independent variables: amplitude (%) (A), solute-to-solvent ratio (g/mL) (B), and pH (C) (Response surface methodology (RSM) is a useful statistical technique for evaluating the influence of different factors and for designing experiments such as the one in this study . An empid pH (C)  with 17 The MOSP yield obtained ranged between 30 and 39%, depending on the combination of the trial. Experimental run 16 showed the lowest yield with an amplitude of 25%, a solute-to-solvent ratio of 1:10, and a pH of 9, while experimental run 10 showed the highest yield with an amplitude of 75%, a solute-to-solvent ratio of 1:20, and a pH of 11. The difference between the measured values and the predicted values is minimal, which shows the rationality of the measured values. Following quadratic polynomial regression, an equation of amplitude (A), solute-to-solvent ratio (B), and pH (C) was developed to obtain the response model of extraction yield:The results obtained were further processed for analysis of variance (ANOVA) with a 95% confidence level in order to check the significance and suitability of the response model .p-value of the overall model is less than 0.0001, which is highly significant, and it indicates the sustainability of the fitted model. Additionally, the F-value documented the significance of the response model. Moreover, the value for determination coefficient (R2) was 0.9999, and the adjusted value (R2 adjusted) for the extracted yield was 0.9998, which shows that the model was correctly interpreted using the measured data. Generally, R2 values higher than 0.75 are considered to be superlative for a good fitted model [The results indicate that the ed model . FurtherAccording to the results obtained, it was observed that the amplitude (A), solute-to-solvent ratio (B), and pH (C) independent factors had a significant effect on the yield of MOSP. The variation in the response factor  due to independent variables in the form of coded levels is also presented in p > 0.05). The interaction between independent variables is also shown in the form of 3D response surface plots (The interactions that occurred between amplitude and solute-to-solvent ratio (AB), amplitude and pH (AC), and solute-to-solvent ratio and pH (BC) had a non-significant effect on protein yield  was predicted at the optimized conditions of an amplitude higher than 75%, a solute-to-solvent ratio of 1:22, and a pH of 11.37, which were rounded off to 75%, 1:20, and 11, respectively, for the ease of experimentation. Validation of the statistical model and the regression equation was confirmed by repeating the experimental run at defined optimized conditions. The measured value of the extraction yield (39.12%) at optimized conditions  confirmed the validity of the model. The obtained values were quite close to the predicted ones. Overall, the results showed that the model for the extraction of MOSP through ultrasonication was fit and acceptable. Furthermore, a comparison of protein yield was made between UAE and conventional extraction (CE). While keeping the other parameters at optimized levels, the sonication treatment resulted in a 150% increase in the protein yield in comparison to CE .M. oleifera seeds. As it was noticed during this study, various studies have also shown an increase in the protein yield with the increase in the power/amplitude of the sound waves, for instance, wampee seed protein [Eurycoma longifolia root protein [Dolichos lablab L. bean protein [Dolichos lablab L. [For a commercial point of view, the yield of protein is highly important, particularly for the efficient utilization of agro-waste and to achieve the goal of sustainable development. Conventional extraction techniques for protein other than ultrasonication are either time-consuming or less productive. Therefore, UAE was applied for the extraction of protein from  protein , lupin s protein , Eurycom protein , and Dol protein . It is k protein . Althoug protein . On the  protein . The pH  protein . Any fur protein  and Doliablab L. .At optimized conditions, different ultrasonic times (0–30 min) were observed for the maximum MOSP yield, and a higher yield was found at 20 min . With fuSolubility is one of the most important functional properties of protein. It is the thermodynamic property which can be elaborated as the ‘protein concentration in a saturated solution’, and it must be at equilibrium with the solid phase at optimal conditions. Solubility of proteins can be altered by any extrinsic and intrinsic factors . At neutThe high amplitude level of ultrasound escalates the protein solubility by altering the conformation and structure of protein; this results in the inside aperture of hydrophilic ends of amino acids toward water . A largeWHC and OHC are important functional properties of protein used for the moderation of the texture and viscosity of the food product and also for the reduction in the processes of dehydration during food storage . The abiLongissmus lumborum [The WHC of MOSP extracted with CE was 0.86 ± 0.009 g/g, and that of the UAE sample was 1.02 ± 0.006 g/g . The inclumborum .Oil-holding capacity (OHC) is the ability of proteins to trap oil or fat within their non-polar chains. The oil-holding capacity of the MOSP sample extracted with CE was 0.91 ± 0.015 g/g . AccordiThe improved WHC and OHC are considered helpful for enhancing the shelf life of processed foods given the fats and water reduced from the surface . Their hThe emulsifying property of protein is its ability to form an emulsion and to maintain the stability of the newly formed emulsion . It is aThe total inter-facial area formed by the whipping of protein is called the foaming capacity (FC) of the protein . The sta−1; the amide-II zone is associated with N–H bonds which are spread over the 1575 to 1480 cm−1 wavelength range; and the amide-III zone representing the N–H bending and C–N is stretched over range of 1400–1200 cm−1. Among these bands, the amide-I is the most responded area to any chemical change in the secondary structure of protein. The FT-IR spectra of MOSP isolated via conventional extraction and ultrasonic-assisted extraction are shown in Moringa oleifera seed protein isolates [The FT-IR spectrum of MOSP can be described in three importance wavelength bands . Generalisolates  treated max of tryptophan < 330 nm shows the occurrence of the amino acid in the non-polar environment, while λmax > 330 nm shows its presence in the polar environment due to conformational changes in the tertiary structure of protein [max for tryptophan, phenylalanine, and tyrosine in the case of MOSP from CE was observed at 380 nm, 380 nm, and 390 nm. After MOSP was obtained by using UAE, no shift in λmax was found except for phenylalanine, which shifted to 360 nm. Moreover, an increase in the fluorescence intensity was found in all of the studied amino residues. The spectra of tyrosine give some noise, but they could be helpful for observing the considerable changes in the chemical structure of MOSP.The fluorescence spectrum is basically a representation of amino acid residues such as tryptophan, tyrosine, and phenylalanine present in a protein, which are, in principal, detrimental to the tertiary structure of protein. Most of the studies related to tertiary structure using intrinsic fluorescence are based on the changes in tryptophan intensity . Usually protein . The pre protein ,54. The The increase in the intensity of amino residues is related to the oxidation of protein by hydroxyl radicals produced during the sonication treatment . A similM. oleifera seeds  of analytical grade were acquired from Sigma-Aldrich. The rest of the chemicals, such as sodium hydroxide, hydrogen chloride, acetone, methanol, and others, were obtained from Duksan, Korea.ra seeds  were proThe UAE method of  was adopg for 20 min, and the supernatant was collected and diluted. Then, 1 mL from each diluted solution was mixed with 5 mL of Coomassie brilliant blue G-250 solution, and the mixture was kept for 2 min at room temperature. The absorbance of each mixture was taken at 525 nm by a UV-Vis Double-Beam spectrophotometer . Bovine serum albumin (0–100 ppm) was used to prepare a standard curve for quantifying the protein contents of the extracts. Based on the protein content of the extract, the protein extraction yield (g/100 g sample) was calculated using following formula:The amount of protein was determined by using the Bradford method . ConciseM. oleifera seed protein (MOSP).Protein from the supernatant obtained during UAE/CE under optimized conditions was isolated by following acid–base methodology . Brieflyg. After the determination of the protein content in the supernatant via the Bradford method, protein solubility was calculated via the following formula:The solubility of the protein samples was estimated by using the method described by . For thig for 15 min. The calculation of WHC and OHC for each sample was performed according to the following formula:The method presented by Yılmaz and Hüriyet  and SahaW), which were taken as shown in the following formula:The emulsion capacity (EC) and emulsion stability (ES) of samples were determined according to the method of Jiang, et al. . The EC The emulsion stability (ES) was observed after 48 h at room temperature by determining the amount of separated water from oil.W is the percentage of water separated.w/v) from each protein solution was taken in a 150 mL beaker and homogenized  for 1 min at 24,000 rpm. The total volume was measured at 0 and 10 min. FC and FS were calculated according to the following formulae:The method adopted by Phongthai, et al.  for the −1 to 400 cm−1. The spectra obtained were processed using the latest OPUS software version 7.0.FT-IR is considered to be one of the best techniques for the determination of the secondary structure of proteins . In thisFluorescence spectroscopy has undergone rapid development due to its enormous technical advances, accuracy, and enhanced methods for analysis based on fluorophore compounds. The fluorescence spectra of protein extracted by using ultrasound and conventional alkaline extraction were taken using a FluoroMax4 Spectrofluorometer  equipped with a xenon lamp. The method described by Mir et al. in 2019 was followed with little modification to acquire spectra. Spectra were taken at the fixed excitation wavelength of 220 nm for tyrosine, 260 nm for phenylalanine, and 280 nm for tryptophan, while the emission range was between 300 and 550 nm with a slit width of 1 nm. Three spectra were taken for each amino acid, and the average spectrum is presented in the results.Based on the principle of central composite design (CCD), amplitude % (A), solute-to-solvent ratio (B), and pH (C) as the studied parameters for the maximum extraction of protein were optimized  via respwhere Y denotes the predicted value of the response variable; β0 denotes the intercepts; and iβ, iiβ, and ijβ are the linear, second order, and interaction regression coefficients predictable by the model, respectively. iX and jX are the values of studied or independent variables.This research revealed that ultrasonic-assisted extraction is significantly better and had beneficial effects in obtaining higher protein yields. During extraction, ultrasound power and pH were among the parameters which significantly affected the protein yield. This treatment altered the secondary and tertiary structure of MOSP, which were evident from FT-IR and fluorescence spectroscopy, respectively. Ultrasonication did not yield any degrading effects on MOSP in terms of functional properties. The functional properties of MOSP such as solubility, water- and oil-holding capacity, emulsion stability, and foaming capacity and stability improved in the beginning and then reduced as the amplitude of sonication further increased. These results would give an understanding of the mechanism and interrelation of structural changes with functional properties observed after the ultrasonic-assisted extraction of MOSP and its further application in the development of advanced sustainable food products.