Brassinosteroid regulates stomatal development in etiolated Arabidopsis cotyledons via transcription factors BZR1 and BES1.

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons.

RGA1 regulates grain size, rice quality and seed germination in the small and round grain mutant srg5.

BACKGROUND: Generating elite rice varieties with high yield and superior quality is the main goal of rice breeding programs. Key agronomic traits, including grain size and seed germination characteristics, affect the final yield and quality of rice. The RGA1 gene, which encodes the α-subunit of rice G-protein, plays an important role in regulating rice architecture, seed size and abiotic stress responses. However, whether RGA1 is involved in the regulation of rice quality and seed germination traits is still unclear. RESULTS: In this study, a rice mutant small and round grain 5 (srg5), was identified in an EMS-induced rice mutant library. Systematic analysis of its major agronomic traits revealed that the srg5 mutant exhibited a semi-dwarf plant height with small and round grain and reduced panicle length. Analysis of the physicochemical properties of rice showed that the difference in rice eating and cooking quality (ECQ) between the srg5 mutant and its wild-type control was small, but the appearance quality was significantly improved. Interestingly, a significant suppression of rice seed germination and shoot growth was observed in the srg5 mutant, which was mainly related to the regulation of ABA metabolism. RGA1 was identified as the candidate gene for the srg5 mutant by BSA analysis. A SNP at the splice site of the first intron disrupted the normal splicing of the RGA1 transcript precursor, resulting in a premature stop codon. Additional linkage analysis confirmed that the target gene causing the srg5 mutant phenotype was RGA1. Finally, the introduction of the RGA1 mutant allele into two indica rice varieties also resulted in small and round rice grains with less chalkiness. CONCLUSIONS: These results indicate that RGA1 is not only involved in the control of rice architecture and grain size, but also in the regulation of rice quality and seed germination. This study sheds new light on the biological functions of RGA1, thereby providing valuable information for future systematic analysis of the G-protein pathway and its potential application in rice breeding programs.

Brassinosteroids regulate rice seed germination through the BZR1-RAmy3D transcriptional module.

Seed dormancy and germination, two physiological processes unique to seed-bearing plants, are critical for plant growth and crop production. The phytohormone brassinosteroid (BR) regulates many aspects of plant growth and development, including seed germination. The molecular mechanisms underlying BR control of rice (Oryza sativa) seed germination are mostly unknown. We investigated the molecular regulatory cascade of BR in promoting rice seed germination and post-germination growth. Physiological assays indicated that blocking BR signaling, including introducing defects into the BR-insensitive 1 (BRI1) receptor or overexpressing the glycogen synthase kinase 2 (GSK2) kinase delayed seed germination and suppressed embryo growth. Our results also indicated that brassinazole-resistant 1 (BZR1) is the key downstream transcription factor that mediates BR regulation of seed germination by binding to the alpha-Amylase 3D (RAmy3D) promoter, which affects α-amylase expression and activity and the degradation of starch in the endosperm. The BZR1-RAmy3D module functions independently from the established Gibberellin MYB-alpha-amylase 1A (RAmy1A) module of the gibberellin (GA) pathway. We demonstrate that the BZR1-RAmy3D module also functions in embryo-related tissues. Moreover, RNA-sequencing (RNA-seq) analysis identified more potential BZR1-responsive genes, including those involved in starch and sucrose metabolism. Our study successfully identified the role of the BZR1-RAmy3D transcriptional module in regulating rice seed germination.

Gene editing of non-coding regulatory DNA and its application in crop improvement.

The development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has provided precise and efficient strategies to edit target genes and generate transgene-free crops. Significant progress has been made in the editing of protein-coding genes; however, studies on the editing of non-coding DNA with regulatory roles lags far behind. Non-coding regulatory DNAs, including those which can be transcribed into long non-coding RNAs (lncRNAs), and miRNAs, together with cis-regulatory elements (CREs), play crucial roles in regulating plant growth and development. Therefore, the combination of CRISPR/Cas technology and non-coding regulatory DNA has great potential to generate novel alleles that affect various agronomic traits of crops, thus providing valuable genetic resources for crop breeding. Herein, we review recent advances in the roles of non-coding regulatory DNA, attempts to edit non-coding regulatory DNA for crop improvement, and potential application of novel editing tools in modulating non-coding regulatory DNA. Finally, the existing problems, possible solutions, and future applications of gene editing of non-coding regulatory DNA in modern crop breeding practice are also discussed.

Glucan, Water-Dikinase 1 (GWD1), an ideal biotechnological target for potential improving yield and quality in rice.

The source-sink relationship determines the overall agronomic performance of rice. Cloning and characterizing key genes involved in the regulation of source and sink dynamics is imperative for improving rice yield. However, few source genes with potential application in rice have been identified. Glucan, Water-Dikinase 1 (GWD1) is an essential enzyme that plays a pivotal role in the first step of transitory starch degradation in source tissues. In the present study, we successfully generated gwd1 weak mutants by promoter editing using CRISPR/Cas9 system, and also leaf-dominant overexpression lines of GWD1 driven by Osl2 promoter. Analysis of the gwd1 plants indicated that promoter editing mediated down-regulation of GWD1 caused no observable effects on rice growth and development, but only mildly modified its grain transparency and seed germination. However, the transgenic pOsl2::GWD1 overexpression lines showed improvements in multiple key traits, including rice yield, grain shape, rice quality, seed germination and stress tolerance. Therefore, our study shows that GWD1 is not only involved in transitory starch degradation in source tissues, but also plays key roles in the seeds, which is a sink tissue. In conclusion, we find that GWD1 is an ideal biotechnological target with promising potential for the breeding of elite rice cultivars via genetic engineering.

Genome-Wide Identification and Expression Analysis of OsbZIP09 Target Genes in Rice Reveal Its Mechanism of Controlling Seed Germination.

Seed dormancy and germination are key events in plant development and are critical for crop production, and defects in seed germination or the inappropriate release of seed dormancy cause substantial losses in crop yields. Rice is the staple food for more than half of the world's population, and preharvest sprouting (PHS) is one of the most severe problems in rice production, due to a low level of seed dormancy, especially under warm and damp conditions. Therefore, PHS leads to yield loss and a decrease in rice quality and vitality. We reveal that mutation of OsbZIP09 inhibited rice PHS. Analysis of the expression of OsbZIP09 and its encoded protein sequence and structure indicated that OsbZIP09 is a typical bZIP transcription factor that contains conserved bZIP domains, and its expression is induced by ABA. Moreover, RNA sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analyses were performed and 52 key direct targets of OsbZIP09 were identified, including OsLOX2 and Late Embryogenesis Abundant (LEA) family genes, which are involved in controlling seed germination. Most of these key targets showed consistent changes in expression in response to abscisic acid (ABA) treatment and OsbZIP09 mutation. The data characterize a number of key target genes that are directly regulated by OsbZIP09 and contribute to revealing the molecular mechanism that underlies how OsbZIP09 controls rice seed germination.

A Connection between Lysine and Serotonin Metabolism in Rice Endosperm.

Cereal endosperms produce a vast array of metabolites, including the essential amino acid lysine (Lys). Enhanced accumulation of Lys has been achieved via metabolic engineering in cereals, but the potential connection between metabolic engineering and Lys fortification is unclear. In mature seeds of engineered High Free Lysine (HFL) rice (Oryza sativa), the endosperm takes on a characteristic dark-brown appearance. In this study, we use an integrated metabolomic and transcriptomic approach combined with functional validation to elucidate the key metabolites responsible for the dark-brown phenotype. Importantly, we found that serotonin biosynthesis was elevated dramatically and closely linked with dark-brown endosperm color in HFL rice. A functional connection between serotonin and endosperm color was confirmed via overexpression of TDC3, a key enzyme of serotonin biosynthesis. Furthermore, we show that both the jasmonate signaling pathway and TDC expression were strongly induced in the late stage of endosperm development of HFL rice, coinciding with serotonin accumulation and dark-brown pigmentation. We propose a model for the metabolic connection between Lys and serotonin metabolism in which elevated 2-aminoadipate from Lys catabolism may play a key role in the connection between the jasmonate signaling pathway, serotonin accumulation, and the brown phenotype in rice endosperm. Our data provide a deeper understanding of amino acid metabolism in rice. In addition, the finding that both Lys and serotonin accumulate in HFL rice grains should promote efforts to create a nutritionally favorable crop.

Abscisic Acid Represses Rice Lamina Joint Inclination by Antagonizing Brassinosteroid Biosynthesis and Signaling.

Leaf angle is a key parameter that determines plant architecture and crop yield. Hormonal crosstalk involving brassinosteroid (BR) plays an essential role in leaf angle regulation in cereals. In this study, we investigated whether abscisic acid (ABA), an important stress-responsive hormone, co-regulates lamina joint inclination together with BR, and, if so, what the underlying mechanism is. Therefore, lamina joint inclination assay and RNA sequencing (RNA-Seq) analysis were performed here. ABA antagonizes the promotive effect of BR on leaf angle. Hundreds of genes responsive to both hormones that are involved in leaf-angle determination were identified by RNA-Seq and the expression of a gene subset was confirmed using quantitative real-time PCR (qRT-PCR). Results from analysis of rice mutants or transgenic lines affected in BR biosynthesis and signaling indicated that ABA antagonizes the effect of BR on lamina joint inclination by targeting the BR biosynthesis gene D11 and BR signaling genes GSK2 and DLT, thus forming a multi-level regulatory module that controls leaf angle in rice. Taken together, our findings demonstrate that BR and ABA antagonistically regulate lamina joint inclination in rice, thus contributing to the elucidation of the complex hormonal interaction network that optimizes leaf angle in rice.

iTRAQ-Based Analysis of Proteins Co-Regulated by Brassinosteroids and Gibberellins in Rice Embryos during Seed Germination.

Seed germination, a pivotal process in higher plants, is precisely regulated by various external and internal stimuli, including brassinosteroid (BR) and gibberellin (GA) phytohormones. The molecular mechanisms of crosstalk between BRs and GAs in regulating plant growth are well established. However, whether BRs interact with GAs to coordinate seed germination remains unknown, as do their common downstream targets. In the present study, 45 differentially expressed proteins responding to both BR and GA deficiency were identified using isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis during seed germination. The results indicate that crosstalk between BRs and GAs participates in seed germination, at least in part, by modulating the same set of responsive proteins. Moreover, most targets exhibited concordant changes in response to BR and GA deficiency, and gene ontology (GO) indicated that most possess catalytic activity and are involved in various metabolic processes. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was used to construct a regulatory network of downstream proteins mediating BR- and GA-regulated seed germination. The mutation of GRP, one representative target, notably suppressed seed germination. Our findings not only provide critical clues for validating BR⁻GA crosstalk during rice seed germination, but also help to optimise molecular regulatory networks.

A residue substitution in the plastid ribosomal protein L12/AL1 produces defective plastid ribosome and causes early seedling lethality in rice.

The plastid ribosome is essential for chloroplast biogenesis as well as seedling formation. As the plastid ribosome closely resembles the prokaryotic 70S ribosome, many plastid ribosomal proteins (PRPs) have been identified in higher plants. However, their assembly in the chloroplast ribosome in rice remains unclear. In the present study, we identified a novel rice mutant, albino lethal 1 (al1), from a chromosome segment substitution line population. The al1 mutant displayed an albino phenotype at the seedling stage and did not survive past the three-leaf stage. No other apparent differences in plant morphology were observed in the al1 mutant. The albino phenotype of the al1 mutant was associated with decreased chlorophyll content and abnormal chloroplast morphology. Using fine mapping, AL1 was shown to encode the PRPL12, a protein localized in the chloroplasts of rice, and a spontaneous single-nucleotide mutation (C/T), resulting in a residue substitution from leucine in AL1 to phenylalanine in al1, was found to be responsible for the early seedling lethality. This point mutation is located at the L10 interface feature of the L12/AL1 protein. Yeast two-hybrid analysis showed that there was no physical interaction between al1 and PRPL10. In addition, the mutation had little effect on the transcript abundance of al1, but had a remarkable effect on the protein abundance of al1 and transcript abundance of chloroplast biogenesis-related and photosynthesis-related genes. These results provide a first glimpse into the molecular details of L12's function in rice.

Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice.

Major ionic compositions of fine particulate matter in an animal feeding operation facility and its vicinity.

Animal feeding operations (AFOs) produce particulate matter (PM) and gaseous pollutants. Investigation of the chemical composition of PM2.5 inside and in the local vicinity of AFOs can help to understand the impact of the AFO emissions on ambient secondary PM formation. This study was conducted on a commercial egg production farm in North Carolina. Samples of PM2.5 were collected from five stations, with one located in an egg production house and the otherfour located in the vicinity ofthe farm alongfour wind directions. The major ions of NH4+, Na+, K+, SO4(2-), Cl-, and NO3- were analyzed using ion chromatography (IC). In the house, the mostly abundant ions were SO4(2-), Cl-, and K+. At ambient stations, SO4(2-), and NH4+ were the two most abundant ions. In the house, NH4+, SO4(2-), and NO3- accounted for only 10% of the PM2.5 mass; at ambient locations, NH4+, SO4(2-), and NO3- accounted for 36-41% of the PM2.5 mass. In the house, NH4+ had small seasonal variations indicating that gas- phase NH3. was not the only major force driving its gas-particle partitioning. At the ambient stations, NH4+ had the highest concentrations in summer In the house, K+, Na+, and Cl- were highly correlated with each other In ambient locations, SO4(2-) and NH4+ had a strong correlation, whereas in the house, SO4(2-) and NH4+ had a very weak correlation. Ambient temperature and solar radiation were positively correlated with NH4+ and SO4(2-). This study suggests that secondary PM formation inside the animal house was not an important source of PM2.5. In the vicinity, NH3 emissions had greater impact on PM2.5 formation.

ABA-mediated regulation of rice grain quality and seed dormancy via the NF-YB1-SLRL2-bHLH144 Module.

Abscisic acid (ABA) plays a crucial role in promoting plant stress resistance and seed dormancy. However, how ABA regulates rice quality remains unclear. This study identifies a key transcription factor SLR1-like2 (SLRL2), which mediates the ABA-regulated amylose content (AC) of rice. Mechanistically, SLRL2 interacts with NF-YB1 to co-regulate Wx, a determinant of AC and rice quality. In contrast to SLR1, SLRL2 is ABA inducible but insensitive to GA. In addition, SLRL2 exhibits DNA-binding activity and directly regulates the expression of Wx, bHLH144 and MFT2. SLRL2 competes with NF-YC12 for interaction with NF-YB1. NF-YB1 also directly represses SLRL2 transcription. Genetic validation supports that SLRL2 functions downstream of NF-YB1 and bHLH144 in regulating rice AC. Thus, an NF-YB1-SLRL2-bHLH144 regulatory module is successfully revealed. Furthermore, SLRL2 regulates rice dormancy by modulating the expression of MFT2. In conclusion, this study revealed an ABA-responsive regulatory cascade that functions in both rice quality and seed dormancy.

Characterization of the spatial and temporal expression of the OsSSII-3 gene encoding a key soluble starch synthase in rice.

BACKGROUND: Starch, the major component of rice grain, consists of amylose and amylopectin. SSIIa, a key soluble starch synthase involved in the biosynthesis of rice amylopectin, is a major factor that controls the gelatinization temperature of rice grain. Extensive work has been done and impressive progress has been made in elaborating the function of the gene encoding SSIIa (OsSSII-3). However, the systematic expression analysis of OsSSII-3 is still rare. RESULTS: In the present study, we performed a comprehensive expression analysis of OsSSII-3 in both the developing seeds and other tissues of indica rice 9311 by using quantitative real-time PCR. The results showed that the gene was dominantly expressed in the developing seeds. In addition, the promoter sequence of OsSSII-3 was cloned and fused with the GUS reporter gene and its expression was carefully monitored in the transgenic rice. The data from both histochemical and fluorometric analyses showed that the OsSSII-3 promoter was capable of driving the target gene to have an endosperm-specific expression, which may be due to the existing of several endosperm-specific motifs in the promoter, including the -300 elements, AACA motifs and GCN4 motifs. This result was quite consistent with that of the endogenous transcription analysis of OsSSII-3. CONCLUSION: This study not only advanced our understanding of the spatial and temporal expression characteristics of OsSSII-3, but also provided a valuable promoter for future application in generating elite rice varieties with high nutritional or medicinal value.

Co-Overexpression of Two Key Source Genes, OsBMY4 and OsISA3, Improves Multiple Key Traits of Rice Seeds.

Optimized source-sink interactions are determinants of both rice yield and quality. However, most source genes have not been well studied in rice, a major grain crop. In this study, OsBMY4 and OsISA3, the key ß-amylase and debranching enzymes that control transient starch degradation in rice leaves, were co-overexpressed in rice in order to accelerate starch degradation efficiency and increase the sugar supply for sink organs. Systematic analyses of the transgenic rice indicated that co-overexpression of OsBMY4 and OsISA3 not only promoted rice yield and quality, but also improved seed germination and stress tolerance. Moreover, since the OsBMY4 gene has not been characterized, we generated osbmy4 mutants using CRIPSR/Cas9 gene editing, which helped to reveal the roles of ß-amylase in rice yield and quality. This study demonstrated that specific modulation of the expression of some key source genes improves the source-sink balance and leads to improvements in multiple key traits of rice seeds.

Field evaluation of particulate matter measurements using tapered element oscillating microbalance in a layer house.

The tapered element oscillating microbalance (TEOM) is one type of continuous ambient particulate matter (PM) monitor. Adsorption and desorption of moisture and semivolatile species may cause positive or negative artifacts in TEOM PM mass measurement. The objective of this field study was to investigate possible uncertainties associated with TEOM measurements in the poultry operation environment. For comparisons of TEOM with filter-based gravimetric method, four instruments (TEOM-PM10, low-volume PM10 sampler TEOM-PM2.5, and PM2.5 speciation sampler) were collocated and tested inside a poultry house for PM2.5 and PM10 (PM with aerodynamic equivalent diameter < or =2.5 and < or =10 microm, respectively) measurements. Fifteen sets of 24-hr PM10 concentrations and 13 sets of 24-hr PM2.5 measurements were obtained. Results indicate that compared with filter-based gravimetric method, TEOM gave significantly lower values of both PM10 and PM2.5 mass concentrations. For PM10, the average ratio of TEOM to the gravimetric method was 0.936. For PM2.5, the average ratio of TEOM to the gravimetric method was 0.738. Particulate matter in the poultry houses possibly contains semivolatile compounds and moisture due to high levels of relative humidity (RH) and gas pollutants. The internal heating mechanism of the TEOM may cause losses in mass through volatilization. To investigate the effects of TEOM settings on concentration measurements, the heaters of two identical TEOMs were set at 50 degrees C, 30 degrees C, or no heating at all. They were collocated and tested for total suspended particle (TSP), PM10, and PM25 measurements in layer house for 6 weeks. For all TSR PM10, and PM2.5 measurements, the internal TEOM temperature setting had a significant effect (P < 0.05). Significantly higher PM mass concentrations were measured at lower temperature settings. The effects of environmental (i.e., temperature, RH, NH3 and CO2 concentrations) and instrumental (i.e., filter loading and noise) parameters on PM measurements were also assessed using regression analysis.

Genes and Their Molecular Functions Determining Seed Structure, Components, and Quality of Rice.

With the improvement of people's living standards and rice trade worldwide, the demand for high-quality rice is increasing. Therefore, breeding high quality rice is critical to meet the market demand. However, progress in improving rice grain quality lags far behind that of rice yield. This might be because of the complexity of rice grain quality research, and the lack of consensus definition and evaluation standards for high quality rice. In general, the main components of rice grain quality are milling quality (MQ), appearance quality (AQ), eating and cooking quality (ECQ), and nutritional quality (NQ). Importantly, all these quality traits are determined directly or indirectly by the structure and composition of the rice seeds. Structurally, rice seeds mainly comprise the spikelet hull, seed coat, aleurone layer, embryo, and endosperm. Among them, the size of spikelet hull is the key determinant of rice grain size, which usually affects rice AQ, MQ, and ECQ. The endosperm, mainly composed of starch and protein, is the major edible part of the rice seed. Therefore, the content, constitution, and physicochemical properties of starch and protein are crucial for multiple rice grain quality traits. Moreover, the other substances, such as lipids, minerals, vitamins, and phytochemicals, included in different parts of the rice seed, also contribute significantly to rice grain quality, especially the NQ. Rice seed growth and development are precisely controlled by many genes; therefore, cloning and dissecting these quality-related genes will enhance our knowledge of rice grain quality and will assist with the breeding of high quality rice. This review focuses on summarizing the recent progress on cloning key genes and their functions in regulating rice seed structure and composition, and their corresponding contributions to rice grain quality. This information will facilitate and advance future high quality rice breeding programs.

Effects of Interactive Dynamic Scalp Acupuncture on Motor Function and Gait of Lower Limbs after Stroke: A Multicenter, Randomized, Controlled Clinical Trial.

OBJECTIVE: To evaluate the effects of interactive dynamic scalp acupuncture (IDSA), simple combination therapy (SCT), and traditional scalp acupuncture (TSA) on motor function and gait of the lower limbs in post-stroke hemiplegia patients. METHODS: A total of 231 patients with post-stroke hemiplegia was randomly divided into IDSA (78 cases), SCT (78 cases), and TSA (75 cases) groups by a random number table. Scalp acupuncture (SA) and lower-limb robot training (LLRT) were both performed in the IDSA and SCT groups. The patients in the TSA group underwent SA and did not receive LLRT. The treatment was administered once daily and 6 times weekly for 8 continuous weeks, each session lasted for 30 min. The primary outcome measures included Fugl-Meyer assessment of the lower extremity (FMA-LE), berg balance scale (BBS), modified barthel index (MBI), and 6-min walking test (6MWT). The secondary outcome measures included stride frequency (SF), stride length (SL), stride width (SW), affected side foot angle (ASFA), passive range of motion (PROM) of the affected hip (PROM-H), knee (PROM-K) and ankle (PROM-A) joints. The patients were evaluated before treatment, at 1- and 2-month treatment, and 1-, and 2-month follow-up visits, respectively. Adverse events during 2-month treatment were observed. RESULTS: Nineteen patients withdrew from the trial, with 8 in the IDSA and 5 in the SCT groups, 6 in the TSA group. The FMA-LE, BBS, 6MWT and MBI scores in the IDSA group were significantly increased after 8-week treatment and 2 follow-up visits compared with the SCT and TSA groups (P<0.05 or P<0.01). Compared with pre-treatment, the grade distribution of BBS and MBI scores in the 3 groups were significantly improved at 1, 2-month treatment and 2 follow-up visits (P<0.05 or P<0.01). The SF, PROM-H, PROM-K and PROM-A in the IDSA group was significantly increased compared with the SCT and TSA groups after 8-week of treatment (P<0.05 or P<0.01). Compared with the SCT group, ASFA of the IDSA group was significantly reduced after 8-week of treatment (P<0.05). SF, SL, PROM-K and PROM-A were significantly increased at the 2nd follow-up visit whereas the ASFA was significantly reduced in the IDSA group compared with the SCT groups at 1st follow-up visit (P<0.05 or P<0.01). The SF was significantly increased in the SCT group compared with the TSA group after 8-week treatment (P<0.05). Compared with the TSA group, PROM-K, PROM-A were significantly increased at the 2nd follow-up visit (P<0.05). CONCLUSIONS: The effects of IDSA on lower-limb motor function and walking ability of post-stroke patients were superior to SCT and TSA. The SCT was comparable to TSA treatment, and appeared to be superior in improving the motion range of the lower extremities. (Registration No. ChiCTR1900027206).

Effect of Interactive Dynamic Scalp Acupuncture on Post-Stroke Cognitive Function, Depression, and Anxiety: A Multicenter, Randomized, Controlled Trial.

OBJECTIVE: To compare the clinical effects of interactive dynamic scalp acupuncture (IDSA), simple combination therapy (SCT), and traditional scalp acupuncture (TSA) on cognitive function, depression and anxiety in patients with post-stroke cognitive impairment. METHODS: A total of 660 patients with post-stroke cognitive impairment who were admitted to 3 hospitals in Shenzhen City between May 2017 and May 2020 were recruited and randomly assigned to the IDSA (218 cases), SCT (222 cases) and TSA groups (220 cases) according to a random number table. All the patients received conventional drug therapy for cerebral stroke and exercise rehabilitation training. Scalp acupuncture and computer-based cognitive training (CBCT) were performed simultaneously in the IDSA group, but separately in the morning and in the afternoon in the SCT group. The patients in the TSA group underwent scalp acupuncture only. The course of treatment was 8 weeks. Before treatment (M0), 1 (M1) and 2 months (M2) after treatment, as well as follow-up at 1 (M3) and 2 months (M4), the cognitive function of patients was assessed using the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment Scale (MoCA) Scales; depression, anxiety, sleep quality, and self-care ability of patients were assessed using Hamilton Depression Rating Scale (HAMD), Hamilton Anxiety Rating Scale (HAMA), Pittsburgh Sleep Quality Index (PSQI), and Modified Barthel Index (MBI), respectively. During this trial, all adverse events (AEs) were accurately recorded. RESULTS: There were no significant differences in the MMSE, MoCA, HAMD, HAMA, PSQI, and MBI scores among the 3 groups at M0 (all P>0.05). In the IDSA group, the MMSE, MoCA and MBI scores from M2 to M4 were significantly higher than those in the SCT and TSA groups, while the HAMD, HAMA and PSQI scores were significantly reduced (all P<0.01). The changes of all above scores (M2-M0, M4-M0) were significantly superior to those in the SCT and TSA groups (all P<0.01, except M4-M0 of HAMD). At M2, the severity of MMSE, HAMD, HAMA, PSQI and MBI in the IDSA group was significantly lower than that in the SCT and TSA groups (all P<0.01). There was no serious AE during this trial. CONCLUSIONS: IDSA can not only significantly improve cognitive function, but also reduce depression, anxiety, which finally improves the patient's self-care ability. The effect of IDSA was significantly better than SCT and TSA. (Trial registration No. ChiCTR1900027206).

[Interactive scalp acupuncture for cognitive dysfunction after stroke: a randomized controlled trial].

OBJECTIVE: To compare the efficacy of interactive scalp acupuncture, scalp acupuncture alone and scalp acupuncture plus cognitive training for cognitive dysfunction after stroke. METHODS: A total of 660 patients with cognitive dysfunction after stroke were randomly divided into an interactive scalp acupuncture group (218 cases, 18 cases dropped off), a scalp acupuncture group (220 cases, 20 cases dropped off) and a scalp acupuncture plus cognitive training group (222 cases, 22 cases dropped off). All the patients were treated with routine medication and exercise rehabilitation training. The interactive scalp acupuncture group was treated with scalp acupuncture on the parietal midline, and contralateral anterior parietal temporal oblique line and posterior parietal temporal oblique line at the same time of cognitive training; the scalp acupuncture group was treated with scalp acupuncture alone, and the scalp acupuncture plus cognitive training group was treated with scalp acupuncture and cognitive training in the morning and afternoon respectively. All the treatments were given once a day, 6 times a week for 8 weeks. Montreal cognitive assessment (MoCA) scale score was used to evaluate the cognitive function before treatment, 4 weeks and 8 weeks into treatment. RESULTS: Compared before treatment, the total score of MoCA was increased after 4-week treatment and 8-week treatment in the three groups (P<0.01), and the score in the interactive scalp acupuncture group was higher than that in the scalp acupuncture group and the scalp acupuncture plus cognitive training group (P<0.05, P<0.01). Compared before treatment, each item score of MoCA was increased after 8-week treatment in the three groups (P<0.01), and the score in the interactive scalp acupuncture group was higher than that in the scalp acupuncture group and the scalp acupuncture plus cognitive training group (P<0.01). Except for the attention, the remaining items scores of MoCA in the scalp acupuncture plus cognitive training group were higher than those in the scalp acupuncture group (P<0.01). CONCLUSION: The interactive scalp acupuncture could significantly improve the cognitive function in patients with cognitive dysfunction after stroke, and the efficacy is superior to scalp acupuncture alone and scalp acupuncture plus cognitive training.