Malus domestica Borkh.). MdAP2-34 expression exhibited a close correlation with carotenoid content in ‘Benin Shogun’ and ‘Yanfu 3’ fruit flesh. MdAP2-34 promotes carotenoid accumulation in MdAP2-34-OVX transgenic apple calli and fruits by participating in the carotenoid biosynthesis pathway. The major carotenoid contents of phytoene and β-carotene were much higher in overexpressing MdAP2-34 transgenic calli and fruit skin, yet the predominant compound of lutein showed no obvious difference, indicating that MdAP2-34 regulates phytoene and β-carotene accumulation but not lutein. MdPSY2-1 (phytoene synthase 2) is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. In addition, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple, and this research is valuable for accelerating the apple breeding process.Color is an important trait for horticultural crops. Carotenoids are one of the main pigments for coloration and have important implications for photosynthesis in plants and benefits for human health. Here, we identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor named MdAP2-34 in apple ( Carotenoids are one of the main pigments for coloration, showing yellow, orange, red, or others in horticultural reproductive organs2. Carotenoids are important components for photosynthesis and are involved in light harvesting and photoprotection, plant growth, and development3. Carotenoids are valuable molecules that are beneficial for human health and animal survival, and carotenoids are also precursors of provitamin A and are antioxidants for reducing various chronic diseases5.Color is an important trait for horticultural crops, such as fruits, vegetables, and flowers7. The structural genes encoding metabolic enzymes in the carotenoid pathway, including phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (ZISO), zeta-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene β-cyclase (LCY-β), lycopene ε-cyclase (LCY-ε), beta-carotene hydroxylase (CHY-β), zeaxanthin epoxidase (ZEP), carotenoid cleavage dioxygenases (CCD), and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been characterized2. Previous studies have shown that PSYs play a significant role in carotenoid accumulation9. Transgenic tomato fruits and potato tubers overexpressing PSY show increased levels of total carotenoids11. Multiple PSY genes modulate carotenoid accumulation in many species, such as maize, rice, loquat, and apple15. CaPSY2 can increase carotenoid content and compensate for the absence of PSY1 in pepper (Capsicum spp.) fruit16. OR/OR-like proteins influence carotenoid content by affecting PSY protein levels through posttranscriptional regulation in Arabidopsis and sweet potato18. For the other limiting genes, the downregulation of LCY-ε increases the β-carotene content in potato19. The expression of the NCED gene results in a low β-carotenoid content in the white apricot cultivar20. Inhibition of BoaCRTISO expression in Chinese kale affected carotenoid pathways and led to decreased carotenoid concentrations21. The Orange gene CmOr regulates β-carotene accumulation in melon fruit by repressing β-carotene turnover and posttranslationally enhancing carotenogenesis in chromoplasts22.The biosynthetic pathway of carotenoids is well known in a variety of plant species, and it is modulated by many factors, such as limiting and metabolic enzyme steps, the availability of storage organs and structures, or transcription factors (TFs)23, and CsMADS6 modulates carotenoid metabolism by directly regulating LCYb1 in sweet orange24. The NAC TFs SlNAC1, SlNAC4, and CpNAC1 regulate carotenoid accumulation during tomato and papaya fruit ripening27. MYB TFs, RCP1 in Mimulus lewisii flowers28, CrMYB68 in citrus29, and AdMYB7 in kiwifruit30, are all involved in carotenoid regulation. The R2R3-MYB TF WP1 together with MtTT8 and MtWD40-1 promotes floral carotenoid accumulation by directly activating the expression of MtLYCe and MtLYCb in Medicago petals31. CpbHLH1/2 regulates carotenoid biosynthesis and is related to carotenoid content during fruit ripening of papaya32. Sl_H2A. Z plays an essential role in the modulation of carotenoid content in tomato33.A number of TFs have been identified to be involved in the carotenoid pathway. AGAMOUS-like 1 and FRUITFULL, which belong to MADS-box TFs, regulate carotenoid content in tomato34. AP2 subfamily proteins contain single or two AP2/ERF domains that are involved in the regulation of development35. AP2/ERF proteins can bind to DNA cis-acting elements, including DRE (CCGAC), GCC box (AGCCGCC), (A/G)CC(G/C)AC, and AA(T)TTCAAA36. The TFs of the AP2/ERF superfamily play important roles in growth, development, and stress response processes in higher plants37. PpERF3 activates ABA biosynthesis by positively regulating PpNCED2/3 transcription in peach38. AP2/ERF TFs have been reported to modulate carotenoid accumulation. SIERF6 negatively regulates carotenoid accumulation in tomato39. In Arabidopsis, ERFs and RAP2.2 promote carotenoid biosynthesis by binding to the PSY promoter40. However, whether and how AP2/ERF TFs modulate carotenoid metabolism in apple fruit are currently unknown.The APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) superfamily contains four subfamilies according to the number of AP2/ERF domains and sequences, including AP2, CBF/DREB, ERF, and RAVMalus domestica Borkh.), which has high nutrient value, is one of the most economical fruit crops worldwide. Fruit flavor and color are major apple traits that determine consumer preferences. Because of differences in carotenoid accumulation, the flesh color varies from white to yellow in different apple genotypes41. Previous studies indicated that MdPSY2 played a dominant role in carotenoid accumulation in apple fruits and was activated by AP2/ERF TFs41. There was a strong positive correlation between the expression of the TF AP2D26 and PSY2 in apple fruit15. In this study, we found that the total carotenoid contents of ‘Benin Shogun’ fruits were significantly higher than those of ‘Yanfu 3’ fruits during the developmental period at 150 and 170 days after full bloom (DAFB). We characterized an AP2 TF named MdAP2-34 and found that it induced increases in carotenoid accumulation. MdAP2-34 was able to promote carotenoid levels by enhancing MdPSY2-1 promoter activity. Moreover, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple.Apple . ‘Benin Shogun’ and ‘Yanfu 3’ are bud mutations of ‘Red Fuji’. Every treatment included six fruits. Leaves of ‘Benin Shogun’ were collected in late spring and used for DNA extraction. Apple fruits of Granny Smith used for the transient transformation were collected at 150 DAFB. The fruits and leaves were stored at –78 °C until use.42. The calli were cultured and subcultured on MS medium  supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid  and 1.0 mg/L 6-benzylaminopurine (6-BA).Apple calli were induced from the ‘Orin’ apple flesh and were used for the transformation42. The open reading frame (ORF) sequence of MdAP2-34 without the stop codon was cloned and inserted into the pRI101:GFP expression vector. The fusion construct 35S:MdAP2-34:GFP was transformed into N. benthamiana leaf epidermal cells. Fluorescence was detected by confocal microscopy after 2−3 days of transfection. DAPI  was used to stain the nucleus.The subcellular localization of MdAP2-34 was conducted as previously described43. Each sample was extracted with acetone-hexane . The total carotenoid content of the extracts was determined using a UV-2550 UV−vis spectrophotometer.The extraction and determination of total carotenoids were performed using a method as described previouslyhttp://www.metware.cn/)33. Carotenoid compounds were detected using the AB Sciex QTRAP6500 LC–MS/MS platform. Three replicates of each assay were performed. Apple fruit skin and apple calli were freeze-dried and then homogenized and powdered in a mill of 50 mg of dried powder. The dried powder was then extracted with a mixed solution of n-hexane:acetone:ethanol. The extracts were analyzed using an LC-APCI-MS/MS system . The analytical and detection conditions were as follows: HPLC: column, YMC C30 ; solvent system, methanol:acetonitrile  :methyl tert-butyl ether (0.01% BHT); gradient program, 100:0 V/V at 0–3 min, 100:0 V/V at 3–6 min, 58:42 V/V at 6–8 min, 20:80 V/V at 8–9 min, 5:95 V/V at 9 min, 100:0 V/V at 9.1–11 min; flow rate, 0.8 mL/min; temperature, 28 °C; injection volume, 2 μL. The α-carotene, antheraxanthin, violaxanthin, neoxanthin, phytofluene, phytoene, β-cryptoxanthin, and rubixanthin standards were purchased from BOC Chemicals. The zeaxanthin standard was purchased from Bide Chemicals. The β-carotene standard was purchased from RHAWN Chemicals. The Lutein standard was purchased from Aladdin Chemicals.Carotenoid components of transformed apple fruit skin and apple calli were measured by MetWare Company , and qRT–PCR (reverse transcription-quantitative PCR) was carried out as previously described45. Each ORF sequence of MdAP2-34 and MdPSY2-1 was cloned and inserted into pRI101-FLAG (overexpression vector). A 728 bp fragment (located between +5 and +732 bp of the ORF) of MdAP2-34 was cloned and inserted into pFGC5941 (RNAi vector). Every recombinant plasmid of 35S:MdAP2-34, RNAi:MdAP2-34, and 35 S:MdPSY2-1 was then transformed into Granny Smith apple fruit skin and Orin apple calli45. All primers are used in Supplemental Table The overexpression and interference plasmids were constructed as previously described45. Anti-ACTIN antibody and anti-FLAG antibody were used, which were obtained from Beyotime Biotech Company.Protein extraction and western blotting assays were performed as previously described45. The ORF sequence of MdAP2-34 was cloned and inserted into the vector pJG4-5 (the effector vector) . Each promoter fragment (upstream of the ATG codon) of MdPSY1 (1725 bp), MdPSY2-1 (1733 bp), MdPSY2-2 (1850 bp), MdPDS (1659 bp), MdZDS1 (1800 bp), MdZDS2 (1683 bp), MdLCY-β (1748 bp), MdCHY-β2-1 (1631 bp), MdZEP (1758 bp), and MdCCD1 (1782 bp) was cloned into the vector pLacZi (the reporter vector). All primers are used in Supplemental Table Y1H (yeast one-hybrid) assays were conducted as previously described45. The ORF sequence of MdAP2-34 was cloned into the vector pGEX-4T-1 (with GST-tag) and then transformed into BL21 (DE3) Escherichia coli cells for MdAP2-34 protein expression. The 5ʹ biotin end-labeled probes were synthesized by Sangon Biotech. The EMSA was performed using the LightShift™ Chemiluminescent EMSA Kit . The primers were used, and the biotin-labeled sequences of the MdPSY2-1 promoter are shown in Supplemental Table The EMSA was conducted as previously described45. The ORF of MdAP2-34 was inserted into the pRI101-FLAG vector (with 3×FLAG), and the fusion MdAP2-34-FLAG was generated. Recombinant pRI101-FLAG-MdAP2-34 was transformed into apple calli, and the MdAP2-34-FLAG protein was extracted from transgenic calli. ChIP-PCR analysis was conducted using the EpiQuik™ Plant ChIP Kit  and anti-DDDDK tag (Binds to FLAG® tag sequence) antibody . Three promoter regions of MdPSY2-1 were used to assess their enrichment. Every ChIP assay was conducted three replicates. The primers used are in Supplemental Table The ChIP-PCR assay was performed following Jia et al.45. The ORF of MdAP2-34 was inserted into pGreenII 62-SK (the effector vector) under the control of the CaMV35S promoter. The promoter fragment of MdPSY2-1 was inserted into pGreenII 0800-LUC (the reporter vector). The vectors of the effector and reporter were cotransformed into 4-week-old N. benthamiana leaves. The luciferase signals of Firefly and Renilla were observed and measured using an Infinite M200 . Every LUC analysis was conducted six replicates. The primers used are in Supplemental Table The LUC analysis was conducted as previously described45 .In the experimental design used, the data were tested and analyzed for three biological replicates, and each biological replicate was analyzed three technical repetitions. Significant differences were determined using GraphPad Prism 5 software34. Based on the annotation of the website (http://bioinformatics.cau.edu.cn/AppleMDO/)46, 12 AP2 TFs were identified that could be expressed in apple fruits  between each AP2 TF transcript levels and total carotenoid content in apple flesh of ‘Benin Shogun’ and ‘Yanfu 3’ indicated that the expression level of MdAP2-34 was significantly positively associated with carotenoid contents  with carotenoid content in ‘Benin Shogun’ and ‘Yanfu 3’ fruit flesh  to contain two conserved AP2 domains  . Phylogenetic analysis showed that MdAP2-34 and AtAIL5 have high homology , and previous results indicated that AtAIL5 could induce a larger floral organ phenotype47. To investigate the subcellular localization of the MdAP2-34 protein, the ORF of MdAP2-34 was fused to EGFP (MdAP2-34:GFP) and transiently expressed in Nicotiana benthamiana leaves. The vector pRI101-GFP (35S:GFP) was used as a control. The results indicated that fusion protein signals were identified in the nucleus  is a typical AP2/ERF TF. MdAP2-34 contains an ORF of 1,101 bp and encodes a predicted 367 amino acid (aa) protein. MdAP2-34 was predicted by NCBI . The MdAP2-34 expression level was remarkably higher in MdAP2-34-OVX transgenic calli than in P101F calli but much lower in the MdAP2-34-RNAi calli than in P5941 calli , which have previously been characterized2. The expression levels of genes related to carotenoid biosynthesis were measured and analyzed in TAC and transgenic Granny Smith fruit skin (TFS) using qRT–PCR  analysis was conducted to detect whether MdAP2-34 directly regulates the transcription of ter Fig. . An EMSAter Fig. . MdAP2-3ter Fig. .Fig. 4MdMdPSY2-1 promoter in vivo, a ChIP-PCR (chromatin immunoprecipitation-PCR) assay was performed. The MdAP2-34-FLAG fusion protein was overexpressed and synthesized in TAC, and P101F was used as a control. The presence of MdAP2-34 enhanced the PCR-based detection of the MdPSY2-1 promoter  transactivation assay in leaves of wild tobacco (Nicotiana benthamiana) through cotransformation with Pro35S:MdAP2-34 and ProMdPSY2-1:LUC constructs was conducted. Three combinations of ProMdPSY2-1:LUC (ProMdPSY2-1) and Pro35S:MdAP2-34 (MdAP2-34) were used, including ProMdPSY2-1 to MdAP2-34 ratios of 1:1, 1:5, and 1:9, respectively. When MdAP2-34 was cotransformed with ProMdPSY2-1, MdPSY2-1 promoter activity was significantly increased. As the ProMdPSY2-1 to MdAP2-34 ratio increased from 1:1 to 1:9, the promoter activity of MdPSY2-1 was significantly increased  than in P101F calli  but high fruit carotenoid concentrations in the rootstock cultivar ‘Aotea’50. However, AP2/ERF TFs have rarely been reported to regulate carotenoid biosynthesis in apple fruits. In this study, based on the previous analysis of the AP2/ERF family34 and the annotation of websites (http://bioinformatics.cau.edu.cn/AppleMDO/)46, we identified 12 AP2 TFs from the AP2 subfamily expressed in apple fruits. By correlation analysis, we characterized MdAP2-34 as a candidate AP2 TF associated with carotenoid accumulation (Supplemental Table Arabidopsis, and AtAIL5 could induce a larger floral organ phenotype47. MdAP2-34 may have a similar function, and further studies are required to test its function.Many TFs play important roles in carotenoid biosynthesis. It is well known that AP2/ERFs participate in many plant development processes and play important roles in regulating fruit quality and pigment biosynthesisPSY promoter in Arabidopsis40. However, SIERF6 represses carotenoid accumulation in tomato, and reduced SlERF6 expression results in increased carotenoid concentration39. This study indicated that overexpressing MdAP2-34 in transgenic calli and fruits enhanced carotenoid accumulation (Fig. 41. In ripening apple fruit flesh, the predominant compounds in ‘Royal Gala’ were violaxanthin and neoxanthin, those in ‘Aotea’ flesh were β-cryptoxanthin and β-carotene, and those in ‘Granny Smith’ were lutein. The study also indicated that lutein was the predominant compound in Granny Smith fruit skin, followed closely by β-carotene and phytoene (Table MdAP2-34-overexpressing transgenic calli and fruit skin, yet lutein showed no obvious difference, indicating that MdAP2-34 regulates β-carotene and phytoene accumulation but not lutein. MdAP2-34 could not modulate lutein-related genes, and other regulators or proteins may contribute to lutein accumulation in apple, which needs further study.It has been reported that AP2/ERF superfamily TFs can modulate carotenoid biosynthesis. With regard to carotenoid regulation, RAP2.2 promotes carotenoid accumulation by binding to the ion Fig. . The conion Fig. . The preMdPSY2-1, MdPDS, MdZDS1, MdLCY-β, and MdCHY-β2-1, which were markedly upregulated in both MdAP2-34-OVX TAC and fruits and were markedly downregulated in both MdAP2-34-RNAi TAC and fruits (Fig. MdPSY2-1 promoter activity. However, MdAP2-34 only directly binds to the MdPSY2-1 promoter but not the other carotenoid-related structural genes (Fig. The structural genes encoding enzymes of carotenoid biosynthesis included nes Fig. . There mPSY, PDS, ZISO, ZDS, CRTISO, LCY-β, CHY-β, ZEP, VDE, CCS, CCD, and NCED, which have previously been characterized2. Multiple PSY genes deserve functional diversity, some of which play important roles in carotenoid accumulation9. Previous studies indicated that PSYs modulate carotenoid accumulation in many fruits of plant species, such as tomato8, potato11, loquat14, apple15, and peppers16. MdPSY2 exhibited higher transcript levels than MdPSY1 in diverse apple cultivars, indicating that MdPSY2 could be mainly responsible for the first carotenoid pathway step and plays a dominant role in carotenoid accumulation in apple fruits41. The different expression levels of MdPSY1 and MdPSY2 in apple were influenced by their promoters and activated by AP2/ERF TFs. There was a strong positive correlation between the expression of the TF AP2D26 and PSY2 in apple fruit, revealing a potential regulatory relationship15. The role of PSYs in the carotenoid pathway was prominent in Arabidopsis, and the PSY promoter was implicated and activated by AtRAP2.240. The results indicated that MdPSY2-1 increases carotenoid content and was directly activated by MdAP2-34 (Figs. 17. In this study, by overexpressing the MdPSY2-1 gene in apple calli, the total carotenoid and phytoene contents were markedly increased (Fig. MdPSY2-1 is a major gene in the carotenoid biosynthesis pathway in apple fruit.The genes encoding enzymes of carotenoid biosynthesis include MdPSY2-1 is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. Furthermore, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. This research is valuable for accelerating the apple breeding process and for further understanding the complex mechanisms of carotenoid synthesis in horticultural plants.Herein, we found that MdAP2-34 promotes carotenoid accumulation by participating in the carotenoid biosynthesis pathway. Supplementary Figures 1-3, Tables 1-2


Magnetic resonance diffusion tensor imaging (DTI) is a new kind of magnetic resonance imaging technology. Its imaging principle is to distinguish different pathological tissues according to the movement of water molecules, which is higher than regular magnetic resonance diffusion-weighted imaging. Magnetic resonance diffusion tensor imaging has exact utility price in medical analysis and sickness evaluation. However, there are few researches on the utility of diffusion tensor imaging in the rehabilitation comparison of patients. This paper explores the utility of magnetic resonance DTI science in evaluating the impact of postoperative patients' exercising rehabilitation. Taking stroke patients as an example, through giving patients rehabilitation training method, using magnetic resonance DTI technology, the motor function rehabilitation of patients was evaluated, and FA changes of the affected side and healthy side and Fugl–Meyer score of two groups of patients before and after rehabilitation were observed. The software outcomes exhibit that, in the contrast of rehabilitation therapy impact of motor feature in sufferers with cerebral infarction, the use of magnetic resonance DTI technological know-how gives a foundation for clinicians to deeply apprehend the CST involvement of patients, which helps to scientifically evaluate the effect and quality of limb motor rehabilitation training of patients and provides a basis for disease treatment. Nuclear magnetic resonance diffusion tensor imaging (DTI) is a new magnetic resonance technological know-how developed from traditional MRI scanning in the current years. It can quantitatively analyse the diffusion movement of water molecules in tissue in three-D space and can be imaged in accordance with the anisotropic traits of the diffusion movement of water molecules in tissue . DiffusiCerebral infarction is one of the common clinical cerebrovascular diseases, which has the characteristics of high mortality and disability. According to the survey, 75% of cerebral infarction patients in China will be accompanied by different degrees of limb dysfunction, of which 10% will be accompanied by severe disability, which directly affects the prognosis and quality of life of patients. Limb function rehabilitation is an effective way to improve motor dysfunction after cerebral infarction . MagnetiThis paper frequently describes the content material of the paper from five parts, and the particular association of every chapter is summarized as follows: Conventional MRI can determine the location and scope of intracranial tumours, which has been consensus, but it still has great limitations for the qualitative and staging of intracranial tumours. In current years, home and overseas students have observed that DTI performs an extraordinarily necessary function in the qualitative identification of intracranial tumours, the differentiation of specific factors of tumours, and the identification of tumour infiltration and Genius enema. Magnetic resonance DTI technological know-how has regularly turned out to be a lookup hotspot of pupils at domestic and abroad. The utility of magnetic resonance DTI science has been extensively involved and developed in the latest years. The utility of magnetic resonance DTI science in the impact of exercising rehabilitation is as follows.It was once determined that the FA price of the tumour aspect was once extensively decreased compared to that of the healthful aspect . The reaRelevant scholars believe that DTI can distinguish the main body of glioma from tumour infiltrating enema area, with a sensitivity of 98% and a specificity of 81%. They can be divided into the following types according to the invasion of white matter fibre tracts displayed by DTI: progressive type: the position and shape of white matter fibre on the diseased side are changed compared with the normal white matter fibre on the opposite side . The FA Conventional MRI can determine the location and scope of intracranial tumours, which has been a consensus, but it still has great limitations for the qualitative and staging of intracranial tumours. Price et al. found that the FA value of the tumour side was significantly lower than that of the contralateral side. The analysis may be due to the invasion of tumour cells, which destroyed the space between nerve fibres and axons. The infiltration of water molecules and the necrosis of the tumour lead to the accelerated diffusion of water molecules in all directions, which reduces the degree of anisotropy. Goebel and other scholars combined DTI and 1H-MRS to study glioma and found that the FA value of peritumoural tissue to tumor core tissue gradually decreased, which reflected the continuous decrease of tissue structure, consistent with the change of NAA, and the changes of both reflected the decrease of neuron structure and fibre bundle integrity. Goebel and other scholars also conducted a DTI study on a total of 23 patients with gliomas. It was found that the fibre tracts around low-grade gliomas were less invaded, whilst most of the fibre tracts around high-grade gliomas were invaded.Dispersion is a form of random, colliding, and surpassing action of dependent molecules in nature, particularly Brownian motion. The dispersion of free molecules in pure liquid is isotropic; that is, the dispersion movement in all instructions is uniform. In human intelligence tissue, it is affected via the traits of tissue cells and the inner shape of cells. In the tissue shape with constant arrangement, mainly in the nerve conduction tract, such as the corticospinal tract, the diffusion of water molecules in a number of instructions is different. Generally, it is extrainclined to diffuse alongside the course of the nerve fibre bundle, however, hardly ever alongside the course perpendicular to the nerve fibre bundle . DiffusiDTI is a new magnetic resonance imaging method based totally on traditional MRI and DWI. The random movement of molecules and other microparticles from high concentration to low concentration is called dispersion, which is one of the ways of material movement. In the homogeneous liquid outside the human body, the water molecules move randomly, and the probability of moving in all directions is the same, which is called dispersion isotropy. In the human body, water molecules are affected by different tissues, structures, and other factors in the process of movement, resulting in different diffusion velocity in three-dimensional space. Diffusion in one direction is more limited than that in the other direction, which has a robust route dependence, which is known as anisotropy . DTI is λ1=λ2=λ3.The action of water molecules in homogeneous media is random and disordered; that is, the chance of transferring in all instructions is the same; that is, it has isotropy. In human tissues, the motion of water molecules is affected by way of the shape of tissues and cells, and the degree of dispersion is different in all directions, which is direction-dependent, that is, anisotropic. In homogeneous media, the free motion of water molecules can be isotropic; that is, the dispersion intensity in all directions is the same. The dispersion tensor D is described as a sphere, and the eigenvalues along the three principal coordinates of MR are λ1=λ2=λ3. The direction corresponding to the maximum eigenvalue is parallel to the fibre bundle passing through the voxel. The exercise rehabilitation model based on DTI is shown in In the white matter, due to the blockading of myelin sheath, the diffusion of water molecules is restrained in the identical route as the fibre, which has a high anisotropy. At this time, the diffusion tensor can be expressed as an ellipsoid with its eigenvalue 2/s. The larger the cost is, the more improved the diffusion potential of water molecules is. The most normally used parameter for analysing anisotropy is the ratio of the anisotropic phase of the dispersion to the whole price of the dispersion tensor, which displays the share of the anisotropic element in the entire dispersion tensor. The price is between zero and 1. Zero represents the most isotropic dispersion, such as the dispersion of water molecules in a totally homogeneous medium, and 1 represents the hypothetical most anisotropic dispersion. FA fee in white be countedused to be positively correlated with myelin integrity, fibre density, and parallelism [The common fee of diffusion amplitude in every course of MR voxel represents the dimension or diploma of diffusion of water molecules in a voxel. In many instances, the used index is the apparent diffusion coefficient (ADC), which represents the change in the diffusion of water molecules per unit time. The unit is mmallelism . RelativTraditional brain tumour surgery focuses on relieving the space-occupying effect of brain tumour. In the operation, it is emphasized to remove the tumour as far as possible on the premise of protecting the cerebral cortex, nerves, and important intracranial vessels. However, with the development and establishment of the concept of microinvasion in recent years, the development of microinvasive neurosurgery for brain tumours has paid more and more attention to the protection of patients' neurological function and the quality of life after operation. The last intention is to enhance the exceptional lifestyles and survival time of patients. The emergence of DTI technology provides the possibility for this purpose. It is a major development of MRI technology in recent years, and it is the only way to display the white matter fibre tracts in vivo. By evaluating the movement state of water molecules in the microenvironment, it can not only infer the degree of infiltration and destruction of white matter fibre tracts in brain tumours from the microscopic perspective but also show the extent of brain tumour invasion and its relationship with important white matter tracts. The key to improving the quality of life of patients after operation is to protect the important cerebral cortex and white matter fibre tracts as much as possible. The emergence of functional magnetic resonance (fMRI) helps neurosurgeons to solve the problem of protecting not only the important cerebral cortex but also the important cerebral white matter fibre tracts, mainly the corticospinal tract, corpus callosum, optic radiation, and so on; the safety of acoustic radiation and different fibre tracts has been perplexing neurosurgeons, which leads to two kinds of adverse mentality: (1) in order to completely remove the tumour tissue as much as possible, the operation is too deep, and the uninvolved pyramidal tract is removed. The postoperative limb dysfunction was aggravated. (2) In order to protect the preoperative limb function, the operation is too conservative. The rate of total tumour resection decreased, and the emergence of DT technology solves this contradiction, because of the white matter fibre bundle. The diffusion direction of water molecules is limited by the fibre sheath of white matter, which has high anisotropy and tends to be the direction of the vessel fibre bundle. Therefore, different nerve conduction and fibre projection directions can be clearly distinguished, especially the corpus corporis bundle and the corpus callosum fibre bundle. Fourth, the anisotropy of the fibre bundle is obvious and the strip structure is high signal. According to the FA colour map and DTT map, we can accurately determine the shape structure, course of the corresponding white matter fibre, and its spatial relationship with adjacent tumours before operation, which is helpful to the preoperative evaluation and operation planning design of brain tumour surgery. In this study, the experimental group patients were treated according to the fibre bundle image information provided by DITI technology before operation . By imprIn this study, we used DTI to monitor the parameters of the focal area and symmetrical normal brain area in patients with newly diagnosed cerebral infarction in acute phase, subacute phase, and chronic phase, mainly including average diffusion coefficient (Drag), volume ratio anisotropy (VRA) and fractional anisotropy, and exponential attenuation. At the same time, the sufferers have been divided into rehabilitation team and nonrehabilitation team in accordance with MBI score, and the parameters of lesion place and contralateral unhurt area, rehabilitation crew, and nonrehabilitation crew have been in contrast at unique time periods, so as to comprehensively analyse the correlation between the above parameters and rehabilitation diploma of sufferers with cerebral infarction with the aid of magnetic resonance diffusion tensor imaging (DTI), to inform the medical improvement of extra optimized rehabilitation treatment.Patients with persistent cerebral infarction admitted to our medical institution from September 2019 to September 2020 have been chosen as the lookup objects, which have been tested by CT or MRI examination and met the diagnostic standards formulated in 2010 Chinese guiding principle for the analysis and cure of acute ischemic stroke. Inclusive criteria include the following: (1) admission for the first time; (2) all lesions being unilateral and involving white matter; (3) with different degrees of motor dysfunction; (4) the clinical data being complete; (5) absence of any other intracranial lesions; (6) providing informed consent. Exclusion criteria include the following: (1) contraindications of MRI examination; (2) combined with mental illness; (3) hearing or language impairment; (4) having serious physical diseases before enrolment, which affected the limb motor rehabilitation treatment; (5) the treatment compliance being poor. The age ranged from 39 to 72 years, with an average of 51.26 ± 2.63 years. The duration of cerebral infarction ranged from 19 days to 76 days (36.31 ± 5.18 d).After the vital signs were stable, all patients received hyperbaric oxygen, brain nerve nutrition, acupuncture and massage, passive limb training, and other rehabilitation treatment programs. Hyperbaric oxygen treatment pressure, pressure time, decompression, and course of treatment were formulated according to clinical standards. Brain nutrition includes the application of brain neurotrophic factor, glial neurotrophic factor, brain protein hydrolysate, traditional Chinese medicine decoction, and so on. For acupuncture, special metal needles are used to penetrate the relevant blood levels, such as Wanigan, Qu chi, Taichung, and USANi, so as to dredge channels and activate collaterals and regulate qi and blood. Massage uses a variety of techniques to stimulate the blood vessels, muscles, nerves, and other parts of the patient, so as to speed up the metabolism and help them recover their functions as soon as possible; passive and active limb training include keeping good posture, elbow extension training, and wrist dorsiflexion training; the patients should be guided to exercise autonomously as far as possible, the affected limb should be encouraged to do lifting activities, and the walking training should be strengthened to adjust the flexion movement of the knee joint of the affected limb; language rehabilitation training includes starting with words and single sentences, then strengthening the practice of complex sentences, and encouraging more communication with family members. The above rehabilitation treatment methods were carried out according to clinical standards under the guidance of doctors and nurses.b = 0, B = 1000 s/mm−2, scanning matrix is 160 × 160, and field of vision is 22 × 22 cm. The scanning range was from skull base to skull top. The scanned image was transmitted to the workstation for processing. FA reconstruction was performed to determine the region of interest with an area of 15 mm2, and FA values of the region and the contralateral normal region were obtained. The relationship between the lesion and CST was observed and divided into three grades: grade 1: the morphology of CST was complete and the trend was normal; grade 2: CST has pressure and trend change; grade 3: CST interrupt.HDX twin speed 3.0TMR scanner produced by GE company of America is used as the inspection instrument, and standard thread end ring is used. The scanning sequences were T1WI, T2WI, FLAIR, and DTI. DTI scanning technology is single-shot spin-echo plane echo imaging (EPI); scanning parameters are as follows: TR/TE is 8000 ms/96 ms, weighted coefficient is In this study, we used the signal HDE 1.5T MRI scanner of GE company to collect the images. The patient took the head first, in a supine position. During the scanning, the patient was asked to rest and relax his limbs. The scanning sequence included axial t1flair ; axial T2WI ; axial t2flair ; axial DWI ; sagittal 2WI ; axial DTI .(1) The objective is to check out the relationship between pathological adjustments and CST. (2) According to the relationship between sickness and CST, sufferers in team A have been grade 2, and sufferers in team B have been grade 3. (3) Two hours earlier than every MRI examination, the motor feature recuperation of the sufferers used to be evaluated by using the easy Fugl–Meyer motor characteristic scale, together with top limb function, finger function, decrease limb function, and so on. The whole rating used to be zero to a hundred. The more the decrease in the score, the more serious the motor dysfunction. The correlation between FA and Fugl-Meyer scores before and after rehabilitation training has been discussed.DCavg, and VRA maps were reconstructed, and the infarction area of 15 mm2in the image was selected as the region of interest (ROI), including the central part and surrounding part of the infarction. FA value, DCavgvalue, VRA value, and Exatvalue were measured in the infarction area, respectively. FA value, DCavgvalue, VRA value, and Exatvalue were measured in the control area. Spss22 software was used to analyse the data. For data (P < 0.05).The collected data were sent to the workstation, FA, ADC, P < 0.05). The FA value of group A was 0.556 ± 0.064, which was significantly higher than that in group B (P < 0.05), as shown in 15 cases of grade 2 were treated as group A, and 21 cases of grade 3 were treated as group B. The FA value of group A was 0.612 ± 0.039, which was significantly higher than that in group B. Compared with before rehabilitation, the difference was significant (P < 0.05). The FA cost of group A (0.556 ± 0.064) was once much larger than that of group B (P < 0.05), as proven in 15 instances of grade two have been dealt with as team A, and 21 cases of grade three had been dealt with as crew B. The FA cost of crew A (0.612 ± 0.039) was once extensively greater than that in team B. Compared with earlier than rehabilitation, the distinction was once huge (P < 0.05), and the FA fee of the nonrehabilitation team in the subacute section used to substantially decrease compared to that in the rehabilitation crew (P < 0.05). In the continual phase, the FA fee of ROI in the rehabilitation team did now not alternate extensively in contrast with that in the subacute phase; however, the distinction was once large than that in the nonrehabilitation team (P < 0.01), as proven in The FA fee of ROI in the rehabilitation team and the nonrehabilitation crew used to substantially decrease compared to that in the contralateral facetin the subacute and persistent segment (P < 0.05). The VRA cost of ROI in the rehabilitation crew was once no longer considerably modified in contrast with that in the acute stage, whilst the VRA cost of ROI in the nonrehabilitation team persevered to decrease, and there used to be a statistical distinction between the two organizations (P < 0.05). In the persistent stage, the VRA fee of ROI in the rehabilitation crew used to be nevertheless now not substantially unique from that in the acute and subacute stages. The VRA price of ROI in the nonrehabilitation crew persisted to decrease, and the statistical distinction between the two businesses was once enlarged (P < 0.01), as proven in P < 0.05). There was no difference in VRA between the rehabilitation group and its acute and subacute stages (P > 0.05), but the VRA of the nonrehabilitation group decreased continuously, and the difference between the two groups was enlarged (P < 0.01).In the acute phase, the VRA price of the two agencies used to considerably decrease compared to that of the contralateral ROI ; however, there was once no widespread distinction in the ROI between the two businesses in the acute phase. In the acute stage, the DCavg fee of the two agencies persisted to increase, and the DCavg fee of the rehabilitation group used to be drastically greater than that of the rehabilitation team (P < 0.05). In the continual phase, the DCavg cost of the rehabilitation team accelerated slowly, whilst the DCavg cost of the nonrehabilitation team persevered to increase, and the enlargement used to be apparently greater than that of the rehabilitation team (P < 0.01), as proven in The Exat value and contralateral ROI of the rehabilitation group were significantly higher than those of the nonrehabilitation group (P < 0.05); after the subacute stage, the Exat value of the rehabilitation group decreased significantly, and there was no statistical difference with the contralateral ROI, whilst the Exat value of nonrehabilitation group decreased slowly, and there was a statistical difference with the contralateral ROI and the ROI of rehabilitation group; after the disease developed to the chronic stage, the Exat value of the rehabilitation group returned to the normal level, whilst the Exat value of the ROI of the nonrehabilitation group decreased slowly, and there was a statistical difference between the contralateral ROI and the ROI of the rehabilitation group (P < 0.05), as shown in The P < 0.05). There was once a terrible correlation between the limit share of CST FA cost and the expanded proportion of Fugl–Meyer rating earlier than and after rehabilitation treatment, as proven in Before rehabilitation, the Fugl–Meyer rating of crew A used to be 57.264 ± 5.821, which was once extensively greater than that of crew B. After rehabilitation, the Fugl–Meyer rating of crew A used to be 85.162 ± 9.861, which used to be extensively greater than that in team B, and the variations had been statistically substantial . At the equal time, the FA price of the nonrehabilitation team used to considerably decrease compared to that of the rehabilitation crew (P < 0.05). It can be determined that the FA price diminished much less in subacute stage, and the sufferers recovered higher in later stage. In the detection of VRA value, as a supplementary index of FA value, it displays quicker than FA value. In the acute stage, the VRA fee of the two agencies lowered significantly, and there was once no massive distinction between the two groups. With the extension of the disorder time, the VRA cost of the two organizations differentiated in the subacute stage. The VRA fee of the nonrehabilitation crew persevered to decline significantly, whilst the VRA cost of the rehabilitation crew diminished slowly, and the distinction of VRA between the two companies used to be statistically sizable (P < 0.05). In the persistent stage, there used to be no distinction in the VRA between the rehabilitation team and its acute and subacute levels (P > 0.05); however, the VRA of the nonrehabilitation crew lowered continuously, and the distinction between the rehabilitation crew and the rehabilitation crew at the identical stage multiplied (P < 0.01), FA fee and VRA cost can song the variations inside the tissue, and DC avg cost is particularly correlated with the diffusion pace of water molecules. In acute stage, the DCavg cost of the two corporations expanded appreciably in contrast with the contralateral facet (P < 0.05). In subacute stage, the DCavg cost of the two companies confirmed the difference. The DCavg fee of the rehabilitation crew elevated slowly, whilst the ROI of the nonrehabilitation crew accelerated continuously. The DCavg fee of the two companies confirmed the sizable distinction (P < 0.05). The DCavg cost of ROI in the rehabilitation crew was once nevertheless extensively special from that in the contralateral side; however the rising vogue was once gentle, whilst that in the nonrehabilitation crew endured to rise, and the distinction between the two businesses grew to become large (P < 0.01). The Exat cost used to be equal to DCavg, which ought to additionally mirror the molecular diffusion characteristics. In the acute phase, the Exat price of ROI in the two organizations reduced extensively at the identical time, and there used to be a full-size distinction with the contralateral ROI (P < 0.05). With the extension of time, the Exat cost started to decrease, particularly in the rehabilitation group, the reduce degree in the subacute segment used to be a lot increased than that in the nonrehabilitation team (P < 0.05). The ROI of the Exat fee for the rehab team is back to normal again, whilst the Exatcost of ROI in the nonrehabilitation crew was once nonetheless above the regular degree (P < 0.05).This confirmed that there used to be no massive distinction in the FA cost of ROI between the rehabilitation crew and the nonrehabilitation team in the acute stage. With the extension of the disease, the FA price of the two corporations was once drastically decreased compared to that of the contralateral crew (The above studies show DTI detection in patients with cerebral infarction in the early stage of a number of abnormal indicators, including FA values in the subacute phase of the difference. The reason for the above changes may be that, in the acute stage of cerebral infarction, neuron cells are first subjected to ischemia and hypoxia, which leads to cell enema and limits the diffusion of water molecules . At thisThe utility effects of this paper exhibit that the software of magnetic resonance DTI science in the assessment of rehabilitation cure impact of motor feature in sufferers with cerebral infarction can supply a foundation for clinicians to deeply recognise the CST involvement of patients, help to scientifically evaluate the effect and quality of limb motor rehabilitation training of patients, and provide a basis for disease treatment. As a new technology, the clinical application of DTI is still in its infancy, and there are some problems, such as the distortion of EPI sequence image and the deviation caused by low resolution. At present, there is no “gold standard” for tracing images of nerve fibre tracts. With the development of MRI technology and software, I believe these problems will be solved gradually in the future. However, DTI can supply precious data about the microstructure and pathological kingdom of tissues. It is the solely noninvasive technique to learn about the morphology and shape of white remember fibretracts in human dwelling brain. It has a very broad application prospect in the diagnosis and treatment of central nervous system diseases, especially neurosurgical diseases; the application of DTI in other systems such as heart, kidney, and skeletal muscle system has been reported. With the further development of DTI technology, DTI will be widely used in clinical work.


The influence ofAr gas additives on ≪black silicon≫formation is shown in this work. The way to achieve the conical shapeof Si texture using low Ar dilution is demonstrated. Also, a possibilityof silicon nanowire width reduction keeping a high density of arrayis shown. No damage to the Si structure caused by Ar plasma was detected.The introduction of Ar into the plasma also does not affect electricalproperties. The lifetime value after cryogenic etching with 5 sccmAr flow remains at the same level of 0.7 ms. The resulting black siliconhas a low total reflectance of 1 ± 0.5% in the range of 450–1000nm in the overall 100 mm Si wafer surface. A short-circuit currentof 42.2 mA/cm2 was reached for such solar cells under AM1.5Gwith an EQE close to 100% in the wavelength range of 300–1000nm being almost independent of the incidence angle. A total reflectanceof 1% can be achieved using black silicon as the texture for the topcontact of the solar cell. Microcrystalline n-type silicon solar cellsobtained in this way demonstrated 21.9% efficiency.13 The rough surface of black silicon acts as recombinationcenters for minority charge carriers, which leads to a decrease inphotovoltaic characteristics and, therefore, requires appropriatepassivation. Excellent surface passivation can be achieved with amorphoushydrogenated silicon (a-Si:H), which provides a record low surfacerecombination rate of 0.7 cm/s.14 Also,well-known materials for surface passivation of silicon are siliconoxide (SiO2) and silicon nitride (SiNx).16 However, in the case of black silicon, theconformal growth of the passivation layers is an important issue.It is especially crucial for the solar cells based on the a-Si:H/c-Siheterojunction, where precise control of undoped a-Si:H layer thicknessis required. Thickness uniformity could be much easily achieved forthe conical shape of Si texture. Black silicon with such a cone-likeshape has demonstrated an extremely high quantum efficiency in theshort-wavelength region, showing evidence of effective passivation.17 Thus, finding ways to achieve Si wire shapecontrol is quite important for further black silicon technology development.Recently, there has been a transitionfrom planar structures tothree-dimensional ones. One of these types of structures is the structureof vertically aligned silicon (≪black silicon≫). Blacksilicon makes it possible to obtain solar cells with an efficiencyclose to 22%.6/O2 gas at cryogenic temperatures, without applying a mask. In the processof plasma etching, the surface of the silicon substrate is bombardedwith SFx radicals (0 ≤ x ≤ 5), formed as a result of the dissociation ofSF6 molecules, with the formation of volatile compoundsSiFx and SiFxOy. Under standard conditions, (at temperatures> −100 °C), the process exhibits isotropic etchingbehavior.With a decrease in temperature, condensation of SiFxOy molecules occurs on the cooledsurface of the silicon substrate, which creates a passivation layeron the silicon surface. Since in plasma etching, active SFx radicals have a strict direction of motion underthe action of the pulling plasma field, the vertical etching rateis much higher than the lateral one. At a sufficiently low temperatureand a high oxygen content in the gas mixture, the mode of passivationof silicon and vertically aligned structures (≪black silicon≫)begins. The addition of inert gases makes it possible to increasethe intensity of the ion bombardment of the etching bottom and thereforepotentially change the shape of Si wires formed during the process.Argon (Ar) is widely used for the ion beam etching process.19 There are a few reports where argon was added for RIE of Si througha mask. Chen et al. reported about the effect of alternating Ar andSF6/C4F8 gas flows in Si plasma etching.The addition of the Ar step increased the etching rate and improvedthe selectivity of Si over a polymer mask.20 However, to the best of our knowledge, argon has not been used toform black silicon by cryogenic unmasked etching. The influence ofAr gas additives on black silicon formation is explored in this paper.To obtain vertically aligned structures on silicon, it is possibleto use the processes of plasma etching in SF+ ionsare extracted from the plasma by an electric field and bombard thesubstrate surface. When Ar+ ions collide with the substratesurface, energy is transmitted to the near-surface atoms of the material.Thereby, Ar+ ions affect the chemical reactions occurringat the Si surface.The scanning electron microscopy (SEM) imagesof the Si surfaceobtained for the processes with a variation of Ar flow from 0 to 7sccm are presented in 6/O2 mixtureby Ar allows one to reduce the width of SiNWs while keeping a highdensity of array. On contrary, Ar plasma is known to have a destructiveeffect on Si. To study the effect of Ar dilution on the SiNW structuralproperties, they were explored by transmission electron microscopy(TEM) measurements. The TEM images of SiNWs obtained without and witha weak (5 sccm) Ar flow are presented in However, weak dilution of the SFThe introduction of Ar into the plasma also does not affect electricalproperties. This was confirmed by the minority charge carrier lifetimemeasurements performed for double-side polished Si wafers passivatedby a-Si:H. The surface of polished wafers remains smooth after cryogenicetching with and without Ar. Thus, any influence of Si wire geometryis avoided. The lifetime value after cryogenic etching with a 5 sccmAr flow determined by photoluminescence decay (PLD) remains at thesame level of 0.7 ms as for the etching process without Ar plasma(0.5 ms). The PLD results correlate well with quasi-steady-state photoconductance(QSSPC) measurements 3, which However, the size of SiNWs 1 with a However, the opticalproperties of the formed array of SiNWs areanother important issue, which should be also verified. The totalreflectance spectra of the SiNWs arrays with different heights andfabricated with and without Ar dilution are shown in 6/O2 mixture by Ar allows one to reduce thewidth of SiNWs while keeping a high density of array. The cone-likeshape of SiNWs could be achieved with Ar dilution being an importantfeature for precise control of passivation layer thickness. No structuraldefect caused by Ar plasma was detected by TEM. The lifetime valueafter cryogenic etching with a 5 sccm Ar flow remains at the samelevel of 0.7 ms as for the etching process without Ar plasma. In addition,the resulting black silicon has a low total reflectance of 1 ±0.5% in the range of 450–1000 nm.The cryogenic unmaskedetching of ≪black silicon≫with Ar gas additives was studied in this work. Weak dilution of theSF100 mm unpolished (100) n-type silicon substrates were used. Thesubstrates were preliminarily cleaned in isopropanol and deionized(DI) water for 5 min to remove organic contaminants. The native oxidewas not removed.6 and oxygen content remained constant for each process with a flowof 15 and 45 sccm for O2 and SF6, respectively.The pressure of 5 mTorr and ICP power of 1000 W were fixed. The RFpower of 40 W (88 mW/cm2) was used, which provides a −220V DC bias. For the first series of processes, the argon flow was variedin the range from 0 to 7 sccm, while the etching time of440 s was fixed. For the second series with 5 sccm Ar and withoutan Ar flow, the etching time was varied in the range from 200 to 440s. We should stress that unpolished Si substrates should be used toachieve a uniform reproducible black silicon formation overall wafersurface.For the maximum effect of over passivation,cryogenic etching wasperformed at a temperature of −150 °C. The SFThe morphology of the resulting structures was studiedusing aSUPRA 25 Zeiss scanning electron microscope and TEM. TEM measurementswere carried out using a Jeol JEM-2100 F microscope . Specimens for TEM wereprepared by the dry mechanical transfer of the wires onto a carbonlacey film supported by a copper TEM grid.21 The native oxide was removed from the Si surface by HF dip priorto the a-Si:H deposition. Plasma-enhanced chemical vapor deposition(PECVD) of a-Si:H layers was performed on both sides of the Si wafer.PECVD was carried out at 250 °C from the SiH4 andH2 gas mixture at 350 mTorr and 11 mW/cm2 RFpower density. This process was successfully used to achieve lifetimevalues of a few ms for polished Si wafers.To estimate the influenceof dry etching on photoelectrical properties,the charge carrier lifetime measurements were performed after passivationof polished Si wafers by a-Si:H. FZ double-side polished (100) n-Siwafers (5–10 Ohm cm) with a bulk charge carrier lifetime ofabout 10 ms were used. Dry etching of polished wafers does not leadto the formation of black Si. The surface remains smooth, allowingone to distinguish the influence of Ar plasma on the charge carrierlifetime independent of the morphology. After plasma etching, thewafers were cleaned by the Shiraki process.14 to 1015 cm–3) was determined by PLD measurements described elsewhere.22 For the higher minority carrier density (1015 to 5 × 1016 cm–3), lifetimewas detected by QSSPC measurements using a Sinton instruments WCT-120.The total reflectance spectra were also obtained using an integratingsphere and AvaSpec SensLine spectrometer.The charge carrierlifetime in the range of low minority carrierdensity (10


The CREB-binding protein (CBP) and p300 are paralogous lysine acetyltransferases that serve as critical co-activators for transcription factors involved in diverse signaling pathways in cancer. Work in the last two decades has firmly established CBP and p300 as important regulators of nuclear hormone signaling mediated by nuclear receptors, such as the androgen receptor (AR) and estrogen receptor (ER). The AR and ER promote tumor growth in hormone-dependent prostate and breast cancer, respectively. Inhibitors of androgen and estrogen signaling are the standard-of-care therapeutics for treating these cancers. However, resistance to current therapies remains a significant clinical problem. Inhibition of CBP and p300 as a means to block the transactivation activity of the AR and ER is an emerging therapeutic strategy for prostate and breast cancers. This review describes how CBP and p300 regulate androgen and estrogen signaling and discusses therapeutic potential of newly discovered potent CBP/p300 inhibitors for treating prostate and breast cancer.The CREB-binding protein (CBP) and p300 are two paralogous lysine acetyltransferases (KATs) that were discovered in the 1980s–1990s. Since their discovery, CBP/p300 have emerged as important regulatory proteins due to their ability to acetylate histone and non-histone proteins to modulate transcription. Work in the last 20 years has firmly established CBP/p300 as critical regulators for nuclear hormone signaling pathways, which drive tumor growth in several cancer types. Indeed, CBP/p300 are critical co-activators for the androgen receptor (AR) and estrogen receptor (ER) signaling in prostate and breast cancer, respectively. The AR and ER are stimulated by sex hormones and function as transcription factors to regulate genes involved in cell cycle progression, metabolism, and other cellular functions that contribute to oncogenesis. Recent structural studies of the AR/p300 and ER/p300 complexes have provided critical insights into the mechanism by which p300 interacts with and activates AR- and ER-mediated transcription. Breast and prostate cancer rank the first and forth respectively in cancer diagnoses worldwide and effective treatments are urgently needed. Recent efforts have identified specific and potent CBP/p300 inhibitors that target the acetyltransferase activity and the acetytllysine-binding bromodomain (BD) of CBP/p300. These compounds inhibit AR signaling and tumor growth in prostate cancer. CBP/p300 inhibitors may also be applicable for treating breast and other hormone-dependent cancers. Here we provide an in-depth account of the critical roles of CBP/p300 in regulating the AR and ER signaling pathways and discuss the potential of CBP/p300 inhibitors for treating prostate and breast cancer. The CREB-binding protein  and p300  are two paralogous lysine acetyltransferases (KATs) that were discovered in the 1980s–1990s ,3,4. CBPCBP/p300 are important regulators of gene expression and are recruited to chromatin through interacting with many DNA-binding transcription factors ,13,14. CEarly work on p300 identified this protein as a potential tumor suppressor because viral oncoproteins, such as the adenovirus E1A protein, inhibit p300 activity to promote cell transformation ,30. In aA significant amount of research now shows that CBP/p300 is critical for pro-growth nuclear steroid hormone receptor signaling in prostate cancer (PCa) and estrogen receptor-positive (ER+) BC. Androgen receptor-positive (AR+) PCa and ER+ BC are dependent on oncogenic hormone signaling mediated by the AR and the ER, respectively ,41. In hPCa and ER+ BC represent large global health burdens and hormone therapies targeting the AR and ER are standard treatments, but clinical resistance to these therapies is common ,41,56,57The realization that CBP/p300 is crucial for AR and ER signaling has led to significant interest in targeting CBP/p300 as an alternative therapeutic strategy to treat PCa and BC. Several specific and potent inhibitors of CBP/p300 function have been discovered. These inhibitors effectively block AR and ER activity and tumor growth. This review aims to provide an in-depth account of the roles of CBP/p300 in regulating hormone signaling and the application of recently developed CBP/p300 inhibitors in hormone-dependent PCa and their potential application in BC. Additionally, common and distinct functions for CBP and p300 in gene regulation and tumorigenesis will be discussed ,11,53,63The modular structure of CBP/p300 consists of eight distinct domains: the nuclear receptor interaction (NRID), C/H1, KIX, bromo, C/H2, the enzymatic lysine acetyltransferase (HAT), C/H3 , and the nuclear co-activator-binding (NCBD) domains A 12,27,,27,64. OCBP/p300 catalyze the transfer of an acetyl group from acetyl-Coenzyme A (acetyl-CoA) to a lysine residue on substrates . CBP/p30Acetylation occurs on thousands of cellular proteins involved in diverse functions, such as the DNA damage response, transcription, cell signaling, and metabolism . CBP/p30The important functional impact of CBP/p300 acetylation on diverse substrates has sparked investigation into the CBP/p300 acetylome. A recent mass-spectrometry study showed that CBP/p300 acetylates over 200 non-histone transcription factors and transcriptional co-activators . Many ofThere have been continuous efforts to discover small molecule inhibitors of CBP/p300 catalytic activity, which have led to a number of chemical probes and drug candidates. These inhibitors include Lys-CoA, curcumin, anacardic acid, C646, A-485, and derivative compounds ,28,81,8250 values of 9.8 and 2.6 nM, respectively ,2322,23]On a genomic scale, Raisner et al. proposed that the CBP/p300 BD is critical for H3K27ac deposition at enhancers because GNE-049 has a significantly greater inhibitory effect on H3K27ac at enhancers in comparison to promoters . These fNotably, some studies have reported that CBP/p300 BD inhibitors do not strongly displace endogenous CBP/p300 from chromatin, despite the BD being able to bind acetylated chromatin ,106. ThiIn summary, the ability of both CBP/p300 BD and HAT inhibitors to block the expression of oncogenes highlights the potential of these inhibitors as promising cancer therapeutics C. NotablProstate cancer (PCa) represents an enormous global health burden, with an estimated 1.6 million cases and 366,000 deaths annually worldwide in men A 41]. T. T41]. TThe AR drives PCa growth through regulating the expression of genes involved in cell cycle progression, metabolism, and survival ,127,128.Mechanisms of treatment resistance in CRPC include AR gene amplification , mutatioVarious alternative strategies for AR inhibition, such as pharmacologic induction of AR protein degradation and targeting the AR N-terminal transactivation domain, have the potential to overcome drug resistance to current hormonal therapies ,150. DruAs a DNA-binding transcription factor, the AR has nearly 200 known co-regulators that can activate or repress AR target genes . TargetiCBP/p300 is recruited to nuclear receptor complexes through an interaction between SRC’s CBP/p300 interaction domain (CID) and CBP/p300’s SRC interaction domain (SID)  51,155,,163,164.This early model of AR/SRC/p300 interaction was further clarified in a recent cryoelectron microscopy (cryo-EM) structural study by Yu et al. . Their wInterestingly, in the cryo-EM structure, SRC-3 does not directly interact with the AR LBD, but instead makes contact with the NTD of one AR monomer . ConcordEarly studies showed p300 was a critical AR co-activator using reporter assays. For example, p300 enhances DHT-stimulated AR transcriptional activity at the androgen-responsive MMTV (mouse mammary tumor virus) promoter . SequestConsistent with their global genomic co-binding, p300 knockdown interferes with the activation of 52.4% of DHT-induced genes in C4-2B cells (including KLK3 and TMPRSS2) . AnotherWhere CBP also interacts with the AR and activates AR-mediated transcription ,167, CBPThe interaction between CBP/p300 and the AR is important for AR protein stability and response to androgens. In PTEN-deficient models of PCa, CBP/p300 regulates AR protein stability through direct acetylation of K630, K632, and K633 on the AR . GeneticAcetylation of K630, K632, and K633 by CBP/p300 also regulates AR response to androgens and strengthens AR interaction with p300 ,171,172.Conversely, androgens and the AR appear to regulate CBP/p300 protein levels ,174. TreIn contrast, a low dose (0.1 nM) of R1881 or androgen deprivation appears to stabilize p300 protein . LNCaP-RAndrogen-independent activation of the AR is a well-known phenomenon and can occur via several different mechanisms, including activation by interleukins ,60. For Constitutively active AR splice variants (AR-Vs) are another well documented mechanism of androgen-independent activation of AR signaling ,179,180.AR-V7 is one of the most highly studied AR-Vs and its expression increases with PCa progression ,179. AR-Increasing evidence shows that p300 promotes PCa tumorigenesis, tumor progression, and treatment resistance ,185,186.A potential role for CBP in PCa appears more complex. Both high expression and downregulation of CBP in PCa have been reported ,174,187.50 of 1.1 nM for CBP and 2.3 nM for p300 in a biochemical BD-binding assay [50 of 4240 nM) [GNE-049 is a potent CBP/p300 BD inhibitor with an ICng assay . This co4240 nM) . GNE-0494240 nM) . In cont4240 nM) . GNE-0494240 nM) . Importa4240 nM) . Therefo4240 nM) . RNA-seq4240 nM) . Signifi4240 nM) . In vivo4240 nM) .Mechanistically, Jin et al. proposed that GNE-049 reduces AR signaling and PCa growth by blocking CBP/p300 co-activator function . Consistd values for BRD2, BRD3, BRD4, and BRDT, and Kd values of 19 and 31 nM for CBP and p300, respectively [50 values of 0.24, 0.61, and 0.17 µM, respectively [Additional CBP/p300 BD inhibitors have been characterized and show efficacy in PCa treatment. NEO2734 is a broad BD inhibitor with single nanomolar Kectively . NEO2734ectively . NEO2734ectively . Yan et ectively . SPOP isectively ,190,191.ectively , AR co-aectively ,195,196,ectively  and MYC ectively . SPOP muectively . NEO2734ectively . NEO2734ectively .50 of approximately 100 nM for the CBP BD [Y08197 is another CBP/p300 BD-selective inhibitor with an ICe CBP BD . Y08197 e CBP BD . Signifie CBP BD . Y08197 e CBP BD . CPI-637e CBP BD . This ine CBP BD ,111.ClinicalTrials.gov Identifier: NCT03568656) was initiated for CBP/p300 BD inhibitor CCS1477 for treatment of metastatic PCa [Collectively, these preclinical studies provide compelling evidence that targeting the CBP/p300 BD represents a promising strategy for treating PCa. Notably, a Phase I/IIa clinical trial  synergizes with CBP/p300 BD inhibitor I-CBP112 to dramatically reduce p300 occupancy genome-wide . This reBC is the leading cause of cancer burden in women and is diagnosed in over 1 million women worldwide each year. BC affects 1 in 20 women globally and as many as 1 in 8 in Western countries. BC is a heterogeneous disease with four main molecular subtypes: Luminal A, Luminal B, Her2-overexpression (HER2-enriched), and basal-like . ERα, orER+ BC constitutes approximately 60–80% of BC cases (depending on the study) and most BC deaths occur in women with ER+ BC A 201,2002. ER siWhile there is clear evidence that current BC treatments have prolonged patient survival , de novoThe full-length ER is a 66 kDa steroid hormone nuclear receptor that shares structural similarity to the AR and other nuclear receptors . Like thER signaling is pro-growth through activation of oncogenes, such as MYC and CCND1, to promote cell cycle progression ,204,218.The ER has a dominant oncogenic role in ER+ BC, and ER-targeted therapeutics are the mainstay therapy for ER+ BC patients. Tamoxifen is a selective inhibitor of ER function in the breast and has been the standard therapy for ER+ BC for decades ,220. UnfThe ER relies on transcriptional co-activators to upregulate its target genes via the well characterized ER activation complex (ER/SRC-3/p300) ,224,225.The SRC proteins, including SRC-3, directly interact with the LBD of the ER via an LXXLL motif in their RID . Depending on the definition of AR positivity, about 70 to 90% of ER+ and 10 to 35% of TNBC express the AR . In ER+ The AR is becoming increasingly recognized as a potential therapeutic target in TNBC, which was previously considered to be hormone-independent. Although TNBC is collectively classified as a major BC subtype, it is highly heterogeneous and can be further divided into six stable subgroups . One subLAR TNBC is not the only subgroup to express or rely on the AR for tumor progression ,259,260.Interestingly, LAR TNBC cell lines are sensitive to CBP/p300 inhibition. The MBA-MB-453 LAR cell line was sensitive to the CBP/p300 HAT inhibitor C646 and BD inhibitors SGC-CBP30 and I-CBP112 . MutatioCBP/p300 also have significant roles in other oncogenic pathways that operate in ER+ and other BC subtypes. Earlier studies have shown that acetylation of STAT3 by p300 is required for its tyrosine phosphorylation and thus its nuclear translocation and activation ,264. IncAdditionally, there is extensive literature documenting the important roles of CBP/p300 in the Wnt/ß-catenin pathway. These KATs physically interact with ß-catenin and serve as co-activators for ß-catenin/TCF-mediated gene expression ,267. CBPCBP/p300 bind specifically to HIF-1α and are critical co-activators for HIF-1α-mediated gene expression under hypoxia conditions, which underlies tumor angiogenesis, invasion, metastasis, and resistance to therapy ,276,277.Of note, several recent studies demonstrated important regulatory roles of histone variant H3.3 in regulating CBP/p300 activity ,281,282.CBP/p300 are critical co-regulators for diverse transcription factors that underlie numerous signaling pathways in normal and cancerous tissues. There is substantial preclinical evidence that CBP/p300 are valid therapeutic targets in PCa, but work remains to be done to determine the precise mechanisms of action of CBP/p300 inhibitors. For example, the CBP/p300 HAT inhibitor A-485 robustly downregulates signaling pathways in PCa that are weakly regulated or unaffected by AR antagonists . These pThere is a compelling rationale for using CBP/p300 inhibitors to treat other hormone receptor-dependent cancers. ER+ BC subtypes, as well as ER-negative BC subtypes expressing the AR, represent largely unexplored opportunities to apply these novel CBP/p300 inhibitors. Rigorous validation of the specificity and mechanisms of action of these compounds will be important in any new cancer model. A combination of small molecule inhibitors with genetic knockdown studies of CBP/p300 will be key to identifying and validating ER target genes that rely on CBP/p300 for their expression. It will be important to identify ER target genes whose expression relies on CBP/p300 in tumor cells expressing WT ER or its mutants that confer resistance to hormone therapy. Such CBP/p300-regulated ER target genes may serve as biomarkers for assessing treatment response to these new compounds.As a future perspective, exploration of CBP/p300 in immune modulations and cancer immunotherapy may lead to new therapeutic strategies. Indeed, p300 is implicated in promoting the expression of CD274 encoding programmed death-ligand 1 (PD-L1), which may confer resistance to cancer immunotherapy in PCa. A-485 inhibits CD274 expression in PCa cell lines . When co


Despite numerous benefits, traditional Pulmonary Rehabilitation (PR) as a resource remains underutilized in chronic lung disease. Less than 3% of eligible candidates for PR attend one or more sessions after hospitalization due to many barriers, including the ongoing COVID-19 pandemic. Emerging alternative models of PR delivery such as home-based PR, tele-rehabilitation, web-based PR, or hybrid models could help address these barriers. Numerous studies have tested the feasibility, safety, and efficacy of these methods, but there is wide variability across studies and methods. We conducted a literature review to help determine if these alternative delivery methods watered down the effectiveness of PR. To evaluate the effectiveness of remotely based PR, the authors performed a literature search for randomized controlled trials (RCTs), cohort studies, and case series using PubMed, CINAHL, and Medline to identify relevant articles through 1 May 2021. Twenty-six applicable studies were found in which 11 compared tele-rehabilitation to conventional clinic-based PR; 11 evaluated tele-rehabilitation using the patient’s baseline status as control; and four compared tele-rehabilitation to no rehabilitation. Despite the different technologies used across studies, tele-rehabilitation was found to be both a feasible and an efficacious option for select patients with lung disease. Outcomes across these studies demonstrated similar benefits to traditional PR programs. Thus the existing data does not show that remotely based PR waters down the effectiveness of conventional PR. Use of remotely based PR is a feasible and effective option to deliver PR, especially for patients with significant barriers to conventional clinic-based PR. Additional, well-conducted RCTs are needed to answer the questions regarding its efficacy, safety, cost-effectiveness and who, among patients with COPD and other lung diseases, will derive the maximum benefit. Pulmonary rehabilitation (PR) is a “comprehensive intervention based on a thorough patient assessment followed by patient-tailored therapies that include, but are not limited to, exercise training, education, and behavior change, designed to improve the physical and psychological condition of people with chronic respiratory disease and to promote the long-term adherence to health-enhancing behaviors” . It is aWe performed a 10-year literature review using PubMed, CINAHL, and Medline to identify relevant articles through 1 May 2021, for studies evaluating effectiveness of remotely-based PR. We reasoned that publications prior to 2011 would likely be less applicable, from a technology standpoint. The search strategy involved the keywords: tele-rehabilitation; pulmonary rehabilitation; rehabilitation; telehealth; chronic obstructive pulmonary disease (COPD); chronic lung disease; and COVID-19. The search was supplemented with articles from our personal files. We sought to identify Randomized Control Trials (RCTs), cohort studies, and case series. A total of 26 studies were identified, 10 of which were RCTs, 15 were cohort studies, and one was a case series  Figure .Out of the total 26 studies conducted between 2011 and 2021; 15 (58%) were cohort studies, 10 38%) were RCTs and one (4%) was a case series . Twenty-% were RCn = 979 study participants with n = 477 participants receiving traditional PR while 502 received a tele-rehabilitation-based intervention.Eleven studies compared tele-rehabilitation to conventional clinic-based PR , seven oModes of Tele-rehabilitation: A variety of modes of tele-rehabilitation were used, including real-time broadcast by a healthcare provider (HCP) to the participant’s home ,23 and tOutcomes: Outcomes from these studies found that, compared with conventional clinic-based PR, the tele-rehabilitation group demonstrated the following improvements: better adherence , upper en = 187 participants were included in these studies.Eleven studies evaluated tele-rehabilitation using the patient’s baseline status as control , ten of Modes of Tele-Rehabilitation: Studies involving tele-rehabilitation without a comparison group used a variety of interventional modes including real-time broadcast by an HCP to the participant’s home ,32,34,36Outcomes: All 11 studies found that tele-rehabilitation was feasible ,34,35,36n = 234 participants, of which n = 115 received a tele-rehabilitation-based intervention. The lone cohort study was both a feasibility and an efficacy study [Four studies  comparedcy study .Modes of Tele-Rehabilitation: When comparing tele-rehabilitation to no rehabilitation, the various modes of delivery included real-time broadcast by an HCP to the participant’s home ,40, pre-Outcomes: Tele-rehabilitation compared with no rehabilitation was found to be feasible ,39 and eRemotely-based PR is a feasible option for people living with chronic lung diseases, especially those with COPD ,36,39,40When compared to no rehabilitation, tele-rehabilitation was effective across studies. It consistently showed statistically significant improvements in exercise capacity ,37,38,40Despite several studies showing feasibility and non-inferiority to conventional PR, the acceptance and implementation of remotely based tele-rehabilitation has been slow and highly variable over the years . The curThe existing data does not show that remotely based PR will water down the effectiveness of conventional PR. Use of remotely based PR is a feasible and effective option to deliver PR, especially for patients with significant barriers to conventional clinic-based PR. Additional, well-conducted RCTs are needed to answer the questions regarding its efficacy, safety, cost-effectiveness and who will derive the maximum benefit among patients with COPD and other lung diseases.