Upon TGF-β stimulation, SMAD2/3 bound to the lnc-UTGF promoter and activated lnc-UTGF expression. In turn, the TGF-β/SMAD signaling was augmented by overexpressing lnc-UTGF, but was inhibited by silencing lnc-UTGF. Mechanism investigations revealed that lnc-UTGF interacted with the mRNAs of SMAD2 and SMAD4 via complementary base-pairing, resulting in enhanced stability of SMAD2/4 mRNAs. These data suggest a novel TGF-β/SMAD/lnc-UTGF positive feedback circuitry. Subsequent gain- and loss-of-function analyses disclosed that lnc-UTGF promoted the migration and invasion of hepatoma cells, and this effect of lnc-UTGF was attenuated by repressing SMAD2/4 expression or by mutating the SMAD2/4-binding sites in lnc-UTGF. Studies using mouse models further confirmed that in vivo metastasis of hepatoma xenografts was inhibited by silencing lnc-UTGF, but was enhanced by ectopic expression of lnc-UTGF. The lnc-UTGF level was positively correlated with the SMAD2/4 levels in xenografts. Consistently, we detected an association of lnc-UTGF upregulation with increase of SMAD2, SMAD4, and their metastasis effector SNAIL1 in human HCC. And high lnc-UTGF level was also significantly associated with enhanced metastasis potential, advanced TNM stages, and worse recurrence-free survival. Conclusion: there exists a lnc-UTGF-mediated positive feedback loop of the TGF-β signaling and its deregulation promotes hepatoma metastasis. These findings may provide a new therapeutic target for HCC metastasis.Aberrant activation of the TGF-β/SMAD signaling pathway is often observed in hepatocellular carcinoma (HCC). Whether lncRNA regulates the TGF-β/SMAD signaling remains largely unknown. Here, we identified an oncogenic lncRNA that was upregulated in HCC and was transcriptionally induced by TGF-β (named lnc-UTGF,  The activated-SMAD2/3 cooperates with SMAD4 to form a SMAD transcription complex, which translocates into nucleus and activates the transcription of its downstream genes.4 The homeostasis of the TGF-β/SMAD signaling is regulated by feedback loops, like TGF-β/SMAD7 negative feedback loop, in which SMAD7 is transactivated by SMAD2/3 and then represses the TGF-β/SMAD signaling by binding to TGFβRI/II.5 It has been shown that disruption of the TGF-β signaling contributes to tumorigenesis, fibrotic disorders, immune malfunctions, etc.7 The TGF-β signaling pathway is frequently activated in different cancer types and facilitates tumor metastasis by inducing the transcription of pro-metastasis genes and promoting the migration and invasion of cancer cells.8 Given the important role of the TGF-β signaling in tumor development, identifying novel feedback loops that modulate TGF-β/SMAD signaling and affect tumor development may provide targets for cancer therapy.Transforming growth factor-β (TGF-β) is a multifunctional cytokine that plays an essential role in cell proliferation and differentiation, and in morphogenesis, tissue homeostasis and regeneration.11 HCC is mainly developed from chronic hepatitis. A high level of TGF-β, as a consequence of chronic liver damage and the activation of fibroblast cells, is detected in HCC tissues and is correlated with poor prognosis of patients.14 Long non-coding RNAs (lncRNAs) are newly discovered non-protein-coding transcripts longer than 200 nucleotides. LncRNAs may interact with DNA, RNA or proteins to regulate various cell activities, like proliferation, apoptosis, and motility.16 Growing evidences indicate that dysfunction of lncRNAs plays vital roles in different physiological and pathological processes, including tumor development.19 To date, whether lncRNA regulates the TGF-β/SMAD signaling in HCC is still unknown.Hepatocellular carcinoma (HCC) is a worldwide common malignancy with high mortality. Early intrahepatic recurrence/metastasis is a frequent event and represents the major cause of the dismal outcome of HCC patients, whereas extrahepatic metastasis occurs much less frequently and is not the leading cause of HCC death.lncRNA upregulated by TGF-β), whereas lnc-UTGF in turn promotes the TGF-β/SMAD signaling by stabilizing the mRNAs of SMAD2 and SMAD4. We further show that upregulation of lnc-UTGF in hepatoma cells results in abnormal activation of this positive feedback loop and thus enhanced HCC metastasis.In this study, we disclose a novel positive feedback loop of the TGF-β/SMAD pathway, that is, the TGF-β/SMAD signaling induces the transcription of lnc-UTGF (representing for lncRNA upregulated by TGF-β) was significantly upregulated as early as 4 h after TGF-β treatment and at a time- and dose-dependent manner , and the intensity of this band increased upon TGF-β stimulation , whereas this promotive effect of TGF-β was blocked by an unlabeled oligonucleotide with two classic SBEs  but remained unchanged in the presence of an unlabeled oligonucleotide with mutant SBE sequence . Antibody supershift assay showed that pre-incubation with anti-SMAD2 or anti-SMAD3 antibody significantly reduced the band intensity of the probe–protein complexes  of lnc-UTGF and characterized lnc-UTGF as a 955-nt transcript that was conserved among human, chimp, and rhesus, but was not conserved between human and mouse or rat  to identify lnc-UTGF-associated pathways by using the transcriptome data of human HCC tissues derived from The Cancer Genome Atlas (TCGA). The results revealed that genes regulating the cellular response to TGF-β stimulus and genes in the TGF-β receptor pathways were significantly enriched in the group with high lnc-UTGF level compared with the group with low lnc-UTGF level  and more metastatic nodules in the liver , although overexpressing lnc-UTGF did not further increase lung metastasis . Consistent with the in vitro findings, the protein levels of SMAD2 and SMAD4 were significantly reduced in lnc-UTGF-silencing xenografts but were increased in lnc-UTGF-overexpressing tumors  TGF-β increased lnc-UTGF expression in a time- and dose-dependent manner; (2) Inhibition of TGFβR1 or silencing of SMAD2/3/4 suppressed the TGF-β-induced lnc-UTGF transcription; (3) Deletion or mutation of putative SBEs in the lnc-UTGF promoter abrogated the role of TGF-β in enhancing the activity of lnc-UTGF promoter; (4) EMSA, antibody supershift and ChIP assays revealed that SMAD2/3 directly interacted with the lnc-UTGF promoter in vitro and in vivo. These data disclose a novel TGF-β/SMAD-transactivated lncRNA.Recent studies have revealed that the levels of four lncRNAs, including lnc-ATB, PVT1, HCCL5, and H19, are affected by TGF-β in HCC cells. However, how TGF-β modulates the levels of lnc-ATB and PVT1 is unexplored, whereas TGF-β indirectly upregulates HCCL5 and reduces H19 via ZEB1 and SOX2, respectively.32 SMAD7 is usually downregulated in tumor cells by extracellular and intracellular perturbations, such as UV irradiation, inflammation, and dysregulation of miRNAs targeting SMAD7.36 Here, we identified a novel positive feedback loop that was mediated by lncRNA and showed that lnc-UTGF could promote the TGF-β/SMAD signaling and in turn hepatoma metastasis. We further revealed that upregulation of lnc-UTGF occurred in various types of human cancers and was associated with higher metastasis potential and worse recurrence-free survival, suggesting that upregulation of lnc-UTGF may represent a universal mechanism that amplifies the TGF-β signaling via the positive feedback loop in cancer cells.The SMAD7-mediated negative feedback loop is essential in preventing abnormal activation of the TGF-β signaling and its dysfunction facilitates cancer metastasis.40 although the underlying mechanism remains unclear. To date, only two lncRNAs have been reported to regulate SMADs’ expression. Mondal et al. showed that maternally expressed gene 3 (MEG3) bound to the promoter-distal GA-rich sequences of SMAD2 and then recruited EZH2 to suppress SMAD2 transcription.41 Wu et al. found that LINC00941 competed with β-TrCP to bind the MH2 domain of SMAD4 protein, and thus prevented the degradation of SMAD4 protein.42 Here, we found that lnc-UTGF directly interacted with the mRNAs of SMAD2 and SMAD4 via highly complementary base-pairing, and thus stabilized their mRNAs, which disclose a new mechanism by which lncRNA regulates the levels of SMADs.Previous studies have shown that the levels of SMAD2, SMAD3, and SMAD4 are significantly increased in HCC,43 However, we found that lnc-UTGF was unable to increase the ZEB1 level in the hepatoma cells we studied (data not shown). Instead, we revealed that lnc-UTGF increased the expression of SNAIL1, which is an important EMT inducer. Notably, lnc-UTGF promoted the TGF-β-stimulated migration of hepatoma cells, whereas this promoting effect was attenuated when SMAD2 and SMAD4 were knocked down. Moreover, mutations in the SMAD2/4-binding sites abrogated the pro-migration effect of lnc-UTGF, suggesting that the pro-metastasis ability of lnc-UTGF in hepatoma cells mostly depends on its effect in increasing SMAD2/4 levels and enhancing the TGF-β/SMAD signaling.Recently, Liang et al. showed that lnc-UTGF promoted EMT in serous ovarian cancer by competitively binding miR-101-3p and then increasing ZEB1 expression.In summary, we identify a novel TGF-β/SMAD/lnc-UTGF positive feedback circuitry and disclose that upregulation of lnc-UTGF augments the TGF-β/SMAD signaling via this feedback loop and thereby promotes HCC metastasis. These findings highlight the importance of lncRNA in regulating the TGF-β signaling and HCC metastasis, which may be exploited for anti-metastasis therapy.Additional information is provided in The following reagents were used: recombinant TGF-β1 , actinomycin-D , SB525334 . Unless otherwise indicated, a final concentration of 2 ng/ml TGF-β1, 5 μg/ml ActD, or 2 μM SB525334 was used.Small interference RNAs (siRNAs) targeting human lnc-UTGF (Ensembl transcript ID: ENST00000428667.1), TGFβR1 (ENST00000374994.9), SMAD2 (ENST00000262160.11), SMAD3 (ENST00000327367.9), and SMAD4 (ENST00000342988.7) transcripts are designated as siUTGF, siTGFβR1, siSMAD2, siSMAD3, and siSMAD4, respectively, and were purchased from GenePharma . The negative control (NC) RNA duplex for siRNAs is non-homologous to any human genome sequence. The sequences of RNA duplexes are provided in Supplementary Table The 5′- and 3′-end of lnc-UTGF transcript was determined by the 5′RACE  and 3′RACE System  respectively, using total RNA from normal liver tissues.Nuclear and cytoplasmic fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagent kit  according to the manufacturer’s instructions.Lentivirus expression vectors pCDH-UTGF, pCDH-UTGF-mut, pCDH-S1m-UTGF, pCDH-shNC, and pCDH-shUTGF were generated using pCDH-CMV-MCS-EF1-copGFP-T2A-Puro , which contained a copGFP expression cassette and was designated pCDH-Ctrl in this study. The pXPR_001-dual-proUTGF was produced based on the lentiviral CRISPR plasmid pXPR_001 vector . pc3-UTGF, pc3-UTGF-antisense, and GFP-fusion protein expression vectors (pc3-ORF-GFP and pc3-GAPDH-GFP) were generated using pcDNA3.0 (Invitrogen). Firefly luciferase reporter vectors pGL3-basic-p(−1.6/+0.1k), pGL3-basic-p(−1.2/+0.1k), pGL3-basic-p(−0.8/+0.1k), pGL3-basic-p(−0.4/+0.1k), pGL3-basic-p(−0.1/+0.1k), pGL3-basic-p(mutSBE), and pGL3-basic-p(delSBE) were constructed based on pGL3-basic vector .For lentivirus production, HEK293T cells were co-transfected with the lentivirus expression vector that contained the target sequence and the packaging plasmid mix  via calcium phosphate precipitation. The lentivirus supernatant was harvested and stored in aliquots at −80 °C until use. Target cells, grown to 30% confluence at 24-well plate, were incubated in 1 ml lentivirus supernatant supplemented with 10 μg/ml polybrene .2 at 37 °C.HEK293T cells and hepatoma cell line SK-HEP-1 were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . Another hepatoma cell line SNU-449 was maintained in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco). All cells were cultured in a humidified atmosphere of 5% COThe stable cell lines were established by infecting SK-HEP-1 or SNU-449 cells with lentivirus that expressed the target sequence. Sublines with stable expression of lnc-UTGF with wild-type sequence  or with mutant SMAD2- and SMAD4-binding sequences (SK-UTGF-mut), S1m-tagged lnc-UTGF (SK-S1m-UTGF) and the control lines , as well as SK-HEP-1 cells with stable silencing (SK-shUTGF) or heterozygous knockout of lnc-UTGF  and the matched control line SK-shNC or SK-UTGF-WT, were constructed.RNA oligos were reversely transfected using Lipofectamine RNAiMAX (Invitrogen). A final concentration of 50 nM RNA duplexes was used. Transfection of plasmid DNA alone or together with RNA duplex was conducted using Lipofectamine 2000 (Invitrogen).The expression level of target genes was analyzed by real-time quantitative polymerase chain reaction (qPCR) and Western blotting.Renilla luciferase expressed by pRL-CMV (Promega) was used as a control to correct the difference in both transfection and harvest efficiency.Luciferase activity was measured using the dual-luciferase reporter assay system (Promega). To characterize the lnc-UTGF promoter, cells were co-transfected with 4 ng pRL-CMV, 100 ng firefly luciferase reporter vector, and 50 nM RNA duplexes for 36 h, followed by incubation without or with 2 ng/ml TGF-β for another 12 h before the luciferase activity assay.To examine the activity of TGF-β signaling, a luciferase reporter plasmid  bearing twelve tandem SMAD-binding elements (SBEs) was used. Cells were transfected with 50 nM RNA duplexes for 12 h and then co-transfected with 100 ng pSBE and 4 ng pRL-CMV for 24 h, followed by incubation in 2 ng/ml TGF-β for 12 h before the luciferase activity assay.44 Briefly, the biotin-labeled probes were incubated with nuclear extracts of SK-HEP-1 cells at room temperature for 30 min and subjected to native-PAGE. For competition assay, nuclear extract was pre-incubated with 100-fold molar excess of unlabeled oligonucleotides prior to adding labeled probe. For antibody-supershift assay, nuclear extract was pre-incubated with anti-SMAD2/3 antibody or isotype-matched IgG before adding to the binding reaction solution that contained labeled probe. Detection of the biotinylated probe in blots was performed using Chemiluminescent EMSA Kit . The sequences of probes are listed in Supplementary Table EMSA and antibody-supershift assays were conducted as described previously.SK-HEP-1 cells were treated with 2 ng/ml TGF-β for 2 h, and then cross-linked by formaldehyde. The chromatin complexes were immunoprecipitated using anti-SMAD2/3 antibody , or isotype-matched IgG (negative control), then collected with Protein A/G MagBeads . The immunoprecipitated DNAs were analyzed by semi-quantitative PCR or qPCR with primers listed in Supplementary Table Immunofluorescence staining assay was performed to examine the expression and localization of SMAD2 and SMAD4.RNA interacted with lnc-UTGF was identified by affinity purification via S1m-tag. S1m-UTGF and their binding RNAs were pulled down by streptavidin Dynabeads . The untagged lnc-UTGF was used as a negative control. RNA was extracted from the precipitates by TRIzol reagent (Invitrogen).RNA pull-down assay was performed using in vitro transcribed biotinylated RNA and streptavidin Dynabeads (Invitrogen). The retrieved RNAs were extracted by TRIzol and analyzed by qPCR with primers listed in Supplementary Table Cell counting and colony formation assays were used to access the in vitro proliferation of tumor cells. Nuclear morphological examination by DAPI staining was used to evaluate the apoptosis of tumor cells.The migration and invasion of tumor cells were analyzed in 24-well Boyden chambers with 8-μm pore size polycarbonate membranes . For invasion assays, the membranes were coated with Matrigel  to form matrix barriers. Briefly, SK-HEP-1 or SNU-449 cells in serum-free DMEM or RPMI were placed into the upper chamber of 24-well Boyden chamber coated without or with Matrigel (R&D Systems), while the lower chamber was filled with 600 μl 10% FBS-containing DMEM/RPMI. After 10 h of incubation, cells were fixed and stained with crystal violet. All the migrated/invaded cells were counted.All procedures for animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and according to the Sun Yat-sen University Institutional Ethical Guidelines for animal experiments.L) and width (W) of the dissected tumors were measured with calipers and the tumor volume (V) was calculated using the formula V = (L × W2) × 0.5. Aliquots of tumor tissues were freshly frozen in liquid nitrogen, or fixed in 10% formalin, and embedded in paraffin. To evaluate the metastasis, serial sections from lungs and livers were stained with hematoxylin-eosin (HE) and screened for metastatic nodules.SK-shNC, SK-shUTGF, SK-Ctrl, or SK-UTGF cells were resuspended in Matrigel (R&D Systems) and then inoculated under the capsule of the left hepatic lobe of male BALB/c nude mice at 5 weeks of age. Four weeks later, the xenografted mice were then applied to evaluate the metastasis. The length .The differences in gene expression levels between the paired HCC tissues and adjacent non-tumor liver tissues were analyzed by paired t test was performed to compare the differences between two groups and one-way ANOVA was applied to compare more than two groups. All statistical tests were two-sided and P < 0.05 was considered to be statistically significant. All analyses were performed using GraphPad Prism version 8.0 .Data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Unless indicated, Student’s Supporting information


EWSR1) encodes a multifunctional protein that can cooperate with the transcription factor ERG to promote prostate cancer. The EWSR1 gene is also commonly involved in oncogenic gene rearrangements in Ewing sarcoma. Despite the cancer relevance of EWSR1, its regulation is poorly understood. Here we find that in prostate cancer, androgen signaling upregulates a 5′ EWSR1 isoform by promoting usage of an intronic polyadenylation site. This isoform encodes a cytoplasmic protein that can strongly promote cell migration and clonogenic growth. Deletion of an Androgen Receptor (AR) binding site near the 5′ EWSR1 polyadenylation site abolished androgen-dependent upregulation. This polyadenylation site is also near the Ewing sarcoma breakpoint hotspot, and androgen signaling promoted R-loop and breakpoint formation. RNase H overexpression reduced breakage and 5′ EWSR1 isoform expression suggesting an R-loop dependent mechanism. These data suggest that androgen signaling can promote R-loops internal to the EWSR1 gene leading to either early transcription termination, or breakpoint formation.Ewing sarcoma breakpoint region 1 ( EWSR1), is ubiquitously expressed in humans and plays an essential role in normal development  family. FET family members have an N-terminal prion-like domain (PrLD) consisting of degenerate SYGQ repeats. This ‘low complexity domain’ has important transcription-related oncogenic properties as the N-terminus of all three FET proteins are found fused in various cancers to transcription factor genes ,12,13. ITMPRSS2/ERG gene fusion found in 50% of prostate tumors  is a ligand-inducible nuclear hormone receptor that is important in development and disease. Once activated by androgens, AR translocates to the nucleus and functions as a transcription factor, controlling genetic programs important for growth and tissue determination . AR prome tumors ,25. Intec arrest , suggestEWSR1 gene to produce different genetic outcomes important for cancer biology. In prostate cancer, we found that AR drives formation of a shortened EWSR1 isoform that promotes cancer-associated phenotypes. We then used prostate cancer cells as a model to show androgen driven formation of a break in EWSR1 at the same breakpoint hotspot that creates the EWSR1/FLI1 oncogene in Ewing sarcoma. While it is known that AR promotes gene fusion formation in prostate cancer, an androgen-dependent mechanism for EWSR1 breakage has not been shown. This is the first study to characterize direct androgen regulation of the EWSR1 gene.In this study, we find that androgen signaling regulates the RNA was extracted using the RNAeasy kit (Qiagen) and DNase treated with the RNase-Free DNase kit (Qiagen) following manufacturer's protocols. 1 μg of total RNA was reverse transcribed using isoform specific primers . RNA wasVCaP, LNCaP and PC3 cells were obtained from ATCC. All cell lines were authenticated by the Oregon Health and Science University DNA Services Core and meet the > 80% STR match threshold . All linWhole cell extracts derived from equivalent cell numbers were electrophoresed on 10% SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% non-fat milk in TBST. Primary antibodies were applied in blocking buffer at manufacturer recommended dilution. Antibodies were tubulin (Sigma Aldrich T9026), HA (Sigma Aldrich H3663), AR (Abcam ab108341), GAPDH (Santa Cruz 0411 sc-47724), EWS (Santa Cruz G5 sc-28327), H3  or ntEWS . Secondary antibodies  were diluted to 1:12,500 in blocking buffer and, after washing, membranes developed with ECL .http://net.net/addgene89080;  RRID:Addgene_89080). See http://n2t.net/addgene:111906; RRID Addgene_111906) was expressed in VCaP cells via transient transfection using TransIT 20–20 (Mirus). Cells were split two days post transfection for downstream experiments.Overexpression constructs were expressed in PC3s by retrovirus and have N-terminal 2xHA tags. Full length EWS was described previously , the N-tThe lentiCRISPR v2 plasmid was obtained from Addgene . gRNAs were designed and cloned into lentiCRISPR v2 according to the protocol provided by Addgene. The gRNA targeting upsteam the FOXA1:AR site was generated by annealing the 5′ primer caccgATCCGGGAGAAGTGATCTGTT and the 3′ primer aaacaacagatcacttctcccggatc . The gRNTranswell migration assays were preformed as previously described . BrieflyProtein precipitation via b-isox was performed as shown previously . BrieflyChIP of indicated proteins was previously described  but aftehttp://ncbi.github.io/sra-tools/). When applicable, sequencing replicates and biological replicates were concatenated. Reads were aligned using Bowtie2 (http://homer.ucsd.edu/homer/motif/HomerMotifDB/homerResults/motif6.info.html), AR half-site (http://homer.ucsd.edu/homer/motif/HomerMotifDB/homerResults/motif7.info.html), and FOXA1:AR motif (http://homer.ucsd.edu/homer/motif/HomerMotifDB/homerResults/motif5.info.html) using the FIMO tool on MEME-suite , for TFs in VCaPs (GSE56086), and for TFs in LNCaPs (GSE83860) were downloaded from the SRA using SRA toolkit  for 30 min to 1 h then assembled in a dot blot apparatus (BioRad). After washing with 100 μl TE buffer, 2 μg and 1 μg of digested genomic DNA in 100 μl of TE buffer was added to appropriate wells and vacuum filtered. Wells were washed twice with 2× SSC buffer (diluted in ddH2O from 20×). The membrane was washed for 30 s with 2× SSC buffer, air dried for 30 min, crosslinked using 0.12 J/m2 UV, then immunoblotted with S9.6 antibody (Abcam) at 1:1000 dilution.Nitrocellulose membrane was soaked in 6× SSC buffer  TCGA dataset housed on UCSC Xena Browser  values for each exon. To determine absolute expression levels, patient samples were separated into two bins: the top 25% of samples (high AR) and the bottom 25% of samples (low AR) and mean expression of each exon was plotted , exon 5a (a brain specific exon), and exons 9–11 . Exons 12 and 13 remained at a similar expression level, regardless of AR expression, while the 3′ most exons were expressed at lower levels when AR was high. Plotting the difference of the two bins  = ∼0.2) difference in 5′ exon expression between the two bins was seen, however the decrease in 3′ exons was still observed. Analysis of published ChIP-seq data , a database of polyA sites (PAS), captures the intronic polyadenylation event that forms ntEWS in 69.2% of samples , we tested whether androgen signaling would increase ntEWS RNA levels. Androgen responsive prostate cancer cell lines VCaP and LNCaP were treated with 10 nM of synthetic androgen R1881 for 24 h and changes in RNA was measured by isoform-specific qRTPCR. Primers detecting ntEWS span the novel exon-exon junction created by the intronic polyadenylation event and the flEWS specific primer set uses the first exon not present in ntEWS. Prostate specific antigen (PSA) RNA expression was used to verify androgen response  using the sequence encoded by the ALE as an epitope. It is important to note that this antibody is not affinity purified and is used in rabbit serum. Since other antibodies are present in rabbit serum, we needed to validate the ability to detect ntEWS using this reagent. The ∼70 kDa band was verified as ntEWS by the presence of a similar migrating band in lysates of ntEWS overexpressing PC3 cells and the absence of this band in lysates of empty vector expressing PC3 cells . FurtherntEWS levels in VCaP, but not LNCaP was due to differences in AR expression levels. In fact, AR is more abundant in VCaP cells than in LNCaP cells . Since AR binding at the intron 5 site is more robust in tumors and VCaP cells, we focused on understanding the importance of AR binding to that site by a CRISPR-Cas9 disruption strategy. To avoid the limitations of targeting a short A:T rich sequence, paired guide RNAs (pgRNA) were targeted to flanking sequences . Three different AR position weight matrices (JASPAR) were run through the FIMO tool from MEME-suite . The moss Figure  as pgRNAs Figure . Cells ws Figure  and prots Figure  level. TSince the function of ntEWS has not been described in the literature, we cloned and overexpressed ntEWS in the androgen insensitive cell line PC3. For comparison, we also overexpressed flEWS and a predicted C-terminal isoform , which is the same length, but includes the amino acids encoded by full-length EWSR1 . InteresEWSR1 . This suntEWS PAS is in close genomic proximity to the sequence in EWSR1 that recurrently rearranges with FLI1 in Ewing sarcoma, the breakpoint hotspot. Therefore, we hypothesized that in addition to regulating ntEWS expression, AR could regulate EWSR1 breakpoint formation. In fact, plotting the AR ChIP-seq dataset from VCaP cells shows AR binding flanks the breakpoint hotspot  was used. Fluorescent probes flank the breakpoint hotspot and create a merged red and green signal (yellow) when the EWSR1 gene is intact and split signal when a break at breakpoint hotspot has occurred  R1881 with TNFα increased breakpoint frequency to near that of cells treated with high dose (100 nM) R1881 (Figure EWSR1 alone at high doses or at low doses in collaboration with inflammation induced stress. To test the importance of R-loops in EWSR1 breakpoint formation, RNase H was expressed in VCaP cells, treated with DMSO or 100 nM R1881 (EWSR1 breakpoint formation.To test the role of androgen signaling in d Figure . Treatmed Figure , with a A damage , we askete cells . We foun1 Figure . These dnM R1881 , and thenM R1881 . This suTranscripts commonly terminate through the formation of transcription dependent R-loops . RNase HEWSR1 to directly upregulate a previously uncharacterized isoform that we have termed ntEWS. Our data indicate that ntEWS is localized to the cytoplasm and can strongly promote phenotypes associated with cancer such as cell migration and clonogenic growth. Further, AR signaling promoted increased R-loop formation in the EWSR1 gene and drove chromosomal breakage at high frequency at the same genomic locus that is rearranged in Ewing sarcoma.We have shown in the prostate cancer setting that AR binds to intron 5 of cis and in trans, and the downstream cellular consequences remain unclear. While 43.5% of AR binding sites are intronic (TSC2 gene is associated with expression of a truncated cytoplasmic isoform of TSC2 that increases cell proliferation (EWSR1, our data indicate that intronic AR binding stimulates early termination. These data suggest that AR functions to regulate isoform expression, however the exact mechanism is still unclear. RNA processing is inherently coupled to transcription, and many transcription factors, including AR, interact with RNA processing factors. For example, the RNA processing factors NONO and SFPQ interact with AR to increase transcriptional output (Alternative RNA processing events can give rise to tissue specific gene isoforms , and theintronic , the exaferation ,61. In tl output . Future ntEWS expression is normally high in the testes. Because this organ is androgen regulated, this may explain why ntEWS expression is under control of AR. Mouse knockout models for EWSR1 and AR are both defective in spermatogenesis due to meiotic arrest and increased apoptosis (EWSR1, did eliminate essential exons for ntEWS (We found that poptosis , indicator ntEWS . Since nEWSR1/FLI1 forms. Ewing sarcoma preferentially affects adolescent males with peak incidence at 15 years of age (EWSR1/FLI1 formation. The origins of the most common fusion in prostate cancer, TMPRSS2/ERG is accredited to intronic AR binding in combination with DNA damage or inflammatory signaling (EWSR1 breakpoint formation followed by EWSR1/FLI1 fusion. Since the EWSR1 breakpoint hotspot is so close to the intronic PAS, we speculate that alternative polyadenylation stress may be related to breakpoint formation. Although we were able to generate EWSR1 breakpoints at high frequencies in a prostate cancer cell line, EWSR1 rearrangements are not common in prostate cancer. A recent study did find a EWSR1/FEV fusion in a prostate tumor from a single patient, but it is unclear whether this tumor should be categorized as prostate adenocarcinoma or Ewing sarcoma (FLI1 is not expressed and is likely in heterchromatin. Our data indicate that high levels of AR in the cell are necessary for AR binding and function within the EWSR1 gene. It is unclear if the cell of origin for Ewing sarcoma would express AR. While mesenchymal stem cells are a likely Ewing sarcoma cell of origin, the exact subset of these cells and their state when EWSR1/FLI1 forms is unknown. Future studies that stimulate both AR and inflammation in a mesenchymal cell setting are needed to test this potential mechanism for gene rearrangement.Unlike many other gene fusions, we do not have a mechanistic explanation for why s of age . This afignaling ,55. Our  sarcoma . We hypoQuantitative PCR complies with MIQE guidelines. See zcab033_Supplemental_FileClick here for additional data file.


Compound 2d directly bound to both CRY1 and 2 in surface plasmon resonance analyses, suggesting that the molecular target is CRY. Effects of compound 5d and 2d on suppressive action of CRY1 on CLOCK:BMAL1-activated E-box-LUC reporter activity revealed that both compounds inhibited the negative feedback actions of CRY on CLOCK:BMAL1. Most importantly, compounds 5d and 2d exhibited significant effects on molecular circadian rhythmicity to be considered circadian clock-enhancers, distinct from the previously developed CRY inhibitors possessing an ethoxypropanoic acid moiety.Circadian dysfunction is closely associated with an increased risk of various diseases. Considering that molecular clock machinery serves as an intrinsic time-keeping system underlying the circadian rhythm of biological processes, the modulation of the molecular clock machinery is an attractive therapeutic target with novel mechanisms of action. Based on the previous structure–activity relationship study of small molecule cryptochrome (CRY) inhibitors possessing an ethoxypropanoic acid moiety, non-ethoxypropanoic acid-type inhibitors have been developed by bioisosteric replacement. They were evaluated as potent and effective enhancers of E-box-mediated transcription, and, in particular, ester  Circadian rhythms of biological processes are evolutionarily well-conserved and ubiquitous biological oscillations with an approximate 24 h daily cycle. The rhythm produced by the intrinsic time-keeping system coordinates internal biological processes to anticipate periodic environmental changes. The intrinsic time-keeping system, also known as the circadian clock, is closely related to behavioral, physiological, and metabolic cyclicities. Numerous studies have revealed that the increased risk of various common diseases is associated with circadian dysfunctions, which are attributed to environmental disruptions and intrinsic misalignment . Thus, tTherefore, attempts for chemical regulation of the circadian clock system have emerged; small molecule modulators of core clock components have also been recently developed. For example, KL001 is the first-in-class activator of cryptochrome (CRY) . Derivat1, KS15 1, , an etho2a, During the structure–activity relationship study of KS15 analogs, bioisosteric replacement of the ethoxypropanoic acid moiety provided a non-ethoxypropanoic acid-type KS15 analog, SJ019 2a,  18]. SJ. SJ2a, 15a–e. Hydrolysis of 5a–e possessing terminal ester provided carboxylic acid 2a–e.Our work commenced with the synthesis of non-ethoxypropanoic acid-type SJ019 analogs. As shown in 2a–e and their methyl ester 5a–e were examined with an expectation of developing prodrugs, but they showed similar potency and efficacy. Following the previous structure–activity relationship study, most of the analogs were potent transcriptional enhancers, except for 2e and 5e with the para-methylsulfonyl group. Among the analogs, those with the para-methoxy (2c and 5c) and the para-nitro groups (2d and 5d) were more effective transcriptional enhancers compared to SJ019 (2a). Notably, KS15 itself with the para-bromo group was the most potent and effective transcriptional enhancer among the KS15 derivatives possessing the 2-ethoxypropanoic acid moiety. Thus, a combination of functional groups and chemical moieties in the analogs tune the transcriptional activities. Finally, compound 5d was chosen for further development in terms of efficacy and potency.With the available selected compounds, their effects on the molecular circadian rhythms were examined. As shown in 5d, inhibitory activities of 5d against five cytochrome P450 (P450) isozymes were examined using a cocktail method with human liver microsomes (HLMs) and P450 probe drugs. KS15 exhibited moderate inhibitory activities against CYP1A2 (IC50 = 14.9 μM), CYP2C9 (IC50 = 9.1 μM), CYP2C19 (IC50 = 20.2 μM), CYP2D6 (IC50 = 23.1 μM), and CYP3A (IC50 = 24.0 μM)  . Resulta5d in HLMs. Rapid hydrolysis of 5d was anticipated because 5d possesses a methyl ester group in its chemical structure. As shown in 1/2 > 60 min), while 5d was time-dependently decreased in HLMs (t1/2 = 2.4 min). The metabolite was determined as the hydrolysis product, i.e., 2d, by mass spectrometry. We also examined the metabolic stability of 2d, which was found to be stable in HLMs (t1/2 > 60 min).Next, we examined the metabolic stability of 5d and 2d  in plasma were measured after the IV or PO administration of 5d, due to its metabolic instability. Pharmacokinetic parameters following IV or PO administration of each compound were summarized for the 2d form in We also conducted pharmacokinetic studies in vivo in rats after intravenous  or an oral  administration of d and 2d . For the5d with that of 2d , clearance (CL), and mean retention time (MRT). Interestingly, the total clearance was slow as the 2d form and Vss was small, indicating the limited tissue distribution of the 2d form in the body. For PO administrations, the absorptions were almost complete  in both cases, but a significant delay in tmax was observed for 5d. Both 5d and 2d might be super-permeable from the gastrointestinal tract to the systemic blood circulation with a negligible first-pass effect on the 2d. Otherwise, 5d might be rapidly converted to a super-permeable 2d, absorbed in the gastrointestinal tract. Indeed, the Cmax values were high . The Tmax for PO administration of 5d was prolonged with a shift of the curve compared with the PO administration of 2d, possibly due to a delay in absorption or due to a metabolic conversion.When we compared the pharmacokinetic characteristics of at of 2d , pharmac2d acts on mammalian CRYs. Therefore, direct binding between compound 2d and CRY1/2 was analyzed by surface plasmon resonance. Previously, biotin-tagged KS15 was reported to directly bind to the C-termini of both CRY1 and 2 in a pull-down assay + . ATR-IR νmax 2918, 2372, 2323, 1761 cm−1.Methyl 2-(3-(1-(((3-trifluoromethylbenzyl)oxy)imino)ethyl)phenoxy)acetate (5c): White solid, 67% yield. Rf = 0.3 (EtOAc:n-hexane = 1:4). 1H NMR  δ 7.36 , 7.29–7.24 , 6.92–6.87 , 5.16 , 4.66 , 3.81 , 3.80 , 2.21 . 13C NMR  δ 169.4 (C=O), 159.5 (OAr), 157.9 (OAr), 154.4 (C=N), 138.4 (Ar), 130.2 (Ar), 130.1 , 129.6 (Ar), 119.9 (Ar), 115.3 (Ar), 113.9 , 112.5 (Ar), 76.1 (O-CH2-Ar), 65.5 (O-CH2-CO2Me), 55.4 (OCH3), 52.4 (OCH3), 13.0 (CH3). HR-EI-MS m/z 343.1420 [M]+ . ATR-IR νmax 2867, 2372, 2326, 1735 cm−1.Methyl 2-(3-(1-(((4-methoxybenzyl)oxy)imino)ethyl)phenoxy)acetate (5d): White solid, 71% yield. Rf = 0.3 (EtOAc:n-hexane = 1:3). 1H NMR  δ 8.12 , 7.45 , 7.24–7.10 , 6.81 , 5.23 , 4.56 , 3.71 , 2.18 . 13C NMR  δ 169.2 (C=O), 157.8 (OAr), 155.4 (C=N), 147.4 (Ar), 145.9 (Ar), 137.7 (Ar), 129.6 (Ar), 128.3 , 123.6 , 119.7 (Ar), 115.3 (Ar), 112.6 (Ar), 74.7 (O-CH2-Ar), 65.3 (O-CH2-CO2Me), 52.3 (OCH3), 12.9 (CH3). HR-EI-MS m/z 358.1165 [M]+ . ATR-IR νmax 2855, 2371, 2324, 1736 cm−1.Methyl 2-(3-(1-(((4-nitrobenzyl)oxy)imino)ethyl)phenoxy)acetate (5e): White solid, 36% yield. Rf = 0.4 (EtOAc:n-hexane = 1:1). 1H NMR  δ 7.96 , 7.61 , 7.34–7.22 , 6.92 , 5.33 , 4.67 , 3.82 , 3.08 , 2.30 . 13C NMR  δ 169.2 (C=O), 157.8 (OAr), 155.4 (C=N), 144.8 (Ar), 139.7 (Ar), 137.8 (Ar), 129.6 (Ar), 128.4 , 127.5 , 119.8 (Ar), 115.3 (Ar), 112.7 (Ar), 74.9 (O-CH2-Ar), 65.3 (O-CH2-CO2Me), 52.3 (OCH3), 44.6 (SO2CH3), 13.0 (CH3). HR-EI-MS m/z 391.1090 [M]+ . ATR-IR νmax 2926, 2371, 2324, 1755 cm−1.Methyl 2-(3-(1-(((4-methylsulfonylbenzyl)oxy)imino)ethyl)phenoxy)acetate  was added to a solution of the compound 5d  in THF (6 mL) and H2O (6 mL). The reaction mixture was stirred for 2 h, then acidified with 1N-HCl. The resulting mixture was extracted with EtOAc. The organic solution was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by SiO2 chromatography to afford the compound 5d as a white solid .LiOH·H2b): Colorless oil, 90% yield. Rf = 0.5 (MeOH:CH2Cl2 = 1:19). 1H NMR  δ 9.82 , 7.57 , 7.51–7.43 , 7.37 , 7.20–7.11 , 6.80 , 5.17 , 4.56 , 2.15 . 13C NMR  δ 172.9 (C=O), 156.1 (OAr), 153.8 (C=N), 137.7 (Ar), 136.6 (Ar), 130.0 (Ar), 129.4 , 128.3 (Ar), 127.5 (Ar), 123.4 , 123.2 , 122.8 , 118.6 (Ar), 114.1 (Ar), 111.1 (Ar), 74.0 (O-CH2-Ar), 63.6 (OCH3), 11.6 (CH3). HR-EI-MS m/z 367.1031 [M]+ . ATR-IR νmax 2936, 2372, 2324, 1735 cm−1.2-(3-(1-(((3-trifluoromethylbenzyl)oxy)imino)ethyl)phenoxy)acetic acid (2c): White solid, 90% yield. Rf = 0.4 (EtOAc:n-hexane = 2:1). 1H NMR  δ 9.76 , 7.37 , 7.32–7.24 , 6.94–6.88 , 5.17 , 4.70 , 3.81 , 2.22 . 13C NMR  δ 174.1 (C=O), 159.4 (OAr), 157.4 (OAr), 154.4 (C=N), 138.4 (Ar), 130.0 , 129.6 (Ar), 120.0 (Ar), 115.3 (Ar), 113.8 , 112.3 (Ar), 76.1 (O-CH2-Ar), 64.8 (O-CH2-CO2H), 55.3 (OCH3), 13.0 (CH3). HR-EI-MS m/z 329.1263 [M]+ . ATR-IR νmax 2906, 2372, 2326, 1733 cm−1.2-(3-(1-(((4-methoxybenzyl)oxy)imino)ethyl)phenoxy)acetic acid (2d): White solid, 71% yield. Rf = 0.4 (MeOH:CH2Cl2 = 1:19). 1H NMR  δ 8.14 , 7.47 , 7.24–7.15 , 6.85 , 5.24 , 4.62 , 2.21 . 13C NMR  δ 173.5 (C=O), 157.4 (OAr), 155.4 (C=N), 147.5 (Ar), 145.8 (Ar), 137.8 (Ar), 129.7 (Ar), 128.3 , 123.7 , 120.1 (Ar), 115.4 (Ar), 112.5 (Ar), 74.8(O-CH2-Ar), 64.8 (O-CH2-CO2H), 13.0 (CH3). HR-EI-MS m/z 344.1008 [M]+ . ATR-IR νmax 2910, 2372, 2324, 1739 cm−1.2-(3-(1-(((4-nitrobenzyl)oxy)imino)ethyl)phenoxy)acetic acid (2e): White solid, 66% yield. Rf = 0.1 (EtOAc:n-hexane = 2:1). 1H NMR  δ 7.94 , 7.65 , 7.29–7.21 , 6.95 , 5.31 , 4.66 , 3.10 , 2.27 .13C NMR  δ 171.2 (C=O), 158.0 (OAr), 155.4 (C=N), 145.0 (Ar), 139.8 (Ar), 137.6 (Ar), 129.2 (Ar), 128.3 , 127.1 , 119.1 (Ar), 115.2 (Ar), 112.0 (Ar), 74.5 (O-CH2-Ar), 64.5 (O-CH2-CO2H), 43.0 (SO2CH3), 11.6 (CH3). HR-EI-MS m/z 377.0933 [M]+ . ATR-IR νmax 2921, 2372, 2324, 1728 cm−1.2-(3-(1-(((4-methylsulfonylbenzyl)oxy)imino)ethyl)phenoxy)acetic acid (2 at 37 °C. Plasmids and transfection experiments for luciferase reporter assay were described in our previous report 5d, or 2d was evaluated using pooled HLMs. Pooled HLMs (1.0 mg/mL), a potassium phosphate buffer, and a test compound (1 μM) were pre-incubated . To initiate the reaction, the NADPH generating system was added to the samples and incubated for specific time points . Then, aliquots were taken and reactions terminated in acetonitrile containing IS. After centrifugation, supernatants were analyzed using LC-MS/MS to determine KS15, 5d, or 2d concentrations in samples. Analyte separation was performed using a Kinetex C18 column . The mobile phase solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile. Gradient separation was performed as follows: 0–3 min (10–90% B) and 3.1–8 min (10% B). The flow rate was 0.2 mL/min, and the run time was 8 min for each sample. Electrospray ionization was conducted in positive ion mode at 4 kV. Optimum operating conditions were as follows: vaporizer temperature, 300 °C; capillary temperature, 250 °C; and collision gas (argon) pressure 17 kPa. Quantitation was performed in the SRM of the [M+H]+ ion and the related product ion for each test compound and IS: KS-15 , 5d , 2d , and IS .The metabolic stability of KS15, 5d or 2d was dissolved in a dosing vehicle consisting of PEG400, normal saline, and dimethylsulfoxide in a ratio of 6:3:1 (v/v/v%) to make a 2 mg/mL dosing solution. This was intravenously bolus-injected into the right femoral vein at a volume of 1 mL/kg (2 mg/kg) for intravenous administration or intragastrically injected using an oral zone at a volume of 2.5 mL/kg (5 mg/kg) for p.o. administration. Blood samples (approximately 100 μL each) were collected in heparinized tubes via the right femoral artery at 0, 1, 5, 15, 30, 60, 120, 240, 360, 480, 1440 min after intravenous administration, and 0, 5, 15, 30, 60, 90, 120, 240, 360, 480, 1440 min after p.o. administration.For in vivo pharmacokinetic studies, Sprague Dawley (SD) male rats (weighing 270–280 g) were purchased from Orient Bio Inc. . Experimental protocols involving animal use were carefully reviewed and approved by the Institutional Animal Care and Use Committee at Gachon University. Prior to studies, animals fasted for 18 h, but with free access to water. Compound 2d were serially prepared in methanol in the 50–10,000 ng/mL concentration range, and 10 μL aliquots were added to 90 μL blank plasma to prepare calibration standards. For sample preparation, 100 μL aliquots of plasma samples or calibration standards were transferred to microcentrifuge tubes and mixed by vortexing vigorously for 5 min. They were then centrifuged at 14,000 rpm for 10 min at 4 °C. Supernatants were transferred to fresh analysis vials and a 2 μL aliquot injected onto the UHPLC-DAD system.To determine drug concentration levels in plasma, the Agilent 1290 UHPLC-DAD system with Polar RP column  was used for chromatographic separations. The mobile phase was composed of 0.1% formic acid in acetonitrile and 0.1% formic acid in DDW in a ratio of 40:60 and was isocratically delivered at a flow rate of 0.2 mL/min. The total run time was 4.4 min for samples which were monitored at 260 nm. Working standard solutions of compound 2d samples (50–800 μM) were prepared in 4% DMSO in TBS-T buffer. Real-time binding kinetics analysis was performed by injecting 100 μL sample aliquots into the channels of the HTE chip for 1 min at 100 μL/min. Then PBS-T buffer (100 μL/min) was flushed over the chip for 200 s to dissociate bound analytes. Recorded resonance units (RU) were normalized and plotted against chemical concentration. We used ProteOn Manager v.3.1.0.6 software for recording, plotting, and analyzing binding curves.We used a ProteOn XPR36 system  equipped with a HTE sensor chip  for surface plasmon resonance analyses. Purified target proteins (6xHis- and GST-tagged hCRY1 and hCRY2) were prepared by a baculovirus expression system . Phosphate buffered saline (PBS) plus 0.005% Tween 20 was used as the running buffer. The 6xHis-hCRY1 and 6xHis-hCRY2 proteins were immobilized onto separate channels of a HTE chip by injecting 210 μL protein sample (2.0 μg/mL) into flow cells (30 μL/min) to allow saturation. Serially diluted compound 5d (20 μM), or 2d (20 μM). Light emission for 2 min at intervals of 20 min was integrated using a dish-type wheeled luminometer . Background-subtracted bioluminescence profiles were analyzed using Cosinor analysis software .For real-time bioluminescence monitoring, we used mouse embryonic fibroblast (MEF) cultures established from Per2-LUC-KI mice ,21. To e50 values for luciferase reporter assay were calculated based on a logistic four-parametric equation using the mean values of intrinsic activation . The IC50 and t1/2 values for the cytochrome P450 inhibition assay and metabolic stability test were calculated by WinNonlin software . Pharmacokinetic parameters were calculated using non-compartmental analysis . Kinetic and equilibrium constants for SPR analysis were obtained by global fitting of the Langmuir 1:1 bimolecular kinetic model . Circadian molecular clock-enhancing activities were expressed as means ± SE and statistically evaluated by Student’s t-test or one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison test for post hoc comparisons. The significance level for the analyses was set at p < 0.05.The EC5d and its hydrolyzed product 2d were potent and effective enhancers on the E-box mediated transcription. Compounds 5d and 2d also exhibited desirable metabolic and pharmacokinetic profiles as promising drug candidates. Furthermore, compound 2d directly binds to CRY1 and 2 in surface plasmon resonance analyses, suggesting that the pharmacological target of 2d is CRYs. Compounds 5d and 2d inhibited the negative feedback actions of CRY on the CLOCK:BMAL1 heterodimer, which also suggested that their molecular target is CRYs. Most importantly, compounds 5d and 2d exhibited significant enough effects on molecular circadian rhythmicity to be considered circadian clock-enhancers, which are distinct from the KS15 possessing ethoxypropanoic acid moiety. Taken altogether, novel cryptochrome inhibitors with circadian clock-enhancing activity have been developed by bioisosteric replacement.Bioisosteric replacement is a useful and prevalent strategy for drug design and molecular modification in medicinal chemistry. Years of cumulative data proved that rational design with such a strategy for modifying lead compounds provided better candidates with improved potency, selectivity, and drug-likeness ,23,24. I


Rehabilitation training and movement evaluation after stroke have become a research hotspot as stroke has become a very common and harmful disease. However, traditional rehabilitation training and evaluation are mainly conducted under the guidance of rehabilitation doctors. The evaluation process is time-consuming and the evaluation results are greatly influenced by doctors. In this study, a desktop upper limb rehabilitation robot was designed and a quantitative evaluation system of upper limb motor function for stroke patients was proposed. The kinematics and dynamics data of stroke patients during active training were collected by sensors. Combined with the scores of patients’ upper limb motor function by rehabilitation doctors using the Wolf Motor Function Test (WMFT) scale, three different quantitative evaluation models of upper limb motor function based on Back Propagation Neural Network (BPNN), K-Nearest Neighbors (KNN), and Support Vector Regression (SVR) algorithms were established. To verify the effectiveness of the quantitative evaluation system, 10 healthy subjects and 21 stroke patients were recruited for experiments. The experimental results show that the BPNN model has the best evaluation performance among the three quantitative evaluation models. The scoring accuracy of the BPNN model reached up to 87.1%. Moreover, there was a significant correlation between the models′ scores and the doctors′ scores. The proposed system can help doctors to quantitatively evaluate the upper limb motor function of stroke patients and accurately master the rehabilitation progress of patients. Stroke is a disease caused by brain nerve injury. It is one of the common diseases among the elderly and the second leading cause of death . Stroke The traditional rehabilitation methods mainly rely on the auxiliary training of rehabilitation doctors. The main task of rehabilitation doctors is to formulate rehabilitation plans to help patients to recover limb motor function . In termAt present, many scholars have made attempts in rehabilitation evaluation. Lambercy et al.  introducIn this study, a desktop rehabilitation training robot with a simple structure and low cost was designed to collect the motion data and mechanical parameters of stroke patients in the process of rehabilitation training. Based on different machine learning algorithms, BPNN, KNN, and SVR, three rehabilitation evaluation models corresponding to the evaluation of the WMFT scale were established respectively. Finally, the performance differences of the three evaluation models were compared through experiments. Compared with the qualitative assessment methods proposed by Lambercy et al. and the single action quantitative assessment methods proposed by Olesh et al., our method can complete the quantitative assessment of the motor function of the entire upper extremity of stroke patients. We also avoid the disadvantages of visual sensors not being able to capture subtle movements and EMG devices requiring tight control over the placement of electrode pads. In addition, the rehabilitation robot proposed in this study is promising for application in home and community.The proposed quantitative evaluation system of upper limb motor function of stroke patients is shown in A desktop upper limb rehabilitation robot is developed to assist patients in active and passive training. The rehabilitation robot has a total length of 0.84 m, a width of 0.64 m, and a height of 0.35 m. The model of the upper limb rehabilitation robot is shown in The hardware system block diagram of the desktop upper limb rehabilitation robot is shown in Subjects were recruited to use the upper limb rehabilitation robot for experiments. The upper limb motor function evaluation scenes are shown in During the upper limb motor function evaluation experiment, the subject sat in front of the rehabilitation robot, as shown in X-axis. The subject then freely pushes the handle along the X-axis and the thrust information of the affected upper limb is collected through the force sensor. The data acquisition interface is shown in Phase1: Firstly, the subject holds the handle and moves it to one end of the X-axis, and the speed data of the subjects’ upper limbs are recorded. The acceleration data are derived from the velocity data.Phase2: The subject pushes the handle freely along Phase3: The subject pushes the handle along the standard trajectory, and the actual motion trajectory of the handle is recorded.The rehabilitation evaluation experiment was conducted in Suzhou Xiangcheng people’s hospital. The experiment was approved by the ethics committee of Suzhou Xiangcheng people’s Hospital ) and the patients’ consent letters were obtained. The inclusion criteria of experimental subjects are as follows: (1) 18–70 years old; (2) first episode of stroke within three months before the experiment; (3) WMFT score 15–75, Brunstrom stage II or above; (4) good mental state and can communicate normally. The exclusion criteria are as follows: (1) more than one stroke (2) the patient has other obvious injuries in the upper limb, such as shoulder subluxation or fracture; (3) the patient has visual defects; (4) participating in other upper limb rehabilitation trials currently. A total of 10 healthy subjects  and 21 stroke patients with upper limb dysfunction  were recruited to participate in the experiment. The patients′ demographic information is shown in N is 200. The comparison of data before and after filtering is shown in Due to the noise in the output signal of the force sensor, a moving average filter  is used To facilitate the subsequent establishment of a quantitative evaluation model based on machine learning algorithms and ensure that all values fall in the domain , the Min-Max method  is used (1)T: the straight line between the start point and the end point is used as the standard trajectory to calculate the deviation between the actual trajectory of the patient’s hand movement and the standard trajectory. The schematic diagram of trajectory offset is showed in Trajectory offset m is the number of trajectory sampling points. The trajectory offset can be used to judge the jitter and muscle coordination ability of the patient’s upper limb.Lee et al.  have pro(2)V: the average value of all instantaneous speeds. The calculation formula is shown in Equation (4).Average speed vN is the total number of seconds taken. The average speed reflects the continuity of strength in the process of rehabilitation training.(3)a: the maximum instantaneous acceleration of the patient’s upper limb. The calculation formula is shown in Equation (5).Maximum instantaneous acceleration tV is the instantaneous velocity variation, and Δit is the instantaneous time difference. The maximum acceleration can reflect the reaction speed of the patient’s upper limb during movement.(4)F:Maximum thrust of upper limb tF is the sampling value and M is the total number of force sampling points. F reflects the muscle strength of the patients’ upper limbs.Using the scores of rehabilitation doctors as labels, the data of 31 subjects were composed into a data set. The 155 samples in the data set were randomly divided into a training set containing 124 samples and a test set containing 31 samples. Because BPNN, KNN, and SVR algorithms have a good nonlinear mapping ability and generalization ability , and they is the output score of the neural network evaluation model. In the process of model training, the stochastic gradient descent is used as the optimization method, and the ReLU function is used as the activation function. The mean square error is used as the loss function, and the number of training steps is set to 1000.BPNN is a multilayer feed-forward neural network with error backpropagation . To obtak nearest neighbors closest to the test points in the training set [X = , where y is the score of the KNN evaluation model. Traverse each point in the training set and calculate the Euclidean distance Le between it and sample X in the test set.KNN regression algorithm aims to find the ning set . The traLe according to the size, and selecting k nearest neighbors jX (1 ≤ j ≤ k) in the training set. The average of the scores corresponding to these k nearest neighbors is used as the score of the KNN evaluation model. In this study, the number of nearest neighbors k is set to five, soSorting the obtained distance x)f( and make it as close to y can then be computed by using Equation (10).ϕ is a nonlinear operator, which maps the input data to a high-dimensional feature space. The kernel function used in the SVR evaluation model is RBF kernel, soσ is a free parameter of RBF kernel.Forming the data of subjects into labeled training sets  problem . The scoIn this study, absolute error was used to reflect the error between the model’s score and the rehabilitation doctor’ score. The absolute error is defined asRMSE), and determination coefficient (R2) were used to evaluate the fitting performance of these models [RMSE, and R2 are shown in Equations (13)–(15). In the experiment, the rehabilitation doctors thought that the error between the model′s score and the doctor′s score should be within 5. So, if the absolute error is less than 5, the score of the model is considered to be correct.To compare the quantitative evaluation results of the above three regression algorithms  on the motor function of patients’ upper limbs, the accuracy, root mean square error  between BPNN model′s scores and rehabilitation doctors′ scores is 0.986, which is bigger than that of the KNN model (0.983) and that of the SVR model (0.945). The p-values of the three evaluation models were all less than 0.05. Therefore, the scores calculated by the three models are significantly correlated with those given by rehabilitation doctors.To verify the effectiveness of the evaluation models, the Pearson correlation test was used to analyze the correlation between the models′ scores and the rehabilitation doctors′ scores in the test set. SPSS  was used for statistical analysis. The results show that the correlation coefficient (RMSE), determination coefficient (R2), correlation coefficient (cR) and absolute error  of the three quantitative evaluation models are shown in The accuracy, root mean square error  and that of the SVR model (0.945). Therefore, the experimental results show that the scores of the three evaluation models are all significantly correlated with the scores of doctors. The reason for the high correlation coefficient may be that the scores of rehabilitation doctors are used as the labels to train these models.Limitations of our current study should also be noted. The total sample size of the data set used to establish the evaluation model is 155, and the relatively small number of samples in this study could limit the evaluation performance of the models. Therefore, more clinical trials are needed to collect more data to improve the accuracy of the evaluation model. There was an imbalance in the severity of the disease, as Brunnstrom stages of patients are mainly concentrated in stage II, V, and VI, but the number of patients in stage III and IV is small. Therefore, it is necessary to recruit more patients in stage III and IV in the future. In addition, this study only considers four features of upper limbs. More features will help to improve the performance of the evaluation model, and thus we need to explore the impact of other features on the performance of the evaluation model in future work.In this study, a quantitative evaluation system was proposed to evaluate the upper limb motor function of stroke patients. Three different quantitative evaluation models based on BPNN, KNN, and SVR regression algorithms were established to evaluate upper limb motor function. Rehabilitation doctors were invited to conduct experiments on 10 healthy subjects and 21 stroke patients in combination with the WMFT scale. Compared with the scores of rehabilitation doctors, it was found that the accuracy of the established quantitative evaluation model reached up to 87.1%. The experimental results show that the BPNN model has the best evaluation performance among the three quantitative evaluation models. Moreover, there was a significant correlation between the models′ scores and the doctors′ scores. The proposed system will help doctors to evaluate the upper limb motor function of stroke survivors, and carry out individualized rehabilitation intervention for different patients. In the future, we will use more evaluation indicators and carry out large-scale clinical trials to improve the quantitative evaluation model. In addition, this study mainly studies the quantitative evaluation of upper limb motor function of stroke patients. In the future, experiments will be carried out to study the rehabilitation effect of the rehabilitation robot on patients.


Triple-negative breast cancers  continue to present a unique treatment challenge and carry unfavorable prognoses. The elucidation of novel therapeutic targets has necessitated the re-evaluation of stratification approaches to best predict prognosis, treatment response and theranostic and prognostic markers. Androgen receptor expression and function have important implications on proliferation, tumor progression, immunity and molecular signaling in breast cancer. Accordingly, there has been increasing support for classification of androgen receptor-negative triple-negative breast cancer or quadruple-negative breast cancer (QNBC). QNBC has unique molecular, signaling and expression regulation profiles, particularly those affected by microRNA regulatory networks. microRNAs are now known to regulate AR-related targets and pathways that are dysregulated in QNBC, including immune checkpoint inhibitors (ICIs), SKP2, EN1, ACSL4 and EGFR. In this review, we explore and define the QNBC tumor subtype, its molecular and clinical distinctions from other subtypes, miRNA dysregulation and function in QNBC, and knowledge gaps in the field. Potential insights into clinical and translational implications of these dysregulated networks in QNBC are discussed. Breast cancers lacking epidermal growth factor receptor 2 (HER2), estrogen receptor and progesterone receptor, are termed triple-negative breast cancers (TNBC) and are distinguished because of a lack of response to therapies targeting these receptors. TNBC is heterogeneous at genomic, transcriptomic levels, showing varying clinical and pathological presentation genetic susceptibility factors and sensitivities for chemotherapeutics . As suchDifferential expression profiles and pathway regulation in breast cancer and TNBC in particular are integrated with the expression and regulation of transcriptional profiles by microRNAs (miRNA) . Importa. There has been increased attention given to the utility of reclassifying TNBC tumors lacking AR expression as QNBC [TNBC has classically been divided into basal-like subtypes (BL1 and BL2), mesenchymal (M) and luminal androgen receptor (LAR) subtypes, and in some systems, according to immunosuppressive versus immune active phenotypes ,11,12,13 as QNBC ,15,16. A as QNBC ,18,19,20 as QNBC . This an as QNBC . In a st as QNBC . EGFR ex as QNBC . PotentiRegardless of some controversy regarding the prognostic significance of AR expression in TNBC , the priAR is a 110 kDa protein residing in the cytoplasm with zinc finger DNA-binding, transcriptional regulation and ligand-binding capabilities . Within Proliferation of tumor cells is generally thought to be increased by AR with evidence of cell-cycle regulation downstream of various androgen-responsive transcriptional activities . AR-ligaAR expression in TNBC may also affect epithelial-to-mesenchymal transition (EMT) leading to altered invasion and metastasis. To this point, nuclear AR was found to be inversely associated with E-cadherin expression and positively associated with increased tumor grade, mesenchymal morphology, metastasis, recurrence and with poor DFS and OS in TNBC ,33. Liu While evidence for angiogenic activity of AR in breast cancer is sparse, regulation of angiogenesis by AR in prostate cancer is implicated through interactions with epigenetic and transcriptional co-activation factors to regulate VEGF, and synergy between anti-androgenic and anti-VEGF therapies .Immune killing of TNBC can also be increased through androgen deprivation therapy using enzalutamide or abiraterone, although this effect was shown to be partially independent of detection of AR expression .Androgen receptor signaling has the potential to regulate DNA repair in QNBC as anti-androgen therapy has been shown to downregulate DNA repair genes that are direct transcriptional targets of AR . In addiGiven that many of the phenotypic effects of AR are pro-tumorigenic and pro-tumor progression, much remains to be discovered regarding mechanisms by which AR negative TNBCs or QNBCs generally have poorer prognoses than AR-positive TNBCs.Subtypes of TNBC, including basal-like, mesenchymal, LAR, and immunomodulatory, are characterized by distinct molecular pathways and profiles  as outliIn addition to direct activity through androgen-response elements, non-DNA-binding mechanisms of AR function exist ,41, inclIn contrast to findings associated with increased EMT with AR expression in TNBC , expressQNBC tumors display distinct molecular profiles including that of transcriptional expression as shown by a study on African American women showing enriched classical basal-like and immune subtype signatures in QNBC . In thisBroad regulation of transcriptional, and subsequently, molecular pathways by miRNAs is known to be important to the understanding of breast cancer pathobiology and markers of the disease. As such, we continue with a detailed review of miRNA regulators of the above pathways, known miRNA dysregulation in QNBC, clinical and translational implications of miRNA regulation in QNBC and unknowns in the field.Since little is known regarding QNBC-specific miRNA regulatory networks, much of the data driving hypotheses for miRNA regulation affecting QNBC pathobiology are extrapolated from the study of TNBC, AR function in breast cancer, and from other cancers. Integration of this existing knowledge, as in this review, provides a jumping-off point for the study of miRNA dysregulation in QNBC. Among the potentially targetable molecular pathways that are known to be altered in QNBC as described above in Other miRNAs that are specifically dysregulated in QNBC have been recently discovered. QNBC was found to exhibit increased expression of miR-135b, which correlated with AR-negativity among TNBCs . DiffereRecently, Bhattari et al. identified increased copy number alterations (CNA), genomic instability and miRNA dysregulation in QNBC compared with AR+ TNBC . They idResearch into such miRNA regulatory networks may provide novel targets and allow the selection of targeted therapies based on the prediction of response of AR-positive TNBC versus QNBC to novel and existing therapeutics. Insights provided by this review and the literature cited herein may provide a foundation for the discovery of miRNA and regulated genes and pathways that may be targeted differentially in QNBC and AR+ TNBC, further justifying the stratification of TNBC according to AR expression. One example may be the tumor promoting miR-135b. Since this miRNA is associated with increased proliferation and TGF-β, Wnt and ErbB signaling in basal-like TNBC , miR-135Neoadjuvant therapy is central to therapy for TNBC given increased response rates compared with other breast cancer types . In the The field of study specific to QNBC is young, leaving much to be discovered about unique pathobiology, regulatory networks, exploitable therapeutic targets and prognostic or theranostic markers. However, there is some understanding of the unique molecular profiles in QNBC. This knowledge can be built on to extrapolate hypotheses regarding the roles of these pathways, their regulation and their targetability with therapeutic interventions. Investigation of miRNA profiles, regulatory networks and pathway analysis of these networks in QNBC compared with AR+ TNBC and other breast cancer subtypes has been sparse so far. Studies using large cohorts comparing these subtypes and additional mechanistic studies of transcript regulation by differentially expressed miRNAs and downstream phenotypic changes relevant to tumorigenesis and progression in QNBC vs AR+ TNBC are needed. Such studies will be required to appreciate the role and potential targeting of these regulators and their regulatory networks in these diseases.There is increasing evidence of unique molecular profiles and targetable pathways other than AR signaling in QNBC versus TNBC and other breast cancer subtypes. These differentiators continue to strengthen the justification for AR expression evaluation of TNBC and classification accordingly. While mutational dysregulation of targetable signaling pathways such as PI3K are more readily identified in other subtypes, these pathways may also be dysregulated in QNBC via transcript modulation by miRNAs. Recent studies have identified consistently and differentially dysregulated miRNAs targeting multiple factors that are relevant to breast cancer pathobiology, including Myc, PI3K, TP53, SOD2 and cell cycle checkpoint factors. Further research into QNBC-specific dysregulation of miRNAs, transcripts and molecular and functional networks can enable the exploitation of these unique factors for the targeted treatment in the face of an otherwise intractable therapeutic challenge.


Stem cell medicine has led to functional recovery in the acute-to-subacute phase of spinal cord injury (SCI), but not yet in the chronic phase, during which various molecular mechanisms drastically remodel the tissue and render it treatment-resistant. Researchers are attempting to identify effective combinatorial treatments that can overcome the refractory state of the chronically injured spinal cord. Regenerative rehabilitation, combinatorial treatment with regenerative medicine that aims to elicit synergistic effects, is being developed. Rehabilitation upon SCI in preclinical studies has recently attracted more attention because it is safe, induces neuronal plasticity involving transplanted stem cells and sensorimotor circuits, and is routinely implemented in human clinics. However, regenerative rehabilitation has not been extensively reviewed, and only a few reviews have focused on the use of physical medicine modalities for rehabilitative purposes, which might be more important in the chronic phase. Here, we summarize regenerative rehabilitation studies according to the effector, site, and mechanism. Specifically, we describe effects on transplanted cells, microstructures at and distant from the lesion, and molecular changes. To establish a treatment regimen that induces robust functional recovery upon chronic SCI, further investigations are required of combinatorial treatments incorporating stem cell therapy, regenerative rehabilitation, and medication. Spinal cord injury (SCI) is a very severe condition with various sequelae represented by motor, sensory, and autonomic disorders, which have an enormous influence on psychosocial aspects of patients’ lives. There are two major classifications of SCI: traumatic SCI and non-traumatic SCI. Traumatic SCI is estimated to have an annual incidence ranging from 8.0  to 246  to 1298 The refractory state of the chronically injured spinal cord is a significant clinical concern. There are 50 times as many patients in the chronic phase than in the acute-to-subacute phase suffering from life-long impairment . SeveralStem cell-based regenerative therapy is an innovative treatment for sequelae of SCI. Significant therapeutic effects have been reported clinically and preclinically in the acute and subacute phases with various cell sources, including neural stem/progenitor cells (NS/PCs), mesenchymal stem cells (MSCs), and olfactory ensheathing cells (OECs). Researchers have elucidated various therapeutic mechanisms, including transplanted cell-mediated neuronal replacement, remyelination, and trophic support, which further induce tissue protection and enhance neuronal plasticity ,21,22. IAlthough the number of studies of regenerative rehabilitation in the acute-to-subacute phase of SCI has increased, very few studies have been performed in the chronic phase ,33,36. IVery few researchers have reviewed regenerative rehabilitation targeting SCI, particularly in the chronic phase. Therefore, the present review aims to organize and summarize the mechanisms and effects of regenerative rehabilitation, including rehabilitative training and physical medicine modalities.In 2010, Ambrosio et al. first suggested the concept of regenerative rehabilitation as the optimized application of rehabilitation science to promote regenerative therapies . In a brThe following rehabilitative training methods have been applied in combination with stem cell therapies to animal models of SCI regardless of chronicity: bipedal treadmill training with body-weight support ,35, actiIt is noteworthy that the status of regenerative rehabilitation differs between clinical and preclinical studies in several respects. First, individuals with SCI routinely undergo rehabilitation in clinical settings and frequently continue rehabilitative training to preserve their joint range-of-motion, muscle strength, mobility, and activities of daily living. Second, rehabilitation is feasible in many clinical settings and is therefore used in combination with stem cell therapies in clinical trials. Third, it is relatively easy to propose appropriate training for patients according to their impairments because there is much knowledge of SCI rehabilitation, and rehabilitation therapists and doctors can receive immediate feedback from patients. By contrast, rehabilitation training is less feasible, very time-consuming, and expensive in preclinical studies , and labVarious combinatorial strategies have been investigated to improve the effects of stem cell therapies on the refractory chronically injured spinal cord . AlthougOn the other hand, the effects of rehabilitation on SCI according to its chronicity have not been determined due to a lack of preclinical studies ,53. WhilDistinct effects of regenerative rehabilitation on chronic SCI have been reported according to the lesion epicenter, transplanted cells, and lumbar enlargement. To delineate the specificity of regenerative rehabilitation in the chronic phase, we summarized the histological, biological, and physiological effects at various sites reported by studies of SCI in the acute-to-subacute and chronic phases. We conclude that most findings in every study of regenerative rehabilitation are independent, i.e., causal relationships are not clearly depicted because this research field is in its infancy. Therefore, we categorized the results according to the nature of the effects, including (1) the direct effect on transplanted stem cells, (2) the effect on the microstructure around the lesion including scar tissue, (3) the effect on the microstructure of spinal cord tissue distant from the lesion, and (4) the mechanisms underlying histological changes . The secIt is frequently reported that combined rehabilitation can change the fate of cell grafts, particularly NS/PCs, which differentiate, proliferate, and migrate after grafting. Two research groups reported identical results showing that NS/PCs differentiated more into neurons and oligodendrocytes, together with a better cell survival rate, when treadmill training was performed in combination with stem cell therapy in rat models of subacute cervical and thoracic SCI ,30. TashSimilar results were reported in studies of physical medicine modalities both in vitro and in vivo. Guo et al. acutely transplanted human umbilical cord blood-derived MSCs (hUCB-MSCs) and applied intermittent rTMS composed of stimulation for 3 s and rest for 6 s at a frequency of 0.5 or 10 Hz. They reported increased proliferation of transplanted cells, consistent with facilitation of differentiation into neurons, oligodendrocytes, and astrocytes. The effect was greater in the high frequency (10 Hz) group . Feng etA massive microenvironmental change in the lesion epicenter restricts neuronal plasticity and the effects of stem cell therapies in various respects, and regenerative rehabilitation seems to attenuate these processes. Histological changes are observed around the lesion epicenter secondary to combined rehabilitation in individuals with acute-to-subacute SCI. Restoration of gross tissue volume, structure, and residual fibers is frequently reported when quadrupedal gait training is performed in combination with transplantation of NS/PCs ,30 or boIn addition to tissue-protective effects, spinal tract regeneration has also been reported. Guo et al. revealed that rTMS in combination with hUCB-MSC transplantation promoted regeneration of the corticospinal tract based on BDA tracing . Younsi Plastic changes in microstructures distant from the lesion, represented by neural regeneration and neuronal circuit reorganization, are frequently reported as the main effects of regenerative rehabilitation. Rehabilitation enhances the activities of various neuronal subtypes. Serotonergic activity, assessed by serotonergic fiber innervation into the lumbar spinal cord, is increased secondary to quadrupedal treadmill training in combination with NS/PC transplantation in the acute/subacute phase . NoradreSachdeva et al.  and TheiThe molecular mechanisms that may induce the abovementioned histological changes can be grossly classified as trophic support and anti-inflammation. Various neurotrophic factors are upregulated secondary to rehabilitative training and physical medicine treatment. Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor, insulin-like growth factor-1 (IGF-1), neurotrophin 3 (NT-3) , BDNF 4, and neuA few studies revealed the involvement of other molecular mechanisms. Hwang et al.  revealedDugan et al.  reportedAlthough there are only a few reports about the mechanisms underlying the changes induced by combinatorial use of treadmill training and stem cell therapy, such research will help to explore more effective approaches and prevent adverse effects . Above mRegenerative rehabilitation elicits various beneficial effects on transplanted cell grafts and the host microstructure around the lesion and in distant areas. However, there is no evidence of its effects on fibrotic and glial scarring or around the lesion of the chronically injured spinal cord. This indicates that a further combinatorial treatment(s), such as medication, is needed to complement the effects of regenerative rehabilitation. It is important to consider if medication and training have overlapping effects because there is a concern that they do not elicit a synergistic effect . TherefoSeveral studies reported significant combinatorial effects using cABC, which digests CSPG, in various experimental conditions, including intensive voluntary forepaw motor rehabilitation of rats with a dorsal corticospinal tract lesion , and quaSerotonin receptor agonists may enhance the neuromodulatory effect of EES. Quipazine, a non-selective 5-HT2A receptor agonist, facilitates motor function when used in combination with robotic locomotor training . CourtinNeurotrophic factors, represented by NT-3 and BDNF, enhance the therapeutic effect of rehabilitation ,71. In aWhile studies have revealed the synergistic effects of regenerative rehabilitation with stem cell therapy on SCI, they seem insufficient to achieve significant functional recovery in the chronic phase. Preclinical studies have delineated the advantages and limitations of regenerative rehabilitation in inducing molecular and histological changes in the chronically injured spinal cord. Thus, it has been gradually revealed under which circumstances further combinatorial treatment with medication should be implemented. To establish a treatment regimen for robust functional recovery upon chronic SCI, further investigations are required of combinatorial treatment with stem cell therapy, regenerative rehabilitation, and medication.


Central coordination disorders (CCD) encompass various abnormalities observed in infants but early therapy may have an impact on their condition. The aim was to seek factors that may affect the early results of therapy of infants with CCD.We analyzed the outcomes of a three-month period of rehabilitation of infants living with CCD. Children were treated at Non-public Specialist Healthcare Institution Medi-Reh in Kalisz in the period from 1 Jan 2014 to 31 Nov 2019. In our retrospective study results of three-month therapy of infants, aged 1 to 6 months, with CCD were analysed regards to the effectiveness and the potential impact of different factors. Therapy and assessment of children were conducted with the use of the Vojta method, which was performed during the first visit (WW) and the follow-up visit (after 3 months- 1WK). The analysis of the influence of various factors on the effect of therapy included: mother's age at the time of delivery, duration of breastfeeding, child APGAR, gestational age in which the child was born, sex of the child, birth weight, age of the child at WW, type of delivery, craniosacral therapy as an additive treatment.Based on the examination results from 66 medical records it was demonstrated that after active period of the therapy, improvement was observed in 54 (81.81%) (p=0.48) children (condition during WW versus 1WK among the group). The sole factor impacting improvement after 3 months was the age of the child at WW, when the child started therapy. This factor significantly (p=0.002) increased the chance of achieving improvement - by 3.2 times . No statistically significant differences were shown for the other studied factors.Prompt implementation of rehabilitation in children with CCD provides a better chance of improving their motor function. The rehabilitation should be started as soon as possible after the diagnosis is constituted. Central coordination disorders (CCD) are a broad term that describes abnormal motor development in children. As CCD is not a specific disease entity, and has only diagnostic and classification value, there are no epidemiological data that could be analysed in the context of its incidence. Evaluation of motor development at an early stage of life should be conducted on a regular basis to diagnose any possible abnormalities as soon as possible and begin rehabilitation .The aim of paediatric interventions is to promptly provide care to a child that has been diagnosed with a medical problem requiring rehabilitation, often as early as from the time of its birth, for example in the case of patients with an increased risk related to pregnancy and birth.In the case of disorders related to immature or damaged nervous system, we apply various methods, jointly referred to as the neurodevelopemental methods .The method developed by prof. Vojta, which is classified as one of early neurophysiological treatments, constitutes a universal method applied in the diagnostic evaluation and rehabilitation of children with movement disorders. It comprises both diagnostic tests that help track the progress made by the child and therapeutic interventions . This diAnother therapy used in rehabilitation of children is craniosacral method originating in osteopathy. In the simplest terms, the method consists in the physical therapist delicately touching specific areas of the infant's body with their hands .This way, the therapist can feel abnormal tension in the tissue structures and mobilize the connective tissue surrounding the organs. The applied touch calms, soothes, relieves tension and gently stimulates the nervous system .Both of these methods are classified as stimulus therapy and are used in rehabilitation of children with CCD. They can mitigate as well as fix the results of their abnormal development resulting from, for example, the labour. Nevertheless, even in children with a similar severity of disorders the response to treatment may differ in terms of time and effect .Vojta therapy is widely used in physiotherapy for the treatment of various pathologies. Scientific evidence indicates positive effects of the Vojta therapy on the treatment of subluxation of the hip joint . The benIt is emphasized that an advanced age of the mother may increase the risk of congenital abnormalities and affect the relationship between the mother and the child, which may strengthen or weaken the reception of the external stimuli applied during the treatment by the child . BreastfKnowledge of such factors and the strength of their impact can be helpful in planning the therapeutic process in all cases involving modifiable factors.The aim of the paper was to seek factors that may affect early results of rehabilitation of infants (three-month observation) and assess the degree of their impact.We analyzed the outcomes of a three-month period of rehabilitation of infants suffering from CCD, aged 1-6 months, which met the below-listed inclusion criteria, and were treated at NSZOZ MEDI-REH in Kalisz. The analysis involved archival materials (a retrospective study) from the medical histories of patients - children rehabilitated by one person - the leader of the study group - in the period from 1 January 2014 to 31 November 2019. The additional treatment, namely craniosacral therapy (if included), was also provided by one person - a certified specialist. Thanks to that it was possible to reduce the impact of the interfering factor, namely the skills of the therapist . Approval for the study was obtained from the Bioethics Committee of the Medical University of Wroclaw (consent number KB-108/2019).a score of 8 - 10 on the APGAR scale in the first minute of life, and no significant deterioration in the health of the child, significantly impairing the function of the neonate (e.g. sudden need for cardiac-pulmonary resuscitation), in the first 10 minutes of its life; changes other than decreasing the score obtained by the child by 1 point in a given time, if the neonate obtained 10 or 9 points in the APGAR scale, were considered to be a significant deterioration of its condition.no major birth defects that would significantly impair the development of the child and/or would require conducting genetic diagnosis.eligibility for rehabilitation confirmed by a medical rehabilitation physician at the centre;the age of the child at the time of the assessment of its eligibility for rehabilitation: 1-6 months (of life), calculated based on completed months of life.abnormal result of the Vojta test on admission to the centre, defined as at least 6 abnormal responses , or delayed (OP) in the test report), with abnormal muscle tension, which indicates moderate to severe CCD;parental consent to the use of the medical history for the purposes of the project;available information from the first medical examination (during the first visit (WW)), and follow-up visit (1WK) on the determined date, maintaining the defined timeframe.Exclusion criteria :children who obtained a score of < 8 points on the APGAR scale in the first minute of life as well as those who obtained 8 - 10 points in the APGAR scale, but their condition deteriorated significantly in the first 10 minutes of life, considerably impairing their function (e.g. sudden need for cardiac and pulmonary resuscitation) and causing a decrease in the APGAR score by more than 1 point, compared to the starting point.diagnosis of major (significant) birth defects, information on a major birth defect, congenital defect syndrome  and/or indications for consultation at a Genetics Clinic,opinion of the physician indicating that there is no need to rehabilitate the child using the Vojta method despite attending the consultation, or selection of other rehabilitation method by the physician;age < 1 month or > 6 months ;< 6 abnormal responses during the Vojta test at first eligibility visit, which indicates mild or very mild CCD;lack of parental consent to the use of documentation for the purposes of the studyThe following inclusion criteria were applied:66 children who met the inclusion criteria were qualified for rehabilitation at the WW visit and completed the 3-month procedure. The rehabilitation and evaluation of children was conducted on the basis of the standard protocol adopted by Non-public Specialist Healthcare Institution Medi-Reh in Kalisz for diagnostic evaluation and treatment. According to the plan, the neurokinesiological examination using the Vojta method is performed at the centre during the first eligibility visit (WW) and during the follow-up visits, the first of which takes place after 3 months (1WK), and each next - after every 3 months following the previous visit .For the purposes of basic characterization and grouping of factors potentially affecting the results of rehabilitation, the so-called "maternal factors" and "child factors" were distinguished in the first place.mother's age at the time of deliveryduration of breastfeeding, from the moment of birth until the completion of the therapyThe following maternal factors were analysed:provided in completed weeks.the APGAR score obtained in the first minute of the child's lifethe week of gestation  in which the child was born (Hbd)the sex of the childbirth weight (BW)the age of the child at first eligibility visit, which was equivalent to the beginning of rehabilitation - calculated based on the months completed since birth.The following were analysed among child-related factors:Subsequently, the authors took into the type of delivery  as well as the rehabilitation method .Statistical analysis was conducted using Statistica 12 and Excel. Descriptive statistics were presented in tables, using measures of location: the mean, the median, the standard deviation, the minimum and the maximum. Distribution of variables was analysed using the Shapiro-Wilk W test. The impact of independent variables, including those encoded using the zero-one system, on variables of dichotomous type was verified using the single-factor logistic regression model, in which the Odds Ratio (OR) plays an important role. A p-value of ≤ 0.05 was deemed to be statistically significant , 26.st follow-up visit. During first eligibility visit, after establishing the diagnosis and assessing the child's eligibility for rehabilitation, the specialist determines the aim of the treatment and the rehabilitation method . Depending on the outcomes, the method can be modified during subsequent visits. If, compared to first eligibility visit, the number of abnormal responses at 1st follow up visit is lower (the Vojta method), it indicates that there has been an improvement.For the purposes of the study, the authors used the results from first eligibility visit and 1The analysed characteristics attributed to the mother and the child are presented in Table In the study group, 27 (40.91%) children were delivered naturally, and 39 (59.09%) were delivered by a Caesarean section. In 52 (78.79%) children, the Vojta therapy was the sole method of rehabilitation, while rehabilitation of 14 (21.21%) infants consisted in combining the Vojta method and the craniosacral treatment, based on the decision of the consulting physician.After 3 months of rehabilitation, improvement was observed in 54 (81.81%) children. There was no difference in the obtained outcomes of rehabilitation between the percentage of children delivered naturally - 21 (38.89%) infants - and those delivered by c-section - 33 (61.11%) infants (p = 0.48).In the group of children rehabilitated with the use of the Vojta method only, improvement was observed in 43 (82.69%) infants, while in the group of patients in whom the Vojta method was combined with craniosacral therapy improvement was observed in 11 (78.57%) patients.The results of the statistical analysis showed no statistically significant effect of the applied therapy on the improvement of the health of the treated patients .Both mother and child-related factors as well as the type of delivery and the rehabilitation method were then analyzed with regard to their impact on the obtained outcomes.The only independent factor affecting the observed improvement after three months of rehabilitation was the child's age at the time of the beginning of the therapy (p < 0.01). This factor increased the chance of improvement by 3.2 times.st follow-up visit. The impact of the evaluated independent factors is presented in Table Other factors were not shown to have an impact on the obtained improvement evaluated at 1We have shown that the earlier age when therapy is started the higher percentage of improvement can be reached among patients. After a three-month-long observation, during the first follow-up visit and based on the assessment of the effects of rehabilitation, it was observed that the faster the treatment is introduced, the better it is for the child (chances of improvement increased by 3.2 times). During the early stage of development, it is possible to inhibit many inherently progressive disorders that without intervention could become established, or even progress over time. By introducing therapy, we can have also an impact on the expansion of synaptic connections , 28. WhaAccording to many researchers, breastfeeding duration may play a significant role with regard to better rehabilitation results –44 as itEven though c-sections should be performed only when medically necessary, the percentage of such deliveries is constantly increasing in many countries . This prLiterature indicates that the outcome of rehabilitation can be affected by a number of variables. According to researchers, it is significantly affected by such factors as: the week of pregnancy in which the child was delivered, birth weight or the condition of the child after its birth  , 70–73. Both therapies applied at the Centre  are classified as stimulus therapies, meaning that external stimulation, in this case applied by a physical therapist, stimulates the nervous system of the child. Therefore, external factors may both strengthen and interfere with the reception of such stimulation and finally affect the outcome of the treatment. It seems that the skills of the therapist as well as their commitment and good contact with parents  who mustThe earlier rehabilitation is started, the better the therapy results. There is a need to assess if the same variable impact rehab effect if the APGAR score is lower. It seems that other studies, focusing on the impact of different variables described in this paper in groups of children with lower APGAR scores are recommendable as such patients may require much more radical action than children with normal scores on the scale.Prompt implementation of rehabilitation in children with CCD provides a better chance of improving their motor function. The rehabilitation should be started as soon as possible after the diagnosis is constituted.We would like to thank the director of Non-public Specialist Healthcare Institution Medi-Reh in Kalisz, Poland, who gave her permission to conduct the study based on the Center records.