The effect of ambient temperature on allergic rhinitis (AR) remains unclear. Accordingly, this study aimed to explore the relationship between ambient temperature and the risk of AR outpatients in Xinxiang, China.2), and nitrogen dioxide (NO2) were controlled as covariates simultaneously.Daily data of outpatients for AR, meteorological conditions, and ambient air pollution in Xinxiang, China were collected from 2015 to 2018. The lag-exposure-response relationship between daily mean temperature and the number of hospital outpatient visits for AR was analyzed by distributed lag non-linear model (DLNM). Humidity, long-time trends, day of the week, public holidays, and air pollutants including sulfur dioxide  percentile, at lag of 0–7 days : 1.05–1.67), and at the 75th (25 °C) percentile at a lag of 0–3 days , respectively. Furthermore, men were more sensitive to temperature changes than women, and the younger groups appeared to be more influenced..Both mild cold and mild hot temperatures may significantly increase the risk of AR outpatients in Xinxiang, China. These findings could have important public health implications for the occurrence and prevention of ARThe online version contains supplementary material available at 10.1186/s12889-021-10671-6. Allergic rhinitis (AR) is triggered by a spectrum of environmental allergens and is considered an immunoglobulin E (IgE) mediated type 1 hypersensitivity illnesses . FurtherIncreasing evidence have revealed that both environmental and genetic factors play important roles in the etiology of allergic diseases, including AR , 12, 13.2) levels has been widely accepted over the past decades. Due to its very large population, even a minor temperature increase could potentially cause huge health losses in China. However, the potential effect of ambient temperatures on AR has not been directly examined in the Chinese population. Consequently, we performed a time-series study to explore the lag-exposure-response relationship between the prevalence of AR-related outpatient visits and ambient temperature.The development of climate change and global warming due to increased atmospheric carbon dioxide (CO2) was approximately 5.67 million at the end of 2014. The city is located in north of Henan province, on the north side of the Yellow River. Our research area was limited to the four traditional urban districts (422 km2). Xinxiang has a typical temperate continental monsoon climate, with prevailing northeasterly winds.The population of Xinxiang, China  and Sulfur dioxide (SO2) pollutants were measured at four fixed-site stations administrated by the Xinxiang Ministry of Ecology and Environment.Daily meteorological parameters, including relative humidity, maximum, minimum, and mean daily temperatures in Xinxiang from January 1, 2015 to December 31, 2018 were collected from the China Meteorological Data Sharing Service System (http//106.37.208.235.20035/). Additionally, daily mean concentrations of nitrogen dioxide  [A generalized additive model was adopted to link the outcome (daily AR outpatient visits) and exposure (daily mean ambient temperature). Additionally, several covariates were incorporated to control for potential confounding effects as previously described . Covaria for AR) . To optiThe 1st (− 2 °C), 25th (7 °C), 75th (25 °C), and 99th (32 °C) daily mean temperature percentiles relative to the median temperature were calculated for the relative risks (RR) and 95% confidence intervals (CIs) of the AR outpatient visits at cumulative lag effects from 0 to 28 days, respectively. These measurements were applied to analyze the effects of cold, mild cold, mild hot, and hot temperatures on AR outpatient visits. Moreover, we conducted sex and age  group stratification analyses to determine their effects on the risk of AR outpatient visits. The statistical significance of differences between effect estimates of the strata by calculating the 95% confidence intervals (CI) as d errors .p value of < 0.05 were considered statistically significant.All statistical analyses were completed by DLNM and MGCV packages in R software (Version 3.3.3). A 2 and SO2 were 48.0 μg/m3 and 33.0 μg/m3, respectively.Between January 1, 2015 and December 31, 2018, there were 14,965 hospital outpatient visits due to AR in Xinxiang, China. A total of 7912 male and 7050 female outpatients (3 records missing) were collected  over different lag times. Non-significant associations between cold temperatures  and AR outpatient visits were observed for all lag times. The heat temperature  had a negative effect on AR outpatient at a lag of 0–14 days, 0–21 days, and 0–28 days. The results show that either mild cold  or mild hot  temperatures could increase the risk for AR. Of note, mild cold temperatures had slightly longer lag times  and the mild hot temperatures had slightly shorter lag times .Table Figure The exposure-response curves of daily mean temperature on AR hospital outpatients at a lag of 0–7 and 0–3 days stratified by sex are presented in Fig. The sensitivity analysis results proved that the acute effects did not change substantially with the adjustment of smoothness of time using alternative df from 4 to 10 per year  may be more sensitive to ambient temperature. To our knowledge, this was one of the few studies to directly investigate the relationship between ambient temperature and AR outpatients among Asian population.2, NO2, and PM10 increased a higher AR risk in the warm season than that in the cold season [Many studies have found positive associations between hot temperatures and AR risk –37. Simid season . Howeverd season . In thisd season .2), may affect aeroallergens through plant distribution, pollen quantity, and season [Other studies have demonstrated that ambient temperature can alter vegetation patterns spatially and temporally, and is associated with allergic symptoms in several ways , 41–43. d season . Evidencd season . In addid season , 47. Neud season .Another noteworthy finding from our study is the significant relationship between AR outpatient visits and mild cold temperatures at a lag of 0–7 days, and also between AR visits and mild hot temperatures at a lag of 0–3 days. One study conducted in Guangzhou, China, also found that the effects of warmer temperatures appeared immediately, whereas the effects of colder temperatures were delayed but persisted longer . Warmer Stronger temperature effects on AR outpatient visits were observed in males than in females, at both mild hot and mild cold temperatures. One possible explanation is that males may have participated in more outdoor work resulting in greater exposure to higher temperatures. However, other studies have shown that females are more susceptible to temperature-related health hazards , 51. ClePrevious studies have observed that adverse effects of temperature on older adults and children are often higher than average . Our stuOur findings will contribute to understanding the influence of ambient temperature on AR, and to predict the impact of increased average temperatures due to climate change on the health of individuals with allergies. In addition, as global average temperatures and the incidence of allergies continue to increase, our results will provide a basis for future studies on the effects of increased temperature specifically on AR, and may also aid in the formulation of relevant health protection strategies.Nevertheless, there are some limitations in our study. First, ecological bias was inevitable. For instance, outdoor fixed-site monitoring data were used as surrogates for individual temperature exposure levels, which may have caused measurement errors since individuals stay indoors most of the time . AlthougIn summary, mild cold and mild hot temperatures both significantly increased the AR hospital outpatient visits in Xinxiang, China. This adverse effect was especially pronounced in males and in the younger population (< 15 years of age). As the global average temperature increases, individuals with AR or other allergies may develop heat-related health problems. Our study may have important public health implications for controlling and preventing adverse health effects associated with exposure to increased air temperature.Additional file 1: Figure S1. Relative risks of allergic rhinitis associated with daily mean temperature at 75th percentile relative to median temperature at lag02 day using different degrees of freedom per year.


To analyze the outcome of repair of aortic valve disease associated with various types of ventricular septal defect.In a retrospective observational study design, data of seventy-two patients of ventricular septal defect (VSD) associated with aortic valve prolapse (AVP) and aortic regurgitation (AR) who was operated in Punjab Institute of cardiology from May 2016 to April 2020 was collected. Depending on presence of AR, all patients were divided in four groups. Group-I (VSD and AVP but no AR) had fifteen patients. Only VSD was closed in this group. Group-II (VSD and Mild AR) had forty patients, only VSD was closed in this group as well. Group-III (VSD and Moderate AR) had ten patients, VSD closure and aortic valve repair was done. Group-IV (VSD and severe AR) had seven patients. Aortic valve was repaired in five patients and replaced (AVR) in two patients along with VSD closure. Associated anomalies were addressed as well.Group-I: Twelve out of fifteen patients (80%) showed no post-operative AR. While two patients (13.3%) showed Trace AR. Single patient (6.6%) showed mild AR. There results were unchanged after mean follow up of 36 months. Group-II: Eight out of forty patients (20%) had no AR, while eight (20%) had trace AR. Twenty-three (57.5%) patients had mild AR. Single (2.5%) patient had moderate AR. After follow up of 24 months the patient with moderate AR progressed to severe AR. We are planning to do Aortic Valve Replacement (AVR) in this case. Rest of cases showed no progression of disease. Group-III: Two out of ten patients (20%) had no AR, four (40%) had trace AR, while four (40%) had mild AR. Mean follow up was 42 months (2.5 years). Neither trace nor mild AR progressed to severe or moderate AR. Group-IV: Among seven patients, five underwent repair while two had AVR. Out of five patients who underwent aortic valve repair, four patients (57.1%) were declared mild AR, while severe AR was converted to moderate AR in single patient (14.28%). Mean follow up was 18 months. The moderate AR patient has progressed to severe AR for last six months and we are planning to do AVR in this patient. Postoperative echo of patients with AVR showed adequately functioning aortic valve with AVPG mean 10 mmHg and 15 mm Hg respectively, with no residual AR.Aortic regurgitation associated with VSD is a congenital lesion with continuously active aortic valve disease resulting in significant morbidity and mortality. Early diagnosis, effective treatment and meticulous follow up decelerate and in most cases arrest the disease process. Pakistan has one of the highest incidences of congenital heart diseases in the world, which is over 9.4/1000 live births.5In a pilot study, we studied the promising results of repair of aortic valve disease in DCSA VSD.DCSA VSD and outlet VSDs 20 and four respectively. VSD without AVP and AR, VSDs associated with tetralogy of Fallot (TOF), right ventricle out flow tract (RVOT) obstruction and other complex congenital cardiac diseases were excluded from study. Ethical and academic committee of PIC’s approval was taken . Proper consent was taken from all patients after explaining all complications. The patients were referred to us by paediatric cardiologists after diagnosing VSD through transthoracic echocardiography and grading the AR by color Doppler. A pressure half time (PHT) of greater than 500 millisecond (ms) was considered mild AR, 500 ms to 349 ms was moderate AR, 300 ms to 200 ms was moderately severe AR while less than 200 ms was considered severe AR. Patients were divided into four groups on the basis of AVP and degree of AR. Group-I comprised of fifteen patients having VSD along with AVP but no AR. All underwent VSD repair only. Group-II had forty patients of VSD with mild AR, all had VSD repair only as there was mild AR preoperatively, associated anomalies were repaired like closure of patent ductus arteriosus (PDA) in two cases, left ventricle outflow tract( LVOT) resection in one and ruptured sinus of valsalva (RSOV) in two patients. Ten patients were in Group-III having VSD with moderate AR. Associated pathologies were RSOV (1patient), aortic cusp perforation (one patient) and moderate tricuspid regurgitation (one patient). VSD closure and Trusler repair was done in seven cases while aortic cusp augmentation was done in two cases. Right coronary cusp (RCC) augmentation was carried out in one patient and non-coronary cusp (NCC) augmentation in one patient. One patient had repair of aortic cusp perforation. Group-IV had seven cases with VSD and severe AR. Cusp augmentation was done in two cases, new cusp formation with pericardium was done in three patients and AVR was done in two cases. Associated pathologies like RSOV repair and LVOT resection were also performed. Trans thoracic Echo was done before discharge followed by Echo at an interval of 3- and 6-months post procedure and then annually. Statistical analysis was done by SPSS version 23. Quantities variables were presented as mean value and the pre-operative and post-operative quantities variables were compared by ANOVA test. P value of ≤.05 was considered statistically significant; F-ratio was also calculated for comparisons.Considering a retrospective observational study design, data of 72 patients with VSD who were operated in Punjab Institute of Cardiology (PIC) from May 2016 – April 2020 was collected. Perimembranous VSD were 48 cases; All the patients were operated by standard median sternotomy followed by establishing routine cardiopulmonary bypass. AR was assessed by (1) Echocardiographic findings pre operatively (2) by observing arterial tracing  prior to bypass and (3)left ventricular distension on initiation of bypass and while giving cardioplegia. Antegrade cardioplegia was used in cases of mild AR and aortic valve prolapse, while coronary ostial cannula was used for cardioplegia in cases of moderate and severe AR after opening the aorta. Topical cooling with ice and moderate hypothermia were used for myocardial protection. VSD was closed with Dacron patch using pledgetted 5/0 prolene sutures. We commonly used right atrial approach for VSD closure while aortic valve repair was done through aortic approach. DCSA VSDs were closed through pulmonary approach. Trusler repair by suspension of RCC and hood plication of commissure with small pericardial patch was done. Cusp augmentation was carried out for shortened cusps by trimming the RCC and NCC in different cases and augmenting them with glutaraldehyde fixed pericardial patch. New cusp formation was done by excising the deformed cusp. Height of structurally normal cusp especially NCC was used as reference to produce the new cusp with pericardium. Structurally deformed aortic valve  had to be excised in two cases and replaced with mechanical valves of 19 mm St- Jude.Mean age of patients was 12.93±6.75 years. There were 18(25%) females and 54(75%) males. Mean weight was 29.04±12.85 kg. Small residual VSD was observed in one patient of Group-I and one patient of Group-II (2.7%), no progression was reported on follow up. Perimembranous VSDs were most common (66%), followed by DCSA (27%) and outlet VSD (5%). No muscular VSD was diagnosed with AR in our study. ICU stay complications i.e.; pneumothorax and chest infections were dealt satisfactorily. Twelve out of fifteen patients (80%) showed no post-operative AR, while two patients (13.3) had trace AR and one patient (6.7%) showed mild AR. The results were unchanged after mean follow up of 36 months.Out of forty patients, twenty-three (57.5%) had mild AR, eight patients (20%) had no AR and further eight (20%) had trace AR. One (2.5%) patient had moderate AR. Mean follow up of this group was two years. The patient with moderate AR showed progression to severe AR. VSD type in this case was perimembranous. We have planned for AVR in this case. Rest of cases showed no progression of disease.Four (40%) patients had mild AR, another four (40%) had trace AR and two out of ten patients (20%) had no AR. Mean follow up was 2.5 years in this group. Neither trace nor mild AR progressed to severe or moderate AR.Four patients (57.1%) revealed mild AR, two (28.5%) had no AR and one patient (14.3%) was found to have moderate AR. Mean follow up was 18 months in this group. Patient with moderate AR had augmentation of RCC cusp which progressed to severe AR during last 6 months follow up so we have planned for AVR in this patient. Again, the VSD type was perimembranous. Moreover, both the patients with AVR have shown adequately functioning aortic valve with AVPG mean 10 mm Hg and 15 mmHg respectively VSD is the most prevailing congenital cardiac lesionFurthermore, RCC was most commonly prolapsed (93%) and NCC was prolapsed in 6% of cases which is similar to findings reported in other local studies.Severe pulmonary hypertension is well known complication of untreated VSDProgression of AR was not observed even in a single patient with mild or trace AR as evidenced by results of Group I and II. Nonetheless moderate AR from Group-II (in one patient) and one patient from Group-IV did progress to severe AR. Likewise, Ishikawa et al. also showed that mild AR do not progress to moderate or severe AR, however moderate AR frequently progress to severe AR, so a careful surveillance is warranted in such cases.12Karpawich et al. and Okita et al. reported that repair of perimembranous VSD defect along with AR is artily, as it involves both RCC and NCC, also, progression of repaired AR associated with perimembranous defect to severe degree is more frequent.Surgical approaches for the aortic valve repair used in our study were Trusler repair, cusp augmentation, cusp perforation repair and new cusp formation with pericardium. Two patients underwent aortic valve replacement . Trusler repair popularized by Trusler and colleagueElgamal et al.Mean age of our study population was 12.93±6.75 years due to late diagnosis and inefficient referral pathway. Neither age nor gender was observed a risk factor for inadequate repair of AR and later progression of disease. Likewise, Trusler et al.20Comparing our results to local studies, Kumari et al.Once developed, AR associated with VSD is a progressive lesion with significant morbidity. However, this study showed that early treatment and effective follow up may regress and even halts the disease process. Delayed referral, lack of comprehensive follow up, and low socioeconomic strata are the reasons because of which it is imperative that government led effective guidance for early diagnosis, timely referral should be well in place to address the potentially avoidable morbidity associated with this condition.The data presented is solely based on single surgeon and single center-based experience so the expertise from other surgeons and experience of other centers is required for validation of results. A small cohort and relatively short follow up periods are also considerable feeble points.Considering the unfortunately very high prevalence of congenital cardiac defects in our country this article provides an evidence of surgical results of our current clinical practice stressing the fact that aortic regurgitation associated with ventricular septal defect is a progressive condition associated with significant morbidity and mortality. Early management of aortic valvular disease slows and, in most cases, halts the progression of disease. Efficient and well-coordinated follow up pathway are imperative for post-operative surveillance.TW: Conceived, designed the research methodology, prepared this manuscript and is accountable for the originality of the research work.MFAR & JAN: Did data analysis, helped in writing the manuscript and reviewed the manuscript.KK: Acquisition of data and helped in writing the manuscript.


As is well known that colorectal cancer is the third most common cancer in the world, and radiation treatment plays a vital role in colorectal cancer therapy, but radiation resistance is a significant problem in the treatment of colorectal cancer. As an important member of the non-coding RNA family, long non-coding RNAs (lncRNAs) have been found that it plays a role in the occurrence and progression of colorectal cancer in recent years. However, little is known about the effect of lncRNA on colorectal cancer sensitivity to radiotherapy. We found that lnc-TLCD2-1 was significantly differentially expressed in radiation-tolerant CCL244 cell lines and radiation-sensitive HCT116 cell lines, suggesting that lnc-TLCD2-1 may regulate the radiosensitivity of colorectal cancer, and the relevant underlying mechanism was investigated. Cell clone formation assay, flow cytometry, and cell counting kit 8 (CCK8) were used to detect radiation sensitivity, apoptosis, and proliferation of colorectal cancer cells, respectively; Quantitative real-time PCR and western blot were used to detect the expression of genes; the direct interaction between lnc-TLCD2-1 and hsa-miR-193a-5p was verified by dual luciferase reporter assays; GEPIA, Starbase, TIMER and DAVID were used to complete expression of lnc-TLCD2-1, miR-193a-5p,YY1 and NF-кB-P65 in colorectal cancer, correlation, immune cell infiltration, GO and KEGG enrichment analysis. Clinical prognostic analysis data were obtained from GSE17536 dataset. After radiotherapy for HCT116, the expression of lnc-TLCD2-1 was increased, and the expression of hsa-miR-193a-5p was significantly decreased, while that of CCL244 was the opposite, and the change range of lnc-TLCD2-1 was relatively small. HCT116 with overexpression of lnc-TLCD2-1 after radiation treatment, the number of cell colonies significantly increased, and cell apoptosis decreased compared with the negative control group. The cell colonies and apoptosis of CCL244 with disturbed expression of lnc-TLCD2-1 were opposite to those of HCT116. Lnc-TLCD2-1 can regulate the expression of YY1/NF-кB-P65 by targeting miR-193a-5p. Lnc-TLCD2-1 can promote the proliferation of colorectal cancer. High expression of lnc-TLCD2-1 independently predicted a shorter survival. Lnc-TLCD2-1 is associated with radiation resistance and short survival in colorectal cancer patients. In addition, Lnc-TLCD2-1 can promote the proliferation of colorectal cancer. Our study provides a scientific basis for targeting lnc-TLCD2-1 in colorectal cancer radiation resistance interventions and selection of prognostic biomarker. Globally, colorectal cancer (CRC) is one of the most common malignant cancers , it is sThere has been a lot of evidence to prove the interaction between lncRNAs and miRNAs, and this is often a negative regulatory relationship –10. Yin To explore the influence of lnc-TLCD2-1 on the sensitivity of CRC to radiotherapy and its related mechanism is of far-reaching significance for clinical treatment of CRC and the prognosis of patients.Tumors and paracancerous tissues from 10 patients with CRC were obtained from the Second Affiliated Hospital of Soochow University and stored in liquid nitrogen immediately until use. Each colorectal cancer patient was diagnosed histopathologically by two independent pathologists, and clinicopathological factors in colorectal cancer patients included gender, age, tumor size, TNM stage, lymph node metastasis, and distant metastasis. All CRC patients signed written informed consent, and this work was approved by the research ethics committee of the Second Affiliated Hospital of Soochow University.2 at 37°C.Human colon cancer cell lines (HCT116 and CCL244) were purchased from American Type Culture Collection . These cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  and 1% Penicillin-Streptomycin  under humidified atmosphere containing 5% COSmall interfering RNA (silnc-TLCD2-1) and overexpressed vector (veclnc-TLCD2-1) targeting lnc-TLCD2-1, miRNA mimic and inhibitor (miR-193a-5p mimic and inhibitor miR-193a-5p) targeting hsa-miR-193a-5p, and the corresponding negative controls  were all synthesized by Genepharma . We transfected the above oligonucleotides or vectors into CCL244 or HCT116 by lipofectamine 2000 reagent  in strict accordance with the instructions.Mix 1.2% agarose and 2×DMEM medium (containing 2% Penicillin-Streptomycin and 20% FBS) at 1:1, add 1.0 mL to each well of the six-well plate, and let it stand at room temperature until set. Mix 0.7% agarose and 2×DMEM medium (containing 2% Penicillin-Streptomycin and 20% FBS) at 1:1, the cells in logarithmic growth phase were digested with 0.25% trypsin and diluted to 10000/ml. The cell suspension was added to the low concentration agarose solution mentioned above to achieve a final cell concentration of 1000/ml. Next, add 1ml of the mixture to each well in a six-well dish. After the upper agarose solidified, culture at 5%CO2 at 37°C for 2 weeks(add 200ul DMEM (containing 1% Penicillin-Streptomycin and 10% FBS) every 2 days and keep it moist). Each well was stained with 0.005% crystal violet (1ml) for 1h, and the number of communities was calculated with Image-Pro Plus6.0 after photographing.® II 1st Strand cDNA Synthesis Kit  done. As for miRNA, cDNA synthesis was performed using the Takara RNA PCR Kit . Subsequently, quantitative real-time PCR was performed to detect the gene expression, using Hieff® qPCR SYBR Green Master Mix (Low Rox) . Relative expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH)or U6 calibration as a reference gene, then use “2 - Δ Δ CT” method to calculate. The primer sequences used are as follows : GAPDH 5ʹ- GAAGTATGACAACAGCCTCAAGA-3ʹ(forward),5ʹ-TCTTCTGGGTGGCAGTGATG-3ʹ (reverse);U6 5ʹ-AAAATTTCTCACGCCGGTATTC-3ʹ(forward),5ʹ-CCTGCAGACCGTTCGTCAA-3ʹ (reverse); lnc-TLCD2-1 5ʹ- ATTAGAGACACTGGCTGGATT-3ʹ(forward), 5ʹ- AGTAAGAGCAGAAGGATGACT-3ʹ (reverse);NF-кB-P65 5ʹ-TGGAGCAGGCTATCAGTCA-3ʹ(forward), 5ʹ-TCGGTTCACTCGGCAGAT-3ʹ (reverse); YY1 5ʹ-AAGAGCGGCAAGAAGAGTTA-3ʹ(forward), 5ʹ-GAATAATCAGGAGGTGAGTTCTC-3ʹ (reverse); hsa-miR-193a-5p 5ʹ-ACGCTGGGTCTTTGCGG -3ʹ(forward); 5ʹ-TATGGTTGTTCACGACTCCTTCAC -3ʹ (reverse).We used Trizol Reagent  to extract the total RNA of cells strictly according to protocal, and the synthesis of complementary DNA (cDNA) was made by using HifairThe proteins were extracted from the transfected cells using RIPA lysis buffers  and then quantified using bicinchoninic acid (BCA) following the manufacturer’s instructions. An equal amount of the extract was separated by electrophoresis with 10% sodium dodecylsulfate polyacrylamide gel and transferred to a nitocellulose membrane. After blocking with 3% non-fat milk for 1 hour, the membrane was incubated with primary antibodies against YY1, NF-кB-P65 and GAPDH and then interacted with secondary antibodies conjugated with horseradish peroxidase. Finally, the immune response signals were observed by enhanced chemiluminescence.2 atmosphere for 72 h. After 72 hours, add 10 microliters of CCK-8 reagent to each well, shake gently and mix well, continue to incubate at 37°C in 5%CO2 for 2 hours. The absorbance at 450 nm was measured with a microplate reader  to determine cell viability. Cell viability * (%) =[A(treatment)-A (empty culture)]/[A (Negative control)-A (empty culture)] ×100%.Proliferative activity of colon cancer cell lines was measured using CCK-8 in a 96-well plate according to standard protocols.HCT116 transfected with vector plasmid and CCL244 transfected with small interfering RNA were transplanted into 96-well plates (1000cells/well), and the cells were incubated at 37°C in 5%COAfter the cells were transfected and irradiated, apoptosis was detected using the Annexin V-FITC/PI Apoptosis Assay Kit  according to the standard protocol. The cells were digested with 0.25% trypsin, and the concentration of the cells to be tested was adjusted to about 5×10^5/ml. 1ml cells were taken, centrifuged at 1000rpm at 4°C for 5 min, and supernatant was discarded. Add 1ml of pre-cooled PBS and gently shake the cells to suspend. The Binding Buffer was diluted 1:3 with sterile deionized water (4 ml Binding Buffer +12 ml sterile deionized water). The cells were resuspend with 250 μl Binding Buffer and the concentration was adjusted to 1×106/ml. Take 100 μl cell suspension into 5 mL flow tube, add 5 μl Annexin V/FITC and 10 μl 20 μg/mL PI solution. Mix well and incubate at room temperature in dark for 15 minutes. Add 400 μl PBS to the reaction tube and analyze by flow cytometry. The blank group and treatment group were set up. The blank control group was negative control without dye treatment, and the treatment group was Annexin V-FITC and PI double staining. Annexin V-FITC and PI positive limits were set in the experimental group according to the negative control group.Partial wild-type (wt) or mutated (mut) DNA sequences of hsa-miR193a-5p binding sites in lnc-TLCD2-1 3’UTR were amplified by PCR. Then, it was cloned into pmirGLO vectors  to obtain lnc-TLCD2-1-wt and lnc-TLCD2-1-mut reporter plasmids. Subsequently, HCT116 and CCL244 were co-transfected with the constructed reporter plasmid and miR-193a-5p mimic or inhibitor, and negative control was set in each group. Cells were collected at 48 h transfection, Dual Luciferase Reporter Gene Assay Kit  was used to detect luciferase activity.http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html) is able to assess the biological relationship between gene expression and clinical prognosis using published cancer gene sequencing data sets (PrognoScan (ata sets . It can ata sets  was perfhttp://gepia.cancer-pku.cn/index.html) is developed by Zefang Tang, Chenwei Li and Boxi Kang of Zhang Lab, Peking University. It has a wide range of functions, including tumor/normal differential expression analysis, survival prognostic analysis, similar gene detection, correlation analysis and dimensionality reduction analysis based on cancer type or pathological stage  recently updated is in March 2017, it is an online bioinformatics resources, aiming at the function of a large number of genes or proteins, annotations, it is the full name of The Database for the Annotation, the Visualization and Integrated Discovery . We used David to carry out GO and KEGG enrichment analysis on 100 genes including YY1 and its similar genes. GO analysis included three aspects: molecular function, biological process and cellular component; KEGG(Kyoto Encyclopedia of Genes and Genomes) is an enrichment analysis of gene molecular signaling pathways.DAVID (iscovery . We usehttps://cistrome.shinyapps.io/timer/) database is Tumor Immune Estimation Resource, it can be used to query tumor immune, clinical and genomic characteristics . One-way ANOVA, two-way ANOVA and Student’s T-test were used to compare the differences between groups. The correlation between lnc-TLCD2-1 and miR-193a-5p was analyzed using Pearson Pearson’s chi-square test. P < 0.05 indicated that the difference was statistically significant.We already reported before in HT29 and LOVO, CACO2, CCL244, HCT116, SW480, HCE8693 these seven kinds of colorectal cancer cell lines, CCL244 for the most resistance to radiation, and HCT116 is the most sensitive to radiation . To idenWe used GEPIA to search for differences in the expression of lnc-TLCD2-1 between CRC and normal colorectal tissue. Compared with normal colorectal tissues, the expression of lnc-TLCD2-1 was decreased in CRC tissues >=0.5 as the selection criteria, GO and KEGG enrichment analysis was performed on the 190 similar genes with co-expression relationship with lnc-TLCD2-1, detailed results of enrichment analysis were given in Colorectal cancer (CRC) is the third most common cancer in the world and accounts for a significant proportion of cancer-related deaths. At the same time, radiation resistance has always been a difficult problem in CRC treatment, especially advanced CRC, and radiation resistance profoundly affects the clinical prognosis of patients with CRC. Therefore, it is of great significance to study the mechanism of radiation resistance in CRC to improve the prognosis of patients. Recent studies have shown that IDO1 inhibition can enhance the radiotherapy effect of CRC , miR-145To our knowledge, lnc-TLCD2-1 is a recently discovered lncRNA, very little is known about lnc-TLCD2-1, especially in CRC. Our study found significant differences in lnc-TLCD2-1 expression levels between radiation-resistant CCL244 and radiation-sensitive HCT116. After CCL244 was irradiated, the expression of lnc-TLCD2-1 was significantly decreased, while that of HCT116 was significantly increased. It is suggested that lnc-TLCD2-1 may have an important correlation with the radiation sensitivity of CRC. Considering that the basic expression of lnc-TLCD2-1 in CCL244 is much higher than that in HCT116, we then reduced its expression in CCL244 and upregulated it in HCT116, and conducted a comparative experiment. Subsequently, we conducted cell coloning experiments and flow cytometry apoptosis experiments, and it was found that after the overexpression of lnc-TLCD2-1, cell apoptosis was reduced; on the contrary, after the decrease of lnc-TLCD2-1, cell apoptosis was increased. This indicates that lnc-TLCD2-1 can cause radiation resistance of CRC. It seems paradoxical that the level of lnc-TLCD2-1 in radiation-resistant CCL244 is decreased after radiation exposure. We believe that after receiving radiation, there are other regulatory pathways in CRC cells that have an impact on the expression of lnc-TLCD2-1. Our study was conducted to identify the lnc-TLCD2-1 effect on the CRC radiosensitivity, as for the regulation of other pathways is the target of our further research in the future.At the same time, bioinformatics analysis of lnc-TLCD2-1 was performed to explore the internal mechanism of lnc-TLCD2-1 leading to radiation resistance in CRC. The expression level of lnc-TLCD2-1 in CRC tissues was significantly lower than that in normal tissues. In addition, high expression of lnc-TLCD2-1 predicted shorter Disease Specific Survival and Overall Survival in patients. This might confirm that CRC radiation resistance closer correlations with lnc-TLCD2-1, because patients with radiation resistance will have a worse prognosis and shorter survival.via sponging miRNA-665 to upregulate POU2F1 , Jiangsu Provincial Health Commission Scientific Research Project (Grant No. QNRC2016873 and No. CXTD-16), the Subject of Suzhou Science and Technology Bureau (Grant No. SYS2018055), and the Gusu Health Talents Project (Grant No. GSWS2020037).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Castration resistance is a lethal form of treatment failure of prostate cancer (PCa) and is associated with ligand-independent activation of the androgen receptor (AR). It is only partially understood how the AR mediates survival and castration-resistant growth of PCa upon androgen deprivation. We investigated integrative genomics using a patient-derived xenograft model recapitulating acquired, AR-dependent castration-resistant PCa (CRPC). Sequencing of chromatin immunoprecipitation using an anti-AR antibody (AR-ChIP seq) revealed distinct profiles of AR binding site (ARBS) in androgen-dependent and castration-resistant xenograft tumors compared with those previously reported based on human PCa cells or tumor tissues. An integrative genetic analysis identified several AR-target genes associated with CRPC progression including OPRK1, which harbors ARBS and was upregulated upon androgen deprivation. Loss of function of OPRK1 retarded the acquisition of castration resistance and inhibited castration-resistant growth of PCa both in vitro and in vivo. Immunohistochemical analysis showed that expression of OPRK1, a G protein-coupled receptor, was upregulated in human prostate cancer tissues after preoperative androgen derivation or CRPC progression. These data suggest that OPRK1 is involved in post-castration survival and cellular adaptation process toward castration-resistant progression of PCa, accelerating the clinical implementation of ORPK1-targeting therapy in the management of this lethal disease. Through comparative genomics using PDX models of androgen-dependent (AD) and androgen-resistant (AR) tumors, Makino et al. identify opioid receptor kappa 1 (OPRK1) as being associated with castration-resistance. Loss of OPRK1 function delays castration-resistance and inhibits castration-resistant growth of prostate cancer cells in culture and in vivo, suggesting OPRK1 as a therapeutic target. Although the majority of PCas are treated with an excellent survival outcome, a subset of patients develop an advanced form of the disease which is ultimately lethal. Since the most of treatment-naïve PCas are dependent on androgen receptor (AR) signaling, the standard treatment for advanced PCa is androgen deprivation and AR-targeting therapy3.Prostate cancer (PCa) is the most frequently diagnosed cancer in men4. However, only a subset of cells undergoes apoptosis or other cell death processes whereas the rest of PCa cells undergo adaptive survival processes5. Biological mechanisms for anti-castration adaptive survival signaling have not been fully understood, although several previous studies reported implication of AKT6 and clusterin7 in this process, being characterized by upregulation upon androgen blockade.Clinically, the initial response rate of PCa to androgen deprivation and targeted AR inhibition therapy is very high. Upon androgen deprivation, AR activity is attenuated and PCa cells go into G1 arrest12. Using chromatin immunoprecipitation with anti-AR antibody (AR-ChIP), Wang and colleagues showed that AR regulated a distinct transcriptional program between androgen-independent and -dependent human PCa cells13. Since then, AR-ChIP using AR-expressing PCa cells has been demonstrated as a useful tool for the understanding of AR-mediated signals in PCa14. In another report by Sharma et al.15, AR-ChIP using human PCa tissues revealed that there were substantial discrepancies in the profiles of AR-binding sites (ARBS) between cell lines and tumor tissues. This indicates that to better recapitulate the processes leading to castration resistance, AR-ChIP for ARBS profiling should be performed using in vivo tumor tissues. However, it is very difficult to obtain sufficient amount of prostate cancer tissue from surgical specimens. Additionally, it is almost impossible to obtain pairwise fresh samples of androgen-dependent (AD) and CR tumors from the same patient in clinical practice.PCa cells that survive under a castrated condition eventually acquire the ability of castration-resistant growth. Amongst various mechanisms previously reported for the development of castration-resistant PCa (CRPC), aberrant activation of AR has been considered as the most important driving force toward castration-resistant (CR) progressionHerein, to overcome the above-mentioned barriers in obtaining comparative genomics data (including gene expression and ARBS profiling) between AD and CR tumors, we used a patient-derived xenograft (PDX) of PCa that recapitulated acquired castration resistance in an AR-dependent manner. Whereas fresh AD and CR tumors from the same patient present a major obstacle, PDX models can circumvent this issue. This work provides useful information on comparative genomics data between AD and CR tumors from the same origin. Critically, the analyses herein have identified OPRK1 as a potential novel therapeutic target; notably, OPRK1 expression was upregulated upon castration, and its loss of function retarded acquisition of CR progression by blocking cell proliferation and tumor growth in multiple CRPC models.16. KUCaP2 tumors grow in intact male mice, stop growing or shrinking upon castration, and start re-growth after ~6 weeks. We confirmed the monotonic growth in non-castrated mice  of 22RV1 cells by approximately two fold18. We observed copy number increases up to 1.5 fold in some of KUCaP2 AD tumors while KUCaP2 CR tumors harbored 2–3-fold increases  and castration-resistant (CR) tumors expressed signals detected by two distinct antibodies against N-terminus (N20) and C-terminus (C19) of AR at the level of 110 kD, which is equivalent to the molecular size of full-length AR  using mass spectrometry. It was revealed that both testosterone  LNCaP1 to 4. AILNCaPs expressed 2–3-fold higher 11. However, a previous report demonstrated that activated AR regulated a distinct transcriptional program from that in the presence of androgen13. Another previous report showed that there was a discrepancy between regulatory programs in cultured AR-driven prostate cancer cells and in vivo human prostate cancer tissues22. To address this issue, we investigated AR-binding sites (ARBS) in KUCaP2 AD and KUCaP2 CR tumors as well as LNCaP and AILNCaP cells using chromatin immunoprecipitation with anti-AR antibody (AR-ChIP).It has been reported that the AR is activated and acts as the driver in the majority of CRPCn = 3 each) were subject to ChIP using anti-AR (N-terminus) antibody, control normal IgG, and anti-histone H3 antibody and abundance of KLK3 enhancer region in immunoprecipitated DNA samples were determined with quantitative PCR. It showed that ChIP using anti-AR antibody enriched KLK3 enhancer region by ~15–80-fold compared with that using control IgG  that were growing in intact and castrated mice, respectively , NKX3.1, TMPRSS2, CAMKK2, FKBP5 (3' end region), FKBP5, HMGCR, SCAP, SGK1, and SLC45A as well as KLK3 (enhancer region)  Fig. , suggestUsing this threshold, we analyzed ARBS detected in KUCaP2 AD and CR tumors. We defined ARBS of KUCaP2 tumors in each status as being called in at least two of the three samples. Then, each ARBS was annotated to a gene located within a 20-Kb range. A total of 11,083 genes were called; 3131 were exclusively for KUCaP2 AD, 1850 for KUCaP2 CR, and 6102 were shared with both AD and CR tumors Fig. . For LNC23. On the other hand, genes harboring ARBS exclusively called in KUCaP2 AD tumors were related to transcription, translation elongation, and cell cycle  for AD and 53.2% (730 of 1371) for CR tumors were shared with PDX tumors, whereas only 14.9% (59 of 396) for AD cells and 13.1% (52 of 396) among ARBS identified in human PCa cells were shared with PDX tumors, highlighting the divergence between AR-targeting genes between in vivo tumors and cultured cells22.We further interrogated ARBS detected in KUCaP2 tumors in comparison with those reported by previous studies on AR ChIP Fig. 13,22. AmWe next investigated potential therapeutic targets in CRPC using KUCaP2, a model that well recapitulated the typical clinical behavior of PCa including androgen-dependent growth, response to castration and castration-resistant progression in vivo. Our AR-ChIP seq strongly indicated that KUCaP2 significantly altered the profile of genes that were transcriptionally regulated by AR in the process of progressing from AD to CR status. However, it did not seem to fully reflect expression of the screened genes in tumors. Therefore, we combined RNA seq analysis using KUCaP2 AD and CR tumors with the results from AR-ChIP seq to narrow candidate genes for a therapeutic target in CRPC. Furthermore, we searched publicly available databases as we considered it important to identify targets that are not specific to KUCaP2 but are more generalized for the treatment of CRPC.24, metastatic PCa25, or metastatic CRPC26. Among the top 30 differentially expressed genes in RNA seq, we identified 12 frequently altered genes according to published NGS data, seven of which were upregulated in CR  while five of which were downregulated in CR  , while the remaining seven genes  showed reproducible differential expressions  Fig. . Of thesaP2 Fig.  and DPP4TRPA1  using RT-PCR. Amongst six that were upregulated in KUCaP2 CR tumors, PA1 Fig.  and CP  and supplementation with 1 nM DHT (LNCaP DHT) counteracted it  cells . We found no significant difference in body weight at least up to 10 weeks after the treatment initiation . Tumors on mice treated with nor-BMI showed significant growth retardation compared with those on vehicle-treated mice . Tumors on vehicle-treated mice acquired the ability to grow under castrated condition in about 4 weeks, whereas those on nor-BNI-treated mice took about 8 weeks . We observed similar significant growth retardation in tumors on nor-BNI-treated mice compared with those on vehicle-treated mice  or siRNA for AR alone (single siRNA) (n = 4 each), and then expression profile of the respective cell line treated with combo-siRNA was compared with that of cells treated with single siRNA. Based on gene expression clustering, cells treated with combo-siRNA and single siRNA were well discriminated  and in AIVCaP (FDR < 0.001) treated with combo-siRNA or single siRNA were screened. As anticipated, there were several gene sets related to G protein-coupled receptor signaling and neuronal signaling, suggesting that OPRK1 expression was effectively altered by siRNA silencing. Among commonly altered pathways, we identified a gene set related to the SMAD6 signaling pathway ion Fig. , suggestP for trend < 0.0001, Fig. P for trend = 0.0008, Fig. P for trend > 0.05, Fig. OPRK1 amplification status using c-BioPortal and found that patients with OPRK1 amplification had significantly shorter survival compared with those without OPRK1 amplification  on human benign prostate and PCa tissues. OPRK1 antibody for IHC was validated by differential stainability between KUCaP2 AD and KUCaP2 CR tissues  that is widely expressed throughout the nervous system and is physiologically activated by endogenous opioid peptide agonists derived from prodynorphin27 and with multiple databases for advanced PCa showing upregulation of OPRK1 in advanced PCa, particularly in those at high risk for death26.Further functional analyses revealed that upregulation of OPRK1 facilitates the survival and progression of PCa under very low androgen conditions. In this regard, it is considered that OPRK1 is induced in response to castration and appears to play a purposeful role as a mechanism of adaptation to the attenuated androgen environment. Indeed, we have demonstrated that pharmacological inhibition of OPRK1 prolonged time to acquisition of castration resistance and retarded castration-resistant tumor growth in multiple in vivo preclinical models of CRPC, suggesting clinical efficacy of OPRK1-targeting treatment for prolonging time to castration-resistant progression of advanced PCa. Furthermore, our RNA seq and IHC analyses showed an increasing trend of OPRK1 expression as PCa progressed from castration-naïve to castration-resistant diseases, which was consistent with the findings from cultured PCa cells35 and in enhancing the aggressiveness of breast cancer36 as a downstream effector of the transforming growth factor-β (TGF-β) signaling pathway. As for downstream effectors of the SMAD6 signaling pathways that were shown to be positively regulated by OPRK1, IL8 has been implicated in castration-resistant progression of PCa37 as well as LAMC2 reported to be involved in progression, migration, and invasion of multiple types of cancer38. It has been reported that IL8 was also negatively regulated by AR binding to the IL8 promoter39, which is consistent with our observation of IL8 upregulation in androgen-independent PCa cells. The present study may provide another mechanism mediated by OPRK1 to upregulate IL8 expression in PCa cells. In line with the reported functions of potential downstream pathways, OPRK1 knockdown decreased both proliferation and migration of CRPC cells including AIVCaP and AILNCaP. We have not elucidated the underlying link between OPRK1 and SMAD6 pathway, which seems to be subject to future studies. Nonetheless, these results collectively strengthen the potential of OPRK1 as a therapeutic target for the prevention of castration-resistant progression and treatment of CRPC.As a consequence of applying ssGSEA and samroc to gene expression profile in castration-sensitive and -resistant VCaP cells with AR ± OPRK1 silencing, it was suggested that OPRK1 upregulated the SMAD6 signaling pathway under suppression of AR activity. It has been reported that SMAD6 plays critical roles in supporting lung cancer cell growth and survival41. It is well known that time to CRPC progression is correlated with OS42 and progression to CRPC is associated with a deterioration in quality of life43. Our findings indicate that OPRK1 inhibition is expected to prolong time to CRPC progression of hormone-sensitive PCa when used in addition to a potent androgen blockade. OPRK1 inhibition can also be expected to suppress tumor growth after CRPC progression.In terms of clinical relevance, the importance of delaying castration-resistant progression in the management of advanced, castration-sensitive PCa has been emphasized in recent years45. The main models used in the present study represented AR-dependent CRPC (ARPC) and OPRK1 blockade did not seem effective for DNPC as evident by inhibitory effect on the proliferation of PC3 and DU145, which are recognized as models of DNPC. Therefore, in the future clinical use of OPRK-targeted therapy, it will be important to distinguish ARPC, DNPC, and NEPC. In this regard, a previous report by Bluemn et al.44 indicated that DNPC could not be distinguished by unique genomic aberrations but could be characterized by FGF and MAPK pathway activation. Additionally, Su et al.45 reported that DNPC could be characterized by CCL2 expression driven by Polycomb Repressor Complex 1 (PRC1) and recruiting M2-like tumor-associated macrophages and regulatory T cells. These unique characteristics will be useful for distinguishing DNPC from ARPC.Recently, as a result of efforts that have been made in clinical and research fields to subtype metastatic CRPCs, a new subtype concept called “double-negative PC (DNPC)”, characterized by the lack of AR pathway activation or neuroendocrine (NE) traits, has emergedThere are, of course, several limitations in the present study. AR-ChIP was not analyzed in combination with mapping of histone methylation or acetylation markers including H3K9me3, H3K27me3, H3K9ac, and H3K27ac. The number of ARBS defined in this study was slightly higher than previous reports due to the lack of narrowing by such markers. We believe that this could be complemented by other integrated omics analyses such as RNA seq. Expression regulation, associated transcriptional programs, and functional relationships between full-length and treatment-induced variant of AR were not dissociated. Since western blotting using anti-OPRK1 antibody could not be optimized by any means, the quantification of OPRK1 protein by gel analysis or proteomics was not available for the present study, and instead, a complimentary approach by extensive qRT-PCR was executed. Anti-tumor effects of nor-BNI could not be confirmed using KUCaP2 since the PDX line was exterminated after repeat passages. We believe that this could be complemented by extensive experiments using multiple other in vivo preclinical models, which rather proved the generalizability of the anti-tumor effect of nor-BNI as a therapeutic agent for CRPC. Genetic knockdown of OPRK1 was not successfully performed in vivo, which we wish to add to pharmacological blockade. Thus, despite several limitations the conclusions of this study have a potential impact on the development of novel treatment strategies for advanced PCa, proposing a new concept of OPRK1 inhibition to block the adaptation pathway for PCa toward CRPC progression. Given those small molecule inhibitors of OPRK1 such as buprenorphine have already been approved by regulatory agencies, novel pharmaceutical advancements or drug repurposing efforts for PCa can be placed on the short-term horizon.OPRK1 inhibitor nor-Binaltorphimine (nor-BNI) was purchased from Abcam . Primers used for PCR in the study are described in Supplementary Data 2. AILNCaP1 to 4, androgen-independent sublines of LNCaP were established as described elsewhere15 and they were cultured in phenol-red-free RPMI 1640 supplemented with 10% charcoal/dextran-stripped FBS (CSFBS). AIVCaPs were established from the parental VCaP cultured in phenol-red-free DMEM medium with 8% CSFBS plus 2% FBS as reported previously27.Human prostate cancer cell lines LNCaP, DU145, PC3, 22RV1, and VCaP were purchased from American Type Culture Collection . LNCaP, DU145, PC3, and 22RV1 were cultured in RPMI 1640, while VCaP was cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), 12.5 mM HEPES, penicillin, and streptomycin at 37 °C in humidified air containing 5% COAll experiments involving laboratory animals were conducted in accordance with policies of the Guideline for Animal Experiments of Kyoto University and Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions by the Ministry of Education, Culture, Sports, Science and Technology of Japan. The experimental protocols were approved by the Animal Research Committee at Kyoto University Graduate School of Medicine .16. The tumor was passaged to BALB/cAJcl-nu/nu nude male mice  before the tumor size exceeded 2 cm in diameter. For cell-based xenografts (CDX), VCaP 2.0 × 106 cells in 150 μL of DMEM medium and Matrigel®  (1:1) were subcutaneously injected to the right flank of BALB/cAJcl-nu/nu nude male mice (6–7 weeks old). AILNCaP xenograft model was prepared in a similar way with 3.0 × 106 cells in RPMI medium and Matrigel® (1:1) to the mice under castration at the same time.BALB/cAJcl-nu/nu mice were purchased from CLEA Japan, Inc. KUCaP2 was previously established from a locally recurrent tumor (CRPC) after radical prostatectomyFor surgical castration, mice were anesthetized with inhaled isoflurane and both testes were gently mobilized to the scrotum, and 7 mm skin incision was made along the midline of the scrotum. The testes were pulled out through the incision and dissected away. The spermatic cord was ligated and the skin incision was closed with 4–0 absorbable suture. Mice in the sham-operation control group were similarly anesthetized, incisions were made, testes were pulled out and put back in the scrotum followed by wound closure.Blood sampling of xenograft mice was taken from the heart under anesthesia and the serum was gathered by centrifugation. PSA in the serum was estimated by chemiluminescent immunoassay (CLIA) method using Architect® PSA  at FALCO biosystems, Ltd . Intratumoral androgens were quantified by liquid chromatography tandem mass spectrometry .For RNA interference in vivo, the siRNA was delivered using the AteloGene® Local Use in vivo siRNA Transfection Kit , and administrated around and intra-tumors following the supplier’s protocol under anesthesia every 6 days for a total of four times.V = L × W2 × 0.5 (mm3), where L is the largest and W is the orthogonal diameter of the tumor.Treatment with nor-BNI in vivo was conducted by intraperitoneal administration (i.p.) to the mice, at a dose of 10 mg/kg for three times a week. Nor-BNI was dissolved in normal saline at a concentration of 1 μg/μL and injected. The control group was treated with vehicle . Tumor growth was monitored once or twice a week by caliper measurements. The tumor volume was calculated according to the formula: For in vitro siRNA silencing, the cells were transfected with specific siRNA oligos (Supplementary Data KLK3 enhancer region (as a positive control locus) and GAPDH promoter lesion (as a negative control locus) before sequencing. Library construction  and high-throughput sequencing using an Illumina Hiseq  and mapping of the resulting reads by Bowtie were performed by Hokkaido System Science Co., Ltd. Japan.AR-ChIP was done using SimpleChIP® Plus Enzymatic Chromatin IP Kit  according to manufacturer’s instruction. For ChIP using tissue samples we preferably used fresh tissues, not frozen. The antibodies used in the experiments are listed in Supplementary Data q-value analysis based on KLK3 controls yielded a threshold (q = 0.129) which defined genes as ARBS. This threshold was applied genome-wide to each sample, identifying target genes based on either in-gene AR-ChIP peaks or peaks in gene promoter regions, defined as being within 20 kb upstream of a gene. The BEDtools package46 was employed to handle the genome arithmetic logic. KUCaP2 AD and CR genes identified through this process were filtered to those present in multiple samples, and then compared to LNCaP-ablated (abl) basal upregulated genes reported in Wang et al.13, and the CRPC tissue upregulated genes reported in Sharma et al.22.Mapped reads were analyzed for peak calls via the MACS software (version 2.0.10.20131216). n = 3 per cell line). Three hundred sixty-two genes were obtained by applying the filter criteria of a one-way ANOVA having a maximum p-value of 0.001, an average expression fold change of >2.5 in abl lines, and no >10% overlap in the 95% expression level confidence intervals estimated. For the comparison with the genes reported in Sharma et al.22, a direct comparison was performed using the gene list provided in the article’s supplementary data.The abl expression data (GSE11428) was analyzed using the microarray probe value matrix format provided by NCBI GEO, where genes were assigned the maximum expression value of constituent probes, and abl-vehicle expression was compared to AR siRNA-treated cell lines  up by filtering out those with a poor probe quality flag of A  and M (high background probe), which resulted in 20,969 flag P  probes retained. Then we downloaded all gene sets version 6.2  from the Molecular Signatures Database . Using these data and gene sets, we performed ssGSEA to generate scores for predefined signature gene sets, as described . For data analyses, the ssGSEA scores were normalized from 0 to 1. Samroc29 was used for detecting significant differential pathways between combo-siRNA and single siRNA. FDR q-values were calculated from Samroc p-values using the R library “p-adjust”.ssGSEA was performed as reported elsewhere49. After transcript coordinate was converted to genomic positions, an optimal mapping result was selected either from transcript or genome mapping by comparing the minimal edit distance to the reference. Local realignment was performed within in-house short reads aligner with a smaller k-mer size (k = 11). Finally, fragments per kilobase of exon per million fragments mapped (fpkm) values were calculated for each UCSC gene while considering strand-specific information.Total RNA was extracted using an RNeasy Mini kit . RNA purity and integrity were evaluated by ND-1000 Spectrophotometer , Agilent 2100 Bioanalyzer . RNA-sequencing (RNA seq) libraries were prepared by TruSeq Rapid PE Cluster Kit and TruSeq Rapid SBS kit . The libraries were sequenced on Illumina HiSeq 2500 using a read length of 2 × 150 bp. RNA seq reads were demultiplexed using CASAVA v1.8.2 and aligned to human transcriptome (UCSC gene) and genome (GRCh37/hg19) references respectively using Burrows–Wheeler AlignerFor cRNA microarray, RNA labeling and hybridization were performed by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol . Briefly, 100 ng of total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The labeled cRNAs were purified by RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 . Then 600 ng of each labeled cRNA was fragmented by adding 5 μl 10x blocking agent and 1 μl of 25x fragmentation buffer and then heated at 60 °C for 30 min. Finally, 25 μl 2x GE hybridization buffer was added to dilute the labeled cRNA. 40 μl of hybridization solution was dispensed into the gasket slide and assembled to the Agilent SurePrint G3 Human GE 8X60K, V3 Microarrays (Agilent®). The slides were incubated for 17 h at 65 °C in an Agilent hybridization oven. then washed at room temperature by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol . The hybridized array was immediately scanned with an Agilent Microarray Scanner D . Microarray results were extracted using Agilent Feature Extraction software v11.0 (Agilent Technologies).4 using primers shown in Supplementary Data For quantitative RT-PCR, RNA was reverse-transcribed into complementary DNA using a ReverTra Ace qPCR RT Kit  according to instructions from each manufacturer. Genome DNA was extracted using QIAamp DNA Mini Kit  and subject to genome-based PCR. Quantitative PCR was performed as reported elsewhere4. For western blot analysis, aliquots of proteins were separated by SDS–PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories). Immunodetection was carried out with the indicated antibodies  and two authors (Y. M. and T.K.) independently scored each staining intensity and discrepant cases were resolved by discussion with all observers.Paraffin sections were cut (5-μm thick), dewaxed in xylene, brought to water down an ethanol gradient, and then subject to hematoxylin and eosin (H&E) or immunohistochemical (IHC) stains. IHC assays for AR and PSA were performed as described previouslyt-test was used for the comparison of numerical data while ANOVA was used for the comparison of repeated numerical data. Chi-square of Fisher’s exact tests were used for contingency analyses. Kruskal–Wallis test was also used for non-parametric comparison between three groups. All statistical analyses were performed using commercially available software . All tests were two-sided and P-values < 0.05 were considered statistically significant.All experiments are triplicate and the data are presented as mean ± S.E.M. unless otherwise indicated. Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data S1Supplementary Data S2Supplementary Data S3Supplementary Data S4Supplementary Data S5Supplementary Data S6Supplementary Data S7Reporting Summary


Children with physical disabilities often have limited performance in daily activities, hindering their physical development, social development and mental health. Therefore, rehabilitation is essential to mitigate the adverse effects of the different causes of physical disabilities and improve independence and quality of life. In the last decade, robotic rehabilitation has shown the potential to augment traditional physical rehabilitation. However, to date, most robotic rehabilitation devices are designed for adult patients who differ in their needs compared to paediatric patients, limiting the devices’ potential because the paediatric patients’ needs are not adequately considered. With this in mind, the current work reviews the existing literature on robotic rehabilitation for children with physical disabilities, intending to summarise how the rehabilitation robots could fulfil children’s needs and inspire researchers to develop new devices. A literature search was conducted utilising the Web of Science, PubMed and Scopus databases. Based on the inclusion–exclusion criteria, 206 publications were included, and 58 robotic devices used by children with a physical disability were identified. Different design factors and the treated conditions using robotic technology were compared. Through the analyses, it was identified that weight, safety, operability and motivation were crucial factors to the successful design of devices for children. The majority of the current devices were used for lower limb rehabilitation. Neurological disorders, in particular cerebral palsy, were the most common conditions for which devices were designed. By far, the most common actuator was the electric motor. Usually, the devices present more than one training strategy being the assistive strategy the most used. The admittance/impedance method is the most popular to interface the robot with the children. Currently, there is a trend on developing exoskeletons, as they can assist children with daily life activities outside of the rehabilitation setting, propitiating a wider adoption of the technology. With this shift in focus, it appears likely that new technologies to actuate the system  and to detect the intention  of children as they go about their daily activities will be required. Mobility and exploration are essential in children’s development and contribute towards cognitive, physical, social and emotional development. However, children with physical disabilities present limitations when performing activities autonomously, which hinders their typical development . OngoingRehabilitation is essential to help the children recover or maintain functionality when interacting with their environment, improving the quality of life and autonomy , 10. FurThe standard therapies to manage the musculoskeletal system’s deterioration and improve and maintain physical ability include passive orthoses, surgery, and physiotherapy , 16. PhyThere is a growing interest in robots that can support the patient, the family and the medical professional in a wide range of activities used for the care of people with physical disabilities, for example, companion robots , 21, monHealthcare robots can be divided into three main categories, clinical robots, assistive robots and rehabilitation robots , 25. CliRehabilitation therapy for the recovery of mobility based on robots has been proposed as a new procedure for children with physical disabilities . This roRehabilitation robots are often classified by their mechanical structure and are generally divided into end-effectors and exoskeletons , 39. EndContrarily, robotic exoskeletons could be termed “wearable machines” that mirror the patient’s skeletal structure; therefore, they only move the joint of the limb where the exoskeleton is worn. This approach allows for independent and concurrent control of specific segments of the limb. However, it is essential to adjust the length of sections of the robot to the lengths of the segments of the patient limb. Moreover, when the joint is in motion, the position of the centre of rotation can change, creating discomfort in the user. Thus, increasing the number of degrees of freedom of the robot increases the control algorithm’s complexity, weight, mechanical complexity, and power requirements, making it unattainable for home use , 41.Apart from mechanical structure, robots possess essential elements to ensure the systems’ reliability and robustness . ActuatoAlthough multiple devices for the robotic rehabilitation of upper and lower limbs have been developed, at least in a proof-of-concept phase , 50, 51,However, to develop technology planned to be used on the paediatric population is not only a matter of reducing the size of the robots. But it should be tailored to their own capabilities and goals that differ from those of the adults. For instance, a simple downscaling of the robots is not enough as the normalised joint torques on adults are greater than those of a child , 55, makConsequently, to address the children’s needs adequately, it is essential to include them and other stakeholders  during the development process, providing feedback to identify possible issues of importance , 43. FurDespite the progressive development of robotic rehabilitation devices, their application to the paediatric population is still scarce. Consequently, the key features to design an optimal robotic rehabilitation device that better enhance children’s abilities with physical disabilities have not been well defined yet. Based on this framework, this review aims to address the following questions: (1) What are the design requirements for paediatric rehabilitation robots? (2) How does the current technology contribute to achieving the paediatric design requirements? And (3) How do the paediatric conditions impact the device design?An in-depth literature search was performed to conduct the review, following the search strategy of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines .A literature search was conducted to identify literature associated with the topic based on searches in PubMed, Scopus, and Web of Science, using the combination of the following keywords: (pediatric OR kid* OR child*) AND (aid OR assist* OR improve* OR augment* OR enhance* OR reinforce* OR therap* OR rehabilitation) AND (active ortho* OR exoskeleton* OR wearable robot* OR portable robot* OR robot* suit OR robot*) AND . To make our search as complete as possible, a search through the university library databases was also conducted.Studies involving robotic devices for robot-assisted rehabilitation therapy,Studies involving robotic devices for assessment of patients with Physically disabilities,Studies involving devices designed for children or utilised with a paediatric population (< 18 years old),written in English,full-text articles.After the preliminary search, the following inclusion and exclusion criteria to narrow the literature search were used. The inclusion criteria were:Studies that only present software solutions or simulations,Studies involving passive devices (do not have actuators),Studies involving postural change,Studies involving only the adult population andStudies involving robots that do not replace the movement itself (e.g. wheelchairs).And the exclusion criteria were:An overview of the literature search,the paediatric robotic rehabilitation design requirements,an analysis of the type of robots used in paediatric robotic rehabilitation;the actuators to drive the robots;training strategy of the robots;the human–computer interface of the assistive systems, andthe treated conditions in children with physical disabilities.The outcome of this literature review is compiled in the following sections:811 publications from Web of Science,547 publications from PubMed,241 from Scopus, and5 from a search on the University library.Based on the keywords mentioned in the methods section, 1604 publications were found, with:First, a check was made for duplicated publications. After this process, the abstracts of 1248 publications were screened, and 301 titles were selected for full-text reading. After carefully applying the inclusion–exclusion criteria to the full read papers, 206 publications were selected. Among the chosen publications, 10 were reviews, 42 only discussed a section of the design process of the rehabilitation robot, 138 presented a clinical application, and 16 included the design process plus a clinical application  or SCI and only investigated their use as part of physical therapy. In Fasoli et al. , Meyer eFifteen different requirements were identified Table . The reqIn paediatric rehabilitation devices, operability is critical as children are in a continuous development phase during which their bodies, cognitive capabilities and physical abilities (e.g. skill levels) are changing, making them a “heterogeneous population” , 70. ConAn important consideration is that the robot’s weight could obstruct the movement pattern of the limb and increase the child’s energy consumption , 73. FurConcerning safety, children often cannot adequately assess the hazards of using complex technological devices . TherefoFinally, motivation is crucial because function recovery is not enough to engage children in the rehabilitation process . ConsequFifty-eight different devices were found that at least had a prototype in action. In Tables In respect of the developmental stage of the devices, the stages were classified into four categories: (1) commercial in the case the robots are available for its commercialisation; (2) clinical trial when the robot undergo a study where the participants were assigned to groups undergoing similar forms of therapy, but at different intensities, using various devices or undergoing various forms of therapy in a different order, aiming to determine the efficiency of therapy ; (3) feaFrom the 58 devices, it was apparent that the majority 67%) were designed or had been redesigned for children. When it comes to the type of robot, more than half were an exoskeleton type structure. In the past five years, there is a trend Tables , 5, 6, 77% were dRehabilitation robots are moved by devices called actuators. Actuators convert a source of energy  into mechanical motion. Commonly rehabilitation assisted robots are powered by electrical actuators. Among the compared systems in Tables In robotic-assisted rehabilitation, the most common actuators are electrical motors with a rigid power transmission element such as a harmonic drive, ball-screws, timing belts, and chains. Unfortunately, their need for transmission negatively affects the back-drivability, efficiency, safety, size, and mass . NeverthSome researchers  used a cFollowing the concept of adding a flexible element in series with the actuator to improve the electric motors’ compliance, serial elastic actuators (SEAs) incorporate an elastic part in series with the electric actuator. This elastic element helps to decrease the actuator’s impedance and inertia and increases the back-drivability allowing better force control, even though they are limited by a large volume, heavy mass and complicated structure , 105. AnSome authors considered that the mechanical linkage of the electric actuators is too heavy and can generate resistance at the joints, making them inadequate for rehabilitation applications , 107. InDevices for robotic rehabilitation may provide different training strategies depending on the type and severity of the patient’s impairment. These can be divided into passive, active, assistive, or resistive . In geneIn passive training, the force/motion is generated by the robot alone to perform the exercise. The advantage of this training is that patients with minimal muscle activity can receive therapy. For instance, through repetition of a movement, ROM can often be maintained with muscles and joint structures (e.g. ligaments) repeatedly stretched, ultimately maintaining their physiological length. Such movement reduces contractures at joints, which can finally be very useful to caregivers making a notable difference to the ease of transfers (e.g. sitting in a wheelchair to lying in bed). Examples of devices using passive training are Innowalk  and InteIn the active training mode, the patient’s muscle can still generate activity on the affected limb. The robot does not help, making the patients perform the exercise by themselves at least partially. The active mode provides data concerning torques and the ROM produced, allowing assessments before and after therapy/surgery. For instance, Kinarm  and LokoFor assistive or active-assistive training, the muscles of the affected body part can still be activated. Therefore, the patient can at least partially perform the exercise or movement without the robot. The assistance will be triggered after a particular event is detected through an HCI, allowing the patients to move further with the robot’s help. Assistive training is relevant as it involves the active participation of the children. Moreover, it improves the physiological responses needed to maintain and increase muscle strength and length, ultimately leading to improved ROM, in which the muscles provide some of the torque required. Due to these advantages, many designers have produced devices that use this training mode. Examples are Pedianklebot  and the As the name implies, the robot applies a force opposing the desired movement in resistive training, making the task more challenging. Resistive training is used to enhance muscle strength in the treated limb. This type of training was employed in the ankle device developed by the University of Arizona  and the The term HCI refers to methodologies to identify the user’s intent to move in the desired direction from different input sources and translate this intention into a command for the robot to move to facilitate the appropriate actions . The desThe main physical interactions used on HCIs to control such robots are Impedance/admittance, body-powered control, and gait phases detectors. Impedance and admittance control are the two most commonly used HCI. They are based on the relation between position and force rather than controlling either force or position explicitly. Impedance control accepts position or velocity as the input and outputs force or torque, and admittance is the opposite of impedance. Hence, force or torque are inputs, and velocity or position the outputs. This method could provide a natural, comfortable, and safe touch interface . Some exWhen the children cannot generate an intention to move with the treated limb, body-powered control is applied. It consists of using the movement of a different body part as the trigger signal to initiate the rehabilitation robot. The main drawback of this approach is that it is hard to control many degrees of freedom due to the activation system’s simplicity. An example of this HCI is the Ekso robot, where the activation was made by moving one’s body weight laterally and then forward to trigger the assistance .In assisted gait, a favoured approach for HCI is the use of gait phase detection. This technique identifies the different gait phases  to apply forces to assist the children’s movement depending on the gait phase. Robots usually perform gait segmentation using inertial measurement units (IMUs) to detect angular velocities of the shank and/or the thigh, or footswitches to detect the foot’s ground reaction forces while the child is walking . The maiAlternatively, for HCI based on physiological signals, Electromyograms (EMG) that measure electrical activity in the muscles and electroencephalograms (EEG), which measure electrical activity in the brain, are the main signals used. They are widely utilised because they can be obtained using non-invasive techniques without the need for medical intervention.Concerning electromyograms, the primary type is surface electromyography (sEMG), a non-invasive and easy-to-configure procedure in which adhesive electrodes are placed on the skin above the muscle of interest. The benefit of using the EMG signal is that it allows detection of the user’s intent before the movement occurs. The electrical activity can be detected even if it is insufficient to generate movement of a joint. However, sEMG can suffer from contamination of the signal by electromagnetic interference, skin perspiration, movement of electrodes and crosstalk artefacts. Also, for each muscle group of interest, a single EMG channel only shows the activation of that group. So, to perform an activity where many muscles fibres are recruited, it is necessary to use multichannel sEMG. Some examples of this technology in paediatric rehabilitation robots are the lower limb exoskeleton HAL  and the The electroencephalogram (EEG) signal is recorded using many small surface electrodes, often configured in a bathing like cap placed over the scalp that detects the underlying electrical signals. The main advantage of the EEG signal is that the physical disability level does not limit it. Even if the patient has lost all their ability to move the limb required for a task, the brain activity thought to be related to the intent to activate the muscles can be recorded. There are two main disadvantages to this system. Firstly, it is unsuitable for children with brain damage as they cannot generate standard brain patterns for limb activation. Secondly, the EEG signal has greater variability within it than the EMG signal, and it is also easily affected by changes in the patient’s mood and attention. Examples of the EEG signal use are the CP walker that used this signal as a part of its HCI to help children with a physical disability move their legs  and the The majority of studies and devices were for children with neurological conditions , CP being the most studied condition . In contrast, other neurological disorders included ABI and strokes. Significantly few researchers investigated other conditions such as neuromuscular diseases  and traumatic injuries to limbs and the spine . The results obtained from the studies that perform clinical trials or a feasibility study suggests that robotic rehabilitation could benefit children with physical disabilities.Concerning CP, there was evidence of improvement in physical disability using assisted rehabilitation robots. The benefits include an increase in muscle activity , 132, enFor paediatric ABI, there were reports in the improvement of the walking ability , improveIn children who suffer a stroke, three studies used rehabilitation robots while performing physical therapy. Marini et al.  demonstrThe neuromuscular disease presented a different scenario than neurological disorder due to the degeneration of their muscles as the disease progresses, making hard the use of rehabilitation robot due to stiffness in the robot’s joints, which can harm children’s weak muscles. Hence they require compliant actuation , 146. JaEven if traumatic injuries are common in the paediatric population, robot rehabilitation has not been applied widely in injuries that differ from those at the head. Only scarce information was found related to these conditions. A study of hands robotic rehabilitation was found, highlighting the possibility of using robotic devices to treat burns . AdditioIt is possible to see that various novel rehabilitation robots have become available to rehabilitation professionals and clients in recent years. And this trend will continue as is possible to incorporate them in activity programs aimed at improving independent function –37 whereThe findings of this review indicate that the design and development of robotic technologies for the physical rehabilitation of children is in a preliminary stage of development, as many of the devices were designed for adult patients. However, there is a trend toward creating robots specifically for children , 57, 148Traditionally, rehabilitation robot designers have focused solely on improving physical function , which cWhen it comes to the type of robot, we can see a trend to migrate from end-effector to exoskeletons. However, most of the devices that had performed clinical trials were end-effector robots designed for adults. This relation could be because the end-effector robot works on the distal part of the limb, guiding the children limb through a movement . This prOn the other hand, exoskeletons work in parallel to the patient limb to perform the activity. Hence, they can be portable devices with the possibility to provide assistive help during activities of ADLs and robotic-assisted rehabilitation therapy in a single device . This adThe paediatric robotic rehabilitation technology is moving from end-effectors to exoskeletons due to their versatility to be used as a rehabilitation tool or an assistive device . ConsequFor the end-effectors robots, actuators are not as critical as with the exoskeletons because they could be placed in external structures. This advantage makes it possible to use bulky and heavy actuators like electric motors. However, using electric motors is hard to achieve compliance that is an important property to increase safety as it is needed to avoid opposing forces that can injure the children. In end-effector robots, compliance was achieved using sensors and a control strategy  or usingOn the other hand, for exoskeletons, the robot design requirements are hard to enhance with the currently used technology. The actuation system components such as motor and rigid elements are designed for industrial applications not to interact with and to be worn by children. However, they are still the standard as they have the advantages of efficiency, are easy to control, and are readably available in the market. Therefore, the choice of the actuation system is crucial to improve the weight, portability and safety of the exoskeleton.The first exoskeletons relied on electric motors with rigid transmissions, making them bulky and heavy, reducing their compliance as they generate high resistive torque from the metallic links of the exoskeleton. Therefore, making it difficult to move and less safe can cause non-desirable inertial movements , 157. FuAs the rehabilitation robots move from rehabilitation therapies inside a medical facility to assist the children during ADLs, new actuation technology is needed. This challenge led to using SEA actuators and cable transmission since they have the advantage of been intrinsically complaints as they incorporate soft materials, making the device safer.Using Bowden cables in the transmissions brings other advantages like simple architecture, low weight on the limb’s distal part, and easy to reconfigure. This last advantage is significant in paediatric rehabilitation as it allows to change the motor easily depending on the abilities and size of the children , 73, eveAnother type of actuator used on the robots was the pneumatic. Their attributes of low weight and easy-to-manufacture actuators of different shapes and sizes , 159 makIn Table In the case of the training strategy, there is no best strategy, but it rather depends on many factors like the abilities and disease that the children have. For example, passive training is suitable for patients with limited mobility; however, when the children are able to generate movements, it tends to decrease children’s participation during the exercise, thus reducing the efficiency of the training . That isAlternatively, some researchers suggest that resistive training could be more beneficial for rehabilitation therapy than an assistive force, as it increases the engagement of the children, which can help drive motor learning , 168. HeThe HCIs are essential in developing robotic rehabilitation robots, as they are the medium for the interaction between the children and the robot, impacting the functioning of the actuators and training strategies directly. Thus, HICs are a crucial factor for safety and motivation needs, as it is how the children “communicate” their intention to the robot. Consequently, If the HCI is complex to use, it could lead to the rejection of the device . FurtherConsequently, selecting the best HCI for every case needs to be evaluated depending on the capabilities of the children. For example, it could be challenging for patients with advanced muscular dystrophy to use EMG and admittance/impedance interfaces as their muscles progressively deteriorate, turning unable to activate the muscles to generate a movement or a detectable signal , 171. OnTo better understand how the technology can positively impact children’s lives, it is essential to analyse how the rehabilitation robot’s technology has addressed the different paediatric conditions that can generate physical disability. Because, even if they share in common the deterioration of the musculoskeletal system, each one of the conditions presents certain specific characteristics that need to be considered.Most of the research has focused on children with neurological conditions, particularly children with CP. However, it seems likely that many of the designed robots that currently work with neurological conditions could also be utilised in traumatic injury scenarios, especially because they have been designed for rehabilitation therapies that can improve common problems across both neurological and traumatic injuries like ROM deficits and a lack of ability to generate muscle force , 150. CoUnfortunately, the outcomes proving the efficiency of rehabilitation robot in children is still scarce, as the pieces of evidence are low and weak. Hence, the information coming from these studies should be asses carefully, as there are very few randomised controlled trials, with small sample sizes and variability in children’s ability, outcomes measures, treatment protocols, and used devices , 65. ThuIt was apparent that some studies were conducted with participants from a wide range of ages; therefore, it was difficult to target all the articles that include paediatric participants. Another problem was the upper bound on the paediatric population’s age as some papers with the term young adults included paediatric participants.While robotic rehabilitation is gaining momentum with increasing numbers of devices being produced for adults, there is a lack of well-designed and effective products available for children. Early examples of robots have often been created by scaling downsize to meet the smaller stature of children. Few robots have been specifically designed and produced, with children being the focus of the project/program. It is apparent that children have special needs, and these need to be incorporated into designs early in the development program. And even if the fulfilment of these needs is closely related to the chosen structural and technological components like the actuator, training strategy and HCI, they go beyond them. Consideration must also be given to the aesthetics that appeal to children and the need for the robot’s structure to be as unobtrusive as possible. Without such needs being met, no matter how effective the robot works from an engineering perspective, it will not be utilised well by the child.It is apparent that there is still a lack of understanding of what the most effective therapy is to improve function and quality of life in specific paediatric conditions (e.g. CP or Stroke). Nevertheless, common impairments  must be addressed across numerous clinical conditions if patients improve function in everyday tasks. Hence, there is much opportunity for robots to play a role in assisting paediatric rehabilitation. A much more difficult goal to achieve is the development of robots to assist children. At the same time, they do function-related tasks like walking, sitting, lying, and assisting when the child moves from one posture to another. This demand increased complexity throughout the various engineering systems of the robot. After that, a further challenge lies in the robot being able to assist indoors within a relatively safe environment and outdoors where the “lay of the land” is notably different and less predictable. Hence, exploring new technologies to actuate the system and detect children’s intentions when they want to move is necessary.