This work contains data on the computational, structural, and electronic characterization of supported ionic liquids phases anchored to copper nanoparticles using Density Functional theory calculations. The data supplement the paper “Interaction of supported ionic liquids phases onto copper nanoparticles: A Density Functional Theory study”  XPS and computational analysis gave mechanistic insights into the Cu NP stabilization pathways, where the anion adsorption onto Cu NP is favored compared to the cation adsorption. The stronger adsorption is observed for Cu@(I)SILP1 complex, which presents the more electron-rich triazole and the higher adsorption value of SILP onto Cu surface (5.18 eV). Computational studies of the adsorption of SILP onto Cu NPs allow evaluating the chemical and physical properties that govern these complexes stability. The schematic representation of Cu@(X)SILPs complexes is displayed in For this work, we designed a series of new triazolium-based supported ionic liquids (SILPs), decorated with Cu NP (Cu@SILPs). The triazoles moieties were functionalized using copper-catalyzed azide-alkyne cycloaddition. Three triazolium cations (T1adsE)  The Cu@SILPs complexes stability was characterized by the adsorption energy (Eads) , where tPREPE) and interaction energy (ΔINTE). The energy decomposition analysis based on absolutely localized molecular orbital (ALMO-EDA), implemented in Q-Chem 5.2 computational package  (0.05 mol%) was added and dissolved in water (5 mL). The reaction was stirred for 4 h at room temperature, and the progress of the reaction was monitored using thin-layer chromatography (TLC). After its completion, the reaction mixture was filtered, and the residue was dissolved in DCM. The combined organic layer was later concentrated in a vacuum to yield the corresponding triazoles.Alkyne , alkyl halide (1.0 eq.), and NaN2.32–I (5 g) was suspended in dry acetonitrile (30 mL), with stoichiometric amounts of triazoles1–3 and refluxed during 24 h. Then, the particles were collected by filtration and repeatedly washed with petroleum ether, and then dried off under vacuum at 110 °C for 4 h to obtain SILP1–3 powder. Based on the amount of IL on the SILP(I)1–3 supports, an excess (1.2 eq.) of NaBF4, KPF6, and LiNTf2 salts were dissolved in deionized water (25 mL) and added to the SILP(I)1–3 (1.0 g) in order to exchange the anions. The suspensions were vigorously stirred for 48 h. The mixtures were washed, centrifuged, and dried to yield the supports SILP(BF4)1–3, SILP(PF6)1–3, and SILP(NTf2)1–3.SiO2.42–I).Initially, 200 g mesh silica gel was soaked in 30% HCl overnight to hydrolyze its surface. Next, activated silica gel (100 g) was suspended in dry toluene (30 mL) in a round-bottom flask equipped with a reflux condenser under nitrogen. While being stirred, 3-iodopropyltrimethoxysilane (0.05 M) was added dropwise. The suspension was refluxed for 72 h. After cooling, the solid was collected by filtration and exhaustively washed by Soxhlet extraction with ethanol and water, and then dried under reduced pressure to yield iodopropyl–silica gel (SiO2.52×2H2O (0.25 mmol) and MeOH (20 mL) was added to SILP(X)1–3 (100 mg) under constant stirring at room temperature for 30 min. A solution of NaBH4 (5 mmol) dissolved in MeOH (3 mL) was added to the reaction mixture dropwise. The reaction mixture turned black due to the formation of Cu NPs that were washed with MeOH (3 × 10 mL) and Et2O (3 × 10 mL). Subsequently, the samples were isolated by centrifugation (4500 rpm) and dried under reduced pressure.A solution of CuCl2.6−9 Torr. Wide energy range survey scans were collected at pass energy of 80 eV in hybrid slot lens mode and a step size of 0.5 eV. High-resolution data on the C 1 s, N 1 s, and F 1 s photoelectron peaks were collected at pass energy 20 eV over energy ranges suitable for each peak, and collection times of 5 min, step sizes of 0.1 eV. The charge neutralizer filament was used to prevent the sample from charging over the irradiated area. The X-ray source was a monochromated Al Kα emission, run at 10 mA and 12 kV (120 W). The energy range for each ‘pass energy’ (resolution) was calibrated using the Kratos Cu 2p3/2, Ag 3d5/2, and Au 4f7/2 three-point calibration method. The data were charge corrected to the reference carbon adventitious signal at 284.8 eV. The transmission function was calibrated using a clean gold sample method for all lens modes and the Kratos transmission generator software within Vision II. The data were processed with CASAXPS (Version 2.3.17).XPS Experiment was collected by powder sample that was mounted on double-sided tape (Sellotape) and pressed to ensure proper coverage of the tape with the powder. X-ray Photoelectron Spectroscopy (XPS) measurements were performed using a Kratos AXIS Ultra DLD instrument. The chamber pressure during the measurements was 5 × 102.7−6 Hartree with no symmetry restriction. Stoll Fragmentation method employing Roothaan-step and exact SCF correction methods after the locally-projected equations. The basis set superposition error (BSSE) and dispersion D3BJ corrections were included.Calculations were calculated using Q-Chem 5.2 computational package ,4. For e2.8−6; criteria for displacement convergence: 1.0 × 10−7; minimal distance between Critical Points of 0.03 Bohr. Skip search if the distance between atoms is longer than the sum of their van der Waals radius multiplied by 1.80; and the criteria for determining if the Hessian matrix is singular of 1.0 × 10−50.The atom in molecules studies (AIM) were developed using the Multiwfn3.6 program 2.9Fragmental charges were calculated using the Charge Model 5 population analysis Kerry Wrighton-Araneda: Conceptualization, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Cristián Valdebenito: Investigation, Writing - original draft, Visualization, Validation. Gabriel Abarca: Investigation, Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing - review & editing, Data curation. Diego Cortés-Arriagada: Investigation, Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing - review & editing, Data curation.The authors declare that they have no known competing for financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Difference of perspective between patients and physicians over integrative medicine (IM) research and service provision remains unclear despite significant use worldwide. We observed an exceptionally low utilisation of IM and potential underreporting in diabetes. We aimed to explore the barriers and recommendations regarding service delivery and research of IM service among diabetes patients and physicians.A 10-group, 50-participant semi-structured focus group interview series was conducted. Twenty-one patients with diverse severity of disease, comorbidities and education levels; and 29 physicians  and 15 Chinese medicine (CM)) with diverse clinical experience, academic background and affiliation were purposively sampled from private and public clinics. Their perspectives were qualitatively analysed by constant comparative method.Seven subthemes regarding barriers towards IM service were identified including finance, service access, advice from medical professionals, uncertainty of service quality, uncertainty of CM effect, difficulty in understanding CM epistemology and access to medical records. Patients underreported the use of CM due to the concern over neutrality of medical advice among physicians. Inconvenience of service access, frequent follow-up, use of decoction and long-term financial burden were identified as key obstacles among patients. Regarding research design, ConM physicians emphasised standardisation and reproducibility while CM physicians emphasised personalisation. Some CM-related outcome measurements were suggested as non-communicable. Both physicians acknowledged the discordance in epistemology should be addressed by pragmatic approach.Key obstacles of CAM clinical utilisation are different between patients. Further assessment on IM should be pragmatic to balance between standardisation, reproducibility and real-world practice. Evidence-based IM programs and research should merge with existing infrastructure. Controversies on incorporating complementary and alternative medicine (CAM) into integrative medical (IM) care continue amid increasing utilisation and volume of evidence worldwide –5. It waHong Kong has a developed health system and the introduction of CM faces challenges similar to the western world , 11. WhiDiabetes is an alarming pandemic affecting 9.5% world adult population, accounting for 9.9% of global all-cause mortality , 21. DiaNevertheless, a service programme in Hong Kong revealed that only 20% of diabetes patients have ever considered CM as an option and only less than 2% have ever used CM as a treatment for diabetes or DKD which was way below the utilisation in other conditions (e.g. around 50% for cancer patients) . InteresWe aimed to explore the barriers and recommendations regarding IM service delivery among patients and physicians of conventional medicine and CAM, and subsequently identify key areas for research, education and service provision.We conducted a 10-group semi-structured focus group interview series on patients and physicians with constant comparative method. Participants were recruited from public and private clinics and teaching hospitals in Hong Kong.Sixty-one invitations were sent out. One patient rejected due to family issues and another 2 rejected due to difficulties in allocating time for the study. Five CM physicians rejected due to work engagement and 3 did not respond to email invitations.Fifty subjects were recruited  from October 2015 to June 2018. Patients aged 18 or above, diagnosed with diabetes / DKD and having follow-up at public outpatient clinics were included and sampled purposively from the consultations of general, renal and family medicine outpatient clinics of Queen Mary Hospital and Tang Shiu Kin Hospital to form 3 groups of 6–8 with diverse age groups, CKD stages, diabetes duration, co-morbidities and education levels to enrich data coverage.ConM physicians formed 3 groups of 3–6, and CM physicians formed 4 groups of 3–4. Physicians with experience in managing diabetes / DKD and were included and purposively sampled with diverse age groups, practicing duration, qualification and experience of ConM and CM, affiliating institutions, service sector (private / public) and involvement in research, administration and teaching.Participants were recruited face-to-face, though telephone or by email. The purpose of interview was explained to the participants by the research coordinator (K.W.C.). The process of recruitment, interview and analysis were iterative until data saturation was observed.The focus group interviews lasted 60–120 min  and were conducted privately in meeting places near participants’ workplace or residence.The interviews were facilitated by a moderator (P.W.L.) with 1) undergraduate public health training, 2) 2 years of in-depth interviews and focus group interviews moderation experience and 3) basic knowledge on CM, ConM and diabetes to ensure adequate skills and knowledge for conducting the interviews. The moderator was not involved in the conceptual design of the study, did not have prior contact with the study subjects, did not have conflict of interests with subjects and the research topic, and was informed about the group composition until 1 week before the interview. The identity of moderator was not disclosed to participants before interviews. He was working in public health administration sector but not involved in integrative medicine or diabetes issues in daily work during the period of study to avoid any potential conflict of interests. One to two clerical staffs were present at different interview sites for administrative support. The moderator recorded participants’ demographics with simple questionnaire and mediated the interviews.The discussion was built around consultation experience, concerns and expectations based on a semi-structured interview guide . The intInterviews were audiotaped and transcribed verbatim. Interviewees’ identity was preserved by using individual codes. Brief notes on interview content and behaviour of interviewees were made real-time during interviews and subsequently combined with the transcripts. The transcripts were cross-checked between 2 independent researchers .The transcripts were analysed using the constant comparative method  aided byCore coding was confirmed for the dataset after observing no new themes at the last round of interview. The finalised coding scheme was applied to index and chart the whole dataset. Charted result was translated by a bilingual investigator (K.W.C.) when used as illustrative quotations to best preserve original meaning. Analysis was completed in October 2018. The analysis process is summarised in Fig. n = 15) had formal credit-bearing ConM education in anatomy, physiology, immunology, pathology and internal medicine as CM undergraduate programmes contained substantial portion of ConM elements in Hong Kong. Table Demographics of subjects are presented in Tables Seven high-level themes  were identified with 25 subthemes. Data on barriers towards IM service and preferred model of integrative service delivery are summarised in Fig. Seven subthemes were identified regarding barriers towards IM service, including finance, inconvenience of access, advice from medical professionals, uncertainty of service quality, uncertainty of CM effect, difficulty in understanding CM epistemology and access to medical records.Finance was identified as a major practical issue against the use of CM / IM by patients. CM service in Hong Kong is financed through insurance and out-of-pocket payment. Government subsidised over 90% for ConM service  and compThe inconvenience of access to CM service, including frequent consultation and the use of raw herbs was also frequently mentioned among patients, although majority of them (61.9%) were unemployed / retired. Patients had a longer follow-up period of 3–6 months for ConM services when compared to 1–2 weeks for CM. Although CM physicians anticipated that inconvenience would affect compliance, they believed it was inevitable due to the personalised nature of CM.“CM is very troublesome. We need to treat the  medicine and attend consultation frequently. ConM doctors give you 3-month medication.” (Patient 5)While some patients were advised against CM by ConM physicians, they hesitated to comply as they believed ConM physicians could not decide the effect of CM. We sought to delineate the underlying mechanism of discouragement and the lack of understanding of CM effect  emerged as the key reason and is further dissected.“They (ConM physicians) do not know which CM physician you consulted and do not know what CM you have taken. How can they give an answer?” (Patient 10)“I advise them (patients) not to take CM as I do not know the interaction between CM and ConM.” (ConM 2)Uncertainty of service quality was a common concern among patients, ConM and CM physicians. Patients doubted CM physicians’ academic qualification and questioned the experience-based consultation of some CM physicians.“They  prescribe medicine based on experience. I do not know the effect after taking their medication.” (Patient 18)“If they (CM physicians) can prove CM does not adversely interact with ConM and they are registered, I do not disagree.” (ConM 12)“I advised them (patients) not to approach CM physicians who are not affiliated with hospitals or NGOs as I am not sure about their understanding on kidney disease or replacement therapy.” (ConM 14)ConM physicians were concerned about the standardisation of CM, interaction between CM and ConM and the background nephrology knowledge among CM physicians.All parties were uncertain about the efficacy of CM. A patient who experienced suspected CM nephrotoxicity suggested that the response to CM was personalised, as he experienced toxicity despite others’ positive feedback. CM physicians also acknowledged that there may not be adequate evidence to inform specific management. However, there were ambivalent opinions on the study design for further research, especially on clinical trials.“For CM, there is limited information on who is more responsive to treatment. You said you (another patient participant) got better after taking CM but I got worse. ConM is different.” (Patient 18)“As CM takes into account many factors including geographical and climatic effect … CM may not be reproducible and may not need to be reproducible.” (CM 15)ConM physicians well-noted that CM has different theory basis and clinical practice when compared to ConM. All parties agreed to emphasise more on the clinical effectiveness.“I studied a short course of CM … the fundamental concept of CM and ConM is very different. For example, when CM refers to heart, lung, liver, spleen and kidney, it is not the organ we understand, they link to five elements and ying yang which is hard to directly translate to ConM.” (ConM 3)“I am not concerned about the principles and methods behind (CM) but we have to be communicable and work on the same  outcomes.” (ConM 6)“(CM theory) is difficult to me. Even if they (CM physicians) wrote consultation notes, I could not understand … I do not understand how they monitor disease progress.” (ConM 1)However, ConM physicians suggested that it is hard to understand and translate CM-related clinical outcomes to their practice.ConM physicians raised concern that the CM medical record, especially hand-written prescription, was often unreadable. This imposed a risk of mishandling of patients under CM service. Also, ConM physicians were worried that the restricted access of investigations among CM physicians may affect clinical management. However, CM physicians who advocated traditional practice were less relied on investigation while other CM physicians suggested that investigations would help alert them critical conditions that require prompt management.“I am worried that CM does not have investigations.” (ConM 14)“I do not want so many consultations. Can you (CM and ConM physicians) both come to offer IM service so that I do not have to separate the consultations?...... I do not mind paying extra if it (IM) really works.” (Patient 1)Four subthemes including organisational management, consultation mode, CM-ConM communication and choice of CM service were identified under the theme ‘Preferred model of IM service delivery’. Data on consultation mode is presented below.vice versa.Majority of participants suggested that there was a need of physically combined service, preferably at the same institution. This would improve the confidence of patients and communication between physicians, leading to more efficient patient management. Patient also expected physicians to offer compromised IM treatment protocol as clinical solutions. Both ConM and CM physicians acknowledged that the combined treatment should be formulated according to the appropriate theory basis, i.e., CM treatment directed by CM theory and “If you reverse the role, you ask all ConM physicians to feel the pulse before prescribing ConM medicine, they will also feel uneasy. … ... We all do not want to follow a protocol that does not match our practice.” (CM 9)“I think collaborative service has a great potential but theory integration almost looks impossible. Even if we have a pharmacy offering both ConM and CM, it does not mean that CM physicians would accept us (ConM physicians) to prescribe CM based on ConM theory. Vice versa, it would be hard for us to accept CM physicians prescribing mycophenolic acid because the patient is having ‘qi deficiency’.” (ConM 7)This is the first focus group study comparing the expectation of patients and physicians regarding the use of IM service for diabetes, with the participation of family medicine, internal medicine and CAM physicians in addition to patients. We demonstrated that there was an expectation mismatch between patients (prefer physicians to design integrative treatment protocol and prefer biomarkers for disease monitoring), CAM physicians  and physicians of conventional medicine (prefer patients to make the choice of treatment).We investigated with a qualitative approach to explore unknown mechanism in the underutilisation and underreporting of CAM among diabetes patients and physicians and conducted focus group interviews to enhance interactions between participants with different experience. The interview guide was designed to explore more practical concerns and actual consultation experience from implementation perspective as existing literature is limited in this aspect. The guide was slightly tilted towards exploring how to integrate CM service into existing ConM system as majority of diabetes patients were receiving ConM as background treatment locally and internationally. The bias of interviewing skills and contents was minimised with an independent experienced moderator. Patients and physicians with different background hypothesised to offer divergent perspectives  were purposively sampled to enrich the content and coverage. As possible underreporting of IM utilisation was previously reported , we sepaData saturation, defined by having no emerged new key themes, was reached in the third round of interview among patients and ConM physicians, and the fourth round of CM physician interview. This reconciled with previous review and empirical studies that 80% of themes could be captured in 2 to 3 rounds of interview and data saturation usually occurred in the 3 to 4 rounds , 43. OnlExisting literature regarding the obstacles of IM emphasised cultural believe and research-related capacity. Our study echoed as uncertainty of service quality and effect of CM were identified as shared concerns among all stakeholders as summarised in Fig. Our findings further highlighted the importance of mutual understanding between physicians of different streams as any advices regarded unreasonable and unexplainable by patients would result in non-compliance and underreporting. In addition to the existing understanding, practical issues including inconvenience of access, frequent follow-up and financial burden were stressed among patients in this series, which were less documented and is likely related to the chronic nature of diabetes. Besides, we found that ConM with exposure to CM knowledge preferred more pragmatic and clinical evaluation of IM service as they recognise there is a fundamental difference in epistemology between ConM and CM. We also documented more practical concerns from the medical community for the consideration of further IM study designs.Convenience of access was frequently quoted by patients as a key barrier while it was not concerned by CM physicians. IM would lead to increased number of clinical visits. While frequent follow-up may provide patients with the best-tailored management, the required effort would compromise patients’ quality of life and increase the economic loss. Combined IM consultation under the same organisational structure was generally preferred by our subjects (both patient and physicians) as it enhances quality control, reduces time cost for consultation and facilitates sharing of medical records among physicians. Integrated clinics are increasingly common globally and provide a platform for physicians from different streams to design and evaluate IM protocols . High quIn contrast to many existing IM models globally that the decision and liability of IM use was shifted to the patients, requiring patients to actively search and critique the evidence themselves, our data suggested that patients expected physicians of different streams to optimise the IM protocol and offer integrated evidence-based recommendations for their informed decision making. This will require centralised and concerted effort in evaluating IM efficacy with parallel mechanism of existing statutory bodies, for instance NICE (UK), FDA (US) and NMPA (China), if not integrating IM procedures into these existing infrastructures.Interestingly, CM physicians from private sector believed the access cost of CM has driven patients to demand extra efficacy and quantifiable evidence when evaluating CM, which amplified the effect of lack of evidence, a well-known barrier. Patients reconciled that cost was one of the key barriers of using IM service. In Hong Kong, CM service is mainly financed through out-of-pocket payment and private insurance whereas ConM service is over 90% publicly subsidised. Comparable CM service of diabetes operated by NGOs is around 100 times more expensive in long run. Government policy on CAM service financing emerged as a decisive factor of implementation.Physicians from both sides called for more basic IM knowledge from their counterparts for better communication. In China, most physicians are double qualified and practise ConM and CM simultaneously. While dual training may provide a comprehensive foundation for IM practice, there are criticisms on the ungrounded integration between ConM and CM on physician level as suggested by the interviewees. As many ConM and CM interventions have been shown to have interactions, IM service requires specifically designed IM-oriented research and evidence instead of simple integration of disaggregated evidence from either side. Also, dual training is difficult to propagate to health systems of other countries and requires substantial resources. The effectiveness and optimal ratio of one physician – dual medical practice model and one administrative system – dual streams of physician model in health systems also require further implementation studies.Regarding research, both ConM and CM physicians acknowledged that research design should formulate according to the corresponding school of theory of ConM and CM to reflect actual practice. Nevertheless, ConM physicians emphasised more on standardisation and reproducibility while CM physicians focused more on personalisation on study design which highlighted the different epistemology between CM and ConM. Both CM and ConM physicians of different specialties proposed pragmatic trial as an agreeable solution which aligned with the trend of establishing real-world comparisons lately , 43, 50.This study has several limitations. Since the aim of this series was to identify detailed barriers, concerns and expectations on IM diabetes management, the findings are context specific, similar to other qualitative studies. However, we believe the major findings could be carefully generalised to the IM management of chronic conditions in places where CAM is not part of the public health system, including most of the developed countries. Also, this series only identified possible mechanisms of social behaviour and further quantitative studies including survey are needed to better determine the magnitude of and prioritisation of the concerns. Lastly, the study period was long due to the availability of funding and difficulties in organising focus groups, especially for physicians, which is a commonly known challenge.Inadequate explanation of CAM effect to patient may lead to underreporting. Practical issues on the access and finance of medical service emerged as major barriers to the delivery of IM service from the patients’ perspective. Inadequacy in evidence of clinical effectiveness and the difficulties in interpreting CM theory remained as the major obstacles in the consideration of CM among ConM physicians. Pragmatic clinical evaluation is mutually accepted by both ConM and CM physicians. Research design should focus on the add-on effect of CAM and consider standardisation, reproducibility and the real-world practice. To enhance the utilisation, IM programmes that are evidence-based should merge with existing infrastructure.Additional file 1.


The aim of this trial was to investigate whether a digital device that provides real-time visualized brushing instructions would contribute to the removal of dental plaque over usual brushing instructions.t-test was used to compare the two groups in accordance with intention-to-treat principles.We conducted a single-center, parallel-group, stratified permuted block randomized control trial with 1:1 allocation ratio. Eligibility criteria included people aged ≥ 18 years, and exclude people who met the following criteria: severely crowded teeth; using interdental cleaning implement; having external injury in the oral cavity, or stomatitis; having less than 20 teeth; using orthodontic apparatus; visited to a dental clinic; having the possibility of consulting a dental clinic; having a dental license; not owning a smartphone or tablet device; smoker; taken antibiotics; pregnant; an allergy to the staining fluid; and employee of Sunstar Inc. All participants received tooth brushing instructions using video materials and were randomly assigned to one of two groups for four weeks: (1) an intervention group who used the digital device, providing real-time visualized instructions by connection with a mobile application; and (2) a control group that used a digital device which only collected their brushing logs. The primary outcome was the change in 6-point method plaque control record (PCR) score of all teeth between baseline and week 4. The p = 0.088, 95% confidence interval −0.70–10.07).Among 118 enrolled individuals, 112 participants were eligible for our analyses. The mean of PCR score at week 4 was 45.05% in the intervention group and 49.65% in the control group, and the change of PCR score from baseline was −20.46% in the intervention group and −15.77% in the control group (A digital device providing real-time visualized brushing instructions may be effective for the removal of dental plaque. Periodontal disease is an inflammatory condition caused by infection with bacteria and is one of the main diseases seen in dentistry . DecayedOnce a tooth is lost from periodontal disease, it causes oral dysfunction and influences the whole body . Many prRemoving dental plaque is important for the prevention and treatment of periodontal disease . The easAppropriate brushing instructions are required to master effective brushing. However, not many people visit the dentist regularly, which is of note as people can get brushing instructions from dentists or dental hygienists. Moreover, one of the tooth brushing methods which is often used in the clinical setting is the Bass method, which requires one to move their toothbrush in small steps ,13. PrevIn addition, although toothbrushing for more than two minutes is recommended to remove dental plaque , a studySunstar Inc.  has developed a digital device that provides visible brushing instructions, linked to a mobile application, to facilitate learning appropriate teeth brushing habits. Many similar applications and devices, which improve oral hygiene or promote oral health behavior, have been developed and there have been reported their effect of only mobile applications or especially with electric toothbrush –21. HoweWe conducted a single-center, parallel-group, randomized control trial (RCT). Randomization was done by stratified permuted block with a 1:1 allocation ratio.th, 2018 to November 30th, 2018. We placed an advertisement about this trial in Kyoto University’s bulletin board from October 3rd, 2018 and recruited participants from October 29th to November 2nd. At the first day of follow-up period, after explaining this trial and gave written informed consents, we registered the participant officially, and followed each participant for one month.The trial was conducted at Kyoto University Health Service from October 29This trial included people aged ≥ 18 years, and we excluded people who met one or more of the following criteria: severely crowded teeth ; using interdental cleaning implement every day; the presence of an external injury in the oral cavity, or stomatitis, that can affect teeth brushing; having less than 20 remaining teeth; using orthodontic apparatus; visit to a dental clinic within the past month; having the possibility of consulting a dental clinic during the study period; having a dental license; not owning a smartphone or tablet device; current smoker; taken antibiotics within the past week; the possibility of being pregnant; an allergy to the staining fluid used for dental plaques; and being an employee of Sunstar Inc.th, 2018, and registered University hospital Medical Information Network (UMIN) Clinical Trial Registry (CTR) (UMIN000034503).The trial was approved by the ethics committee of Kyoto University (C1390) on June 27Before the trial, participants were measured plaque control record (PCR) score and underwent brushing for dental plaque removal by a dental hygienist. The PCR score was measured by O’Leary’s method , where t® device, which had three functions linked with a mobile application: (1) to visualize the toothbrush position and provide a guide for the brushing sequence; (2) to ensure the brushing time lasted more than 3 minutes; and (3) to visualize whether the user is moving the toothbrush in small steps or not  during the trial and were restricted from using other dental materials for plaque removal, such as dental floss and mouthwash. The PCR score was measured 2 and 4 weeks after the baseline measurement by the dental hygienist. The participants were prohibited from eating, drinking and tooth brushing two hours before the oral examination, so that the time and the oral conditions at the three examinations were as similar as possible.All participants used a standard toothbrush  and toothpaste  followed by brushing instructions from a dental hygienist. A week later, PCR was re-measured (B). On the same day, the digital device provided visualized brushing instructions was supplied to the participants, and after receiving instructions on how to use it, they performed teeth brushing with use of the application and digital device for a week, prior to a further set of PCR measurements being performed (C). The PCR score (A–B) of the control group in this study decreased by 9.4% from baseline, while in the intervention group, the score (A–C) decreased by 16%. Thus, assuming that the use of the brushing-instruction digital device results in a 6% decrease in the PCR score, with the level of significance (α) set at 5% (two-sided level) and the detection power (1–β) at 80%, the required participants’ number in one group was 63. Moreover, we estimated a drop-out rate of 10%, a power calculation indicated that a sample of 70 participants in each group was required to detect intergroup differences.For the randomization process, the stratified block randomization method  was used, where participants were randomly assigned to either of the two groups in a 1:1 ratio using stratification methods according to gender  and baseline PCR score (≥ 60%/< 60%) with a computer-generated randomization list, by a trial staff who only took charge of randomization. Randomization code was prepared in advance by the trial statistician. When an eligible participant was identified, the trial staff checked his/her teeth report and allocated using the randomization list.Both the assignment process and the explanation of the digital devices were conducted in a protected area, and randomization concealment for the group was maintained for the dental hygienists who measured PCR score and for the analysts of the data. On the other hand, we could not blind for the participants.t-test for continuous variables and the Chi-square test for categorical variables. In evaluating the primary and secondary outcome, changes in PCR score between baseline and week 4 were assessed using the t-test. In addition, we divided the whole tooth into three surfaces parts  and two teeth parts (anterior teeth and posterior teeth), and calculated the mean PCR score at baseline and week 4, and the presence of changes in each part. The differences in the consciousness of brushing and the time spent brushing between the two groups were evaluated using the Mann-Whitney U test.Participants characteristics were compared between the two groups using the We also calculated the number of participants with plaque on each tooth surface at baseline, and the number of participants who still had plaque after 4 weeks, as well as the decrease ratio, which was classified into 10% groups. Moreover, we calculated the number of the tooth surfaces of each group of the decrease ratio and the proportion of tooth surfaces relative to all 192 teeth surfaces.All statistical analyses were performed on an intention-to-treat basis. All p values were two-sided, and p values < 0.05 were considered statistically significant. We used R statistical software  .th, 2018 to November 30, 2018 (the last day of the follow-up for the participants). The trial was conducted from October 29p = 0.088, 95% confidence interval [95%CI] −0.70–10.07). The change of mean PCR score from baseline using the 4-point method as the secondary outcome was significantly greater in the intervention group (−18.09%) compared to the control group . The change of mean PCR score from baseline to week 2 was −13.53% in the intervention group and −10.26% in the control group. Moreover, the mean PCR score at week 4 decreased from baseline to a larger extent in the intervention group compared to the control group across all three surfaces parts  and two teeth locations . Furthermore, the distribution of each consciousness and brushing time were different in the two groups at week 4. Some participants in the intervention group reported that, "I could learn the appropriate brushing methods." and that " The digital device gave me an opportunity to review my brushing" at week 4.During this trial, adverse events and serious harms were not observed.In this RCT, we evaluated the effects of the digital device, which offers real-time visualized brushing instructions. We found that the 6-point method PCR score decreased 4.69% more after four weeks by using the digital device with visualized brushing instructions compared to usual brushing instructions alone, although the difference was not statistically significant. The PCR score calculated using the 4-point method as the secondary outcome measure was 4.98% lower through the use of the digital device with visualized brushing instructions compared to usual brushing instructions alone, and this difference was statistically significant. In buccal center surfaces, lingual center surfaces and the anterior teeth, the PCR score at week 4 had a tendency to show a greater decrease in the intervention group compared to the control group. Our questionnaire survey data indicated behavioral change more in the intervention group participants compared to the control group participants. These results have important implications for the removal of dental plaque and habituation of appropriate brushing techniques.Several previous studies have investigated the effect of plaque removal by brushing, providing instructions about brushing methods, and also the association between brushing time and plaque removal –11. SchlWe used the same video material for all participants receiving tooth brushing instructions in this trial, therefore we were unable to personalized instructions based on each participant’s oral condition. It may be more effective, even in difficult parts of the mouth, to learn an individual’s oral condition and provide suitable brushing methods under professional instruction.Plaque control is recognized as an effective approach to prevent periodontal disease, and the prevention of periodontal disease can result in the prevention of many lifestyle-related diseases –6. RecenThe PCR score tended to decrease more from baseline in the intervention group compared to the control group. The decrease ratio of PCR score on each tooth surface part tended to be greater in the intervention group than the control group. However, in several parts most participants could not remove the plaque and it was difficult to achieve the appropriate PCR score regardless of the use of the brushing-instruction digital device. Guidelines recommend keeping the PCR score between 10% and 20% in order to prevent periodontal disease , howeverIn the questionnaire survey at week 4, most participants in the intervention group stated that they are “always conscious” of the procedure of teeth brushing, of brushing each surface of the tooth, and moving the toothbrush in small steps. This suggests that participants may have an increased awareness of appropriate brushing techniques due to instructions from the digital device. It is important to continue appropriate teeth brushing every day to enable plaque control, and motivation as well as repeated brushing instructions are required for this. As can be seen from our results of decreasing the mean of the PCR score at week 4 from week 2, the use of the digital device, which provides repeated real-time visualized brushing instructions, may be effective for people to get habituation of continuous brushing their teeth properly. However, it remains to be seen whether participants can maintain the consciousness of appropriate teeth brushing after this trial, and further investigations are needed to evaluate this.This trial has several inherent limitations. First, the number of participants did not satisfy the sample size we calculated beforehand, because of the difficulties in recruitment. Second, masking participants was impossible in our trial because of the different brushing approaches in the intervention and control groups. Third, the Hawthorne effect may have influenced our trial results, with participants performing better with teeth brushing because of awareness that they are being monitored in a trial setting. Fourth, the follow-up period of this trial was restricted. It is assumed that people lose interest for the digital device as they use it for long time. Therefore, we cannot discuss whether the effect of this digital device continues, if people use it for a long time. Finally, the generalizability of our results is limited by the fact that this was a single-center trial with several inclusion criteria. This shows that we only investigate the restricted population, moreover, the most participants were university students and the mean of their age was 20s. Generally, the proportion of the people who have periodontal disease is said to be high in elderly persons compared to that of young persons. Therefore, the findings of this trial may not apply to elder population.We observed a non-significant reduction in the 6-point method PCR score after 4 weeks of using the digital device with visualized brushing instructions, when compared to usual brushing alone. However, the consciousness of teeth brushing at week 4 improved more in the intervention than control group. The use of a digital device providing real-time visualized brushing instructions may be somewhat effective for the removal of dental plaque and habituation of appropriate brushing, and may be useful as a trigger for self-care.S1 Fig(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S2 Protocol(DOCX)Click here for additional data file.


Correction of calcium (Ca), phosphorus (P), and parathyroid hormone (PTH) disorders is the standard of treatment in non-dialysis patients with chronic kidney disease-mineral and bone disorder (CKD-MBD), but the side effects and adverse reactions brought by western medicine (WM) are still the main problems. More importantly, the lack of protection of kidney function in the treatment greatly affects the health of patients. Although traditional Chinese medicine (TCM), specifically tonifying kidney and strengthen bone (TKSB) therapy is wildly applied for patients with CKD-MBD in China, the evidence of TKSB therapy in the treatment of CKD-MBD is limited. Thus, we pretent to conduct this protocol to evaluate the efficacy and safety of TKSB therapy combined with WM for non-dialysis patients with CKD-MBD.A system research of randomized controlled trials (RCTs) of TKSB therapy for CKD-MBD will be conducted by 2 investigators from 7 electronic databases. Methodological quality evaluations will be performed by using the Cochrane collaboration tool and data analysis will be conducted by RevMan V5.3 software and STATA v15.0.The results of this paper will be submitted to a peer-reviewed journal for publication.This research will determine the safety and efficacy of TKSB therapy in treating non-dialysis patients with CKD-MBD.INPLASY2020120086 Such a high incidence and prevalence have brought a great burden to society. Chronic kidney disease-mineral and bone disorder (CKD-MBD) is one of the most common complications of chronic kidney disease. With the progression of CKD, the incidence of CKD-MBD is increasing. When it progresses to end-stage renal disease, almost all patients have renal bone disease (RBD). As early as CKD stage 3, due to the decline of renal function and the decrease of glomerular filtration rate, the renal phosphate excretion is impaired and the phosphate in the blood is retained. Such high phosphorus in the blood can directly inhibit the secretion of 1α-hydroxylase by the kidneys, leading to obstacles in the production of renal active vitamin D3. However, active vitamin D3 is an important component of intestinal absorption of calcium into the blood, so lack of it will cause hypocalcemia. The reduction of activated vitamin D3 can make bones have varying degrees of resistance to parathyroid hormone (PTH), which will increase the compensatory secretion of PTH in the blood. At this time, the increased PTH will mobilize a large amount of bone calcium into the blood to maintain the balance of Ca and P, but it will cause bone calcium loss in bone tissue, leading to osteoporosis, fractures, bone pain, and other bone diseases, which is the so-called CKD-MBD. These diseases have severely affected the quality of the patients’ life and shortened their survival time.Chronic kidney disease (CKD) is a major disease threatening human health. In the past 3 decades, the global incidence and prevalence of CKD have increased by 89% and 87%, respectively.: reduce phosphorus intake and promote phosphorus clearance to control hyperphosphatemia; supplement calcium and active vitamin D3, adjust blood calcium to maintain a safe level and avoid the occurrence of hypercalcemia; prevention and treatment of secondary hyperparathyroidism; if the effect of drug therapy is not good, parathyroid surgery can be considered. These therapies listed above are effective in controlling calcium, phosphorus, and parathyroid hormone in the blood, but there are many adverse reactions. For example, the oral administration of aluminum hydroxide can easily cause the accumulation of aluminum in the body, and the improper grasp of the dosage and treatment course of calcium can easily cause hypercalcemia. Once in end-stage of CKD-MBD, parathyroidectomy for the treatment of tertiary hyperparathyroidism not only brings irreversible damage to the body, but also causes a heavy economic burden on the patient. More importantly, western medicine (WM) ignores the improvement of renal function, as well as the lack of early prevention of CKD-MBD. Patients often take treatment measures only when they have severe bone pain, bone deformity, and other clinical symptoms, but it is very difficult to reverse the course of the disease at this time. Therefore, it is essential to seek treatments which can both improve renal function and regulate Ca, P, and PTH levels.At present, the main clinical treatment methods for CKD-MBD are as follows The combination of TKSB therapy with WM can not only improve renal function, but also regulate the levels of Ca, P, and PTH. For example, Zheng et al found that Yishen Jiangzhuo Granule, an effective TCM for TKSB, combined with WM treatment, could significantly improve renal function by increasing CCr and decreasing SCr when compared with WM alone. Zhou showed that the combination of WM with TKSB therapy could improve the symptoms of renal function and reduce bone loss by increasing Ca levels in blood, reducing P and PTH levels in the blood, increasing bone density, and reducing ALP content. The therapeutic effect of combining treatment group was superior to the WM treatment group. All the above studies have shown that TKSB therapy pays more attention to improving renal function in the treatment of CKD-MBD, that is, it uses herbal drugs to match the kidney bias to improve the vitality of the kidney to help the body restore the normal metabolic function of the kidney, which is the essence of TCM in the treatment of CKD-MBD. It does not only focus on regulating the abnormal trace elements in the blood as doing like this will ignore the analysis of pathogeny, leading to the gradual decline of renal function and ultimately resulting in an irreversible situation of dialysis treatment. Inspired by these findings, we wonder if the treatment is focused on improving renal function, perhaps the trace elements in the blood will return to normal, which is what TCM is good at and pursues. At present, a large number of literature have reported the clinical efficacy and drug safety evaluation of TKSB therapy in the treatment of CKD-MBD, including dialysis patients and non-dialysis patients. Since metabolism is not carried out through the kidney in dialysis patients, the effect of TCM on renal function improvement cannot be reflected in the body of dialysis patients. Based on this point, we believe that the therapeutic effect of TKSB therapy on non-dialysis patients is more significant. However, there has been no meta-analysis to evaluate the clinical efficacy of TKSB therapy in the treatment of CKD-MBD in non-dialysis patients. Consequently, our study is to lay a foundation for promoting the clinical application of TKSB in the treatment of CKD-MBD in non-dialysis patients.Traditional Chinese medicine (TCM) is widely used in the prevention and treatment of kidney diseases and bone metabolism diseases. According to the theory of TCM, renal bone disease belongs to the category of “bone impotence” in TCM. Bone is closely related to kidney. Bone loss is the main characteristic of kidney deficiency. Kidney deficiency and bone atrophy are the basic pathological features of RBD. Therefore, the classic therapy of TCM for RBD is tonifying kidney and strengthen bone (Bushen Jiangu).2This protocol is registered in INPLASY (INPLASY2020120086). This protocol was performed in accordance with the preferred reporting items for systematic reviews and meta-analysis protocol. Ethical approval is unnecessary because this is a literature-based study.2.1http://www.chictr.org.cn/), and international clinical trial registry . We will contact the original investigators for more complete details of the study to solve questions about eligibility if necessary.We will comprehensively search the following 7 databases: PubMed, EMBASE, Cochrane Library, Wanfang database, Chinese National Knowledge Infrastructure (CNKI), China Biological Medicine (CBM), and VIP Journals Database. The basic terms were performed as follows: (bushen OR yishen OR tonifyingkidney OR bugu OR jiangu OR zhuanggu OR strengthen bone) AND . Ambiguous literature will be manually searched to avoid missing eligible trials. Ongoing registered clinical trials will be searched on the websites of the Chinese clinical trial registry  combined with one type of WM .2.2.3The control group should receive WM treatment alone. If the control group contains other TCM therapy or dialysis treatment, it will be excluded.2.2.4Included studies should contain at least 4 of the following evaluated outcomes: clinical effective rate; serum Ca level; serum P level; parathyroid hormone (PTH); blood urea nitrogen (BUN) level; serum creatinine (SCr) level; adverse events from TKSB therapy or WM.2.2.5Randomized controlled trial and quasi- randomized controlled trials (RCT) including combination therapy of TKSB will be included. Articles will be excluded if they are case reports, letters, editorials, and nonhuman studies. The flow diagram of the study selection is shown in Fig. 2.3For eligible studies, the following data will be independently extracted by 2 authors (GLW and LL) according to predefined criteria: first author, publication year, region, sample size, detail of intervention, treatment courses, and outcome parameters. For any disagreement between the 2 authors, it would be settled through discussion with a third author (YQX). The reasons for exclusion were recorded. The data were extracted from the included RCTs to a predefined Excel table  and cross-checked by the 2 reviewers . In the event of missing data, we will attempt to contact the corresponding authors for details.2.4 Disagreements, if any, were resolved by discussion and reached consensus through a third reviewer (JL). The assessed items included 6 domains: random sequence generation; allocation concealment; blinding method; integrity of data; selective reporting; other bias. Selective reporting bias was judged according to the published protocols for the registered clinical trials that were contained on the Chinese clinical trial registry (http://www.chictr.org) and international clinical trial registry of the US National Institutes of Health  websites. We compared the outcome measures between the study protocol and the final published trial.Two authors  independently assessed the methodological quality of each trial according to the standards advised by the Cochrane Handbook.2.5I2 test. The result of I2 test above 50% will be considered to indicate significant heterogeneity, and the meta-regression model will be applied to find out the sources of the heterogeneity. Additionally, if necessary, a sensitivity analysis will be carried out to evaluate the stability and reliability of the results by removing individual studies at a time.Review Manager 5.3 , compiled by the Cochrane Collaboration, will be employed to pool and analyze data. Dichotomous variables will summarize as risk ratios (RR) with 95% confidence intervals (CI), while continuous variables will summarize as mean difference (MD) with 95% CI. Heterogeneity will be examined by the 2.6The funnel plot to evaluate the potential publication bias will be used when there are >10 studies included in the meta-analysis. And quantitative analysis of publication bias will measured by the method of Egger test or Begg test.3 However, the optimal strategy for the treatment of CKD-MBD remains controversial. For example, Gross et al believe that there is still no convincing RCT evidence to confirm whether reducing the level of P in the blood can improve the clinical prognosis and reduce the mortality of patients with CKD-MBD. Block et al believed that the use of calcium-phosphorus binder or non-calcium-phosphorus binder in patients with CKD stage 3–4 could accelerate the rate of vascular calcification, and it was difficult to obtain satisfactory clinical efficacy. In this context, the current treatment of CKD-MBD only focuses on regulating the metabolic levels of Ca, P, and PTH in the blood, while ignoring the improvement of renal function and considering its adverse effects. At present, TCM is widely used to improve the symptoms and signs of patients with CKD-MBD, and it is especially good at improving patients’ physical conditions and improving their quality of life. According to the theory of TCM, the incidence of CKD-MBD is usually in the kidney and bone, and its pathogenesis is closely related to kidney deficiency and bone impotence, which mostly belong to deficiency syndrome. Thus, TKSB therapy is the main method for TCM treatment of CKD-MBD. In recent years, increasingly RCTs have been reported on the treatment of CKD-MBD with TKSB therapy, which provides an opportunity for the objective and comprehensive evaluation of this method. In order to clarify the efficacy and safety of this treatment strategy in a scientific and reasonable way so as to promote its clinical application, we pretent to conduct a meta-analysis in the future. Through the forthcoming meta-analysis, we will conduct a statistical analysis of the herbs used at a high frequency to provide a better therapy.RBD is one of the most common and serious complications of CKD. Metabolic disorders such as Ca, P, and PTH in vivo caused by chronic renal impairment will lead to cardiovascular diseases and fractures, posing a great threat to human life and health.As the systematic review is based on the secondary research of published literature, there are undeniable methodological defects. In addition, the quality of the included studies determines the quality level and reliability of the final results. We will begin to conduct the review when the necessary trials are met, and all operating procedures will be performed in accordance of Cochrane Handbook to ensure that the provided information is helpful for clinicians and patients.The authors would like to thank Professor Youping Li, Director of Cochrane Center in China; Professor Rui Peng, Director of College of Acupuncture and Orthopedics of Hubei University of Chinese Medicine in China; Professor Jia Li, Director of Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and Moxibustion in China for their training on Cochrane system evaluation and knowledge of statistical analysis.Data curation: Liang Li.Formal analysis: Guiling Wu.Methodology: Jia Li.Software: Youqiong Xie.Supervision: Rui Peng.Writing – original draft: Zi Jian Wu.


Mesenchymal stem cell therapy (MSCT) has been shown to be a new therapeutic option for treating alopecia areata (AA). Outer root sheath cells (ORSCs) play key roles in maintaining the hair follicle structure and supporting the bulge area. In human ORSCs (hORSCs), the mechanism for this process has not been extensively studied. In this study, we aimed to examine the influence of human hematopoietic mesenchymal stem cells (hHMSCs) in the hORSCs in vitro model of AA and determine the mechanisms controlling efficacy. Interferon-gamma (IFN-γ) pretreatment was used to induce an in vitro model of AA in hORSCs. The effect of MSCT on the viability and migration of hORSCs was examined using co-cultures, the MTT assay, and migration assays. We investigated the expression of molecules related to the Wnt/β-catenin pathway, JAK/STAT pathway, and growth factors in hHMSC-treated hORSCs by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. hHMSCs increased hORSC viability and migration when they were co-cultured. hHMSCs reverted IFN-γ-induced expression—including NLRP3, ASC, caspase-1, CXCL-9 through 11, IL-1β, and IL-15—and upregulated several growth factors and hair stem cell markers. hHMSCs activated several molecules in the Wnt/β-catenin signaling pathway, such as in the Wnt families, β-catenin, phosphorylated GSK-3β and cyclin D1, and suppressed the expression of DKK1 induced by IFN-γ in hORSCs. hHMSCs suppressed the phosphorylation of JAK1 to 3, STAT1, and STAT3 compared to the controls and IFN-γ-pretreated hORSCs. These results demonstrate that hHMSCs increased hORSC viability and migration in the in vitro AA model. Additionally, MSCT definitely stimulated anagen survival and hair growth in an HF organ culture model. MSCT appeared to be associated with the Wnt/β-catenin and JAK/STAT pathways in hORSCs. Alopecia areata (AA) is an autoimmune disease that targets hair follicles (HFs) during the anagen stage and is primarily caused by the disruption of immune privilege. ,2. AA isAccording to the concept of pathology, the prevention or recovery of immune privilege disruption of HFs can ultimately lead to the more effective management of AA. Recently, research studies have reported that treatments using mesenchymal stem cells (MSCs) can be useful for treating AA. Mesenchymal stem cell therapy (MSCT) or stem cell therapy using umbilical cord blood-derived cells for AA has been reported in several refractory cases ,7. A recThe mechanism by which MSCT is associated with improvement of hair growth and the immune environment is not yet well known. According to our previous study, MSCT reverted an AA-like environment in human dermal papillar cells (hDPCs) via the Wnt/β-catenin and Janus kinase (JAK)/signal transducers and activators of transcription protein (STAT) signaling pathways. In addition, various growth factors and cytokines associated with anagen re-entry induction were found to be enhanced by MSCT . ActivatHFs are composed of cells of multiple origins, are single independent mini-organs, and have a characteristic periodic hair cycle . The haiThe hORSCs of HFs are multilayered tissues predominantly made up of undifferentiated keratinocytes . hORSCs The effects of MSCT on hORSCs have not yet been studied. To study the therapeutic mechanism of MSCT in AA, we investigated the effect of MSCT on hORSCs, with or without pretreatment with IFN-γ. We examined the effect of MSCT on the viability and migration ability of hORSCs. We focused on the activation of the Wnt/β-catenin and JAK/STAT signaling pathways and changes associated with the expression of several cytokines and chemokines, inflammasomes, growth factors, hair stem cell markers, and migration in response to MSCT.4 cells/well, according to a previous study [First, we examined whether MSCT could increase the cell viability of hORSCs. The concentration of hHMSCs used was 5 × 10us study . To deteNLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β at the mRNA levels. As shown in NLRP3, ASC, caspase-1, and IL-1β were significantly increased in the IFN-γ-treated group compared to the controls. There was no significant difference in the expression levels in the MSCT group compared to the controls, whereas the expression of inflammasome components was upregulated in the IFN-γ-treated group, and downregulated by additional MSCT.To investigate whether NLRP3 inflammasome activation was related to MSCT in hORSCs, we examined the expression of of CXCL9, CXCL10, CXCL11, TNF-α, IL-10, IL-15, IL-18, IFN-γR, and Major histocompatibility complex (MHC) class I chain-related protein A (MICA) at the mRNA level  and AXIN2 compared to the controls. Additionally, IFN-γ-hHMSC-treated group increased the mRNA expression levels of WNT3, WNT5, WNT10, GSK-3β, β-catenin, Cyclin D1, LEF1 and AXIN2, which was decreased by IFN-γ. The mRNA levels of WNT7 were significantly increased only in the MSCT group.We measured the expression of Wnt/β-catenin genes by real-time PCR to investigate the roles of MSCT . The resDKK1 in the MSCT group with and without IFN-γ treatment compared to the controls. The change in TGF-β2 expression was not significant in the MSCT group compared to the controls. The expression levels of DKK1 and TGF-β2 were increased in the IFN-γ-treated group and were suppressed by MSCT.As shown in We also investigated the effects of MSCT on the WNT/β-catenin pathway at the protein level by Western blotting . The levKeratin15 genes by qPCR , fibroblast growth factor 2(FGF2), fibroblast growth factor 7(FGF7), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), sonic hedgehog (SHH), bone morphogenetic protein 2 (BMP2), and BMP4 are representative growth factors associated with hair growth.IGF1, FGF2, FGF7, VEGF, PDGF, SHH, BMP2 and BMP4 in response to MSCT were significantly upregulated compared to the control or IFN-γ-treated groups in this study.As shown in We examined the effects of MSCT on the JAK/STAT signaling pathway at the protein level using Western blotting . OverallThe level of phosphorylation of JAK1-3 was significantly increased in IFN-γ-treated cells compared to the controls. Phosphorylation was significantly suppressed by MSCT compared to the IFN-γ-treated controls B. The leNext, we also investigated the effect of MSCT on IFN-γR and IL-15, which are the key cytokines in JAK/ STAT pathway activation. The level of IFN-γR was increased in the IFN-γ-treated group compared to the control and was suppressed by MSCT. IL-15 was only expressed in the IFN-γ-treated group and MSCT blocked the expression of IL-15 .Next, the effect of MSCT on hORSC migration was examined by the crystal violet assay using six-well polycarbonate Transwell inserts. As shown in To examine the effect of MSCT at the organ level, we performed an ex vivo culture of mouse vibrissae HFs. In this study, 20 HFs were cultured individually with control, IFN-γ, IFN-γ plus MSCT, or MSCT. Each hair shaft was measured every third day.As shown in IL-10, which contributes to the immunomodulatory effects of MSCT, was increased by MSCT. Hair growth factors were upregulated by MSCT  attack MICA positive HFs, which results in apoptosis and hair loss . AccordiThe Wnt/β-catenin signaling pathway is important for hair morphogenesis and anagen re-entry . β-catenTGF-β2 mRNA was only increased by IFN-γ treatment and not by MSCT in this study. Most of the Wnt/β-catenin-related molecules were increased by MSCT conditions in vitro. This suggests that MSCT could help induce anagen re-entry by stimulating the Wnt/β-catenin signal pathway.In this study, MSCT significantly induced Wnt family proteins, β-catenin, GSK-3β and cyclin D1 expression. Among the Wnt family proteins, Wnt3a and 10b are related to the hair cycle and regeneration, and increase anagen gene expression. Kishimoto et al. generated Wnt3a-overexpressing keratinocyte feeder cells and co-cultured the cells with freshly isolated murine DPCs. In this experiment, Wnt3a was found to increase β-catenin expression and increase hair growth in nude mice that underwent skin remodeling composed of DPCs and keratinocytes . Wnt5a iSOX9 is characteristically expressed in CD34-positive hORSCs in the bulge area and is involved in the regulation of bulge formation and stem cell transcription ,34,35. CIn AA, dystrophic HFs are often observed instead of normal HFs because hORSCs are attacked by inflammatory cells around the HFs and the immune privilege of the HFs is disrupted. The main role of hORSCs is to secrete and react to inflammatory substances. hORSCs are important immunocompetent cells that express NLRP3 inflammasomes constitutively in the HFs . TherefoIL-10 is an anti-inflammatory cytokine that has the opposite effect. These results can be the outcome of JAK/STAT pathway activation. Recent studies have shown that increased JAK-STAT signaling suppressed HF stem cell function in vitro and that STAT5 signaling regulated HF stem cell quiescence [We also found that the IFN-γ-induced proinflammatory cytokines and chemokines of AA  were supiescence ,40. EvenIn this study, the phosphorylation of JAK1-3 and STAT1 was significantly decreased in hORSCs by MSCT, which reverted the IFN-γ-induced proinflammatory changes. Although the downregulation of STAT3 in the IFN-γ-hHMSCs treated group was not significant compared to the IFN-γ-treated groups, the changes were significant compared to the controls in Western blotting analysis. We suggest that MSCT suppress IFN-γ signaling through downregulation of the JAK/STAT pathway. This result suggests that the MSCT-suppressed activation of JAK1/2 reversed catagen induction and induced a potential mechanism involved in the new hair cycle. TGF-β is well known as an inducer of regression . In accoIGF1, FGF2, FGF7, PDGF, VEGF, SHH, BMP2 and BMP4 mRNA expression was suppressed by IFN-γ, and MSCT had a stimulatory effect on the transcription of growth factors. Our results indicate that MSCT increased hair growth factors to facilitate the viability and migration of hORSCs  were seeded into six-well culture plates in Dulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin  for 24 h. hORSCs at passage three to five were used for the experiments. Then, hORSCs were seeded on culture dish plates with serum-free DMEM, and the plates were co-cultured with upper chamber containing hHMSCs.We obtained human bone marrow-derived mesenchymal stem cells . We purchased human outer root sheath cells (hORSCs) from ScienCell  and cultured them, as previously described . hORSCs 5 cells per well. hHMSCs were added to the upper chamber of the Transwell in separate plates at a density of 5 × 104 cells per well. The upper chambers were coated with polyethylene terephthalate . After 24 h, the media was changed to serum-free DMEM and the upper chamber containing hHMSCs was transferred to the wells where the hORSCs were cultured to produce the co-culture. The IFN-γ group was treated with recombinant human IFN-gamma at 100 ng/mL, obtained from Peprotech . After different times , the upper chamber was removed and washed with Dulbecco’s phosphate-buffered saline . Next, the hORSCs were harvested and used for analysis. The experiment was repeated three times.The co-culture of hORSCs and hHMSCs was performed as previously described . hORSCs Cell viability was measured via the 3--2,5-diphenyltetrazolium bromide (MTT) assay. After co-culturing for different times, the MTT reagent was added and reacted for 2 h. The reagent was removed, and the resulting formazan crystals were dissolved in dimethylsulfoxide (DMSO) . Then, tTotal RNA was isolated from the hORSCs using TRIzol reagent , and cDNA was synthesized with the MG cDNA Synthesis Kit  according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed with primers and Moloney-murine leukemia virus (M-MLV) reverse transcriptase (RTase) . Real-time PCR was performed using the SYBR Green Master mix . The gene expression levels were quantified using analysis software . The primer sequences are listed in hORSCs were harvested with RIPA lysis buffer supplemented with Protease and Phosphatase Inhibitor Cocktail . The amount of protein was measured using the BCA protein assay kit  and compared to bovine serum albumin (ThermoFisher) standards. Cell lysates containing the same quantity of total protein were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBST for an hour at room temperature and incubated with primary antibodies in 5% BSA at 4 °C overnight. Primary antibodies against phospho-JAK1-3, total-JAK1-3, phospho-STAT1-3 total-STAT1-3, DKK1, IL-15, phospho-GSK-3β, GSK-3β, β-catenin, SOX9, CD34, CD200, and GAPDH were purchased from Santa Cruz Biotechnology  and Cell Signaling Technology, Inc. . On the following day, the membranes were washed with TBST several times and incubated in peroxidase-conjugated secondary antibody  for 2 h at room temperature. The immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) detection system (ThermoFisher) and were obtained using a chemiluminescence imaging system  after incubation with horseradish peroxidase.5 hORSCs that were already treated with IFN-γ or hHMSCs. The number of hHMSCs and density of IFN-γ were the same as those used in the co-culture procedure. DMEM containing 10% FBS was added to the lower chambers. After incubation for 15 h at room temperature, the cells in the upper chamber were carefully removed using a cotton swab and the membranes were fixed with 4% paraformaldehyde for 20 min. The cells that had migrated under the filter were stained with 0.5% crystal violet for 20 min and then observed under a light microscope [The migration of hORSCs was measured using Transwell plates that were 6.5 mm in diameter with 8 μm pore filters. The Transwell system was separated from the lower segment by a permeable membrane coated with polyethylene terephthalate. The upper chambers were loaded with 1 × 10croscope . The cel4 cells per well as described in the co-culture method. At least 20 HFs were cultured individually with control, IFN-γ, IFN-γ plus MSCT, or MSCT. The culture medium was changed every three days, and images of individual follicles were filmed before and after hHMSCs treatment. All procedures were carried out under the Institutional Animal Care and Use Committee—approved protocols .Six-week-old female C57BL/6 mouse vibrissae follicles were micro-dissected under sterile conditions, as previously described . IndividThe length of vibrissae HFs from the top of hair to the bottom of the hair bulb in organ culture was measured with a microscope every third day for 10 days. Changes in hair length were calculated from the photographs and expressed as mean ± SEM of 20 vibrissae HFs.t-test were used for the statistical analyses with SPSS (v.7.0) software . All tests were one-sided, and a p-value of less than 0.05 was considered statistically significant.All data are expressed as the mean ± SEM. An ANOVA test and paired Student’s


Peptide receptor radionuclide therapy (PRRT) is a systemic treatment consisting of the administration of a tumor-targeting radiopharmaceutical into the circulation of a patient. The radiopharmaceutical will bind to a specific peptide receptor leading to tumor-specific binding and retention. This will subsequently cause lethal DNA damage to the tumor cell. The only target that is currently used in widespread clinical practice is the somatostatin receptor, which is overexpressed on a range of tumor cells, including neuroendocrine tumors and neural-crest derived tumors. Academia played an important role in the development of PRRT, which has led to heterogeneous literature over the last two decades, as no standard radiopharmaceutical or regimen has been available for a long time. This review focuses on the basic principles and clinical applications of PRRT, and discusses several PRRT-optimization strategies.Peptide receptor radionuclide therapy (PRRT) consists of the administration of a tumor-targeting radiopharmaceutical into the circulation of a patient. The radiopharmaceutical will bind to a specific peptide receptor leading to tumor-specific binding and retention. The only target that is currently used in clinical practice is the somatostatin receptor (SSTR), which is overexpressed on a range of tumor cells, including neuroendocrine tumors and neural-crest derived tumors. Academia played an important role in the development of PRRT, which has led to heterogeneous literature over the last two decades, as no standard radiopharmaceutical or regimen has been available for a long time. This review provides a summary of the treatment efficacy , impact on patient outcome and toxicity profile of PRRT performed with different generations of SSTR-targeting radiopharmaceuticals, including the landmark randomized-controlled trial NETTER-1. In addition, multiple optimization strategies for PRRT are discussed, i.e., the dose–effect concept, dosimetry, combination therapies , new radiopharmaceuticals , administration route  and response prediction via molecular testing or imaging. The evolution and continuous refinement of PRRT resulted in many lessons for the future development of radionuclide therapy aimed at other targets and tumor types. Peptide receptor radionuclide therapy (PRRT) is a systemic treatment consisting of the administration of a tumor-targeting radiopharmaceutical into the circulation of a patient. The radiopharmaceutical will bind to a specific peptide receptor leading to tumor-specific binding and retention. Examples of receptors that have been studied include the somatostatin receptor (SSTR), glucagon-like peptide-1 receptor (GLP-1R), bombesin receptor, cholecystokinin type 2 (CCK2R) and melanocortin receptor -polymerase 1 (PARP-1) activity is required for the DNA damage repair of single strand breaks that can be caused by PRRT. When single-strand breaks are not repaired, they will lead to replication fork arrest and double-strand break formation during replication, and ultimately to cell death. PARP inhibitors are currently used in the treatment of several solid tumors . As such, an increase in preclinical research using PARP inhibitors as a radiosensitizer has emerged over the last few years. Several preclinical studies have shown that the combination of PARP inhibitors with ll death ,135,136.177Lu-OPS201  to 177Lu-DOTATATE, have shown that 177Lu-OPS201 exhibits a higher tumor uptake, higher number of double-strand breaks, longer tumor residence time and improved tumor-to-kidney dose ratio [177Lu-OPS201 was performed in four patients with advanced neuroendocrine neoplasms (NENs) and chronic grade 2 or 3 kidney disease [177Lu-DOTATATE, 177Lu-OPS201 showed a longer tumoral residence time and higher tumor uptake resulting in 1.7–10.6 times higher tumor doses. Toxicity was minor and reversible. Moreover, a phase-I trial including 20 patients with progressive well-differentiated NETs using 177Lu-OPS201 led to a high ORR of 45% and SD in 40% of patients [177Lu-DOTA-LM3 in 51 patients with advanced NENs [177Lu-DOTATOC or -TATE. Promising results were achieved with a high DCR of 85%. The occurrence of hematological toxicity was low, contrary to the previously described phase-I trial with 177Lu-OPS201 [68Ga-DOTATOC or -TATE PET/CT (insufficient for agonist PRRT) [Over the last few decades, the PRRT-paradigm for effective tumor targeting consisted of using receptor agonists which internalize after receptor binding, hereby causing tracer accumulation in the tumor cells. However, SSTR-antagonists are slowly emerging in the SSTR imaging and PRRT scene, with several preclinical studies showing their superiority over SSTR-agonists, despite the very slight amount of internalization . It is pse ratio ,140. A p disease . Comparepatients . Nephrotced NENs . Sixty-nu-OPS201 . This isst PRRT) . Several177Lu-DOTA-EB-TATE was conducted in five patients with advanced metastatic NETs [177Lu-DOTATATE (n = 3 patients), 177Lu-DOTA-EB-TATE achieved an extended blood-circulation and a 7.9-fold increase in tumor dose delivery. No adverse effects were noticed which was excepted as the administered activity was subtherapeutic, however, the dose delivery to the kidneys and bone marrow was significantly higher in patients receiving 177Lu-DOTA-EB-TATE compared to 177Lu-DOTATATE  [max) after treatment in the 177Lu-DOTA-EB-TATE group was achieved compared to the 177Lu-DOTATATE group , in a selection of lesions with a comparable baseline SUVmax (range 15–40). Furthermore, the safety and efficacy of administering up to three cycles 177Lu-DOTA-EB-TATE has been evaluated in 32 NET patients  [177Lu-DOTA-EB-TATE and 177Lu-DOTATOC was conducted in 5 patients with progressive SSTR positive disease [177Lu-DOTA-EB-TATE compared to 177Lu-DOTATOC. Therefore, all 5 patients were eventually treated with 177Lu-DOTATOC. The higher tumor dose delivered with this agent comes at the price of an even higher increase of the bone marrow dose, and thus it remains uncertain if using this compound is superior to higher activity/more cycles of established radiopharmaceuticals. More prospective studies are needed to evaluate the toxicity/treatment efficacy balance of this new compound.An attempt to improve the pharmacokinetics of the known radiolabeled SSAs was made by conjugating an Evans blue analog onto octreotate (EB-TATE). This results in a reversible binding of EB-TATE to serum albumin through the EB moiety, hereby extending its biological half-life in blood . The firtic NETs . In compctively) . These fctively) . A signi0.5 GBq) . Hematot disease . The ratThe use of alpha-emitters  in PRRT is an emerging strategy, however, the availability of these alpha-emitters is still limited world-wide. To date, preclinical and clinical research studies with actinium-225 or bismuth-213 were predominantly conducted by the extraction of actinium-225 from thorium-229 sources, arising from the decay of fissile uranium-233. However, these thorium-229/uranium-233 stocks are limited due to legal requirements related to fissile materials. This led to the investigation of several accelerator-based production routes over the last years, which will increase the availability of these alpha-emitters in the future . 213Bi-DOTATOC to eight patients with progressive NETs refractory to nonradioactive octreotide and tandem therapy with 90Y/177Lu-DOTATOC  [213Bi-DOTATOC was able to overcome resistance against beta radiation and induce long-term tumor remission. Nephrotoxicity and acute hematotoxicity were in the acceptable range. A prospective study was performed with 225Ac-DOTATATE in 32 patients with metastatic GEP-NENs who had SD after completing 177Lu-DOTATATE (n = 14) or PD on 177Lu-DOTATATE therapy (n = 18) [177Lu-DOTATATE, revealed PR (n = 8) or MR (n = 4) in the 12 patients who were assessed. In addition, in the patient group that had PD on 177Lu-DOTATATE, no PD was seen during the response assessment after two cycles of 225Ac-DOTATATE. No grade III/IV hematotoxicity, nephrotoxicity or hepatotoxicity occurred [212Pb-DOTAMTATE in 20 patients are promising [212Pb-DOTAMTATE at the highest dose level of 2.50 kBq/kg/cycle, have completed all four cycles of treatment. A high ORR of 83.3% (5/6 patients) via RECIST 1.1 was achieved and toxicity was low. In conclusion, these preliminary clinical results provide proof-of-principle evidence that α-PRRT can overcome resistance to β-PRRT.A first-in-human study was conducted by administering tration) . 213Bi-D(n = 18) . The meaoccurred . Furtherromising . At pres68Ga-DOTATOC compared with intravenous (IV) administration in 15 patients with GEP-NETs. IA administration of 68Ga-DOTATOC resulted in an average increase in SUV of 3.75-fold higher in liver metastases and 1.44- to 7.8-fold higher (dependent on the catheter placement) in the primary tumor, all compared with IV administration [Given the fact that NETs often metastasize to the liver, it can be hypothesized that intra-arterial (IA) administration of the radiopharmaceutical in the hepatic artery can lead to higher radiopharmaceutical concentrations and hence and improvement in treatment response, with a potential reduction in treatment toxicity . A high stration .90Y-DOTATOC and/or 177Lu-DOTATOC in 15 patients with liver metastases from GEP-NETs [111In-DOTATOC was also performed in this study. The time-activity curve of the intra-arterial administration of 111In-DOTATOC revealed a 3.5-fold higher uptake ratio just after termination of the infusion compared with IV administration. However, the time-activity curve of the IA administration also showed a saturation phase followed by a washout phase; this washout phase was not visible in the IV administration. This can be explained by receptor saturation, which depends on the total amount of administered peptide and the route of administration of the radiopharmaceutical (IA versus IV). Despite the washout effect, a higher tumor uptake was visible at 4 h (2-fold increase in uptake ratio) and 72 h post-injection (1.3-fold increase in uptake ratio) with the IA method, compared with the IV administration [Kratochwil et al. performed a pilot study investigating the efficacy of hepatic IA GEP-NETs . An ORR stration . FurtherAnother way of PRRT optimization can be achieved by pretreatment patient stratification. This strategy is gaining popularity over the last few years, given the fact that 15–30% of the patients show progression during PRRT and 10–20% of patients progress within the year after termination of PRRT ,64,152. The PRRT predictive quotient (PPQ) allows pre-PRRT patient stratification into PRRT-responders (PPQ positive) and PRRT-non-responders (PPQ negative). PPQ is based on serum-circulating gene clusters combined with tissue-tumor grading . The cirmax of more than 17.9, derived from 68Ga-DOTATOC PET, can predict responders from non-responders after PRRT with 90Y-DOTATOC, with a sensitivity of 100% and specificity of 95% [max of more than 16.4, derived from liver metastases on 68Ga-DOTATOC PET, can predict treatment response after PRRT with 90Y/177Lu-DOTATOC with a sensitivity of 95% and specificity of 60% [max of a single lesion and the average of up to five lesions (maximum two target lesions per organ) on 68Ga-DOTATATE PET/CT predicted response after 177Lu-DOTATATE [max cut-off of 13.0 was most optimal . This cut-off value was also associated with longer PFS (median 45.1 months if >13.0 compared to 19.9 if <13.0) with HR 2.5 (95%CI: 1.06–6.09). Ortega et al. investigated the relationship between multiple quantitative parameters, derived from 68Ga-DOTATATE PET/CT at baseline and after 1 cycle of PRRT, and treatment response in 91 patients with progressive metastatic NETs [max of malignant lesions on baseline and interim PET and mean higher SUVmax tumor-to-liver ratio of malignant lesions on baseline PET were predictive of therapy response . Higher values of kurtosis, a first-order heterogeneity parameter, derived from the malignant lesions on baseline PET, were observed in non-responders compared to responders . Further, Haug et al. investigated the role of 68Ga-DOTATATE PET/CT in early response prediction after PRRT (90Y/177Lu-DOTATATE), by evaluating changes in SUVmax and tumor-to-spleen SUV ratio (SUVT/S) between baseline and interim 68Ga-DOTATATE PET 3 months after the first cycle [T/S after the first cycle of PRRT was a significant predictor of time-to-progression in multivariate analysis. To the contrary, several studies did not find a predictive relationship between SUV and treatment response [max, the total tumor burden should also be assessed via SUVmean or total lesion activity [Several attempts have been made to discover a predictive relationship between quantitative PET-derived parameters on SSTR-imaging and treatment response after PRRT. Öksuz et al. found that a pretherapeutic SUVy of 95% . Moreovey of 60% . Sharma DOTATATE . A baseltic NETs . A highest cycle . Only a response ,161. Theactivity . Further177Lu-DOTATATE results in a pronounced longer PFS and a significantly higher response rate compared to high-dose octreotide long-acting-release [The phase-III NETTER-1 RCT has proven that PRRT with -release . In addi-release ,73. The