In this study, we evaluated real-world data on radium-223 plus abiraterone/prednisone or enzalutamide. Previously, the ERA 223 trial (NCT02043678) demonstrated increased fracture risk with concurrent treatment with radium-223 and abiraterone plus prednisone/prednisolone in patients with metastatic castration-resistant prostate cancer (mCRPC).We used the Flatiron Health database to perform a retrospective study of patients with mCRPC treated with radium-223. Treatment with radium-223 plus abiraterone/prednisone or enzalutamide was defined as concurrent if both drugs started within 30 days of one another, or layered when the second drug started ≥30 days after the first. The index date was defined as the day of the first radium-223 dose. Outcome measures included symptomatic skeletal events (SSEs), overall survival (OS), and patterns of treatments received.Of the 625 patients treated with radium-223, 22% received it together with abiraterone/prednisone and 27% with enzalutamide. When these agents were combined, they were often initiated in a layered fashion . Prior or concomitant bone health agents (BHAs) were received by 67% and 55% of patients, respectively. Median follow-up was 9 months. Overall, incidence rates for SSEs and pathologic fractures were 0.35 and 0.11 patients per person-year, respectively. Median OS from mCRPC diagnosis was 28.1 months.In this real-world setting, combination treatments with radium-223 and abiraterone/prednisone or enzalutamide were common. These agents were more commonly given in a layered than a concurrent fashion. Incidence rates for SSEs were reduced when BHAs were used; however, BHAs were underutilized. The approval of several agents that prolong survival in patients with metastatic castration-resistant prostate cancer (mCRPC) has provided physicians with the opportunity to select treatment based on patient preference, disease characteristics, accessibility, cost, and clinician experience –11. BecaRadium-223 is a targeted alpha therapy that prolonged overall survival (OS) and time to first symptomatic skeletal event  in patients with mCRPC and bone metastases in the phase 3 ALSYMPCA trial , 12. EarAs the treatment landscape for mCRPC continues to change with the emergence of new phase 3 data, there remains a lack of information regarding treatment patterns and outcomes in the real-world setting. Consequently, treatment decisions are often based on personal clinical judgment and expert opinion. A retrospective real-world study of patients with mCRPC in the US using the Flatiron Health database showed a substantial variation in the choice of first-, second-, and third-line treatments for mCRPC, with abiraterone/prednisone, enzalutamide, and docetaxel being the most common treatment options in each respective setting. Combination regimens containing radium-223 were frequently used in the second- and third-line settings .To expand on the findings from clinical trials and to provide further insights into the real-world use of combination therapies with radium-223 and abiraterone/prednisone or enzalutamide, retrospective analyses were performed using the Flatiron Health database. Two treatment approaches were evaluated: a concurrent approach (treatments starting within 30 days of each other)  and a laA retrospective study of patients whose health records were included in the Flatiron Health database was conducted. This longitudinal database, which is demographically and geographically diverse, is derived from structured and unstructured de-identified electronic health record data, curated via technology-enabled abstraction. As of June 2019, the Flatiron database included de-identified data from over 280 cancer clinics across the US (~800 sites of care), representing more than 2.2 million patients with cancer. Data from the subset of patients with mCRPC who were treated with radium-223 were used to examine real-world outcomes with this agent, including treatment patterns, skeletal events, and survival. A general description of the Flatiron Health database and the methodology for analyzing the mCRPC subset of this database, together with a detailed description of the methods for data gathering (including patient selection) and handling , have been published elsewhere .Records of patients with a confirmed diagnosis of mCRPC who were treated with radium-223 between January 1, 2013, and June 30, 2017, were included in the overall cohort. Records were excluded if patients participated in any clinical trial or had missing start dates for radium-223, enzalutamide, and abiraterone/prednisone.For the purpose of this analysis, a concurrent treatment approach was defined as both radium-223 and either enzalutamide or abiraterone/prednisone starting within 30 days of each other, whereas a layered approach was defined as one drug starting ≥30 days after the other, giving four sub-cohorts in total . The indThe primary objective was to assess the rate of SSEs . Secondary objectives were to assess OS from diagnosis of mCRPC and from initiation of radium-223. Exploratory objectives were to describe treatment patterns and duration of treatment with radium-223, enzalutamide, or abiraterone/prednisone.Descriptive statistical analyses were used for baseline patient demographics and disease characteristics, prior therapies, bone health agent (BHA) use, and clinical outcomes (including SSE rates and OS). The Kaplan–Meier method was used for time-to-event analyses (OS). No formal comparisons between sub-cohorts were conducted. To account for variations in follow-up time, incidence rates for SSE and pathologic fracture (number of patients per person-year) were calculated. Statistical analysis was performed using SAS Enterprise Guide Version 7.12.The dataset included 625 patients with mCRPC who had received at least one dose of radium-223 outside of a clinical trial setting. Forty-six patients had missing administration dates for radium-223, abiraterone/prednisone, or enzalutamide and were excluded only from the sub-cohort analysis. A total of 303/625 patients (48%) received radium-223 in combination with either abiraterone/prednisone or enzalutamide. Layered or concurrent treatment with radium-223 and abiraterone/prednisone was administered to 136/625 patients (22%), and radium-223 and enzalutamide to 167/625 patients (27%). Of those who received combination treatment, most  received it as a layered regimen.The median age of patients at the start of treatment was 73 years Table . The medAt baseline, 314/625 patients (50%) in the overall cohort had documented prior SSEs, with similar proportions across all four sub-cohorts. In the overall cohort, 110/625 patients (18%) experienced prior pathologic fractures, compared with 10% and 13% in the concurrent and layered abiraterone/prednisone sub-cohorts, respectively, and 18% in the enzalutamide sub-cohorts  in the overall cohort had experienced an on-treatment SSE, ranging from 20 to 36% across each of the four sub-cohorts  is investigating the efficacy and safety of concurrent treatment with radium-223 and enzalutamide compared with enzalutamide alone as first-line therapy in patients with asymptomatic or mildly symptomatic, chemotherapy-naïve mCRPC, and bone metastases. An interim safety analysis showed that although the risk of fractures was higher when enzalutamide was given in combination with radium-223 than when enzalutamide was given alone, this risk was almost eliminated when continuous BHA use was mandated . In comb42 is invLimitations of our study include that, while the Flatiron Health network has broad coverage, it does not include urologists or urology centers, and it is unknown if it fully reflects prostate cancer care and outcomes. In addition, the study was descriptive in nature, so no formal statistical comparisons were made between groups, in part because of small sample sizes; thus, there were no adjustments for potentially confounding factors. Regarding treatment, the dates on which treatment was discontinued may not always have been properly recorded. For oral therapies, data were derived both from prescriptions and pharmacy orders in the electronic health record, but whether these prescriptions were filled or refilled cannot be verified, so analysts may have considered a business rule that extended the use of an oral therapy to the start date of the next therapy. The median follow-up time of 9 months for the overall cohort and 10–13 months across all four sub-cohorts is dependent on the median OS of this patient population, and therefore is unlikely to exceed 1 year unless long-term survival increases in this patient population over the observation period. However, the observation period for the study commenced in 2013, meaning that data were collected over several years. Furthermore, there is a potential survival bias for OS, because the treatment groups in this study are defined by the treatments received after mCRPC diagnosis, but time zero is diagnosis of mCRPC. Thus, all patients had to survive long enough to receive treatment.In summary, in this retrospective study of real-world data from patients with mCRPC who received radium-223, combination therapy with radium-223 and either abiraterone/prednisone or enzalutamide was common. Layered therapy was more common than the concurrent approach used in the ERA 223 trial. Real-world data cannot be directly compared with data from phase 3 trials, but an indirect comparison shows that reported pathologic fracture rates appeared to be lower in general in the real-world setting than in clinical trials. Overall, SSE incidence rates and pathologic fracture rates were reduced with BHA use, but only 67% and 55% of patients received prior or concurrent BHAs, respectively. As experience with the use of radium-223 grows, real-world evidence can provide valuable additional information on outcomes across a range of settings to complement the findings from controlled clinical trials.


Depressive disorders in University students have risen dramatically in the past few decades to the extent that students’ mental health has become a current global public health priority. Obtaining information from University students about their mental health is challenging because of potential embarrassment of disclosing one’s concerns and fear of stigmatization. Self-rated health might be a good solution to evaluate mental health state by a simple and neutral indicator. The aim of the study is to investigate the association between depressive symptoms and self-rated health by sex among University students in France and Japan.n = 5655 in Bordeaux, France and n = 17,148 in Kyoto, Japan). Depressive symptoms , Likert scale of self-rated health, socio-demographic characteristics and health habits were collected through self-administered questionnaires. Multivariate logistic regression models were performed to describe the association between depressive symptoms and other variables including self-rated health, stratified by sex.A cross-sectional study was conducted by using two large cohorts of students aged ≥18 years (p < 0.001), it was similar in Japanese students , who reported less depressive symptoms than French students. The association between depressive symptoms and poor self-rated health was greater in Japanese females  than in males , whereas the strength of the association was almost similar in French students .A high score of PHQ-2 (high depressive symptoms) was associated with poor self-rated health in both cohorts independently of all other variables . Although the prevalence of depressive symptoms between sexes was different in French students (males 15.4%, females 25.0%, Depressive symptoms were associated with self-rated health among University students in both countries with slightly differences in sex. Self-rated health would be a simple, reliable and universal indicator for healthcare professionals and University staff to identify students at risk of depression.The online version contains supplementary material available at 10.1186/s12888-020-02948-8. Depressive disorders among University students have risen dramatically in the past few decades to the extent that students’ mental health has become a current global public health priority , 2. UnivRecently, the WHO World Mental Health Survey (WMH) Initiative  has collObtaining information from University students about their mental health is challenging because of potential embarrassment in disclosing one’s concerns and fear of stigmatization . TherefoMental health problems are twice as common in women as in men across most socio-economic contexts and regions , 21. HowThe purposes of the current comparative study were (1) to investigate the association between depressive symptoms and self-rated health by sex in two large cohorts of University students from two different countries, France and Japan, and (2) to assess whether self-rated health could be used as a simple indicator for screening University students with potential depressive symptoms across countries. We hypothesized that lower levels of self-rated health would be significantly associated with higher depressive symptoms. As a result, making a simple question on self-rated health would allow detecting students in distress, without using larger and more intrusive psychological assessment scales.This is a cross sectional study using data from two independent cohorts of University students: i-Share in France and the Kyoto University students’ cohort in Japan.www.i-share.fr) is an ongoing prospective population-based cohort study on the health and wellbeing of French University students. The Universities of Bordeaux and Versailles Saint-Quentin were the first higher education institutes to recruit voluntary participants from February 2013; other French Universities have further joined the project. Inclusion criteria are: being ≥18 years and able to read and understand French. Recruitment strategies comprise the use of flyers, information stands at University campuses, classroom-based interventions, e-mailing and social media. The i-Share project is based on an online questionnaire containing items on physical and mental health , socio-demographic characteristics and healthy habits. Three months after the completion of this questionnaire, a supplementary online questionnaire focused on mental health status is addressed to students. This questionnaire, which is still completed on a voluntary basis, asks specific information on depressive symptoms. For this comparative study, we used data available as of September 3rd, 2018. The i-Share project is carried out in accordance with the Declaration of Helsinki and has been approved by the National Commission of Informatics and Liberty , number DR-2013-019. Every student is requested to sign an online informed consent form before completion of the baseline questionnaire, according to the Committee of Protection of Persons . No further local ethical approval was needed  and 84 (6.5) for [3–6]; p value was calculated without missing values. Table S3. Multivariate logistic regression analysis stratified by Sex. Abbreviation; BMI, body mass index. Association between PHQ-2 and Self-rated health, stratified by Sex, adjusted for Sex, Age, Year of study, Sleep, BMI, Physical activity, Smoking and Alcohol consumption. Figure S1-1. Correlation between self-rated health and depressive symptoms, PHQ-2 score and numbers and percentages of students with elevated depressive symptoms in each of the five categories of self-rated health: i-Share cohort, Spearman correlation coefficient 0.261, P value < 0.001. Figure S1-2. Correlation between self-rated health and depressive symptoms, PHQ-2 score and numbers and percentages of students with elevated depressive symptoms in each of the five categories of self-rated health: Kyoto cohort, Spearman correlation coefficient 0.339, P value < 0.001.


Posttraumatic stress disorder (PTSD) is a commonly reported and serious complication among people living with HIV (PLWH). PTSD may significantly increase unintentional non-adherence to antiretroviral therapy. In this systematic review and meta-analysis, we aimed to pool the observational studies exploring the association between PTSD and medication adherence among PLWH.Comprehensive searches were conducted in PubMed/Medline, Web of Science, PsycINFO, Google Scholar, and ProQuest to identify relevant articles and dissertations. A random effects meta-analysis with inverse variance weighting was used to summarize the odds ratio (OR) across studies. Meta-regression and subgroup analyses were also carried out to assess the moderation effects for potential factors.p = 0.02). No significant publication bias was detected by Egger’s test . Factors moderating the association were mean age of participants, depression adjustment, and depression .By synthesizing 12 studies comprising 2489 participants, the pooled odd ratio of non-adherence to antiretroviral therapy was 1.19 , 1.03–1.37, This meta-analysis supports that PTSD is related to adherence in PLWH. The hypothesized mechanisms (avoidant behavior and cognitive impairment) underlying this association need further investigation. Overall, this study highlights that clinicians should thoughtfully integrate timely mental health intervention into routine care. Compared with the general population, posttraumatic stress disorder (PTSD) is a commonly reported psychiatric disease among people living with HIV (PLWH) –4. The eOne previously published systematic review has summarized the impact of different mental health disorders including PTSD on ART non-adherence . No quanPrevious studies exploring the association between PTSD and ART nonadherence adjusted several demographic and psychological confounders. A large number of studies reported that depression and substance were associated with lower level of ART adherence in PLWH –27. We tIn summary, we conducted a systematic review and meta-analysis to quantitatively pool the observational studies exploring the association between PTSD and ART adherence among PLWH. We hypothesized PLWH with PTSD would be more likely to be non-adherent to their ART. We also tried to figure out potential moderators that influence the association.This meta-analysis complied with the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) checklist .Comprehensive searches were conducted in PubMed/Medline, Web of Science, PsycINFO, Google Scholar, and ProQuest to identify relevant articles and dissertations. The search terms included an intersection of PTSD- and participant- terms. PTSD terms included “Posttraumatic stress disorder,” “PTSD,” and “trauma.” Participant terms included “HIV,” “AIDS,” and “human immunodeficiency virus.” All searches were restricted to English articles and dissertations. Additional searches were conducted in the reference lists of included articles. The detailed information for searching process is attached in Studies were considered as eligible if they met all of the following inclusion criteria:P: people living with HIV;I: not available;C: PTSD vs no PTSD;O: the association of PTSD and ART adherence (coefficient or odd ratio);S: cohort or cross-sectional studyWe used Endnote version 7 to assist the process of study selection. First, titles and abstracts were preliminarily screened to exclude irrelevant studies by JHH and TYJ. Second, full-text versions were assessed independently to ensure that all inclusion criteria were met by JHH and TYJ. Disagreement in the process of study selection was resolved by discussion with TZ. The two researchers used standardized Microsoft Excel spreadsheets to extract the following information: authors, year of publication, country of study, number of participants, study design, age, gender, education level, PTSD assessment, and adherence assessment.p<0.05 were considered as statistical significance.The primary outcome of interest was the association between PTSD and ART adherence among PLWH. Risk estimates were standardized to express odds ratios of non-adherence. A random effects meta-analysis with inverse variance weighting was used to summarize the odds ratio (OR) in CMA 2 . The extent and significance of between-study heterogeneity was assessed by I-square and Cochrane Q test, respectively . Egger’sWe used the Agency for Healthcare Research and Quality (AHRQ) checklist with a maximum score of 11 to assess the study quality for observational studies . Each itThe comprehensive search yielded 3425 items  or its revised version (IES-R); the PTSD Checklist (PCL) or its Stressor Specific (PCL-S); the Trauma Awareness and Treatment Center (TATC) PTSD Scale; the Posttraumatic Diagnostic Scale (PDS); the Startle, Physiological arousal to reminders, Anger, and Numbness (SPAN) and Mississippi (Echelle) PTSD measure.Ten studies  used self-report, and four articles  used various objective adherence measurement. Seven studies adopted Medication Adherence Questionnaire of AIDS Clinical Test Group (ACTG). Two studies adopted a three-item self-report questionnaire or a five-point Likert scale, respectively. Sauceda’s team systematically assessed the missing doses in past 7 days and past month, due medication doses, frequency of taking all scheduled doses for one month, ability to take all medicine in five questions. Two studies used the pill-counting method by the following formula: Medication adherence=number of pills actually used by the patient/number of pills that the patient should have used*100%.p = 0.02, p<0.01). No significant asymmetry was detected by Egger’s test , indicating that compared to younger participants, older PLWH was associated with lower odds of non-adherence for individuals with PTSD. Adjustment of depression status was also significantly related to PTSD-adherence association (Q(1) = 5.48, p = 0.019), indicating that compared to depression-unadjusted studies, PLWH was associated with lower odds of non-adherence for individuals with PTSD in depression-adjusted studies. The level of depression was associated with higher odds of non-adherence among participants with PTSD (Q(1) = 7.594, p = 0.006). Other factors did not significantly moderate this association. Only three studies reported substance use proportion, and we thus did not carry out moderator analysis for this variable (Mean age of participants was significantly related to the PTSD-adherence association (Q(1) = 5.59, variable –17. The As far as we know, this is the first systematic review and meta-analysis that explores the PTSD-ART adherence association among PLWH. The present meta-analysis of 12 studies found that there was elevated odds of ART non-adherence amongst HIV-infected participants with PTSD. Although the effect size for this association was small, from the perspective of public health, the result is still meaningful in making public health policy in terms of high prevalence (28%) of PTSD among PLWH worldwide . MoreoveUnderlying the relationship between PTSD and ART adherence in PLWH relates to cognitive impairments and PTSD symptoms. PLWH may already have potential impairment in multi-cognitive domains that have been linked to ART non-adherence . SpecifiAdditionally, avoidance, a specific PTSD symptom, may lead to intentional non-adherence . HIV-relAs aforementioned, our moderator analyses also shed lights on roles of participants’ age and depression. Within this narrow age range in this meta-analysis (38–55.4), older participants may develop more systematic coping strategies to confront the traumatic events. Conversely, biological ageing process and HIV infection are associated with severe prospective memory deficit which may lead to medication nonadherence , 43. We Several limitations should be mentioned. First, limited articles in potential moderators  make it hard to draw conclusion on all related factors. In addition, the reported association in this study should also interpreted in cautious due to limited studies. Second, the interactive effects between all these factors necessitate more complicated mathematical modeling methods (such as SEM). Third, only ¼ articles were categorized as high quality, which may result in potential bias. Furthermore, there was a high between-study heterogeneity. However, no group difference was detected between high vs. low quality studies. Next, we cannot draw causal relationship from this meta-analysis and exclude potential confounding effects due to the cross-sectional nature of included studies. Future studies adopting randomized controlled trials are also needed to verify the causal relationship between PTSD and ART nonadherence. Furthermore, only studies in English were accepted. And the cutoff for the p-value was not corrected for multiple comparisons, which increases the risk of false positive.In conclusion, this is the very first meta-analysis suggesting that PTSD is related to ART nonadherence among PLWH. The age of participants and depression adjustment our statistical models moderated this association. Some hypothesized mechanisms (avoidant behavior and cognitive impairment) underlying this association need further investigation. PTSD is one of the most commonly reported mental disorders among PLWH, so clinicians should thoughtfully integrate timely mental health intervention into routine care.zt_doc@ccmu.edu.cn) upon request.The data supporting this meta-analysis are from previously reported studies and datasets, which have been cited. The processed data are available from the corresponding author Dr. Tong Zhang ; the Beijing Municipal of Science and Technology Major Project ; and the Beijing Key Laboratory for HIV/AIDS Research (BZ0089). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This work was supported by the National Science and Technology Major Project: the National 13The 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.


As an emerging clinical problem, locally advanced drug-resistant gastrointestinal stromal tumors (LADRGISTs) has relatively few therapeutic schemes. Although radiotherapy is not often considered for GISTs, it could be a valuable contributing modality. The aim of our study is to explore a safe and effective radiation regimen for LADR-GISTs.Three patients with LADR-GISTs were treated with simultaneous integrated boost intensity-modulated radiation therapy (SIB-IMRT) plans. In the SIB-IMRT plans, gross target volume (GTV) was divided into GTV-outer, GTV-mid, and GTV-center. And the prescribed dose of planning gross target volume (PGTV) and GTV-outer were both set to 50.4 Gy in 28 fractions. GTV-mid and GTV-center were simultaneously boosted to 60–62 Gy and 62–64 Gy respectively. For comparison purposes, conventional IMRT (Con-IMRT) plans with uniform dose distribution were generated for same optimization objectives without a dose boost to GTV-mid and GTV-center. All plans were optimized to make sure that deliver at least 95% of the prescription dose was delivered to PGTV. Isodose distribution, dose profiles, conformity indexes (CIs), monitor units (MUs), and dose volume histogram (DVH) was evaluated for each individual patient. After the three patients were treated with SIB-IMRT plans, the relative changes in the tumor size and CT values by CT scanning were also tracked.95 to D2 of the GTV. With steeper dose gradients in the dose profiles, SIB-IMRT plans had GTV-mid and GTV-center accumulated with higher dose mainly by delivering extra 93 MUs in average. However, there was no significant difference in CIs and organs at risks (OARs) DVH. The relative changes in tumor size and CT values of the three patients in follow up were up to the Choi criteria and the three patients were all assessed as partial response.Compared with Con-IMRT plans, SIB-IMRT plans saw a significant increase from DThe proposed SIB-IMRT may be a potential technique for achieving objective response and prolonging survival of selected GISTs patients. Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm of the gastrointestinal tract, arising from the interstitial cells of Cajal. The relevant researches reported the pathogenesis of GISTs are mainly related to mutations in the tyrosine kinase receptor (KIT) and/or platelet-derived growth factor receptor alpha (PDGFRA) gene –3. CurreHistorically, GISTs have been considered to be relatively insensitive to radiotherapy, just as most other soft issue sarcomas. So far, a significant number of publications have demonstrated that GISTs are not uniformly radioresistant and radiotherapy could be beneficial to the management of GISTs. Pollock et al.  presenteAccording to the previously mentioned, radiotherapy could be regarded as a promising and viable option for GISTs. However, radiotherapy for GISTs was still limited to dose-limiting toxicity of the adjacent small bowel. In recent years, intensity-modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT) and other technological advances has realized dose escalation in target volume and potential reduction in acute and delayed toxicity by facilitating treatment delivery and normal tissue protection . MoreoveIn current clinic practice, locally advanced drug-resistant GISTs (LADR-GISTs) that are technically unresectable and failed in systemic TKIs therapies have emerged as a common clinical problem, with relatively few therapeutic schemes. To the best of our knowledge, there is no report about SIB-IMRT for LADR-GISTs. In this study, we designed a novel SIB-IMRT plan and the dose was gradually escalated from the peripheral region of GTV to the center region. The focus of this study was to compared efficacy and toxicity between conventional IMRT (Con-IMRT) plans and SIB-IMRT plans and explore a safe and practical radiation regimen for LADR-GISTs.0 resection of the primary tumor as soon as the disease was detected, and then started on systemic TKIs therapies. After a period of time (median time: 3 years), their tumors recurred due to drug resistance. Moreover, the progression of lesions were detected in all enrolled patients. Due to the resistance to TKIs therapy and lack of surgical options, they received radiation therapies to relieve symptoms . From 2016 to 2019, three patients with LADR-GISTs were treated with SIB-IMRT. The enrolled patients are 62, 50, and 56 years old at diagnosis. They underwent REach patient underwent computed tomography simulation in the supine position using GE CT scanner . The CT scan covered the total abdomen and pelvic cavity. Moreover, all patients were instructed to drink 400 ml of water and empty the rectum in one hour prior to the CT scan, and they were advised to follow the same instructions in daily radiotherapy. The gross tumor was defined as GTV. Due to a rare (1-2%) lymph-node metastases in GISTs, the lymphatic drainage of the gross tumor were not irradiated as the clinical target volume (CTV) –26. The In this study, Con-IMRT plan and SIB-IMRT plan were offered to each patient. In SIB-IMRT plan, GTV was divided into three parts  to obtain inhomogeneity dose in target volume. Therefore, the prescribed dose of PGTV and GTV-outer were both set to 50.4 Gy in 28 fractions. However, the presibribed dose of GTV-mid and GTV-center were boosted to 60–62 and 62–64Gy respectively. The detailed dose objectives of different patients were listed in 20, V30, V40, V45, V50, D1cc, and D2cc, was also compared.To perform a better analysis and evaluation, both Con-IMRT plans and SIB-IMRT plans were normalized by having 95% of the PGTV receive 100% of the prescribed dose. Dose-volume histograms (DVH) was applied for calculation and evaluation of GTV, PTV and OARs. The dose profile . Meantime, the corresponding CT values was extracted from the same area with abundant blood supply at the arterial phase. Tumor response to radiotherapy was assessed by Choi criteria. Choi criteria includes the following four response categories: complete response , partial response (PR: Decrease in tumor size ≥10% or decrease in tumor density ≥15% on CT), stable disease  and progressive disease (PD: Increase in tumor size ≥10% and does not meet PR criteria by tumor density).As shown in 30, V50, D1cc, and D2cc of SIB-IMRT plans are better than Con-IMRT plans. Although other bladder DVH indexes of Con-IMRT plans is a little bit better than that of SIB-IMRT plans, the difference is too small to be clinically significant. Similar results are seen in the rectum and the intestines. On the whole, most of OARs DVH parameters in SIB-IMRT plans were superior to that in Con-IMRT plans.DVH indexes of each OAR are listed in During applying SIB-IMRT plans, three patients were well tolerated and their symptoms caused by abdominal mass compression were gradually alleviated. Their abdominal discomfort and deleterious effect disappeared after the end of treatment. As shown in Rare intra-abdominal tumors, localized GISTs are typically treated with surgical resection. So, TKIs therapy is a recommended option for recurrent, metastatic or unresectable patients. However, it is well-known that about 40–50% of GISTs recurs after surgery. In addition, resistance to TKIs therapy is also a known clinical problem . The posHistorically, radiotherapy has been less commonly considered in GISTs due to two reasons: the moderate radiosensitivity in GISTs and the dose-limiting toxicity to adjacent intra-abdominal organs.First of all, radiotherapy is mainly used for local control of abdominal metastases and relief of symptoms . ConventSecondly, the radiotoxicity of healthy tissue is another factor of concern in GISTs. For one thing, the gastrointestinal location, patterns spread and tumor size would potentially require large abdominal fields . For anoThere are also some limitations in our study. Firstly, our sample size was small, only three patients, and the follow-up was short. That is why we reached the conclusion through observation and comparative analysis rather than statistical analysis. Secondly, despite the fact that some patients with LADR-GISTs have acquired efficacy through radiotherapy, further research is needed to make certain the optimal radiation dose schedule. Finally, although three patients did not take TKIs therapy after radiotherapy for personal reasons, radiotherapy influenced by TKIs therapy is a noticeable problem and will be an attractive topic. To sum up, it was just our preliminary study, and we will expand the sample size to continue our exploration in the future.A novel SIB-IMRT technique was designed for locally advanced drug-resistant GISTs and a heterogeneous dose distribution was escalated from the peripheral region of GTV to the center region. Compared to the Con-IMRT plans, the SIB-IMRT plans had the potential to improve the tumor response without significant increase in the radiotoxicity of the adjacent normal tissue in LADR-GISTs. Radiotherapy may be underutilized for GISTs, and SIB-IMRT technique may provide a new method for achieving objective response and prolonging survival in selected GISTs patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by the Ethical and Scientific Committees of the First Affiliated Hospital of Chongqing Medical University . The patient gave written informed consent in accordance with the Declaration of Helsinki. Written, informed consent was obtained from the patient individual(s) for the publication of any potentially identifiable images or data included in this article.YL, LL, and XY: drafting of work, analysis and interpretation of trials and literature, drafting of manuscript, and manuscript review. HC and XZ collected the data, reviewed the literature, and wrote the paper. JD and QJ prepared the figure and contributed in the revision of the literature. All authors contributed to the article and approved the submitted version.This work is funded by the Beijing Medical and Health Public Welfare Foundation “Medical Science Research Fund” project (No.: YWJKJJHKYJJ-B184084-Q14) and the Chongqing Yuzhong District Science and Technology Commission key project (No.20190101).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.


The amplitude modulation was independent of the phase relationship between the tACS signal and alpha oscillations, and the state of the visual system manipulated via closed- and open-eye conditions. It was also absent in conventionally analyzed single-channel and multi-channel data from an average parieto-occipital region. The fact that the tACS modulation of oscillations was phase-independent suggests that mechanisms driving the effects of tACS may not be explained by entrainment alone, but rather require neuroplastic changes or transient disruption of neural oscillations. Our study also supports the notion that the response to tACS is subject-specific, where the modulatory effects are shaped by the interplay between the stimulation and different alpha generators. This favors stimulation protocols as well as analysis regimes exploiting inter-individual differences, such as spatial filters to reveal otherwise hidden stimulation effects and, thereby, comprehensively induce and study the effects and underlying mechanisms of tACS.Non-invasive brain stimulation (NIBS) techniques such as transcranial alternating current stimulation (tACS) have recently become extensively utilized due to their potential to modulate ongoing neuronal oscillatory activity and consequently to induce cortical plasticity relevant for various cognitive functions. However, the neurophysiological basis for stimulation effects as well as their inter-individual differences is not yet understood. In the present study, we used a closed-loop electroencephalography-tACS(EEG-tACS) protocol to examine the modulation of alpha oscillations generated in occipito-parietal areas. In particular, we investigated the effects of a repeated short-time intermittent stimulation protocol  applied over the visual cortex (Cz and Oz) and adjusted according to the phase and frequency of visual alpha oscillations on the amplitude of these oscillations. Based on previous findings, we expected higher increases in alpha amplitudes for tACS applied in-phase with ongoing oscillations as compared to an application in anti-phase and this modulation to be present in low-alpha amplitude states of the visual system  but not high . Contrary to our expectations, we found a transient suppression of alpha power in inter-individually derived spatially specific parieto-occipital components obtained  Non-invasive brain stimulation (NIBS) technology has gained increasing attention in the last few years from the scientific community  took part in this experiment and received monetary compensation for their participation. None of the participants had a history of psychiatric or neurological diseases and none were on any current medication affecting the central nervous system. Participants were informed about all aspects of the study and gave their written informed consent before the experiment. The study protocol was approved by the local ethics committee . None of the participants have claimed that the feeling of stimulation was unpleasant, and none of them have experienced phosphenes.Each experimental session consisted of a preparation and information part, as well as the actual stimulation and EEG recording. During the preparation and information part, participants were introduced to the aim, set-up, and procedure of the study as well as the technical background of the stimulation. Individual contraindications for tACS were checked and the consent form was given, explained, and signed. tACS electrodes as well as EEG electrodes were then set up. Participants were subsequently instructed to sit relaxed, avoid movements, and later to keep their eyes opened or closed cued block-wise. The experiment included one session, which was completed by all participants. In the beginning, four 1-min resting-state EEG data  were collected to determine individual alpha frequencies (IAFs) by contrasting peaks in fast Fourier transform (FFT) spectra between EC and EO states at channel POz. Approximately five short stimulation sequences were then applied to test the correct functioning of closed-loop model units. Thereafter, 10 blocks  of 50 tACS-trials were executed consecutively with short breaks between each block. Each trial consisted of a 1 s pre-stimulation interval, 1 s of stimulation (with phase adjusted by prediction from pre-stimulation), a 1 s post-stimulation interval, and an inter-trial interval (ITI) . The rantACS electrodes (two conductive rubber 4 × 4 cm) were attached over standardized Cz and Oz channel locations Jasper,  underneaEEG data were recorded using Brain Products amplifier BrainAmp MRplus  with 31 Ag-AgCl electrodes mounted in a passive EEG EasyCap using a standard 10-20 system layout without Oz and Cz electrodes, with reference and ground electrode positioned at FCz and AFz and applying a sampling rate of 500 Hz. The low sampling rate was used to reduce data transfer delays through components of the model, and during recording, the data was streamed from BrainVision Recorder through its RDA client to BCI2000 , which then transmitted it to the Closed-Loop application for analysis and optimal phase prediction.via its the Hilbert-transform. Depending on the experimental condition a stimulation signal of length 1 s was generated that was either in-phase with the previous 250 ms of data or in the opposite phase  the state (EO vs. EC); and on (2) the phase relationship of the applied tACS signal and ongoing visual alpha-band activity (in phase vs. opposite phase).via FFT of the detrended EEG data (zero-padding up to 512 samples). Afterward, individual alpha power values were calculated by averaging power values in the range of IAF − 1 Hz to IAF + 1 Hz  for pre- and post-stimulation time windows and averaging across trials of each condition. Alpha-power values were extracted conventionally from a single parieto-occipital channel  and a parietal-occipital cluster . Also, we used a novel analysis approach tailored to account for inter-individual differences in signal representations by extracting alpha-signals, not from single channels but spatial filters weighing all electrodes to maximize signal representation (see RM) comprising the factors TIME (pre- vs. post-stimulation), STATE (EO vs. EC) and STIMULATION (in-phase vs. anti-phase) separately for all signal sources: POz data, POC data, CSP(pre) data, and CSP(post) data.The 1 s long pre- and post-stimulation data intervals for each stimulation interval were extracted and bandpass-filtered with a 5–40 Hz 4th order Butterworth zero-phase filter, to reduce the impact of unspecific high-and low-frequency noise in our data. Pre-stimulation and post-stimulation alpha-band power were extracted from 500 ms before and 500 ms after each tACS interval  data (see “Results” section) change across the time course of the experiment as responsiveness to the stimulation may change. For this purpose, pre-to post-stimulation power modulations in percent were calculated for each trial and averaged separately for each experimental block and condition. In an ANOVARM the factors BLOCK, STATE, and STIMULATION were then tested.In an exploratory post hoc analysis we wanted to investigate the time scale of post-stimulation alpha power decreases. For this purpose, we examined the temporal evolution of the decrease by comparing alpha power of a pre-stimulation time window [(−600 to −100) ms] to four different 500-ms long, overlapping, post-stimulation time windows . Pre-to-post modulations of alpha power values for the CSP(pre) data were modeled with an ANOVARM comprising the factors TIMEBIN, STATE, and STIMULATION.In a second RM models were corrected for multiple comparisons using Bonferroni correction, when necessary. In the case of a violation of the homoscedasticity, degrees of freedom were corrected based on the Greenhouse–Geisser correction. Statistical analysis was performed in R ] than post-stimulation power values . Additionally, there is a trend for an interaction of factors TIME × STATE (p = 0.058), with larger pre- to post-stimulation decreases when eyes are closed  as compared to EO . As revealed by the main effects STATE, for all signals , but are not significantly modulated by tACS or any interaction between the experimental factors  derived from the single electrode POz as well as the parieto-occipital cluster (POC), in contrast, are also modulated by the factor STATE -derived alpha power values by tACS changed across the experimental blocks. As visible in RM model revealed the factor BLOCK to be insignificant (see p = 0.031) and was larger when eyes were closed  as compared to eyes open .In an exploratory cant see . Therefop = 0.018; see Post hoc linear contrasts revealed pre- to post-stimulation decreases to be modeled best by a linear decrease (t(57) = 3.497; p = 0.001). Overall, independent of the experimental condition  the pre-to post-stimulation decreases are largest right after the stimulation and then decay across subsequent time windows , disrupted ongoing alpha oscillations. This finding is difficult to reconcile with a stimulation effect mediated by entrainment. While in animal studies online effects were found to be directly related to entrainment of ongoing neural activity by the applied electric oscillation . Effects related to neuroplastic changes seem to depend on the stimulation duration. For instance, neural excitability is longer modulated the longer tDCS was applied , this method also can be useful for discriminating between activity in the same spatial region, which was previously shown for alpha oscillations with standard and deviant visual stimuli  were utilized over the whole duration of the stimulation block or experiment  and focused our phase extraction-, stimulation- and analysis-regimes to capture and modulate parieto-occipital alpha-generators, this may not capture the richness of the underlying dynamics that may stem from an interplay of thalamic and multiple cortical alpha generators (Bollimunta et al., Closed-loop tACS and, in general, adaptive NIBS in comparison to conventional stimulation protocols have several advantages, which could be potentially beneficial for the whole brain stimulation research field and transform it into a more reliable and clinically applicable approach. As mentioned by Zrenner et al. , these aIn summary see , we founThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Ethical Committee at the Medical Faculty, Leipzig University, under the protocol “Modulation neuronaler Oszillationen mittels transkranieller Wechselstromstimulation und ihr Effekt auf die somatosensorische Wahrnehmung,” 12.08.2014, Reference number: 218-14-14072014. The patients/participants provided their written informed consent to participate in this study.GZ, CG, AV, and MB: conceptualization. GZ and CG: data curation. GZ, CG, and VN: formal analysis. AV and MB: funding acquisition. GZ, CG, and VN: investigation. GZ, CG, VN, and MB: methodology. GZ and CG: project administration, visualization, and writing—original draft. MB and AV: resources. GZ, CG, VN, and MB: software. VN, AV, MB, and CG: supervision. GZ, CG, and VN: validation. GZ, CG, VN, AV, and MB: writing—review and editing.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.


The impact of protein therapeutics in healthcare is steadily increasing, due to advancements in the field of biotechnology and a deeper understanding of several pathologies. However, their safety and efficacy are often limited by instability, short half-life and immunogenicity. Nanodelivery systems are currently being investigated for overcoming these limitations and include covalent attachment of biocompatible polymers  as well as protein nanoencapsulation in colloidal systems (liposomes and other lipid or polymeric nanocarriers). Such strategies have the potential to develop next-generation protein therapeutics. Herein, we review recent research progresses on these nanodelivery approaches, as well as future directions and challenges. Over the last few years, several therapeutic proteins have been approved for clinical usage, and others are in the process of development . NowadayFrom a therapeutic perspective, the success of therapeutic protein products is related to their increased specificity and high potency, longer duration of their effect due to the slower clearance from the body, and reduced intrinsic toxicity . These cDespite these advantages, these products must overcome the typical drawbacks of short half-life, instability, and immunogenicity, and limited permeability through the biological barriers, due to their high molecular weight . SeveralMost efforts have been devoted to the modification of the protein structure, either by mutation or by covalent attachment of moieties, including Fc-fusion , albuminThe intent of this review is to highlight the recent advances in developing nanosized delivery systems to improve safety and efficacy of protein therapeutics. This includes the areas of polymer conjugates (such as PEGylation and more recent technologies), liposomes, as well as alternative strategies based on protein nanoencapsulation in lipid-based and polymer-based nanocarriers. The advantages and limitations of systems that have reached the clinical stage are discussed, and advanced delivery strategies are also examined, aiming to provide useful insights for future development.Protein-polymer conjugates are widely used as therapeutics, since these nanosystems display a unique combination of properties derived from both materials , which can be individually tuned to obtain the desired effects . PolymerPoly(ethylene glycol) (PEG) is a synthetic, hydrophilic and FDA-approved polymer, typically synthesized using a ring-opening polymerization of ethylene oxide to produce a broad range of polymers with targeted molecular weight, narrow molecular weight distribution, and desired terminal functional groups . Due to in vivo ] and carbonates (PEG p-nitrophenyl carbonate) target the ε-amino groups of lysines, due to their abundance on the protein surface. This conjugation is generally non-selective, and other groups  can also be modified to a minor degree  and the N-terminal α-amino group of proteins (7.6 to 8) . At optiAnother functional group used for PEGylation is the thiol of cysteine residues. In this case, PEG functionalised with electron-poor olefins  are frequently used to form a thioether bond by Michael-type addition. In order to avoid non-selective coupling with amines, the reaction pH should be carried out at range of 6.5–7.5, values below lysine residues pKa . A methoO-glycosylation is a post-translational modification which occurs when a saccharide is covalently bound to a protein through a hydroxyl group of a serine or threonine. O-glycosylated proteins can be conjugated to sialic acid- functionalised PEG by sialyltransferase  and Oncaspar® (pegaspargase) are actually complex mixtures of various PEGylated species for the treatment of severe combined immunodeficiency, and adequate asparagine depletion in leukemia patients, respectively , via an unstable urethane bond that slowly releases the free protein , was approved in 2003 for the treatment of acromegaly  PEGylated erythropoietin with an extended half-life  was FDA approved in 2011 as adjuvant treatment of melanoma (melanoma  and it iAdynovate® is a PEGylated recombinant factor VIII (rFVIII) approved for hemophilia A and characterized by the prolonged circulatory half-life  is a phenylalanine ammonia-lyase (rAvPAL) conjugated with linear 20 kDa PEG-NHS. It was recently used in the clinic to treat phenylketonuria, a genetic disorder caused by a lack of phenylalanine hydroxylase causing neurotoxic phenylalanine accumulation (mulation .PEGylation diminishes immunogenicity and improves pharmacodynamic stability .ADI-PEG 20 is a arginine deiminase (rhArg) conjugate with 10–12 chains of 20 kDa SS-PEG, which has been used against glioblastoma tumor (GBM). Preliminary tests showed that ADI-PEG20 efficiently depleted blood arginine and significantly reduces the growth of GBM in mice, with the advantage that this approach does not require overcoming the blood brain barrier. Although ADI-PEG20 is still under development and not in the market, it is in phase III clinical trials for the treatment of hepatocellular carcinoma, and in phase II studies for acute myeloid leukemia/non-Hodgkin’s lymphoma and for the treatment of metastatic melanoma and some other tumors (r tumors .Filgrastim is an unglycosylated recombinant methionyl human granulocyte colony-stimulating factor (G-CSF), which regulates the production and release of functional neutrophils from the bone marrow.Lonquez® and Neulasta®) have been recently approved against neutropenia (O-glycosylation (Two similar products (tropenia . In Lonqsylation . In Neulsylation .Cimzia® (certolizumab pegol) is a PEGylated anti tumor necrosis factor (TNF) recombinant antibody Fab fragment approved for the treatment of rheumatoid arthritis, Crohn’s disease, and axial spondyloarthritis  moiety at the N-terminus via reductive amination (clerosis . Glycosymination .Refixia® (nonacog beta pegol), a PEGylated factor IX (rFIX), is used against hemophilia B. The protein is modified by a selective glycoPEGylation  and Esperoct®  are site-specific PEGylated (rFVIII) approved for the treatment of hemophilia A  cause an unexpected immunogenic response, also known as the “accelerated blood clearance (ABC) phenomenon . The preIn a recent study, pre-existing anti-PEG was identified in over 25% of healthy blood donors , in contin vitro studies demonstrated that OH-PEG is a stronger complement activator than mPEG, since the hydroxyl group is able to covalently bind to the complement component C3  (PVP) and Poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) are non-biodegradable, nonionic and non-immunogenic polymers, well-established as biocompatible drug carriers. They have been recently synthesized via Reversible Addition Fragmentation Chain Transfer (RAFT) to obtain narrow molecular weight distributions, which are ideal for bioconjugation (jugation . PVP- cojugation . HPMA cojugation .Polyglycerol (PG) showed similar characteristics to PEG in terms of non-degradability, protein repellence, and superior biocompatibility and toxicity profile ( profile . Linear  profile .Polyoxazolines (POZs) are biocompatible polymers with ‘stealth’ properties and easy renal clearance (in vivo (learance . Bioconj(in vivo . POZs wi(in vivo  obtaininN-acryloylmorpholine) (PNAM)Poly, also known as columinic acid, is a linear small polysaccharide containing α-2,8-linked sialic acid (neurominic acid) with (n = 8 to >100) residues. PSA-conjugated L-asparaginase, obtained by reductive amination, reduced the antigenicity of asparaginase and prolongs the circulation half-life in mice   , have becid (HA) . The parcid (HA) , 2012. Acid (HA) . In diabcid (HA) .Hydroxyethyl starch (HES) is a biodegradable FDA approved polymer, whose non-immunogenicity is possibly attributed to structural similarities with glycogen (in vitro and in vivo activities to PEGylated-EPO (Mircera)  . The conMircera) . Furthertectants .poly(ethyl ethylene phosphate) (PEEP) was also reported  and RAFT have been recently explored for site-specific polymer growth in aqueous solvent, ambient temperature, and physological pH, i.e., conditions that are well tolerated by biomolecules . The mai® survived for over 58 days  methyl ether methacrylate) (POEGMA), was used to treat a murine cancer model. The POEGMA-INFα conjugate completely inhibited and eradicated tumors of 75% mice without appreciable systemic toxicity, whereas at the same dose, no mice treated with the PEGASYS 58 days .N-[3-propyl] acrylamide (DMAPA) was grafted-from by Continuous Activator Regeneration (ICAR) ATRP  ATRP . The actN-isopropylacrylamide) linked with BSA (N-isopropylacrylamide)-b-poly block copolymer conjugates  polymerisation of DMAPA was successfully used on TL lipase without affecting its activity . RAFT powith BSA  and lysonjugates .Liposomes are phospholipid vesicles which consist of at least one lipid bilayer enclosing a discrete aqueous domain. While hydrophobic compounds can be inserted into the lipid membrane, hydrophilic molecules can be entrapped in the aqueous core, and this characteristic enables low and high molecular weight biomolecules to be encapsulated and later released at the targeted site . Liposomin vivo, and may be conjugated with active ligands to provide active targeting (in vivo delivery of high molecular weight compounds (such as protein/peptide drugs and nucleic acids), their nanoencapsulation is often hindered by instability issues during the liposome preparative process and storage, as well as by the low encapsulation efficiencies  as a therapy against flu. It is similar to Epaxal as it consists of unilamellar bilayer liposomes of about 150 nm, made of phosphatidylcholine, and the mixture of three monovalent virosome pools, each formed with one influenza strain- specific hemagglutinin and neuraminidase glycoproteins  is a product (FDA approved in the ’90s) composed by sterile suspension for endotracheobronchial instillation of animal-derived lipids used for the treatment of neonatal respiratory distress syndrome. This product is composed of phosphatidylcholine, dipalmitoylphosphatidylcholine and the small hydrophobic surfactant proteins SP-B (8.7 kDa) and SP-C (3.7 kDa). Through a reorganization of the lipids present in the fluid that covers the lung, the alveoli can swell more easily, thus preventing the alveolar collapse. The two proteins are essential to reduce the surface tension at the air-water interface by the formation of a surface-active film highly enriched in dipalmitoylphosphatidylcholine (lcholine . The parlcholine .T4N5 liposome lotion (Dimericine) is based on T4 endonuclease V enzyme loaded into egg lecithin liposomes. The T4 endonuclease V enzyme repairs the damaged DNA preventing the first stage of skin cancer (n cancer . Phase In cancer .Hepatic-directed vesicles-insulin (HDV-1) is a liposomal delivery system for diabetes treatment via oral and subcutaneous routes, which have been tested in phase II clinical trials  -infected tissues, to deliver IFN-α into the skin and mucosal tissues. It regards with the encapsulation of the therapeutic protein in multilayered, lipid-based submicronvesicles , synthetic, or semi-synthetic  .in vivo, since they are quickly uptaken and eliminated by mononuclear phagocyte system. PEGylation is also used in liposomes to inhibit the opsonization, thus extending blood-circulation. This effect can be modulated by the molecular weight of the PEG chains and the grafting density at the liposome surface  liposomal formulations have been studied for protein delivery, for instance as safe and effective means to deliver protein antigens (tetanus toxoid (TT), ovalbumin) to potent antigen-presenting dendritic cells for the induction of CD4+ and CD8+ T-cell response in vivo . Hemogloin vivo .Similarly to PEG-protein conjugates, PEGylation also presents undesirable effects in liposomes. For example, PEG steric effects reduce the interaction of liposomes with the cell membrane or tissue extracellular matrix when specific targeting is required . LigandsDifferent methods of liposomes preparation have been reported to optimize the drug encapsulation and to obtain a homogenous particle population, such as mechanical dispersion methods, solvent dispersion methods, and detergent removal methods . The pooin vivo applications, with results showing significant differences in terms of size distribution and encapsulation efficiency .The encapsulation of granulocyte colony-stimulating factor (rhG-CSF) was obtained by using three different preparative methods . Results showed that encapsulation efficiency increased with the size of the nanocarriers, and that these liposomes were successful in releasing rhG-CSF in rats . In thisThe encapsulation of ovalbumin (OVA), tetanus toxoid (TT), bovine serum albumin (BSA), glutathione S-transferase (GST), human gamma-globulin (hIgG) by different techniques also showed marked differences in size distribution and encapsulation efficiency . More sp2) as a non-toxic substitute for organic solvents have led to some potential applications in the pharmaceutical industry for the micro- and nano-encapsulation of drugs  higher than conventional methods  and presented smaller and homogenous particle size between 60 and 100 nm .The low encapsulation efficiency in small-sized liposomes represents a major challenge in the development of liposomal drug delivery systems for therapeutic proteins . The nanThe effect of protein interactions with the lipid membrane on the encapsulation efficiency is still a point of discussion among scientists. In fact, the protein may be surrounded by the lipid membrane or occupy the hydrophobic transmembrane region, depending on the nature of the proteins and the lipids involved, which are capable of forming electrostatic and hydrophobic interactions, hydrogen bonds, due to the polar and hydrophobic groups present in their complex structure . ComputaIn liposomes, the release of proteins is generally controlled by physicochemical mechanisms such as lipid dissociations and simple diffusion . RecentlO-loaded thermosensitive immunoliposomes were developed to release the payload when heated slightly above body temperature (Thermosensitive liposomes (TSL) are another example of “smart” nanocarriers as temperature changes can be used as “trigger” at the diseased site. Such liposomes are composed of phospholipids that present a gel-to-liquid crystalline phase transition temperature (Tm) slightly above the physiological temperature. When mild hyperthermia  is applied, the lipid bilayer will ‘melt’ to a fluid state upon arrival in the heated targeted area, and in that process liposomes rapidly release their payloads . Severalperature . Small uperature .Beside protein-polymer conjugates and liposomes, alternative nanosized systems are under development for the delivery for therapeutic proteins . AdvanceLipid-based micro- and nanocarriers such as emulsions, exosomes, non-ionic surfactant vesicles, solid lipid particles and micelles and have been studied for nanoencapsulation and transport of therapeutic proteins .Emulsions are colloidal dispersions composed of oil, water and surfactants. Depending on the formulation and manufacturing conditions, the oil-in-water or water-in-oil droplets can be small in size (microemulsion and nanoemulsions) and employed for the delivery of proteins by non-parenteral routes, such as oral and transdermal delivery  with a native membrane composition. These natural vesicles are involved in cell-to-cell communication and play an important role in the biomolecule transfer pathways. The similarities between exosomes and liposomes include the presence of the lipid bilayer (rich in cholesterol and diacylglycerol), the minimal toxicity, biocompatibility, the nanometric size and the internal volume where several biomolecules can be entrapped (in vitro (less that 40% in 24 h)  . The comin 24 h) .Niosomes are non-ionic surfactant vesicles principally composed of non-ionic surfactants and cholesterol. The particle size (from 10 nm to 20 μm) depends on the preparation method and the composition  and slow kinetics of dissociation, they do not undergo immediate dissolution after extreme dilution after intravenous injection -b-poly(l-glutamic acid) (PEG-PLE), were proposed to stabilize and to improve BDNF delivery throughout the brain .Polymersomes composed of block or graft amphiphilic copolymers have properties similar to those of liposomes, with the advantage of a higher membrane stability. The hydrophobic domain of the polymeric membrane can incorporate hydrophobic proteins/drugs, whereas the aqueous core can encapsulate hydrophilic proteins -poly(propylene sulfide) block copolymers and low molecular weight PEG was used to obtain polymersomes by a direct hydration method . EncapsuPolymer networks may be used to encapsulate hydrophilic proteins within their matrix (in vivo (r matrix . Hydroge(in vivo . Nanosiz(in vivo . Dendrim(in vivo . Recentl(in vivo . In this(in vivo . Similar(in vivo . Self-as(in vivo , and lay(in vivo  have alsNanomedicine has already demonstrated its ability to overcome some critical limitations of protein therapeutics, and we expect to provide more examples of clinically validated technologies in the upcoming years. While protein-polymer conjugates and liposomes are well-established nanosystems with a list of therapeutically approved products, various forms of protein-loaded nanocarriers of different sizes, shapes, and compositions have been explored. The use of different nanodelivery methods and the design of nanomaterials of tunable physicochemical properties, release mechanisms and targeting strategies make these alternatives very attractive. Each of these technologies has its own advantages and disadvantages. Although some of them have successfully reached the market, the delivery of therapeutic proteins at the right concentration to the right site of action, without provoking adverse side effects, still remains a major challenge. Moreover, the development of more sophisticated nanomaterials needs a deeper understanding of their physicochemical and biological properties, and of their pharmacokinetic and pharmacodynamic effects. All these requirements, together with the need of a higher control of the manufacturing process, scale-reproducibility, and the final quality of the product, pose additional challenges in regulatory terms, which need to be addressed to achieve the maximal impact in healthcare.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


P = 0.002) and cardiac death . Moreover, cystatin C-based eGFR significantly improved the risk reclassification of event with a high value of net reclassification improvement and integrated discrimination improvement. This study may prove that cystatin C-based eGFR is a better predictor of both all-cause mortality and cardiac death than other equations in populations with CTO undergoing PCI.Renal function estimated by various biomarkers predicting for adverse cardiovascular events has not been well-identified in received percutaneous coronary intervention (PCI) for chronic total occlusion (CTO), the advanced stages of atherosclerosis. We aim to determine whether the serum cystatin C-based-estimated glomerular filtration rate (eGFR) can have an improved predictive value in patients with CTO lesions undergoing PCI as compared with multiple creatinine-based estimates of kidney function. Six hundred and seventy-one patients received CTO PCI were retrospectively included in the study. The eGFR was calculated by modification of diet in renal disease equation for Chinese (cMDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations at baseline, respectively. Then, the cohort was categorized into three groups according to standard KDIGO kidney stages based on eGFR. The primary endpoint was all-cause mortality, and the secondary endpoint was cardiac death. Strikingly, cystatin C-based eGFR showed a better performance with the greater area being under the receiver operating characteristic (ROC) curve  and a better assessment for survival free from adverse event across renal levels among four eGFR equations. Compared with eGFR calculated by other formulas, serum cystatin C-based eGFR showed the highest prognostic value for both all-cause mortality (adjusted HR 3.6, 95% CI 1.6–8.1,  Renal insufficiency has been found to increase the incidence of both cardiovascular diseases and adverse outcome . EstimatRecently, a number of endogenous biomarkers, especially cystatin C, have been established and used to estimate GFR. Cystatin C is a 13-kDa protein and a member of the family of competitive lysosomal cysteine protease inhibitors. It can be freely filtered by the glomerulus. Compared to creatinine-based eGFR, cystatin C depends less on health status, muscle mass, or other demographic characteristics and appears to be more consistent across cohorts. It has been proved to be a more reliable and sensitive marker for estimating GFR in patients with normal creatinine-based eGFR  or with Renal dysfunction can be estimated by various equations and has been identified as an important predictor of mortality for patients with coronary heart disease after percutaneous coronary intervention (PCI) and surgical revascularization. Chronic coronary total occlusion (CTO) is advanced atherosclerotic lesions and represents a challenging subset of coronary artery disease. However, the value of eGFR in predicting adverse cardiovascular events has not been well-established within the patients with CTO after PCI. This deficiency forms the basis for this study, aiming to determine whether or not the decreased eGFR calculated by cystatin C can have a higher risk predictive value for assessing risk and risk grading in terms of all-cause mortality and cardiac death in CTO cohort available in our Cardiology Department.A total of 762 patients with CTO lesions confirmed by coronary angiography in the Cardiology Department of the First Affiliated Hospital of Xi'an JiaoTong University  undergoing PCI between June 2013 and October 2017 were included in the study. The follow-up for all-cause mortality and cardiac death was carried out via telephone contacts between 2018 and 2019. The study was approved by both Research and Ethics Committees of the First Affiliated Hospital of Xi'an JiaoTong University. Recanalization of CTO lesion was performed according to current guidelines  by the h2) was calculated on the basis of weight and height. Blood pressure (BP) was measured three times or more by a nurse while patients were seated after 5 min of rest. Patients either with persistent BP>140/90 mmHg or those currently taking antihypertensive drugs were considered hypertensive. Presence of type 2 diabetes mellitus (T2DM) was determined by means of a medical history of diabetes, fasting glucose levels >126 mg/dL (7.0 mmol/L), and/or glycated hemoglobin (HbA1c)>6.5%. Several parameters, including low-density lipoprotein-cholesterol (LDL-c), HDL cholesterol (HDL-c), and C-reactive protein, were measured. Serum levels of cystatin C were determined by an automated particle-enhanced immunoturbidimetric method on a Siemens Dade Behring BN II Nephelometer. Renal function was assessed by means of serum levels of creatinine and cystatin C at baseline and calculated by MDRD for the Chinese equation   as shown in The cohort was categorized according to its prognostic category, defined by an existing classification system—the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines —that is,https://www.r-project.org) were used for conducting statistical analyses, and a two-sided P < 0.05 was considered statistically significant. There is no adjustment for multiplicity.Continuous variables are presented as the mean ± standard deviation if normally distributed or median  otherwise, and Shapiro–Wilk was used for normality test. Categorical variables are presented as numbers and percentages. Differences in the parameters among groups were analyzed using analysis of variance (ANOVA) for normally distributed variables, the Kruskal–Wallis test was used for non-normally distributed continuous variables, and the chi-square test was used for categorical variables. We assessed performance through the receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC). Clinical event rates were compared with the Kaplan–Meier method using the log rank test for comparisons between groups. Cox proportional hazards regression models were used in univariate analyses and multivariate analyses to determine the prognostic value of different kidney stage levels estimated by each eGFR equation. Multivariate analyses adjusted for significant baseline variables and the factors closely related to the outcome of patients with cardiovascular disease, such as age, sex, smoking, BMI, diabetes mellitus (DM), hypertension (HT), and low-density lipoprotein cholesterol (LDL-c), left ventricular ejection fraction (LVEF), C-reactive protein (CRP), and procedural success. Net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were performed to analyze the degree to which eGFR improved the predictive ability of the eGFRcys. Both NRI and IDI values were analyzed with their 95% CI. SPSS version 25.0.0  and R version 3.6.1  with fewer men (p < 0.001) and had a higher prevalence of smoking (p < 0.01), a lower  ejection fraction (p < 0.001), and increased levels of CRP as well. Procedural success rate, prevalence of DM and HT, serum levels of LDLc, and other baseline parameters did not differ among the three groups.The baseline characteristics of the cohort are shown in Levels of renal function estimated by each eGFR equation  were classified according to the risk event of all-cause mortality and cardiac death . All theThe accuracies of eGFR-cMDRD, eGFRcys, eGFRcre, and eGFRcre-cys for predicting all-cause mortality and cardiac death are shown as the AUC in Kaplan–Meier curves were used to illustrate the survival free from adverse events. Overall, patients at a lower eGFR levels had a significantly worse outcome of survival free from all-cause mortality during the follow-up period . eGFRcysP = 0.002) was a stronger predictor than eGFRcre , eGFRcre-cys , and eGFR-cMDRD  for the risk of all-cause mortality for moderate-to-severe reduction in GFR (eGFR <60 ml/min/1.73 m2). Likewise, cystatin C-based eGFR  was able to predict the risk of cardiovascular mortality better than eGFRcre , eGFRcre-cys , and eGFR-cMDRD  for moderate-to-severe reduction in renal function.We performed Cox regression hazard models on eGFR calculated by each equation as the categorical variable grouped as previously mentioned. The risk of each adverse outcome was assessed by comparing Group 1 (reference) with Groups 2 and 3 in every equation. Cystatin C-based eGFR . On the other hand, eGFRcys showed an increased risk rate of 3.6-folds, respectively, as compared with that of the population without CKD. This means that cystatin C is able to improve the predictive value by 111.7% (for the eGFRcys) in patients with moderate-to-severe reduction in GFR in terms of all-cause mortality. Likewise, cystatin C-based eGFR may improve the estimation of risk by 26.1% (for the cystatin C-based equation) in terms of cardiovascular mortality, as compared with those estimated with other equations. A forest plot summarizing these data is shown in The above data showed that in terms of outcome of all-cause mortality, eGFRcre was able to estimate an increased risk that was 1.7 times higher than that of the population without CKD . Overall, the reclassification of other equations performed worse both on all-cause mortality and cardiac death . CystatiThis study showed an increase in all-cause mortality and cardiac death after CTO PCI as baseline renal function declined. Patients with renal insufficiency represent a high-risk patient subset. The values of the four measures showed similar accuracy using the area under the receiver operating characteristic curves. The classification into standard kidney stages showed significant differences in the survival curves and Cox regression models for both endpoints.p < 0.001) and increased levels of CRP. We also found some HR p-values which are significant in the univariate analyses become non-significant after adjusting for confounders, and this statistical phenomenon mostly occurred in creatinine-based eGFR. The true causal effect between eGFR values and outcome is affected by confounders  when compared with the individuals with normal renal function (>90 ml/min/1.73 m2). The main finding in our research is that the eGFRcys performed better than other three eGFR equations in assessing the adverse cardiovascular risk in both cardiac death and all-cause mortality in our CTO population.The main novelty of our research centers on the fact that it was specifically performed in patients with CTO. To the best of our knowledge, this is the first study to address the prognostic usefulness of eGFRcys, by comparing it with eGFRcrea, eGFRcre-cys, and eGFR-cMDRD in patients that received CTO PCI. The analysis of our series of patients suggested that eGFRcys might be a better predictor for cardiovascular events in patients with renal impairment (<60 ml/min/1.73 m2) and in patients with a mild renal insufficiency (60–89 ml/min/1.73 m2). Recently, Shlipak et al. (2). Pedro et al. (Consistent with our findings, Shlipak et al. , 20 founo et al.  providedo et al.  and Lim o et al.  found tho et al.  also fouo et al. . Thus, aOne strength of our research was the use of eGFRcys to estimate the risk of cardiac death and all-cause mortality with standard statistical methods, which could be better than the use of the other equations. It should be noted that of more significance to our study was the assessment of a distinct subset of population, i.e., CTO patients. Our results may provide physicians with a support for the need of reconsidering which eGFR equation is the best one for assessment of long-term adverse events in CTO patients.The research is a single-center retrospective observational design and is restricted in generalization of the findings. Future multicenter prospective study with a larger number of patients and a longer follow-up is required.This study has demonstrated that all-cause mortality and cardiac death are increased across stages of renal dysfunction, and the decreased eGFR calculated with cystatin C has a higher risk predictive value to assessing risk and risk-grading in terms of all-cause mortality and cardiac death in our CTO population than eGFR calculated by other three equations.The data generated during this study are included in this article. Raw data are available upon reasonable request.The studies involving human participants were reviewed and approved by Research and Ethics Committees of the First Affiliated Hospital of Xi'an JiaoTong University. The patients/participants provided their written informed consent to participate in this study.BL: conceptualization, project administration, writing - original draft, and writing - review and editing. JR: methodology, project administration, data analysis, and writing - review and editing. BW, KG, Y-MH, SL, and ZJ: investigation and data curation. XW: data curation and writing - review. HL: resources and data curation. LC: writing - review and editing. YZ and HH: writing - review and editing and data curation. YP: data curation. YW: resources, funding acquisition, and writing - review and editing. X-ZZ: supervision, project administration, and writing - review and editing. All authors contributed to the article and approved the submitted version.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.


Klebsiella pneumoniae is one of the most important pathogens concerned with multidrug resistance in healthcare-associated infections. The treating of infections caused by this bacterium is complicated due to the emergence and rapid spreading of carbapenem-resistant strains, which are associated with high mortality rates. Recently, several hypervirulent and carbapenemase-producing isolates were reported that make the situation even more complicated. In order to better understand the resistance and virulence mechanisms, and, in turn, to develop effective treatment strategies for the infections caused by multidrug-resistant K. pneumoniae, more comprehensive genomic and phenotypic data are required. Here, we present the first detailed molecular epidemiology report based on second and third generation (long-read) sequencing for the clinical isolates of K. pneumoniae in the Russian Federation. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as the virulence and plasmid profiling for 36 K. pneumoniae isolates. We have revealed 2 new multilocus sequence typing (MLST)-based sequence types, 32 multidrug-resistant (MDR) isolates and 5 colistin-resistant isolates in our samples. Three MDR isolates belonged to a very rare ST377 type. The whole genome sequences and additional data obtained will greatly facilitate further investigations in the field of antimicrobial resistance studies. Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacterium, being one of the six most important healthcare-associated pathogens (ESKAPE) which often exhibit multidrug resistance and/or are highly virulent, thus causing major healthcare concerns. The virulence of K. pneumoniae is determined by numerous factors including the polysaccharide capsule that allows the bacterium to evade phagocytosis by the host [K. pneumoniae carry genes associated with life-threating invasive diseases [the host , type 1 the host . Severaldiseases . K. pneumoniae remains unknown [K. pneumoniae isolates resistant to this drug have already been reported [The treating of infections associated with this bacterium is also complicated due to the emergence of carbapenem-resistant strains revealed in the mid-2000 s. This infection has spread around the world, and it represents a serious problem due to its high mortality rate, which can sometimes exceed 50% . Making  unknown . Colistireported , and morreported . Five suK. pneumoniae resistomes determined by traditional and in silico methods were conducted [The development of novel antimicrobial drugs and therapies is hindered by the fact that many resistance mechanisms have not yet been revealed, although the increasing availability of whole-genome sequencing (WGS) greatly facilitates such investigations. Thus, additional data linking antimicrobial resistance determined by traditional methods with genomic traits are required. A number of studies comparing onducted ,10,11,12onducted ,10. HoweK. pneumoniae isolates belonging to various sequence types (ST), including two novel ones not described before. Among these isolates, 32 were multidrug-resistant (MDR) according to the commonly used definition [In this paper, we provide the comprehensive data for 36 clinical finition . We provK. pneumoniae developed in 2005 [gapA , infB (translation initiation factor 2), mdh , pgi (phosphoglucose isomerase), phoE (phosphorine E), rpoB (beta-subunit of RNA polymerase) and tonB (periplasmic energy transducer), and a comparison of their allelic profiles with the ones from public databases [K. pneumoniae are contained in the Institut Pasteur MLST system [The MLST scheme for  in 2005  includesatabases . CurrentT system .K. pneumoniae included the ones based on capsular  loci and oligosaccharide (O) loci. The current versions of the corresponding databases contain about 100 KL-type profiles and 12 O-profiles [Additional isolate typing schemes for profiles . KL1–KL7There are many other available typing schemes, for example, based on sequence properties , but in The data also comprises antimicrobial resistance information, including plasmid determination, virulence genes’ presence and several typing schemes. The resistance/susceptibility data were acquired by both traditional methods and bioinformatics-based methods for the bacterial whole genomes obtained by second and third generation (long-read) sequencing.In Three isolates  possessed novel combinations of MLST-gene alleles, i.e., a new sequence type (ST), between which P-212 and P-259 had the same ST. The last two samples were taken from the same patient, but from different sites and in different times to ensure the presence of a novel ST. We have submitted these isolates to the Institut Pasteur MLST system , and newIn order to obtain additional information regarding bacteria similarity, a maximum likelihood tree was built based on the core genome of 36 isolates containing 3888 genes. The tree is presented below in It is worth noting that the KL-type provides additional classification narrowing in comparison with the sequence type alone. For example, P-183 forms a standalone clade since it possesses the KL-type (KL23) different from its neighbors’ type (KL24), although they share the same ST (ST11). Thus, such a hybrid typing scheme could be applied for a precise distinguishing of the isolates in clinical settings, which is an important task in epidemiological surveillance and infection prevention.We have tested the isolates for antimicrobial drug resistance both by empirical (disc diffusion) and theoretical (searching for known acquired resistance genes in genomic sequences) methods. Although the empirical methods are more reliable, they undoubtedly require more time and resources. When the time is a critical factor, e.g., for patients with a severe unidentified bacterial infection, a quick bioinformatics scan for resistance based on a whole-genome sequence may become a promising option for future studies using third generation sequencers like MinION. However, currently, this technology cannot be readily implemented in routine clinical diagnostics since it allows only detecting the presence of some gene, but cannot reveal its expression or activation state. K. pneumoniae isolates. The data obtained are summarized in Thus, in order to facilitate the development of such novel point-of-care resistance determination systems, a comprehensive comparison of empirical and theoretical susceptibility determination is required. We have performed such a comparison for all 36 Klebsiella bacteriophages, and were susceptible only to moxifloxacin  and nalidixic acid . P-28 was resistant to all antimicrobials from the extended panel.Phenotype studies have shown that 32 of the 36 isolates were MDR. Three isolates  were resistant to 12 antimicrobial drugs that have been tested for all the isolates. Additional studies have shown that P-28, P-29 and P-237 were resistant to many other drugs, including commercially available K. pneumoniae [K. pneumoniae infections [Five isolates, including P-28, P-29 and P-237 mentioned above, were colistin-resistant, which is rather rare for fections , this seK. pneumoniae [blaCTX-M-15 (21 isolates), blaTEM-1B (20 isolates), blaOXA-1 and blaOXA-48 . In addition, 26 isolates were resistant to ciprofloxacin, and this resistance is reflected in the presence of the aac(6′)-lb-cr gene, which is known to provide fluoroquinolone resistance in Enterobacteriaceae [All isolates were ampicillin-resistant, which corresponds well with their possession of SHV-type beta-lactamase genes revealed by bioinformatics methods. These genes are known to be chromosome-encoded and provide ampicillin resistance for eumoniae . Most oflaTEM-1B  isolatesDespite the fact that the antimicrobial resistance phenotypes and genotypes correlated in general, some discrepancies were found. This is not surprising because the presence of a resistance gene in a genome cannot be considered as sole evidence for its expression and activity in the given isolate. In addition, colistin resistance genes were not revealed by bioinformatics methods. However, ColRNAI plasmids known to carry such genes  were fouSince plasmids play a key role in acquired antimicrobial drug resistance, it is important to reveal the plasmid sequences in the WGS data. This information will greatly facilitate the investigations of resistance mechanisms. We have performed an in silico search for plasmids in all genomic sequences of the isolates. In addition, sequencing with long reads usually provides necessary resolution to separate genomic and plasmid sequences during reads’ assembly to contigs , so we hThe plasmid data correspond with the antimicrobial resistance data obtained by the traditional and in silico methods. For example, IncF-type plasmids revealed in most isolates are commonly reported worldwide to carry carbapenemase genes and quinolone resistance genes , which wmcr-1 and mcr-3 plasmid-mediated colistin resistance genes in E. coli [mcr genes have not been revealed in P-26, P-28, P-29 or P-200, which suggests some different resistance mechanism. In P-237, the plasmid has not been revealed either. However, the long-read sequencing of the selected samples confirmed the possibility of the plasmid mechanism of antimicrobial resistance acquisition to other antibiotics in most isolates.The ColRNAI plasmid carrying colistin resistance genes was revealed in three MDR  isolates belonging to the ST377 sequence type. This plasmid was also found in hypervirulent P-108 and P-140, but their phenotypic resistance/sensitivity to colistin was not confirmed, as well as for P-152, since we have not performed the serial dilution approach to confirm their sensitivity. Nevertheless, the isolates having this plasmid require additional attention. At the same time, the other colistin-resistant isolates P-26 and P-200 had an IncH plasmid that is associated with the  E. coli ,24. HoweIn general, although the long-read sequencing allowed to obtain the reliable whole-plasmid sequences, it did not provide additional information regarding plasmid typing. Deeper investigations of plasmid sequences and structure are required to get additional insights in resistance transfer mechanisms, but such studies lie beyond the scope of this manuscript.Besides the antibiotic resistance data, another important piece of information is the virulence of the investigated isolates that represents the degree of damage caused by a bacterium to its host. Although the bacteria of some sequence types, especially ST23, were considered to be hypervirulent [K. pneumoniae has many virulence factors, for example, lipopolysaccharide, fimbriae, outer membrane proteins and determinants of iron acquisition and nitrogen source utilization [lization .magA) and K2 (wzi) serotypes, together with seven other virulence factors  are considered important [K. pneumoniae virulence include outer membrane proteins (porins) and the allantoin metabolism system [Several panels of genes encoding the virulence factors were proposed. Usually, the genes specifying the K1  and entB (enterobactin), while mrkD  and ybtS  were rather common as well. In addition, most isolates (from 26 to 33 for different genes) contained other genes of the mrk cluster, which are involved in type 3 fimbriae formation [rmpA (regulator of mucoid phenotype A) gene was rare and was revealed only in two isolates  belonging to the hypervirulent ST23 type. This high degree of virulence was also supported by the fact that only these two isolates possessed 6 of 7 virulence genes studied. Genes from the kfu cluster (iron uptake system) were revealed in four isolates, including two (P-212 and P-259) possessing the novel sequence type ST4061. The aerobactin transporter gene (iutA) was found in 10 isolates. A half of all isolates contained four or more virulence factors, suggesting that they could be dangerous in clinical settings, as they are also MDR, and require further investigations.All isolates possessed K. pneumoniae in the Russian Federation. In this study two new MLST-based sequence types represented by one and two isolates, respectively, were revealed. Both sequence types were deposited to the MLST database of the Institut Pasteur  [To the best of our knowledge, this is the first detailed molecular epidemiology report based on second and third generation whole-genome sequencing for clinical isolates of  France)  as ST406Our study has confirmed the negative tendency of carbapenemase-producing pathogens’ spread in various Russian high-technological medical centers described recently  and in eIt is also interesting that no ST258 isolates were revealed, although this sequence type is rather common in Europe, North America and Australia . Howevermcr genes in colistin-resistant isolates did not reveal any results, but the P-29 isolate carried a C->A mutation in position 29 of the mgrB gene, leading to a C39F amino acid substitution, which, in turn, could lead to function deactivation according to the PROVEAN server (http://provean.jcvi.org/index.php). Other isolates exhibited a wild-type mgrB, so the mechanisms of their resistance remain to be elucidated. This partially complies with the results obtained in recent colistin resistance investigations of K. pneumoniae from Greece [Thirty-two of 36 (89%) isolates expressed an MDR phenotype, 18 of them (50%) were resistant to carbapenems, and 5 to colistin (14%), while 3 were resistant to the whole panel of 12 antibiotics belonging to various classes. The in silico screening for the m Greece . In addiIn most cases, there was a good compliance between the phenotypic and genotypic antibiotic resistance, but some discrepancies were revealed, in particular, for colistin, which suggests a possible resistance mechanism based on the chromosomal gene inactivation in such cases.K. pneumoniae genomes (down to one contig for each genome), including the distinguishing of the plasmid sequences. The data obtained will facilitate future studies of antimicrobial resistance mechanisms in Enterobacteria.Long-read sequencing on MinION  allowed to greatly improve the assembly of 14 K. pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM) and oxacillinases-48 (OXA-48) types of carbapenemases, antibiotic-resistant hvKP isolates have begun to emerge in recent years [In general, the prevalence of antibiotic resistance in the isolates with the hypermucoviscosity phenotype  is rare compared with the high prevalence of antibiotic-resistant isolates ,32. Howe™ DNA Sample Prep Kit , and the WGS of all 36 isolates was performed on Illumina® Hiseq or MiSeq platforms. Thirty-six patient samples were obtained from 34 patients  in various sources and clinical departments see  of a mulThe WGS was also performed using the Oxford Nanopore MinION sequencing system . DNA was used to prepare the MinION library with the Rapid Barcoding Sequencing kit SQK-RBK004 . The amount of initial DNA used for the barcoding kit was 400 ng for each sample. All mixing steps for the DNA samples were performed by gently flicking the microfuge tube instead of pipetting. All libraries were prepared according to the manufacturer’s protocols. The final library was sequenced on an R9 SpotON flow cell. The standard 24 h sequencing protocol was initiated using the MinKNOW software . Fourteen of 36 isolates considered interesting according to various criteria were sequenced.Base calling of the raw MinION data was performed with the Guppy Basecalling Software version 2.3.1 . Demultiplexing was made using Deepbinner version 0.2.0 . AssemblThe statistics showing the parameters of the isolate sequencing and assembly are shown in Ethical approval was not required as human samples were routinely collected and patients’ data remained anonymous. The planning conduct and reporting of the study was in line with the Declaration of Helsinki, as revised in 2013.http://www.antibiotic.ru/minzdrav/files/docs/clrec-dsma2015.pdf) which are based on EUCAST 2015.All isolates were identified down to a species level by time-of-flight mass spectrometry (MALDI-TOF MS) using the VITEC MS system . The susceptibility was determined by the disc diffusion method using the Mueller–Hinton medium  and disks with antibiotics , and by the boundary concentration method on a VITEK2Compact30 analyzer . The isolates were tested for susceptibility/resistance to the following drugs: amikacin, ampicillin, aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, colistin, doripenem, gentamicin and tigecycline. Imipenem and meropenem were also tested, but yielded the same results as doripenem in terms of resistance/susceptibility, so they are not included in the output results. The panel of antimicrobial compounds included for testing in this study reflected those agents used for human therapy in the Russian Federation. To interpret the results obtained, we used the clinical guidelines “Determination of the susceptibility of microorganisms to antimicrobial drugs”, version 2015-02   was perfcharides , which acharides  to searcTo build the phylogenetic tree representing the relations between the isolates, Prokka  was usedK. pneumoniae isolates from Moscow, Russia, among which 32 isolates exhibited an MDR phenotype. Although WGS in general, and long-read sequencing in particular, allows getting some insights into the resistance transmission and spreading of pathogenic isolates across hospital departments, more research, which is currently underway, is needed to analyze the mechanisms of the resistance acquisition and virulence of these isolates.In conclusion, here we presented a general report containing various types of molecular-based typing, antibiotic resistance profiling, virulence factors and plasmid descriptions for 36 clinical