This study aimed to evaluate both publication and authorship characteristics in Knee Surgery, Sports Traumatology, Arthroscopy journal (KSSTA) regarding knee arthroplasty over the past 15 years.PubMed was searched for articles published in KSSTA between January 1, 2006, and December 31st, 2020, utilising the search term ‘knee arthroplasty’. 1288 articles met the inclusion criteria. The articles were evaluated using the following criteria: type of article, type of study, main topic and special topic, use of patient-reported outcome scores, number of references and citations, level of evidence (LOE), number of authors, gender of the first author and continent of origin. Three time intervals were compared: 2006–2010, 2011–2015 and 2016–2020.p < 0.001). Interest in navigation and computer-assisted surgery decreased, whereas interest in perioperative management, robotic and individualized surgery increased over time (p < 0.001). There was an increasing number of references  while number of citations decreased . LOE showed no significant changes (p = 0.439). The number of authors increased between each time interval (p < 0.001), while the percentage of female authors was comparable between first and last interval (p = 0.252). Europe published significantly fewer articles over time (56% vs. 47% vs. 52%), whereas the number of articles from Asia increased .Between 2016 and 2020, publications peaked at 670 articles (52%) compared with 465 (36%) published between 2011 and 2016 and 153 articles (12%) between 2006 and 2010. While percentage of reviews (2006–2010: 0% vs. 2011–2015: 5% vs. 2016–2020: 5%) and meta-analyses (1% vs. 6% vs. 5%) increased, fewer case reports were published (13% vs. 3% vs. 1%) (Increasing interest in the field of knee arthroplasty-related surgery arose within the last 15 years in KSSTA. The investigated topics showed a significant trend towards the latest techniques at each time interval. With rising number of authors, the part of female first authors also increased—but not significantly. Furthermore, publishing characteristics showed an increasing number of publications from Asia and a slightly decreasing number in Europe.IV. Although knee arthroplasty is a successful procedure to treat end-stage osteoarthritis, 20% of patients are not satisfied with their outcome . TherefoKnee surgery, sports traumatology, arthroscopy (KSSTA) journal represents an orthopaedic high-impact journal which requires high research standards for publication achieving an impact factor of 3.166 in 2019  in KSSTA journal between January 1, 2006, and December 31, 2020. The following search terms were used:“Knee surgery, sports traumatology, arthroscopy: official journal of the ESSKA”[Journal] AND ((“2006/01/01”[Date—Publication]: “2020/12/31”[Date—Publication])) AND (knee arthroplasty).n = 309) were excluded. An overview of the remaining 1288 articles  is shown in Fig. In total, 1597 articles were identified, representing 25% of all 6289 published articles in KSSTA within the chosen time interval  AND ((“2006/01/01”[Date—Publication]: “2020/12/31”[Date—Publication])))). Articles not related to the topic of knee arthroplasty  was recorded . FurtherContinent of origin, number of authors and gender of the first author were analysed. A Google search of all first authors was performed to identify their gender. If proper author identification was not possible, the Google database was used to analyse common name patterns. If the author’s gender could not be identified with either method, the gender was marked as “unknown”. Authorship characteristics were, additionally to interval analysis, evaluated by year to detect even minimal changes in publishing characteristics especially regarding the number and gender distribution of authorship.The number of used references, number of times an article was cited (= citations) in Google Scholar search. The level of evidence (LOE) reported by KSSTA was documented as well. Additionally, the number of cases and study design  were documented for every original research article.If reported, the type of patient-reported outcome measurement (PROM) was documented, namely the Knee Society Score (KSS), (Oxford Knee Score (OKS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Short Form-12 (SF-12), Short Form-36 (SF-36), EuroQol 5D (EQ-5D), Visual Analogue Scale (VAS), Hospital for Special Surgery Score (HSS), American Knee Society Score (AKSS), Knee Injury and Osteoarthritis Outcome Score (KOOS), Forgotten joint Score (FJS), University of California at Los Angeles (UCLA), Knee Society Knee Score, Knee Society Function Score (KSKS & KSFS) and others.p < 0.05 was considered significant.SPSS Version 26 (IBM®) was used for statistical analysis. Comparison of the publication characteristics of nominal variables was performed using chi-squared or Fisher’s test. The Kruskal–Wallis test was used for comparisons of publication characteristics over time involving count variables. The data distribution of each metric parameter was checked using the Kolmogorov–Smirnov test. If data showed no normal distribution, statistical evaluation was expressed as medians [with ranges]. If the data showed a normal distribution, the results were presented as means KSSTA showed an increasing number of publications concerning knee arthroplasty-associated themes over the last 15 years , followed by meta-analyses , systematic reviews , and case reports . Editorials made up 1.6% (n = 21), errata 1.1% (n = 14), letters to the editor 0.9% (n = 11) and instructional letters 0.5% (n = 6) of all knee arthroplasty related publications between 2006 and 2020.Most of the articles published within the last 15 years were original articles  and meta-analyses (0.7% vs. 5.2%) while fewer case reports (13.1% vs. 1.5%) were published (n = 749), followed by biomechanical/kinematic studies , clinical prognostic studies  and clinical diagnostic studies . Anatomical studies represented 5.7% (n = 74), clinical economic studies 0–7% (n = 9) and 0.5% were basic sciences studies (n = 6). Nine studies did not meet any of this topic (0.7%). Whereas the percentage of clinical therapeutic studies slightly decreased from 59.5% (n = 91) between 2006 and 2010 to 57% (n = 382) between 2016 and 2020, the raw numbers of published clinical therapeutic articles on knee arthroplasty increased tremendously. There was an increase in clinical prognostic studies from 5.9% (n = 9) to 12.7 (n = 85) whereas clinical diagnostic studies  and biomechanical/kinematic studies  showed no relevant changes over the three time intervals , followed by UKA  and patellofemoral joint replacement . Overall, 5.6% of the published studies in the KSSTA journal focused on revision surgery in knee arthroplasty. Comparison of the three time intervals revealed that the topic of TKA was covered in 78.4% (n = 120) of the articles from 2006 to 2010, 77.8% (n = 362) between 2011 and 2015, and 71% (n = 476) between 2016 and 2020. Regarding UKA, 11.1% (n = 17) of all the articles published between 2006 and 2010 treated this topic, 11.4% (n = 53) between 2011 and 2015, and 12.4% (n = 83) between 2016 and 2020. These changes did not reach statistical significance (p = 0.187).Most studies evaluated the field of TKA  of the articles researched navigation and computer-assisted surgery, and 4.9% (n = 33) focused on that topic from 2016 to 2020. Additionally, interest in perioperative management increased from 0.2% (n = 3) to 4.9% (n = 33), interest in rehabilitation and general outcome increased from 13.1% (n = 20) to 24.7% (n = 165) and interest in patient-specific instrumentation and implants increased from 0.7% (n = 1) to 6.3% (n = 42) between 2006 until 2010 and 2016 until 2020 (p < 0.001). Covid-19 associated research reached 1% (n = 5) of all publication within the latest interval of 2016 until 2020. The topic of robotic-assisted surgery also just arose within the last interval between 2016 and 2020 .Figure n = 651), followed by Asia with 39.7% (n = 511) and North America 6.9% (n = 89). The number of publications from Europe decreased from 56.2% (n = 86) of all publications in 2006–2010 to 51.8% (n = 347) in 2016–2020 (Table n = 54) to 45.4% (n = 211) between 2011 and 2015 and again returned to 36.7% (n = 246) between 2016 and 2020. North America increased from 3.9% (n = 6) of all publications between 2006 and 2010 to 9% (n = 60) between 2016 and 2020. Those changes were significant (p = 0.005). Table Most of the publications were from Europe . This significant rise is demonstrated in detail by Fig. The average number of authors was 5 [1–9] between 2006 and 2010, 5 [1–14] between 2011 and 2015 and 6 [1–19] between 2016 and 2020 (n = 1090) of the remaining authors publishing in KSSTA journal about knee arthroplasty within the last 15 years were male. The percentage of female authors decreased between 2006 and 2010  and between 2011 and 2015  and increased significantly between 2016 and 2020 .In 85 cases (6.6%), gender identification of the first author was not possible. Most  whereas no significant change in the number of references was seen between 2011 and 2020 (p = 0.192). Number of times an article was cited significantly decreased between each time interval , only a comparison between the time ranges of 2011–2015 [2.7 ± 1.1 (1–5)] and 2016–2020 [2.8 ± 1 (1–5)] was performed. The LOE showed no significant change over time (p = 0.439).Because the LOE was not reported regularly from 2006 to 2010 (p = 0.001). Between 2006 and 2010 average of 115 [5–1474] cases were included. 2011 and 2015 193 cases [6–14785] and 2016 and 2020 1565 cases [1–290601].Evaluating the number of cases examined in the original articles, a significant increase was observed over all intervals (n = 34) vs. 43.6% (n = 222) whereas percentage of prospective study design decreased meanwhile from 67.5% (n = 77) to 51.3% . Cadaver studies constantly increased from 2.6% (n = 3) between 2006 and 2010 to 5.1% (n = 26).A significant increase was found in the percentage of retrospective study designs between 2006–2011 and 2016–2020 (29.8% (n = 527) of the publications reported the use of PROM. The overall use of PROM increased over time (2006–2010: 35.9% (n = 55); 2011–2015: 37.2% (n = 173); 2016–2020: 44.6% (n = 299); p = 0.018). In total, the KSS was the most used PROM , followed by the WOMAC score  and OKS . While the use of the KSS (32–25%) and HSS (12–3%) decreased over the three time intervals, the use of the OKS (5–14%), WOMAC score (13–15%), KSKS & KSFS (2–5%) and KOOS (1–6%) increased , offering researchers more academic visibility. Furthermore, publication in KSSTA became more attractive over time regarding the increasing impact factor within the last 15 years , resulting in a higher prestige of published articles and better academic visibility.The British Medical Journal in 2015 [Although case reports have become rare within the last 15 years, the publication of systematic reviews as well as meta-analyses has increased considerably. This trend was not only observed in KSSTA but also was already described in an editorial in  in 2015 . The num in 2015 . These fAmong the original articles, a notable shift concerning the special topics of published articles was observed. Although interest in patient-specific implants and instrumentation, TKA positioning and alignment have increased constantly, and research in the field of robotics has only emerged within the last 5 years, interest in navigation, as well as computer-assisted surgery, has nearly vanished. Due to the outstanding pandemic situation beginning at the end of 2019 the topic of COVID-19 just arose within the last year. Although some foci have changed throughout the years, research issues concerning the TKA outcome and improving rehabilitation have remained important topics throughout all the time intervals. This observation is critical because a proportion of patients (up to 20%) are unsatisfied with their outcome after TKA . TherefoThe Bone and Joint Journal between 2004 and 2018 (7.9–16.7%). The increasing number, particularly of Asian publications, was also described by other authors [Lancet 2017 [The percentage of European publications has shown a continuous decrease over the last 15 years, while that of North America demonstrated a major increase. Although Asian publication increased between 2006 and 2015 the recent study revealed a major decrease within the last interval. This drop might be caused by a pandemic situation starting in Asia in 2019 and might have influenced Asian countries earlier than European or American countries also in research submission. Bradley et al. described, for example, a significant increase in publications from Asia in  authors . Zhi et  authors . The grecet 2017 . To impr2018 7.9–.7%. The The Journal of Bone and Joint Surgery (JBJS) and confirmed a significant increase in authorships per orthopaedic publication over time [Increases in the number of authors per publication have been found in multiple fields of medicine. A previous study evaluated publication characteristics in the American ver time . Multiplver time , 9. Hanzver time . Anotherver time , 14, 18.JBJS, The American Journal of Sports Medicine and Clinical Orthopaedics and Related Research between 2006 and 2017 [Throughout the whole period, most of the articles were published by male first authors. Although the absolute number of first female authors increased over time, no significant increase was observed in the percentage of women gaining visibility as a first author in the field of knee arthroplasty between first and latest interval. The positive trend reported by other authors, evaluating publishing characteristics in the topic of foot and ankle as well as hand surgery, was not confirmed by the recent study , 9. In cand 2017 . They foAn increasing quality of published papers in KSSTA can be assumed due to the increasing number of cases evaluated in original articles. This development is probably also impacted by the increasing number of register studies which are published over the last years . The conTo assess postoperative outcomes, the use of PROM has become an essential part of prospective and retrospective study designs. Because many established PROMs, such as KSS or HSS, show ceiling effects after arthroplasty and are not suitable to access an increasing number of active and demanding patients, a shift occurs towards more modern PROMS, such as KOOS or FJS, which provide higher responsiveness and lower ceiling effects than traditional PROMs .The current study has several limitations. First, some studies related to the topic of knee arthroplasty might were sorted out by the filtering process because they did not contain the keyword or any related description in either title, keywords nor abstract. Second limitation represents the classification procedure concerning the main and special topic of publications. In order to decrease selection bias, every publication was classified by two independent reviewers and in case of any differences, the classification was set in a consensus approach. Another limitation represents the simplified origin of publication to the continent instead of country. As KSSTA reaches out to a lot of different countries resulting in a large number of data authors decided to simplify publication origin. Furthermore, identified publishing and author characteristics are findings of KSSTA analysis and might not represent orthopaedic or knee arthroplasty related research in general. However, various topics were carefully reviewed, and several important trends were identified over a 15-year period.Increasing interest in the field of TKA-related surgery has arisen within the last 15 years in KSSTA, accepting an increasing number of articles in each time interval. The main topics showed a significant trend towards the latest techniques at each time interval. While interest in computer-assisted surgery or navigation vanished, interest in optimized perioperative management, rehabilitation and robotic increased. The overall number of references and authors increased which might indicate increased collaboration and globalization over time. Although the absolute number of female first authorships increased, their proportion did not change significantly compared to the first-time interval. There was an increasing number of publications from Asia and North America in KSSTA. The authors hope that the identified publication characteristics might help to better interpret the literature, helps to identify future research topics and serve as a benchmark where knee arthroplasty related research currently stands in KSSTA.


We presumed that there was a pathophysiological overlap between refractory ITP and refractory thrombocytopenia (RT): a subtype of refractory cytopenia of childhood (RCC). Immunosuppressive therapies including anti-thymocyte globulin and cyclosporine (CsA) have been adopted for children with RCC in Japan. Thus, from 2009 onwards, we changed the diagnosis from refractory ITP to RT and introduced CsA for refractory ITP/RT. Nine of 42 patients developed refractory ITP in the 1998–2008 group, who received conventional treatments such as IVIG and steroid therapy. Eight of 45 patients developed refractory ITP in the 2009–2017 group, who received CsA with or without IVIG therapy. The response rate at three years after diagnosis was significantly higher in the 2009–2017 group (98%) than in the 1998–2008 group (83%) (p = 0.019). In conclusion, our strategy of introducing CsA for refractory ITP/RT contributed to better outcomes.Treatment of children with refractory immune thrombocytopenic purpura (ITP) is challenging and poorly established. We retrospectively reviewed the clinical data of 87 patients under the age of 16 years who were diagnosed with ITP from April 1998 to March 2017 in our institution. Refractory ITP was defined as a platelet count of < 50 × 10 In contrast to adults, ITP in children is self-limiting. However, 20–30% of newly diagnosed ITP patients become refractory to high-dose intravenous immunoglobulin (IVIG) and glucocorticoid therapy3. Rituximab (RTX) as a second-line treatment was also approved for patients including children with chronic ITP in 2017; however, only 14–26% of children showed a lasting response with a platelet count of > 100 × 109/L5. The incidence of complete remission in children with chronic ITP who received IVIG, glucocorticoid therapy, or splenectomies was 30–52% at five years7.Treatment of children with refractory ITP is challenging and appropriate treatment strategies remain largely unestablished. In 2010, thrombopoietin receptor agonists were authorized as a second-line treatment for adults with chronic ITP in Japan. Forty percent of children with chronic ITP responded to eltrombopag in western countries8. Further, splenectomy increases the late risks of venous and arterial thrombosis as well as sepsis from Streptococcus pneumoniae9. In 2008, refractory cytopenia of childhood (RCC) was introduced as a tentative subtype of pediatric myelodysplastic syndromes (MDS) in the fourth edition of the World Health Organization’s classification of myeloid neoplasms and acute leukemia10. We presumed that there was a pathophysiological overlap between refractory ITP and refractory thrombocytopenia (RT), which is a subtype of RCC. Immunosuppressive therapies including anti-thymocyte globulin and cyclosporine (CsA) have been adopted for children with RCC in Japan. From 2009 onwards, we changed the diagnosis from refractory ITP to RT and introduced CsA treatment for such patients instead of glucocorticoid therapy and splenectomy. In this study, we retrospectively analyze the long-term outcomes of 87 children with ITP in our institution and aim to evaluate whether the strategy of introducing CsA for refractory ITP/RT was appropriate.Prior to 2008, patients with refractory ITP underwent administration of glucocorticoid or splenectomy in our institution. However, long term administration of glucocorticoid in children affects bone density as well as growth11. Informed consents to participate in the study were obtained through providing patients and their guardians with the opportunity to opt-out because this study was a retrospective nature. This procedure was approved by the Institutional Review Board of Nihon University Itabashi hospital and conformed to the revised Declaration of Helsinki, 7th version.We retrospectively reviewed the clinical data of children who were newly diagnosed with ITP and treated from April 1998 to March 2017 at the Nihon University Itabashi hospital. We excluded patients > 16 years old or with a follow-up duration less than three months from this study. We retrieved the following patient characteristics: age at diagnosis, sex, follow-up time, bleeding manifestations at diagnosis, bone marrow findings, karyotype of bone marrow cells, flow cytometric data on bone marrow, anti-nuclear antibody, platelet count, treatment history, medication history, and outcome at three years after diagnosis. We also classified ITP as newly diagnosed (< 3 months from diagnosis), persistent (3–12 months from diagnosis), and chronic (lasting for > 12 months from diagnosis) according to the criteria of an International Working Group11. In this study, we defined a platelet count of < 50 × 109/L at 14 days after receiving IVIG and prednisolone (PSL) as refractory ITP. Treatment response was defined as either complete response (CR) with a platelet count sustaining > 100 × 109/L or partial response (PR) with a platelet count between 50 and 100 × 109/L.An international Working Group defined refractory ITP as unresponsive or relapsing after a splenectomy, and requiring treatment to minimize the risk of clinically significant bleedingFrom 2009 onwards, we changed the diagnosis from refractory ITP to RT, and introduced CsA treatment for refractory ITP/RT. We collected the following data: the interval from diagnosis to CsA therapy, the interval from CsA therapy to PR and CR, additional therapies after CsA therapy , relapse, the duration of CsA therapy, and the outcome at three years after diagnosis.12. Chi-square test and Wilcoxon rank sum test were used to compare patients’ characteristics between the two period groups. Chi-square test was used to compare the incidence of CR and PR between the two period groups. The level of statistical significance was set at p < 0.05.To evaluate whether the strategy of introducing CsA for refractory ITP/RT contributed to better outcomes, we compared the incidence of CR and PR in ITP at three years after diagnosis between two period groups: 1998–2008 and 2009–2017. Not all patients had been followed up for three years because this study had a retrospective design. If there was no sign of relapse after completion of follow-up, the patient status was defined as CR at three years after diagnosis. Statistical analysis was performed using Jmp, version 14 A total of 87 children (50 boys and 37 girls) with ITP were included in this study, of which 42 children were in the 1998–2008 group and 45 children were in the 2009–2017 group. Forty-two children in the 1998–2008 group were classified as follows: 30 patients with newly diagnosed ITP, three patients with persistent ITP, and nine patients with chronic ITP. Further, 45 children in the 2009–2017 group were classified as follows: 36 patients with newly diagnosed ITP, three patients with persistent ITP, and six patients with chronic ITP. The median age at diagnosis was 3.1 years . The median follow-up time was 16 months . Bone marrow findings in 86 patients (99%) showed an increased number of mature or immature megakaryocytes, which was consistent with ITP. Bleeding manifestations were present at diagnosis in all patients: skin purpura (99%), nasal bleeding (22%), gingival bleeding (11%), melena (5%), hematuria (2%), intracranial hemorrhage (1%), and genital bleeding (1%). The patients’ characteristics are shown in Table 9/L of platelet count and bleeding symptom. All patients with the indication of the first-line treatments received them during the study period. IVIG (1–2 g/kg/day) for 57 patients or PSL (2 mg/kg/day) for 68 patients was administered as the first-line therapy. In the 1998–2008 group, 41 out of 42 patients received PSL as an initial treatment, and 30 patients achieved CR. Out of 11 patients with no response to PSL, 10 patients received IVIG, and two patients achieved CR. The remaining one patient received IVIG as an initial treatment but did not respond to IVIG or addition of PSL. In the 2009–2017 group, 43 out of 45 patients received IVIG as an initial treatment, and 19 patients achieved CR. All 24 patients with no response to IVIG received PSL, and 16 patients achieved CR. The remaining two patients received PSL as an initial treatment and achieved CR by adding IVIG. We found nine patients (21%) in the 1998–2008 group and eight (18%) in the 2009–2017 group with refractory ITP; treatment flow charts of the 17 patients are shown in Fig. In our institution, the first-line treatments were used for patients presenting with ≤ 20 × 109/L at three years after diagnosis.We changed the diagnosis of refractory ITP for all eight patients in the 2009–2017 group to RT, and initiated treatment with CsA. CsA was administered orally at a dose of 4–6 mg/kg/day and the trough level was adjusted between 100 and 150 ng/mL. Characteristics and outcomes of eight patients who received CsA are presented in Table 9/L at three years after diagnosis, of whom three (50%) underwent splenectomy, and CR was achieved at the last follow up. Characteristics and outcomes of the nine patients with refractory ITP in the 1998–2008 group are presented in Supplementary Table p = 0.019).In contrast, only two of the nine patients (22%) with refractory ITP achieved CR without splenectomy at three years after diagnosis in the 1998–2008 group. One patient was lost to follow-up. The remaining six patients had a platelet count of < 50 × 1013. The median administration time of CsA therapy was 9.3 month , and 11 (37%) and six patients (20%) achieved CR (platelet count > 100 × 109/L) and PR (platelet count between 30 and 100 × 109/L), respectively. Seven out of the 17 patients with CR or PR maintained the response after CsA discontinuation, with the median time of 41.7 months  from CsA discontinuation. An Italian group demonstrated that 12 adults with refractory ITP received CsA alone, with the median administration time of 14.5 months 14. The response (platelet count > 80 × 109/L) was achieved in 10 patients (83%), of whom six patients sustained the response after CsA discontinuation, with the median time of 29.5 months  from CsA discontinuation. An Australian group showed a six-month response rate to a more intensive regimen with dexamethasone + RTX + CsA (platelet count > 30 × 109/L) in 12 out of 20 adults (60%) with persistent or chronic ITP15. A more probable response to CsA with or without IVIG for refractory ITP/RT was shown in the current cohort. The dose of CsA was reduced by 10% every month after the platelet count was sustained (> 100 × 109/L) for more than three months. This slowly tapering method was according to that commonly used in patients with aplastic anemia in our country, which might contribute to the high CR rate even after CsA discontinuation.We reported eight pediatric cases with refractory ITP/RT treated with CsA as a salvage therapy with or without IVIG. Surprisingly, seven patients (88%) achieved CR and PR, and six patients (75%) have maintained CR after CsA discontinuation. A Hong Kong group demonstrated that 30 children with persistent or chronic ITP received a salvage therapy with CsA alone10. Interestingly, a common pathogenesis is believed to exist between aplastic anemia and RCC, because immunosuppressive therapies including anti-thymocyte globulin and CsA have been successful for 53–74% of children with RCC18. RT is a subtype of RCC and is characterized by > 10% morphologic dysplasia limited to the megakaryocytic lineage10. However, RT has often been confused with ITP due to the lack of distinguishable dysplasia19. Moreover, a case study reported a patient diagnosed with RT using a complementary DNA microarray analysis despite no dysplasia in megakaryocytic lineage20. Therefore, we presumed that there was a pathophysiological overlap between refractory ITP and RT.RCC is characterized by persistent cytopenia with < 2% blasts in the peripheral blood and < 5% blasts in the bone marrow. Moreover, bone marrow films show dysplastic changes in at least two cell lineages, or > 10% within one cell lineage21. Our results also indicate that refractory thrombocytopenia is partially mediated by T cells.Autoreactive CD4+ T cells as well as macrophages and B cells are involved in the production of platelet-associated antibodies in adult patients with ITP, which supported our treatment strategy9/L after splenectomy23. Splenectomy has been a conventional treatment for ITP. However, it is not currently preferred because it increases the late risks of venous and arterial thrombosis as well as sepsis from Streptococcus pneumoniae9. Thus, from 2009 onwards, we avoided splenectomy for refractory ITP owing to these risks. Our study showed that CsA with or without IVIG therapy is feasible and effective in children with refractory ITP to avoid splenectomy.In this study, three patients with refractory ITP in the 1998–2008 group underwent splenectomy, and all the patients achieved CR. A retrospective study in pediatric cases showed 85–87% of children with chronic or refractory ITP had a platelet count of > 50 × 10Our study has some limitations. Firstly, this study followed a retrospective design. Secondly, the study was restricted to a single institution. Moreover, the optimal trough level and treatment duration of CsA remains unestablished.In conclusion, our strategy of introducing CsA for refractory ITP/RT contributed to better outcomes. Further research is required for optimizing the trough level and duration of CsA.Supplementary Information.


The bioconjugate molecular weight (50 kDa < Mw < 75 kDa) was obtained by SDS-PAGE, while the spectroscopic characteristics have been studied in order to reveal the presence of grafted sunitinib. In both FT-IR and UV/Vis spectra, signals corresponding to sunitinib functional groups have been identified. Since sunitinib is an anticancer drug characterized by low bioavailability and low permeability, the bioconjugation aimed at their enhancement. In vitro studies demonstrated that bioavailability has been increased to almost 74%, compared with commercial formulation. Also cell membrane permeability has been augmented in in vitro tests, in which membrane models have been used to determine the lipid membrane/physiological fluid partition coefficient (Kp). The log(Kp) value of the bioconjugate was increased to over 4. This effect resulted in a three-fold decrease of IC50 value against MCF-7 cells.Sericin is a natural protein that has been used in biomedical and pharmaceutical fields as raw material for polypeptide-based drug delivery systems (DDSs). In this paper, it has been employed as pharmaceutical biopolymer for the production of sunitinib–polypeptide conjugate. The synthesis has been carried out by simple click reaction in water, using the redox couple  Biopolymer–drug conjugates have recently attracted much attention as drug delivery systems (DDSs). Bioconjugates generally aim at the exploitation of the properties of the biopolymeric material at which the drug is attached , a by-product of textile industry. Silk filaments, produced by the silkworm Bombyx mori, are made of a double strand of fibroin, held together by a gummy coating mainly constituted by SER. During silk processing, the silk filaments are treated with boiling water. In this way, SER is removed and discarded (degumming).In the present work, a bioconjugate was obtained Sericin’s molecular weight, ranging from about 10 to over 400 kDa, strongly depends on the extraction and isolation methods used. Molecular weight also influences the solubility in aqueous solutions: smaller polypeptides having a molecular weight less than 60 kDa are more soluble in cold water; on the other hand, SER fractions having molecular weight over 60 kDa are poorly soluble in water at room temperature, but still soluble in hot water  has been chosen as model drug.Small molecular tyrosine kinase inhibitors (smTKIs) are powerful anticancer drugs that are experiencing rapid growth. SmTKIs include imatinib, gefitinib, erlotinib, afatinib, dasatinib, bosutinib, ponatinib, etc., divided in first-, second- and third-generation TKIs  as a class IV drug  bioconjugate was obtained Conjugation with SER could be applied to a variety of drugs that are similar to SUT, such as bosutinib, crizotinib, nilotinib, vemurafenib among smTKIs, but also amphotericin B, chlorothiazide, colistin, ciprofloxacin, mebendazole, methotrexate, neomycin, furosemide, hydrochlorothiazide. They are all classified as Class IV drugs by BCS and possess similar properties to SUT , l-ascorbic acid (AA), hydrochloric acid (37% w/w), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium hydrogen carbonate, pepsin from porcine gastric mucosa, esterase from porcine liver, α-amylase from porcine pancreas, pancreatin from porcine pancreas, sodium cholate, bile extract porcine and l-α-phosphatidylcholine from egg yolk were purchased by Sigma-Aldrich .Sunitinib malate, hydrogen peroxide .Dialysis tubes MWCO: 3500 Da and 12 000–14 000 Da were provided by Spectrum Laboratories Inc .IR spectra were recorded as films or KBr pellets on a Jasco FT-IR 4200 . Absorption spectra were recorded with a Jasco V-530 UV/Vis spectrometer .The water in which the silkworm cocoons are boiled during silk production has been provided by a local silk manufacturer. The water soluble fraction (sericin) dissolved in the solution was collected after centrifugation at 7000 rpm for 20 min. The light colored supernatant was then dialyzed for 72 h against distilled water using 3.5 kDa MWCO dialysis membrane. After 24 h of lyophilization, the sericin powder was obtained and used for further experimentations.l-ascorbic acid as redox pair, was carried out as follows: in a suitable beaker 40 mL of water and 15 mL of ethanol were poured. 200 mg of sericin and 40 mg of sunitinib malate were dissolved in the reaction mixture. After complete dissolution, 2.5 mL of H2O2 (30% v/v) containing 83.5 mg of l-ascorbic acid were added. The mixture was maintained under magnetic stirring at 25 °C for 24 h under atmospheric pressure. The obtained SER–SUT conjugate was purified by dialysis (3.5 kDa MWCO) for 12 h against ethanol/water and for further 48 h against water. The solution recovered was then frozen and lyophilized to a powder.Single-step grafting of sunitinib onto sericin, by employing hydrogen peroxide/All chemicals used in SDS-PAGE and gel staining were obtained from Bio-Rad Laboratories S.r.l. or Sigma-Aldrich  and the solutions were prepared in deionized water. The protein samples were solubilized in loading buffer and gels were run on a Protean III mini-electrophoresis unit  according to Laemmli . After tin vitro bioavailability study was carried out in a simulated gastric and intestinal environment through the previously reported method of the dialysis tubing procedure  with 1.0 mL of a 0.85 N HCl solution, 3.0 mL of a sodium cholate solution (2% w/v in distilled water) and 24 000 U of porcine pepsin per mL. The dialysis bag was carefully sealed on each end with clamps and immersed in 10 mL of a 0.85 N HCl solution (pH 1.0). The system was then left for 2 h into a water-bath at 37 ± 0.5 °C.3 solution, 11 mg of amylase, 11 mg of esterase and 11 mg of porcine pancreatin to the bag content. The dialysis bag was then sealed again and placed into 10 mL of a phosphate buffer saline (PBS) solution at pH 7.0. The incubation was repeated for further 4 h, into the shaking water bath at 37 ± 0.5 °C.After 2 h, the dialysis bag was recovered and carefully opened in order to allow the addition of 1.3 mL of a 0.8 M NaHCOin vitro bioavailability of the sample, 3 mL of the medium used to mimic gastric and intestinal environment (0.85 N HCl solution pH 1.0 and PBS pH 7.0) were withdrawn at the time points of 2 and 6 h, respectively. The concentrations of the samples were determined by UV/Vis spectroscopy (at 426 nm) and calculated by using the equations obtained from the calibration curves of SUT standard solutions at pH 1.0 and 7.0, respectively. For this purpose, all the prepared standard solutions were analyzed by UV/Vis spectrophotometer and the correlation coefficient (R2) and slope of the regression equations obtained by the method of least square were calculated at pH 1.0 and 7.0, respectively.In order to evaluate the The same procedure was applied to evaluate the bioavailability of sunitinib pharmaceutical formulation, commercially available under the name of Sutent®, used as control for data comparison. Each experiment was repeated three times.Giant unilamellar vesicles (GUVs) were prepared according to the method reported by Moscho et al.  and WaldSUVs size and distribution were determined by dynamic light scattering (DLS) analysis using 90 Plus particle size analyzer  at 25.0 ± 0.1 °C by measuring the autocorrelation function at 90°. The laser was operating at 658 nm. The distribution size and the polydispersity index were directly obtained from the instrument.SUVs obtained have been used as a cell membrane model to calculate the membrane/buffer solution partition coefficient of SER–SUT conjugate, using derivative spectrophotometry, as reported by Takegami et al. .9 sample solutions containing SUT (20 μM) and various amounts of the SUV suspension with a concentration of PC in the range of 0–1200 μM were prepared. The reference solutions were prepared without SUT. Each vial containing the sample was shaken and incubated for 2 h in order to reach the equilibrium state.The absorption spectra were measured using 1 cm light-pass length cuvette, in the wavelength range between 400 and 550 nm with interval of 0.5 nm. The second-derivative spectra were calculated using “spectra manager” software v. 1.53.01, based on the Savitzky–Golay method in which the second-order polynomial convolution of 21 points was employed , 1% l-Glutamine, 1% penicillin/streptomycin, 0.5 mg/ml hydrocortisone, 20 ng/ml hEGF  and 0.1 mg/ml cholera enterotoxin  and 10 μg/ml insulin. SUT was dissolved in dimethylsulfoxide (DMSO)  at a concentration of 50 mM and diluted in DMEM/F12 medium supplemented with 1% FBS (MCF-7) or 1% HS (MCF-10A) to obtain the working concentration. SER–SUT conjugated and SER were directly dissolved in DMEM/F12 medium supplemented with 1% FBS (MCF-7) or 1% HS (MCF-10A). Before the treatment, cells were serum deprived for 24 h, then treated with six different concentrations of SUT, SER–SUT or SER for 72 h in medium containing 1% FBS (MCF-7) or 1% HS (MCF-10A). At the end of the treatment, fresh MTT re-suspended in PBS was added to each well to a final concentration of 0.2 mg/mL. After 2 h incubation at 37 °C, cells were lysed with a solution containing 50% (v:v) N,N-dimethylformamide and 20% (w:v) SDS, pH of 4.5, and then optical density was measured at 570 nm with a reference wavelength of 620 nm, using a microplate reader. MTT experiments have been performed in sestuplicate and repeated three times. Absorbance values were used to determine the IC50 using GraphPad Prism 5 Software . Data are representative of three independent experiments; standard deviations (SD) were shown.Cell viability was determined using the 3--2,5-diphenyltetrazolium  assay as reported by Saturnino et al.  with somp < 0.05, **p < 0.01.Statistical significance between control and treated cells was analyzed by the means of GraphPad Prism 5.0  software, using the analysis of variance (ANOVA) with Kruskal–Wallace test and post hoc Dunn’s Multiple Comparison Test. Significance was defined as *Bombyx mori strain were boiled in hot water, at atmospheric pressure. Sericin was then isolated and purified according to the procedure described in the experimental session. The yield of extraction was calculated comparing the dry weight of sericin purified to the dry extract of the cocoon boiling water. The yield obtained ranged between 19% and 22%. Molecular weight and biological activity on healthy tissue were evaluated and reported in the following sections.Sericin extracted and isolated from silk can vary depending on extraction method and on source type. These two factors can highly influence the yield of extraction, as well as sericin molecular weight and its biological activity  conjugate was synthesized using a click chemistry approach. The reaction involved the direct free radical grafting of SUT onto a biopolymeric chain, such as the protein sericin. According to synthetic procedure used for the achievement of the SER–SUT conjugate, a covalent bond has been introduced between the protein and the drug.SER is a hydrophilic polypeptide characterized by fine water solubility as well as by low cytotoxicity towards living tissues. SER, indeed, has been successfully applied in various fields, especially in pharmaceutical and biomedical ones.l-ascorbic acid redox pair . On the contrary, the same peak is absent in the sericin spectrum.FT-IR analyses were performed aiming at the identification and verification of the grafting of the drug on SER. Three different spectra were recorded : sunitinmax = 273 nm and λmax = 426 nm, respectively. As confirmation of the occurred grafting, both peaks can be observed in the SER–SUT conjugate spectrum.For further evidence, UV/Vis spectra of sericin, sunitinb malate and sericin–sunitinib conjugate were also recorded between 250 nm and 700 nm (data not shown). Sericin and sunitinib malate showed typical absorbance at λWith the help of UV/Vis spectroscopy the amount of grafted SUT was determined, through the equation of the calibration curve of sunitinib malate. The content of SUT calculated was 6.5 mg of SUT per g of SER–SUT conjugate.Literature data reported that sericin from silk may exist in different molecular weight forms, depending on the extraction processes, processing time, temperature or pH  and SER–SUT conjugate. Data reported shows a good bioavailability percentage after six hours for both samples, slightly improved in the case of the conjugate. However, a significative increase is yet observable after 2 h, meaning a ready dissolution of the sericin protein–sunitinib conjugate in the gastric environment.In our study, the bioavailability evaluation was performed comparing formulated sunitinib, also commercially known as Sutent® .However, liposomal model is a more reliable system, able to mimic the cell environment. Liposomes are closed membranes made of amphiphilic lipids, characterized by a liquid crystal structure, that take into account of the surface charges involved in the drug/cell electrostatic interactions, particularly important for charged and polar drugs. In addition, the complexity of the bilayer structure and steric forces can contribute, either positively or negatively, in drugs bioaccessibility. For such reasons, the partition coefficient obtained by anisotropic membrane/buffer solution system better predicts drugs behavior in biological environment than an isotropic 2-phase solvent system . SUVs size was measured by DLS analysis showing a homogeneous population of liposomes with a mean diameter in the range of 170–180 nm. The absorption spectra of 20 μM SUT and SER–SUT conjugate with equal SUT concentration, recorded in suspensions containing various concentrations of PC liposomes, are shown in For the experimental procedure small unilamellar vesicles (SUVs) were produced The presence of isosbestic points in the overlaid spectra at different concentrations of PC confirmed the existence of an equilibrium between drug dissolved in the polar aqueous phase of the buffer solution and in the non-polar PC bilayer phase.max is the maximum ΔD value assuming all drug is partitioned in PC SUVs. Partition coefficient was expressed as log(Kp) for a ready interpretation of the data. Results obtained are reported in The membrane/buffer solution partition coefficient (Kp) of both SUT and SER–SUT conjugate were calculated using the following n-octanol/water, should be less than 5. From the scientific literature, we know that log(Kp)o/w for sunitinib is 5.2  or the log of the ratio of the solubility of the drug in Using this model, SUT showed a good log(Kp) value see , while S50 values, reported in 50 > 76 μM, In order to evaluate the antitumor activity of SER–SUT conjugate, estrogen receptor positive (ER+) MCF-7 cells were used. Concentrations of SER–SUT conjugate have been chosen taking into account the content of SUT conjugated to SER from UV/Vis experiments (see “sericin–sunitinib conjugate synthesis” paragraph), so that same concentrations of SUT alone and SER–SUT conjugate were tested. Cells were grown up to 50–60% confluence and treated with increasing doses of both SUT and SER–SUT conjugate  and, after 72 h, cell viability was determined by MTT assay  has been employed in order to initiate the reaction. The covalent attachment of sunitinib has been obtained by hydroxyl radical action generated during the reaction. It was confirmed by FT-IR and UV/Vis spectroscopy, after protein purification.In the present research paper, we have reported the free radical grafting of sunitinib onto sericin polypeptidic chain, In vitro gastrointestinal bioavailability experiments have highlighted an improved percentage of sunitinib that can cross the simulated GI lumen, compared with the commercially available drug (Sutent®). This effect is certainly due to augmented water solubility of the sericin–sunitinib conjugate and it is evident from the very first two hours, in which we observed the ready dissolution of the functionalized material.Second derivative spectroscopy has given the opportunity to evaluate the interaction between sericin–sunitinib conjugates and cell membrane models, such as phosphatidylcholine small unilamellar vesicles. The study demonstrated an improvement of sericin–sunitinib membrane permeability, expressed as an increase of membrane/buffer solution partition coefficient.in vitro cytotoxicity tests showed that the newly synthesized sericin–sunitinib conjugate is three times more effective than free sunitinib, thanks to the characteristics acquired by conjugation with the protein.In the end, the Thanks to these results, we can state that the production of a sericin-grafted biomaterial is a valuable method to improve the bioavailability and permeability of drugs like sunitinib.


Prostate leiomyosarcoma is a rare, aggressive neoplasm.2, doxorubicin 60 mg/m2, ifosfamide 7500 mg/m2, and dacarbazine 900 mg/m2) at 4‐week intervals. The tumor shrank by 28% and exhibited necrotic changes. He underwent total pelvic exenteration with en bloc resection of the prostate, bladder, rectum, and anus. Pathological surgical margin was negative. The patient is alive with no disease at 5 years postoperatively.A 52‐year‐old man presented with worsening frequent micturition and painful urination. Rectal examination revealed a significantly enlarged prostate. Magnetic resonance imaging showed a large prostate tumor with urinary bladder and bilateral seminal vesicle invasion. A prostate biopsy revealed diffuse proliferation of pleomorphic atypical cells. Immunohistochemistry confirmed the diagnosis of prostrate leiomyosarcoma. The patient received three cycles of the mesna, doxorubicin, ifosfamide, and dacarbazine regime (mesna 6000 mg/mNeoadjuvant chemotherapy and surgical resection are essential to achieve a long‐term survival of patients with localized prostate leiomyosarcoma. Complete resection with negative margins is essential, but surgery alone cannot improve its prognosis. The MAID  regimen administered as preoperative chemotherapy is effective.Sarcomas of the prostate are rare and account for 0.1–0.2% of all malignant prostatic tumors. Rhabdomyosarcomas of prostate are known as the most frequent tumors in childhood, whereas leiomyosarcomas are the most common sarcomas involving the prostate in adults.A 52‐year‐old man presented with a complaint of frequency and difficulty in urination since February 2016. The patient visited a urologic clinic and received antibiotics and an α1‐blocking agent. However, the perineal pain and difficulty in urination worsened. The patient was referred to our hospital in May 2016. Rectal examination revealed a significant enlargement of the prostate with unclear margins. The prostate specific antigen level was 0.8 ng/mL. MRI (T2 weighted image) showed a heterogeneous, large, prostate tumor (10.6 × 9.3 cm) with invasion into the urinary bladder and bilateral seminal vesicles without invasion of the rectal wall or internal obturator muscle, as shown in Figure 2, doxorubicin 60 mg/m2, ifosfamide 7500 mg/m2, and dacarbazine 900 mg/m2, every 4 weeks from June 2016. Grade 3 febrile neutropenia was observed after every cycle, despite subcutaneous pegfilgrastim injections. CT after three cycles of chemotherapy showed 28% tumor shrinkage and obvious necrotic changes in the tumor, as shown in Figure After consulting the oncologist at our hospital, we decided to apply preoperative chemotherapy with MAID regime. The patient underwent three cycles of MAID regime comprising mesna 6000 mg/mLMSOP is very rare, and <200 cases have been reported to date.vs 20 months, P = 0.018). The OS of patients with incomplete resection was worse than that of those showing negative surgical margin .et al. reported there was no significant correlation between the status of surgical margin and OS.Clinical outcomes for patients with LMSOP are poor. A review of 34 patients with LMSOP demonstrated that median survival is 17 months (95% CI 20.7–43.7 months) and the survival rates at 1,3,5 years are 68%, 34%, and 26%, respectively. The OS of patients with metastasis at initial diagnosis was worse than that of those with no metastasis (median OS, 5 et al.et al.et al.Sexton et al.Ogura A multimodal approach, including systemic chemotherapy and radical surgery, should be considered to achieve a long‐term survival of patients with localized prostate leiomyosarcoma.Toshiaki Kawaguchi: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; project administration; resources; software; validation; visualization; writing‐original draft; writing‐review & editing. Toshikazu Tanaka: Data curation; resources; software; validation; visualization. Masaru Ogasawara: Data curation; project administration; resources; supervision; validation. Iwabuchi Ikuya: Funding acquisition; methodology; project administration; supervision; validation.The authors declare no conflict of interest.Not applicable.Not applicable.Not applicable.


Cross‐sectional incidence testing is used to estimate population‐level HIV incidence and measure the impact of prevention interventions. There are limited data evaluating the accuracy of estimates in settings where antiretroviral therapy coverage and levels of viral suppression are high. Understanding cross‐sectional incidence estimates in these settings is important as viral suppression can lead to false recent test results. We compared the accuracy of multi‐assay algorithms (MAA) for incidence estimation to that observed in the community‐randomized HPTN 071 (PopART) trial, where the majority of participants with HIV infection were virally suppressed.HIV incidence was assessed during the second year of the study, and included only individuals who were tested for HIV at visits 1 and 2 years after the start of the study (2016–2017). Incidence estimates from three MAAs were compared to the observed incidence between years 1 and 2 . The mean duration of recent infection (MDRI) used for the three MAAs was 248, 130 and 180 days, respectively.p<0.01).The study consisted of: 15,845 HIV‐negative individuals; 4406 HIV positive at both visits; and 221 who seroconverted between visits. Viral load (VL) data were available for all HIV‐positive participants at the 2‐year visit. Sixty four (29%) of the seroconverters and 3227 (72%) prevelant positive participants were virally supressed (<400 copies/ml). Observed HIV incidence was 1.34% (95% CI: 1.17–1.53). Estimates of incidence were similar to observed incidence for MAA‐C, 1.26% (95% CI: 1.02–1.51) and the LAg+VL MAA, 1.29 (95% CI: 0.97–1.62). Incidence estimated by the Rapid+VL MAA was significantly lower than observed incidence (0.92%, 95% CI: 0.69–1.15, MAA‐C and the LAg+VL MAA provided accurate point estimates of incidence in this cohort with high levels of viral suppression. The Rapid+VL significantly underestimated incidence, suggesting that the MDRI recommended by the manufacturer is too long or the assay is not accurately detecting enough recent infections. Cross‐sThe accuracy of cross‐sectional incidence assays is influenced by several factors, most notably antiretroviral therapy (ART) , 11, 12.We evaluated the performance of MAAs in a community‐randomized trial that evaluated the impact of a combination prevention package, including universal testing and treatment on HIV incidence: the HIV Prevention Trials Network (HPTN) 071 (PopART) trial. HIV incidence estimates from MAAs were compared to observed HIV incidence based on annual HIV testing. Three MAAs were evaluated: an MAA optimized for incidence estimation in subtype C epidemics (MAA‐C); the widely used MAA that includes the LAg assay plus VL (LAg+VL); and an MAA that includes a point‐of‐care assay [the Asanté HIV‐1 Rapid Recency Test plus VL (Rapid+VL)], currently being used in the TRACE program , 16. Inc22.1The sample set evaluated originated from the HPTN 071 trial (conducted between 2013 and 2018) in 21 communities in Zambia and South Africa . ParticiThe HPTN 071 trial enrolled a randomly selected Population Cohort (PC) of 48,302 adults age 18–44), who were followed for up to 3 years with four annual surveys  . Partici–44, who 2.2Plasma samples from participants who were HIV positive at PC24 were tested using the HIV‐1 LAg‐Avidity EIA  and the Johns Hopkins modified BioRad‐Avidity assay , 19. The2.3Incidence estimates from the three MAAs were compared to observed HIV incidence. Observational incidence was measured by dividing the number of observed seroconversions over the number of years of follow up for participants who were HIV‐uninfected at the first visit (PC12). HIV incidence is expressed as the number of infections per 100 person years. MAA‐C classifies infections as recent if they have a LAg result <2.8 ODn, an AI <95% and a VL >400 copies/ml. The mean duration of recent infection (MDRI) for this MAA is 248 days . The LAg3The analysis included: 221 individuals who acquired HIV infection between PC12 and PC24 ; 15,845 individuals who were HIV negative at PC12 and PC24; 4189 participants who were HIV positive at enrolment ; and 217 individuals who were negative at PC0, but positive at PC12 and PC24. VL data were obtained in the parent study , 1.29 [95% CI: 0.97–1.62] and 0.92 [95% CI: 0.69–1.15], respectively . All data analysed are available in the Supporting Information.Incidence estimates were also generated for participant sub‐groups, based on study arm, country, sex and sex in younger participants (aged 18–24) Figure . In thesIn the Rapid+VL algorithm, a total of 10 samples did not have a reactive HIV band. None of these individuals had reactive long‐term bands either. The median LAg value for these samples was 0.17 ODn, though four samples did have values above 0.5 ODn. Nine of 10 samples were from virally suppressed individuals. Among the 100 samples from individuals infected >2 years with VLs <1000 copies/ml, 32 did not have a visible long‐term infection band. Five of these samples were among those appearing to be HIV uninfected. All of these individuals were sero positive by the ARCHITECT HIV Ag/Ab COMBO test and Geenius HIV‐1/2 Supplemental assay .Our study demonstrated that the MAA‐C and the LAg+VL MAA provided accurate point estimates of HIV incidence in a population with a high frequency of viral suppression. In a previous study, MAA‐C and LAg+VL MAA also provided accurate point estimates of incidence in a subtype C epidemic setting where ART was not widespread . AlthougThe Rapid+VL MAA was the only MAA that yielded an incidence estimate that was consistently and significantly lower than observed incidence. This MAA significantly underestimated HIV incidence overall and in all sub‐groups except for male participants, the population with the lowest observed incidence. The poor performance of this MAA suggests that it may not be as effective as the other MAAs to identify clusters of recent infections. The Consortium for the Evaluation and Performance of HIV Incidence Assays (CEPHIA) estimated an MDRI of 83 days for this MAA in a non‐peer reviewed study . The lowOur study has limitations. Observed incidence was assessed over 1 year of follow‐up, while the incidence estimates were calculated using MAAs with window periods <1 year. However, the time periods assessed by the two methods are overlapping, and incidence was not likely to have changed significantly during the 1‐year period. The HPTN 071 cohort was conducted in Zambia and South Africa, where most infections are caused by HIV subtype C . Further4As ART becomes more widely available across the globe, and as universal ART is more widely implemented, it is especially important to identify MAAs that perform well in populations with high rates of early ART and viral suppression. Our study demonstrates that the MAA‐C and LAg+VL MAA can provide accurate incidence estimates in current universal treatment settings.The authors have no competing interests to declare.EK, DD, SHE and OL designed the study. AM, BK, JB, NB‐M, AJ, HA, PB and SF had primary responsibility for the blood specimen and data collection. EK, EP‐M and REF performed laboratory testing. EW, OL, BK, AJ and DD supervised data collection. OL, EP‐M, SHE and REF supervised laboratory testing. OL, EW and DD performed data analyses. EK, EW, SHE and OL interpreted primary results. EK, SHE and OL primarily drafted the manuscript. AM, BK, JB, NB‐M, AJ, HA, PB, DD, SF and RH performed critical editing of the manuscript. EK, EW, REF, EP‐M, AM, BK, JB, NB‐M, AJ, HA, PB, DD, SF, RH, SHE and OL reviewed and approved the manuscript. Click here for additional data file.


Land‐use and land‐cover change associated with agriculture is one of the main drivers of biodiversity loss. In heavily modified agricultural landscapes, grazing lands may be the only areas that can provide essential resources for native grassland species. Management decisions, such as choice of livestock species, affect the extent to which grazing lands provide suitable habitat for native species such as pollinators.Our study compared how sheep versus cattle herbivory affected floral resources and butterfly abundance across low‐diversity, former Conservation Reserve Program (CRP) pastures managed with patch‐burn grazing.Across all years (2017–2019), flowering species richness and abundance were significantly higher in cattle pastures than sheep pastures. On average, we recorded 6.9 flowering species/transect in cattle pastures and 3.8 flowering species/transect in sheep pastures. The average floral abundance per transect was 1278 stems/transect in cattle pastures and 116 stems/transect in pastures grazed by sheep.Colias philodice was the most abundant (61% of observations). Speyeria idalia and Danaus plexippus, two butterflies of conservation concern, comprised less than 0.5% of butterfly observations.Similarly, we observed higher butterfly species richness, diversity, and abundance in cattle than in sheep pastures. In cattle pastures, we observed an average of 75 butterflies and 6.75 species per transect, compared with an average of 52 butterflies and 3.37 species per transect in sheep pastures. However, the butterfly community composition did not significantly differ between grazing treatments likely because agricultural‐tolerant, habitat generalists comprised the majority of the butterfly community. Five generalist butterflies comprised 92.3% of observations; Our results, which are among the first attempt quantifying butterfly use of post‐CRP fields grazed by livestock, show that increased precipitation and cattle grazing promoted higher forb abundance and richness. However, additional interventions may be needed to enhance floral resources to sustain and improve pollinator diversity in these landscapes. Sheep grazing resulted in significantly lower floral abundance compared with cattle grazing. However, butterfly communities were fairly homogenous and did not differ between grazers. Cattle and sheep production are major economic enterprises in the Northern Great Plains , resulting in $21.7 billion in cattle sales, $276 million in sales from sheep meat, and an additional $12.7 million from wool production in 2017 . During the study (2017–2019), the average temperatures were 12°C in May, 18°C in June, 21.9°C in July, and 19.3°C in August NDAWN, . Thirty‐Elymus hispidus [P. Opiz] Melderis), alfalfa (Medicago sativa L.), crested wheatgrass (Agropyron cristatum [L.] Gaertn), and yellow sweet clover  , and common bindweed (Convolvulus arvensis L.), field pennycress (Thlaspi arvense L.), and herb sophia (Descurainia sophia (L.) Webb ex Prantl) were also common.Our study sites are former Conservation Reserve Program fields, planted with Natural Resource Conservation Service Conservation Planting 1 (CP1) “introduced grasses” in the late 1980s , and the other cattle pasture and sheep pasture were 3 km south of Hettinger (site 3). We weighed and sorted animals to target a moderate stocking rate of 0.5–0.6 ha/AUM  in all pastures , we counted all flowering ramets within 1 m of either side of the transect to quantify how different grazers affected floral resources , we used the transformation suggested by  + 0.5]/N, where y is the response variable and N is the sample size to transform the floral diversity data. We selected distributions for each model by using “descdist” from fitdistrplus to assess the skewness and Pearsons's kurtosis values for the response variable , our dry year versus our near‐average year 2017 vs. 2018), and our wet year versus our near‐average year (2019 vs. 2018) to compare the relative effects of precipitation versus grazer species. We obtained confidence intervals for the pairwise comparisons from the previous GLMMs using the “confint” function  is that 2019 was the only year that contained two year since fire observations. Since there was strong yearly variation in floral characteristics, it seemed preferable to use data from the patch that remained unburned throughout the study and the most recently burned unit in each pasture to assess whether there was an effect of fire on floral variables. We created a model for each floral characteristic  in “glmmTMB” using burned/unburned as the main effect. Again, we used “descdist” from 2.4.3fitdistrplus  was incorporated as a random effect (strata) within “envfit”. We grouped plant species into the following functional groups: native forbs, introduced forbs, native grass, introduced grass, and native shrub. Origin of plants (non/native) was verified using the NRCS PLANTS database  had sufficient observations each year (50+) to get robust predicted densities  using package 3Colias philodice, a disturbance‐ and agricultural‐tolerant species was the most abundant, accounting for 61.3% of observations (8449 detected). The top five most abundant species (including C. philodice) were all agricultural‐tolerant, diet generalist species whose caterpillars can subsist on non‐native/weedy mustards or alfalfa: Plebejus melissa (21.3% of observations), Vanessa cardui (4.3% of observations), Pontia protodice (3.7% of observations), Colias eurytheme (1.7% of observations). Species of conservation concern such as Speyeria idalia  and Danaus plexippus (monarch) represented <0.5% of observations , but all other pairwise comparisons between grazers‐years were significant at α = 0.05 level . The effects of grazer species and precipitation were smaller for floral richness; grazer and a wet year had a positive influence and a drought year had a negative effect . Unlike floral abundance and richness, grazer species and a wet year did not have a significant effect on floral diversity. Instead, lower precipitation had a medium to strong negative effect on floral diversity .There was a strong year effect on floral abundance in cattle pastures, but floral availability in sheep pastures remained low regardless of year Figure . Both ca3.2χ2 = 6068.9, df = 1, p < .001). There were more flowers in unburned units than recently burned units . Floral richness was not significantly different between unburned and recently burned units , nor was floral diversity .Floral abundance was significantly different between recently burned and unburned units  = 15.054, p < .001; butterfly richness: χ2 (1) = 25.94, p < .001, butterfly diversity: χ2 (1) = 25.315, p < .001, Figure df = 207, p = .997, Figure p < .0.001, Figure 2 (2) = 12.840, p = .0016). For butterfly richness per transect, there was no significant difference between cattle and sheep pastures in 2018  = 1.453, p = .4836). Across all years, butterfly diversity was significantly higher in cattle pastures and there was not a significant grazer‐year interaction (Grazer: year: χ2 (2) = 2.470, p = .2908, Figure Overall, pastures grazed by cattle had significantly more butterflies, butterfly species, and higher butterfly diversity than those grazed by sheep (butterfly abundance: χ3.4k = 4). Grazer species was associated with less than 2% of the variance in the butterfly community . Year was most strongly associated with distinct patterns in the butterfly community . Time since fire intervals for unburned, recently burned, and one‐year post‐fire were clustered around the origin, meaning there was minimal variation between sites based on those time since fire . Two years since fire was further from origin, however, 2019 was the only season with 2 years since fire data, so that result is likely an artifact of limited sample size rather than a biological response to two years since fire. To see whether year was overshadowing effects of time since fire, we created separate NMDS ordinations for each year, but within each year, time since fire intervals overlapped and were not associated with a distinct pattern in the butterfly community , but were associated with minimal variation in butterfly communities (R2 = 0.02–0.07).Grazer species, TSF, floral attributes, and site characteristics showed minimal association with patterns in the butterfly community, with only year associated with distinct dissimilarities in butterfly community. We found considerable overlap in butterfly communities regardless of grazer treatment  is less beneficial to pollinator conservation than cattle grazing. Similar to other studies, we found lower floral and butterfly abundance and richness in sheep pastures compared to pastures with cattle Carvell, . It is uFloral abundance and richness responded positively to a wet year and floral richness and diversity responded negatively to a drought. High annual variability in precipitation is common in grasslands  at all sites may have limited the ability of the ordination to discern differences between pastures. Additionally, the majority of these studies occurred in wetter climates , which may influence relationships between site characteristics and butterfly community variation. Our study experienced high precipitation variation and showed low diversity in plant and butterfly communities.Although there was minimal variation in ordination space between time since fire intervals and the butterfly community, this lack of a trend suggests that fire was not detrimental to the butterfly community. We did not observe a strong association between species of concern and unburned units, which would have suggested that those species preferred the habitat characteristics associated with no fire or longer fire intervals. We burned three‐fourth of each pasture by the end of this study, but a full application of patch‐burn grazing would mean that each unit in the pastures have received fire at least once  and less than 0.5% of observations were Hopwood, .Agri‐environmental schemes such as the CRP are generally thought to be important refuges for wildlife within agricultural landscapes ; Formal analysis ; Investigation (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Torre Hovick: Conceptualization (lead); Funding acquisition (lead); Methodology ; Project administration ; Resources (lead); Supervision (lead); Writing‐review & editing (supporting). Devan McGranahan: Conceptualization ; Formal analysis ; Funding acquisition (supporting); Methodology (supporting); Visualization . Jason Harmon: Formal analysis (supporting); Writing‐review & editing (supporting). Ryan Limb: Conceptualization (supporting); Funding acquisition (supporting). Jonathan Spiess: Formal analysis (supporting); Writing‐review & editing (supporting). Benjamin Geaumont: Conceptualization (lead); Funding acquisition (lead); Investigation (supporting); Methodology (lead); Project administration (lead); Supervision (lead); Writing‐review & editing (supporting).Appendix S1Click here for additional data file.


Circular RNAs (circRNAs), a type of non‐coding RNA, are single‐stranded circularized molecules characterized by high abundance, evolutionary conservation and cell development‐ and tissue‐specific expression. A large body of studies has found that circRNAs exert a wide variety of functions in diverse biological processes, including cell cycle. The cell cycle is controlled by the coordinated activation and deactivation of cell cycle regulators. CircRNAs exert mutifunctional roles by regulating gene expression via various mechanisms. However, the functional relevance of circRNAs and cell cycle regulation largely remains to be elucidated. Herein, we briefly describe the biogenesis and mechanistic models of circRNAs and summarize their functions and mechanisms in the regulation of critical cell cycle modulators, including cyclins, cyclin‐dependent kinases and cyclin‐dependent kinase inhibitors. Moreover, we highlight the participation of circRNAs in cell cycle‐related signalling pathways and the clinical value of circRNAs as promising biomarkers or therapeutic targets in diseases related to cell cycle disorder. CircRNAs exert a wide variety of functions in diverse biological processes, including cell cycle. The cell cycle is controlled by the coordinated activation and deactivation of cell cycle regulators, including cyclins, cyclin‐dependent kinases and cyclin‐dependent kinase inhibitors. This review summarizes the functions and mechanisms of circRNAs in cell cycle regulation and highlights the clinical value of circRNAs as promising biomarkers or therapeutic targets. These RNA transcripts function as natural microRNA (miRNA) sponges or competing endogenous RNAs (ceRNAs) that communicate with and co‐regulate each other by competing or interacting with shared miRNAs. Deeper understanding of this novel RNA crosstalk will help shed light on gene regulatory networks and broaden the horizon on the study of circRNAs in human development and disease.The mammalian cell cycle is precisely regulated by diverse cyclins and cyclin‐dependent kinases (CDKs) as well as RB and p53‐related pathways.22.1The widespread use of sequencing technologies and bioinformatics has facilitated the detection of circRNAs in diverse species, including humans, fungi, plants and other organisms. Emerging evidence demonstrates that circRNAs are widely expressed in different cells and tissues.Currently, different circRNAs are being constantly discovered and recognized as hotspots in diverse diseases, such as cancer and Alzheimer's disease.2.2CircRNAs play a diverse range of roles in multiple phenotypic aspects, including cell proliferation, apoptosis, invasion and metastasis.miRNA sponges: Different sponge types can act as competing endogenous RNAs (ceRNA) to sequester miRNAs and dampen the gene expression of miRNA targets.Binding proteins: CircRNAs affect cell cycle progression by binding to RNA‐binding proteins (RBPs), thereby affecting RNA processing and mRNA stability.1/S transition. CDK2 binds to cyclin A and cyclin E to promote cell cycle entry.Transcriptional regulation: CircRNAs function as epigenetic and transcriptional regulators to regulate gene expression. For example, circNOL10 facilitates transcription factor sex comb on midleg‐like 1 (SCML1) expression by suppressing its ubiquitination, thereby influencing the modulation of the humanin polypeptide family and suppressing cell cycle progression.Translation regulation: Recent studies uncovered that circRNAs act as mRNA translation brake and protein scaffolds to regulate gene expression. Circ‐MALAT1 serves as both microRNA sponge and mRNA translation brake to facilitate self‐renewal of hepatocellular cancer stem cells.Herein, we summarize the multiple regulatory functions of circRNAs in cell proliferation and apoptosis via diverse mechanisms Figure . (i) miRTogether, these reports indicate that circRNAs play a diverse range of roles for regulating cell cycle progression by different molecular mechanisms Table . Therefo3CircRNAs can specifically interact with different partner molecules, including DNA, RNA and proteins, and function as key regulators for mediating a diverse range of phenotypic changes. In this section, we focus on the expanding landscape of circRNAs in cell cycle regulation, including regulation of cyclins, CDKs, CKIs and related pathways Table .3.11/S phase and G2/M phase transitions. The current classification of cyclins and CDKs is based on functional relevance and evolutionary conservation.1/S transition point, cyclin E–CDK2 promotes G1/S progression by phosphorylating pRB as well as several DNA replication‐related proteins.2 into M phase. Although cyclin B (with subtypes B1 and B2) is expressed in G1, S and G2 phases, its peak expression and function are found in the M phase.Cyclins and CDKs play versatile roles as regulators in G1 to S transition. CircRNAs reportedly promote the cell cycle transition by interacting with cyclin D1 and corresponding regulators. For example, Yang et al. identified abnormal overexpression of circRNA circCHFR in the ox‐LDL‐mediated vascular smooth muscle cells (VSMCs); circ‐CHFR knockdown inhibited VSMC proliferation and migration via circCHFR/miR‐370/FOXO1/CyclinD1 axis, implicating circRNAs in VSMCs and atherosclerosis.et al. found that circMYLK was markedly up‐regulated in laryngeal squamous cell carcinoma (LSCC). Gain‐of‐function experiments indicated that circMYLK facilitated LSCC cell proliferation and G1/S cell cycle transition. Thus, circMYLK elevated cyclin D1 expression by sponging miR‐195.et al. identified high expression of hsa_circ_000984 in non–small cell lung cancer (NSCLC) tissues and cell lines, which was linked to advanced TNM stage and lymph node metastasis. Functional assays demonstrated that hsa_circ_000984 silencing inhibited NSCLC cell proliferation, migration, invasion and EMT. In addition, hsa_circ_000984 exerts oncogenic effects by regulating the activation of Wnt/β‐catenin pathway, as determined by detecting its influence on the expression levels of β‐catenin, c‐myc and cyclin D1.2/M phase transition by sponging miR‐1299 and up‐regulating CDK6 expression.Current studies are focused on the effect of circRNAs on tumour progression via regulation of diverse cyclins/CDKs and their downstream genes. Mounting evidence indicates that circRNAs function as molecular sponges that absorb miRNAs to regulate cell or tumour progression. Numerous lines of evidence have proven that miRNAs may serve as pivotal modulators of the cell cycle, including G0/G1 phase. Moreover, hsa_circ_0000096 alters protein levels of CDK6, cyclin D1, MMP‐2 and MMP‐9.1/S transition by directly or indirectly affecting cyclins.On the other hand, several circRNAs inhibit the cell cycle transition via diverse molecular mechanisms. For example, circNR3C1 significantly suppresses cell proliferation and cell cycle progression by sponging miR‐27a‐3p, thereby inhibiting cyclin D1 expression; it was found to be significantly down‐regulated in bladder cancer.et al. found that high expression of circAGFG1 in triple‐negative breast cancer (TNBC) was linked to clinical stage, pathological grade and poor prognosis. Functional assays confirmed that circAGFG1 could promote TNBC cell proliferation and tumourigenesis in vivo. CircAGFG1 acts as an miRNA sponge; its binding to miR‐195‐5p increases cyclin E1 expression, indicating that circAGFG1 could serve as a diagnostic biomarker and therapeutic target in TNBC.et al.et al.There are, of course, other ways to influence tumour progression, such as by modulating activation of transcription factors, affecting DNA damage or overcoming chemoresistance, or through yet unknown mechanisms.et al. identified circMMP9 up‐regulation and its oncogenic role in glioblastoma multiforme (GBM). They found that eukaryotic initiation factor 4A3 (eIF4A3) physically interacts with matrix metallopeptidase 9 (MMP9) mRNA transcript to induce its cyclization, promoting circMMP9 production. CircMMP9 enhanced GBM cell proliferation and tumourigenesis via CDK4 and aurora kinase A (AURKA) by sponging miR‐124.et al.Accumulating evidence indicates that tumour cells require specific interphase CDKs for proliferation.3.21/S transition.Considering that most cyclins facilitate CDK activity, CKIs repress CDK activity.et al. identified down‐regulation of circ‐ZKSCAN1 in bladder cancer tissues and cell lines; circ‐ZKSCAN1 suppresses cell proliferation by sponging miR‐1178‐3p to increase p21 expression.Emerging studies suggest that circRNAs regulate cell proliferation and progression by affecting the circRNAs/miRNA/p21 axis. Bi Furthermore, circRNAs regulate CKIs through other mechanisms. CircRNAs target miRNAs that in turn target tumour suppressor genes. This axis is used to regulate cell cycle progression and considered an indicator of healing. For example, circ_0021977 was found to suppress CRC proliferation, migration and invasion by modulating the miR‐10b‐5p/p21 and p53 axis. Low circ_0021977 expression in CRC patients was correlated with higher TNM stage and poorer prognosis.3.3In addition to the p53 pathway, many other signalling pathways are regulated by a range of circRNAs in the cell cycle. Here, we discuss two circRNA‐mediated signalling pathways involved in cell cycle regulation: RB‐E2F pathway and PI3K/AKT pathway.3.3.11 to S phase is regulated by the interplay between CDKs and retinoblastoma protein (Rb) phosphorylation, thereby releasing E2F transcription factors to facilitate the expression of S phase genes. It is widely recognized that pRB targets members of the E2F family.1/S transition.et al. reported aberrant circSDHC expression profile in renal cell carcinoma (RCC) patients; it promoted RCC proliferation and invasion by competitively binding miR‐127‐3p to up‐regulate the downstream gene, CDKN3 and the E2F1 pathway.The transition from G3.3.21/S transition via inhibiting phosphorylated Rb:Rb ratio. By contrast, circ‐ZNF609 expression promotes cell proliferation in rhabdomyosarcoma, highlighting circ‐ZNF609 as a new regulator of cell proliferation‐related pathways that abrogates p‐Akt proteasome‐dependent degradation.The phosphoinositide 3‐kinase (PI3K) pathway plays an integral role in many cellular processes and is frequently altered in cancer, contributing to tumour growth and survival.4Emerging studies have shown that certain circRNAs act as promising biomarkers and therapeutic targets in various diseases. The expression patterns and characteristics of circRNAs make them perfect candidates as valuable biomarkers to improve diagnostic efficacy. Furthermore, circRNAs are abundant in human body fluids, such as blood and saliva, which makes them easily accessible and easy to detect, and thus, viable biomarkers for cancer detection, particularly via liquid biopsies. Moreover, a series of circRNAs, such as F‐circEA, are more suitable for developing biomarkers than the linear RNA transcripts.4.1Considering that circRNAs exert key roles in disease progression and cell cycle as well as feature abundance, conservation stability and prevalence,4.2et al.Several studies have focused on the clinical utility of circRNAs as promising therapeutic targets in many diseases.An understanding of gene overexpression or knockdown mechanisms may provide new clues for the therapeutic targeting of circRNAs. Currently, RNA‐based therapeutic approaches mainly include RNA interference (RNAi) and antisense oligonucleotides (ASOs), which can be designed to target a large and heterogeneous class of transcripts.1/S arrest exclusively.in vivo can pave the way for their utilization in cell cycle‐related diseases. However, more clinical trials are needed to drive the development of ncRNA‐based diagnostic tests and therapeutic interventions for cancer patients.Currently, several specific CDK4/6 inhibitors targeting CDK‐Rb‐E2F pathway are either approved or in clinical trials for the treatment of different cancer types.51/S phase transition and involve miRNA sponging, protein interaction, transcription and translation. Currently, circRNAs have been recognized to have distinct expression profiles in different diseases and might exert multifunctional roles, making them promising biomarkers for diagnosis, prognosis and treatment.In this review, we emphasized the expanding functions of circRNAs in cell cycle regulation and their potential diagnostic and therapeutic application in diverse diseases. Significantly, accumulating evidence indicates that circRNAs regulate cell cycle by directly or indirectly interacting with cyclins/CDKs. Moreover, circRNAs orchestrate cell cycle progression via distinct molecular mechanisms by interacting with RNA, DNA or proteins. The functions and mechanisms of circRNAs in cell cycle regulation are largely focused on the GDespite these recent advances, the exact mechanisms of action of circRNAs remain largely unknown, certain challenges and limitations in this field need to be overcome. Therefore, the following key questions should be answered: (i) As sequence conservation was identified in most cyclin/CDK families, are circRNAs that regulate these cyclins/CDKs also conserved? (ii) Considering that cyclins are periodic, are the circRNAs that regulate these cyclins also periodic? (iii) In light of the diversity of cyclin families and encoded peptides, is circRNA coding heterogeneous from the same source DNA, and what are the main factors and mechanisms underlying this dynamic regulation?CircRNAs are dynamic as well as specific to tissue type, genetics and stage, suggesting their potential diagnostic and prognostic significance in multiple types of cancer. The immunomodulatory activity of CDK4/6 inhibitors focuses on their regulatory effects on the proliferation and activation of immune cells. CDK4/6 inhibition resulted in significant positive therapeutic effect in patients with advanced hormone‐positive breast cancer. However, specific mechanisms of resistance for tumour cell evasion remain largely unknown.The authors declare no conflict of interest.W.X. and J.L. collected the related paper and drafted the manuscript. J.H. and L.W. helped to modify the manuscript and figures. Z.M. conceived the review article Z.M., G.S. and J.R.H designed the review and revised the manuscript. All authors read and approved the final manuscript.


Muscle weakness and exercise intolerance contribute to reduced quality of life (QOL) in Barth syndrome (BTHS). Our group previously found that 12 weeks of resistance exercise training (RET) improved muscle strength, however, did not increase muscle (lean) mass or QOL in n = 3 young adults with BTHS. The overall objective of this pilot study was to examine the safety and effectiveness of RET plus daily protein supplementation (RET + protein) on muscle strength, skeletal muscle mass, exercise tolerance, cardiac function, and QOL in late adolescents/young adults with BTHS.Participants with BTHS  performed 12 weeks of supervised RET  and consumed 42 g/day of whey protein. Muscle strength, muscle mass, exercise capacity, cardiac function, and health‐related QOL were assessed pre‐post intervention.RET + protein was safe, increased muscle strength and quality of life, and tended to increase lean mass.RET + protein appears safe, increases muscle strength and quality of life and tends to increase lean mass. Larger studies are needed to confirm these findings and to fully determine the effects of RET + protein in individuals with BTHS. We hypothesized that RET plus supplemental protein would increase skeletal muscle strength and mass and improve the quality of life subdomains associated with physical activity/fatigue.22.1TAFAZZIN mutations: exon 2: frameshift deletion, exon 10: nonsense, exon 2: nonsense, and exon 2: splicing defect. Baseline and follow‐up testing were performed at the Washington University Institute for Clinical and Translational Sciences (ICTS) Clinical Research Unit and included a medical history and physical, body composition analysis, and fasting blood chemistries including a complete blood count (CBC) and comprehensive metabolic panel (CMP). Postintervention testing occurred 48‐72 hours after the final exercise session. Studies were approved by the Human Studies Committee at Washington University in St. Louis and all participants and parents  provided written informed consent.Five , sedentary (no routine exercise ≥2x/week) male participants with BTHS were recruited from the Barth Syndrome Foundation Registry located at the University of Florida and entered this 12‐week study. Medications used by participants included beta blockers , ACE inhibitors/angiotensin receptor blockers , cardiac glycosides , and granulotcyte colony stimulating factor . Participants  had the following 2.2Body composition measurements were performed in n = 4 participants using dual‐energy X‐ray absorptiometry (DXA) . Regional and whole body fat, lean mass, bone mineral content, and bone mineral density were assessed. A certified technician performed both pretraining and posttraining tests. Body composition was performed in one participant by using air displacement plethymosgraphy  due to technical difficulties with DXA at the time of the baseline visit.2.3Two‐dimensional (2D), M‐mode, pulsed‐wave Doppler, tissue Doppler echocardiography, and 2D speckle‐tracking global longitudinal strain was performed on all participants  as previously described : 1‐RM is the maximal weight a participant can lift, through the full range of motion, just once using proper form and not substituting other groups of muscles to complete the lifting motion. 1‐RM weight was recorded for the leg press, bench press, biceps curl, seated row, knee extension, and shoulder press according to guidelines established by the American College of Sports Medicine (2000) by using the Hoist Fitness Systems  at both pretraining and posttraining testing supervised by a trained research team member.2.5Participants completed the Minnesota Living with Heart Failure Questionnaire (MLWHFQ), a 21‐item survey examining the effects of heart failure on their QOL (Rector and Cohn2.62), carbon dioxide production (VCO2), ventilation (VE), and respiratory exchange ratio (RER)  were continuously collected during the test. Achievement of peak exercise was determined by attainment of ≥85% predicted peak heart rate (220 − age) and/or RER ≥ 1.10 according to the American College of Sports Medicine.Exercise tolerance was assessed before and after the intervention by performing a graded exercise test using a ramped protocol on a recumbent cycle ergometer . Exercise intensity  was increased by 10 W/min while cycling at 60 rpm until volitional exhaustion was reached. 12‐lead ECG, blood pressure, ratings of perceived exertion, oxygen consumption . The protein powder could be added to liquid (flavored) or sprinkled over food (unflavored). Each protein bar had approximately 21 g of protein. The amino acid composition of the protein powder and bars are provided in Table 2.9P ≤ .05 .Differences between pretesting and posttesting for all outcomes were determined using Wilcoxon Rank Sum test. Significance was determined at 33.1P = .76), total protein , total carbohydrate , and total fat  did not change pre‐post intervention. As per participant report, compliance with the supplemental protein intake was >90%. The intervention was well tolerated among participants with no adverse events or complications. Plasma concentrations of glycine and glutamic acid tended (P = .07) to increase following the intervention  dietary intake including total calories . Muscle strength increased for leg press, bench press, seated row, and shoulder press on 1‐RM testing  and tended to increase muscle strength for biceps curl  lifted from session 1 to session 36 increased from 4206 ± 1776 kg to 7074 ± 2420 kg  or respiratory exchange ratio during graded exercise testing did not change with in the intervention (Table Systolic and diastolic blood pressure decreased following RET + protein Table . Echocaron Table .3.5P = .06) in the physical domain appeared to be in walking or climbing stairs  participants with BTHS.without protein supplementation in individuals with mitochondrial myopathyThe findings of improved muscle strength with resistance exercise and supplemental protein in the current study are consistent with current literature on the effects of resistance training and protein supplementation in healthy individuals without BTHS, although the increase of 1.04 kg in lean mass in our study appears to be approximately half of increases seen in healthy adults.Reassuringly, we found that the intervention did not increase markers of cardiac muscle damage or heart failure. Additionally, we found that the intervention did not change plasma markers of renal function . Moreover, the intervention in the current study did not change left ventricular systolic function but reduced both systolic and diastolic blood pressure in the participants with BTHS. The blood pressure lowering effect with resistance training is consistent with other studies in healthy individuals.Our previous pilot study examining resistance training alone did not find an improvement in quality of life however was not powered to do so. In contrast, we found improved quality of life following resistance training plus protein supplementation. The mean overall 15‐point decrease  on the Minnesota Living with Hearth Failure questionnaire in BTHS is consistent with the reported effects of exercise training and protein supplementation on quality of life in patients with non‐BTHS related heart failure. Two studies in patients with chronic systolic heart failure examining resistance exercise training alone on quality of life reported that a program of 8‐12 weeks resulted in a mean decrease of −14Our study has a number of limitations. Our findings are limited by the pilot nature of the study, the small number of participants, and that we did not perform a statistical correction for multiple comparisons and therefore future larger studies are warranted to fully elucidate the impact of this resistance training and protein supplementation in individuals with BTHS. Further, we are not able to directly assess the relative contributions of RET and/or protein supplementation on muscle size/strength. Future cross‐over study designs should be considered to determine the independent effects of RET and protein supplementation in populations with limited number of participants such as BTHS. All of our participants ingested 42 g/day of the whey protein isolate; however, the participants may have received greater benefit from individualised protein amounts based on body weight. Due to technical difficulties, air displacement plethymosgraphy was used to determine pre‐post resistance training body composition vs dual x‐ray absorptiometry for n = 4 participants however this method appears to be rigorous and reliable, providing values that are consistent with DXA.In conclusion, 12 weeks of RET with 42 g/day whey protein supplementation was safe, improved muscle strength and quality of life and tended to increase muscle mass in late adolescents and young adults with BTHS. Future larger studies are needed to confirm these findings and examine the mechanism(s) through which each of these interventions benefits strength and body composition changes in people living with BTHS.All authors declare that they have no conflict of interest.W. Todd Cade, Kathryn L. Bohnert, Barry J. Byrne, Carolyn Taylor, and Dominic N. Reeds designed the study. W. Todd Cade, Kathryn L. Bohnert, Adam J. Bittel, Lisa de las Fuentes, and Dominic N. Reeds conducted the study. W. Todd Cade, Kathryn L. Bohnert, Barry J. Byrne, and Dominic N. Reeds interpreted the data. Kathryn L. Bohnert, Grace Ditzenberger, and W. Todd Cade wrote the manuscript. All authors edited the manuscript.Studies were approved by the Human Research Protection Office at Washington University in St. Louis.All minor participants provided assent and all participants and/or parents provided written informed consent.