The as‐prepared PCN shows excellent porosity and mesoporous feature with an average pore size of 26.8 nm. It also shows superior light absorption of 88% and better thermal insulation . Based on these characteristics, the as‐prepared PCN can be used as a promising solar receiver. Under 1 sun, 2 sun, and 3 sun irradiation, the PCN‐based solar receiver shows high energy conversion efficiencies of 88%, 86%, and 84%, respectively. Taking advantage of the abundant, low‐cost, and commercial availability of pitch as well as its simple and cost‐effective manufacture method, the PCN‐based solar receiver may hold great potential for a broad variety of solar‐steam generation applications, for instance, fresh water production, power generation, desalination, and so on.Photothermal‐material‐assisted solar‐steam generation has recently attracted intensive attention due to its superior evaporation rate with high energy conversion efficiency for desalination. In this work, a simple approach for fabrication of porous carbon nanofoam (PCN) is reported, which is prepared by the carbonization of pitch using a combination of CaCO 3 nanoparticle/pitch mixture is first prepared as a novel photothermal material, which shows a high evaporation efficiency of 88% under 1 kW m−2 illumination, thus showing great potential for practical solar steam generation.Porous carbon nanofoam derived from the pyrolysis of a CaCO Though various modern technologies, for example, microfiltration, ultrafiltration reverse osmosis, multi‐effect distillation, multi‐stage flash, and adsorption, have been employed for desalination to this end, usually the need of huge energy consumption makes them unsuitable for large‐scale applications, especially in those un‐developed regions or countries.The severe issues of the scarcity of water resources or water pollution have seriously restricted the sustainable development of modern society. Therefotration, ultrafiltration reverseosmosis, multi‐ellation, multi‐se flash, and adsorption, have be The most impressive feature of solar steam generation is its high energy conversion efficiency, which is benefited from its unique “interfacial‐evaporation” manner, by comparison with the traditional bulk water vaporization. According to these design principles, a wide range of photothermal materials have been created so far, including carbon‐based materials, metallic nano‐particles, natural materials, inorganic silica‐based materials, and synthetic porous polymers. For example, plasmonics have quite extensive applications in the area of solar energy conversion as the local plasmon resonance on the surface of metal nanoparticles results in a significant increase in magnetic field strength, and thus localization of heat around the nanoparticles, the method of generating steam require high light intensities , which gets a relative low vapor generation efficiency  and increases the surface heat loss. To improve the light absorption ability of a solar receiver, the common approach to this end is to create a light absorption layer  on the surface, a porous layer with better thermal insulation. Along this line, a number of solar receivers are derived from natural products, polymers, clay, or composites and so on. Alternatively, carbon aerogel materials (such as carbon nanotube aerogels and graphene aerogels) have a highly developed pore structure, low thermal conductivity, and a wide range of light absorption, making them potential solar photothermal materials for desalination. Compared with those double‐layered solar receiver usually suffering the drawbacks of multi‐step or complicated process, carbon aerogel materials themselves have excellent light absorption due to their carbon in nature, thus showing advantage of simple fabrication. On the other hand, rich resources of carbon materials or their precursors with low‐cost make them promising candidates as solar photothermal materials especially for practical applicationsRecently, solar steam generation, which employs green and inexhaustible solar energy as energy input mediated by solar receivers, has emerged as one of the cutting‐edge technologies with great potentials for a wide range of applications such as wastewater treatment, fresh water production, power generation, desalination, and so on., 13, 14iciency,, 19, 20terials,, 22, 23rticles,, 25, 26terials,, 28 inoterials, and synolymers., 31 Forrticles,, 35, 36on layer, 38  as efficient solar receiver for solar steam generation. The as‐resulted PCN has good thermal stability, excellent light absorption, low apparent density, and thermal conductivity, which render it an ideal candidate as a solar photothermal material. Based on these merits mentioned above, the PCN shows superior solar photothermal performance with a high evaporation rate of 1.68 kg m−2 h−1 achieved under 1 sun irradiation. The solar energy conversion efficiency was measured to be as high as 88%, 86%, and 84% under 1 sun, 2 sun, and 3 sun irradiation, respectively. Taking advantages of their high solar steam generation performance, low‐cost raw chemicals, and simple manufacture process, the PCN may have great potentials as a kind of promising solar receiver for solar steam generation.Inspired by the previous works reported by others and us,, 45, 4623 nanoparticle/pitch mixture is shown in Figure3 nanoparticle was uniformly mixed at a ratio of 1:14, and a saturated NaCl solution of 10 mL was added thereto and stirring was continued. Thereafter, as shown in Figure −1. The color of the sample changed from brown to black, PCN was washed with 1 m HCl to remove CaCO3 nanoparticles. Partial electron microscope image of PCN finished products is shown in Figure A detailed procedure for preparing a CaCOFigure3 and pitch are roughly uniform and spherically clustered together to form roughly uniform micropore and mesopore arrangement. SEM showed that micropore diameters are about 100 µm, 5 nm, or bigger. As shown in Figure As shown in Figure3, nanoparticles in the sample were decomposed into CaO by heat, the crystal characteristic peak appeared. Saturated saline NaCl was also used in the experiment, which was also an ionic crystal, so the diffraction peak appeared. The specific surface area analysis of BET is shown in Figure 2‐adsorption/desorption measurement is 77.3 k, and the BET specific surface area of PCN is 16.0740 m2 g−1. The single‐point total pore volume of the pores at P/P0 = 0.9916 was found to be 0.1077 cm3 g−1 for PCN. Average pore width of adsorption (4 V A−1): 284.250 A; average pore width of desorption (4 V A−1): 225.361 A.Thermogravimetric analysis (TGA) was used to characterize and decompose. TGA was performed on the surface of pitch samples, and the results are shown in FigureA = 1 − R − T, where R and T are the reflection and transmission efficiency, respectively. The optical absorption of the PCN studied using a UV‐3600 spectrophotometer exhibited excess 86% light absorption in a wide wavelength range from 200–2500 nm, indicating the excellent light absorption of the PCN. The X‐ray photoelectron spectroscopy (XPS) revealed that PCNs were largely composed of hydrophilic materials containing oxygen‐containing groups such as C=O and C—O  was added to dichloromethane (10 mL) and magnetically stirred for 20 min to completely dissolve it. Then nanometer CaCO3 powder (7 g) and saturated salt water (10 mL) were added and stirred evenly for 30 min. The sample was molded at room temperature; it was carbonized in an N2 protected tubular furnace. The carbonization temperature was 600 °C, the heating rate was 2 °C min−1, and the insulation time to obtain PCN was 2 h. Finally, PCN was washed with 1 m HCl to remove the CaCO3 nanoparticles.In this study, pitch and nanometer CaCOThe steam generation experiments were performed under a homemade optical system, with a solar simulator, and the temperature of the PCN surface was observed with the assistance of an IR thermal camera . The experiments were typically conducted at an ambient temperature of 20 °C and humidity of ≈40%. The PCN floated on the water with the help of foam; the mass change was measured by a high accuracy balance (0.0001 g in accuracy) and then real‐time communicated to a computer for the evaluation of the evaporation rate and solar‐thermal conversion efficiency.The morphologies of the PCN were examined by SEM . The PCN surface was illuminated with a solar simulator  . Thermal conductivity values for the samples were measured with a multifunction rapid thermal conductivity tester . An energy dispersive spectrometer was equipped to detect elements and analyze their contents in samples . The powder XRD patterns were recorded on a D/Max‐2400 X‐ray diffractometer  using Cu‐Ka radiation, operated at 40 kV and 100 mA from 10° to 80°. XPS analyses were performed using a PHI‐5300ESCA spectrometer (Perkin–Elmer). The adsorption and desorption curves of PCN were determined by the Brunauer–Emmett–Teller  specific surface area test method. The optical transmittance and reflectance spectra of the PCN were measured in the range of 200–2500 nm with a Shimadzu UV3600 spectrophotometer attached to an integrating sphere.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Due to extensive clinical and genetic heterogeneity of intellectual disability (ID) syndromes, the process of diagnosis is very challenging even for expert clinicians. Despite recent advancements in molecular diagnostics methodologies, a significant fraction of ID patients remains without a clinical diagnosis.Here, in a prospective study on a cohort of 21 families (trios) with a child presenting with ID of unknown etiology, we executed phenotype‐driven bioinformatic analysis method, PhenIX, utilizing targeted next‐generation sequencing (NGS) data and Human Phenotype Ontology (HPO)‐encoded phenotype data. This approach resulted in clinical diagnosis for eight individuals presenting with atypical manifestations of Rubinstein–Taybi syndrome 2 (MIM 613684), Spastic Paraplegia 50 (MIM 612936), Wiedemann–Steiner syndrome (MIM 605130), Cornelia de Lange syndrome 2 (MIM 300590), Cerebral creatine deficiency syndrome 1 (MIM 300352), Glass Syndrome (MIM 612313), Mental retardation, autosomal dominant 31 (MIM 616158), and Bosch–Boonstra–Schaaf optic atrophy syndrome (MIM 615722). Due to extensive clinical and genetic heterogeneity of intellectual disability syndromes (ID), the process of diagnosis is very challenging even for expert clinicians. Despite recent advancements in molecular diagnostics methodologies, significant fraction of ID patients remains without clinical diagnosis. Here, in a prospective study on a cohort of 21 families (trios) with a child presenting with ID of unknown etiology, we executed phenotype‐driven bioinformatic analysis method, PhenIX, utilizing targeted next generation sequencing (NGS) data and Human Phenotype Ontology (HPO)‐encoded phenotype data. This approach resulted in clinical diagnosis for eight individuals presenting with atypical manifestations of syndromes with intellectual disability. The process of diagnosis is very challenging even to expert clinicians, owing to the clinical and genetic heterogeneity of multiple ID disorders. The introduction of next‐generation sequencing (NGS) has revolutionized diagnostics for intellectual disability patients  with a child presenting with intellectual disability, we have implemented into our diagnostic clinical workflow the bioinformatic tool, PhenIX  for DAG Panel sequencing and SureSelect Human All Exon V6 (Agilent) for WES. Each library was qualified using Bioanalyzer (Agilent) and quantified using Qubit (Life Technologies). Before the sequencing run, cluster amplification was carried out on cBot (Illumina) with TruSeq PE Cluster Kit v4 (Illumina). HiSeq4000 (Illumina) paired‐end sequencing (2 × 100 bp) was performed using TruSeq SBS Kit v4 (Illumina) with the required mean sequence coverage × 140 (GE20 ≥ 98%).Genomic DNA was extracted by an automated method  from peripheral blood samples of the patients and their parents. Next‐generation sequencing of the disease‐associated genome targeting ~2,800 genes known to be associated with Mendelian disorders  was performed for all patients .http://varnomen.hgvs.org/). The human cDNA sequence of the appropriate gene is according to the Genebank (https://www.ncbi.nlm.nih.gov/genbank/).The nomenclature of molecular variants follows the Human Genome Variation Society guidelines , Spastic Paraplegia 50 (MIM 612936), Wiedemann–Steiner syndrome (MIM 605130), Cornelia de Lange syndrome 2 (MIM 300590), Cerebral creatine deficiency syndrome 1 (MIM 300352), Glass Syndrome (MIM 612313), Mental retardation, autosomal dominant 31 (MIM 616158), and Bosch–Boonstra–Schaaf optic atrophy syndrome (MIM 615722).All 21 ID patients, before enrolment in the study, underwent detailed clinical and phenotypical examination and were classified as “Nonspecific intellectual disability” patients. In Table Patient 1, the boy diagnosed with Rubinstein–Taybi syndrome Figure , was borPatient 2 Figure , diagnosPatient 3, the girl diagnosed with Wiedemann–Steiner syndrome Figure  was bornPatient 4, the girl diagnosed with Cornelia de Lange syndrome 2 Figure  was refePatient 5 Figure  diagnoseMoreover, scoliosis and gait imbalance were noted. The proband's craniofacial phenotype was marked by posteriorly rotated ears, synophrys, diastema and crowded teeth, short philtrum, long and prominent nose. She had a friendly and happy personality.Patient 6, the boy diagnosed with Bosch–Boonstra–Schaaf optic atrophy syndrome Figure  was refePatient 7 Figure  diagnosePatient 8 Figure , diagnos4During the last decades, the clinical model of dysmorphic syndrome recognition has changed diametrically. Before the introduction of microarray chromosomal analyses or, the more, next‐generation sequencing, molecular tests were ordered based on clinicians’ suspicion from clinical examination and patients’ history (“phenotype‐first” approach). We have currently entered the era of reverse dysmorphology, where “genotype‐first” model of genetic assessment is favored  terms Table , which pBy utilizing the diagnostic workflow based on PhenIX, we established the diagnosis for 8 of 21 patients  with Rubinstein–Taybi syndrome (RTST2) had no classical features, which are broad toes/thumbs or long columella extending below the alae nasi. Besides, narrow and short palpebral fissures distorted his facial features. Because of somewhat broad tips of his fingers and downwards position of the palpebral fissures in differential dysmorphological diagnostics, we did consider Rubinstein–Taybi syndrome, and analysis of the During our observation, the facial phenotype of the girl diagnosed with Wiedemann–Steiner syndrome (WSS), Patient 3, has been notably changing and become more specific for WSS, which is in line with the literature  was definitely mild, as compared to classical phenotype of SATB2 reported at the time of the patient's examination. One feature frequently published for Glass syndrome patients is hand cleft palate, which was not noted in the patient , in Patient 5, was not taken into consideration as there were only a few cases with heterozygous mutations in the The phenotype of the patient (Patient 6) with Bosch–Boonstra–Schaaf optic atrophy syndrome (BBSOAS) was not specific enough to allow for diagnosis based on dysmorphic features. Analysis of medical records, after the molecular result, let to clinical verification and confirmation of the disorder.The diagnosis of Spastic Paraplegia 50 , Cerebral creatine deficiency syndrome 1 , as well as Mental retardation, autosomal dominant 31 , would not be possible without the application of next‐generation sequencing. None of these diseases is manifesting with specific dysmorphic features. The case of the Patient 2, diagnosed with SPG50, was even complicated by the presence of pes equinovarus also in his mother, as well as perinatal complications .5Based on the current state‐of‐the‐art, and in line with the data presented in this paper, de novo mutations constitute the most important cause of intellectual disability.Given the authors’ long‐standing experience in clinical dysmorphology, and the ability to recognize many of classical dysmorphic syndromes, it is still very challenging for them to recognize their mild/unusual presentations. In the absence of recognized phenotypic features specific for a particular monogenic syndrome, the application of unbiased genome‐wide next‐generation sequencing is mandatory to facilitate clinical diagnosis.A significant milestone in the clinical diagnostics of genetic disease was the implementation of recently developed bioinformatics tools like PhenIX (Zemojtel et al., The authors declare no conflict of interest.E.C., D.J., M.K., and T.Z, implemented molecular diagnostics, A.J.‐S., M.K.‐W., and T.Z. designed the study, A. J.‐S., M.K.‐W., and T.Z. analyzed the data, K.H.C., C.E., D.J., A.J.‐S., M. K.W., and T.Z. wrote the manuscript, M.K.‐W., J.M., and K.H.C. examined patients.Table S1Click here for additional data file.


Cancer‐related cognitive impairment (CRCI) among adolescent and young adult (AYA) cancer patients with noncentral nervous system (CNS) cancers has not been well studied. In this study, we aimed to describe CRCI‐associated trends and characteristics among AYA cancer patients.In a longitudinal cohort of AYA cancer patients without CNS disease, CRCI was evaluated over 1 year using the Functional Assessment of Cancer Therapy‐Cognitive Function Instrument, a self‐reported cognitive outcome measure. CRCI prevalence was quantified using the previously established minimal clinically important difference. CRCI‐associated longitudinal trends and factors were evaluated with mixed‐effects model analysis.P = .02), Indian ethnicity (P < .01), current smokers (P < .01), anxiety/depressive symptoms (P < .01) and fatigue (P < .01) were found to be associated with poorer cognitive function among AYAs.Ninety‐one patients (mean age = 28.4 ± 6.7 years) were included. Approximately one‐third (34.1%) experienced CRCI at least once during the study follow‐up. Female gender (Although AYA cancer patients were relatively young and without CNS disease involvement, a significant proportion of them experienced clinically important decline in cognitive function. With improved understanding of this subject, effective strategies can be formulated to promote awareness of CRCI and mitigate its negative effects among AYA cancer patients. The International Cancer and Cognition Taskforce (ICCTF) defines CRCI as a decline in memory, attention, concentration, and executive function among cancer patients.Preliminary data suggest that AYAs are encumbered by CRCI at work or in school. The AYA Health Outcomes and Patient Experience study reports that up to 53% of working cancer survivors within this age range face problems with memory or paying attention at work or school.We have previously conducted a longitudinal assessment of symptom burden and distress in a cohort of AYA cancer patients.22.1This was a secondary analysis of self‐reported cognitive outcomes collected from a longitudinal cohort study at the National Cancer Centre Singapore. The study was conducted from September 2015 to October 2018. Ethics approval was obtained from SingHealth Centralized Institutional Review Board (2015/2011).2.2Recruitment was carried out at outpatient oncology clinics. Patients who were newly referred to a medical oncologist and fulfilled the eligibility criteria were referred to the study team by their physicians. The inclusion criteria were: (a) between 15 and 39 years old and (b) an official cancer diagnosis. Those who were unable to provide informed consent, had serious comorbid illnesses , or would not have any scheduled visits subsequently were ineligible. For the purpose of this analysis, patients with organic causes of cognitive decline, such as primary and secondary CNS tumors, were excluded. All participants provided written informed consent prior to joining the study.2.3Participants were assessed at four time points over 1 year: (a) baseline at recruitment (T1); (b) 1 ± 0.5 month later (T2); (c) 6 ± 2 months later (T3); and (d) 12 ± 2 months later (T4). The three follow‐up time points were intended to correspond to the active treatment period, when treatment had just completed and during the survivorship phase. At baseline, participants provided demographic information via a self‐administered questionnaire. Information included age, gender, ethnicity, education level, smoking status, and alcohol consumption. Medical history and clinical parameters, such as diagnosis, disease staging, Eastern Cooperative Oncology Group (ECOG) performance status, and treatment details were extracted from electronic medical records. At each time point, participants were also requested to complete a series of self‐administered patient‐reported outcome (PRO) questionnaires described below.2.3.1The Functional Assessment of Cancer Therapy‐Cognitive Function (FACT‐Cog) version 3 is a 37‐item self‐reported questionnaire designed to assess CRCI.2.3.2Distress thermometer (DT) is a brief screening tool that measures distress. A problem checklist is also provided for respondents to identify items that have posed difficulties for them in the past week, including memory or concentration. The Rotterdam Symptom Checklist (RSCL) is used to assess the symptom burden of cancer patients. In this study, RSCL was also used to measure anxiety/depressive symptoms2.4t‐tests and the Chi‐square test, respectively. Longitudinal trends and factors associated with cognitive function were assessed using linear mixed‐effects models with the assessment time point  incorporated as a fixed effect and the intercept varied as a random effect for each individual participant. In order to assess the influence of treatment modality on cognitive function, the exposure of each participant to chemotherapy, surgery, or radiotherapy at each time point was forced into the model as a fixed effect. Other demographic and clinical parameters were incorporated into the model in a stepwise manner with Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) used to guide model building. Lower AIC and BIC values indicated model stability. A sensitivity analysis was conducted using the PCI score as the dependent variable in the final model. In order to compare the concordance between DT and FACT‐Cog when determining CRCI, percentage agreement and Cohen's kappa coefficient were calculated. For all statistical tests performed, a P‐value of <.05 was considered statistically significant. Adjustment for multiple comparisons was not carried out due to the exploratory nature of our study.Statistical analyses were conducted with STATA Version 15 . Descriptive statistics were used to describe the demographic and clinical characteristics of the study population and to illustrate self‐perceived CRCI trends as reported by the participants. Differences in continuous and categorical variables between participants with and without complete data were compared using two‐tailed Student's 33.1A total of 91 cancer patients with a mean age of 28.4 years old (range: 16‐39 years old) completed cognitive evaluation at baseline (Supporting Information SEighty‐two (90.1%) participants had complete data for at least one follow‐up time point while 47 participants (51.6%) had complete data for all study time points. The demographic characteristics of participants who had complete data at each study time point were largely similar to those with missing data. Of note, no significant difference between baseline self‐reported cognitive function was observed between participants who had complete and incomplete data at all study time points , Indian descent (P < .01), and smoking (P < .01) were associated with a higher likelihood of reporting cognitive‐associated complaints. Psychosocial factors, such as anxiety/depressive symptoms (P < .01) and fatigue (P < .01), both measured using the RSCL, were also associated with worse self‐perceived cognitive function  were significantly associated with self‐reported cognitive function. Based on model diagnostics Nevertheless, in view of the significant proportion of AYA cancer patients who experienced clinically important CRCI, it is vital to identify individuals who are susceptible to CRCI. This is because cancer‐related complications have been suggested to reduce the performance of AYA cancer survivors in mental tasks at work,Interestingly, higher baseline FACT‐Cog scores were noted among patients who exhibited CRCI at subsequent follow‐up time points. This suggests that patients who eventually demonstrated significant CRCI were likely to have fewer cognitive complaints or better self‐reported cognitive function at baseline. It has previously been found  that a low, rather than high, cognitive reserve increases the risk of developing CRCI.Our study has also provided an opportunity to evaluate DT as a screening tool for CRCI. Although validated PRO measures, such as FACT‐Cog, have been widely recommended for monitoring and improving symptom management in the cancer population,5Similar to older cancer patients, AYA cancer patients experience CRCI. If left unaddressed and unmanaged, CRCI can lead to devastating economic impact on AYA cancer patients and society at large. Therefore, with improved understanding of this subject, effective strategies can be formulated to promote awareness of CRCI and mitigate its negative effects among AYA cancer patients.6Our study had several limitations. Firstly, only 52% of participants had complete data at all time points. Low retention rates are characteristic of clinical studies involving AYA cancer patients.ClinicalTrials.gov identifier:NCT03476070) has been launched for this purpose, incorporating recommendations from the ICCTFThis report represents the secondary analysis of data from a longitudinal study investigating distress among AYA patients. Hence, further work is necessary to validate our findings. A prospective cohort study (7Our study addressed an important and neglected research gap within CRCI research. Although patients were relatively young and without CNS disease involvement, one‐third of AYA cancer patients experienced significant decline in cognitive function. Demographic characteristics, such as gender, ethnicity, and smoking status were also found to be linked to poor self‐reported cognitive function. Nevertheless, given the exploratory nature of our analysis, future work is needed with adequately powered studies which incorporate noncancer controls and neuropsychological assessments.The authors have declared no competing interest.AppendixS1. Supporting Information.Click here for additional data file.AppendixS2. Supporting Information.Click here for additional data file.AppendixS3. Supporting Information.Click here for additional data file.AppendixS4. Supporting Information.Click here for additional data file.AppendixS5. Supporting Information.Click here for additional data file.AppendixS6. Supporting Information.Click here for additional data file.


Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycin™, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy.Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg L− 1) and concentration-dependent tobramycin (up to 80 mg L− 1), respectively. Two common bacteriological growth media, tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB), were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin T™ removed 72 h and 168 h P. aeruginosa biofilms after 1 day treatment, while few 72 h S. aureus biofilms survived after 2 days treatment with vancomycin containing OSTEOmycin V™.The commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin™ products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment. To improve diagnosis, treatment and prevention of infections, it is necessary to differentiate between acute infections with primarily planktonic microorganisms and biofilm infections with overweight of clusters of microbial cells –4. Most Staphylococcus aureus and Pseudomonas aeruginosa, and their MIC and MBEC values determined in several studies. As shown in Tables S. aureus varies from 1 to more than 8000 mg L− 1. Similarly, the MBEC of tobramycin towards P. aeruginosa varies from 2 to 2560 mg L− 1. This large discrepancy in MBEC values is surprising, especially in light of some studies using the same strain. We hypothesize that the different test parameters and lack of standardization contributed to the large disparity.An early and correct diagnosis is necessary for proper antibiotic administration. The minimum inhibitory concentration (MIC) is defined as the lowest concentration of the antibiotics preventing visible bacterial growth, while minimum bactericidal concentration (MBC) is the lowest concentration required to kill the bacteria. MICs are used by diagnostic laboratories mainly to confirm resistance. Determination of MIC and MBC is based on planktonic cells, whereas the minimum biofilm eradication concentration (MBEC) is defined as the lowest concentration of antibiotic required to eradicate the biofilm. MBEC has not been implemented in the clinical setting yet, and the published MBEC data are often incomparable because of different experimental conditions. Tables Biofilm infections such as prosthetic joint infections and chronic osteomyelitis are difficult to treat by oral or parenteral antibiotic therapy alone and debridement is needed for physical removal of biofilms , 25. ManS. aureus and P. aeruginosa biofilm removal using vancomycin (up to 3000 mg L− 1) and tobramycin (up to 80 mg L− 1), respectively. These two antibiotics were chosen because they are recommended for serious and life-threatening infections caused by Gram-positive bacteria and Gram-negative bacteria. TSB and CaMHB were chosen enabling comparison with studies in Tables S. aureus strains DSM 110939 2+ and Mgan blood  have alsan blood , 54, butan blood , or salian blood , or humaan blood , 58. BesS. aureus biofilms  were not specific for a distinct biofilm infection and more suitable for initial testing of antibiofilm product. It was by no means meant as a standardization or guideline for clinical application. The purpose was to raise the awareness that biofilm eradication depends on many factors, including the ones mentioned here, but also pH, oxygen level, temperature, shear, and complicated by polymicrobial community interactions and the presence of human factors such as the human immune systems. For specific biofilm infections, we think it is necessary to develop assays with in vivo like environment and validate obtained results by comparing with clinical outcome.This study showed biofilm removal efficacy was influenced by media, biofilm age and antibiotics treatment duration. It is therefore necessary to take these parameters into consideration when designing experiments. We recommend choosing the conditions most similar to the in vivo situation and explaining the rationale when reporting. This study also showed that in vitro biofilms were possible to be eradicated when treated with long-term high concentrations of antibiotics. This finding needs to be confirmed by in vivo studies.S. aureus strains DSM 110939 [P. aeruginosa strains PA14 and ATCC 15442 were tested with tobramycin (Sigma-Aldrich). Both tryptic soy broth (TSB) (Sigma-Aldrich) and cation-adjusted Mueller Hinton broth (CaMHB) (Sigma-Aldrich) media were employed in susceptibility testing.M 110939  and ATCC6 colony-forming unit (CFU) mL− 1. Then, 100 μl of the diluted culture was added into each well of a 96-well-plate containing 100 μl of antibiotics at concentrations from 0.31 to 80 mg L− 1 of tobramycin or from 1.25 to 3000 mg L− 1 of vancomycin). The plate was covered and inoculated at 37 °C with shaking at 150 rpm for 24 h. After that, OD595 of each well was measured by Infinite F200 Pro  to determine MIC.The broth microdilution method was used to determine the MIC of each strain according to the procedures described in Wiegand et al. . Briefly4 CFU was added into the wells of 96 well microtiter plate (Thermo Fisher Scientific) and then covered with peg lid The biofilms were allowed to grow in TSB or CaMHB media at 37 °C with shaking at 150 rpm for 24 h or 72 h. After incubation, the lid with biofilms was transferred to a rinse plate containing 200 μl saline in each well and incubated for 1 min. The rinsed lid was then transferred to a challenge plate containing 200 μl antibiotics solution in each well. The antibiotics were prepared in the media used for growing biofilms. The plates were challenged for 24, 48, 96 or 168 h at 37 °C with shaking at 150 rpm. After challenged in the antibiotics solution, the lid containing biofilms was rinsed twice with fresh saline each time and then transferred to a recovery plate containing 200 μl sterile media followed by sonication at 40KHz for 5 min.CBD  was usedAfter removal of the lid, the recovery plate was inoculated for another 24 h at 37 °C with shaking at 150 rpm and OD595 measured by Infinite F200 Pro to determine the biofilm removal efficacy. All tests were repeated at least on two occasions with minimum 10 replicates each time. Percentage of the surviving replicates was calculated and presented as biofilm survival ratio.S. aureus ATCC 49230 and P. aeruginosa PA14 were chosen for this test. Seventy-two hours (3-days) or 168 h (7-days) biofilms were challenged with OSTEOmycin™ for 1, 2, 4 or 7 days following the method described above. S. aureus ATCC 49230 biofilms were challenged with 280 g L− 1 OSTEOmycin V™ in CaMHB, while P. aeruginosa PA14 biofilms were subjected to 220 g L− 1 OSTEOmycin T™.OSTEOmycin™ samples were obtained from European Cell and Tissue Bank. Two clinical strains ANOVA was used to calculate the difference between biofilm formation on CBD pegs. Binary logistic regression model was used to compare biofilm removal efficacy under different conditions.Additional file 1: Figure S1. S. aureus biofilm formation on CBD. After 24 or 72 h of growth, biofilms were removed from the pegs, transferred into the recovery plate and harvested by sonication. Six random wells of each row were selected for CFU count, in total 48 wells per plate. The number of CFUs per peg were different under different conditions. Generally 3 days incubation resulted in more CFUs per peg. Figure S2. P. aeruginosa PA14 biofilm formation on CBD. After 24 or 72 h of growth, biofilms were removed from the pegs, transferred into the recovery plate and harvested by sonication. Six random wells of each row were selected for CFU count, in total 48 wells per plate. The number of CFUs per peg were different under different conditions. Generally 3 days incubation resulted in more CFUs per peg. Figure S3. Flow diagram of the MBEC assay.


CBZ) causes life‐threating T‐cell‐mediated hypersensitivity reactions, including serious cutaneous adverse reactions (SCARs) and drug‐induced liver injury (CBZ‐DILI). In order to evaluate shared or phenotype‐specific genetic predisposing factors for CBZ hypersensitivity reactions, we performed a meta‐analysis of two genomewide association studies (GWAS) on a total of 43 well‐phenotyped Northern and Southern European CBZ‐SCAR cases and 10,701 population controls and a GWAS on 12 CBZ‐DILI cases and 8,438 ethnically matched population controls. HLA‐A*31:01 was identified as the strongest genetic predisposing factor for both CBZ‐SCAR (odds ratio (OR) = 8.0; 95% CI 4.10–15.80; P = 1.2 × 10−9) and CBZ‐DILI  in European populations. The association with HLA‐A*31:01 in patients with SCAR was mainly driven by hypersensitivity syndrome  rather than by Stevens‐Johnson syndrome/toxic epidermal necrolysis cases, which showed an association with HLA‐B*57:01. We also identified a novel risk locus mapping to ALK only for CBZ‐SCAR cases, which needs replication in additional cohorts and functional evaluation.Carbamazepine ( WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?HLA‐B*15:02 and HLA‐A*31:01) consistent with the immune pathogenesis.☑ Carbamazepine (CBZ) is associated with serious, and sometimes fatal, cutaneous and liver adverse reactions. Genomewide profiling has shown that these predisposing factors largely reside in the HLA region (WHAT QUESTION DID THIS STUDY ADDRESS?☑ What are the genetic predisposing factors in Northern and Southern European populations for CBZ‐induced hypersensitivity reactions affecting both the skin and liver?WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?HLA‐A*31:01 predisposes to serious cutaneous adverse reaction (SCAR) in both Northern and Southern European populations. HLA‐A*31:01 also seems to predispose to CBZ‐induced liver injury. In addition, an uncommon variant in the ALK gene was associated with an increased risk of CBZ‐SCAR.☑ Genetic profiling confirmed that HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?HLA‐A*31:01 in predisposing to a variety of CBZ hypersensitivity phenotypes and highlights the need to implement its pre‐prescription and preemptive use in clinical settings.☑ Our study adds to the overwhelming evidence of the role of Carbamazepine (CBZ) is prescribed for epilepsy, trigeminal neuralgia, and bipolar disorder.HLA‐B*15:02 in Han Chinese, Thais, and Malays,HLA‐A*24:02 has also recently been identified as a risk factor in Han Chinese.HLA‐A*31:01 is associated with a variety of CBZ‐SCAR phenotypes and maculopapular exanthema in Japanese,HLA‐A*02:06,HLA‐B*15:11,HLA‐B*51:01,CBZ‐induced SJS/TEN is strongly associated with HLA‐A*31:01 stratifying by European subpopulations and clinical phenotypes, including SCAR and DILI.The purpose of our study was to perform a meta‐analysis between our previously published CBZ hypersensitivity cohortGWAS) studies: British GWAS  and broadly European GWAS . The British GWAS included Northern European patients from the United Kingdom, which we have reported previously.TableFigureTableOur meta‐analysis included a total of 43 CBZ‐SCAR cases and 10,701 population controls from two genomewide association studies :After quality control of the imputed data, we retained 5,271,349 single nucleotide polymorphisms (SNPs) for association analyses. Our meta‐analysis identified two loci attaining genomewide significance  = 18.1; 95% CI 8.03–40.90; ALK gene was observed outside the MHC  and similar across platforms . The HLA class I allele also reached genomewide significance in the meta‐analysis  with A*31:01 r2 = 0.75.A*31:01 r2 = 0.75.HLA‐A*31:01 association was less significant in the broadly European cases than in the British cases, probably because of greater heterogeneity in their geographic origin . There were no differences in allele frequencies of the GWAS variants in control groups despite the different genotyping arrays used.The in Figure1. A loHLA‐A*31:01 represent the same underlying association signal (P < 10−5) with CBZ‐SCAR, mapping to MUC22 . Joint carriage of the three alleles had a stronger signal compared with HLA‐A*31:01 alone , but could not better explain the CBZ‐SCAR association than HLA‐A*31:01. The AA is shared by the A*33 and A*31 alleles.73 or to HLA‐A*31:01 revealed that isoleucine80 in the B locus is a new independent factor, and it is shared with HLA‐B*57:01 among other HLA B alleles.MUC22 gene  residues in different HLA alleles. Therefore, we performed an aggregated investigation by analyzing the polymorphic AA residues at these proteins. The strongest association was observed for isoleucineC22 gene Table S4HLA‐A*31:01.We stratified the CBZ‐SCAR cases by clinical phenotypes . Consistent with the literature,HLA‐A*31:01 showed a stronger effect with CBZ‐DRESS than with SJS/TEN . HLA‐B*57:01 was in strong LD (r2 = 0.8) with the most highly associated SNP in the MHC region  but was independent of HLA‐A*31:01  after conditioning on HLA‐A*31:01. The polymorphic AA position 97 showed an association when valine was present . Valine97 is specific for HLA‐B*57:01, HLA‐B*57:02, and HLA‐B*57:03 and other very rare B57 alleles.The effect of rs187926838 in the subtypes Table S5HLA‐A*31:01 carriers , although the OR was less than that seen with DRESS (OR = 12.9). Our prediction of the association with HLA‐A*31:01 was fully confirmed by HLA sequencing in 11 cases (DNA was no longer available in one patient). CBZ‐DILI cases did not show an association with rs187926838 (in the ALK gene), distinct from that seen with CBZ‐SCAR cases  and threonine9  at the HLA‐A locus. The two residues were in LD . Moreover, there was enrichment of HLA‐A*31:01 carriers in the 10 patients with CBZ‐SCAR with liver involvement compared with the remaining cases .GWAS analysis of 12 European CBZ‐DILI cases against 8,438 European controls did not identify a genomewide association because of limited power. However, 33% of cases were AR cases Table S5HLA‐A*31:01  together, the risk of developing either DILI or SCAR when given CBZ was eightfold higher in cases carrying HLA‐A*31:01 is the strongest predictor of CBZ‐SCAR in a European‐ancestry population, extending our previous study in Northern EuropeansHLA‐A*31:01 seems to predispose to CBZ‐induced liver injury, and (ii) a variant in the ALK gene was associated with an increased risk of CBZ‐SCAR but not CBZ‐DILI.In the current study, we show that HLA‐A*31:01, initially reported in 2011,et al.et al.HLA‐A*31:01 was a stronger risk factor for DRESS than SJS/TEN, in line with Genin et al.et al.HLA‐A*31:01 is the most important shared risk factor across CBZ hypersensitivity phenotypes, with the strongest predisposition being to DRESS. Indeed, our meta‐analysis shows that HLA‐A*31:01 carriers have eightfold higher risk of developing CBZ‐SCAR than noncarriers. Interestingly, a recent prospective study in Japan was able to show that pre‐prescription genotyping for HLA‐A*31:01 reduces the incidence of CBZ hypersensitivity reactions.HLA‐A*31:01 in predisposing to CBZ‐SCAR shows that there is a need to implement its use in clinical settings, as outlined in the recent Clinical Pharmacogenetics Implementation Consortium guideline.HLA‐A*31:01 association, we identified a residual effect in the MHC region mapping to MUC22 gene (lead SNP rs116071718). MUC22 codes for panbronchiolitis‐related mucin‐like protein 1 and has been associated with SJS/TEN caused by a variety of drugs, including CBZ in European patients.HLA‐B*51:01 was the second most significant HLA allele. HLA‐B*51:01 has already been reported to be marginally associated with CBZ‐SCARHLA‐B*51:01 together with HLA‐A*31:01 and HLA‐C*15:02 constitute a haplotype that has a larger OR than each single allele  differs between the skin and liver given the major role of the liver in drug metabolism and its ability to form chemically reactive intermediates.We have, we believe, for the first time evaluated whether ALK gene, which codes for the anaplastic lymphoma kinase gene. Somatic mutations in the ALK gene have been identified in different cancers,ALK gene, a receptor tyrosine kinase belonging to the insulin receptor family, seems to be important for the balance between proliferation and apoptosis.http://www.proteinatlas.org/ENSG00000171094-ALK/tissue), it may have an important role in T‐cell proliferation and keratinocyte death, both important in the pathogenesis of SCAR.A tantalizing association that we have identified in our meta‐analysis (that did not pass genomewide significance after permutation) that was observed in the SCAR but not in the DILI cases was with uncommon variants in the ALK and MUC22 genes, which require further validation and experimental investigation to determine the mechanisms. It is possible that there are also other genetic factors outside the MHC region, but we may not have had the statistical power to detect them in this study. It is, therefore, important that further work also involves a trans‐ethnic meta‐analysis of the GWAS in diverse populations, which have been undertaken to date, to fully assess differences in the genetic architecture of CBZ adverse reactions between ancestries and, more precisely, localize the underlying causal alleles.In conclusion, we have provided further data regarding genetic factors predisposing to different CBZ adverse reaction phenotypes, which are thought to have an immune pathogenesis, namely SCAR and DILI. We have extended our studies to include Southern Europeans in addition to our previous study in Northern EuropeansOur study combines results from two GWAS conducted in subjects with ancestry from Northern Europe, Italy, and Spain. All participants provided written informed consent and each study was approved by the appropriate national or institutional ethical review boards. Because the reactions have a very low prevalence, we used general population samples as study controls.et al.n = 7) originally described were excluded either because of population stratification issues or because two patients had DILI rather than skin involvement .The study included 14 patients with CBZ‐hypersensitivity of Northern European descent of the 21 recruited at the University of Liverpool, as described by McCormack TableThe study included a total of 29 CBZ‐SCAR samples obtained from two available sources: 3 cases were from the PGX40001 studyhttp://www.hypergenes.eu), the National Spanish DNA Bank cohort (http://www.bancoadn.org/), the Swedish Twin Registry cohort (http://ki.se/en/research/the-swedish-twin-registry), LAM30004 study,For this group, a total of 8,438 population controls of European descent were selected from the Wellcome Trust Case Control Consortium (not overlapping with samples from the British cohort), the population reference sample (POPRES),As part of the phase II study, we also collected 12 patients of European descent with CBZ‐DILI, in which the liver was involved by itself. The cases were recruited from two large international DILI consortia: iDILIC and DILIN,Supplementary Materials.Out of the cumulative 43 CBZ‐SCAR cases, three PGX40001 cases were previously genotyped with Illumina 1M Duo chip.z‐score fixed‐effects meta‐analysis, implemented in METAL.Q statistic,P < 0.05) with the same direction of effect on CBZ‐SCAR in both GWAS phases . Furthermore, we tested for association of each SNP with CBZ‐SCAR clinical subtypes: SJS/TEN in phase II GWAS and DRESS across both phases in the same meta‐analysis framework. We also tested for association of each SNP with CBZ‐DILI in phase II GWAS, in a logistic regression framework, under an additive genetic model, with adjustment for the principal components to account for population structure. Genomewide significance was defined using a common threshold of P < 5 × 10−8. To account for the small sample size and the disproportionate case/control ratio, we applied a permutation approach for genomewide significant signals. In particular, we randomly permuted genotypes among individuals within the same phase of the design, tested for association, and then meta‐analyzed. We applied 5 × 108 permutations to demonstrate genomewide significance. All detailed analyses and Manhattan plots were performed with R version 3.0.2.We tested for association of each SNP with CBZ‐SCAR, separately in British and broadly European GWAS, in a logistic regression framework, under an additive genetic model, with adjustment for the principal components from smartPCA to account for population structure using PLINK version 1.07.P < 0.00025 accounting for 200 observed HLA alleles across loci (0.05/200). Conditional analyses in the MHC region were undertaken and the genotypes at the conditioning SNP(s) were included as covariates under an additive model. Fixed‐effects meta‐analyses across the two phases of GWAS were performed using the methods described above for unconditional analyses. High‐resolution genotyping of HLA loci was performed on all DILI cases by Histogenetics , as previously described.For each batch, HLA alleles were inferred using HLA genotype imputation with attribute bagginghttps://dilin.org/) is supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (NIH) as a Cooperative Agreement (U01s). The Spanish DILI Registry is partly funded by the Spanish Medicine Agency, Fondo Europeo de Desarrollo Regional . CIBERehd is funded by Instituto de Salud Carlos III. The Swedish case collection SWEDEGENE (www.swedegene.se) has received support from the Swedish Medical Products Agency, the Swedish Society of Medicine (2008‐21619), Swedish Research Council (Medicine 521‐2011‐2440 and 521‐2014‐3370), and Swedish Heart and Lung Foundation (20120557 and 20140291). The EUDRAGENE collaboration received support from the EC 5th Framework program (QLRI‐CT‐2002‐02757). M.M. is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St. Thomas’ NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.This work was supported by the International Serious Adverse Events Consortium (iSAEC). The iSAEC is a nonprofit organization dedicated to identifying and validating DNA‐variants useful in predicting the risk of drug‐related serious adverse events. The Consortium brings together the pharmaceutical industry, regulatory authorities, and academic centers to address clinical and scientific issues associated with the genetics of drug‐related serious adverse events. The iSAEC's current funding members include: Abbott, Amgen, AstraZeneca, Daiichi Sankyo, GlaxoSmithKline, Merck, Novartis, Pfizer, Takeda, and the Wellcome Trust. M.P. is an NIHR Senior Investigator. M.P. and A.A. thank the MRC Centre for Drug Safety Science for support (MR/L006758/1). P.N. was supported by iSAEC. A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (under award WT098017). This is a summary of independent research partly (the DILIGEN and iDILIC sample collection) funded by the National Institute for Health Research (NIHR) Nottingham Digestive Diseases Biomedical Research Unit at the Nottingham University Hospitals NHS Trust and University of Nottingham. The DILIN (P.N. is an employee of Sema4 Mount Sinai venture, Stamford, CT, USA. M.R.N. is an employee of GlaxoSmithKline. All other authors declared no competing interests for this work.P.N., A.K.D., M.R.N., G.A., A.P.M., A.A., and M.P. wrote the manuscript. P.N., A.P.M., M.R.N., M.J.D., A.K.D., G.A., Y.S., A.A., and M.P. designed the research. P.N., A.S., and A.P.M. analyzed the data. M.P., A.K.D., P.B.W., S.B., L.M., G.A., M.I.L., R.J.A., M.W., P.H., C.S., E.S.B., P.F., K.K., T.L., A.M., M.M., E.P., W.P., A.R., N.S., G.S., L.K.T., and A.F. contributed new reagents/analytical tools.Supplementary Methods, Figures, and Tables.Click here for additional data file.


Remarkably, a high concentration of the genistein (811.57 μg/g) was found in the Cuminum cyminum. Solvent mixture (50 mL Methanol+25 mL Dimethyl sulphoxide+25 mL Water (v/v/v)), temperature (80°C), and time (1 h) were found to be the optimum extraction conditions. The concentration of genistein before optimization was 226.67 μg/g and after optimization is 811.57 μg/g. This shows the efficiency of the extraction method in the extraction of genistein without the need for hydrolysis. Novel source for genistein is identified in regular human food can be consumed in a regular diet which increases wellness of human health along with enhancing the taste of the food. The developed extraction method coupled with high throughput, sensitive, and selective UPLC-APCI-TOF-MS technique facilitates rapid quantification (8 minutes of run time) without primary purification of complex extract.Genistein has been shown to have a broad spectrum of health advantages. Only legumes were reported to have a significant amount of genistein with the highest concentration in Soybean. Soybean was found to cause allergies in children with atopic dermatitis and in adults. Limited food sources have hindered the use of genistein in daily diets, medications, and nutraceuticals. The main objective of the current research work was to discover the novel source for genistein by the simple method of extraction and quantification. Genistein was extracted by solid-liquid extraction technique. Extraction parameters were optimized by a single factor test. Identification and quantification of genistein from the selected seeds of Apiaceae were carried out using UPLC-APCI-TOF-MS. UPLC-APCI-TOF-MS method was successfully developed, validated (linearity (R Primary prevention of chronic and degenerative diseases through lifestyle interventions is the best choice for preventing morbidity and mortality. Diet is one of the important contributing factors for many chronic diseases . One parin vitro studies have reported genistein to induce proliferation in breast cancer cells [α/β ER isoforms [κB, MMPs, Bax/Bcl-2, and interaction with ERαb [Genistein  is often referred to as phytoestrogen, which is a plant-derived nonsteroidal estrogen mimic , 7, pancer cells , 14, butisoforms . Epidemiisoforms –18. Clinisoforms . Genisteith ERαb .On the other hand, genistein was experimentally proven as the most active flavonoid which has a capacity to stimulate enzymatic cleave of DNA ~10 fold , and eviGenistein content of commonly eaten food sources has been evaluated and found substantial amounts only in leguminous plants with the highest concentrations in Soybean . ResearcGenista tinctoria [Many methods have been reported for quantification of genistein including UV-Visible spectrophotometric method, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography –28. Thesinctoria  and in Minctoria , to dateAnalytical standard genistein is purchased from Sigma-Aldrich (U.S.). HPLC grade Formic acid, Acetonitrile, Ethanol, Methanol, and Dimethyl Sulphoxide (DMSO) were procured from Merck . Milli-Q water was generated using a Milli-Q purification unit  and used throughout the study.Coriandrum sativum, Apium graveolens, Cuminum cyminum, Foeniculum vulgare, Nigella sativa, Carum carvi, and Anethum graveolens, were purchased from local supermarket ; voucher specimen was numbered and deposited in the Dept. of Biotechnology, SJCE, Mysuru. Seeds were milled to fine powder, sieved (80 mesh), and refrigerated in airtight container at 4°C until analysis.Seven seed samples, namely All seven pulverized seed samples (1 g) were extracted with 100 mL of 90% Methanol at 70°C for 1 h with continuous shaking in hot water bath shaker. Filtered extract solutions were concentrated to 10 mL by keeping it in a hot air oven at 70°C . This extract solution was directly injected to the UPLC-APCI-TOF-MS system for screening and quantification of genistein.The analytical separation and quantification analysis were performed on a Waters ACQUITY UPLC system coupled with APCI-TOF mass spectrometer. Waters acquity UPLC system  consisted of column manager, binary solvent manager, and sample manager to facilitate solvent and sample delivery. Gradient elution method was followed for better separation, where mobile phase A consisted of 0.1% Formic acid in water and mobile phase B was Acetonitrile.18 (1.7 μm 1.0 × 50 mm) maintained at 50°C. The sample manager was maintained at 5°C, and the typical injection volume of extract was 2 μL. The run time was fixed as 8 min to allow full chromatographic separation which is necessary to avoid matrix effects. The concentration of analyte and standard were detected using a Waters mass spectrometer  equipped with time of flight (TOF) as mass analyser and atmospheric pressure chemical ionization (APCI) as ionizing source. The quantification analysis was achieved by operating the mass spectrometer in negative ion APCI mode, and mass spectrometry data was represented as base peak intensity (BPI) chromatogram. MS system was operated under nebulizer pressure 60 psi, vaporizer temperature 475°C, sheath gas flow rate 90 arbitrary units, discharge current 5 V, capillary temperature 170°C, capillary voltage –30 kV, and electrospray voltage 4.5 kV.The analytes were separated by the following gradient : 0 min 98% A, 4 min 2% A, 6 min 2% A, 7 min 9% A, and 8.0 min 98% A, and delivered with a flow rate of 0.2 mL/min on a ACQUITY UPLC BEH Cx-axis vs. concentration (μg/g) as y-axis.Isoflavone genistein in selected plant seed samples was identified by comparing the retention time and mass spectra of unknown peaks with the retention time and mass spectra of the reference standard. The quantification of genistein in test samples was done using an external calibration curve of standard. The standard solution of genistein at varying concentrations was injected into UPLC-APCI-TOF-MS, and calibration curve was constructed by plotting peak area as There are several factors influencing the extraction yield, of which the key factors are extraction solvent, extraction time, and extraction temperature. One of the objectives of this work was to propose suitable method conditions to extract maximum genistein from the sample. Optimization studies were carried on by following a method called single factor test, which is performed by varying one factor/parameter at a time by keeping the rest of the parameters constant.v/v), (2) Methanol : Water : Dimethyl sulphoxide (90 : 5 : 5 v/v), (3) Ethanol : Water , (4) Ethanol : Water : Dimethyl sulphoxide , (5) Acetonitrile : Water , and (6) Acetonitrile : Water : Dimethyl sulphoxide . Other conditions of extraction were kept constant (extracting temperature 70°C and extraction time 1 hour). Each extract solution was centrifuged at 2000 g, for 10 min, filtered through Whatman filter paper grade No. 1, and concentrated to 10 mL at 80°C in hot air oven. These aliquots of extracts were filtered through a 0.45 μm PVDF syringe filter prior to analysis of genistein by UPLC-APCI-TOF-MS. From each parameter study, the highest yielding factor is considered for further parameter optimization studies.Genistein is generally soluble in polar organic solvents, and subsequently, the concentration of these solvents is also very important in extracting the maximum amount of genistein from the sample. Initial extraction was performed by taking 1 g of sample in 100 mL of solvent at 70°C for 1 h. Extraction was carried out using six different solvent mixtures (1) Methanol : Water  was prepared and was fed individually in triplicate to the UPLC system. The calibration curve was established by plotting peak area versus concentration where the square of the correlation coefficient The precision of an analytical method is defined as the multiple aliquots of analyte yielding a same/close reading which is when measured repeatedly from a single homogeneous system. Relative standard deviation (% R.S.D.) application was used to determine the precision of the method. Three independent determinations at three different concentration levels  were measured on the same day at different interval of time that gives intraday precision and were measured on the consecutive days that give intermediate precision.n = 9)  was added to sample extracts, and the recovery percentage was calculated.The accuracy of the method was determined by the application of the standard addition method. Genistein at three different concentration levels ..2). (1)Lσ is the standard deviation of the response or standard deviation of y-intercepts.s is the slope of the calibration curve.Cuminum cyminum seed sample was found to contain genistein (226.67 μg/g), and the remaining seed samples did not show any presence of genistein in the mass spectrum and chromatogram. Both negative and positive ion modes were tested for standard as well as for sample. Both standard and Cuminum cyminum samples were well ionized in the negative ion mode compared to positive ion mode − ion of m/z 268 . The presence of genistein in individual seed sample was confirmed by comparing retention time and mass spectra with that of the standard genistein. Among the seven varieties of seeds, the sample containing the highest concentration of genistein is considered for further studies. The quantification of genistein was done using the external calibration curve of the analytical standard. Only the  Figures  and 2. Atrometer . The fra m/z 268  that for m/z 269 . In gene m/z 271 . Peak wiCuminum cyminum seeds with different extraction solvent mixtures is shown in P < 0.0001) among the concentrations of genistein and ranged from 201.67 μg/g–280.03 μg/g. The highest amount of genistein (280.03 μg/g) was obtained with 90% Methanol and dimethyl sulphoxide solvent system. As shown in Cuminum cyminum and extracts a remarkably high amount of genistein at a concentration of 25% from the sample compared to all other solvents alone. Concentration of dimethyl sulphoxide solvent beyond 25% was not considered in this study due to the high-temperature requirement for evaporation of dimethyl sulphoxide.The relative amount of genistein extracted from v/v/v), and hence, it was chosen as the best solvent for extracting genistein from Cuminum cyminum and used in the following experiments.The maximum amount of genistein as measured by the UPLC peak areas was obtained when extraction was performed with Methanol : Water : Dimethyl sulphoxide . As can be seen from Cuminum cyminum are summarized in v/v/v), temperature (80°C), and time (1 h) were found to be the ideal extraction conditions for extraction of genistein from Cuminum cyminum.The efficiencies of the various solvents system, concentration of DMSO, temperature degrees, and time limits for the extraction of the genistein from The linearity of the calibration curve of analytical standard genistein was constructed by plotting the area under the curve (AUC) of the main peak versus concentration. The calibration curve was found to be linear over the concentration range of 5–125 ng/mL with a correlation coefficient of 0.999.n = 9) and interday precision analysis (n = 9), the values were found to be R.S.D. = 3.6% and R.S.D. = 4.8%, respectively. The obtained R.S.D. (%) values were lower than 5.0%, which attested the precision of the method.Both intraday and interday precisions of the analytical method were determined. In the intraday precision (n = 9). The mean recovery of 107.23% (R.S.D. = 1.29%) assured the method accuracy.It was investigated by means of a standard addition experiment, at three concentration levels in triplicate . Other legumes such as Pueraria lobate/kudzu, Lupines luteus/lupin, Phaseolus vulgaris/black turtle bean, Phaseolus lunatus/baby lima bean), Phaseolus lunatus/large lima bean, Phaseolus vulgaris/red kidney bean, and Glycine max/Soybean were reported to have 316 μg/g, 86.7 μg/g, 45.1 μg/g, 40 μg/g, 34.4 μg/g, 29.3 μg/g, and 24.1 μg/g, respectively [Many plant products have been tested for genistein content from many years, including legumes, cereals, vegetables, fruits, and nuts. Legumes are known to be good sources for genistein as they contain a significant amount of genistein than other plant-based sources. Early study on phytoestrogen content of legumes reported a remarkably high amount of genistein in ectively .μg/g of genistein but less than 1 μg/g, whereas White rice, Corn flour, and Maize meal have 10 ng/g but less than 150 ng/g [μg/g and 5.1 μg/g [A variety of cereals have been screened for genistein content by Liggins et al., 2002. They found Wholegrain brown rice and shredded wheat to have 0.5 150 ng/g . Later s150 ng/g . Geniste5.1 μg/g .μg/100 g. Fig (dried), Grape , and Grape  contained 12 μg/100 g, 4 μg/100 g, and 3 μg/100 g, respectively. Berries and currants like Blackberries, Raspberries, Cranberries, and Red currant were shown to have less than 1 μg/100 g. With respect to vegetables, a variety of vegetables in both raw and cooked form were tested for genistein content. Vegetables like Cabbage, Carrot, Radish, Cauliflower, Onion, Cucumber, Lettuce, Potato (boiled), Pumpkin, and Sweet corn (boiled) were reported to have less than 1 μg/100 g. Beans and other vegetables have shown to have a considerable amount of genistein which includes Green bean (16 μg/100 g), Red lentils (19 μg/100 g), Parsley leaves (57 μg/100 g), Butter beans (21 μg/100 g), French beans (35 μg/100 g), Haricot beans (14 μg/100 g), Kidney beans (26 μg/100 g), Runner beans (78 μg/100 g), Bean sprouts (225 μg/100 g), and Chickpeas (79 μg/100 g). Among all the tested vegetables, Soybean and Soybean-derived foods have been found to contain the highest concentration of genistein and vary from 5540 to 13770 μg/100 g [Kuhnle et al., 2009, had conducted research to find out the concentration of genistein in commonly eaten vegetables and fruits in the U.K. They have found that fruits such as Apple, Banana, Apricot, Gooseberry, Lemon, Kiwi, Lychee, Mango, Watermelon, Papaya, Passion fruit, Peach, Pear, and Avocado have less than 1 μg/100 g . The majμg/100 g , 39.Genista tinctoria [Psoralea corylifolia [Glycine max [Cuminum cyminum, a member of Apiaceae, containing 811.57 μg/g of genistein as a novel food source of genistein. Cuminum cyminum has been identified as a therapeutic molecule from ancient time and reported to be beneficial against a variety of disorders including diabetes, cancer, and hypolipidemia. The Cuminum cyminum has been reported to have good inhibitory activity against α-glucosidase with IC50 value of 100 μg/mL, whereas chemical drug, Acarbose, has an IC50 value of 25 μg/mL suggesting the comparable efficiency of Cuminum cyminum against diabetes [Cuminum cyminum in daily diet could serve beneficial against the development of various diseases.To date, only legumes have been the main source for genistein, of which inctoria , Psoraleylifolia , and Glycine max  were foudiabetes . Hence, Many chromatographic methods such as thin-layer chromatography (TLC)/high-performance thin-layer chromatography (HPTLC); HPLC with MS, UV, or PDA detector; and Gas chromatography-mass spectrometry (GC-MS) have been reported for quantification of genistein, but these methods were found to have poor resolution, lower sensitivity/selectivity, and longer run time, and due to this extended run time, methods demand more material resources. HPLC method employing ultraviolet (UV) detector led to improper separations of peaks and showed a poor range of linearity. LC combined with fluorescent detectors is limited as a number of isoflavones that are naturally fluorescent are rare and the acidification of the mobile phase has a quenching effect which in turn it reduces detector sensitivity . GC-MS rIn order to facilitate high specificity and sensitivity, MS detection is a choice for the best detection system. Ultraperformance liquid chromatography (UPLC) coupled with MS system is one of the trending and preferred methods over other methods for detecting and quantifying analytes present as it is capable of providing the accurate retention time, high mass resolution, and UV-Vis absorption data for the identification. The elemental composition can be calculated with minimum (±5 ppm) mass error by the high-resolution mass. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the two most commonly used ionization sources in mass spectrometry coupled liquid chromatography. Chen et al., 2002, analysed isoflavones content in soy nutrition supplement using HPLC-MS technique and compared ESI and APCI modes. A better separation and quantification with high sensitivity has been recorded with APCI mode for analysing isoflavones in soy nutrition supplement . APCI mov/v/v)), temperature (80°C), and time (1 h) are the best suitable condition for extracting genistein from Cuminum cyminum. The concentration of genistein before optimization was 226.67 μg/g and after optimization is 811.57 μg/g. This proves the efficiency of the method in the extraction of genistein without hydrolysis step, and the obtained optimized condition is in agreement with many other research works. Although the use of DMSO has been reported to have negative effects on chromatographic resolution [The information obtained above from the optimization study illustrate that solvent mixture (50 mL Methanol+25 mL Dimethyl sulphoxide+25 mL Water (Study on comparison of extraction solvents used for the assay of isoflavones from Soybean shows correlating results by showing a high amount of genistein, when extraction is performed with the addition of Dimethyl sulphoxide . GriffitBased on these evidences and results obtained, it can be said that the composition of solvent and extraction temperature greatly influences the yield of genistein during extraction depending upon the type of plant material.Cuminum cyminum was discovered as a natural novel source for genistein, containing 811.57 μg/g of genistein, and can be consumed in the regular human diet which increases wellness of human health along with enhancing the taste of the food.Diet is predicted to contribute to approximately 50% of cancers and other major chronic diseases in the advanced world. The incorporation of food rich in phytoestrogen in the human diet has proved to lower the risk of the development of several severe diseases including cancer, cardiovascular diseases, neurological disorders, and other degenerative diseases. In this study, Cuminum cyminum. Further research on isolation of genistein compound from the crude extract of Cuminum cyminum and in vivo and in vitro studies of genistein on various disease forms are essentials. A simple and easy method of extraction accompanied with rapid and highly sensitive UPLC-APCI-TOF-MS technique is successfully developed and validated. The system requires only 8 min of run time for complete separation of molecules and to display results. The high throughput, sensitive, and selective techniques facilitate the accurate quantification of many other potential isoflavones in complex matrices such as plant extracts without primary purification in a short duration.Based on these studies, the concentration of DMSO and extraction temperature had the most profound effects on the extraction of genistein from


In the self‐administration (n = 621) vs HCP (n = 133) groups, respectively, median age was 53 vs 58 years, median time since primary ITP diagnosis was 3.7 vs 2.5 years, and median baseline platelet count at ITP diagnosis was 19.0 vs 20.0 × 109/L. In the self‐administration and HCP‐dosed groups, median romiplostim treatment duration was 89 vs 52 weeks and median total number of doses was 81 vs 50, respectively. In the self‐administration and HCP groups, respectively: 95.0% and 100.0% of patients achieved ≥1 platelet response (defined as weekly platelet count ≥50 × 109/L without rescue medication in previous 4 weeks); the median percentage of weeks with a response was 94.5% and 95.9%; and rescue medication was used in 36.7% and 39.8% of patients. Self‐administration did not adversely affect safety; duration‐adjusted rates for all treatment‐emergent adverse events (TEAEs) and bleeding TEAEs were numerically lower with self‐administration. Romiplostim self‐administration appears effective and well tolerated in eligible patients with ITP.Romiplostim self‐administration by patients or caregivers may offer time/cost savings to healthcare professionals (HCPs) and convenience for patients who avoid weekly clinic visits. We performed an integrated analysis of five clinical trials to evaluate the efficacy and safety of romiplostim self‐administration. Data were analyzed from adults with immune thrombocytopenia (ITP) who received weekly romiplostim via self‐administration or from an HCP. Patients who achieved a stable romiplostim dose for ≥3 weeks (HCP group ≥5 weeks to provide an appropriate index date to enable comparisons with the self‐administration group) with platelet counts ≥50 × 10 We describe an integrated efficacy and safety analysis of five clinical studies in which romiplostim self‐administration was used in adult patients. Furthermore, we compare patients who self‐administered romiplostim with those patients who had all their doses administered by an HCP and who had received a stable dose for ≥5 consecutive weeks, and who had achieved a weekly platelet count ≥50 × 10The results of this study should provide a clearer understanding of the safety and efficacy of romiplostim self‐administration in eligible, adequately trained patients with ITP.22.19/L to be achieved without romiplostim dose adjustment for ≥3 or ≥4 consecutive weeks  the fifth dose would represent the equivalent time point at which self‐administration could have started.The analysis set comprised the self‐administration group and the HCP comparator group. The self‐administration group included any patients who self‐injected romiplostim in any of the studies included in the integrated analysis. For these patients, the index date (day 1) was the first day a subject self‐administered romiplostim across all studies. The HCP group included patients who had received a stable dose of romiplostim for ≥5 consecutive weeks and who had achieved a platelet count ≥50 × 102.3The primary objectives of the integrated analysis were to examine the efficacy and safety of romiplostim self‐administration.9/L without the use of any rescue medication within the 4 weeks preceding platelet measurement. This was assessed at 4‐week intervals and excluded platelet counts measured following on‐study splenectomy. Rescue medication was defined as any medication or transfusion used for the purposes of increasing platelet counts. Analysis of platelet response included evaluation of the incidence of platelet response, percentage of time with a platelet response, and percentage of patients with platelet counts <50 and ≥50 × 109/L. Also included in the efficacy analysis was the incidence of rescue medication use. In addition, the total number of romiplostim doses was assessed. Finally, the number of dose adjustments (defined as observing different stable doses following ≥1 dose change in between) within 8 weeks and 6 months of the index date, as well as the duration‐adjusted rate of dose adjustment (number of dose adjustments per 100 subject‐weeks) were evaluated. Dosing algorithms used for each study are described in Table The efficacy analysis evaluated platelet response to romiplostim, defined as a weekly platelet count ≥50 × 10Safety data were collected throughout the entirety of each study and comprised the nature, frequency, severity, seriousness and relationship to treatment of treatment‐emergent adverse events (TEAEs). The TEAEs that led to withdrawal from romiplostim treatment or study withdrawal were recorded. Events of special interest included hypersensitivity, cardiac disorders, drug‐related hepatic disorders, thrombotic/thromboembolic events, hemorrhages, immunogenicity, malignancies, myelofibrosis, pulmonary disorders, renal disorders, thrombocytosis, cytopenias, anemia, and leukocytosis. Also, TEAEs were coded according to Medical Dictionary for Regulatory Activities (MedDRA) version 20.1 by Standardized MedDRA Query (SMQ) and their preferred term. Bleeding events were identified using a pre‐defined list of preferred terms from the “Hemorrhages” (narrow search) SMQ.2.4Data were analyzed using descriptive statistics with no hypothesis testing. Continuous variables were described by number (n), mean, SD, median, interquartile range , minimum and maximum. Numbers and percentages were used for categorical variables.Safety parameters were adjusted for differences in romiplostim treatment duration in the self‐administration and HCP groups and presented as duration‐adjusted event rates .33.19/L in the self‐administration group vs 20.0 × 109/L in the HCP group).The analysis set comprised 754 patients, of which 621 patients were in the self‐administration group and 133 were in the HCP group. Baseline demographics and patient characteristics are shown in Table A higher proportion of patients in the self‐administration group (38%) vs the HCP group (21%) came from a long‐term, open‐label extension study, which analyzed the clinical effects of up to 5 years of romiplostim dosing.One patient (0.2%) in the self‐administration group and 27 patients (20.3%) in the HCP group experienced dose adjustment within 8 weeks of the index date. Within 6 months of the index date, 149 (24.0%) patients in the self‐administration group and 63 (47.4%) patients in the HCP group underwent dose adjustment. From 4 weeks to 1 week prior to the index date (weeks −4 to −1), duration‐adjusted dose adjustment rates were numerically higher in the self‐administration group compared with the HCP group . In the weeks following the index date (week 1 until the end of treatment), dose adjustment rates were 1.24 vs 1.60 per 100 patient‐weeks in the self‐administration vs HCP groups, respectively.Overall, the median number of visits for assessment of platelet counts was 47 vs 53 in the self‐administration group and HCP group, respectively.3.29/L without any use of rescue medication) at least once. From week 1 until end of treatment, 95.0% (95% CI: 93.0%‐96.6%) of the self‐administration group and 100.0% (95% CI: 97.3‐100.0%) of patients in the HCP group achieved platelet response (weekly platelet count ≥50 × 109/L without any use of rescue medication in the prior 4 weeks) at least once.During weeks −4 to −1, 86.9%  of the self‐administration group and 100.0% of the HCP group (95% CI: 97.3%‐100.0%) achieved a platelet response  of these weeks with a platelet response (≥50 × 109/L) compared with 95.9% (IQR: 81.3%‐100.0%) of weeks for patients in the HCP group. Rescue medication was most commonly given during the first 6 months of the study period  and 96.2% (n = 128) of self‐administration and HCP‐dosed groups, respectively. When adjusted for romiplostim duration, the rate of observed TEAEs was numerically lower in the self‐administration group (735.7 events/100 patient‐years compared with 918.4 events/100 patient‐years in the HCP group) (Table The most frequent TEAEs were headache (38.5%), nasopharyngitis (30.9%), and arthralgia (26.6%) in the self‐administration group and headache (30.1%), fatigue (22.6%), and nausea (22.6%) in the HCP group Table . The mosWhile duration‐adjusted rates of bleeding TEAEs were similar between groups , the duration‐adjusted grade ≥3 bleeding TEAEs and SAEs associated with bleeding were numerically lower for self‐administration, vs HCP‐dosed patients at 9.4 vs 15.2 (grade ≥ 3), and 4.6 vs 8.7 (SAEs) events/100 patient‐years, respectively  compared with the HCP group , two‐thirds of whom self‐administered their medication.9/L at any visit. Similarly, only 2.2% of self‐administration patients and 4.7% of HCP‐dosed patients had high platelet levels (>400 × 109/L).Patients who self‐administer romiplostim do not seem to be at additional risk of thrombocytosis or thrombocytopenia. Although not accurate predictors of thrombosis or bleeding, thrombocytosis and thrombocytopenia are sensitive indicators of dosing accuracy. In our analysis, very few patients (less than 4% in each group) had low platelet counts of <20 × 10Dose adjustments appeared to be numerically less common with self‐administration than HCP administration between week 1 and end of treatment. The proportion of patients experiencing dose adjustments within 6 months of the index date in the self‐administration group (24%) was lower than that observed in an open‐label extension study of romiplostim self‐administration where 35% of patients (N = 292) had one or more dose adjustments over the first 6 months of self‐administration.Self‐administration did not appear to affect the safety profile of romiplostim compared with patients in the HCP group. The most commonly observed TEAEs in the self‐administration group ‐ headache, nasopharyngitis, and arthralgia ‐ are congruent with those reported in open‐label extension studies of extended romiplostim treatment.Patients with ITP are known to visit their physician more often and to have poorer work and productivity scores than healthy age‐ and sex‐matched controls.The feasibility of expanding the use of romiplostim self‐administration is strengthened by the availability of dedicated Home Administration Training (HAT) materials for HCPs and patients that are distributed with self‐administration kits.Our analysis provides some insights on which patients tend to be selected by HCPs for self‐administration of romiplostim. While statistical testing was not performed in this descriptive analysis, the self‐administration group had a trend towards a lower median age than the HCP group , suggesting that younger patients may be deemed to be more suitable. Additionally, patients in the self‐administration group appeared to be more experienced with managing their condition than those in the HCP group, with a numerically longer median time since primary diagnosis of ITP (3.7 vs 2.5 years in the HCP group), and a higher incidence of splenectomy (34.0% vs 27.8% in the HCP group).By integrating data from five romiplostim self‐administration trials, this study provides a comprehensive analysis of self‐administration compared with HCP administration in adult patients with a stable platelet count for ≥3 consecutive weeks. This study has an important strength compared with previous studies assessing romiplostim self‐administration,9/L under HCP supervision and who are willing to undergo training. Romiplostim self‐administration may lead to time savings for patients and HCPs and, therefore, potential cost savings for healthcare systems.In conclusion, these findings suggest that romiplostim self‐administration is efficacious and does not compromise safety in patients with ITP who have achieved stable platelet counts ≥50 × 10D.J.K. has received research funding from Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BristolMyers Squibb, Kezar, Principia, Protalex, Rigel, Takeda (Bioverativ), and UCB, and acted as a consultant for Actelion (Syntimmune), Agios, Alnylam, Amgen, Argenx, BristolMyers Squibb, Caremark, Daiichi Sankyo, Dova, Kyowa‐Kirin, Merck, Sharp & Dohme, Momenta, Novartis, Pfizer, Platelet Disorder Support Association, Principia, Protalex, Protalix, Rigel, Rubius, Sanofi, Genzyme, Shionogi, Shire, Takeda (Bioverativ), UCB, Up‐To‐Date, and Zafgen. D.M.A. has received research funding from Novartis and BristolMyers Squibb, and acted as a consultant for Rigel, Principia, Novartis, and Amgen. F.R. has acted as a consultant for, received lecture fees from, and received research funding from Amgen and Novartis. A.J. has participated on advisory boards, and received travel grants and speakerʼs fees for AbbVie, Amgen, Celgene, Janssen, Novartis, Roche, and Sanofi Genzyme. D.S. has acted as a consultant for and participated in speakerʼs bureau for Amgen. R.B. has been part of a speaker faculty (honoraria declined) for Amgen and Novartis, and acted as a principal investigator in clinical trials for Amgen, Novartis, CSL, BristolMyers Squibb, Principia Pharma, and Rigel. A.N. has acted as a consultant for Amgen, Angle, Argenx, Dova, Novartis, Ono, Rigel, and Shionogi, received research funding from Amgen, Novartis, and Rigel, received honoraria from Amgen, Angle, Argenx, Dova, Novartis, Ono, Rigel, and Shionogi, and provided paid expert testimony for Argenx and Rigel. J.M. has received research funding from Amgen, Dova Pharmaceuticals and Rigel. K.M. and R.O. are employed by Amgen.https://wwwext.amgen.com/science/clinical-trials/clinical-data-transparency-practices/.There is a plan to share data. This may include de‐identified individual patient data for variables necessary to address the specific research question in an approved data‐sharing request; also related data dictionaries, study protocol, statistical analysis plan, informed consent form, and/or clinical study report. Data sharing requests relating to data in this manuscript will be considered after the publication date and (a) this product and indication (or other new use) have been granted marketing authorization in both the US and Europe, or (b) clinical development discontinues and the data will not be submitted to regulatory authorities. There is no end date for eligibility to submit a data sharing request for these data. Qualified researchers may submit a request containing the research objectives, the Amgen product(s) and Amgen study/studies in scope, endpoints/outcomes of interest, statistical analysis plan, data requirements, publication plan, and qualifications of the researcher(s). In general, Amgen does not grant external requests for individual patient data for the purpose of re‐evaluating safety and efficacy issues already addressed in the product labeling. A committee of internal advisors reviews requests. If not approved, requests may be further arbitrated by a Data Sharing Independent Review Panel. Requests that pose a potential conflict of interest or an actual or potential competitive risk may be declined at Amgenʼs sole discretion and without further arbitration. Upon approval, information necessary to address the research question will be provided under the terms of a data sharing agreement. This may include anonymized individual patient data and/or available supporting documents, containing fragments of analysis code where provided in analysis specifications. Further details are available at the following: Appendix S1. Supporting information.Click here for additional data file.