To evaluate the association of the pancreatic exocrine function estimated by cine-dynamic magnetic resonance cholangiopancreatography (MRCP) using a spatially selective inversion-recovery (IR) pulse with the pancreatic endocrine function estimated by the T1 relaxation time of the pancreatic parenchyma and HbA1c values.U tests.Forty-three patients with suspected hepatobiliary or pancreatic diseases were included. Patients were classified into three groups: HbA1c < 5.7% , 5.7% ≤ HbA1c < 6.5% (prediabetes group), and HbA1c ≥ 6.5% (diabetes group). The frequency of the secretory flow of the pancreatic juice was observed within the area of the IR pulse, and the moving distance (mean secretion grade) of the pancreatic juice inflow within the area of the IR pulse on cine-dynamic MRCP, and the T1 relaxation time of the pancreatic parenchyma on the T1 map images were assessed. The MR imaging measurements were compared using Spearman’s rank correlation coefficient analysis and the Kruskal–Wallis and Mann–Whitney r = − 0.335, p = 0.028 and r = − 0.305, p = 0.047, respectively) and HbA1c values . Both the mean secretion grade and frequency of the pancreatic secretory inflow in the elevated HbA1c (prediabetes and diabetes) group were significantly lower than those in the normal group .Both the mean secretion grade and frequency of the pancreatic secretory inflow had a significant negative correlation with the T1 relaxation time of the pancreatic parenchyma (The pancreatic exocrine function estimated by cine-dynamic MRCP was significantly lower in patients with prediabetes and diabetes than in controls. Cine-dynamic MRCP with a spatially selective IR pulse may be useful for the early diagnosis of pancreatic exocrine insufficiency in patients with pancreatic endocrine insufficiency. The endocrine and exocrine tissues of the pancreas reciprocally interact to coordinate the activities of digestion, absorption, and glucose metabolisms . InsulinDiabetes mellitus (DM) is one of the most common endocrine disorders, resulting from insulin resistance and insulin deficiency and accompanied by an impaired exocrine function that manifests as an insufficient activity of digestive enzymes. Pancreatic exocrine insufficiency has been reported to be present in a considerable proportion of patients with DM (26%-74% of type 1 DM patients and 10%-56% of type 2 DM patients) . PatientTo facilitate the early diagnosis of pancreatic exocrine insufficiency, indirect pancreatic function tests, especially the measurement of fecal elastase-1 (FE-1) in spot stool, have been used in clinical practice. FE-1 has demonstrated good sensitivity and specificity in moderate and severe pancreatic exocrine insufficiency ; however, in mild cases of pancreatic exocrine insufficiency, the sensitivity is inadequate  .Magnetic resonance imaging (MRI) have been increasingly used to evaluate the impairment of the pancreatic endocrine and exocrine function in patients with pancreatic diseases, such as DM and chronic pancreatitis (CP) –11. A prTherefore, the present study evaluated the association of the pancreatic exocrine function estimated by cine-dynamic MRCP using a spatially selective IR pulse with the pancreatic endocrine function estimated by the T1 relaxation time of the pancreatic parenchyma and HbA1c values.The institutional review board of our hospital approved this retrospective study, and the need for patient informed consent was waived. Using our radiology reporting database system, we searched for patients suspected of having hepatobiliary or pancreatic diseases who underwent abdominal MRI, including T1 mapping and cine-dynamic MRCP with a spatially selective IR pulse, between March 2019 and January 2020.n = 1); unclear visualization of the main pancreatic duct (n = 2); large tumors of the pancreatic head that hampered the evaluation of the pancreatic juice inflow on cine-dynamic MRCP (n = 2); and severe fatty infiltration (n = 1). Thus, 43 consecutive patients  were included in our study. These included patients with pancreatic diseases (n = 19), such as branch-duct intraductal papillary mucinous neoplasms , pancreatic cyst (n = 1), pancreatic ductal dilatation with unknown causes (n = 4) and chronic pancreatitis (n = 2); patients with hepatobiliary diseases (n = 22) such as acute cholecystitis (n = 4), biliary stones (n = 5), cholangitis (n = 3), adenomyomatosis (n = 2), choledochal cyst (n = 1), peribiliary cyst (n = 1), intrahepatic bile ductal dilatation with unknown cause (n = 1), gallbladder polyp (n = 1), gallbladder cancer (n = 1), hepatocellular carcinoma (n = 1), hepatic hemangioma (n = 1) and fatty liver (n = 1); and patients with no diseases (n = 2). The patients were classified into three groups based on the American Diabetes Association criteria using HbA1c values as follows: normal group, HbA1c < 5.7% (n = 12); prediabetes group, 5.7% ≤ HbA1c < 6.5% (n = 15); and diabetes group, HbA1c ≥ 6.5% (n = 16).Among these patients, we identified 49 patients who underwent an HbA1c test within 1 month before or after MRI because of the clinical requirement for the assessment of glucose tolerance. Six of these patients were excluded for the following reasons: incomplete cine-dynamic MRCP  equipped with a 16-channel body coil. Patients were required to fast for at least 4 h before MR examinations. At the MR examination, 36 mg of manganese chloride tetrahydrate  was ingested orally to reduce the signal from the bowel. Cine-dynamic MRCP with a spatially selective IR pulse and T1 mapping were routinely performed as part of our hepatobiliary and pancreatic MR examination protocol. First, a breath-hold, thick-slab 2D MRCP was obtained as a reference image to depict the main pancreatic duct in the coronal plane. The imaging parameters were as follows: repetition time (TR)/echo time (TE), 5000/507 ms; echo train spacing, 6.5 ms; slice thickness, 50 mm; acquisition matrix, 512 × 512; field of view (FOV), 350 × 350 mm; and bandwidth, 558 Hz/pixel. Then, using the same MR sequence, a spatially selective IR pulse  with a width of 20 mm was placed on the pancreatic head perpendicular to the main pancreatic duct to null the static pancreatic juice signal. With this method, the static pancreatic juice in the areas subjected to a spatially selective IR pulse appeared dark, while the inflow of the pancreatic juice in these areas was observed as a high signal intensity. MRCP with a spatially selective IR pulse was repeatedly performed every 15 s (5 s of scanning and 10 s of rest) during 5 min , and a series of these MRCP images was defined as cine-dynamic MRCP with a spatially selective IR pulse  who were blinded to any clinical information of the patients, and any interpretation discrepancies were resolved by reaching a consensus. MR images were evaluated for (a) the frequency of the pancreatic juice secretion, defined as the frequency of the detection of a high signal inflow of the pancreatic juice within the area with a spatially selective IR pulse, and (b) the secretion grade of the pancreatic juice, which was assigned according to the distance that the pancreatic juice flowed into the area of a spatially selective IR pulse using the 5-point secretion grading score  . A Spearman’s rank correlation coefficient analysis was performed to evaluate the correlation between the MRI measurements and HbA1c values. Kruskal–Wallis and Mann–Whitney r = − 0.308, p = 0.044) . In contrast, the HbA1c values had a significant positive correlation with the T1 relaxation time of the pancreatic parenchyma . In addition, the T1 relaxation time of the pancreatic parenchyma had significant negative correlations with both the mean secretion grade  .In the HbA1c test, the mean HbA1c value was 6.3% ± 0.9% range 4.7%-8.2%) in all patients. The median T1 relaxation times of the pancreatic parenchyma on the T1 map images was 899 ms . In cine-dynamic MRCP with a spatially selective IR pulse, the median frequency of the pancreatic juice inflow in all patients was 7 times  in a series of 20 images. The median mean secretion grade of the pancreatic juice in all patients was 0.35 . The interobserver agreement for the detection of the inflow of the pancreatic juice and the grading of the moving distance of the pancreatic juice with cine-dynamic MRCP was excellent . Regarding the relationship of the HbA1c values with the mean secretion grade and frequency of the pancreatic secretory inflow, the HbA1c values had significant negative correlations with both the mean secretion grade (.7%-8.2% p = 0.034). In cine-dynamic MRCP findings, no significant differences were noted in the mean secretion grade (p = 0.095) and frequency of the pancreatic secretory inflow (p = 0.093) among the 3 groups. However, there were trends toward significances in both the mean secretion grade  and the frequency of the pancreatic secretory inflow  between the normal group and the prediabetes group although the differences were not statistically significant. Comparing the prediabetes group and the diabetes group, the differences in T1 relaxation time, mean secretion grade and frequency of the pancreatic secretory inflow were not significant .In the comparison among the 3 groups Table , the T1 n = 31) (Table p = 0.029). In addition, the mean secretion grade was also significantly lower in the elevated HbA1c group than in the normal group , indicating that the pancreatic juice secretion was significantly decreased in the patients with elevated HbA1c.In the comparison between the normal group and the group with elevated HbA1c  Table , the freOur findings showed that both the mean secretion grade and frequency of the pancreatic secretory inflow in cine-dynamic MRCP with a spatially selective IR pulse had a significant negative correlation with the HbA1c values. In addition, both the mean secretion grade and frequency of the pancreatic secretory inflow in the elevated HbA1c group were significantly lower than those in the normal group. A previous study demonstrated that the cut-off value of 0.7 for the mean secretion grade in cine-dynamic MRCP was the criterion of pancreatic exocrine dysfunction . This inIn this study, the T1 relaxation time of the pancreatic parenchyma showed a significant positive correlation with the HbA1c values, in accordance with a previous study . In addiIn this study, both the mean secretion grade and frequency of the pancreatic secretory inflow tended to be lower in the patients with prediabetes and DM than in those with a normal pancreatic endocrine function. This suggests that the pancreatic exocrine function might already be decreased in patients with prediabetes. Previous studies on the pancreas histology of diabetic patients showed many histopathological findings and structural changes, such as atrophy with decreased acinar and beta-cell, chronic inflammation, fibrosis due to microvascular complications, arteriosclerosis, nerve fiber loss and morphological changes in the pancreatic duct –14, and While we expected that the pancreatic exocrine function would be reduced with the progression of pancreatic endocrine dysfunction, no significant differences in the mean secretion grade or frequency of the pancreatic secretory inflow were observed between the patients with prediabetes and those with DM. In a cohort study of 667 patients with DM, Larger et al.  found noSeveral limitations associated with the present study warrant mention. First, because this study was retrospective and the number of patients was relatively small, there may have been potential selection bias. Second, the evaluation of the pancreatic secretory inflow within the area with a spatially selective IR pulse in cine-dynamic MRCP images may be subjective. However, the interobserver agreement for the assessment of the inflow of the pancreatic juice with cine-dynamic MRCP was excellent. Third, study population included patients with branch-duct intraductal papillary mucinous neoplasms which could increase the viscosity of pancreatic juice. However, the size of IPMNs was relatively small, and no patients showed the dilated pancreatic duct suggesting stagnant pancreatic flow. Therefore, it is less likely to affect the flow dynamics of pancreatic juice in these patients with IPMNs, and we believe that the validity of this study will not be compromised. Finally, we classified patients into three groups according to the HbA1c values. Although the measurement of HbA1c is adequate for the diagnosis of diabetes as well as the measurement of fasting plasma glucose and 2-h plasma glucose value during a 75-g oral glucose tolerance test, HbA1c is an indirectly measured value of the average blood glucose levels and may be influenced by a number of factors, such as age, race and presence of anemia . TherefoIn conclusion, the pancreatic exocrine function estimated by cine-dynamic MRCP with a spatially selective IR pulse was significantly decreased in patients with prediabetes and DM, suggesting the interrelation between the pancreatic exocrine and endocrine functions. Cine-dynamic MRCP with a spatially selective IR pulse may contribute to the diagnosis of pancreatic exocrine insufficiency in patients with pancreatic endocrine insufficiency.


HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L  using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation.Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin ( Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: No**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: In this paper, Peerapittayamongkol and colleagues build upon prior studies where they found correlation between a SNP in HBS1L and level of fetal hemoglobin (HbF) as well as disease severity in patients diagnosed with HbE β-thalassemia. As expression of HBS1L was reported to be reduced in culture differentiated erythroblasts from individuals with hereditary persistence of fetal hemoglobin (HPFH), the authors hypothesized that shRNA-mediated knockdown of HBS1L may lead to increased production of HbF in culture-differentiated erythroblasts of heathy volunteers and HbE β-thalassemia patients. That said, studies conducted on erythroblasts from HPFH individuals did not account for variations in genetic architecture of the globin locus or differential effects of known HbF repressor proteins. Also to the contrary, Sankaran and colleagues reported that complete loss of all major isoforms of HBS1L had no impact on hematological parameters or HbF levels  122 (23): 3845–384).Here the authors, found that expression of HBS1L in culture-differentiated erythroblasts of heathy volunteers and HbE β-thalassemia patients was essentially indifferent, however, this experiment was only designed to assess total HBS1L rather than discriminate between total and alternative splice variants of this gene product . This is particularly important as the authors provide an argument for significance of the V3 isoform in the discussion as a contradiction to prior published findings  122 (23): 3845–384). They went on to show that shRNA knockdown of HBS1L increased levels of gamma-globin mRNA and HbF in differentiated erythroblasts of heathy volunteers and HbE β-thalassemia patients without significant impact on erythroid differentiation or cell morphology. That said, they did not evaluate the expression of fetal hemoglobin per cell by flow cytometry, ability of these cultured cells to reach full maturity as demonstrated by enucleation, or compare total hemoglobin for HBS1L KD β-thalassemia cells versus healthy donors. Finally, the authors do not expand upon these initial finds by delving into the potential responsible mechanism(s) to include changes in expression of: 1) known HbF repressors or activators, 2) chromatin modifiers, or 3) erythroid-specific transcription factors that may be affected by loss of a protein involved in mRNA stability, which limits the overall significance of these findings.Major commentslines 65 – 68: The description of HBS1L function is difficult to appreciate from the provided text. Please revise these two sentences to clarify this information.Supplementary Table 1. It is preferable to use “healthy donor” as an alternative to “normal”. Patients in this study are also “normal” they just have thalassemia. Please replace N = normal with HD = Healthy donor throughout the manuscript to include figures and figure legends.lines 116 – 141. The description of the shRNAs, construction of lentiviral vector, and production and transduction can be combined into a single section.lines 148 – 162. The description of RNA isolation, reverse transcription and qPCR can be combined into a single section.Figures 1 and 2. Please combine these data into a single figure where panel A demonstrates results for the 3 healthy donors and panel B for the 4 thalassemia patients. It would be beneficial if the scale of the Y-axis were the same for panels A and B so that differences in mRNA expression can be easily appreciated by the reader. This figure also needs a descriptive legend summarizing the information presented in each panel. Please replace N = normal with HD = Healthy donor for the reason described above. Finally, it would be beneficial to place into context the mean expression level ± S.D. for the healthy donors vs. patients at each time point. Were any of the differences in expression statistically significant?Figures 3 and 4. Please combine these data into a single figure where panel A demonstrates results for HBS1L mRNA level and panel B protein level. This figure also needs a descriptive legend summarizing the information presented in each panel and could benefit from a schematic diagram shown the timing of lentiviral transduction, cell expansion and differentiation as well as collection of samples for isolation of mRNA and protein. It is odd that levels of HBS1L mRNA were unchanged relative to untreated cells by qPCR but protein levels appear to be reduced in both the cytoplasmic and nuclear fractions even though equivalent amounts of protein appear to be loaded as demonstrated by lamin and actin. Do the authors have any explanation for this result?Figures 5 and 6. Please combine these data into a single figure where panel A - D demonstrates flow cytometry results and panels E and F photomicrographs of cell cytospins. This figure also needs a descriptive legend summarizing the information presented in each panel.Figures 7, 8, and 9. Please combine these data into a single figure where panel A and B demonstrates results for alpha, beta and gamma globin expression for the healthy donors and thalassemia patients, respectively. It would be beneficial if the scale of the Y-axis were the same for panels A and B so that differences in expression can be easily appreciated by the reader. Figure 8 can be presented as panel C with results plotted as bars demonstrating mean ± SD for each condition as an alternative to the line graph which is somewhat confusing. Finally, HPLC results can be presented as panels D and E, respectively. This figure also needs a descriptive legend summarizing the information presented in each panel.Minor commentsline 29: remove space in “Hb E” to read “HbE” and remove the word “erythroid”line 30: replace “on” with “of”line 39: Change “Asea” to “Asia”line 45: remove space in “Hb E” to read “HbE”line 47: citation (6) appears to have a reduced font sizeline 47: remove space in “Hb F” to read “HbF”line 49: replace “transplantation” with “transplant”line 54: replace “amending” with “amend”lines 45-57: combine text into a single paragraphline 59: remove the word “in” and replace “reveals” with “revealed”line 61: remove space in “Hb E” to read “HbE”line 62: remove “the” and remove space in “Hb F” to read “HbF”line 64: remove “the” and remove space in “Hb F” to read “HbF”line 70: remove space in “Hb E” to read “HbE”line 75: remove space in “Hb F” to read “HbF”line 84: remove the word “normal”line 85: remove space in “Hb E” to read “HbE”line 86: replace “normal” with “volunteers” or “subjects”line 90: “Mmononuclear” should read “Mononuclear”line 121: % appears to have a reduced font sizeline 282: replace “expressions” with “expression”lines 283 and 286: replace “normal” with “healthy controls” or “healthy subjects”line 302: replace “normal” with “healthy”Reviewer #2: Hemoglobin disorders including beta-thalassemia can be cured by hematopoietic stem cell (HSC) gene therapy with beta-globin gene addition or short hairpin RNA (shRNA)-based fetal hemoglobin (HbF) induction. BCL11A gene, LRF gene, HBS1L-MYB region, and other HPFH  mutations are main targets for HbF induction. In this study, the authors demonstrated that shRNA-based knockdown of the HBS1L gene can induce HbF expression in CD34+ cell-derived erythroid cells in vitro ; however, the HbF induction levels were relatively low at 5-10% in healthy donor cells (1% at the baseline) and 35-40% in patients’ cells (20% at the baseline).Major points1. The authors should demonstrate the shRNA-target sites (shRNA1 and shRNA2) on a schema of the HBS1L gene and RNA variants. In addition, the authors should demonstrate the target sequences of shRNA1 and shRNA2.2. The authors should compare HbF amounts at the protein level in CD34+ cell-derived erythroid cells between HBS1L knockdown and BCL11A knockdown.3. The authors should evaluate erythroid-specific transcriptional factors  in erythroid cells with or without knockdown of HBS1L.4. The authors should demonstrate a schema of methods regarding cell type, conditions of lentiviral transduction, and methods of erythroid differentiation for Figures 1-2, Figures 3-4, Figures 5-6, and Figures 7-9.5. In Figure 1-2, the authors should demonstrate whether erythroid cells are sufficiently matured for hemoglobin analysis, such as cell surface analysis for CD71 and CD235a.6. In Figure 3, the authors should demonstrate HBS1L RNA amounts in un-transduced cells.7. In Figure 4, the authors should explain why HBS1L protein amounts were reduced in the shNTC control.8. In Figures 3-4, Figures 5-6, and Figures 7-9, the authors should demonstrate transduction efficiency (such as vector copy number) in transduced cells.9. In Figure 5, the author should demonstrate vector copy number (or HbF induction) among each fraction, if lentiviral transduction efficiency in bulk cells is not ~100%.10. In Figure 6, the authors should measure the percentage of enucleation among groups.11. In Figure 7, the authors should demonstrate gamma-globin RNA amounts in un-transduced cells.12. In Figure 8, the authors should demonstrate a graph of the average and standard deviation with statistical analysis regarding the absolute amounts  of HbF, HbA, and HbA2/E.Minor pints1. In Methods, the authors should define the pLL3.7 lentiviral vector.2. In Methods, the authors should explain the more detailed methods for HPLC.3. The authors should combine Figures 1 and 2, Figures 3 and 4, Figures 5 and 6, as well as Figures 7, 8, and 9.4. In the Figure 3 legend, the author should explain how to calculate HBS1L RNA amounts.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  27 Aug 2022We sincerely appreciate the Editor's, Reviewer 1's, and Reviewer 2's feedback and ideas.AttachmentRespoonse to Reviewers.docxSubmitted filename: Click here for additional data file. 16 Nov 2022
 PONE-D-21-35741R1

 Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells.

 PLOS ONEDear Dr. Peerapittayamongkol,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.The authors have properly addressed the comments raised during the first round of revision and have substantially improved the overall quality of the manuscript.However, before being considered for acceptance, the manuscript should be revised (minor revision) in order to address the comment raised by Reviewer 3: "Interactions of the erythroid-specific transcription factor GATA-1 have been demonstrated with several sites in the HBS1L-MYB interval. Since the authors mention that the expression of GATA-1 was relatively unchanged, this has to be incorporated and explained in the discussion."plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Dec 31 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.
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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments:The authors have properly addressed the comments raised during the first round of revision and have substantially improved the overall quality of the manuscript.However, before being considered for acceptance, the manuscript should be revised (minor revision) in order to address the comment raised by Reviewer 3: "Interactions of the erythroid-specific transcription factor GATA-1 have been demonstrated with several sites in the HBS1L-MYB interval. Since the authors mention that the expression of GATA-1 was relatively unchanged, this has to be incorporated and explained in the discussion."[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #3: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #3: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #3: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #3: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #3: It is well designed and writen paper that presents the ability of shRNA-induced HBS1L knockdown to increase HbF in thalassemic erythroblasts and furthermore to slightly improve erythroid differentiation.One suggestion: Interactions of the erythroid-specific transcription factor GATA-1 have been demonstrated with several sites in the HBS1L-MYB interval. Since the authors mention that the expression of GATA-1 was relatively unchanged, this has to be incorporated and explained in the discussion.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #3: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  6 Dec 2022I want to thank Reviewers 1 and 2 for addressing the important issues in the earlier submission. I sincerely appreciate Reviewer 3's remarks on our reviewer response and the interest to discuss about the relationship between GATA-1 and the HBS1l-MYB intergenic region. Additional discussion has been added to line number 375-390 of the present revision.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 17 Jan 2023Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells.PONE-D-21-35741R2Dear Dr. Peerapittayamongkol,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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Knowledge about research priorities enables researchers to align their work with the needs of people with diabetes.To investigate (i) the importance and priorities of research objectives for people with type 1 (T1DM) and type 2 diabetes (T2DM); (ii) subgroups with specific research priorities; (iii) associated factors  of the subgroups. The cross-sectional survey was conducted in 2018 using data from 869 respondents  from a German statutory health insurance population. Diabetes-related research priorities were assessed with a questionnaire. Subgroups and associated factors were identified using latent class analysis. Three subgroups were found in T1DM: (1) high priority for the research topic ‘healing diabetes’ and moderate priority for the research topic ‘prevention of long-term complications’, (2) priorities for simplifying handling (high) and stress reduction (moderate), (3) priorities for healing diabetes (high) and simplifying handling (high). Three subgroups were found in T2DM: (1) priorities for simplifying handling (moderate), diabetes prevention (moderate) and prevention of long-term complications (moderate), (2) priorities for stress reduction (high) and diabetes prevention (moderate), (3) priorities for simplifying handling (high) and stress reduction (high). Classes differed in age and HbA To provide high quality care for people with DM, a patient-centred approach with a particular attention to patient and public involvement is a fundamental principle3. In early stages of diabetes research processes, patient-centredness can be achieved by incorporating the concerns, needs, and values of the stakeholders in the research agenda6.Diabetes mellitus (DM) is a chronic health condition that comprises different groups of heterogeneous diseases and has an increasing global prevalence8, which remain insufficiently addressed9. A cluster-randomized controlled trial showed that when patients are involved, priorities for healthcare improvement can change and a higher level of agreement between patients and physicians can be achieved10. According to the Patient-Centered Outcomes Research Institute, the term ‘research priority setting’ includes the following engagement activities: ‘provide input on the research topic, prioritization/agenda setting and how to frame the research question’11. Patients are seen as experts on the disease through their individual experiences in the healthcare system12.The research priorities of physicians and scientists do not always match the needs and preferences of patients14. A recent review by Harris et al.15 investigated health outcomes in patient and community involvement in diabetes research projects. The main benefits of patient and public involvement on the research agenda were ‘initiating the research topic’ and ‘identifying different research questions’. Furthermore, several studies involved different groups of stakeholders (e.g. people with type 1 diabetes (T1DM), type 2 diabetes (T2DM) or other types of diabetes; relatives and health care providers) and used different study designs (e.g. mixed-methods and participatory approaches) to identify priorities in diabetes research27. However, it is still unclear how diverse these priorities are within the DM community. Brown et al.17 suggest that the individual perspectives of patients (e.g. determined by age26) have an influence on research priorities. Depending on the method used, there would be a risk for under-representing particular groups when setting research priorities (e.g. people with multiple comorbidities)25. In Germany, there was only one questionnaire study in 201123 that analyzed the research priorities of people with DM. However, it used a convenience sample from the readership of a popular German news magazine and did not investigate the differences between groups of people with different sociodemographic or diabetes-related characteristics.Previous studies have shown that people with DM have a high interest in diabetes researchThe present questionnaire study aims to investigate (i) the importance and priorities of future diabetes-related research objectives for people with T1DM and T2DM in Germany; (ii) different subgroups of people with specific diabetes-related research priorities; and (iii) associated characteristics of the identified subgroups, such as sociodemographic and diabetes-related characteristics.pronova BKK’). The inclusion criteria for a diabetes diagnosis based on health insurance data were adapted from other studies29. People between the ages of 18 and 80 years were included if they had ‘(i) a regular documentation of 10th International Classification of Diseases (ICD-10) diagnosis “diabetes” (E10–E14) in three of four quarters in 2016 or (ii) regular prescription of antihyperglycaemic drugs  at least two prescriptions in 2016 or (iii) a single prescription of an antihyperglycaemic drug 2016 and a diagnosis of “diabetes” or a single prescription of an antihyperglycaemic drug within 2016 and a blood glucose or hemoglobin A1c (HbA1c) measurement in the same quarter’28. Patients who had a caregiver, a care level 2–5, ICD F70–79 code  or ICD Z51.5 code  were excluded.This quantitative cross-sectional study was conducted from September 2018 to November 2018. Data were collected using a postal questionnaire among a nationwide random patient sample of a German statutory health insurance . The questionnaires were returned to the Institute of Health Services Research and Health Economics by means of an enclosed prepaid envelope. Finally, a total of 869 people responded (29% response).30 was based on a literature review and five focus groups consisting of people with T1DM and T2DM30. The focus group data were analyzed by a multidisciplinary, professional team  using qualitative inductive content analysis. As a result, the authors developed a questionnaire that included 18 diabetes-related research objectives. In a pretest phase, experts in the field of DM and questionnaire design were consulted, and cognitive pretests were conducted with patients treated by specialists in diabetes and with people from a diabetes self-help group. In addition, a final standard field pretest was conducted (n = 27). The final questionnaire can be found in Supplementary Material.The questionnaire intended to measure the importance of and priorities for future diabetes research objectivesIn the first section of the questionnaire, people with DM were asked the following: ‘How important is it for you personally that diabetes research accomplishes the following results in the next few years?’. Each of the 18 objectives could be answered on a 4-point Likert scale from ‘not very important’ to ‘extremely important’. In the second section of the questionnaire, the respondents were to indicate up to three of the 18 research objectives as their priorities. In our study, these 18 objectives were grouped into the following seven overarching topics  and whether they were primarily treated by a general practitioner or a diabetologist.We collected age (in years), sex and education as sociodemographic factors. Education was assessed by reference to the highest school graduation, which was dichotomized and coded as ‘other graduation’ (including those without a school graduation) or ‘university entrance qualification’. In addition, we included the type of diabetes coded as ‘type 1 diabetes’ and ‘type 2 diabetes’. The code ‘unknown’ was excluded from statistical analysis. Diabetes duration was measured by the question ‘How long have you had diabetes?’, and HbAWe obtained descriptive summaries for participant characteristics depending on the distribution of the variables by frequencies, percentages, means (M), standard deviations (SD), median and interquartile range. Data were analyzed and presented separately for the two types of diabetes, T1DM and T2DM. Latent class analyses (LCAs) were performed with Mplus Version 8.4 ; the remaining quantitative analyses were performed with SAS 9.4.33, we chose the best model by analyzing whether the classes were meaningful and had a minimum class prevalence of 5%. In addition, we also took into account the ‘law of parsimony’34 and considered the following indicators for model fit: Lower values of the Bayesian information criterion (BIC) indicated better fit. A (relative) entropy close to ‘one’ indicated high separation of classes.LCA is a statistical approach used to identify a finite set of mutually exclusive and exhaustive unobserved (latent) classes of individuals based on their pattern of observed multivariate categorical variables. To identify the optimal number of subgroups with specific diabetes-related research priorities and associated characteristics, LCAs without covariates were performed. We modelled latent classes with one to six classes based on the merged seven topics. In line with Lanza et al.1c values (including the answer ‘I don't know’), insulin injection and primary practitioner. The variables ‘insulin injection’ and ‘primary practitioner’ were excluded in models involving participants with T1DM, as T1DM patients are generally treated with insulin and in specialized practices by diabetologists.Finally, we performed LCA models for T1DM and T2DM with the favored number of classes including the following variables: age, sex, education, diabetes duration, HbAThe present study was approved by the ethics committee of the Heinrich Heine University Düsseldorf . The study was performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.Table Respondents with T1DM have most often rated the objective ‘diabetes complications are detected early or, better yet, prevented’ as very or extremely important for future diabetes research (96.3%)  and ‘technical systems think and act independently when measuring blood sugar and injecting insulin’ (32.9%)  Fig. . FurtherWhen asked to identify the top three research objectives, the majority of respondents with T2DM selected the following two: ‘drugs allow for stable blood sugar levels’ (34.0%) and ‘diabetes complications are detected early or, better yet, prevented’ (33.1%)  among the top three Fig. . ResearcMost respondents with T2DM also selected at least one research objective on ‘simplifying diabetes handling’ (65.2%). Research objectives concerning ‘information and personal responsibility’ (21.2%) and ‘prevention of acute complications’ (13.6%) were mentioned less frequently.The analysis showed that a three-class model exhibited the best fit for people with T1DM and T2DM. This model had the lowest BIC value and the best entropy score. The model fit indicators are described in detail in Supplementary Table High priority for the research topic ‘treatment with the aim to cure diabetes’ and moderate priority for the research topic ‘prevention of long-term complications  and preventing long-term complications’ (moderate)): This class included participants most likely to prioritize research objectives concerning ‘treatment with the aim to cure diabetes’ (96.0%). Here, the topic ‘prevention of long-term complications’ had the probability of 55.3%. For the other topics, the probability was between 0.0% and 48.2%. Overall, the estimated class prevalence was 17.1%.High priority for the research topic ‘simplifying diabetes handling’ and moderate priority for the research topic ‘stress reduction’ (class with priorities for simplifying handling (high) and stress reduction (moderate)): This class included participants most likely to prioritize research objectives concerning ‘simplifying diabetes handling’ (79.1%). For ‘stress reduction’, the probability was 48.0%. For other topics, it lay between 0.0% and 36.5%. The estimated class prevalence was 53.3%.High priority for the research topic ‘treatment with the aim to cure diabetes’ and for ‘simplifying diabetes handling’  and simplifying handling (high)): This class included participants who prioritize research objectives on the topics ‘treatment with the aim to cure diabetes’ and ‘simplifying diabetes handling’ (100.0% respectively). For other topics, the probability was between 1.2% and 20.0%. The estimated class prevalence was 29.6%.For people with T2DM, we identified the following three research priority profiles , diabetes prevention (moderate), and preventing long-term complications (moderate)): This class included participants most likely to prioritize research objectives on the topic ‘simplifying diabetes handling’ (68.9%). For the topics ‘diabetes prevention’ and ‘prevention of long-term complications’, the probability was 45.4% and 43.7%. For the others, the probability was between 0.0% and 29.8%. The estimated class prevalence was 54.7%.High priority for the research topic ‘stress reduction’ and moderate priority for the research topic ‘diabetes prevention’ (class with priorities for stress reduction (high) and diabetes prevention (moderate)): This class included participants who prioritize research objectives on the topic ‘stress reduction’ (100%). In addition, half prioritize research objectives concerned with ‘diabetes prevention’ (50%). For the others, the probability was between 0.0% and 30.8%. The estimated class prevalence was 17.9%.High priority for the research topics ‘simplifying diabetes handling’ and ‘stress reduction’ (class with priorities for simplifying handling (high) and stress reduction (high)): This class included participants who prioritize research objectives on the topics ‘simplifying diabetes handling’ and ‘daily stress’ (100% respectively). For other topics, the probability was between 5.0% and 19.3%. The estimated class prevalence was 27.4%.For people with T1DM, we identified the following three research priority profiles Fig. :High pri1c-values. In T1DM, the class ‘priorities for healing diabetes (high) and preventing long-term complications (moderate)’ was younger than the class ‘priorities for simplifying handling (high) and stress reduction (moderate)’  and stress reduction (moderate)’ and was more likely to have intermediate or higher (≥ 7.5%) than lower HbA1c values compared with the class ‘priorities for healing diabetes (high) and simplifying handling (high)’. The class ‘priorities for simplifying handling (high) and stress reduction (moderate)’ was older than the other two and more likely to have lower than intermediate/unknown HbA1c values compared with the classes ‘priorities for healing diabetes (high) and preventing long-term complications (moderate)’/ ‘priorities for healing diabetes (high) and simplifying handling (high)’.We found significant associations between belonging to one of the identified subgroups and age as well HbA)’ Table . In addipriorities for simplifying handling (high) and stress reduction (high)’ was younger than the class ‘priorities for simplifying handling (moderate), diabetes prevention (moderate) and preventing long-term complications (moderate)’. Respondents’ characteristics stratified by classes are presented in Table In T2DM, classes differed significantly in age. The class ‘class with priorities for healing diabetes (high) and prevention of long-term complications (moderate)’ , ‘class with priorities for simplifying handling (high) and stress reduction (moderate)’ (53.3%), ‘class with priorities for healing diabetes (high) and simplifying handling (high)’ (29.6%). They differed significantly in age and HbA1c values. People with T2DM could be assigned to the following three classes: ‘class with priorities for simplifying handling (moderate), diabetes prevention (moderate), and prevention of long-term complications (moderate)’ (54.7%), ‘class with priorities for stress reduction (high) and diabetes prevention (moderate)’ (17.9%), and ‘class with priorities for simplifying handling (high) and stress reduction (high)’ (27.4%). Two of them differed significantly in age.Most respondents rated all future research objectives as at least very important, especially ‘diabetes complications are detected early or, better yet, prevented’. Research priorities primarily concerned research objectives related to the topic ‘simplifying diabetes handling’. Comparing T1DM and T2DM, people with T1DM assigned higher priority to the research topic ‘treatment with the aim of curing diabetes’ and people with T2DM to the research topic ‘diabetes prevention’. The following three classes of people with T1DM with different research priority profiles were identified: ‘25. The present results showed that while this objective pertaining to the research topic ‘prevention of long-term complications’ matter greatly, other research topics were given even higher priority when objectives were grouped. In particular, respondents reported research priorities on ‘simplifying diabetes handling’, e.g. simple ways to measure blood glucose, which were also clearly expressed in previous work30. As discussed by other authors30, one explanation could be that research priorities of people with DM reflect basic needs and are therefore predominantly focused on short-term solutions that directly affect their current daily lives. A dialogue about different research priorities can increase the relevance of diabetes research, e.g. by raising awareness of everyday issues15. The findings support the recommendations of other studies6 that research institutes and funders should involve patients more in decision-making about the research direction.A high importance of the research objective ‘diabetes complications are detected early or, better yet, prevented’ was also found in other studies on T1DM and T2DM2. In T1DM, prioritization of research objectives on the research topic ‘treatment with the aim to cure diabetes’ was also mentioned by others18 and may occur due to frequent life-threatening complications2 and lifelong insulin treatment35. In future, T1DM could be cured through biological approaches such as pancreas transplantation37. Respondents’ research priorities may be influenced by new scientific advances expected in the search for a cure in the coming years36. In T2DM, prioritization of research objectives on the research topic ‘diabetes prevention’ may be influenced by knowledge of several effective (lifestyle) interventions that have been shown to prevent T2DM40. Therefore, prevention is also of great importance in medical care39. Our findings were new compared to the previous German study where differences between diabetes types were not investigated23. Comparing the research priorities of the two studies, we see that the priorities identified by Arnolds et al.23 in relation to the research topics ‘treatment with the aim to cure diabetes’ and ‘simplifying diabetes handling’ are more in line with the present profiles for T1DM. Arnolds et al.23 found that research on psychological aspects and trainings were prioritized less frequently, while these had a higher priority in our study, especially for people with T2DM . In the light of our findings, we recommend considering the research priorities of people with T1DM and T2DM separately.Differences in research priorities between respondents with T1DM and T2DM may be due to the heterogeneous health conditions, e.g., the underlying cause of increase in blood glucose levels1c values). Therefore, this study was not able to identify ‘types’ of people41 with specific research priorities and characteristics. The question arises whether the differences can be explained by other factors not collected in the present study, e.g., cultural background17 and comorbidities25. Nevertheless, being aware of the different research priorities is important when involving people with T1DM and T2DM more specifically in research projects.Although different research priority profiles were identified in people with T1DM and T2DM, the classes did not differ significantly in the characteristics collected  and stress reduction (moderate) rather than for healing DM, as in the other two classes. This is surprising given that Gadsby43, achieving adequate HbA1c levels is challenging for people with T2DM. Although no significant differences in HbA1c values were found among priority classes, problems during the course of treatment leading to a change in treatment strategy may have affected the priorities reported. Further longitudinal studies should investigate possible changes in priorities over time, such as when DM treatment is adjusted .In T2DM, two of three classes that included most participants had priorities for simplifying DM handling, although insulin use was less common in T2DM than in T1DM. Nevertheless, those research objectives were of key priority, particularly the wish that ‘drugs allow for stable blood sugar levels’. As has already been shown44; 8.1%45) among members of statutory health insurance. However, we performed a responder/non-responder analysis. The age and sex distribution were similar for the responders and the entire sample. But there are more people with T2DM who are treated with insulin in our sample compared to a representative study in 2010 (36.6% vs. 25.2%)46. Therefore, people who have received treatment specifically targeting lifestyle changes may be underrepresented. This could be due to the fact that people with DM who are treated with insulin and are therefore more likely to be affected by DM in their daily lives are more interested in participating in the study. Another limitation is the language. The questionnaire was provided only in German. A few recipients responded apologetically that they could not fill out the questionnaire due to insufficient comprehension. In future studies, the questionnaire could be translated into multiple languages to reach more participants from diverse cultural backgrounds. Another limitation is that we possibly did not capture the relevant associated factors  that explain the differences among the identified research priority profiles for T1DM and especially for T2DM.The major limitation is the low response rate (29.0%), which could have led to a non-responder bias. We are aware of studies with higher, but also with lower response rate  when dealing with the challenge covering clinical and non-clinical research questions as broadly as possible5. We see our work as a first step towards a joint dialogue between patients and researchers as reported by Abma and Broerse47. Further discussions can build on our results.A strength of the study is the questionnaire used, which was developed using a qualitative and participatory approach based on the views of people with DM. In addition, the survey enabled us to reach a large number of people with T1DM and T2DM with different sociodemographic and clinical backgrounds nationwide. Other studies use various participatory and collaborative methods to set priorities, for example the SEED methodKnowledge of diabetes-related research priorities enables researchers to improve their research on behalf of people with DM and to align research with their needs and wishes. In particular, objectives related to simplifying DM handling are important for patients. This study has shown that groups of people with T1DM and T2DM have divergent research priorities, focusing to different degrees on healing diabetes, prevention of long-term complications, diabetes prevention, stress reduction and simplifying handling. This highlights the importance of including both people with T1DM and people with T2DM more specifically in research projects. In addition, we join the current calls for a diverse group of people (with different research priority profiles) to be actively involved in setting research agendas to ensure that their varied interests are considered in research programs overall. Further research should investigate possible changes of research priorities during the course of the disease  and include the research priorities of other relevant stakeholders  to promote a broad collection of research priorities and a joint, cross-perspective dialogue. Research pathways can then be determined based on a reconciliation of what people with DM desire and what researchers deem important. In this way, both needs-based research on potentially short-term solutions and basic research or research on medium- and long-term solutions that may be prioritized by a group of scientists can be included equally in a common prioritization process.Supplementary Information.


This adhesion factor scales the edge length of the cubic inclusions, thus reducing the stress transfer area between matrix and filler. Fitting the experimental data with the modified Paul model provides reasonable kadh for PA66, PBT, PP, PE-LD and BR which are in line with their surface energies. Further analysis showed that stiffening only occurs if kadh exceeds EM, filler modulus EF, filler volume content vF and adhesion factor kadh. Thus, finite element analysis (FEA) simulations of any particle filled polymer parts as well as materials selection are significantly eased. FEA of cubic and hexagonal EV arrangements show that stress distributions within the EV exhibit more shear stresses if one deviates from the cubic arrangement. At high filler contents the assumption that the property of the EV is representative for the whole composite, holds only for filler volume contents up to 15 or 20% (corresponding to 30 to 40 weight %). Thus, for vast majority of commercially available particulate composites, the modified model can be applied. Furthermore, this indicates that the cube in cube approach reaches two limits: (i) the occurrence of increasing shear stresses at filler contents above 20% due to deviations of EV arrangements or spatial filler distribution from cubic arrangements (singular), and (ii) increasing interaction between particles with the formation of particle network within the matrix violating the EV assumption of their homogeneous dispersion.The cube in cube approach was used by Paul and Ishai-Cohen to model and derive formulas for filler content dependent Young’s moduli of particle filled composites assuming perfect filler matrix adhesion. Their formulas were chosen because of their simplicity, and recalculated using an elementary volume approach which transforms spherical inclusions to cubic inclusions. The EV approach led to expression of the composites moduli that allows introducing an adhesion factor  Sustainability demands on performance of polymer parts are steadily increasing. Not only polymeric systems from renewable sources are currently developed, but contemporary established materials commodities are recycled. To meet these requirements, polymers are often modified by blending, copolymerization and reinforcement with particulate fillers and fibers. The access of these materials to emerging applications brings necessity of fast availability of relevant physical properties as stiffness, strength, and thermal properties e.g. thermal length expansion or heat conductivity.1, and their performance has to be determined with respect to varying or even undefined mechanical properties of raw materials. This can be achieved by an elaborate thermo-mechanical analysis of a particular composite system (which is expensive and time consuming) or via accurate predictions of composite properties using analytical approaches.In this perspective, use of reinforced composites will increase substantially2. A filler-matrix adhesion can be modified by coupling agents. He and Jiang3 showed that glass bead reinforced polyepichlorhydrin pretreated with a coupling agent yielded in an enhanced stiffness behavior over the whole filler volume range (5–35%). Demir et al.4 investigated the effect of different coupling agents on the performance of luffa fiber/polypropylene composites and found out that the moduli of the composites containing 15 wt% fiber increased by 52 to 98% after modification. They concluded that the addition of the coupling agents was accompanied by the decrease in water absorption due to a better adhesion between fibers and matrix. Similar conclusion was presented by Jacob et al.5, which studied the effect of various silane coupling agents on viscoelastic properties of sisal/oil palm hybrid fiber reinforced natural rubber composites, and reported that the chemical modification of fibers with respect to the used coupling agents led to improved wettability and consequently to an increase in storage and loss moduli of the composites.Performance of composites is determined by properties of a matrix, dispersed phase and interface between filler and matrix6 reviewed an effect of various pretreatments and coupling agents on tensile properties of natural fiber reinforced polymer composites. They summarized the studies, which confirmed the increase in Young’s modulus due to the enhanced interfacial adhesion caused by the pretreatment of the fillers and the coupling agents. On the other hand, Dekkers et al.7 and Dibendetto et al.8 used glass beads as fillers for polystyrene and epoxy resin matrices and found that the Young’s moduli were not affected by the surface treatment. Wang et al.9 observed increasing moduli of polypropylene/barium sulfate composites using stearic acid, silane and maleic anhydride as surface modifications, but they attributed it not to the improved interfacial adhesion, but rather to higher crystallinity and the formation of the crystal lattices in the matrix.Ku et al.11. Three-phase models are not considered in this work because their higher complexity requires additional materials parameters14.Numerous models have been developed to calculate Young’s moduli of particulate polymer composites considering elastic properties of fillers and matrices, volume content and aspect ratio, Table 15 and Reuss16 represent the upper bound for the uniform stress distribution and the lower bound for the uniform strain distribution, respectively, and differ significantly. In most cases the measured Young’s moduli lie in between. This indicates that they can only serve as a rough estimation, and that real loading states are more complex than uniform stress or uniform strain. Guth17 proposed a two-phase model based on Einstein’s approach18 to determine the viscosity of a suspension with spherical inclusions.The models of Voigtcube in cube models (4–10) take into account a dispersed structure of particle filled composites. Takayanagi et al.19 combined the models of Voigt and Reuss in a way that allows for introducing a dependency on filler volume content. Paul20 assumed uniform stress states with a perfect filler-matrix adhesion, whereas Ishai and Cohen21 assumed uniform strain states. Their models represent more precise upper and lower bounds for composites moduli than those of Voigt and Reuss. More complicated models require additional parameters, which are determined by a fitting procedure:22 combined the models of Voigt and Reuss balancing their contributions with a factor χ.Hirsch23 developed a simple model for concrete systems assuming perfect filler matrix adhesion; it coincides with the Hirsch model for χ = 0.5.Counto24 developed a model to describe anisotropic properties of fiber and filler reinforced composites with one equation in which an efficiency factor ξ takes into account fiber or filler geometry and spatial orientation of fibers or platelets; due to its simplicity it became popular regardless of limited accuracy.Halpin and Tsai25, Lewis and Nielsen26 and Nielsen27 proposed an alternative model based on Halpin–Tsai24 and Kerner28 in which mechanical properties depend additionally on fiber or filler geometry and load direction, maximum filler content vF,max; moreover, Nielsen assumed a Poisson ratio-dependent Einstein coefficient KE.NielsenThe M and filler EF, filler volume content vF, parameter χ determining stress transfer between fiber and matrix, geometry factor ξ, Einstein coefficient KE and maximum volume fraction vF,max.Moduli of matrix Ecube in cube models. The aim of this study is to introduce an adhesion factor having values between “0” and “1”to the EV models of Paul and Ishai-Cohen to express adhesion quantitatively, and to investigate how the adhesion factor and spatial arrangement of EV affect the filler content dependent composites moduli.All models depicted in Table D + a is shown with a spherical inclusion of diameter D consisting of matrix M and filler, Fig. k D with the efficiency factor k. It takes into account that less than the maximum cross-section contributes to the stress transfer, and thus depends on particle shapes. For spheres it is determined by the condition in series or parallel to account for microscopic mechanical properties, Fig. If filler particles are homogeneously dispersed in a matrix, one can define an elementary volume (EV) containing a single particle that is to be representative for composite properties. A cube of length in series arrangement “perfect” adhesion is assumed over the entire cross-section (D + a)2 perpendicular to the load direction between matrix part and composites part, and “zero” adhesion is required between filler and matrix of the composite part along the load direction. If the EV cube is strained to εEV by the load F0, the matrix part experiences the strain εM and the composites part the strain εC.For the parallel arrangement “perfect” adhesion is assumed only over the cross-section (k D)2 of the composite part between the filler and the matrix perpendicular to the load direction, and “zero” adhesion is required between the composite part and the matrix part along the load direction. If the EV cube is strained to εEV by the load F0, the matrix of the composite part experiences the strain εC,M and the filler of the composite part the strain εC,F.For the This allows calculation how the modulus of the EV depends on the arrangements of matrix part and composites part. Furthermore, the analysis of the EV separation shows that certain assumptions were made with respect to the adhesions acting between matrix and filler:If the aspect ratio is not close to “1”, one has to distinguish the extremes “along the long axis” and “perpendicular to the long axis” e.g. with a square column consideration. In this consideration a reduction of adhesion can only apply to “perfect” adhesion, whereas “zero” adhesion leads to neglecting of shear stresses.The first step is to define the elastic stress–strain-relation of the EV to represent the macroscopic mechanical behavior:EF to the stress–strain-relation of the composites does not contribute to full extent. In this context the EV representation of stress–strain-relation (kadh that ranges from “0” (no adhesion) to “1” (perfect adhesion). In Eq.  an an13) ank is related to the filler volume content vF byThe efficiency factor and  becomes:kadh = 1, Eq.  a a25b) anvF using 29\documekadh = 1, the Eq. (28) coincides to Ishai-Cohen’s relation, Table For perfect interfacial adhesion with ER is calculated with EM = 3200 MPa and EF = 63,000 MPa, respectively, and varying kadh between 0 and 1, Fig. From Eq.  also folentclass1pt{minimakadh causes a decrease of Young’s moduli. Therefore, only Eq. : RADIPOL A45 (0 wt% GB), AKROMID A3 GK 30 1 natur (30 wt% GB), AKROMID A3 GK 40 1 natur (40 wt% GB)polybutyleneterephthalate (PBT): Ultradur B 2550 (0 wt% GB), Ultradur B 4300 K4 (20 wt% GB), Ultradur B 4300 K6 (30 wt% GB)MN = 2 to 3 * 105, cross-linked with dicumyl peroxide,0, 15, 30 and 45 vol% GB unsized and sizedbutadiene rubber (BR): isomer ratio cis:trans:vinyl = 20:60:20, polyethylene (PE-LD): Nova Chemicals LDPE LA-0219-A, Canada with 0, 20, 40, and 60 wt% GBisotactic polypropylene (iPP): Petoplen MH418 with 0, 10, 20 and 30 vol% GBFive kinds of polymer matrices filled with various contents of glass beads (GB) as spherical inclusions were used in this study Table :polyamid® A45, 2021) and PBT  composites were determined experimentally, whereas the properties of nonpolar BR32, PE-LD33 and iPP34 were taken from literature. As in Lohrmann32 two compound series—one with unsized GB and one with sized GB—were investigated giving the chance to check how surface treatment of GB affects the stiffness. Glass beads filler has density ρF = 2.5 g/cm3, Young’s modulus EF = 63 GPa and Poisson ratio µF = 0.22.The properties of polar PA66  were tested after annealing for 4 h at 180 °C to minimize effects of a thermal history.n = 5, equipped with a 10 kN load cell (resolution: 0.12%) and a multiXtens extensometer (resolution: 0.1 µm), an initial sample length of 50 mm, a preload of 0.1 MPa. Differing to ISO 527, the tensile tests of PA66 and PBT compounds were performed with a strain rate of 10%/min. The stress–strain-curves were evaluated using the viscoelastic stress strain function (VSSF)35:E and relaxation strain εR. Equation  was used to perform tensile tests according to DIN EN ISO 527-1 with 32, digitized and evaluated correspondingly to include an example of a rubber matrix composite.The stress–strain curves of BR compounds were taken fromEEV according to the Eq. .If a filler matrix adhesion is reduced, a composite modulus decreases. As the modulus  the Eq.  represenTensile test bars of PA66 and PBT compounds (type 1A according to ISO 527-2) were fractured under cryogenic conditions in a liquid nitrogen after approximately 5 min of storage. The fracture surfaces were sputtered by gold layer for 90 s at 20 mA, 0.1 mbar in an argon atmosphere. SEM  equipped with a tungsten cathode was used to investigate the structure of the fracture surfaces at 5.0 kV under vaccuum conditions.y- and z-directions with a reference node using a kinematic coupling constraint. For the reference node at the bottom all translational and rotational degrees of freedom were fixed to u1 = u2 = ….. = u6 = 0. For the reference node at the top, all degrees of freedom except for the translation in y-direction were fixed as well. The displacement in the positive y-direction (u2) was defined at the top node to apply a uniaxial tensile load. Static linear analysis was carried out by implementing materials properties as a filler modulus EF and a matrix modulus EM, a filler Poisson ratio µF and a matrix Poisson ratio µM, and a filler volume content vF. Filler content dependent EV arrangements in a cubic and a hexagonal lattice were investigated, Fig. FEA using the software SIMULIA/ABAQUS2020 was performed in 2D for the middle plane of the EV, where maximum stresses occur. A 3D simulation would provide the same stress distribution for the middle plane. A mixed mesh of triangle (S3) and rectangle (S4) shell elements with linear shape functions were used. The total number of elements for each arrangement was approximately 53,000. The nodes at the top and bottom were coupled in ε1 = 0.05% and ε2 = 0.25%.The tensile properties of PA 66 and PBT composites were determined according to ISO 527 and evaluated using the VSSF, Table kadh were determined by a fitting procedure that brings calculated moduli EEV according to the Eq.  and 0.5 (PBT). Both reproduce well the measured Young’s moduli, but only the EV provides the quantitative information concerning the filler-matrix adhesion.The comparison of measured Young’s moduli of GB filled PA66 and PBT composites to calculated ones, Fig. SEM images of PA66 and PBT composites show a fracture surface with embedded GB, which are partly covered by the matrix indicating rather poor filler-matrix adhesion, Fig. kadh. Small fibrils partly visible on the fractured surfaces of both the PA66 and PBT composites correspond to ductile failure of the matrices. It seems that the fracture behavior becomes more ductile, the higher the GB content is.Better adhesion seen for the PA66 composite correlates with its higher kadh between 0.02 and 0.05 for vF = 0.15 and 0.30. For vF = 0.45 all models generate too small Young’s moduli. This can be attributed to the fact that at high filler contents, particle interactions become stronger going along with the formation of a particle network with significantly higher modulus36. On first sight this issue could be eliminated by allowing adhesion factors exceeding 1. However, it turned out that the models of Ishai-Cohen and Paul limit the filler volume content dependent stiffening factors to:Similar increases of filler content dependent Young’s moduli of elastomer (BR) composites were found for all models, Table σy,GB >  > σy,M. For all cubic arrangements the maximum stresses occur between the GB in the matrix forming the parallel stress lines in the composite part along the load direction, whereas in the interjacent matrix parts the stresses remain smaller. With the increasing filler content, the stresses in tensile direction increase leading to a failure of the interface, and cracks propagation perpendicular to the load. Nevertheless, the stresses in both parts are always oriented in the parallel and uniaxial manner in tensile direction.From Fig. vF = 0.40. Both reduces moduli of the hexagonal arrangement in the load direction, Table The hexagonal arrangement of EV shows stress pattern with in-load direction oriented oval “stress islands” around the GB particles. The minimum stresses occur perpendicular to the load direction in the matrix between the GB. The matrix stresses in the load direction between the GB seem to be more homogeneous compared to the cubic arrangement providing more deformability. With increasing the GB content, the stress islands unify and form shear stress band under an angle of 45° seen in Fig. kadh is under-estimated if the Eq.  and Ishai-Cohen (lower bound) determine the moduli of particle filled composites assuming perfect filler-matrix adhesion. To take into account reduced adhesion between filler particles and matrix polymer their models were recalculated using an EV approach, and extended by an adhesion factor kadh that scales the edge length of the cubic inclusion. It leads to the reduction of the filler content dependent composites moduli EC(vF). The modified Paul model in EV version as the upper bound of EC(vF) was used to fit experimental moduli of glass bead filled polymers and provided reasonable adhesion factors: 0.6 for polyamide (PA66), 0.5 for polybutylene terephthalate (PBT), 0.2 for polypropylene (iPP), 0.13 for low density polyethylene (PE-LD) and 0.05 for butadiene rubber (BR), which are in line with the corresponding surface energies. The modified model allows for design engineers to calculate realistic modulus of any particulate composite using only matrix and filler moduli, filler content and adhesion factor without performing tests. Further analysis of the modified model elucidated that stiffening only occurs if kadh exceeds EM and filler modulus EF. This means almost all technical relevant filler contents lead to stiffening of the polymer. Furthermore, the EV approach shows directly that the moduli of particulate composites depend on particle shape—quantified by the efficiency factor k—and the dimensions within the EV—expressed by the normalized filler distance d which depends on a filler volume content.The cube in cube model: first, the occurrence of increasing shear stresses if EV arrangements deviate from cubic arrangements, and second, increasing interaction among filler particles with the formation of particle networks within the matrix disturbing the EV assumption of homogeneously dispersed particles. The modulus increase of rubber composite by factor 2 or 3 if the filler content changes from 30 to 45%, respectively, cannot be explained within the cube in cube model anymore. From a practical point of view, where commercially available particle filled polymers have filler volume contents less than 20%, it is positive that the EV arrangement affects calculated composites moduli within the experimental error of 3 to 5%.Additionally, finite element analyses of cubic and hexagonal EV arrangements show that the spatial EV arrangement leads to differences of the filler content dependent composites moduli especially for high volume contents. Thus, the assumption “The properties of the EV are representative for the whole composites” only holds for filler volume contents up to 15 or 20%. The increasing difference between composites moduli calculated for cubic and hexagonal EV arrangement can be attributed to two limits of the Supplementary Information.


This is a case of eosinophilic myocarditis which had large left-ventricular thrombus seen on echocardiography and cardiac magnetic resonance imaging which was successfully treated with a highly selective direct Factor Xa inhibitor—rivaroxaban. Trans-thoracic echocardiography demonstrated a dilated left ventricle (LV) and globally impaired systolic function and apical LV thrombus. Further examination by cardiac magnetic resonance imaging demonstrated an LV thrombus in the apex measuring 28 × 14 mm and sub-endocardial apical fibrosis on late gadolinium enhancement imaging. Endomyocardial pattern of fibrosis seen in the LV (‘V sign’) is typical for eosinophilic myocarditis. He was treated with Prednisolone 10 mg for 2 months and standard heart failure therapy. For the LV thrombus a highly selective direct Factor Xa inhibitor, rivaroxaban 20 mg was initiated instead of warfarin in light of logistic issues due to COVID-19 pandemic. After 9 months of treatment, the eosinophil count went down to 0.03 × 109/L and the LV thrombus resolved to leave a fine rim of organized thrombus only, which has a relatively lower risk for systemic embolization .A 76-year-old gentleman with hypereosinophilia (6.21 × 102 Even though we are not the first to report successful thrombus resolution with this treatment strategy, it is worth reporting our imaging findings to further add to the evidence database so that there is a motive for future prospective design studies, which explore the role of glucocorticoids and rivaroxaban in eosinophilic myocarditis.This case highlights that a multi-faceted personalized treatment using disease-modifying agents and anticoagulation has a synergistic role in the resolution of myocardial inflammation and of the thrombus. This treatment strategy has been shown in limited cases previously.


A long-term drift in polar motion (PM) has been observed for more than a century, and Glacial Isostatic Adjustment (GIA) has been understood as an important cause. However, observed PM includes contributions from other sources, including contemporary climate change and perhaps others associated with Earth’s interior dynamics. It has been difficult to separate these effects, because there is considerable scatter among GIA models concerning predicted PM rates. Here we develop a new method to estimate GIA PM using data from the GRACE mission. Changes in GRACE degree 2, order 1 spherical harmonic coefficients are due both to GIA and contemporary surface mass load changes. We estimate the surface mass load contribution to degree 2, order 1 coefficients using GRACE data, relying on higher-degree GRACE coefficients that are dominantly affected by surface loads. Then the GIA PM trend is obtained from the difference between observed PM trend (which includes effects from GIA and surface mass loads) and the estimated PM trend mostly associated with surface mass loads. A previous estimate of the GIA PM trend from PM observations for the period 1900–1978 is toward 79.90° W at a speed of 3.53 mas/year (10.91 cm/year). Our new estimate for the GIA trend is in a direction of 61.77° W at a speed of 2.18 mas/year (6.74 cm/year), similar to the observed PM trend during the early twentieth century. This is consistent with the view that the early twentieth-century trend was dominated by GIA and that more recently there is an increasing contribution from contemporary surface mass load redistribution associated with climate change. Our GIA PM also agrees with the linear mean pole during 1900–2017. Contributions from other solid Earth process such as mantle convection would also produce a linear trend in PM and could be included in our GIA estimate. Polar motion (PM), movement of Earth’s rotational axis relative to the crust, is a geophysical phenomenon excited by relative motion and mass redistribution within the Earth system. Sources include the atmosphere, hydrosphere, cryosphere and solid Earth Gross . PM obseRecent studies have examined PM as a measure of contemporary climate change and found causes of interannual and longer period PM variations. For example, Youm et al.  showed tA dominant contributor to a linear PM drift is recognized to be Glacial Isostatic Adjustment (GIA). GIA reflects redistribution of mass in the mantle, as a viscoelastic response to the last glacial maximum .Recently, GRACE data were used to estimate the contemporary surface mass load contribution, and compared with PM observations, which involved adoption of a GIA model to remove this contribution and to correct GRACE data  in the PM trend (secular PM) has been difficult although it is critical to understanding the viscoelastic response of the mantle and rotational theories, as well as contemporary global change driven by a warming climate. In this study, we estimate separate contributions to secular PM associated with GIA and contemporary mass redistribution using GRACE (CSR RL06) degree 2, order 1 SH coefficients. We refer to observed coefficients as (1), A13–Peltier18 (R2) and Peltier18–Purcell16 (R3), are the red, blue and green lines in Fig. 1, R2 and R3, respectively. These GIA model differences can be taken as measures of model error, including different realizations of rotational theories. We compare these GIA model errors with surface mass load signals. Figure It has been a common practice to correct GRACE SH coefficients using GIA models. These corrected SH coefficients are presumed to be due to contemporary surface mass loads, but may also include residual GIA effects due to imperfect GIA models. An estimate of the size of these residuals can be understood by examining variations among GIA models, which provide predictions of SH coefficient time rates of change. We first consider three models: A13 A et al. , PurcellSynthetic data can be useful to understand uncertainty in mentclass2pt{minim1, R2 and R3) to the synthetic data to simulate GIA model error. Left panels of Fig. 21 and S21 SH coefficients. Gray lines in the left panels represent ‘true’ 1, R2 and R3 GIA model errors. It is clear that GIA model errors dominate trends in S21 SH coefficients, indicating large error in GIA model and resulting uncertainty in the estimation of We add GIA model differences R1, R and R3 tHere we develop a new method to estimate entclass1pt{miniman et al. . They esn et al. . We exteFollowing Sun et al. , the sixHere values in the column vector mentclass2pt{minimThe where whose components areHere, In Eq. , \documementclass2pt{minimmentclass2pt{minimmentclass2pt{minimWe apply the same method to real GRACE data. Figure entclass1pt{minimaentclass1pt{minima–11, − 0.71 × 10–11), very close to GRACE values in Fig. –11, − 0.64 × 10–11, red lines in left panels).Similar results are obtained using satellite laser ranging (SLR) SH coefficients   8) and iThe iterative and direct methods provide almost identical estimates of Using the four GIA error corrected time series as in the right panel of Fig. 21 and S21 (with A13) is shown by the dashed-red arrow. The dashed-blue and dashed-red arrows include common effects of surface mass loads and A13 uncertainties, but differ due to different pole tide corrections in CSR RL06 and RL05. The dashed-red arrow (the difference between CSR RL05 C21 and S21 with A13) and the red arrow from Adhikari and Ivins  and red arrows  from Adhikari and Ivins . This has been assumed to be due to GIA, but it must also include effects from climate-driven contemporary surface mass loads and possibly mantle convection or other interior sources. GIA model predictions confirm its importance, but there are significant model-to-model differences, making it difficult to separate GIA from other causes. The linear mean pole, the average PM trend for 1900–2017 has been also assumed to be GIA PM and used in GRACE pole tide corrections. It shows an evidently different direction (64.10° W). We use GRACE gravity data to correct GIA models for contemporary surface load contributions by assuming that changes in SH coefficients above degree 2 are dominated by surface mass load effects, and that mass exchanges between land and oceans are associated with ocean mass distribution that conforms to SAL theory. Four different GIA models were corrected including A13, Purcell16, Caron18 and Peltier18. Estimated GIA PM based on the four corrected GIA models has a mean speed and direction of 2.18 mas/year (6.74 cm/year) and 61.77° W, respectively. The GIA PM direction estimated in this study is very close to the observed PM trend during the early twentieth century when surface mass load redistribution associated with climate warming was probably less important than GIA. Our new GIA PM also agrees with the linear mean pole during 1900–2017. Average PM trend over such a long period of time would suppress multi-decadal effects associated with climate changes and thus represent the GIA PM. However, in the future, the linear mean pole trend would differ from the GIA PM because the contributions from AIS and GrIS have emerged. A revised estimate of the GIA trend has implications for the processing of GRACE data; in particular, this would revise the correction made for the pole tide. GRACE data provide unique information in understanding causes of PM, and further contributions can be expected from GRACE-FO as the gravity field time series is extended.