Background: Polymer infiltrated ceramic networks, or hybrid ceramics, are a combination of infiltrating polymerizable organic monomers into a pre-sintered porous ceramic matrix. In addition to having good mechanical properties, the polymer infiltrated ceramic network must comply with the possibility of adequate bonding to the resinous cement. The surface conditioning of this hybrid material must be carefully considered due to its organic composition and ceramic network. The purpose of this research is to evaluate the effect of hydrofluoric acid and a self-etching ceramic primer, under two different application times, on the bond strength of a polymer infiltrated ceramic network.Methods: Blocks of a polymer infiltrated ceramic network were cut to obtain sheets, and these were randomized into five groups. For the group termed AAS, airborne-particle abrasion with Al2O3  of 50µm was used. For groups HF2 and HF6, hydrofluoric acid was used for 20 and 60 seconds respectively, and for the groups MB2 and MB6, a self-etch ceramic primer was applied for 20 and 60 seconds respectively. A silane was applied to the groups AAS, HF2, and HF6 after the treatment. After 24-hour storage in distilled water, a micro-shear bond strength test was performed using a universal mechanical testing machine. All samples were evaluated in a stereomicroscope at 40x and 50x to determine the type of failure.Results: The highest and lowest values of bond strength were reported by groups MB6 and AAS, respectively. Groups HF2, HF6, MB6, and MB2 did not report statistically significant differences. The predominant failure pattern was a mixed failure.Conclusions: With the limitations of the present investigation, the treatments of self-etching ceramic primer and hydrofluoric acid followed by silane were reported to be statistically equal at 20 and 60 seconds. The Editorial Team is currently requesting further information from the authors regarding this. This Editorial Note will be updated as new information is provided.Editorial Note . It presents mechanical properties comparable to CAD-CAM (computer-aided design/computer-aided manufacturing) polymers with nano-ceramic particles, lower than lithium disilicate, but superior to CAD-CAM feldspathic ceramics.– Hybrid ceramics, in addition to having good mechanical properties, must comply with the possibility of adequate bonding to the resinous cement.PICN (polymer infiltrated ceramic network), or hybrid ceramics, were introduced in dentistry as a restorative material, which, by infiltrating polymerizable organic monomers into a pre-sintered porous ceramic matrix, promises to obtain mechanical properties like dental enamel and a density equivalent to that of dentine. Due to its high ceramic content, this type of material requires surface conditioning prior to resinous cement to ensure an adequate bond quality. Etching with 5% hydrofluoric acid for 60 seconds is the PICN manufacturer's recommended surface conditioning. However, other treatments such as hydrofluoric acid at 9% for 90 seconds, silanes, airborne-particle abrasion with aluminum oxide (30-50 μm particles) and tribochemical silica (CoJet System - 30 μm particles) have been reported., The association of hydrofluoric acid or air abrasion  with a silane agent can achieve considerable values of bond strength, due to the structure with a high content of feldspathic ceramic present in this material (>80%wt), compared to other CAD-CAM materials with polymeric matrix and dispersed ceramic particles, with lower values of bond strength using this same combination of surface treatments. However, given the risk of excessive dissolution of the vitreous matrix due to the attack of hydrofluoric acid, an etching agent and silane have been proposed in a single step, known commercially as Monobond Etch & Prime. With a chemical in its composition less aggressive than hydrofluoric acid, tetrabutylammonium dihydrogen trifluoride, this self-etching ceramic primer has reported considerable values of bond strength in lithium disilicate and feldspathic ceramics.Adhesive cementation increases the surface energy and the retention of the restoration reinforces its structural strength and maintains marginal integrity.The objective of this study was to evaluate the effect of hydrofluoric acid and a self-etching ceramic primer, under two different application times, on the bond strength of a polymer infiltrated ceramic network. The null hypothesis was that the mean values of bond strength of the groups treated with Monobond Etch & Prime would be equal to the groups treated with hydrofluoric acid and silane.We obtained ethical approval for this study from Faculty of Dentistry, Universidad Nacional de Colombia . No patients or biological material obtained from patients were involved in this investigation.In the present study, 15 sheets (8 mm wide and 10 mm high) were made from three VITA ENAMIC EM14 blocks  by means of cuts every 2 mm using a precision diamond disc with constant cooling with distilled water . These sheets were included in cylinders of self-curing acrylic resin , 10 mm high, leaving an exposed face of the hybrid ceramic to receive surface treatment. The exposed surfaces were sanded with #600, #800, #1000, and #1200 grain size silicon carbide abrasive paper for approximately 1 minute under manual pressure. The samples were washed in distilled water with ultrasound for 15 minutes.2O3 of 50 μm applied for 10 seconds with a pressure of 1 bar, it was then washed with an air-water syringe for 20 seconds followed by drying with pressurized air for 10 seconds. A silane  was applied with a microbrush rubbing for five seconds and leaving it to act for 60 seconds. Group MB2 used a self-etch primer  which was applied for 20 seconds and then washed with water from an air-water syringe and dried with pressurized air for 10 seconds. Group MB6: like group MB2, but with the only difference that the primer was left to act for 60 seconds. Group HF2 used 9.6% hydrofluoric acid  which was applied for 20 seconds, it was then washed with an air-water syringe for 20 seconds, and dried with pressurized air, silane  was applied with a microbrush rubbing for five seconds and it was left to act for 60 seconds, then air was gently applied for 10 seconds. Group HF6 was similar to group HF2, but with the difference that the etching with hydrofluoric acid was carried out for 60 seconds. The hydrofluoric acid residues were neutralized in a supersaturated solution of calcium carbonate.The sheets were randomized into five groups, with three sheets per group (n = 15) according to the surface treatments to be received. The group termed AAS had airborne-particle abrasion with Al2. The adhesive area (A) was determined by π.r2, where π is a constant and r is the radius obtained from the internal diameter of the medical-grade silicone tube.On the conditioned surfaces, 0.75 mm internal diameter and 0.8 mm high medical grade silicone tubes were positioned and carefully filled with dual resinous cement , finally, each cylinder was cured with the high setting for 20 seconds with a LED lamp . In total, 5 tubes were placed for each hybrid ceramic sheet (n = 15). All samples were stored at 37°C for 24 hours in distilled water . Once the storage was completed, the mold tubes were carefully removed with #12 and #15 scalpel blades, exposing the resinous cement cylinders. All the samples were taken to a universal mechanical testing machine , and by means of a steel wire handle, gauge 0.22 mm, with traction force, at a speed of 0.5 mm/min crosshead and a 50N load cell, the micro-shear test was performed. The bond strength was calculated using the equation: R = N/A, where R is bond strength given in MPa, N force in Newtons necessary for failure, and A is the area of the resin-cement-hybrid ceramic joint, measured in mmAll samples were evaluated in a stereomicroscope  at 40× and 50× to determine the type of failure, these were classified as: adhesive failure, i.e. where there is separation of the union between cement and ceramic, leaving an intact ceramic surface; cohesive failure in the ceramic, i.e where separation occurs only in ceramic; cohesive failure in the resin cement, i.e. where separation occurs only in cement; and mixed failure, which is a combination of the cohesive and adhesive patterns.R-project version R-3.6.3 for Windows was used for the statistical analyses .For the statistical analysis, normality tests were carried out (Shapiro Wilk), and for the comparison of the groups, one-way ANOVA  tests and Tukey's test were executed. A significance level of p < 0.05 was set. TheDescriptively, the results of the groups can be understood as follows: a graphical similarity in the mean values of bond strength the groups MB2, MB6, HF2 and HF6 was observed. the highest data dispersion was obtained in the AAS group, and the lowest graphic dispersion of data were obtained by both groups MB. The highest and lowest mean values of bond strength were reported by groups MB6 and AAS, respectively .Underlying data.In the statistical comparison by pairs (Tukey’s test) the groups that used Monobond Etch & Prime and hydrofluoric acid were considered equal, while the group AAS was statistically inferior compared to the other four groups . The preIn the present study, the bond strength between a PICN and a resinous cement after the use of hydrofluoric acid, a self-etching ceramic primer, and airborne-particle abrasion was compared. The airborne-particle abrasion group was significantly inferior to the two groups of acid etching agents. Therefore, the hypothesis that raised the statistical equality of the mean values of bond strength in the three surface treatments was not accepted. Parameters such as distance, time, pressure, and particle size can result in different roughness patterns., Abrasion with 30 μm size aluminum oxide has been associated with high roughness values and acceptable bond strength mean values on PICN. Although in the literature it is also reported that in CAD-CAM materials with polymeric matrix and dispersed ceramic particles, airborne-particle abrasion with aluminum oxide has shown superior results to acid etching. In PICN an opposite phenomenon generally occurs, this is perhaps based on the microstructural organization of this material. The use of airborne-particle abrasion on the surface of a hybrid ceramic creates roughness through impact microcracks, unlike acid etching that creates micropores due to the partial dissolution of the vitreous matrix. Etching with hydrofluoric acid and airborne-particle abrasion with aluminum oxide, followed by silane, has been reported with surface energy values in Enamic of 147 mJ/m2 and 96 mJ/m2 respectively. This suggests that the amount of roughness in a surface is not predictive of higher bond strength. Another possible explanation is that VITA ENAMIC, unlike other hybrid CAD-CAM materials, contains a porous network of feldspathic ceramic reinforced with alumina and only one infiltrate of urethane dimethacrylate polymer, allowing a better action of the etching agent and assuming the increased risk of surface damage and unevenness if airborne-particle abrasion is used. This mechanism probably explains why, even though both methods can create comparable values of surface roughness in a hybrid ceramic, the bond strength results can be higher after a chemical attack by acid., Only one protocol of air abrasion with aluminum oxide was contemplated in this study, which could be considered as a limitation of the research.Airborne-particle abrasion with aluminum oxide particles is an alternative to increase the roughness of the ceramic substrate by impulsing and impacting the surface with abrasive particles, with the potential risk of microcracks and excessive damage to the treated surface., Both etching agents can produce similar roughness values, but self-etching ceramic primer was introduced to the market as a less aggressive alternative to hydrofluoric acid., The physical-chemical action is due to an acid etching with tetrabutylammonium dihydrogen trifluoride and a chemical interaction through trimethoxy propyl methacrylate, without diminishing the mechanical resistance and fatigue of the ceramic substrate. Additionally, a formation of a hydrophobic silane layer that is theoretically more resistant to hydrolytic degradation has been associated with the use of this self-etching ceramic primer. The etching depth of 9% hydrofluoric acid for 20 and 60s is greater than 290 μm compared to a depth of 7 μm of Monobond Etch & Prime for 40s in VITA ENAMIC. Therefore, the indication of which surface treatment to use does not only depend on the bond strength results obtained. Hydrofluoric acid on VITA ENAMIC is dependent on the time and concentration of use. Hydrofluoric acid concentrations of 5% between 30 and 90 seconds do not seem to significantly affect flexural strength, on the contrary, the use of hydrofluoric acid at 10% after 30 seconds reports a significant decrease in flexural strength of this hybrid ceramic. In this study a concentration of 9.6% was used, with similar bond strength results between 20 and 60 seconds. A consistent finding that hydrofluoric acid etching of VITA ENAMIC for more than 30 seconds does not significantly improve bond strength by shear test. This analysis may be somewhat helpful in supporting the recommendation of the lowest possible time and concentration of hydrofluoric acid to treat the surface of VITA ENAMIC. However, the present investigation had the limitation of only evaluating one hydrofluoric acid concentration.On the other hand, the self-etching ceramic primer in the literature has reported bond strength results comparable to hydrofluoric acid and silane on polymer infiltrated ceramic network., In the present investigation, the predominant pattern was mixed for the hydrofluoric acid and self-etching ceramic primer groups, consistent with the highest values of bond strength in the study, and an almost exclusive pattern of adhesive failures for the airborne-particle abrasion group, in which the lowest values were obtained. The results of this investigation should be considered with caution since the bond strength test was carried out after the short-term storage of the samples. Bond strength can be affected by hydrolytic degradation of the materials in the small adhesive area, storage in water for long periods of time, or thermal cycling.,Adhesive failure patterns have been associated with low values of bond strength, cohesive and mixed patterns seem to be considered more acceptable because they suppose a better infiltration of the resinous cementing agent in the conditioned surface of the PICN.With the limitations of the present investigation, the self-etching ceramic primer and hydrofluoric acid followed by silane were reported to be statistically equal at 20 and 60 seconds. The 20 second time seems to be a more reasonable option than 60 seconds applying these surface treatments on a PICN. This is because, if the same bond strength result is obtained at 20 and 60 seconds, it can increase the surface energy in less time. Airborne-particle abrasion is not recommended as the first choice of surface treatment in a PICN.https://doi.org/10.17632/dfm9wr4bwn.4.Mendeley Data: Micro-shear bond strength of different surface treatments on a polymer infiltrated ceramic network.–Acronym manuscript Micro-shear bond strength of different surface treatments on a polymer infiltrated ceramic network.docx (description of acronyms used within the data spreadsheets).–Statistical Data.xlsx .–Description groups.xlsx .–Comparison adhesive resistance groups.xlsx .This project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The authors investigated the micro-shear bond strength of different surface treatments on a polymer infiltrated ceramic network, especially of VITA Enamic. Schematic illustrations are required to differentiate the groupsConclusion can be rephrased in line with the objective of the study. Flowchart of methodology to be included. Were the samples thermocycled ?Discussion can include the results of similar studies and how this study stands out from the rest?Mechanism of action of sandblasting and acid etching can be illustrated.  The following are my comments:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Dental ceramicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. et al. The authors show the interesting results. They evaluate the effect of hydrofluoric acid and a self-etching ceramic primer, considering two different application times. Also, they evaluate the influence of both surface conditions on the bond strength of a polymer-infiltrated ceramic network. The manuscript is well organized, and the references used are fine in number and actual. The authors describe the preparation of samples, and they describe the methodology to determine the bond strength. The current work is complemented by the determination of the type of failure  considering the stereomicroscope technique. The predominant failure pattern was a mixed failure. In general, the statistical analyses and their interpretation seem to have been performed properly and the conclusions reached are in line with the results presented. The methods and analyses provided in this manuscript can be replicated by others. Certainly, the theme of this manuscript is suitable for the journal and surely of wide interest to the reading community of F1000Research.I read the manuscript entitled “Micro-shear bond strength of different surface treatments on a polymer infiltrated ceramic network” written by WahjuningrumThe scale bar in the stereomicroscope images should be clearer . Atomic force microscopy (AFM) analysis should be implemented. The authors mention, in the discussion section, "different roughness patterns, high roughness, and surface roughness", which needs to be verified (including micrographs) and quantified. Additionally, AFM analysis can provide information about physical defects such as microcracks. Also, this technique allows us to evaluate the presence of micropores.The authors focus on the effect of airborne particle abrasion on the surface of a hybrid ceramic. However, the average particle size  influence on roughness is not investigated. Please conduct transmission electron microscopy analysis. The paper is acceptable after minor revisions for indexing after the comments below are addressed.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?NoReviewer Expertise:Ceramics (ferroelectric and ferromagnetic), Raman, and EELS spectroscopies.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. In Vitro study, which has potential implications for clinical applications in restorative dentistry using the PICN. The clarity and organization of the manuscript are commendable. I have only minor suggestions for improvement as follows:This study examines the micro-shear bond strength of various PICN surface treatments. This is anTo improve the coherence of the abstract, it is recommended to align the sequence of description with the order of experimentation and results. Specifically, the abstract currently discusses HF2 and HF6 before MB2 and MB6, while the presentation of experimental details and results showcases MB2 and MB6 before HF2 and HF6. To enhance consistency, consider revising the abstract to ensure that the order of describing treatments aligns with the order in which the experimentation and results are presented. Introduction:The introduction section is well structured. However, to improve this section, explain and emphasize existing knowledge that has been studied and the gap of knowledge related to surface treatment of PICN. Methodology: Abstract:Please explain more about how the manual pressure is uniformly controlled for polishing the specimens.It is typically preferred to prepare samples with width and length matching the block dimensions, but the sample in this study has dimensions of 8x10x2 mm. It was prepared by cutting the VITA ENAMIC EM14 block with dimensions of 12x14x18 mm. Please explain the cutting method. To prepare additional samples in the content or use figures. 1. Research design:The introduction states, "Etching with 5% hydrofluoric acid for 60 seconds is the PICN manufacturer's recommended surface conditioning." However, the testing involved the use of 9.6% HF. Could you provide the rationale for this choice?In this study, all samples underwent evaluation using a stereomicroscope  at both 40x and 50x magnifications to ascertain the type of failure. Why was testing conducted at both 40x and 50x, especially considering that study results are only presented at 50x magnification?     2. Experimental procedure:The order of abbreviation descriptions in Tables 1 and 2 should be modified to reflect the order of information presented in the tables. Result:This study on the effects of 9.6% hydrofluoric acid, therefore, the statement "This analysis may be somewhat helpful in supporting the recommendation of the lowest possible time and concentration of hydrofluoric acid to treat the surface of the VITA ENAMIC." is inaccurate and does not align with the outcomes of this study. Please correct this statement. Discussion:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:digital dentistry, esthetic dentistry, dental materialsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


This article was migrated. The article was marked as recommended.Introduction:The University of Texas Southwestern Medical School (UT Southwestern) developed a required, pass/fail Step 1 preparation course in conjunction with their new curriculum to support students with structure, encouragement, and accountability. This study examined the Step 1 performance of students who have taken the prep course after completing pre-clinical coursework and factors which predicted outcomes.Methods: Data were collected between January 2017 and July 2018 from 453 2nd-year medical students enrolled in the six week course. Multilinear regression and Chi-squared analysis were performed to compare pre-clinical course performance with Step 1 readiness and outcomes.Results: Average course final exam scores and CBSSA results were significantly and directly associated with Step 1 scores. Pre-clinical exam performance, practice exam scores at the beginning of the Step 1 preparation period and an increase in practice exam scores over the study period correlated with Step 1 performance. The percent pass rates and mean scores in 2017 were at 97% and 235, but in 2018, no student failed Step 1 (100% pass rate), and the mean score rose to 239.Discussion and Conclusion: A structured Step 1 Prep Course was well-accepted by students. Exam performance can be predicted from pre-clerkship performance and progress made during the course on practice exams and early intervention for at-risk students can improve performance of at-risk students. The United States Medical Licensing Examination (USMLE) Step 1 exam is the first and arguably most important medical licensure exam in the United States for obtaining a desired residency position . The posFirst Aid for Step 1 andUWorld to be completed before lecture sessions. This approach at the University of Illinois College of Medicine in Chicago raised mean USMLE Step 1 exam scores by 8.82 points and first attempt pass rates by 8%.Therefore, most medical schools provide support and dedicated time for students to prepare for the Step 1 exam. Typically, students develop their own study plan to take Step 1 . At AlbeUT Southwestern Medical School (UT Southwestern) admits approximately 230 student per year. The current pre-clinical phase transitioned in August of 2015 from a classic 24-month, departmentally-based curriculum to 18 months of pass/fail courses with topics integrated by concepts and organ systems. The pre-clinical curriculum does not overly emphasize preparation for the USMLE Step 1 exam and students are encouraged to focus on course work as a foundation for success with both Step 1 and the clinical phase of their education. The Medical School developed a required, pass/fail Step 1 preparation course (“Step 1 Prep Course”) as part of a revised curriculum implemented in August 2015. This study examines whether the current curriculum and Step 1 Prep Course at UT Southwestern is as effective as our previous curriculum in preparing students for Step 1, and identifies factors that correlate with success on the examination.Course design:The six-week Step 1 Prep Course is offered to second-year students via 5 blocks in the first semester (January to June) of the clinical phase. The Step 1 Prep Course has been required since 2017, and is based on a 2016 pilot version offered in the previous curriculum. The Director of Student Academic Support Services (SASS), an experienced educational advisor, serves as course director. At-risk students, who perform poorly in their pre-clinical courses or who have wellness concerns, are advised to request clerkship schedules easily adapted for additional Step 1 study time; other students make choices based on personal preferences. Thus, placement into the prep course blocks is neither random nor based on student preference alone.UWorld QBank practice questions scheduled for each week, completion dates for three passes throughFirst Aid for Step 1, dates for a minimum of three practice tests, and the date of their Step 1 exam.All students are invited to workshops to enhance understanding of the preparation process, and to cover topics such as time management, development of personal study plans, relative benefits and efficient usage of study resources, and exploration of basic strategies for stress management and overall wellness. They must take a school-provided NBME Comprehensive Basic Science Self-Assessment (CBSSA) and submit a study plan by the first day of the course. The study plan must include the number ofTracking progress: SASS tracks each student’s progress in their study plan using weekly, confidential, web-based surveys. Students are encouraged to ask questions and share concerns about their progress in the survey comment section. They may seek counseling at any time, whether they perceive a problem or simply desire to maximize their performance. Students are required to pass the USMLE Step 1 exam to pass the course.Qbank questions after the third week of the course. In addition, the course director reaches out periodically to all students, even those doing well, to provide motivation and encouragement. Two-week extensions are granted if a student is diagnosed with a significant medical condition  and are also considered for students who fail two or more practice tests in the last two weeks of the course. The decision for granting extensions requires agreement of the course director and the Associate Deans for Student Affairs.The course director tracks individual survey responses and intervenes if students request assistance, are making slow progress, report practice exam scores below passing (194), or complete less than 50% ofComprehensive Basic Science Self-Assessment (CBSSA) scores for 2017 and 2018, converted to Step 1 equivalent scores, and first time USMLE Step 1 Exam scores for UT Southwestern students for 2016, 2017 and 2018  were obtained directly from the National Board of Medical Examiners (NBME) website. The percentage of students in a score range were interpolated from the NBME Score Histogram plots. Final exam averages were calculated from first-attempt final exam scores from the fifteen pre-clinical courses . The difference between categorical data was determined using Chi-squared analysis and between means by Student’s T-test. Linear regression analysis was used to determine relationships between USMLE Step 1 Exam scores, average final exam scores for pre-clinical courses and results of the CBSSA exam. Finally, logistic regression analysis utilizing SAS Software Version 9.4 was employed to model factors that influenced the odds of specific levels of performance on the Step 1 exam.From January 2017 to July 2018, 453 University of Texas Southwestern medical students completed the Step 1 Prep Course and sat for the Step 1 exam, and 446 had average final exam scores from the current curriculum. Seven students, whose pre-clinical course work was during the previous curriculum, are not part of this study. The probability of obtaining a Step 1 score below 210 rose from 1% in students with exam averages greater than 70%, to 6%, 8%, 17%, and 25% if they scored less than 70% in one, two, three, or more than three courses as noted inThe percentages of students failing the Step 1 exam were 11%, 6%, 1%, and 0% of students with final exam averages in the ranges 65%-69%, 70%-74%, 75%-79% and greater than 80%, respectively. The relationships between the initial CBSSA exam scores (designated as Week 0) and subsequent scores were analyzed by sequencing practice exams in the order in which they were taken , since students were quite variable in the timing. The upper curve (squares) inNote the significant improvement from the CBSSA exam to first practice exam followed by continued improvement on practice exams 2 and 3. Mean scores then plateaued in both groups. Students with Step 1 scores above 220 had significantly higher CBSSA scores than those with scores less than 220, and although both groups improved during the course, students scoring lower than 220 never reached practice exam performance of students who scored higher than 220.Subgroup analysis: The results of multiple linear regression are summarized in2 value of 0.604, F value of 333.76, and an F significance < 0.001. Thus, measures of pre-clinical performance predicted about 60 % of the variability in Step 1 exam scores.Average course final exam scores and CBSSA exam scores were significantly and directly associated with Step 1 scores. The multiple regression model predicted Step 1 scores with an Rmax, the score on the last practice exam minus the score on the initial practice exam, was inversely related to likelihood of scoring less than 210 on the step exam with a log Odds Ratio of 0.979. This calculates to about a 10 % decrease in the odds of obtaining a Step 1 score less than 210 for each 5 unit increase in Dmax. Stated alternatively, a student scoring less than 180 on the first practice exam could on average decrease the odds of scoring less than 210 from 26- to 18-fold if they increased practice exam scores 15 points during the course.Logistic regression was then used to model the likelihood of a student scoring above and below 210 on Step 1. Students who scored below 210 increased exam scores with more weeks of practice, but were not affected by the total number of practice exams reported. The higher the first practice exam score, the higher the chance that the student would score greater than 210. For example, student with an initial practice score above 180 was 26 times more likely to score above 210 than a student below 180. Dmax did. When compared to the students scoring less than 200 on the first practice examination, students scoring between 200 and 220 are 14.9 times, and those scoring above 220 are 55.3 times, more likely to score greater than 220 on the Step 1 exam compared to colleagues who scored less than 200. Dmax was positively related with scoring above 220 with a log Odds Ratio for Dmax of 0.0526 indicating student could double their odds of scoring greater than 220 on the Step exam if they increased their practice scores 15 points during the course.Data was remodeled to examine factors associated with obtaining a Step 1 exam greater than 220, a common screening value used to screen for residency interviews. The number of practice examinations did not significantly impact the likelihood of student scoring above 220 but a higher score on the initial practice examination and a larger DComparison of UT Southwestern performance with national benchmarks:The overall performance of the UT Southwestern students on the USMLE Step 1 exams was benchmarked with NBME-reported national outcomes from 2016 through 2018 inThe percent of students who scored below 200 and above 245 are plotted by year. For each year, UT Southwestern students had a lower percentage of scores below 200 and higher percentage of scores greater than 245 than the national mean. The percentage of students scoring greater than 245 increased from 33% in 2016 to 40%  in 2018. By 2018, only 1% of students scored below 200 compared with a national mean of 10%. The percent pass rates and mean scores for UT Southwestern students in 2016 and 2017 were essentially the same at 97% and 235, but in 2018, no student failed Step 1 (100% pass rate), and the mean score rose to 239.UT Southwestern Human Research Protection Program (HRPP) determined that this project does not meet the definition of research under 45 CFR 46.102 and, therefore does not require IRB approval or oversight.The UT Southwestern Step 1 Prep Course was designed to support students as they tackle their first high-stakes NBME licensure exam. The perception of the importance of the Step 1 exam by students is currently magnified beyond concern for licensure because residency program directors commonly use the Step 1 score to screen students for residency interviews . ConsideSince January 2017, second-year medical students in our current curriculum participate in the required Step 1 Prep Course. The course allows for considerable self-directed study while SASS provides guidance, support, and oversight during their study period. The course is well accepted by our medical students, who report that they feel emotionally supported during the prep period and that it fosters their success on the Step 1 exam: post-course surveys revealed that 70% thought the course was helpful versus 20% who did not perceive benefit. Most importantly, monitoring students identified those progressing slowly and allowed for helpful intervention before the exam was taken.The data from this study has been quite informative as we assess and modify the Step 1 Prep Course. During the second year of the course, we increased email interaction, phone contact, and face-to-face consultation with students. At-risk students are now matched with an experienced upper-class student to aid with development of effective study strategies. In addition, students are now more liberally referred to the Student Wellness and Counseling center for testing and treatment of anxiety and stress disorders. The performance of students on the Step 1 exam reassure us that the current 18-month pre-clerkship curriculum and Step Prep Course prepared them for the exam. These data also validate findings in the literature that pre-clinical course performance and CBSSA exam scores are predictors of Step 1 performance , a subgrThe conclusions that can be made from this study are limited. It was a non-randomized study, and there were no control groups. In addition, the heterogeneity of a large medical school class and the nuances of managing a complex curriculum generated many hidden factors which cannot be controlled for or measured. Nonetheless, several conclusions can be made. First, Step 1 performance is strongly dependent on a strong knowledge base developed during pre-clinical courses. This influence had a lasting effect since low and high performers on course exams separated into two distinct groups, despite a common 6-week study period. A Step 1 Prep Course is not a substitute for mastering the core of medical knowledge to pass Step 1, but rather an adjunct to it. Second, these findings are not to be interpreted as evidence that at-risk students cannot obtain excellent scores on the Step 1 exam or that students who do well in pre-clinical courses are certain to avoid low scores. Many students either out- or underperformed their predicted score from curriculum performance data. Third, since the majority of progress occurred with the first 3 practice exams, decisions concerning interventions should be made before the midpoint of the prep course. Finally, although in general students do not benefit from extension of the study period, a subset of high-risk individuals benefited from a 2-week extension of the course. This primarily included students with medical and mental health problems amenable to treatment.In summary, at UT Southwestern a structured Step 1 Prep Course is well-accepted by students and can serve in a supportive role in increasing student success on Step 1. Pre-clinical exam performance, practice exam scores at the beginning of the Step 1 preparation period and progress on practice exams over the study period correlate with Step 1 performance. Proactive utilization of these data during the Step 1 study period can be effective methods of identifying at-risk students and providing supportive interventions.•Step 1 performance is strongly dependent on a strong knowledge base developed during pre-clinical courses.•Curriculum performance data is not a definite indicator that a student is at risk of underperforming on the Step 1 assessment.•Successful curriculum performance should not be used to indicate if a student does not need guidance.•Decisions of intervention during Step 1 preparation can and should be made before the half way mark of a 6 week Step prep course.•A combination of a student’s self-study and the guidance of a mentor reduces failure rate for Step 1.Arlene Sachs, PhD is Director of Student Academic Support Services, and is Assistant Professor of Psychiatry, UT Southwestern Medical Center, Dallas, Texas.Blake Barker, MD, is Associate Dean of Students, and is Associate Professor of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas.Angela Mihalic, MD, is Dean of Medical Students, Associate Dean of Student Affairs, and is Professor of Pediatrics, UT Southwestern Medical Center, Dallas, Texas.Dorothy Sendelbach, MD, is Distinguished Teaching Professor of Pediatrics and is Assistant Dean of Undergraduate Medical Education, UT Southwestern Medical Center, Dallas, Texas.Carol Wortham, MEd, is Manager of Student Academic Support Services, UT Southwestern Medical Center, Dallas, Texas.Robert Rege, MD, is Associate Dean for Undergraduate Medical Education,Associate Dean Continuing Medical Education, and is Professor of Surgery, UT Southwestern Medical Center, Dallas, Texas. This review has been migrated. The reviewer awarded 4 stars out of 5 This paper reports an approach at one medical school to the challenging issue of increasing the success of Step 1 candidates in the national Step 1 exam. It reads well, uses appropriate methods and reports success in getting students 'over the line'. This is not a surprise - students love practice exams, and if the practice exams are well-aligned with the external exam, success should predict success. This of course requires a lot of work by the faculty to generate a sound examination with the same blueprint and item quality. For that, the authors are to be congratulated. However, the paper also raises important issues about assessment practice design and philosophy and the impact on learners. Should progress be based on more than just a single test score when specific exam coaching achieves 100% progress? What is the impact on other exams and learning throughout the curriculum? Was this a retreat to 'teaching for the test"? Did the practice exam approach look similar to progress testing testing, with Step 1 calibration but broader coverage to include other, perhaps non-core, topics? Some comment on these issues might add interest, but the paper should be of interest to other medical schools whose students face national licensing exams.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 4 stars out of 5 An interesting paper on a study to examine the effects of structured step 1 support within a curriculum.The paper is well-written, beginning with a good overview of the problem and a comprehensive description of the course. A danger of an intervention course like this is that it threatens to become simply a teaching-for-the-exam exercise, in which (through the practice exams) students are essentially resitting the exam until they pass. In this course, though, the institution has taken a much broader approach than simplistic exam preparation, and has simultaneously addressed all aspects of the students’ work, increasing their students’ overall preparedness, and their chances of becoming better health professionals.Two small points:• The Discussion mentions data from post-course surveys. These data (and other numbers about them) should be given in the Results, and the surveys need to be described in the Methods, otherwise they should not be discussed here.• The limitations are mentioned only in the Conclusion, and should really be at the end of the Discussion.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 5 stars out of 5 this article is informative and educative. the authors have adequately shared their experiences in student preparations for step 1 licentiate examinations.Reviewer Expertise:NANo decision status is available


Background: Priapism induces regulation of Transforming Growth Factor-β1 (TGF-β1) expression and collagen-type-1 deposition. This will replace the normal corpora cavernosa with fibrotic tissue which eventually resulted in erectile dysfunction. It is also known that the fibrosis process of corpora cavernosa is related to Renin-Angiotensin II System (RAS). Angiotensin II receptor blockers (ARB), especially losartan, inhibit the inflammation process and fibrotic tissue formation. This study evaluated the effect of losartan in reducing fibrosis in priapism by evaluating TGF-β1 and collagen-type-1 in cavernous tissue and determined the effect of losartan in preventing fibrosis in priapism model of Wistar rats assessed by the metavir score.Methods: A total of eighteen male Wistar rats mean were divided into five groups. For the priapism models, we applied negative pressure on the penis to make an artificial erection to mimic the priapism process. The control groups were observed and the treatment groups were orally given losartan 15 mg/kg/day.Corpora cavernosa was harvested for TGF-β1 and collagen-type-1 measurement using an enzyme-linked immunosorbent assay (ELISA). The fibrotic tissue of each rat was then collected and assessed histopathologically with the metavir scoring system.Results: Penile TGF-β1 concentration in the losartan-treated group was not significantly different on day 10 and day 28 of observation . Meanwhile, collagen-type-1 concentration was significantly lower compared to control group . There was a significant difference in metavir scores in rats that received losartan and those who did not .Conclusion: Losartan could suppress the fibrosis process in the priapism model. It could decrease the collagen type 1 deposition duringcorpora cavernosa tissue regeneration. Based on the metavir score, the group receiving losartan therapy was better than the control group. Based on its pathophysiology, priapism can be classified into the high and low flow., Low flow priapism correlated with venous occlusion which increases internal corpus cavernous pressure and prevents arterial blood flow.,, Intervention more than 48 hours prevent structural recovery which leads to erectile dysfunction.,,Priapism is defined as prolonged erection more than four hours after sexual stimulation with orgasm or without any sexual stimulation.– Hypoxia-induced growth factors increase during hypoxia environment such as Transforming Growth Factor 1 (TGF beta 1). Overexpression of TGF beta 1 leads to tissue injury and scar formation., TGF beta 1 recruits fibroblast to form molecular matrices such as fibronectin and collagen to build extracellular matrix and increase protease inhibitor production which inhibits protease degrades the extracellular matrix.During hypoxia condition, in the first 12 hours, histological examination showed interstitial edema in the corporal specimen. This condition is followed by endothelial sinusoid injury, inflammation cell infiltration to basal membrane, and platelet aggregation after 24 hours. In 48 hours, thrombus can be found in sinusoids with smooth muscle necrosis and fibroblast proliferation. Unfortunately, both medical and surgery focuses more on restoring erectile function than preventing the formation of fibrotic tissue which causes Erectile Dysfunction (ED).A study by Costa et al showed that corporal cavernous tissue in the adult penis with a history of priapism had a decreased smooth muscle component with increased type 1 collagen compared to them without priapism history. Various therapy has been carried out to manage the fibrotic condition in the corpora cavernosa, with the hope of treating erectile dysfunction conditions which include medical and surgical therapy. Phosphodiesterase-5 inhibitor (PDE5-I) and pentoxiphylline were an example of medicine while penile implants are one of the surgical options.corpora cavernosa can be a factor that initiates fibrosis in the corpora cavernosa via the angiotensin II-Smad pathway which is initiated by activation of the renin-angiotensin II system (RAS). Angiotensin II will bind to the AT1 receptor and cause an increase in TGF-β1 and activate the smad pathway. AngII through the AT1 receptor also increases tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) to inhibit matrix metalloproteinase (MMP) which functions in the degradation (turnover) of ECM proteins in the remodeling process. Changes in the histological picture of the corpora cavernosa in the form of a decrease in the number of smooth muscle cells replaced by collagen caused a decrease in the ratio of smooth muscle and collagen and an increase in fibroblasts which could be assessed by Masson’s Trichrome staining with the metavir scoring system.The hypoxic state in theCurrently, there are several ways of assessing fibrosis in the corpora cavernosa such as the ratio of smooth muscle to collagen, but none of them have assessed the lumen of the cavernosa which is the most important part of the penile erection process, so the metavir scoring system is considered the best for assessing the purpose of this study.,, Several studies have demonstrated the ability of ARBs as tissue antifibrosis by inhibiting angiotensin II receptor type 1, thereby reducing the formation of TGF-β1. This can be seen in the study by Yamamotoet al. regarding the effect of ACE-I and ARB in reducing fibrosis in the heart ventricles that occurs due to hypertension. Losartan is one of the first-generation ARB drugs that have good antifibrotic properties. The administration of losartan in various studies has been shown to reduce the incidence of heart, liver, and kidney fibrosis.–Angiotensin II receptor blockers (ARBs) are antihypertensive drugs widely used to inhibit the inflammatory process and the formation of fibrotic tissue. The types of ARBs commonly used are losartan, candesartan, eprosartan mesylate, irbesartan, telmisartan, and valsartan.The antifibrotic ability of losartan gives hope to overcome the fibrotic process that occurs in various pathological conditions in the human body, including the penis.This study aimed to evaluate the antifibrotic effect of losartan in reducing TGF-β1 and type 1 Collagen in the priapism model and to determine the effect of losartan administration on the process of corpora cavernous fibrosis in the priapism model of Wistar rats assessed by the metavir score.•Group I (without ARB therapy): Priapism model rats, sacrificed on day ten•Group II (without ARB therapy): Priapism model rats, sacrificed on day 28•Group III (ARB therapy): Priapism model rats given orally losartan, sacrificed on day ten•Group IV (ARB therapy): Priapism model rats given orally losartan, sacrificed on day 28•Group V : Untreated mice, sacrificed on day 28This was an experimental study. Sample size calculation was done using Federer formula (T-1) (N-1)corpora cavernosa was harvested for TGF-β1 and collagen type 1 measurement using Enzyme-Linked Immunosorbent Assay (ELISA).The inclusion criteria were as follows: male Wistar rat, rat bodyweight 300–350 grams, has no congenital or acquired disease, and is active. Other confounders other than our inclusion criteria were not controlled. For the priapism models, we applied negative pressure on the penis to make an artificial erection and hold the base of the penile shaft using a rubber band to mimic the priapism process. After 12 hours, the rubber band was released. The control groups were observed and the treatment groups were orally given losartan 15 mg/kg body weight per day. Half of the groups were euthanized on day ten and the rest on day 28. Following euthanasia,et al.The procedure for making a low flow type of priapism model was carried out on 18 adult male Wistar rats included in the study. The cages were cleaned daily and the temperature was maintained between 26–29 degrees Celsius. The temperature was measured using a thermometer and monitored every morning. Every morning the rat cage was left exposed to the sun from 8 to 10 am, and the cage was cleaned at the same time. After being given the same treatment and resting for a week, the samples were divided into five groups. The manufacture of the priapism model was carried out in collaboration with experienced and certified laboratory personnel using the method that has been carried out and has been published by Sanli,corpora cavernosa of the rat penis resembling priapism.This method was performed on 16 male Wistar rats group I to IV which were anesthetized intraperitoneally by injection of ketamine 75 mg/kg body weight. Meanwhile, two rats in group V were not given any treatment. Furthermore, we performed negative pressure on the penis by placing the tip of the 50 mL catheter tip around the penis and slowly pulling the syringe until the penis is fully erect. The negative pressure exerted was equal to the pull at 20 ccs. Then ligation at the base of the penis by tying a piece of 16 Fr rubber catheter that had been cut 2 mm long. This was done so that hypoxia occurs in theThe rubber band was then removed after 12 hours to restore blood flow to the corpora cavernosa penis of the rat. Following the removal of the rubber band, groups III and IV were given ARB losartan 15 mg/kg body weight/day orally with the help of NGT number 6 Fr for 10 days and 28 days. The mice in groups I and III were sacrificed after ten days and group II, IV, and V mice were sacrificed after 28 days. Rats were sacrificed by euthanasia injection of ketamine 90 mg/kg body weight intraperitoneally. Surgical removal of the corpora cavernosa was performed in each group. Rats were then exterminated using an incinerator.°C or frozen at -20°C if it was to be stored beforehand. Before the procedure, the sample tissue was allowed to reach room temperature and then centrifuged at 2000–3000 RPM for 20 minutes.The levels of TGF-β1 and type 1 collagen in the corpora cavernosa tissue samples were measured using the ELISA technique. The corpora cavernosa from the penile tissue was separated from other structures such as the urethra, corpora spongiosa, and tunica albuginea. Type 1 collagen in the tissue would stick to the plate which had been coated with type 1 collagen antibody. Before processing, the tissue was rinsed using Phosphate Buffer Saline (PBS) solution (pH 7.4) to remove excess blood and tissue. The tissue was then smoothed and homogenized in PBS solution (pH 7.4) with a glass homogenizer on ice. The tissue could be thawed at 2-8The corpora cavernosa tissue samples were fixed in 10% formaldehyde solution. The sample is then processed and made into paraffin blocks. Paraffin-embedded tissue was sliced into 5-m sections and placed on an object-glass, followed by deparaffinization and rehydration. Hydrogen peroxide was administered for 10 minutes to eliminate endogenous peroxidase activity. The sample was then rinsed using PBS-Triton X 100 (Tx). Hematoxylin-eosin (HE) staining was performed to assess structural changes in the corpora cavernosa. To determine smooth muscle and collagen, cross-sections were stained with Masson’s trichrome (MT) using the MT staining kit. Areas of smooth muscle (red stain) and collagen (blue-green stain) were evaluated in 200× magnification images of tissue using a microscope. Corpora cavernous fibrosis was assessed after Masson’s trichrome staining with the metavir scoring system.The data were processed using SPSS version 20.0 with Windows as the operating system. The normality test of the data was carried out with the Shapiro-Wilk test. Because the number of samples was less than 50. The p-value that was considered significant is 0.05. Parametric statistical test to assess the comparison of type I collagen and TGF-β1 between groups receiving losartan therapy with controls at each observation time using the unpaired T-test if the data was normally distributed or the Mann-Whitney test if the data were not normally distributed. Non-parametric statistical tests were used to assess meta var scores using the Kruskal-Wallis statistical test with posthoc analysis using the Mann-Whitney U test. This study has been approved by the Health Research Ethics of Universitas Padjadjaran Bandung with ethical protocol number: 329/UN6.KEP/EC/2021.Rattus norvegicus) with an average weight of 328.1 grams at the beginning of the study. The samples were divided into five groups, namely four rats in group I (a model of priapism without therapy was sacrificed on day ten), four rats in group II (a model of priapism without therapy which was sacrificed on day 28), four rats in group III (a model of priapism with the ARB losartan therapy sacrificed on day ten), four mice in group IV (a model of priapism with the ARB losartan therapy sacrificed on day 28), and two mice in group V . All lived until the end of the study and none of the mice’s penises was necrotic. Each group was selected randomly and there was no significant difference in body weight in all rats ; p < 0.05).This study involved 18 male Wistar rats . However, due to the small number of samples, all of them were considered normally distributed.Analytical comparative study between TGF-β1 concentration in both groups based on observation time using unpaired T-Test (p < 0.05). FromStatistical analysis of the comparison of the concentration of type 1 collagen in the two groups based on the time of observation using the unpaired T-test (p < 0.05). Fromcorpora cavernosa treated with Losartan, the metavir score was lower.Changes in metavir scores in the losartan therapy group and the control group are shown incorpora cavernosa of rats compared to controls that were assessed histopathologically with metavir scores.Based on– The process of tissue fibrosis can occur due to chronic diseases such as chronic disorders, heart failure, liver cirrhosis. Molecularly, this occurs because tissue hypoxia will increase the expression of angiotensin II which stimulates the AT1 receptor causing the synthesis of TGF-β1. TGF-β1 will stimulate fibroblasts to turn into myofibroblasts to produce type I collagen so that type 1 collagen deposits occur in the extracellular matrix and form fibrotic tissue.,–In low-flow priapism, prolonged hypoxia occurs, causing histopathological changes of endothelial damage, exposure of the basement membrane, platelet aggregation, and changes in smooth muscle cells to fibroblasts. This mechanism caused the process of fibrosis in the corpora cavernosa tissue.et al, evaluation of ARB administration in liver fibrosis model mice on days seven, 14, and 28 showed that fibrosis improvement occurred at week two (day 14) and week four (day 28). In general there are three phases in the occurrence of wound healing, namely: the hemostasis phase which lasts from the first day to the 5th day, the proliferative phase from day five to 12, and the remodeling phase which lasts for months. Fibroblast and collagen recruitment was assessed in this study. Therefore, sampling was carried out on day ten during the proliferative phase and day 28 during the remodeling phase.This research was conducted in vivo on male Wistar rats. The priapism penis model is made by applying negative pressure to the penis by placing the tip of a 50 mL catheter tip around the penis and gently pulling the syringe until the penis is fully erect. The penis is then ligated by attaching a 16 Fr rubber catheter to create a priapism-like condition and then removed after 12 hours. According to research by HarnIn this study, there was an increase in TGF-β1 and type 1 collagen in the groups modeled for priapism (control group and the losartan ARB therapy group). Then we can also note that the increase in the concentration of TGF-β1 in the group receiving losartan therapy was lower than the control group on both the 10th and 28th-day observations. In addition, the concentration tends to decrease on the 28th day of observation compared to the 10th day.et al that can be helped by several antifibrotic agents, one of which is losartan. In this study, giving losartan 10 mg/kg/day is believed to be able to modulate the expression of fibrosis genes and effectively inhibit AT1 receptors and prevent the formation of TGF-β1 to prevent type 1 collagen deposition, prevent fibrosis, and enhance the tissue regeneration process. Administration of losartan as antifibrotic drugs can be used in most chronic inflammatory diseases, especially in heart and kidney disease., The use of the ARB losartan to prevent fibrosis in acute conditions such as priapism can be a new finding and an attractive alternative in the management of daily practice.This decrease in the concentration of TGF-β1 is in line with research by GargAlthough the levels of TGF-1 on day ten and day 28 in the group receiving losartan were lower than the control, this was not statistically significant when compared to the control group . Many factors may have an effect, one of which is the dose and duration of monitoring. If you look at the tendency for TGF-1 concentrations to continue to decrease on day 28 compared to day ten in the treatment group, the administration of losartan with a longer duration can reduce the level of TGF-β1 to a statistical significance.et al, that administration of angiotensin inhibitors can inhibit type 1 collagen deposits and restore the process of tissue fibrosis.The concentration of type 1 collagen also appeared to decrease with the administration of losartan on both the 10th and 28th days of observation. Then through calculations, it was proven that this was statistically significant between the group receiving losartan therapy when compared to the control group (p day 10 = 0.002 and p 28th day = 0.01). This research is in line with research by Varoet al. In his study, it was found that total circulating levels of TGF beta did not change with ARB administration but type 1 collagen levels decreased significantly. It is interesting to note in this study that the decrease in type 1 collagen levels is believed to occur due to a change in the ratio of active and inactive TGF beta of up to 90% which results in a decrease in type 1 collagen deposition and reduced fibrosis.There was no statistically significant difference in TGF beta levels in this study in line with research conducted by FrimpongIn addition, as we know the formation of type 1 collagen is a result of balancing various fibrotic and anti-fibrotic pathways. One of the cytokines that play a role in type 1 collagen deposition is the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1). Meanwhile, the cytokine that causes the degradation of collagen is Matrix Metalloproteinase (MMP). MMP consists of collagenase, stromelysin, and gelatinase. TIMP-1 is generated by signals from inflammation, while MMP is produced by tissues constantly. TIMP-1 forms a balance with MMP in regulating type 1 collagen levels in tissues. If TIMP-1 is overexpressed, fibrosis will occur. Angiotensin II has a role in increasing TIMP-1 so that giving Losartan as an angiotensin inhibitor, can reduce TIMP-1 concentrations and restore MMP/TIMP-1 balance and able to reduce type 1 collagen deposition and prevent fibrosis by various pathways.In this study, the approach used was the ability of losartan to suppress the action of profibrotic cytokines such as TGF-β1, but its ability to increase antifibrotic cytokines such as MMP which can degrade collagen has not been assessed. In addition, the effective dosage and duration of the use of losartan may need to be studied further to obtain maximum results. This can be a good suggestion for further research to determine the effectiveness of losartan in preventing fibrosis in priapism. In a retrospective human study, ACE-I/ARBs were able to reduce liver fibrosis in patients with chronic liver disease. In patients with hepatitis C virus infection who had undergone liver transplantation, the fibrosis stage and fibrosis progression index on liver biopsy were significantly lower in patients treated with ACE-I/ARB than in those not treated with ACE-I/ARB.Several studies have shown a reduction in hepatic fibrosis due to hepatitis C with losartan therapy.Histopathological examination of the liver is the gold standard for fibrosis. In one meta-analysis, there were two randomized controlled trials (RCTs) evaluating fibrosis scores. The results showed that ARB therapy had a trend of positive effects on liver fibrosis as assessed by the Metavir score.ARB studies in cardiac fibrosis were performed using high doses of Telmisartan (5 mg/kg/day) and low doses (0.5 mg/kg/day) for five weeks. Myocardial fibrosis was significantly decreased in both groups (high dose and low dose). Perivascular fibrosis is also prevented by ARB. The use of ARBs does not decrease the serum angiotensin II concentration. However, aldosterone concentrations were significantly decreased. This suggests that aldosterone changes may be another influential factor in the prevention of cardiac fibrosis and hypertrophy.Several experimental studies demonstrated the inhibitory effect of AngII on reducing skeletal muscle fibrosis. Losartan can reduce fibrosis and improve skeletal muscle strength in animal models of congenital muscular dystrophy and Marfan syndrome.,The role of AngII in fibrosis is seen in the presence of excessive induction of renin and AT. In addition, the synthesis of the extracellular matrix increases and AngII induces an increase in the concentration of collagen and fibronectin. Connective tissue growth factor (CTGF) appears as a profibrotic cytokine. CTGF is a mediator that stimulates TGF-β1 resulting in proliferation and activation of fibroblasts.The fibrogenic effect of AngII is related to the activation of TGF-β1 signaling. Over-activation of TGF-β1 causes fibrosis in almost all tissues. TGF-β1 stimulates myofibroblast differentiation and extracellular matrix synthesis. TGF-β1 also acts to maintain extracellular matrix proteins by inhibiting the activity of matrix metalloproteinases (MMPs) and inducing the synthesis of tissue inhibitor metalloproteinases (TIMPs).AngII inhibitory mechanism can block TGF-β1 signaling. Losartan antifibrotic effect occurs through the mechanism of suppression of TGF-β1 levels via the AngII type I receptor. Using a dose that had minimal effect on mean arterial blood pressure (MABP), it was shown that losartan reduced collagen I levels. Long-term administration of ACE-I increases compensation. The accumulation of bradykinin due to ACE inhibition by ACE-I can lead to the development of fibrosis and activate TGF-β1, as well as synthesize extracellular matrix proteins.Another study also demonstrated a superior antifibrotic effect of ARBs compared to ACE-I on liver tissue fibrosis.,However, findings related to the effect of ACE-I/ARB on fibrosis are still contradictory. Another study on liver fibrosis showed that losartan could increase the stage of liver fibrosis in chronic hepatitis C patients. Long-term use of ACE-I/ARBs for hepatitis C patients has not been shown to inhibit the development of liver fibrosis.Contradictions have also been found in studies of the use of ARBs in interstitial lung disease. A study suggests a possible harmful role of renin-angiotensin inhibition in interstitial lung disease. The use of ARBs was associated with a 1.9-fold increase in the likelihood of having lung interstitial abnormalities at ten-year follow-up. It is suspected that ARB-induced compensation causes an increase in renin levels increasing angiotensin II. Renin itself increases the synthesis of collagen and TGF-β1 via an angiotensin II-independent mechanism. However, studies are still limited.There are some limitations of the research. In this study, there was no comparison between the use of losartan and proven anti-fibrotic such as phosphodiesterase-5 inhibitors and pentoxifylline. It is necessary to compare with other anti-fibrotic agents to measure the effectiveness of losartan as an anti-fibrotic. In addition, further research is needed on the minimum dose required to provide anti-fibrotic effects in humans. The side effects of losartan anti-fibrotic doses in humans also need to be evaluated to prevent toxicity and unwanted side effects.Oral administration of the ARB losartan can reduce the concentration of TGF-β1 in male Wistar rats model priapism although this is not statistically significant and oral administration of the ARB losartan decreased the concentration of collagen type I statistically in male Wistar rats model priapism. Losartan administration also can prevent corpora cavernosa fibrosis in priapism model Wistar rats assessed by metavir score.https://doi.org/10.17632/7dpb82y8j5.1Mendeley Data. The Role of Angiotensin Receptor Blocker (Losartan) on Decreasing Fibrotic Process of Corpora Cavernosa in Priapism Model of Wistar Rats. DOI:-Methods: A total of eighteen male Wistar rats mean were divided into 5 groups. For the priapism models, we applied negative pressure on the penis to make an artificial erection to mimic the priapism process. The control groups were observed and the treatment groups were orally given Losartan 15 mg/kg/day. Corpora cavernosa was harvested for TGF-β1 and collagen-type-1 measurement using an enzyme-linked immunosorbent assay (ELISA). The fibrotic tissue of each rat was then collected and assessed histopathologically with the Metavir scoring systemThis project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of thehttps://doi.org/10.17632/7dpb82y8j5.2Mendeley data. ARRIVE checklist. DOI:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The author present a manuscript that describes an antifibrotic effect of angiotensin receptor blockers in a rat model of priapism. They derive their conclusions from decreased levels of collagen I, which were established by ELISA, and the metavir score, which is typically reserved for assessment of hepatic fibrosis.How was the drug dose selected? Is this dose achievable and safe in human subjects?Despite presenting this as an exploratory study, the n-numbers are quite low, which is a limitation that should be acknowledged.The manuscript needs minor text changes throughout to improve the quality of writing.The authors use both: antifibrotic and anti-fibrotic, please choose on and use throughout the entire manuscript.How was the losartan administered? Oral gavage or in drinking water? If drinking water, how was uptake/serum level confirmed?The ketamine dose seems very low at 90mg/kg for euthanasia, given that this is a safe dosage to anaesthetise pregnant rats (PMID:521380).In the TGF-beta1 and collagen assessment section in the methods: was 2,000 or 3,000rpm used? Why is there a range?The metavir scoring is not explained sufficiently. If this forms such a major part of their results and conclusions, then the methods need to reflect this and include a lot more detail on how the scoring was performed.Was the observer blinded during metavir scoring?TGF-beta1 shows no decrease after 28 days, it stays virtually the same as day 10 and is still above control levels. The authors presentation of this as a decrease is confusing.Tables 3&4 should also include the healthy control. The authors should also consider using ANOVA with multiple comparisons for statistical analysis here.Table 6 shows that there is no statistically significant difference between losartan and sham groups. Again, ANOVA with multiple comparisons might be more appropriate to analyse and interpret these results.The discussion says that proliferation and remodelling happen at days 10 and 28, respectively and make reference to ref 19. Reference 19 does not support these statements. Further, are days 10 and 28 applicable to the human or rat situation?The discussion then mentions the Garg paper, which is ref 21, not ref 20. Please correct, as ref 20 does not support the statement made.Can the authors comment on the use of losartan in erectile dysfunction and how this fits with their data?The metavir score section in the discussion needs to be reworded. Specifically: "Histopathological examination of the liver is the gold standard for fibrosis". This might apply for hepatic fibrosis, but examination of the liver is not the gold standard for all fibrosis. There are numerous issues that need to be addressed/clarified.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:AndrologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The sample size is too small, there is only a total 18 animals in 5 groups. Only 2 rats in normal control group. The current research findings are not reliable.Please provide detail information about the groups, including the animal number in each group.Please provide the full name of the abbreviation the first time it appears in the manuscript.Please provide the statistical analysis information of the table, such as P value, N, etc.Please provide images in high resolution >300 dpi.Figure 3 should be re-organized, please provide a professional Figure.Figure 4 is unqualified. Please compare the same area of penile erectile tissue and use the same magnification. Please add the scale bar. More importantly, please provide images for each group. Professional English editing is recommended.The manuscript in title of “The role of angiotensin receptor blocker (losartan) on decreasing fibrotic process of corpora cavernosa in priapism model of wistar rats” aims to explore the effect of losartan in reducing fibrosis in priapism by evaluating TGF-β1 and collagen-type-1 in cavernous tissue and determined the effect of losartan in preventing fibrosis in priapism model of Wistar rats assessed by the metavir score. This is a very interesting study. However, there are several issues need be revised extensively.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?NoAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?NoReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Federer formula to decide the minimum sample size of each group .This study uses the In the normal control group (sham group) we only used 2 samples because we consider the animal welfare law from the Institutional Animal Care and Use Committee (IACUC) guidance.Group 1: sample 1.1, 1.2, 1.3, 1.4 (a model of priapism without therapy which was sacrificed on day ten)Group 2: sample 2.1, 2.2, 2.3, 2.4 (a model of priapism without treatment which was sacrificed on day 28).Group 3: sample 3.1, 3.2, 3.3, 3.4 (a model of priapism with the ARB losartan therapy sacrificed on day ten).Group 4: sample 4.1, 4.2, 4.3, 4.4 (a model of priapism with the ARB losartan therapy sacrificed on day 28).Group 5: sample 5.1, 5.2 .Full name of the abbreviation in this manuscript has been added when it appears for the first time.Tables 1 and 2 don’t need statistical analysis. while Table 3-6 already have their respective statistical analysis results.Figures have been replaced with better resolutions images.Figure 3 has been revised as per the instructions.Figure 4 has been revised as per the instructions.Greetings, I am very sorry for the late response. Congratulations and well done to all the authors for finishing a well-written manuscript. The authors presented an experimental animal study on the effect of Losartan on TGF-β1 expression and collagen type-1 deposition due to low-flow priapism in rats. The topic chosen by the authors is novel and intriguing. The current approach to low-flow priapism has not considered using ARB to prevent fibrosis. Thus the finding of the study will provide novelty to the body of medical literature. The whole manuscript is clear and concise. The title and the abstract adequately represent the study's concept and finding. The objectives of the study are very clear and the methodology is also sound and reproducible. All the findings were correlated to the previous study. The figures are clear and legible. However, further research will still be needed to confirm its finding due to the small number of samples and the nature of the animal study. Nevertheless, this research will open a new horizon and possibility to a medical or non-surgical approach for low-flow priapism, at least for adjunctive treatment to prevent fibrosis due to priapism.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Andrology, male infertility, male sexual dysfunction, priapism, erectile dysfunction, ejaculatory and orgasmic problem, endoruology, urinary tract stone disease,I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for the review, yes we hope this research will make a pilot study with larger sample to other research in the future, Many thanks


While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni Surface plasmon resonance (SPR) was first used for biosensing in 1983  and in tThe technique relies on the phenomenon of surface plasmon resonance. At a specific angle of incident light, the energy of the exciting photon couples with electrons in the sensing surface, which is typically a metal coating ,16. The When the sensing surface contains nanoparticles at a size less than or equal to the wavelength of light in use, the free electrons have a collective oscillation defined as localized surface plasmons ,20. LocaThere are a wide variety of strategies to immobilize ligands to the sensor chip surface, ranging from covalent coupling via carboxyl or thiol chemistry to affinity tag immobilization methods, including 6xHis-Ni-NTA linkage or streptavidin–biotin interactions. In particular, the 6xHis-Ni-NTA coupling strategy has numerous benefits that make it widely used. The small size of the 6xHis peptide tag results in minimal steric hindrance of the ligand compared to larger tags like glutathione S-transferase (GST), maltose binding protein (MBP), or streptavidin. It also offers the ability to control the orientation of immobilization  and the Our group has used the Nicoya OpenSPR-XT instrument and Nicoya NTA sensor chips to characterize the binding affinity of clinically used antibodies to recombinantly expressed repeat regions of biomarker protein MUC16 . Our expThis study is a systematic characterization of the variability of commercially available NTA sensor chips for SPR and offers a more thorough understanding of the sources of sensor chip variability and strategies to combat them.Techniques that rely on nanoparticle-sensing surfaces, such as LSPR or surface enhanced Raman spectroscopy (SERS), are popular detection methods. A growing area of research is developing new types of nanoparticle surfaces to improve sensing applications, such as the development of a SERS substrate based on diatom frustules from unicellular algae , upconveE. coli , and clones were grown to exponential phase in Luria-Bertani (LB) broth  with 100 μg/mL of ampicillin at 30 °C. Protein expression was induced by adding 400 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Cells in phosphate-buffered saline (PBS) buffer with cOmpletetTM protease inhibitor (Thermo) were harvested and lysed via freeze–thaw cycles. Ni2+-NTA beads (Thermo) were used to purify the 6xHis-tagged repeat proteins. Protein identities were verified via 6xHis antibody Western blots and mass spectrometry as described in our previous work [The MUC16 sequence cited by O’Brien et al.  was sourous work  and as d280 mode (set to 1 Abs = 1 mg/mL). Protein was then diluted to 125 nM in PBS-T with the exception of repeat 6 (R6), which required a higher concentration (500 nM) to achieve acceptable immobilization levels. The surface was regenerated with two injections of 10 mM Glycine-HCl (pH 1.5) and one injection of 350 mM EDTA . This regeneration method was used to clean the surface before the start of each experiment and after each ligand–analyte pair was injected. Regeneration was optimized during experimental design to ensure ligand was completely removed prior to next immobilization.All samples were run on a Nicoya Lifesciences OpenSPR-XT 2-channel instrument  with NTA-functionalized sensors from the same vendor. PBS-T  was used as immobilization and running buffers. Default experimental temperature was set to 20 °C for autosampler tray and sensor. Protein concentration was estimated before each experiment using a NanoDrop 2000  in Protein A2 , and recombinant repeat was immobilized on the surface. Recombinant 6xHis-tagged streptavidin  was used as a blocking molecule at a concentration of 0.75 μM. Following blocking, the antibodies were injected, and the affinity was measured. M11  was injected at stock concentration. OC125  was injected at a dilution of 1:200. M11-like  and OC125-like  were injected at a dilution of 1:2000. After each antibody injection, the surface was regenerated, resulting in four cycles of ligand immobilization followed by analyte injection for each experiment. Three replicates of each repeat were run, with an extra replicate for R5. Three different repeats were run per chip, with the order of repeat immobilized on the chip scrambled between chips. All reagent injections were done at a flow rate of 20 μL/min, except for Glycine-HCl (150 μL/min) and EDTA (100 μL/min). All injections had a dissociation time of 270 s. The average signals from Channel 1 (reference) and Channel 2 (ligand-immobilized) and the Corrected signal (Channel 1 subtracted from Channel 2) were calculated over the interval of 500–525 s at the end of the dissociation phase using TraceDrawer Analysis 1.9.2 . Because the experiment was designed as a semi-quantitative comparison of binding analysis and only used one concentration of each analyte, binding kinetics were not characterized. We used nine different sensor chips from two different lots to run the entire set of repeats in triplicate. Chips 1–5 were from lot SND0927, and chips 6–9 were from lot SNE0111.The surface was activated for immobilization using 40 mM NiClThe characterization of binding affinity between monoclonal antibodies and repeat proteins (described above) used three replicates analyzed across three separate sensor chips. To compare reproducibility with replicates collected on the same chip, we collected additional replicates of R58. On the chip used to collect the third replicates of R58 and of two other repeats, we ran fourth and fifth replicates of R58, for a total of three replicates of R58 on different chips and three replicates on the same chip with one experiment common to both. To assess reproducibility of an additional repeat, three replicates of R9 were run on a single chip. Experimental parameters were held the same for additional replicates, except for the determination of R9 concentration, which used a bicinchoninic acid (BCA) concentration assay rather than NanoDrop.To establish the maximum protein immobilization on a chip, we injected a saturating concentration of HE4  followed by a lower concentration (250 nM). We first immobilized His-SA (0.75 μM) and regenerated the surface. Three cycles of high–low protein immobilization were performed per chip, with regeneration between sets. The corrected signal from both injections was added together, and the total immobilization for all three cycles was averaged. This experiment was run on three different sensor chips from two different lots. The first chip was from lot SND0927 and the second two were from lot SNE0111. HE4 concentration was measured using a BCA assay.We performed a variety of calibrations with different protein–antibody pairs. A general experimental design for these calibrations is given in The order of sample concentrations immobilized on the surface was randomized for all tests. For the tandem repeat proteins, we immobilized 25, 50, 100, 150, 200, and 250 nM protein, and antibody concentrations corresponded with those used for the tandem repeat binding affinity experiments. Flow rates, blocking, and dissociation time also matched the experimental conditions, with an additional EDTA injection during the regeneration cycle for a total of two Glycine-HCl injections and two EDTA injections. Tandem repeat protein concentration was estimated using the NanoDrop.For the HE4 system, we immobilized 250, 500, 750, 1000, 1250, 1500 nM samples and held antibody concentration constant at 75 nM. The flow rates and blocking were the same as other experiments, and regeneration cycles used two Glycine-HCl injections and one EDTA injection. All signals were averaged over the 500–525 s interval, but the antibody samples had an extended dissociation time of 290 s instead of 270 s. HE4 and anti-HE4-mAb concentrations were measured with a BCA assay.2 = 0.99) was chosen from the calibration and those HE4 concentrations were used in the following tests. We immobilized 250, 500, 750, and 1000 nM HE4 protein and injected antibody at a constant concentration of 75 nM. The regeneration conditions, flow rate, blocking steps, and extended dissociation time matched the conditions of the HE4 calibration experiments. Signal was averaged over the 500–525 s interval for both channels and the corrected output.A linear range of ligand immobilization level and antibody signal (RWe used four MUC16 antibodies to characterize the variability in non-specific binding (NSB) on different sensor chips. We averaged the signals resulting from injections of antibodies over a streptavidin-blocked sensing surface (Channel 1) from the four antibodies run in triplicate on each chip and collected the averages for all nine chips utilized in the experiment. Maxima and range data are shown in There was high variability in NSB observed on different chips, measured by changes in the reference channel signal, as shown in The reference channel is intended to separate the effects of non-specific interactions from binding to the immobilized ligand, and thus we subtract it from the signal generated in the ligand-immobilized channel. We expect this subtraction to make the data more closely reflect the signal resulting from binding, but if the amount of NSB measured on different chips varies widely, it has the potential to affect how much signal we report for binding in the corrected signal after this subtraction. Because repeat protein is only immobilized in the ligand channel, it is possible to compare the reference channel signal for antibody injections across the three different experiments per chip. However, given that the experiments had only one replicate each for the binding of a given repeat with the four antibodies, and the magnitude of binding signal in channel 2 was very different for different repeats, a relative comparison of the ratio between signal in both channels cannot be made between different chips here. The range of total NSB observed from multiple runs on a single chip is smaller than the range observed across different chips, suggesting that replicates should be collected on a single chip whenever feasible.When we compared the experiments run on the same chip to the experiments run on different chips, we saw lower variability for both immobilization and antibody binding signals on the same chip versus different chips. The lowest immobilization variability was observed for R9 experiments on the same chip for which the RSD was 60.8 parts ppth. In contrast, the RSD of immobilization signal variability on different chips was 105.1 ppth. All RSD values are found in The lowest variability was observed for R9 samples, for which protein concentration was determined using BCA  and were run on the same chip A. The RSWe saw different maximum signals for immobilization of the same concentration of ligand on three sensor chips . The secThough it lacked the same second injection to verify sensor saturation, a comparison of His-SA immobilization in Channel 1 throughout the tandem repeat binding affinity experiments offers a useful insight into maximum possible immobilizations on a chip, as all injections reflect the same concentration of His-SA immobilized . There iIt is also useful to consider the above chips in terms of lot variability. The first five chips were from one lot and display much higher variability in their average immobilization level and the spread of measurements, with an RSD of 170.2 and SEM values for each chip ranging from 118.8 to 393.4. However, the last four chips were from a separate lot and display a much more consistent average and lower overall variability when considered either in terms of individual chips or as a whole lot. This second set had an RSD of 61.4, and the SEM values for the individual chips range from 36.5 to 209.0. For circumstances in which all experiments cannot be done on a single chip, there appears to still be a benefit to doing experiments on only chips derived from the same lot.Also supporting the reproducibility of performing tests on the same chip, Nicoya has demonstrated immobilizing His-SA on an NTA chip up to 27 cycles, with only 2.9% variability .In addition to differences in the performance of the sensor chips with regard to the functionalized nanoparticle sensing surface, we also observed variability in the thickness of the glass sensor chips. Using digital calipers, we measured 88 sensor chips  and saw a range of 0.1 mm between the thickest chip and the thinnest see . The docWe have seen that different chips achieve different possible immobilization maxima, and that the chips with more consistent immobilizations also had more consistent antibody-binding responses. We performed calibration experiments to determine a more exact relationship between the two parameters see  and obseDifferent ligands have different maximum immobilization signals and ranges where analyte binding signal correlates positively with immobilization level. However, ligands that are similar (such as the tandem repeats) appear to converge on a similar linear positive correlation range, even if the binding past saturating immobilization levels has much more variation. For a set of ligands with similar properties, this would likely enable calibrating with a few ligand–analyte pairs to determine the best immobilization range instead of characterizing every single pair across the full immobilization range. It is important to understand the relationship between the immobilization level and analyte signal to ensure that the binding is being measured in the range where immobilization and analyte binding signal are positively correlated. Attempts to normalize a set of experiments without this initial calibration did not lead to more consistency in the data, and the variety of possible responses observed during the example calibrations sheds light on why this variability persisted. Using an immobilization level within the linear range is recommended because if immobilization level is too high and the surface is oversaturated, steric hindrance can obscure potential binding. By calibrating before performing affinity characterizations, the investigator can choose a ligand immobilization level that allows for the accurate measurement of binding and enables the normalization of the data for analysis.We used the calibration approach described in the previous section to characterize the relationship between HE4 immobilization and subsequent binding of its antibody D. We theThe present work demonstrates a variety of sources of possible variability when performing experiments using NTA chips, and we suggest corresponding controls that researchers can use to minimize the observed variation. By running experiments on the same chip, the effect of variable non-specific binding and different immobilization capacities can be minimized. It is also critical to calibrate the relationship between immobilization and binding signal of a given ligand–analyte pair, because calibration removes variability from possible different immobilization levels and allows for more consistent data when binding signal drops over time. Calibration also helps to ensure that tests accurately reflect any possible analyte binding and that potential binding has not been obscured by a ligand immobilization level that is too high or too low. Future work will assess more quantitative binding analysis methods, including studying how chip variability affects the fitting of kinetic parameters and increasing the number of replicates performed to make violin plots and to better understand sensor chip variability on a broad scale. In addition, there are insights to be gained about the physical properties of the chip by characterizing the surface morphology of the nanoparticles and quantitatively assessing how the thickness of the sensor chips affects data quality and reproducibility. Identifying sources of variability in NTA-based sensing experiments offers the opportunity to correct those sources of variation, improving reproducibility and yielding a more accurate measurement of ligand–analyte binding.