The age of patients referred for kidney transplantation has increased progressively. However, the precise influence of age on transplant outcomes is controversial.Etrospective study in which graft and recipient survival were assessed in a cohort of ≥75 years old kidney recipients and compared with a contemporary younger one aged 60-65 years through a propensity score analysis.We included 106 recipients between 60-65 and 57 patients of ≥75 years old with a median follow-up of 31 [13-54] months. Unadjusted one- and five-year recipient survival did not significantly differ between the older (91% and 74%) and the younger group . In the IPTW weighted Cox regression analysis, recipient age was not associated with an increased risk of death . Unadjusted one- and five-year death-censored graft survival did not significantly differ between both groups . After IPTW weighted Cox Regression analysis, recipient age ≥75 years was no associated with an increased risk of graft loss .These results suggest that recipient age should not be considered itself as an absolute contraindication for kidney transplant Kidney transplantation has demonstrated to improve the survival of patients with end-stage kidney disease (ESKD) compared to those who remain on dialysis , 2. DuriThe benefit of kidney transplantation in elderly patients is controversial, although there is increasing evidence that age should not be considered as an absolute contraindication for kidney transplant –11. In fThe aim of the present study was to evaluate the impact of the recipient age on patient and kidney graft outcomes in a cohort of ≥ 75 years-old patients, but using as a reference a cohort of recipients with an age difference of at least 10 years (60-65 years). Furthermore, since the majority of older recipients received a kidney from an elderly donor, the influence of the donor’s age was also evaluated.Single-center, longitudinal and retrospective study in which kidney transplant recipients between 60 and 65 and ≥ 75 years old performed in our center from January 1st, 2010 to June 30th 2019 were included. Follow-up and data collection were performed until June 30th, 2020. The study protocol was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Research Ethics Committee of the Hospital Clinic.® screening. Solid phase single bead antigen was performed in the presence of a positive class I and/or II Luminex® screening. Expanded Criteria Donor (ECD) was defined as donor age ≥ 60 years or 50 to 59 years and two of the following: [1] Cause of death is cerebrovascular accident; [2] pre-existing history of systemic hypertension; and [3] terminal serum creatinine >1.5 mg/dl  with respect to the younger one (60-65 years) were compared. Pre-transplant patient assessment included cardiologic evaluation , and CT-scan of splanchnic and iliac vessels. Immunological workup included complement dependent cytotoxicity (CDC), panel reactive antibodies (PRA) and solid phase Luminex.5 mg/dl .Primary outcomes included recipient and kidney graft survival at one and five years after transplantation. As secondary outcomes, we evaluated one-year biopsy-proven acute rejection (BPAR), new onset neoplasms, the rate of post-transplant infection during follow-up, kidney delayed graft function (DGF), which was defined as the need for at least one session of hemodialysis during the first week following kidney transplantation, as well as death-censored kidney graft failure, which was defined as return to dialysis or re-transplantation.® 1,25mg/Kg/day or ATG® 2,5mg/Kg/day) for 5 consecutive days in immunological risk recipients  and in kidney transplants from a Donor After Circulatory Death (DCD). Maintenance immunosuppression protocol was based on triple therapy with tacrolimus, mycophenolate or mTOR inhibitors, and steroids .Induction immunosuppression therapy was used in all patients. Induction therapy consisted in two doses of anti-IL2 monoclonal antibody (basiliximab) of 20 mg at day 0 and at day +4 after surgery or rabbit anti-human lymphocytes polyclonal antibodies  for parametric variables and median (interquartile range [IQR]) for the non-parametric ones. The corresponding tests used were T-test or Mann-Whitney U test as appropriated. Categorical data were compared using the Chi-Square test.Inverse probability of treatment weighting (IPTW) was used to account for covariate imbalance between older and younger recipients. IPTW was estimated from a propensity score from a logistic regression model to be assigned to the older and younger group. The model included: donor age, transplantation from DCD donors, cold ischemia time, dialysis before kidney transplant, diabetes mellitus (DM) before kidney transplant, ECD, Major Adverse Cardiovascular Event (MACE) before transplant, induction and maintenance immunosuppression.A stabilized weighting method was performed by multiplying the IPTW by the proportion of recipients with ≥ 75 and 60-65 years old. Check for adequate balance of covariates after IPTW analyses was performed by calculation of standardized differences and an absolute difference greater than 0.1 represented a meaningful imbalance , 15. AllStatistical analysis was conducted using IBM SPSS Statistics 22.0  software for Windows. Graphical representation of Kaplan-Meier survival curveswas designed with GraphPad v.5 . All tests were two-tailed and the significance level was defined as a P value <0.05.A total of 163 kidney recipients were included. Of these, 106 were between 60 to 65 years old (mean age 63 ± 1.7 years) and 57 were ≥75 years (78 ± 2.1 years). Median follow-up was 31 [13-54] months.Prevalence of pre-transplant recipient comorbidities as dyslipidemia, smoking, hypertension and diabetes were not significantly different between both groups, as well as prevalence of Ischemic Heart Disease (IHD), Cerebrovascular Accident (CVA), dialysis modality and dialysis vintage Table 1.In the ≥ 75 years old group, donors were older  andwere more frequently ECD  Table 1.After IPTW adjustment, no significant differences in donor and recipient characteristics were observed (except for recipient age). Recipient survival was not significantly different for recipients of ≥75 years when compared to those between 60-65 years, with 1- and 5-year recipient survival of 95% and 82% for the group 60-65 years , and 91% and 74% for the group of ≥75 years , respectively (P = 0.06)  in the group of 60-65 years, and solid organ and hematological malignancies ≥ 75 years group (1% for each)  and 2 patients (4%) in the younger and older group developed or each) Table 2.In the Cox regression analysis after IPTW weighting, recipient age ≥75 years was not independently associated with lower recipient survival .Kidney graft function at one year did not differ significantly between both age groups, with 1-year serum creatinine (SCr) of 1.78 ± 0.88 and 1.60 ± 0.58 mg/dL (P = 0.23), and estimated glomerular filtration rate (eGFR) of 42.80 ± 16.31 and 42.41 ± 15.87 mL/min/1.73m2 for the 60-65 and ≥ 75 years old group, respectively (P = 0.76). At last follow-up, there also were not significant differences in the SCr  or in the eGFR between both age groups  Table 2.One-year Biopsy-Proven Acute Rejection (BPAR) rate was of 16% in both age groups , although in the younger one the most frequent was T-Cell Mediated Rejection (TCMR) and in the older group was Antibody-Mediated Rejection (ABMR) . There were no statistically significant differences in the overall rate or in the rejection type between both groups (P = 0.97) Table 2.Moving to death-censored graft survival, 1- and 5-year survival rate was of 99% and 89% for the younger group, and 96% and 83% for the older group, respectively, without significant differences between them (P = 0.08)  Table 3.In the present study we analyzed recipient and kidney graft survival in a cohort of older recipients with ≥75 years using a stabilized IPTW weighting method for donor and recipient covariates, comparing their outcomes with those of a cohort of recipients with 60-65 years and, thus, with at least a 10-year difference in recipient age between both groups. In summary, we observed that the recipient and graft survival up to 5 years after transplantation was not significantly different between both age groups.Kidney transplantation has demonstrated to be the treatment of choice for end-stage kidney disease (ESKD) , 6, 16. In 2016, Lønning et al.  comparedAfter IPTW weighing for donor and recipient covariates, age was not independently associated with an increased risk of recipient death, a result which is in line with some already published, although using as reference groups patients with a closer age range to the evaluated one . Our resDeath-censored graft survival at 1 and 5 years was not significantly different when compared to the reference younger cohort, and was also similar to that previously reported , 10, 11.The authors acknowledge that the study has some limitations that should be taken into account when extrapolating the results. This is a retrospective and single-centre study in which a cohort of older recipients that has been considered suitable for kidney transplantation has been compared with a younger group. This could have induced a selection bias since the included recipients in the waiting list have probably been patients with only few comorbidities and a good condition to be transplanted, as well as the donor has been selected according to the recipient risk to minimize the consequences of a non-functioning kidney graft. Nevertheless, the IPTW method partially reduces the effect of this potential selection bias by approaching baseline characteristics between both groups. Another limitation to take into account is the relatively small sample size of older recipients with the short number of events that make necessary to be cautious with the conclusions, especially when extrapolating to other populations to avoid bias.Despite these limitations, we believe that our study provides more evidence about the impact of the recipient age in kidney transplantation by comparing older recipients with a younger cohort whose age range is clearly separated from its older counterparts and showing the lower mortality we have nowadays in this kidney recipients.In conclusion, in the present study we suggest that recipient age by itself should not be a criterion to contraindicate kidney transplantation. A complete evaluation of both donor and recipient  should guide kidney transplant indication in older recipients. Thus, with a judicious selection of both the recipient and the donor, kidney transplantation can be safely performed in elderly recipients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study of human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.EC-P analyzed, interpreted the data and prepared the manuscript. EM-M collected, analyzed, and interpreted the data and prepared the manuscript. JC-U collected, analyzed and interpreted the data. JR-Z collected, analyzed and interpreted the data. DR-E collected, analyzed and interpreted the data, JC collected, analyzed and interpreted the data, CA collected, analyzed and interpreted the data, DC collected the data and critically revised the manuscript. PV-A interpreted the data and critically revised the manuscript. IR interpreted the data and critically revised the manuscript. GP interpreted the data and critically revised the manuscript. NE interpreted the data and critically revised the manuscript. FC interpreted the data and critically revised the manuscript. JU critically revised the manuscript. JMC. interpreted the data and critically revised the manuscript. FO interpreted the data and critically revised the manuscript. J-VT analyzed and interpreted the data and prepared the manuscript. FD interpreted the data and critically revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Back pain negatively impacts a person’s quality of life and can cause major disability or even death. The measurement of spinal stiffness can be utilized as a promising tool to guide therapeutic decisions regarding physical therapy that result in effective back pain management. This study aimed to determine the reliability of instrumented postero-anterior (PA) stiffness assessment of the lumbar spine in asymptomatic participants by novice assessors using a portable algometer and the Kinovea program.Thirty asymptomatic participants aged 18–25 years were enrolled in this study. Two novice assessors examined the participants for lumbar spinal stiffness at L1–L5 for two consecutive days. The algometer was applied to measure the PA force that applied to each lumbar. The stiffness assessment of each lumbar region was recorded as a video. The 600 data sets of assessment videos were imported into the Kinovea program to perform displacement measurements of each lumbar level. Spinal displacement values at 15 N were defined by graph plotting between force and displacement. The spinal stiffness values were defined by slope calculation. Both variables were analyzed for inter-rater and test-retest reliabilities using intra-class correlation coefficients (ICCs) and standard error of measurement (SEM). Bland-Altman analysis was applied to assess the inter-rater and test-retest systematical bias and limits of agreement of measuring displacement and stiffness.The inter-rater reliability of measuring the displacement and the stiffness of L1–L5 was moderate to good . The test-retest reliability of measuring the displacement and stiffness of L1–L5 were moderate to good, ICCs: 0.57–0.86 and ICCs: 0.51–0.88, respectively. The inter-rater analysis’s Bland-Altman plot showed that the systematic bias was 0.83 when measuring displacement and 0.20 when measuring stiffness and the bias of both parameters were in both directions. While the test-retest systematically biased measurements of displacement and stiffness were −0.26 mm and 0.22 N/mm, respectively, and the bias of both parameters were in both directions.The moderate-to-good inter-rater and test-retest reliabilities of the portable instrumented spinal stiffness assessment using a digital algometer and the Kinovea program by novice assessors were demonstrated in this study. Bland-Altman analysis showed that measuring stiffness was more stable and had less systematic bias than measuring displacement. To figure out how reliable the device is in general, more comprehensive studies should be comparatively conducted in the future on subgroups of patients with normal vertebra, hypomobile or hypermobile conditions. Low back pain is a health problem related to the musculoskeletal system . The mosPhysical therapists can use manual passive intervertebral movement (PIVM) in the back-to-front direction or postero-anterior (PA) direction to measure how stiff the spine is in people with low back pain. Spinal stiffness of the lumbar spine is one of the symptoms of low back pain, and it plays an important role in clinical decision-making and effective treatment . The phyHowever, mechanical spinal stiffness measurement devices are not widespread due to their lack of portability, high cost, and lack of user friendliness . TherefoThis cross-sectional study was conducted during September to December 2020. The test-retest and inter-rater reliabilities of lumbar stiffness were conducted in the laboratory room at the Physical Therapy Department, Walailak University, Nakhon Si Thammarat province, Thailand. During the testing period, the laboratory room’s temperature, light, and noise were controlled. The study was approved by the Ethics Committee of Walailak University and followed the Declaration of Helsinki .A total of 30 male and female students of Walailak University aged between 18 and 25 years with asymptomatic low back pain were recruited by a convenience sampling method. In order to identify rater dependence in spinal stiffness measurement and to rule out potential confounding factors of dysfunction or disease, healthy volunteers were then chosen to participate in the study. The exclusion criteria were: (1) pregnancy; (2) diagnosis of inflammation or a spinal infection; (3) history of spinal surgery; (4) spinal deformities such as scoliosis, (5) discomfort in the lower back, and (6) a percentage of body fat higher than the normal range.α) 0.05 (The sample size was estimated for intraclass correlation observed by two raters using the formula derived from α) 0.05 . The samvia the algometer. The camera was set so the distance between the participant and the camera was 3 m, which could focus the lumbar region and algometer during the testing period. Finally, the Kinovea software version 0.9.5 , which can be applied the force from 0 to 110 N, was used to monitor the PA force on each lumbar segment. Previous studies demonstrated the high reliability of measuring the force . The algon 0.9.5  which ison 0.9.5 , was appTwo novice raters were undergraduate physical therapy students. The raters were recruited from the senior undergraduate physical therapy students who passed the manipulation course and clinical practice under clinical instructors. The raters were female, 22 years old. All raters participated in a two-hour training for five sessions to be experience trained personnel on how to perform spinal stiffness measurements using a digital algometer. The training covered the use of the digital algometers on the lumbar region, palpating and marking the locations of the body parts, they also provided an explanation of the study’s protocols to the pain-free spinal participants who were not included in the main study.All volunteers were provided detailed information about the study’s objective and protocol. Participants were given informed consent before participating in the study and were assessed for exclusion criteria. They were also recorded demographic data, including gender, weight, height, percentage of body fat, underlying disease, and medicines.The eligible participants were randomly allocated to Rater 1 or Rater 2. Rater 1 was assigned to determine the lumbar stiffness first if the participant drew Rater 1. On the first day, the participants were measured by two raters; the interval between the raters was 15 min or until the participant was allowed to be evaluated by the other rater. The next day, the participants were examined using the same protocol as the first day.The two raters were assigned to apply PA pressure on each lumbar spine to determine the lumbar displacement. The participants lay on an electric adjustable bed in prone, arms at the sides. They exposed the cloth to the waist and attached the force display to the right wrist. The rater applied adhesive tape to fix the skin under the lumbar area, marking the five lumbar segments. The study was designed to randomize the sequence of the lumbar segment to determine the lumbar displacement. Then, each segment was applied PA pressure while participants used the algometer and held the pressure at the end range for 5 s. Each segment was determined only once, as the same protocol was used the next day. The measurement for each segment was recorded as a separate video clip. Two raters were blinded to the results of each test. The assumption that spinal stiffness would typically not change over two days led to the choice to assess the individuals twice . Due to ®, Redmond, WA, USA) to make a graph between force (N) and displacement (mm)  . Then, wTwo-way mixed effects with absolute agreement and multiple raters and measurements of an Intraclass Correlation Coefficient  were calculated to determine the inter-rater and the test-retest reliabilities for displacement and stiffness measurements at each lumbar segment individually. We selected a two-way mixed-effects model because the selected raters were the only raters of interest . We seleThe standard error of measurement (SEM) was calculated to demonstrate the absolute reliability of the measurement using the following equation : \documFurthermore, the Bland-Altman plot analysis was performed to determine the systematic bias and agreement range of spinal displacement and spinal stiffness measurements. The difference in spinal displacement and spinal stiffness values between raters (rater1–rater2) and between days (day 1–day 2) were plotted against the means of two raters and 2 days, respectively .p-value less than 0.05. The following criteria were used to evaluate the Intraclass Correlation Coefficient values: values less than 0.50 were considered poor, 0.50–0.75 were considered moderate, 0.75–0.90 were considered good, and 0.90–1.00 were considered excellent  to record, edit and enter all of our data for statistical analyses. The significance level was set at a xcellent .2, were enrolled in this study.Thirty asymptomatic participants, 28 females and two males, with mean age of 21.46 (SD = 0.62) years, and mean body mass index of 20.11 (SD = 1.97) kg/mNo participant was excluded; however, 11 videos were excluded from the total of 600. Seven videos did not start at force application at 0 N, and two videos could not be performed line calibration due to obstructions at the algometer’s shaft. Consequently, the sample size for reliability analysis in each reliability category ranged from 24 to 30 participants, as demonstrated in The inter-rater reliability analysis showed that the ICC ranged from 0.67 to 0.80 for measuring displacement on day 1 and from 0.60 to 0.83 for measuring stiffness. The ICCs of measuring displacement and stiffness on day 2 ranged from 0.68 to 0.83 and from 0.63 to 0.80, respectively. These results show moderate to good reliability of displacement and stiffness measurements between the two raters on both days. On day 1, the SEM for measuring displacement ranged from 1.18 to 1.80 mm and from 0.45 to 0.74 N/mm for measuring stiffness. On day 2, the SEM for measuring displacement ranged from 1.00 to 1.68 mm and from 0.47 to 0.78 N/mm for measuring stiffness.The test-retest reliability analysis showed that the ICC for measuring rater 1’s displacement ranged from 0.59 to 0.86, and the ICC for measuring stiffness ranged from 0.72 to 0.88. The ICCs for measuring displacement and stiffness for rater 2 ranged from 0.57 to 0.70 and from 0.51 to 0.63, respectively. These results show moderate to good reliability of displacement and stiffness measurements between the two days of rater 1, whereas measurements by rater had moderate reliability. The SEM for measuring the displacement of rater 1 ranged from 1.41 to 1.98 mm, and from 0.47 to 0.78 N/mm for measuring stiffness. The SEM for measuring the displacement of rater 2 ranged from 1.42 to 1.99 mm and from 0.60 to 0.77 N/mm for measuring stiffness.The Bland-Altman plot of the inter-rater analysis revealed the mean difference between raters for measuring displacement was 0.83 (95% CI [−4.34–6.00]) mm and 0.20 (95% CI [−1.90–2.31]) N/mm for measuring stiffness . Figure In this study, we investigated the inter-rater and test-retest reliabilities of spinal stiffness assessments of the lumbar spine in asymptomatic individuals adopting a portable algometer device and the Kinovea program conducted by novice assessors. In addition, we demonstrated two variables for representing the spinal stiffness, which were displacement at 15 N of force applied and average stiffness calculated by the slope of every 2 mm displacement change in the graph between force and displacement. We discovered moderate to good reliability for both measurements.in vivo study  were lower than the previous study’s (ICCs of 0.75 to 0.96) . The maiHowever, our study has some limitations. First, the mechanically assisted device cannot control the direction and speed of force application, which are major factors that might interfere with the reliability of measurements . HoweverThe current study aimed to determine the inter-rater and test-retest reliabilities of mechanically assisted spinal stiffness testing devices using a portable algometer and the Kinovea program. The novice assessors performed the measurement at five lumbar segments in asymptomatic individuals. Although this study used novice assessors, it could also provide moderate to good reliability. The Bland-Altman analysis indicated that the inter-rater and test-retest measurements of stiffness illustrated less systematic bias and were more stable than the measurements of displacement. Consequently, we suggest that our mechanically assisted spinal stiffness testing device  is better suited for clinical than research applications, and the spinal stiffness variable is recommended for both inter-rater and test-retest measurements. To figure out how reliable the device is in general, more comprehensive studies should be comparatively conducted in the future on subgroups of patients with normal vertebra, hypomobile or hypermobile conditions.10.7717/peerj.16148/supp-1Supplemental Information 1Click here for additional data file.


Among all the pharmaceutical dosage forms, tablets are still the most preferred and the most commonly used option because of their advantages. The direct compression method of tablet preparation exempts several steps needed in the granulation method. Therefore, the pursuit of better direct compression tablet excipients is evident in contemporary research endeavors. Pregelatinized Taro Boloso-I starch has comparable flow properties and higher compressibility and compactibility than Starch 1500®. However, there is no evidence in the literature regarding the lubricant sensitivity and dilution potential of pregelatinized Taro Boloso-I starch. This study was aimed at performing the Results and Discussion. PGTBIS had a significantly lower amount of amylose than Starch 1500®. In the ATR-IR spectra of the mixture of the paracetamol and pregelatinized PGTBIS, all the major absorbance peaks of the drug were maintained indicating the absence of chemical modifications. PGTBIS showed better flow properties than Starch 1500®. The modified starch was shown to withstand magnesium stearate up to 0.5% concentration.  Taro Boloso-I starch was pregelatinized, and its properties including amylose to amylopectin ratio, densities, flow properties, swelling power, water solubility index, particle morphology, moisture content, and moisture sorption profile were evaluated. Furthermore, the lubricant sensitivity test, dilution potential study, and compatibility test with the paracetamol drug using ATR spectroscopy were performed. The properties of the directly compressed tablets prepared accordingly were evaluated. The majority of evaluations were performed in comparison with Starch 1500®.  PGTBIS could accommodate higher drug cargo than Starch 1500® with acceptable tablet properties. Accordingly, PGTBIS starch could be taken as a potential direct compression excipient. While there are several types of pharmaceutical dosage forms, tablets are still the most preferred and the most commonly used option , 2. The Direct compression overcomes these problems. It is more economical as it requires fewer unit operations. It also reduces the chances of contamination and steps to be validated and documented. It avoids stability problems of especially moisture and heat-sensitive drugs, drugs whose dissolution profile is likely to change on storage, and drugs at risk of microbial growth. Furthermore, it favors faster dissolution as the tablet disintegrates directly into API particles than into granules. It reduces the wear and tear of punches due to the exemption of high compaction pressure involved in the production of tablets by slugging or roller compaction.Flowability, low friction tendency, compressibility, and fast disintegration capacity are some of the features and criteria of directly compressible excipients –7. ThereWhile starch is one of the leading polymers for use as a pharmaceutical excipient of several advantages , native Pregelatinization enhances flow property of Taro Boloso-I starch. There is a report in the literature  that wasThere is no evidence in the literature regarding the compatibility of the PGTBIS with paracetamol, its lubricant sensitivity, and dilution potential by using specific drugs. This study was aimed at performing the in vitro evaluation of paracetamol tablets prepared using pregelatinized Taro Boloso-I starch as a direct compression excipient.Taro Boloso-I was obtained from Areka Agricultural Research Institute, located at Areka (300 km south of Addis Ababa), Wolaita, Ethiopia. Pure paracetamol  was donated by Ethiopian Pharmaceutical Manufacturing Share Company (EPHARM). Sodium hydroxide and magnesium stearate , sodium chloride , Ac-Di-Sol® , iodine resublimed , hydrochloric acid 37% , and potassium iodide  were used as obtained from the Laboratory of School of Pharmacy, Addis Ababa University.μm aperture sieve. Finally, the samples were stored separately in tightly sealed glass containers. The amylose to amylopectin ratio was determined by the colorimetric assay method, and the morphological study was performed using scanning electron microscopy (SEM) [Taro Boloso-I starch (NTBIS) was extracted as per the methods described elsewhere in the literature . Then, ipy (SEM) . Swellinpy (SEM) , 13.The lubricant sensitivity study was performed using the method used elsewhere by Svačinová et al. . TabletsTablets of 300 mg weight containing 20%, 30%, 40%, and 50% paracetamol were prepared using PGTBIS or Starch 1500® by the direct compression method . In brieD, and T stand for tensile strength, the breaking strength, diameter, and thickness of tablets, respectively.To determine thicknesses, 10 tablets were taken and their thicknesses were measured using a sliding caliper scale . The tablet bulk density was determined from the weight, thickness, and diameter data according to the methods described elsewhere . To measIn order to evaluate the friability of compressed tablets, 20 tablets of each batch were placed into the friability tester. The friability tester was rotated for 4 min at 25 rpm, letting the tablets fall a distance of 6 inches. Then, the tablets were taken out and dusted, and the percent weight loss was calculated. The disintegration and dissolution tests were performed according to the methods described in USP-NF  on a dis−1 at a resolution of 4 cm−1. For data presentation, Origin version 7  was applied.The attenuated total reflectance (ATR)-IR spectra of pure paracetamol, PGTBIS, and paracetamol-PGTBIS physical mixture (1 : 1) were obtained with an infrared spectrophotometer  in the ATR mode. For each run, 16 scans were performed in the range of a wave number of 4000–500 cmπ ± σ). The target limit of the significance of statistical data was 95% CI.All the results of direct measurements were presented as the arithmetic mean ± standard deviation  of PGTBIS are presented in The moisture content of PGTBIS was comparatively determined with that of NTBIS. The moisture content of NTBIS, PGTBIS, and Starch 1500® were 9.11 ± 0.25%, 10.43 ± 0.42%, and 9.49 ± 0.39%, respectively.Moisture sorption of starches can affect the physicochemical properties of solid dosage forms containing starches. The moisture sorption profiles of NTBIS, PGTBIS, and Starch 1500® determined at relative humidity values of 32.7%, 65.4%, 75.6%, 85.1%, and 100% by using saturated salt solutions of magnesium chloride hexahydrate, sodium nitrite, sodium chloride, potassium chloride, and distilled water, respectively, are shown in To study the compatibility of the starch with paracetamol, functional groups that define paracetamol were assessed using the attenuated total reflectance (ATR) spectra of PGTBIS, 1 : 1 ratio mixture of paracetamol and PGTBIS, and pure paracetamol .The TBF, friability, and radial tensile strength values of tablets of pure PGTBIS and lubricated at different concentrations of Mg stearate (0–2.00% w/w) were investigated .The dilution potential of PGTBIS was tested in paracetamol tablets compressed at a force of 17 kN with variable concentrations of the drug at 20, 30, 40, and 50% w/w. The weight variation, TBF, TS, friability, and disintegration time were determined. Tablets of the same formulation and processes were repeated substituting PGTBIS with Starch 1500® as comparators . SimilarAs the results show, PGTBIS had a significantly lower amount of amylose than Starch 1500®. Moreover, the process of pre/gelatinization did not have any significant effect on the amylose to amylopectin ratio of NTBIS. The reason is that pregelatinization is a physical modification and that a physical modification does not change the amylose to amylopectin ratio as described in the literature .The swelling power of the three starches followed the order: PGTBIS > Starch 1500® > NTBIS at 20, 37, 50, and 65°C . A possiAs it is clear from the figure, the pregelatinized starch particles had slightly smoother polygonal shapes than native granules. The morphological change might be because of partial pregelatinization that had taken place which resulted in more aggregated granules, having less physical integrity compared to NTBIS. More spherical shape, aggregation, and loss of physical integrity make changes similar to that of heat moisture-treated low amylose rice starches reported elsewhere , 25.p < 0.05). This implies that the pregelatinization of NTBIS decreased the moisture content for unknown reasons, indicating its better potential stability.Commonly, dry starch contains 6–16% moisture. The moisture content, if high, can result in microbial deterioration of the product . PGTB1S p < 0.05). At the RH values of 75.4% and higher, the moisture sorption of PGTBIS was higher than that of NTBIS. The likely reason for the increased moisture sorption of PGTBIS compared to that of NTBIS was the decrement of crystal phases due to hydrothermal disruption accompanied by pregelatinization [The moisture sorption profile of PGTBIS was higher than the corresponding values of Starch 1500® at RH values of 65.4% and beyond  of the mixture were coinciding with those of pure paracetamol. The sharp absorption band at 3321.29 cm−1 and 1650.70 cm−1 were corresponding to the symmetric stretching and out-of-plane (OOP) bending bands of -NH- bonds, respectively. Similarly, the strong peaks at 3164.36 cm−1 and 3108.69 cm−1 represent CO-H stretching vibration bands. The presence of a strong band in the range 3162–3035 belongs to the stretching vibration of CH3. The presence of aromatic rings was evidenced because there are the doublets (1562.20 cm−1 and 1505.14 cm−1), possible weak overtone, and combination bands between 2000 cm−1 and 1700 cm−1. The broad background absorption around 3350.00–3108.69 cm−1 (OH-stretches) with the consideration of the finger print region claiming the phenolic -OH group. The presence of the acetyl group was supported as there were strong bands at 2930.38 cm−1, 2887.58 cm−1, and 1369.60 cm−1 of the methyl C-H bonds. It was reinforced considering the presence of the strong protruding band at 1650.66 cm−1, suggesting CO stretching vibration. The presence of the peaks at 1256.90 cm−1 and 1224.10 cm−1 is common to C-O/C-N stretching vibrations [The ATR of pure PGTBIS, pure paracetamol, and PGTBIS with paracetamol in a 1 : 1 ratio (w/w) is presented in p < 0.05). This is likely due to increasing bond inhibition [The weight variation of all the tablets was quite acceptable which is below 7.5% . The TBFhibition . The TBFhibition . The tenhibition . In all hibition . All thehibition .The weight variation of all the tablets was quite acceptable which was below 7.5% . The TBFThe weight uniformity of PGTBIS-containing paracetamol tablets satisfies compendial requirements, i.e., within ±5% of the mean. The TBF of paracetamol tablets was shown to reasonably increase with the increasing proportion of the starches. The tablets of the PGTBIS paracetamol tablets were acceptably hard (TS = 1.19 MPa), up to 30% of the drug. In contrast, Starch 1500® equivalent was hard and attrition resistant enough up to only 20% (w/w). At all the paracetamol levels, the tablets of PGTBIS had higher mechanical strength (TS) than those of Starch 1500®. The paracetamol tablets of both PGTBIS and Starch 1500® beyond the respective paracetamol concentration of 30% and 20% (w/w) had low tensile strength, higher friability (>1%), or capping and lamination . The DTsAccording to the compendial requirements, the dissolution profile of paracetamol tablets is tested at a pH of 5.8. . In viewThe findings of the present study showed that PGTBIS has comparable flow properties with standard Starch 1500®. As far as ATR spectroscopy is concerned, the pregelatinized starch did not chemically interact with paracetamol when mixed for tablet preparation. When mixed with equal respective amounts of the magnesium stearate lubricant and the paracetamol drug cargo, PGTBIS performs better than Starch 1500®. It was shown to accommodate higher drug cargo (30%) than Starch 1500® (20%) with better performance. In terms of both the lubricant sensitivity and the dilution potential, it shows higher tablet-breaking strengths and lower percent friability values. Accordingly, the PGTBIS starch could be taken as a potential direct compression excipient calling for further investigations towards its application including the brittle fracture index, Young's modulus, toughness, and stability studies both in paracetamol and other drugs.


This study explores the impacts of the Council on Medical Student Education in Pediatrics (COMSEP) Journal Club, a unique means of providing monthly professional development for a large international community of pediatric undergraduate medical educators. In particular, we sought to establish member engagement with the Journal Club, identify factors impacting member contributions to the Journal Club, and determine perceived benefits of and barriers to participation as a Journal Club reviewer.Using an established Annual Survey as a study instrument, six survey questions were distributed to members of COMSEP. Items were pilot tested prior to inclusion. Quantitative data were analyzed using descriptive statistics and chi-square analysis..Of 125 respondents who completed the survey, 38% reported reading the Journal Club most months or always. Level of engagement varied. Reasons for reading included a topic of interest, keeping up to date on medical education literature, gaining practical tips for teaching and implementing new curricula. Motivators for writing a review included keeping up to date, contributing to a professional organization, and developing skill in analyzing medical education literature, with a minority citing reasons of enhancing their educational portfolio or academic promotion. The most commonly cited barriers were lack of time and lack of confidence or training in ability to analyze medical education literature.As a strategy to disseminate the latest evidence in medical education to its membership, the COMSEP Journal Club is effective. Its format is ideally suited for busy educators and may help in members’ professional development and in the development of a community of practice.The online version contains supplementary material available at 10.1186/s12909-023-04550-4. Staying current with the medical literature can take more time than busy clinicians are able to afford. It is equally important for those involved with health professions education (HPE) to stay abreast of the literature in the HPE field, to ensure their teaching and assessment practices are also evidence based . AlthougAlthough journal clubs have been used in varying formats as a means of faculty development by regularly reviewing the academic literature in the clinical domain, the medical education literature may be underutilized as a tool to enhance educator development. While smaller institutional journal clubs have been described, –4 includcommunities of practice. The theory of community of practice holds that learning does not occur simply through acquisition of new knowledge but rather through participation in a group with a common purpose and a mutual enterprise (or practice). Individuals become members of the group by adopting the language, norms, and culture of the group as well as by participating in group activities.Others have considered the education of physicians in general , and jouThe Council on Medical Student Education in Pediatrics (COMSEP) is an international professional organization of pediatric clinician educators dedicated to pediatric undergraduate medical education. For more than a decade, COMSEP has electronically disseminated a monthly Journal Club that provides brief reviews of articles published in the medical education literature. The impact of this approach, engaging members and targeting a larger community of practice, has not been previously studied. As a unique means of providing regular professional development for a large community of educators, we sought to explore the benefits and challenges of this initiative in promoting educator development with the following objectives: (1) to establish current COMSEP member engagement with and perspectives on the current COMSEP Journal Club format; (2) to identify factors that motivate COMSEP members to read the Journal Club; (3) to establish benefits perceived by COMSEP members who have completed reviews for the Journal Club; and (4) to identify barriers to contributing to the Journal Club, as perceived by COMSEP members.The COMSEP Journal Club started in its current format in 2011 as a monthly review of the medical education literature for the edification of its members. Reviewers select a recently published article (within the last 2 years) from the medical education literature on a topic felt to be of interest to the COMSEP membership, with a focus on pediatric medical student education. Reviewers then write a brief 300 to 350 -word summary of the article in a standard format, with the following four sections: (1) What was the study question?; (2) How was it done?; (3) What were the results?; (4) What are the implications? (how can this be applied to my work in education). These reviews are edited by the COMSEP Journal Club editors, who provide brief additional commentary to foster readers’ reflection on applicability of the study to their educational contexts. Three to four reviews are curated and published each month. The Journal Club editors oversee the process of recruiting reviewers, assist with and ensure suitability of article selection, edit reviews, and disseminate in electronic format the monthly Journal Club to the general COMSEP membership. An example of a Journal Club review is included as Supplemental File The COMSEP Journal Club was established to meet the needs of its members in the following ways: (1) to give reviewers experience examining and applying evidence in medical education; (2) to provide the general COMSEP membership a brief synopsis of important and relevant articles in the medical education literature in order to improve the evidence behind their medical education decisions; and (3) to give COMSEP members an opportunity to enhance their educational portfolios by serving as reviewers and giving back to the COMSEP community.Review of our data on COMSEP members’ contributions to the journal club, along with primary journal sources and review topics, demonstrates that over a three-year period, 58 reviewers contributed a total of 129 article reviews, with the majority of reviewers contributing more than one review  and R Core Team for chi square analysis .One hundred twenty-five respondents (27%) completed the annual survey, with 90 (19%) responding to Journal Club survey questions.. Respondents to the annual survey included 35 core clerkship directors (31.5%), 19 associate or assistant clerkship directors (17.1%), and 17 faculty associated with the dean’s office of office of medical education (15.3%). There were 77 women (69.4%). Forty-four percent were assistant professors, 25% were associate professors, and 25% were full professors.Almost all respondents (92%) indicated they read the Journal Club; thirty-four (38%) respondents reported that they read the Journal Club most months or always and 49 (54%) reported that they read the Journal Club occasionally (a few times a year).The majority (71.1%) reported that they only read those articles that interested them. Readers’ level of engagement with the process varied with 28 (31.1%) indicating that they skimmed the review for the main points, 44 (49%) reporting that they read the entire review and 14 (15.6%) reporting that they read the review and the article it referenced.Respondents’ reasons for reading the Journal Club were multiple, and also varied. Respondents could select as many responses as were applicable. Seventy-five respondents (83.3%) reported that they read the reviews if the topic interested them. Seventy-two respondents (80%) read the reviews to keep up to date on the medical education literature. Thirty-eight (42.2%) read to get practical tips on their own teaching and 25 (27.8%) to develop and implement new curricula. Other motivators included developing skill in analyzing the medical literature, to stimulate one’s research ideas, and if a colleague had written a review. Further reasons cited included to share with colleagues who are clerkship directors in other fields, or to share with colleagues who are doing research or developing a project in a related area.We also asked respondents about the impact of alternative formats on the likelihood that they would engage in the Journal Club. The majority of respondents (60%) reported that a theme issue  would make them neither more nor less likely to engage. When it came to the use of virtual technology to enhance the Journal Club experience, a majority (63%) reported that a brief virtual discussion would make them less likely to engage and a substantial minority (40%) reported that they would be less likely to engage in a podcast version of the Journal Club as well.p < 0.05) and that they read every review in a given month . Other responses were similar between the two groups.When comparing the experience of those who had previously written a review with those who had not, we found that reviewers were more likely to report that they read the Journal Club most months (57.9% versus 32.3%), p < 0.05). These perspectives of benefits and barriers to writing a review are depicted in Fig. Nineteen (15%) respondents reported that they had written a review in the past. Among them, there were a wide range of perceived benefits. Fourteen (73.7%) reported that they did so to keep up to date with the medical literature, and the same number reported that they did so to contribute to COMSEP. Thirteen (68.4%) reported that they did so to develop their own skill in reading and analyzing the medical education literature. Ten 52.6%) reported that they did so in order to apply it to their own teaching. A minority of respondents reported benefits such as adding the review to their educational portfolio or curriculum vitae, using the review for academic promotion, coaching of trainees, or stimulating their own research ideas. On the other hand, the most common barrier among all respondents was perceived lack of time to do so (85.6%). Other barriers included lack of confidence in one’s ability to analyze and critique medical education literature (38.9%), lack of training (25.6%) or lack of value by one’s institution or for promotion (13.3%). Those who had contributed reviews were less likely to report that article selection or lack of training were barriers  of respondents reported that they read the Journal Club at least a few times a year, and more than one third of respondents 38%) read it most months. There are several examples of journal clubs focused on medical education % of resp, 8–12, b% read itJust as with publications in clinical research, there are variable reasons that members choose to read a particular review. Some are looking to address a particular question based on their own needs in teaching or curriculum development, others are perusing the reviews to keep ‘up to date’ in general, while a minority are using the reviews as a springboard for their own research. A recent study suggested that, analogous to the practitioners of clinical medicine, educators prefer synthesized or mediated reviews rather than accessing the primary literature themselves . The forFor reviewers themselves, a majority listed contributing to COMSEP as a large professional organization as a key benefit of reviewing. Many also reported that the activity helped them develop their skills in critical appraisal of the medical education literature. Both reasons suggest that serving as a reviewer creates a sense of belonging to a community of practice, allowing reviewers to both adopt the language of and contribute to the enterprise of undergraduate medical educators. It is not surprising that those who contribute reviews read Journal Club reviews more frequently than non-contributors, and are also less likely to see issues like article selection and training as barriers to writing. Contributing to the project both as consumers and producers likely indicates movement from peripheral to full participation within the community of practice as described by Cruess et al. .Others have made the connection between journal clubs and communities of practice , 12, desOur study is limited by the response rate and potential biases of those COMSEP members who responded to the Annual Survey and specifically the Journal Club questions; the respondents may reflect those members who are more engaged in COMSEP as an organization and may not reflect the entire membership. Future studies may explore in more detail the way in which engagement with similar formats lead to actual change in educational practice.Brief reviews of recent articles in medical education can be effective in engaging clinician-educators both as readers and as authors. Previous studies have examined the use of the journal club format to disseminate evidence about medical education, but this is the first to describe the use of an asynchronous format for a large membership organization.For others looking to encourage professional development and professional identity formation among a community of medical educators separated both geographically and by institution, the experience of the COMSEP Journal Club might serve as a useful model. The demographics of the respondents suggest that educators in a variety of roles and at different levels of experience find the format engaging.Additional file 1.Additional file 2.


Arabidopsis mutants, we effectively fitted the experimental data to the model. The system-level assessment revealed that genetic defects in both circadian clock components and sugar sensing pathway hindered the appropriate adjustment of the starch degradation rate, particularly under long-day conditions. These findings not only confirmed the previous empirical findings but also provide the novel insights into the role of each gene within the gene regulatory network on the emergence of carbon homeostasis.Starch serves as an important carbon storage mechanism for many plant species, facilitating their adaptation to the cyclic variations in the light environment, including day–night cycles as well as seasonal changes in photoperiod. By dynamically adjusting starch accumulation and degradation rates, plants maintain carbon homeostasis, enabling continuous growth under fluctuating environmental conditions. To understand dynamic nature of starch metabolism at the molecular level, it is necessary to integrate empirical knowledge from genetic defects in specific regulatory pathways into the dynamical system of starch metabolism. To achieve this, we evaluated the impact of genetic defects in the circadian clock, sugar sensing and starch degradation pathways using the carbon homeostasis model that encompasses the interplay between these pathways. Through the collection of starch metabolism data from 10  For photosynthetic organisms like plants, the adjustment of their physiological states to periodic fluctuations of light environments is crucial, as their survival, growth and reproduction rely on the photoassimilates synthesized solely during light period. To adapt to the periodic fluctuations of light environments, many plant species have evolved the ability to store a portion of the photoassimilates as transient starch in leaves during light period and to degrade it into sugars for energy provision during dark period in order to maintain carbon (C) homeostasis ,2. The rbidopsis ,3–5. Whebidopsis ,7.Arabidopsis thaliana with shorter or longer circadian period are unable to properly modulate the rate of starch degradation, resulting in C depletion or excess at the end of night [circadian clock associated/late elongated hypocotyl (cca1/lhy) with a free running period of 17 h [pseudo-response regulator 7/9 (prr7/9) , unravelled that plants with a dysfunctional clock are unable to adjust the starch accumulation rate, but they can still adjust the pace of starch degradation [Previous experimental and theoretical studies have identified the importance of the circadian clock in the regulation of starch metabolism. Clock mutants of of night . For ins of 17 h  exhausts of 17 h . This ap of 17 h . By contf 29.4 h ) delays radation . This meradation .sweet11/12 [PSEUDO RESPONSE REGULATOR7 (PRR7) gene, which is involved in the circadian oscillator and is highly expressed in the morning [A crucial signal conveyed in this complex network is the sugar status. When C availability surpasses the growth capacity, sugar signals are incorporated into the regulation of pacing starch degradation. One example has emerged from the role of the sugar signal trehalose 6-phosphate (T6P) in regulating starch degradation in plants under conditions of excess C availability, such as in the sucrose efflux protein mutant eet11/12 –15. Modueet11/12 ,17. The eet11/12 . Intereseet11/12 . Indeed, morning . These pExperimentally, mutant analyses have been used to investigate the functional roles of specific genes involved in sugar sensing, starch metabolism and the circadian clock. However, to understand the dynamic nature of starch metabolism adjustment, it is necessary to develop a framework capable of assessing the impacts of genetic defects in specific pathways on the overall system-level behaviour of starch metabolism. To achieve this, the use of a mathematical model that formalizes the interplay between sugar sensing, starch metabolism and circadian clock becomes advantageous. Several mathematical models have been proposed to explain the potential mechanism for rapid adjustment of the rate of starch degradation in response to photoperiod . Among tArabidopsis plants and 10 different mutants affecting clock components, sugar sensing pathways and starch-degrading enzymes. By leveraging the experimental data obtained in this study, we fitted the carbon homeostasis model to the data. This allowed us to assess how each parameter value of the model was affected by genetic defects in each regulatory pathway. Through these system-level assessments of the genetic defects on starch metabolism, we demonstrate the significant role of synergistic regulation from the circadian clock and photosynthetic sugars in optimizing the starch degradation process within a fluctuating photoperiodic environment. This framework facilitates the study of starch metabolism dynamics and its regulation by the circadian clock and sugar signalling pathways at a system level, contributing to a deeper understanding of the complex interplay within this biological system.This study aims to evaluate the impact of genetic defects on potential regulatory processes of starch metabolism at the system level. To this aim, we first obtained starch metabolism data using both wild-type . 2. 2.1Arabidopsis plants and the carbon homeostasis model describing the dynamics of starch and sugar in a plant leaf [tC and sucrose tS. Carbon is fixed by photosynthesis at a rate a and partitioned into starch and sucrose during the light period. The partitioning rate to starch is represented as γ, and the increment of starch and sucrose per unit time under light is given as aγ and a(1–γ) respectively (a). The accumulated starch is then broken down at the degradation rate tβ, which is dependent on time of day due to the clock regulation and light–dark cycle of the environment (a).To assess the impacts of genetic defects on starch metabolism, we performed an integrated analysis of the time series data of starch metabolisms in ant leaf –23. In tectively a. The acironment a.FiguretκC. The value of κ depends on the shape of a granule, and we assumed spherical shape, setting κ = 2/3. Using this formulation, the amount of starch broken into sucrose per unit time is expressed as H (a). Overall, the dynamics of starch and sucrose concentration is described as the following set of differential equations:Because starch forms a granule packed in high density within chloroplasts, starch degradation occurs at the surface of the granule. Thus, starch degradation rate is assumed to be proportional to the surface area of the starch granule, which is represented as a rate H a. OveralIn equation (2.1), time scale is standardized with the length of a day as 1. Previous studies have demonstrated that when temporal variations in sucrose is minimized , the diel profile of starch always becomes linear and vice versa.tβ that realizes sucrose homeostasis is predicted to be a nonlinear function of time that has a peak at dawn , as formalized asAs reported in the previous study , the sha at dawn b,c, as fpβ and Lτ. pβ represents the degradation potential, which determines the maximum starch degradation rate at dawn. When pβ is small, starch degradation rate is low , and starch is likely to remain after dawn. Subjective photoperiod Lτ represents the diel cycle of the starch degradation rate. In this model, it is assumed that plants adjust their starch degradation rate so that sucrose concentration becomes constant through a day–night cycle, which leads to the synchronization of the starch degradation profile with the external day–night cycles [Lτ becomes equal to the external one and when the sucrose is depleted or excessed due to the excessive or reduced starch degradation, the Lτ becomes longer or shorter than the external photoperiod, respectively. Therefore, Lτ reflects how accurately the diel profile of starch degradation is synchronized with the external photoperiod. Here, we examine whether such an adjustment is observed in real plants by estimating and comparing the values of Lτ in Arabidopsis plants under short and long photoperiod. Note that Seki et al. [tβ) becomes 0 under light period to minimize the discrepancy from the carbon homeostasis [tβ becomes 0 under light period in extremely short photoperiod. The impact of changes in each parameter value on tβ, tC and tS is illustrated in electronic supplementary material, figure S1.The starch degradation function (equation (2.4)) has two parameters t cycles . When thi et al.  demonstreostasis . Therefo. 2.2A. thaliana (b) under long- and short-day conditions. The plant lines used in this study are listed in Arabidopsis seeds were spread onto 12 cm pots filled with Arabidopsis basic medium composed mainly of white peat, pre-soaked with 0.15% Previcur (fungicide) and 0.10% boron in water and placed at 4°C in the dark for 3 days to synchronize seed germination. After stratification, the pots were transferred into a controlled growth chamber, in short day  or long day  at a temperature of 21/19°C light/dark cycles and relative humidity of 75%. The differences in irradiance used in SD and LD conditions aim to minimize differences in plant developmental stages at the time of the harvest. The whole rosette was harvested 30 days after sowing at ZT0, 1, 2, 4, 8, 12, 15, 16, 20, 23 and 24 for LD and ZT0, 1, 2, 4, 7, 8, 12, 16, 20, 23 and 24 for SD, snapped freeze into liquid nitrogen and stored at −80°C until processing. Pools of three and eleven rosettes were harvested to constitute a biological replicate for LD and SD, respectively. In total, three or five biological replicates were collected for plants growing in SD and LD conditions, respectively. Fifty milligrams of frozen ground rosettes were used for starch and metabolite quantification as described by da Silva et al. [Here we explain the methods to obtain starch metabolism data used to assess the impacts of genetic defects on starch metabolism. We measured maltose, sucrose, and starch from wild-type and 10 mutants of thaliana b under l. 2.3a). Our mutants are involved in starch turnover, sucrose transportation, the circadian clock and the sugar sensing pathway (a). We grouped the 10 mutants into four categories and explain the function of each gene within each group.In order to evaluate the effectiveness of our methodology using the carbon homeostasis model, we specifically chose 10 mutants that are potentially associated with each model parameter based on previous studies a. Our mu pathway a. We gro. 2.3.1gwd, pwd, bam1, bam2 and amy3/bam1. gwd and pwd are the mutants of the starch phosphorylation enzymes, glucan, water dikinase (GWD)[PWD [bam1 and bam2 are the β-amylases that catalyse the hydrolysis of the oligosaccharides, which produces maltose [amy3/bam1 is a double mutant of the α-amylase and β-amylase. AMY3 and BAM1 are mainly localized in guard cells and regulating stomatal opening in response to light environment [The group includes five mutants whose function is associated with the starch degradation: ase (GWD) and PWD GWD)[PWD . These eGWD)[PWD ,32. The GWD)[PWD . bam1 an maltose ,27. The ironment .. 2.3.2sweet11/12 is the mutant of the sucrose transporters SWEET11 and SWEET12 which plays a significant role in the phloem loading of source–sink transportation in plants [sweet11/12 shows significantly higher sucrose concentration in a leaf tissue than wild-type and it does not oscillate between day and night. Because in the carbon homeostasis model, plants are assumed to sense the deviation of sugar concentration rather than the concentration itself to synchronize the starch degradation to the external diel cycle, the phenotype of this mutant, high concentration but relatively small deviation of sugar, is suitable for our analysis, compared with other sucrose transporters such as sucrose-H+ cotransporters (SUCs) [The group includes the mutant associated with the sucrose flux in a leaf. n plants . sweet11s (SUCs) . These gs (SUCs) .. 2.3.3prr7 and prr7/9. The transcription of PRR7 is known to be sensitive to sugar concentration and the main candidate of the sugar-dependent phase modulation of the circadian clock [prr7/9 has extremely longer circadian photoperiod [prr7/9 delays the onset of starch degradation [Lτ and pβ) which reflect the time-dependent profile of starch degradation . Particularly, we can test the contribution of the clock regulation for the adjustment of starch degradation tβ.The group includes the mutants of the sugar-sensitive components of the circadian clock: an clock ,29. The toperiod . It has . 2.3.4kin10 and bzip63-1. KIN10 is a catalytic subunit of Snf1-related protein kinase (SnRK1) and bZIP63 is a sugar-sensitive transcription factor that regulates many pathways including the transcript level of the starch degrading enzymes. These genes are involved in the sugar-dependent phase modulation of the circadian clock, in which SnRK1 activates bZIP63 under starvation and bZIP63 transcriptionally regulates the expression of PRR7 [Lτ This group includes the mutant associated with the sugar sensing pathways particularly involved in interaction between sugars and clock:  of PRR7 ,19. If tPRR7 [τL b,c. 2.4tβ exhibits diel fluctuations with a peak at dawn [tβ can be calculated by dividing maltose concentration by the surface area of starch granule as follows:The previous theoretical study predicted that the starch degradation rate  at dawn . By usintβ is a relative value, the scale of tβ measured in maltose data and that measured in the model setting may not always align. To account for this discrepancy, we standardized both empirical and theoretical estimates so that their maximum values equal 1. The measured concentration of maltose is displayed in electronic supplementary material, figure S2.We compared observed and predicted starch degradation rates by examining a decreasing trend during the light period and an increasing trend during the dark period in a diurnal cycle. As the estimate of . 2.5X = {X1, X2, … , nX} includes four variables at each time point t that range from 1 to n,t and superscript L = LD or SD indicates long- or short-day condition, respectively. Our primary objective is to find the probability density of the model parametersH) depends on photoperiod because previous studies demonstrate that photoperiod affects the export of C from the source leaves [Lτ in LD and SD respectively to examine whether its value is adjusted by photoperiod in Arabidopsis plants.To fit the model to time series data of starch and sucrose, we applied a Bayesian estimation. The time series data e leaves . We alsoP represents the posterior probability distribution of the parameter vector θ, given the model M and observed data X. P represents the likelihood function, and P(X) represents the probability distribution of observed data. P(θ) is the prior probability distribution of the parameters, determined by prior knowledge about the parameter before the experiment is conducted. Bayesian inference based on the Markov chain Monte Carlo (MCMC) algorithm approximates the sampling of the parameter from the target posterior probability distribution P by utilizing the information of P(θ)P. From the samples generated by the MCMC algorithm, we can estimate the posterior probability distribution of the parameters and compute posterior quantities such as the posterior mean, variance and other relevant statistics.The Bayesian framework practically implements the posterior inference of parameters by incorporating prior knowledge through a tailored likelihood function. The Bayesian inference is generally grounded on Bayes' theorem as follows:P, which manifests the probability of observing the actual data, is computed by assuming that measurement noise follows a normal distribution with mean μt and variance σ2. Here, μt is the true concentration of starch and sucrose in a leaf tissue under a specific time and photoperiod, which is derived from the model M with a given set of parameters θ. It is assumed that the variance σ2 differs between starch and sucrose and is denoted as μt is calculated by solving the differential equations defined above numerically using the fourth order Runge–Kutta method. The initial condition was set as C(0) = 1/pβ and S(0) = Laτ/H, which is the steady solution when the starch degradation is perfectly adjusted [μt for a duration equivalent to six days and used the latter three-day portion of it for the model fitting to mitigate the impact of the initial condition.The likelihood adjusted . We calcP(θ). From the biological constraints, we restricted the range of the parameters to be positive, specifically between 0 and 1 for the parameters associated with the ratios. Given P(θ)P, we applied the Hamiltonian Monte Carlo (HMC) method through the use of a probabilistic programming language, Stan (Rstan v. 2.21.3) to obtain samples of parameters drawn from the posterior distribution. We ran four independent Markov chains, initialized at distinct random starting points, with a burn-in phase lasting 1000 iterations, followed by a sampling phase of an additional 1000 iterations. After computing the posterior distribution of each parameter, we assessed the convergence and robustness of the distribution by evaluating the maximum R hat and effective sample size, as recommended by the Stan reference guide. We also computed the point estimate of the parameter value from the posterior samples. We adapted the maximum a posteriori (MAP) estimate as the point estimate. To do this, we first applied kernel density estimation to the posterior samples using the ‘density’ function of R software (v. 4.1.2 (2021-11-01)) and then calculated the MAP estimates as the values corresponding to the maximum density. Our model fitting to the time series data of wild-type was performed independently for 13 batches.As we lack prior information about the specific parameter values, we employed a uniform distribution for . 2.6θmt) is given as the sum of two terms: the estimate for the wild-type (θwt) and the deviation from the wild-type (Δ). Here, we used the posterior estimates for θwt. If the parameter value for the mutant does not deviate significantly from that of the wild-type, the respective element of the vector Δ equals to 0. Given that our model has seven parameters, the number of possible combinations of elements of Δ with zero values is 27 = 128. We treated these combinations as distinct models and select the optimal model by evaluating the widely applicable information criterion (WAIC). After estimating the posterior probability distribution of a mutant through Bayesian estimation for each model, we calculated WAIC using the following formula:jθ is a sampled parameter from the estimated posterior distribution (HMC sample). The first term is a sum of mean likelihood for data iX with parameter sample jθ from posterior distribution. The second term is the sum of the variance of the likelihood. The model with the lowest WAIC was selected as the optimal model.To estimate the parameter values of the model for each mutant, we assume that values of all parameters are not necessarily different between the wild-type and the mutant. Rather, some parameter values are equivalent between the wild-type and the mutant because of conserved genetic background. In this context, we assume that the vector representing the parameter values for the mutant mutant and (Lτ)wt are the estimated values of the subjective photoperiods in the mutants and wild-type, respectively. We numerically calculated the distribution of ΔLτ by extracting Lτ randomly from the MCMC samples of the wild-type and the mutants. The negative sign of the To evaluate the impact of genetic defects on starch metabolism we compared the estimated parameter values between the mutant and the wild-type. We calculated the MAP estimates from MCMC samples for each parameter and for each mutant. Then, from the posterior estimate, we computed log2 foldchange from wild-type for each mutant. 3. 3.1a). The measured levels of starch and sucrose differed from batch to batch, possibly due to subtle differences in experimental condition or material preparation that could not be controlled. The carbon homeostasis model successfully explained these variations, which were mainly reflected as variations in photosynthetic activity (a) and sucrose consumption parameter values (LDH and SDH).To evaluate the impact of genetic defects on potential regulatory processes of starch metabolism at the system level, we first applied the carbon homeostasis model to the wild-type data. Model fitting to the starch and sucrose data of wild-type was overall successful. The prediction of the model depicted more rapid accumulation and slower degradation of starch under SD compared with those under LD, which was consistent with the observations a. The meLτ between LD and SD condition. If starch degradation rate is properly adjusted to realize the C homeostasis, the value of τL should equal to the external photoperiod. We found that the estimated subjective photoperiods under LD (b), suggesting that wild-type plants properly adjusted their starch degradation rates to maintain the C homeostasis. In SD conditions, mean of estimated values for H) exhibited notable differences between LD and SD conditions (b), with higher values observed under LD compared with SD conditions.The ability of the adjustment of starch degradation in response to photoperiod changes in wild-type plants was evaluated by comparing the estimated values of the subjective photoperiod parameter operiods b, suggesnditions b, with htβ of wild-type plants given in equation (2.4). The predicted diel profile of starch degradation rate has a sharp peak at dawn . To avoid over-fitting and to characterize the phenotypes with a small set of parameters, we first applied model selection (see Methods). The analysis of model selection revealed that most of the physiological processes associated with starch metabolism are affected by defects in at least three of the 10 genes examined here .To assess the impacts of the genetic defects on starch metabolism using the carbon homeostasis model, we next performed the model fitting to the data obtained from the mutants potentially associated with each model parameter according to previous studies. The mutants were included in the following four groups: (I) starch-degrading enzymes, (II) sucrose transporter, (III) clock components, and (IV) sugar sensing b. To avopβ which controls the maximum level of the starch degradation rate. It was affected by the defect of most of genes from all groups (a). The starch phosphorylation enzymes gwd and pwd exhibited a marked deviation from the wild-type in pβ, and the starch degradation rate tβ was estimated to be low throughout the day without a peak around dawn observed in the wild-type (b). By contrast, other degradation enzymes, bam1, bam2 and amy3/bam1, have little effects on pβ compared with gwd and pwd (a). It indicates that the impact on starch degradation rate varies even among the starch degrading enzymes. Additionally, in a mutant of sugar-sensitive transcription factor bzip63-1, the starch degradation rate and starch degradation potential (pβ) slightly decreased compared with the wild-type (a). By contrast, prr7, one of the regulatory targets of bZIP63, did not show such a decrease in starch degradation (a).Across seven parameters of the carbon homeostasis model, the most sensitive parameter to the genetic defect was the starch degrading potential l groups a. The stild-type b. By con and pwd a. It indild-type a. By conradation a.sweet11/12 showed excessive starch accumulation and high concentration of sucrose (b). The estimated sucrose consumption rate H was extremely lower than the one in the wild-type in both photoperiods (a), which is in agreement with its molecular function as a sugar transporter that exports sugars from source to sink organs. In addition to the sucrose consumption, starch degradation rate was also estimated to be lower in sweet11/12 (b), which is consistent with the previous experiments, showing that the starch degradation is inhibited by sucrose via T6P signalling [γ) was less sensitive to the genetic defect because only three mutants in the Group I (starch-degrading enzymes) showed diminished values compared with the wild-type . The photosynthesis rate (a) was also less sensitive to the genetic defects (|log2(θmutant /θwt) | < 1.5) (a).The sucrose transporter  sucrose b. The esoperiods a, which eet11/12 b, which gnalling  and by ggnalling . Carbon | < 1.5) a.Figure. 3.3gwd and pwd , which can be explained by the direct role of these enzymes in starch degradation, rather than the regulatory role of the enzyme activity for starch degradation. In contrast to gwd and pwd, other starch degradation enzymes had less effects on the response to photoperiod (figure 5a). Among the other candidate mutants for the regulatory roles in starch degradation, the top two mutants that showed significant deviation from the wild-type were a sugar-sensitive clock component prr7 (−1.4 h) and its transcriptional regulator bzip63-1 (−1.1 h) . These mutants exhibited a subjective photoperiod that was estimated to be more than 1 h shorter  and the circadian clock (prr7) affect the proper adjustment of the starch degradation rate . The desynchronization between the subjective and external photoperiod was predicted only under LD conditions but not for SD conditions. This suggests that the response to SD conditions is more robust and more tightly regulated compared with the one to LD conditions, probably due to the higher requirement for carbon homeostasis under SD conditions, where there is a greater risk of carbon depletion. This robustness to the shorter photoperiods is consistent with the observation that the response of the clock transcript abondance is restricted to the extremely low light condition or low CO2 condition [We also found that loss-of-function mutations in nditions a. The deondition . Furtherondition . In addiondition , which iPRR7 only showed the defects in the subjective photoperiod Lτ, which corresponds to the timing of trough in diel oscillation of starch degradation, whereas it did not show significant changes in the other parameters including pβ, which represents the peak level of starch degradation. This result indicates that the phase modulation of the clock is specific to temporal control of the starch degradation for adjusting the timing of trough and peak of the oscillation pattern to the photoperiod of the surrounding environment. On the other hand, bZIP63, an upstream regulator of PRR7, showed not only the defects in the subjective photoperiod but also the slight decrease of the degradation potential pβ. This may suggest that bZIP63 also regulates the activity of starch-degrading enzymes in different ways as well as the transcriptional regulation of PRR7. This suggestion is in line with an observation that the bzip63 mutant shows reduced activation of starch-degrading enzyme GWD [Our findings highlight the specialized role of the clock-phase modulation in the regulation of starch degradation in response to changes of the photoperiods. The mutant of zyme GWD .Our approach integrating the mathematical model and mutant experiments enabled us to quantify the impacts of genetic defects on the adjustment of starch degradation. The approach can be extended to analyse the data collected in different genetic background and different environmental conditions such as natural environments where photoperiod and light intensity change continuously. Future research may uncover limitations and areas for improvement in this approach. Application of this study to field data would shed new light on the adaptive significance of the feedback regulation of starch metabolism in plants growing in natural conditions.


Accurate localization of retinal holes is essential for successful scleral buckling (SB) surgery. We aimed to verify the feasibility of using ultra-wide-field (UWF) imaging for preoperative estimation of retinal hole location.We observed 21 eyes from 21 patients with rhegmatogenous retinal detachment (RRD) who underwent successful SB. They were treated at the Department of Ophthalmology of the Second Hospital of Hebei Medical University between November 2020 and November 2021. UWF fundus photography using an Optos device was performed at different steering positions 1 day before, 1 day after, and 1 month after SB. Using the preoperative fundus images, we measured the transverse diameter of the optic disc (D1) and the distance from the centre of the retinal holes to the ora serrata (D2). The accurate transverse diameter of the optic disc (Dd) was measured preoperatively using optical coherence tomography. The same surgeon measured the scleral chord lengths intraoperatively from the limbus to the located retinal hole marked on the sclera using an ophthalmic calliper. Statistical software was used to analyze the consistency of scleral chord length between the retinal hole and the limbus, which was estimated by preoperative UWF imaging and was measured using an ophthalmic calliper intraoperatively.There was no statistically significant difference in the scleral chord length between the retinal holes and the limbus, which was estimated by preoperative UWF fundus photography and was measured by the calliper during surgery.It is feasible to locate retinal holes using UWF fundus photography before SB, which is helpful for quick localization, thereby reducing the learning curve of SB surgery. Preoperative ultra-wide-field imaging can provide abundant information about retinal holes and is helpful for assessing their location before surgery.In this prospective cohort study of 21 patients, 25 retinal holes in four quadrants were observed.Axial length and the position of the holes have little impact on preoperative ultra-wide field imaging assessment. Rhegmatogenous retinal detachment (RRD) is the most common type of retinal detachment; blindness may occur in the affected eye unless surgery is performed. The primary surgical methods include scleral buckling (SB), pars plana vitrectomy (PPV), and pneumatic retinopexy (PR) . SB is aAccurate localization of retinal holes during surgery determines the success of SB. Currently, most intraoperative localization methods require an indirect ophthalmoscope to observe the hole. The difficulty in mastering this technique is an important reason for the long learning curve of SB. New SB surgery, which is assisted by chandelier endoillumination, makes it easier for surgeons to observe the fundus and locate holes, but it also requires additional scleral incisions and instruments . AlthougThe Optos ultra-wide-field (UWF) imaging system is the latest generation of fundus examination instruments with non-contact and ultra-wide-field characteristics . SurgeonThis prospective cohort study included patients diagnosed with RRD and treated with SB in the Department of Ophthalmology of the Second Hospital of Hebei Medical University between November 2020 and November 2021. Patients with retinal holes detected by UWF fundus photography were included. The following patients were excluded: those who had undergone or needed cataract surgery and/or vitrectomy; those with PVR C2 or above, giant retinal tears, or dialysis of the ora serrata; and those with RRD where holes could be detected using traditional methods  but not by UWF images. A total of 21 eyes from 21 patients were included. This study adhered to the tenets of the Declaration of Helsinki, and the protocols were approved by the Ethical Committee of The Second Hospital of Hebei Medical University. Written informed consent was obtained from the patients enrolled in the study.Ultra-wide-angle fundus images were taken using an Optos Ultra-Widefield Fundus Camera . These images were taken 1 day preoperatively, 1 week postoperatively, and 1 month postoperatively. Pupils were dilated to the greatest extent before taking images. The images of the central, superior, inferior, nasal, and temporal retina of each eye were taken by the same experienced technician. During the process, patients’ eyelids were lifted by the technician, and they had to try their best to stare in the corresponding directions. Images of the retinal holes were taken in a similar manner. All images were taken when the ‘green in-focus’ signal was obtained on the Optos machine.The images of the inner surface of the eye, which is nearly spherical, cannot be mapped to a flat surface without distortion. The size and shape of some structures can be greatly distorted with further eccentricity in taking images. Therefore, we standardized the images to make them more accurate. We exported images from the Optos and converted them to grayscale in PhotoshopCS3 . Images centred on the posterior portion of the eye were inspected for sharpness and lack of systemic distortion across the image. We selected the best image and considered it to be the best image of the periphery. To standardize the resultant images, histogram stretching was performed. Peripheral images were transformed using elastic deformation, and the posterior portions of peripheral images matched the base reference images. Warped images were merged into a montage . The UWFThe macular centre was considered the centre, and the horizontal position was determined through the connection between the macular centre and the optic disc centre. Based on the location of the hole, the combined standardized fundus image was divided into four regions: the nasal side (Z1), the superior side (Z2), the temporal side (Z3), and the inferior side (Z4) . We measThe optic disc transverse diameter was measured by an optical coherence tomography scanner . Automatic software with automated measurement was used to determine the transverse diameter of the optic disc of each eye. We used the transverse diameter values of the optic disc . A ZeissBased on the measured AL, patients were divided into two groups: AL ≥ 26 mm and AL < 26 mm. The optic disc transverse diameter measured using optical coherence tomography was labelled Dd. The preoperative scleral chord length between the limbus and retinal hole was calculated as follows: D = ral side . HoweverLocating and marking of retinal holes on the sclera were performed by the same experienced surgeon (Duan). The statistics calculated from the images were not shared with the surgeon who performed the surgery. After locating the hole in the surgery, an ophthalmic calliper was used to measure the scleral chord length between the limbus and scleral markers of the retinal holes . Holes wp values < 0.01 were considered statistically significant. Calculation of preoperative scleral chord length and statistical analysis were completed by another researcher (Zhou) who did not participate in data collection.The preoperative estimated scleral chord length was calculated and compared with the measured scleral chord length during surgery. All analyses were performed using SPSS statistical software . Continuous, normally distributed data were expressed as mean ± standard deviation and were compared using a paired t-test. The data and images of 21 eyes of 21 patients  were collected. The average age of the patients was 27.1 ± 8.6 (range: 16–53) years; 25 retinal holes were observed. The average AL of all eyes was 26.32 ± 1.30 (range: 24.29–29.23) mm. The mean preoperative best-corrected visual acuity (BCVA) was 0.36 ± 0.35 LogMAR, ranging from 1.4 to −0.1 LogMAR. All included eyes were phakic .p = 0.445 > 0.01). There were 9 holes in the AL < 26 mm group. The average estimated preoperative scleral chord length was 14.01 ± 1.59 mm, and the average actual intraoperative chord length was 13.89 ± 1.76 mm; the difference between them was 0.12 (range: −1.51–1.66) mm and not significant (p = 0.731 > 0.01). Comparing the partitions, there was no statistically significant difference between the preoperative and intraoperative estimated chord lengths in the Z2 (p = 0.080 > 0.01), Z3 (p = 0.751 > 0.01), and Z4 (p = 0.522 > 0.01) regions. There was only one case that involved the Z1 region, which was Case 9. The estimated preoperative chord length was 16.24 mm, and the actual intraoperative measurement was 15 mm. This data was too small to be analyzed.There were 16 holes in the AL ≥ 26 mm group. The average estimated preoperative scleral chord length was 13.99 ± 1.18 mm, and the average actual intraoperative chord length was 13.84 ± 1.19 mm; the difference between them was 0.14 (range: −0.99–1.28) mm and not significant  . InformaIn our research, 21 patients were included, and 25 retinal holes were observed. The inner surface of the eye is a three-dimensional sphere, which is difficult to map to a two-dimensional plane without distortion. Deviations measured directly from the wide-angle image and the actual data of the eyeball are uncertain . Therefop =   0.445 > 0.01) and the AL < 26 mm group (p  = 0.731 > 0.01). Regarding the different zones of the holes, statistical analyses based on the location of the retinal holes showed that there was no statistically significant difference in the retinal tears in the Z2, Z3, or Z4 regions. However, only one case involved the Z1 region, and the data was insufficient for analysis. We also observed on the postoperative images that all the retinal holes were on the buckle, suggesting the feasibility of locating retinal holes using UWF photography preoperatively.From our results, the average difference between the estimated chord length of retinal tears and the actual measured chord length was 0.13 (range: −1.51–1.66) mm. There was no statistically significant difference between the estimated chord length and the actual measured chord length in the AL ≥ 26 mm group . According to resident training in our study site, our method improves the learning curve of localization of retinal holes skill in SB surgery.In clinical practice, we used a relatively simple method of calculation. We used the Optos software to acquire the optic disc transverse diameter (D1) and the distance between the centre of the retinal hole and the ora serrata (D2). The optic disc diameter was estimated to be 1.5 mm. We then used the formula D = There are few studies on calculations of the retinal hole positions using a UWF image. Ishikawa et al. calculated the distance between the posterior edge of the scleral buckle and cornea on a UWF image after SB and compared it to the distance measured during surgery. A regression equation was obtained based on the data. The position of the retinal hole in the preoperative image was brought into the regression equation, and the results fell within the range of the scleral buckle . Their sHowever, our study has some limitations. First, the error caused by the distortion of the wide-angle image could not be completely avoided. Single-shot imaging of the Optos cannot cover the entire retina. We obtained images of the peripheral retina by guiding the direction of the patient’s gaze. Therefore, even a small deviation may significantly impact the final plane image. Second, the chord length from the ora serrata to the limbus that we used in our study  was based on estimation and may have individual variations. Third, our data was insufficient for nasal position analysis. Last, the patients in our study might not be representative of all RRD populations; thus, generalizability based on different phakic statuses, large retinal tears, and bullous retinal detachment may be limited, which warrants future investigation.There was no statistically significant difference in the scleral chord length between the retinal holes and the limbus estimated by UWF fundus photography used preoperatively and measured intraoperatively. Preoperative analysis using UWF imaging helps surgeons estimate the scleral chord length between retinal holes and the limbus. Thereby, it may help to improve the learning process for SB surgery.Click here for additional data file.