In contrast to C3 plants where starch is mainly degraded by the hydrolytic route, different observations suggested the phosphorolytic route to be a major pathway for starch degradation in CAM plants. To elucidate the interplay and relevant contributions of the phosphorolytic and hydrolytic pathways for starch degradation in CAM, we assessed diel patterns for metabolites and enzymes implicated in both the hydrolytic route  and the phosphorolytic route (starch phosphorylase) of starch degradation in the CAM orchid Phalaenopsis “Edessa.” By comparing the catalytic enzyme activities and starch degradation rates, we showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by β-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways in higher plants and is characterized by high water use efficiency. This mainly relies on major nocturnal CO It is convenient to recognize four distinct phases of gas exchange in CAM plants, which are also used to describe the photosynthetic performance. Sequestration of CO2 at night (Phase I) occurs via the enzyme phosphoenolpyruvate carboxylase (PEPC) with the 3-C substrate phosphoenolpyruvate (PEP) provided by the glycolytic breakdown of carbohydrate. The final 4-C product, malic acid, is stored in a large central vacuole. At the start of the next day, in Phase II, stomata will gradually close and external CO2 is mainly fixed by ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco). Gas exchange is curtailed by stomatal closure during the middle of the day (Phase III), thereby reducing transpirational water losses and improving WUE. During this phase, malic acid exits the vacuole and decarboxylation releases CO2 which is re-fixed by rubisco. In Phase IV stomata open again towards the end of the day and external CO2 is mainly sequestered via rubisco (Crassulacean acid metabolism (CAM) is one of the three photosynthetic pathways present in higher plants and is characterized by an optimized water use efficiency (WUE) by taking up CO rubisco .2 fixation  is able to switch from C3 to CAM under drought or salt stress and has emerged as a useful model for exploring CAM. By shifting to the CAM mode, a considerable export of Glc6P out of the chloroplast was observed whilst maltose export dramatically diminished  from the non-reducing ends of glucan chains. Subsequently, phosphoglucomutase (PGM) catalyses the conversion of Glc1P to Glc6P which is exported to the cytosol via the plastidic Glucose 6-Phosphate: Phosphate Translocator (GPT). GPT transcript abundance has been found to show a > 70-fold upregulation in M. crystallinum upon CAM induction  to yield glucose and a spectrum of larger, linear oligosaccharides which can further be metabolized again by β-amylase. Notwithstanding α-amylase also possesses the capacity to degrade transitory starch, no phenotype has been detected in knockout mutants of Arabidopsis  demonstrated the upregulation of transcript abundances and activities of a range of starch-degrading enzymes implicated in both the hydrolytic and phosphorolytic routes of starch degradation ) following CAM induction  or the phosphorolytic route (starch-phosphorylase) of starch degradation in the CAM orchid Phalaenopsis “Edessa.” These analyses were further complemented with measurements of diel leaf gas exchange and important metabolite dynamics . By comparing in vitro catalytic activities and starch degradation rates we were able to gain more insight about the relative contributions of both pathways and postulated a bottleneck limiting hydrolytic starch conversion.Several studies on the common ice plant  with photosynthetic photon flux density (PPFD) of 100 µmol m-2 s-1. Watering was performed twice a week; once with a nutrient solution Peters 20N-8.7P-16.6K of 1 mS cm-1 and once with water. After six weeks, leaf samples (n = 5) were taken from the upper one-third of young fully expanded source leaves during a cycle of 24 h starting from 08.00 h (ZT0) every 2 h until 08.00 h the next morning (ZT24). The samples from 08.00 h (ZT0 and ZT24) were taken when the lights were turned on whilst the samples taken at 20.00 h (ZT12) were taken in the dark under green safety light.Kalanchoë fedtschenkoi in order to provide comparison with the Phalaenopsis enzyme activity determinations. These plants were also grown in a growth room with a temperature of 25 °C and 19 °C during the light and dark periods respectively, with a relative humidity of 75% and a 12-h photoperiod (zeitgeber time ZT0-ZT12) with photosynthetic photon flux density (PPFD) of ∼250 µmol m-2 s-1. Plants were grown under these conditions for at least 8-10 weeks before sampling at which time all plants had around 10 to 12 leaf pairs.At ZT12 samples were also taken from leaf pair six of the obligate model CAM plant All samples were immediately frozen in liquid nitrogen, powdered and stored at -80°C.2 exchange was measured on the youngest fully expanded leaves, using a LCi Portable Photosynthesis System . The top part of the leaf was enclosed in a broad leaf chamber (6.25 cm2) and the incoming air was passed through a 20-l bottle to buffer short-term fluctuations in the CO2 concentration. After six weeks, gas exchange data were collected over a 24-h period with measurements obtained at 15-min intervals (n = 3).Net COSoluble sugars  were extracted using hot water (80°C), as described by 4. The mixture was allowed to thaw slowly on ice for 30 min. The resulting suspension was then centrifuged at 4°C for 10 min at 16,200 g. The insoluble residue from the perchloric acid extraction was used to determine starch content spectrophotometrically at 340 nm as glucose equivalents , following digestion with a mix of amyloglucosidase (EC 3.2.1.3) and α-amylase (EC 3.2.1.1). The analyses were conducted as earlier described by 4 extraction was neutralized at 4°C with 5 M K2CO3, and the resulting potassium perchlorate precipitate was removed by 10 min centrifugation at 16,200 g and 4°C. Five mg activated charcoal was added to the supernatant, and after 15 min at 4°C, removed by new centrifugation. The supernatant was used for measurement of malic acid, Glc6P, and Glc1P. Malic acid was measured in a 500-µl reaction mixture (EnzytecTM code n° E1215) containing: glycylglycinebuffer, NAD, glutamate oxaloacetate transaminase . Analysis was performed spetrophotometrically by determining the change in absorbance at 340 nm after adding L-malate dehydrogenase . The phosphorylated sugars Glc6P and Glc1P were measured in a 500-µl reaction mixture containing: 100 mM HEPES-KOH, pH 7.6, 4 mM MgCl2, 0.2 mM NADP and 1 unit glucose-6-phosphate dehydrogenase  for Glc6P, then 1 unit glucose phosphate isomerase  and 1 unit PGM (EC 5.4.2.2) for Glc1P  was performed as described by or Glc1P . Analysi2, 1 mM EDTA, 1 mM EGTA, 4.6 mM DTT, 80 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM Triton X-100.All extraction steps were performed by homogenizing leaf material at 4°C in extraction buffer containing: 0.3 M N-(2-hydroxyethyl)piperazine-N′-(2-ethane sulphonic acid) , 20 mM MgCl2, 1.2 mg ml-1 ATP, 1.0 mg ml-1 NAD, and 2 units hexokinase (EC 2.7.1.1). Analysis was performed spectrophotometrically  by determining the change in absorbance at 340 nm after adding 4 units G6PDH.The extraction of β-amylase was based on the method described by 2, 1.2 mg ml-1 ATP, 1.0 mg ml-1 NAD and 2 units hexokinase (EC 2.7.1.1). Analysis was performed spectrophotometrically by determining the change in absorbance at 340 nm after adding 4 units G6PDH.The extraction of maltase (α-glucosidase) and plastidic D-enzyme (4-α-glucanotransferase: DPE1) was based on the methods described by The extraction and assay of cytosolic D-enzyme (DPE2) was based on the method described by -1 soluble potato starch, and 16 mM phosphate mix Na2HPO4/KH2PO4. The reaction was stopped by boiling for 1 min and the supernatant was assayed in a 500 µl reaction mixture: 0.1 M HEPES (pH 7.6), 4 mM MgCl2, and 1.5 mg ml-1 NADP. The resulting Glc1P was determined spectrophotometrically by calculating the change in absorbance at 340 nm after addition of 3 units PGM (EC 5.4.2.2) and 3 units G6PDH.The extraction of starch phosphorylase (α-glucan phosphorylase) was based on the method described by 3. The homogenate was centrifuged for 2 min at 16,200 g. The extract was then desalted by passing twice through a 0.5-ml column of Sephadex G-25, equilibrated with 100 mM Tris-HCl (pH 7.5 at 4°C), 1 mM DTT, 1 mM benzamidine, and 5% (w/v) glycerol. The Ki of PEPC for malic acid was estimated using different malic acid concentrations  in a reaction mix (500 µl) containing: 65 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.2 mM NADH, 10 mM NaHCO3, and 2.5 mM PEP. Production of oxaloacetate by PEPC was coupled to oxidation of NADH by the high endogenous NAD-dependent MDH activity in the extracts. The reaction was initiated by the addition of 50 µl of extract and change in absorbance at 340 nm was measured for 4 min at 25°C. Preliminary experiments confirmed a linear decrease of NADH for during at least 6 min.The extraction and assay of PEPC was based on the method described by Where appropriate, data were analyzed using the statistical software package IBM SPSS Statistics V23. Before carrying out statistical tests, normality of the data was checked by means of the Kolmogorov-Smirnoff statistic (p > 0.05). Means are compared by independent sample t-test (α = 0.05). All replicates considered in our study were independent biological replicates originating from different plants.2 = 0.998) with a mean rate of 67 ± 13 nmol g-1FW min-1, providing 32 ± 7 µmol Glc eq. g-1FW which is equivalent to the provision of 64 µmol g-1FW of PEP. Moving closer to dawn, from ZT20 to ZT24, nocturnal starch degradation was also linear but  proceeded with a significantly lower rate of 38 ± 6 nmol g-1FW min-1 and provided 9 ± 2 µmol Glc eq. g-1FW which is equivalent to 18 µmol g-1FW of PEP. In each of the considered periods, starch degradation provided enough substrate to sustain nocturnal malic acid synthesis  of the dark period whilst a decreasing CO2 assimilation rate was noticed during the final three hours (ZT21 – ZT24) of the dark period   and remained relatively stable during the remainder of the night, until a strong decrease during the last two hours of the dark period (ZT22 –ZT24). A similar diel pattern was observed for Glc1P .The diel pattern of sucrose showed a rather stable diurnal phase followed by a significant (p < 0.05) nocturnal decrease to a minimum value of 3 ± 1 µmol gk levels Figure 2AW (ZT22) Figure 2DW (ZT22) Figure 2Eor Glc1P Figure 2F-1FW min-1 to 48 ± 6 nmol g-1FW min-1 (-1FW min-1 (-1FW min-1 and 19 ± 6 µmol g-1FW min-1 were observed during both day and night. The chloroplastic disproportionating enzyme (DPE1) showed an increasing activity during daytime from 44 ± 16 nmol g-1FW min-1 (ZT0) to 200 ± 28 nmol g-1FW min-1 (ZT10) (-1FW min-1), nocturnal DPE1 activity was quite stable around a mean value of 156 ± 12 nmol g-1FW min-1. For cytosolic disproportionating enzyme (DPE2) high activities were observed during the photoperiod at ZT2 and ZT4, i.e. 124 ± 16 nmol g-1FW min-1 and 112 ± 4 nmol g-1FW min-1 respectively. However, during the remainder of the day and night, the enzyme showed an overall low activity of only 8 ± 4 nmol g-1FW min-1 (-1FW min-1 (-1FW min-1 (ZT12) to a maximum activity of 44 ± 5 nmol g-1FW min-1 (ZT24).In order to gain more insight into the nocturnal starch degradation process, total extractable activities of a range of enzymes implicated in starch degradation were measured over the complete diel cycle Figure 3.FW min-1 Figure 3AFW min-1 Figure 3AFW min-1 Figure 3B1 (ZT10) Figure 3CFW min-1 Figure 3DFW min-1 Figure 3Ein vitro activities of the enzymes involved in starch degradation  were considered relative to the upper and lower limits of the observed starch degradation rate i.e. 67 ± 13 nmol g-1FW min-1 from ZT12-ZT20 and 38 ± 6 nmol g-1FW min-1 from ZT20-ZT24. Registered minimum, maximum, and mean activities were all considered for each enzyme in comparison with the calculated nocturnal starch degradation rate and symbols were used to indicate whether enzymatic capacity was theoretically sufficient (√) or insufficient (X) to meet the observed starch degradation rates  and a positive control CAM species such as K. fedtschenkoi . The data clearly indicated that starch phosphorylase activity in the model CAM plant K. fedtschenkoi could also easily accommodate the observed starch degradation whilst DPE2 + maltase fell short. These observations strongly contradicted with Arabidopsis showing a completely different situation with high DPE2 activity, supporting starch hydrolysis, and insufficient starch phosphorylase activity. Moreover, high DPE2 and low starch phosphorylase activities have also been reported by M. crystallinum (in the CAM mode) under identical growth conditions.To strengthen our study, we further compared our mean values of enzyme activities from Phalaenopsis “Edessa” we considered two starch degradation rates in our study i.e. (1) 67 ± 13 nmol g-1FW min-1 from ZT12 to ZT20 and (2) 38 ± 6 nmol g-1FW min-1 from ZT20 to ZT24 . This is consistent with an important role for starch phosphorylase in starch degradation, since Glc6P is the major export product from phosphorolytic starch degradation. Glc6P is not only considered an allosteric activator of PEPC but can also easily be converted to PEP in the cytosol providing ATP by substrate-level phosphorylation and offering energetic advantages  or maltase. DPE2 showed only very low nocturnal activities in Phalaenopsis and could theoretically meet only about 15% of the observed starch breakdown. Low DPE2 activities have also been reported in M. crystallinum and have previously been hypothesized to potentially represent a possible bottleneck in the hydrolytic starch degradation process  than required to account for the observed nocturnal starch degradation  to 47 ± 13 nmol g-1FW (ZT22) in Phalaenopsis leaves.The other starch degrading enzymes residing in the chloroplast and belonging to the hydrolytic starch conversion pathway, i.e. β-amylase and DPE1, showed  2 to 80 Table 1.  process . This isradation Table 2. radation Table 2, radation Table 1. limiting Table 2. in vivo nocturnal starch degradation and in vitro enzyme activity measurements during the whole night in leaves of the CAM orchid Phalaenopsis “Edessa” to shed more light into the interplay of the phosphorolytic and hydrolytic processes of starch degradation in CAM. We showed that the phosphorolytic pathway is the major route to accommodate nocturnal starch degradation and that measured activities of starch phosphorylase perfectly matched calculated starch degradation rates in order to avoid premature exhaustion of starch reserves before dawn. The hydrolytic pathway seemed hampered in starch processing not by β-amylase but through insufficient catalytic capacity of both DPE2 and maltase. These considerations were further corroborated by measurements of enzyme activities in the CAM model plant Kalanchoë fedtschenkoi and strongly contradict with the situation in the C3 plant Arabidopsis. The data support the view that the phosphorolytic pathway might be the main route of starch degradation in CAM to provide substrate for PEP with additional hydrolytic starch breakdown to accommodate mainly sucrose synthesis.In conclusion we compared All datasets generated for this study are included in the article/supplementary material.NC, WV and JC proposed the conceptual framework for the study and performed the data collection and analysis. NC and MF performed the experimental analyses. NC and JC interpreted the data and wrote the manuscript.This research was supported by the Research Fund KU Leuven.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.


This viewpoint celebrates the accomplishments of the Journal of Medical Internet Research (JMIR) on its twentieth anniversary and reviews accomplishments around research publications, journal innovation, and supporting people. Reflecting on the twentieth anniversary of the Journal of Medical Internet Research (JMIR), “form follows function” also well explains the success and impact of the journal in promoting high quality research, supporting innovation in publication, and supporting a generation of young investigators.In the 20 years JMIR has represented the field of digital health, the nature of digital health research has evolved and transformed. Today’s papers often focus on machine learning, virtual reality, smartphone apps, and other topics that were nonexistent or nascent in health care research in 1999 when the journal began, or in 2001 when the journal outlined a clear definition for electronic health  , and onlHowever, JMIR is more than high quality research. The interdisciplinary nature, rapid paradigm shifts, and global impact of digital health requires a journal to adopt a unique form to meet its function in this space. For 20 years, JMIR has been rethinking how a journal can support the digital mental health community with leading efforts in open access publication, preprints, an easy to access Web and mobile layout, social media outreach and dissemination, crowdfunding, and offering peer reviewers publication credits, among others. The journal’s adaptable online format can support research from not only the health fields but also engineering, computer sciences, design, implementation, policy, and other unique disciplines. Its unique functioning has created a form that offers the ability to publish unique special issues  on topicEven beyond high quality research and innovative publishing models, JMIR’s legacy of 20 years includes supporting people. The journal has forged a reputation for offering patients a voice in the medical world ,7. It alReflecting on the twentieth anniversary of JMIR, it is clear that “form follows function” only begins to describe the innovation and impact of the journal. From leading efforts in research, innovation in publication, and investment in people, the field has grown because of JMIR and its support for digital health. While the format of JMIR will continue to adapt to lead the field, its function as a leading force will remain constant and it will continue to act as a beacon of certainty in the always evolving world of digital health.


Added sugar intake was assessed by a food frequency questionnaire, and arterial stiffness measures included pulse wave velocity (PWV) and augmentation index. Separate multivariate linear regression models were used to evaluate the association between added sugar and arterial stiffness. Separate interaction terms were included to test for effect modification by body mass index (BMI) z-score and physical activity (PA). Overall, there was no association between added sugar and arterial stiffness (P > 0.05); however, the association between added sugar and arterial stiffness differed by BMI z-score (P for interaction = 0.003). For participants with lower BMI z-scores, added sugar intake was positively associated with PWV trunk measurements, whereas there was no association for those who had a higher BMI z-score. PA did not significantly modify the association between added sugar and arterial stiffness. Further research is needed to determine the longitudinal relationship and to confirm that obesity differentially affects this association.The relationship between added sugar and arterial stiffness in youth with type 1 diabetes (T1D) has not been well-described. We used data from the SEARCH for Diabetes in Youth Study (SEARCH), an ongoing observational cohort study, to determine the association between added sugar and arterial stiffness in individuals diagnosed with T1D <20 years of age ( Current recommendations from the Dietary Guidelines for Americans and the World Health Organization advise individuals to consume less than 10% of their total caloric intake from added sugars . HoweverArterial stiffness is a measure of subclinical CVD and has been associated with markers of atherosclerosis, stroke and coronary heart disease, and mortality in adult populations ,16,17,18We aimed to determine the association between added sugar and arterial stiffness, which is an independent predictor of CVD events and measures subclinical disease  in indivData for this study were derived from the SEARCH Study and the SEARCH Nutritional Ancillary Study. SEARCH ascertained cases of diabetes among individuals diagnosed before the age of 20 years from 2002 onward. A detailed description of SEARCH study methods has been published elsewhere . BrieflyAt baseline, 12-, and 60-month follow-up, and outcome study visits, dietary intake was obtained via a food frequency questionnaire (FFQ) for youth aged 10 years and older; the majority of youth completed the FFQ without assistance after receiving staff instruction . The detAt the SEARCH Outcome visit conducted between 2011 and 2015, noninvasive measures of arterial stiffness, including pulse wave velocity (PWV) and augmentation index (AIx), were ascertained using a SphygmoCor-Vx device and tonometer. Measurements were obtained in a stable room temperature after 10 min of rest. The utility and details of the arterial stiffness measures in youth including their reproducibility and validity are described elsewhere ,33,34,35www.cdc.gov/healthyyouth/data/yrbs/index.htm), and individuals self-reported the number of days per week they participated in physical activity that made them breathe hard or sweat for at least 20 minutes.At each study visit, questionnaires were used to obtain demographic  and diabetes-related information . Fasting blood samples were obtained under conditions of metabolic stability, defined as no episode of diabetic ketoacidosis during the previous month. Samples were analyzed for glycated hemoglobin (HbA1c), lipid measurements, and glutamic acid decarboxylase-65 (GAD65) and insulinoma-associated-2 (IA-2) diabetes autoantibodies. A Hitachi 917 autoanalyzer  was used for assays of plasma cholesterol, triglyceride, and HDL cholesterol. The Friedewald equation was used to calculate LDL cholesterol if triglyceride concentration was <400 mg/dL (4.52 mM/L) and by the Beta Quantification procedure if triglyceride was ≥400 mg/dL (Hainline). Height, weight, waist circumference, and blood pressure (BP) were measured according to standardized protocol by trained and certified staff. Body mass index (BMI) was calculated as weight (kg)/height squared (m²) and converted to an age and gender-specific BMI z-score . Insulinn = 1539). Of these eligible individuals, 1517 had PWV trunk data, 1470 had PWV leg data, and 1277 had Aix-75 data, which comprised our sample sizes for cross-sectional analyses.We included individuals with T1D , plus a positive diabetes autoantibody test result (GAD65 or IA-2) who had completed the FFQ at the outcome visit (n = 553), we had at least two or more measurements of added sugar from the FFQs at the baseline visit and/or follow-up visits. Additional analyses were performed on this subsample to examine the relationship between longer-term estimates of added sugar intake and the outcomes.In addition to the cross-sectional sample at the outcome visit, for some participants  . DescripP < 0.1). For significant interactions, we conducted post hoc tests to examine the effect of added sugars on the outcomes at selected levels of the potential effect modifier. As part of sensitivity analyses, we also examined the interaction of added sugar with HbA1c.Because threshold effects were not evident, separate, multiple linear regression was used to obtain regression (β) coefficients representing the cross-sectional associations between continuous added sugar intake and arterial stiffness. Outcome variables with skewed distributions were log-transformed to improve normality (PWV trunk). For PWV outcomes, Model 1 was adjusted for heart rate, mean arterial pressure, and calories. For Aix-75, Model 1 was adjusted for height, mean arterial pressure, and calories. For all outcomes, Model 2 adjusted for Model 1 covariates, demographic  variables, and diabetes-related variables (diabetes duration and insulin regimen). Model 3 adjusted for Model 2 covariates and overall diet quality using the HEI-2015 excluding the added sugar component. Given evidence ,41 that In addition to cross-sectional associations, it was of interest to understand how long-term added sugar intake is associated with arterial stiffness. For the participants with multiple measurements of added sugar, we constructed a longitudinally assessed summary of average sugar intake . To compute the time-weighted average, we used a stepwise approximation, assuming added sugar intakes were constant for half the time interval before and half the time interval after the visit an intake was reported . We fit Our sample consisted of youth with T1D who were predominately non-Hispanic White with a mean age of 18 years (17.7 ± 4.1 years) and had diabetes for 8 years (7.9 ± 1.9 years) at the outcome visit. Mean added sugar intake was 47.0 g/d (interquartile range (IQR) = 26.9–66.4 g/d), and accounted for 12.4% of total calories consumed. Added sugar intake primarily came from sweets and desserts (18.4 ± 16.8 g/d) and sugar-sweetened beverages (17.1 ± 27.4 g/d). Other notable food sources were meal replacement/sports bars (4.1 ± 6.7 g/d), yogurt (3.0 ± 3.9 g/d), and low-fiber grain products (2.2 ± 2.2 g/d).Demographics and clinical characteristics of the study population at the outcome visit by quartile of added sugar intake are shown in The multivariate associations between added sugar intake and arterial stiffness measurements after adjustment for covariates are shown in P for interaction = 0.003). Post hoc tests revealed that at higher BMI z-scores (BMI z-score = 2), there was no association between added sugar intake and PWV-trunk ; however, for lower BMI z-scores, there was a significant, positive association . To facilitate interpretation on the scale of the original variables, model-predicted log-PWV trunk values were back-transformed at various levels of added sugar intake and displayed by BMI z-score (P for interaction ≥ 0.10). Additional analyses exploring potential effect modifications by glycemic control by including an interaction of HbA1c with added sugar was not significant (P for interaction ≥ 0.10).Given our interest in examining the potentially heterogeneous effects of diet on cardiovascular disease, we examined whether BMI z-score and physical activity modified the relationship between added sugar intake and arterial stiffness measures using our full sample. Multivariate analysis adjusting for potential confounders revealed that the association between added sugar intake and log-PWV trunk was modified by BMI z-score ( z-score . As showP for interaction = 0.001), but not other measures of arterial stiffness. Long-term physical activity did not modify the association between long-term added sugar intake and arterial stiffness.In the subsample of participants with multiple measurements of added sugar, parallel multivariate analysis revealed similar findings. Long-term added sugar intake was not associated with any measure of arterial stiffness in either the minimally adjusted or fully adjusted models (results not shown). Long-term BMI modified the association between long-term added sugar intake and log-PWV trunk , which observed that added sugar intake accounted for 16% of total calories in the adolescent (12–19 years) diet . Our datIn our study, the association between added sugar and arterial stiffness was nuanced. There was no association overall between added sugar and arterial stiffness in unadjusted or adjusted models. However, because research suggests that diet may have a heterogeneous effect on CVD risk ,45,46,47Although BMI z-score differentially affected the association between added sugar and PWV trunk, the effect was not observed across all measures of arterial stiffness in our analyses. BMI z-score did not modify the relationship of added sugar with PWV-leg or AIx. The differential associations between added sugar intake and the arterial stiffness measures may be expected given that PWV and AIx are not interchangeable measurements, and obesity may have a greater impact on central arterial stiffness measures than peripheral measures or on Aix , which mPhysical activity is shown to have a cardioprotective effect  and is aThere are some limitations to our study. Arterial stiffness was only measured at one point in time, precluding a longitudinal analysis. The cross-sectional analysis allows associations to be identified, but not causality or directionality. However, we did examine how long-term added sugar measurements affected arterial stiffness and we observed similar results. SEARCH is an ongoing study, and future analysis will allow a thorough longitudinal examination of the relationship between changes in added sugar intake and changes in arterial stiffness. Other limitations of the study include the use of a food frequency questionnaire, which was validated but relies on retrospective self-recall and is prone to error. In addition, while individuals with diabetes have higher levels of arterial stiffness, it is possible that, given the age of the participants, the impact of diabetes on arterial stiffness was not severe enough to allow us to detect an association; however, we did use continuous measures of both added sugar and arterial stiffness to increase the power to detect an association. We also used BMI z-score as a measure of adiposity in these analyses. While other measures may more accurately capture adiposity, BMI is recommended for large epidemiologic studies and adequately correlates with total body fat . FinallyOur study also has several important strengths including a large, well-characterized multiethnic sample of youth with T1D who are understudied yet a medically vulnerable population. To our knowledge, this study is the first to assess the relation of added sugar to arterial stiffness in youth with diabetes, and we include multiple measures of arterial stiffness. PWV-trunk measurements predict cardiovascular events above and beyond measurements of traditional cardiovascular risk factors, measure subclinical cardiovascular disease , and proIn conclusion, our findings indicate that youth with T1D consume similar levels of added sugar to youth in the general U.S. population, which underscores the need to improve dietary adherence in this at-risk population. Further, our study suggests a nuanced relationship between added sugar and CVD risk. Youth with T1D who are lean or less physically active may benefit from a reduction in added sugar consumption. Future studies should explore factors that differentially affect the role of added sugar on CVD and confirm that obesity and physical activity modify this association.


Journal club holds a well-respected place in medical education by promoting critical review of the literature and fostering scholarly discussions. Journal clubs are often not available to trainees with niche interests due to the geographic limitations of subspecialty programs such as simulation, medical education, disaster medicine, ultrasound, global health, and women’s health.A recurring online journal club was held on a quarterly basis to connect simulation fellows. An online conferencing program with screen-sharing capabilities served as the platform for this scholarly exchange. Articles were presented by fellows supported by more seasoned mentors. We surveyed participants to evaluate the program and provide feedback to the presenter.The first eight sessions drew participants from across the United States and Canada. The program was highly rated by participants who commented specifically on its value. Presenters were also highly rated, suggesting that fellows, with online support and mentoring, were effective in providing a quality program.Online synchronous journal clubs can fill an educational niche for subspecialists and their trainees, as demonstrated with this curriculum piloted with simulation fellows. Challenges of scheduling across time zones, distribution of materials, and recruitment of participants can be overcome by a dedicated team of facilitators aided by readily accessible technology. The practice of journal clubs at academic medical centers began over 100 years ago. Since then, journal clubs have grown to hold a well-respected role in continuing medical education.3After residency training, some trainees will continue to build a niche through fellowship training. The educational innovation described in this report was piloted with simulation fellows. Simulation fellows dedicate their fellowship training to learning the theory behind and practical strategies to implement successful simulation-based medical education and research. Due to the niche of simulation training, fellows are often isolated from like-minded scholars. Local interaction with others in simulation may be limited due to low numbers of faculty trained in simulation, and few passionate simulation experts at their institution. A baseline needs assessment from participants demonstrated that 73% of the participants had, on average, two or less communications per month with simulation colleagues at other centers, and 55% reported they did not have access to a local simulation journal club.One might extrapolate that a similar need exists in other subspecialties such as medical education, disaster medicine, ultrasound, women’s health, and global health, to name a few. Furthermore, medical students and other trainees may attend institutions where local experts do not exist. For example, a medical student may have an interest in global health but attend a school that does not have a global health program or faculty with this expertise. A synchronous online journal club, as described below, can provide exposure and networking opportunities not locally available. Program goals include exposing simulation fellows to advances in simulation-based education and research outside of their locality and clinical specialty, improving knowledge and critical appraisal of current research, and increasing communication and collaboration among simulation fellows and professionals at different geographic sites.Through regular participation in this activity, learners will be able to do the following: 1) Identify advances and new trends in their field, occurring outside of their locality; 2) demonstrate critical appraisal of simulation literature; and 3) exhibit increased scholarly communication with colleagues at distant sites.In-person journal clubs typically begin with a brief presentation of the article followed by an analytical discussion of methods and how to interpret the results and conclusions. This same structure was followed for the synchronous online journal club. A different simulation fellow presented at each session. He or she chose the article and prepared PowerPoint slides to visually aid the presentation. Presenters connected with a more experienced mentor to help screen appropriate articles and serve as a resource for the trainee preparing the presentation. The article chosen by the presenter was announced to the participants by e-mail 1–2 weeks prior to the live session. The full citation and a link to the article were often included. Unless the article was open access or freely available, the PDF was not included. The other participants in the session were other fellows and scholarly-minded simulation educators/researchers not in a fellowship role.Presenters were provided with a slide template and encouraged to present using a standardized format. The first 20 minutes consisted of a factual presentation of relevant background, methods, results, and the author’s conclusions. The next 30 minutes was used for discussion. Presenters were encouraged to have specific questions and discussion topics to help facilitate and guide the discussion. In lieu of a traditional in-person meeting, the conversation was through an online conferencing program, thus eliminating geographic barriers. This recurring program met quarterly.What do we already know about this issue?Journal club is a common educational activity that fosters critical literature appraisal. Subspecialty fellows may not have local access to this opportunity.What was the research question?Can an online journal club connect geographically distant trainees for discussion of subspecialty literature?What was the major finding of the study?An eight-session pilot with simulation fellows had 83 learner encounters. It was feasible and well received.How does this improve population health?Online journal club may be applied to other subspecialties to complement education, provide presentation opportunities, and increase networking.An online conferencing system was used to facilitate the online discussion and allow the presenter to share his or her screen. Several similar platforms exist; some are free while others require an initiation fee for the presenter. A synchronous online journal club could use any platform that allows online group chat, video and audio streaming, and screen sharing. Many institutions have paid subscriptions to one of these services. GoToMeeting was used for the first eight sessions based on availability of an institutional subscription. Zoom and Google Hangouts have also been used with equal success. None of these platforms incur any cost for the attendee. Sessions can be easily recorded with these platforms. However, we decided not to record the journal club discussions, as we felt participants could have been more hesitant to speak and share their analysis and opinion if they were being recorded.A few ground rules were reviewed prior to each session . The rulDue to the nature of the problem this educational innovation solves, the learner group may not be immediately available or easy to contact. Therefore, some advertising and recruitment of learners is necessary. A network of simulation fellows and scholarly professionals were invited to participate by using a multiplatform approach. Advertisement strategies included directly e-mailing fellowship program directors, posting to discussion boards, and announcing events on social media . Fellowship directors were targeted as the initial person of contact as they have more visible and stable contact information. Fellows and trainees frequently have changing contact information due to institutional changes during their early career. Senior faculty were also invited to serve as faculty mentors.In an effort to enhance the educational value of this initiative, the participants were given a program evaluation survey after each session. Additional data collected included demographics and educational opportunities available locally. Participants were also asked to provide feedback to the presenter. Initial data was collected using an online survey tool through the learning management system at the Winter Institute for Simulation, Education, and Research  .The synchronous online journal club is an educational innovation connecting simulation fellows across geographic barriers. This electronic education activity allows scholarly exchange using existing technology that is cheap and readily available. The broad geographic and subspecialty participation demonstrates the easy accessibility of this format. The initial eight sessions were hosted by WISER. In this article, we focus on presenting data from these eight sessions, which ran from October 2014 to June 2016. Average attendance at these sessions was 10, with a range of 6–15.Participants were from various clinical specialties including emergency medicine, anesthesia, surgery, pediatrics, biomedical engineering, critical care, internal medicine, nursing, and pharmacy. The majority of respondents identified as simulation fellows, with a minority identified as researchers, administrators, or simulation center directors. All participants had a particular interest in simulation. The geographic spread included participants from California, Washington State, Massachusetts, New York, Pennsylvania, Florida, Texas, Ohio, Michigan, Illinois, North Carolina, Washington DC, and Canada. Participants were asked to rate the overall journal club  and provThis innovation broadened the exposure of fellows to journal club, as many participants did not have this available locally. Participation in journal club has been shown to increase scholarly reading behavior.am might work great for those in Eastern Standard Time (EST), but this translates to 5 am in Pacific Standard Time. When scheduling journal club events, the leader of the program needs to choose a time that is reasonable to the greatest audience. Through experimentation and informal feedback, we found that 2 pm EST worked the best for participants across U.S. time zones. It is late enough in the day for those on the West Coast to attend after working a late evening shift the night before, but still during the “normal office hours” for those on the East Coast.While this program provides great value, there are some challenges to creating a synchronous online journal club. As a live video conference, audience participation is somewhat limited by times zones. For example, 8 The distribution of articles can pose a challenge. Due to copyright concerns, we only distributed citations and links to PubMed. Most participants are able to access the articles for no cost, as they belong to academic institutions with subscriptions to most journals. However, this must be recognized as a potential limitation. Another potential concern is the recruitment of presenters. While any academically minded simulation educators or researchers are welcome to attend the sessions and contribute to the conversation, fellows are identified as presenters. There is generally a steady stream of fellows willing to present. This pool of fellows is refreshed every year or two, as they graduate and are replaced by new trainees. For the fellows, presenting during journal club is an opportunity to increase their visibility in front of a diverse audience while providing a platform for networking and allowing for honing of presentation skills. The journal club also provides publicity for their training program.Several limitations of this study need to be addressed. In the first eight sessions, we had a total of 83 learner encounters. We collected 32 survey responses, for a response rate of 39%. Feedback forms were completely anonymous; thus, because attendance records did not allow for us to track the percentage of repeat attendees, they could not be excluded from the data. The feedback form was locally developed, and no formal validation occurred. The qualitative comments shared in this article are descriptive and representative of the feedback received; however, no formal thematic analysis was performed and was beyond the scope of this project.Now that the program has been established, it began to be supported by the Society for Simulation in Healthcare (SSH) as an affinity group in 2018. This allows for easier communication to schedule and promote upcoming events through a common website and discussion board. Anyone can create a free account, as it does not require membership in SSH. Current feedback is collected using Google Forms. Dates are now announced annually, at the beginning of the academic year to allow ample time for schedule requests.In conclusion, the educational experience of subspecialty trainees can be enhanced by using low-cost, existing technology to connect peer learners and passionate experts with an online journal club. Niche training programs, such as simulation fellowships, are not ubiquitous and therefore geographically dispersed. This synchronous journal club was trialed using simulation fellows, and would likely have success in other subspecialties that face similar challenges.


Endometrial Cancer (EC) is an important cause of death in women worldwide. Despite early diagnosis and optimal treatment of localized disease, relapsed patients have few therapeutic options because after first line therapy, currently no standard of care exists. On the basis of endocrine positivity of most endometrioid ECs, Endocrine Therapy (ET) is a reasonable and widely accepted option. Better knowledge of molecular mechanisms involved in cancer highlighted the deregulated activity of Cyclin-Dependent Kinases (CDKs) in the cell cycle as a hallmark of carcinogenesis supporting the development of a new class of drugs: CDK inhibitors (CDKis). The aim of this review is to give an overview on CDKis preclinical, early clinical activity and future development in EC. Use of CDKis has a strong preclinical rationale but we have poor clinical data. Similar to breast cancer, most ongoing trials are investigating synergistic associations between CDKis and ET. These trials will probably help in defining the best clinical setting of CDKis in ECs, which are the best partner drugs, and how to manage CDKis toxicities with a focus on potential biomarkers of response. In 2018, endometrial cancers (ECs) accounted for about 63.230 new cases and 11.350 deaths in women in the United States . GeneticEC is usually diagnosed at an early-stage, with vaginal bleeding being the most frequent clinical manifestation . PatientSeveral endocrine therapies have been developed, among which progestins, Selective ER Modulators (SERMs), Selective ER Degraders (SERDs) and Aromatase Inhibitors (AIs) are used in clinical practice ,19.As underlined before, prognosis of advanced ECs is still poor. Treatment of advanced, relapsed and metastatic patients is still an unmet clinical need, and the role of ET in ECs has to be elucidated. Moreover, EC patients usually have several comorbidities, being obesity and diabetes important risk factors . All theThe aim of this review is to describe the rationale behind CDKis development, CDKis data of activity in ECs and future perspectives for development of these drugs. In this review, we highlight EC molecular and pathological classifications that play a role in defining prognosis but will probably also drive therapeutic choices in the near future , focusing on frequently altered pathways which may be relevant for CDKis treatment. Moreover, we discuss the general state of the art on CDKis, their preclinical activity and future perspective in ECs. EC has been historically classified into type I and type II on the basis of clinical characteristics and histological features ,26. TypeType II EC accounts for about 30% of all ECs. It is predominantly serous, clear cell or grade 3 endometrioid, with low or absence of ERs and PgRs. It is associated with endometrial atrophy and has poor prognosis with 5-year OS of 59% . Type IIThis model has some limits, because it is difficult for some tumours to be histologically classified, and some mutations recur in both types of EC. This classification has been recently enriched and will probably be replaced by The Cancer Genome Atlas (TCGA). POLE-ultramutated malignancies, accounting for 17.4% of high-grade endometrioid tumours, are characterized by a high mutational burden with somatic mutations in the exonuclease domain of POLE which encodes the catalytic subunit of polymerase epsilon. Despite the histological grade, this group is associated with good prognosis ,30,31.MSI-hypermutated tumours represent 28.6% of low-grade and 54.3% of high-grade endometrioid EC. They are characterized by MSI and a high mutation rate; they are related to alteration of Mismatch Repair (MMR) system  ,28,32.Copy number low subgroup is typically composed by low-grade endometrioid tumours with low mutation rate and microsatellite stability (MSS). PTEN and PIK3 are mutated in 77% and 90% samples, respectively. This subgroup is characterized by high expression of ER and PgR ,28.Copy number-high serous-like tumours have serous or mixed histology. They have a low mutation rate and a small load of copy number aberrations. TP53 is commonly mutated (91.7%). They show HER2-amplification and deregulation of the cell cycle (in particular with CCNE1 overexpression ). PrognoIt identifies 4 subgroups on the basis of copy number alteration, mutations and Microsatellite Instability (MSI): POLE-ultra-mutate, MSI-hyper-mutated (MSI-H), copy-number low and copy-number high subgroup ,28:POLE-The cell cycle is the set of phases in which DesoxyriboNucleicAcid (DNA) and cellular components double and divide into daughter cells . The celThe cell cycle is carefully regulated in each phase by cyclins, CDKs, and CDKis ,36. CDKsAt present we know that the CDK family is composed of 20 molecules , while 1Lastly, M checkpoint is where the attachment of the spindle fibers to centromeres is assessed. Only if this is correct mitosis will proceed . All theFor an overview of the cell cycle and the most important Cyclin-CDK complexes, see The cell cycle consists of four phases  regulated by cyclins, Cdks, and Cdkis. G1/S phase transition is controlled by Cdk4/6-cyclin D and Cdk2-cyclin E complexes. During G1 phase, Cdk4/6 and cyclin D form a complex that phosphorylates Rb causing the dissociation of E2F factor from Rb, thus inducing DNA transcription and S phase progression in association with the CDK2-cyclin E complex. During S phase, DNA is duplicated. During G2-phase, Cdk2-cyclin A and Cdk1-cyclin B complexes activate the expression of FoxM1 target genes that are essential for transition to M-phase. Cdk1 activity is controlled by the balance between WEE and Cdc25 phosphatases. Also, Cdk7 has an important regulatory role across different cell-cycle phases. Cdk7 phosphorylates and activates other Cdks, in combination with Mat1 and Cyclin H.First generation : characterized by low potency (pan-CDK), lack of specificity and off target toxic effects;Second generation : characterized by a non-selective inhibition of a wide range of CDKs  and equiThird generation : characterized by selectivity for both a subset of CDKs and tumor cells compared to untransformed cells, as reported below;Fourth generation : characterized by higher potency than first generation and by a broad spectrum of activity against other pathways such as angiogenesis. These compounds are under investigation in preclinical and clinical settings.Four generations of CDKis have been currently developed :First geFirst generation CDKis, in particular alvociclib, are characterized by an important cytotoxic effect in vitro and in vivo, both in tumour and normal cells, with acute tumour lyses as the main adverse event (AE) in chronic lymphocytic leukemia (CLL) . MyelosuIn second generation CDKis, the most common G3 and G4 AEs registered in patients treated with dinaciclib  were neuFor a summary of CDKis generations and features see The setting in which CDKis have been clinically developed is HR-positive BC. In HR-positive BC, ER activates the promoter of CCND1, a gene encoding for cyclin D. Indeed, cyclin D is amplified or overexpressed in 20% and 50% of cases, respectively, while mutations of RB are rare ,52. On tIndeed ER-positive cell lines were more sensitive to palbociclib than Triple negative BC . MoreoveThird generation CDKis  have been tested in large phase III trials in BC patients and have been recently approved in clinical practice .PALBOCICLIB: Palbociclib is a selective inhibitor of CDK4 and CDK6 . The cheIn combination with fulvestrant, for the treatment of HR-positive, HER2-negative advanced or metastatic BC that progressed after ET on the basis of MONARCH 2 ;As monotherapy for patients with HR-positive, HER2-negative advanced or metastatic BC that progressed after ET and previous chemotherapy;In combination with an AI as first-line ET for postmenopausal women with HR-positive HER2-negative advanced or metastatic BC on the basis of MONARCH 3 .ABEMACICLIB: Abemaciclib is a selective inhibitor of the complexes CDK4/cyclin D1 and CDK6/cyclin D1 . The cheRIBOCICLIB: Ribociclib is a small molecule that selectively inhibits CDK4 and CDK6 . The cheNowadays, we have no predictors of response to CDKis in BC with the exception of ER positivity . Indeed,The cell cycle and its checkpoints play an important role in endometrial proliferation and cancer. Indeed, a high expression of cyclin A, cyclin D and cyclin E is frequent in EC. Moreover, estrogens and progesterone show opposite activity in the cell cycle as described below. This explains the rationale for the development of CDKis above all in type 1 EC.EC shares with BC several features, suggesting that CDKis could be exploited in this malignancy. Indeed, EC expresses HR; ET is a therapeutic option and it shows a deregulation of the CDK pathway . Cyclin On the other hand, serous carcinoma of the uterus (USC) is an interesting setting for CDKis. It accounts for only 10% of all endometrial cancers but has a poor prognosis and few therapeutic options . Almost ECs patients were enrolled in phase I dose escalation studies of the main CDK 4/6 inhibitors, reporting a manageable safety profile ,85.Palbociclib toxicity profile was evaluated in a phase I trial that enrolled 41 patients. Dose Limiting Toxicity (DLTs) was neutropenia (12%) with Maximum tolerated Dose (MTD) and Recommended Phase II Dose (RP2D) of 125 mg once daily. Among patients evaluated for tumour response, 27% had stable disease with 6 patients receiving at least 10 cycles .CCND1 compared with patients who received ribociclib for <8 weeks and three of the four patients (75%) who had the longest clinical benefit were cyclin D amplified [Infante and colleagues evaluated the safety of ribociclib in 132 patients with different solid tumours and lymphomas expressing RB. MTD was established at 900 mg/day while RP2D was 600 mg/day in a 3 weeks on/one week off schedule. The most frequent G3-G4 Adverse Events (AEs) were neutropenia (27%), leukopenia (17%), while asymptomatic corrected QT prolongation was specific to doses ≥600 mg/day  . Initialmplified .Abemaciclib shows a manageable profile of toxicity in a phase I trial with expansion cohorts that enrolled 225 patients. The only DLT was fatigue . The MTDAs for other CDKis, seliciclib, an orally bioavailable inhibitor of several complexes , has been evaluated in a phase I trial . MTD wasOngoing clinical trials are evaluating CDKis activity in ECs. Exploiting the crosstalk between the two pathways downstream, HR and CDKis in EC, CDKis can be used to potentiate activity of ET. Indeed, combinations of AI with one of three main CDK4/6 inhibitors are under development  in phase II trials. NCT03643510 is evaluating activity of fulvestrant plus abemaciclib in metastatic EC patients that could have received only one prior line of ET and prior chemotherapy. NCT02657928 recruits both ECs and Ovarian Cancer patients receiving ribociclib and letrozole. A translational explorative analysis of this study is searching potential predictive biomarkers and is evaluating response in PDXs. NCT02730429 is a double blind placebo controlled phase II trial that evaluates activity of letrozole with or without palbociclib in patients that could have received no more than one prior ET. Interestingly, an inclusion criterion is that the tumour must be ER positive (with immunohistochemistry ≥10%). On the other hand, even more powerful combinations are under study, for example, the triplet of CDKis, ET and PI3K-inhibitors . These combinations are also under development in BC. Indeed, preclinical evidence suggests that endocrine resistance is mediated, at least in part, by interactions of ER with CDK4, resulting in PI3K hyperactivation . MonitorLastly, despite interesting preclinical activity of CDKis in cyclin E amplified USC, nowadays we have no ongoing study in this specific setting.For a list of the main ongoing clinical trials developing CDKis in ECs, see Metastatic or recurrent ECs are still an important cause of morbidity and death in women worldwide and despite important efforts and new drugs, median OS has not significantly changed over the last years. In this context, better knowledge of molecular alterations led us understand that cell cycle checkpoint deregulation plays an important role in carcinogenesis and could represent an interesting strategy in different settings. On this basis, new-generation CDKis, with a specific target and a more manageable toxicity profile, have demonstrated outstanding activity in BC , prolonging PFS in HR-positive BC patients. This paved the way for the employment of this class of drugs in other malignancies.What role will CDKis play in ECs?What are the best partner drugs?How can we select patients that could benefit from CDKis?Are toxicities manageable in these patients?In EC, ET is an option in pretreated patients, above all in Type I EC that is characterized by strong HR-positivity, being a safe treatment in a setting of poor therapeutic options and in patients that usually have several comorbidities . NeverthAlthough different combinations are under development, doublet with an AI and a CDKi has the strongest preclinical rationale on the basis of cyclin D1 activity, which facilitates ER transcriptional activity, inducing ER-related gene expression. On the other hand, activation of cyclin D1 is downstream of the ER pathway ,80. MoreAs described before, nowadays we have no clear predictor of response to CDKis with the exception of ER positivity in BC. Unfortunately, in ECs, ERs and PgRs, although having a role as prognostic factors, are not clear predictors of response to ET ,93,94. MDose limiting toxicities have been the major concern of first and second generation CDKis. A growing experience in management of CDK4/6 inhibitors in BC patients demonstrates that these are long-term well tolerated drugs in this setting with transient hematologic toxicities or diarrhea that recover with dose reduction . UnfortuHowever, several questions are still open:Use of CDKis in ECs has a strong preclinical rationale and preclinical data on their activity are interesting. Moreover, EC intrinsic endocrine sensitivity, and its frequent deregulation of cell cycle suggest that CDKis could play a role in these patients, enhancing activity of ET. On the other hand, we have few clinical data, and no phase III trial is ongoing in this setting. Ongoing studies are evaluating the activity of CDKis in EC, focusing above all on combinations with AIs. Optimizing ET without adding unacceptable toxicities is the main goal of future CDKis development, but several questions are still open. Firstly, although ET and CDKis seem to be the most interesting combination, preclinical data on CDKis in CCNE1-expressing USC and on CDKis activity on TME suggest that CDKis development could be extended to MSI-Hypermutated EC in combination with immunotherapy and USC, but clinical trials are scarce. Secondly, we have few studies evaluating biomarkers of activity in EC and development of CDKis could be an opportunity to evaluate biomarkers of response in homogeneous populations. Thirdly, toxicities of CDKis in BC suggest that they have manageable side effects in patients that have several comorbidities, but further data are needed for EC patients.In conclusion, ongoing trials will probably define the best settings and partner drugs of CDKis and will clarify AEs of CDKis in EC patients and how to manage these AEs.


Purpose. to report malignant glaucoma and infectious crystalline keratopathy as complications after an uneventful Descemet Membrane Endothelial Keratoplasty (DMEK), and corneal clearance despite graft detachment after the surgery in a patient with pseudophakic bullous keratopathy. Method. A 81-year-old patient with high Intraocular Pressure (IOP) and flat anterior chamber with patent iridotomies after DMEK was diagnosed of malignant glaucoma. The medical approach being insufficient, the patient required a pars-plana vitrectomy, capsulo-hyaloidectomy, and surgical iridectomy. Results. The IOP was reduced and anterior chamber was repositioned after surgical management. Corneal clearance was observed despite graft detachment. The patient developed an infectious crystalline keratopathy after the resolution of malignant glaucoma. Conclusions. malignant glaucoma is a rare complication following DMEK. Corneal clearance can be attained despite graft detachment after DMEK probably due to an unintentional Descemet Membrane Endothelial Transfer (DMET). However, in low dosage, steroid treatment remains a risk factor for developing ICK. Abbreviations: PBK = Pseudophakic Bullous Keratopathy, DMEK = Descemet Membrane Endothelial Keratoplasty, DMET = Descemet Membrane Endothelial Transfer, IOP = Intraocular Pressure, BCVA = Best Corrected Visual Acuity, AC = Anterior Chamber, MG = Malignant Glaucoma, ICK = Infectious Crystalline Keratopathy  Nevertheless, DMEK is not without complications [2], graft detachment being the most common complication. Either air or expandable gas is used both in the straightforward surgery and in the “re-bubbling” procedure to treat graft detachment. The main cause of raised Intraocular Pressure (IOP) in the early postoperative period is usually a reverse pupillary block secondary to a full air or gas fill that pushes the iris backwards .Pseudophakic Bullous Keratopathy (PBK) is a common indication of corneal transplantation. Descemet Membrane Endothelial Keratoplasty (DMEK) is increasingly performed due to its excellent results, low graft rejection rate and reduced need of steroids . A careful differential diagnosis of MG should be undergone. Pupillary block presents a moderate depth of the central AC and iris bombée. Choroidal detachments and effusions are typically hypotonic. Suprachoroidal haemorrhage usually presents severe pain and is often preceded by hypotony [5]. Medical approach of MG is recommended during the first five days using mydriatic and cycloplegic agents in order to relax the ciliary muscle and retracting the lens-iris diaphragm. This can also be achieved by dehydrating the vitreous using hyperosmotics. Aqueous suppressants are used to decrease posterior pooling of aqueous humour. In refractory cases, Nd:YAG anterior hyaloidotomy should be considered in pseudophakic and aphakic eyes, allowing movement of fluid between the posterior and anterior segments of the eye. If ineffective, pars plana vitrectomy with capsulo-hyaloidectomy and lensectomy should be considered [5]. MG is defined as a uniform flattening of the AC in an eye with raised IOP in the presence of a patent iridotomy. It is thought that the cause is a misdirection of the aqueous humour towards the vitreous cavity, shifting the lens-iris diaphragm forward. MG is described as a complication of most intraocular surgeries, especially glaucoma surgery in patients with prior angle closure, being more frequent in hyperopic eyes . In the case we presented, we hypothesized that the irido-corneal contact, in the context of MG after DMEK, caused an almost fully detached graft, hence, the procedure ended up being an unintentional DMET. Interestingly, the corneal oedema improved in the following months. Several cases of corneal oedema resolution in presence of DMEK graft detachment, or even descemetorhexis with no posterior graft transplant have been reported. Dirisamer et al. proposed that contact between donor and host cornea is required to achieve corneal clearance as they reported successful results in patients with partial or large detachments, but not in eyes with a “free-floating graft roll” in the anterior chamber . Our case matched these findings, as the graft was not completely detached. Descemet Membrane Endothelial Transfer (DMET) is performed by injecting the donor graft consisting in Descemet Membrane and endothelium into the anterior chamber after descemetorhexis, without fully unfolding it, but with some contact area between graft and host cornea [8]. Shah et al. reported an increase in endothelial population despite the extraction of the graft and in descematorrhexis-alone procedures, suggesting a possible mechanism of host endothelial cell migration [9]. Dirisamer also had similar results and hypothesized that the recipient endothelium must be primarily involved due to the difference in clinical outcome of DMET in PBK and FED. Hence, DMET is ineffective in eyes with total deficiency of endothelial cells (PBK), yet if performed after descematorrhexis in patients with a pathologically altered subcellular matrix (FED) results in re-endothelialization and corneal clearance [9]. However, in our patient with severe PBK the corneal oedema improvement was evident despite the graft being detached. Although the mechanism is uncertain, we reason that DMET might have contributed to recover partially the corneal transparency [6].Birbal et al. reported unsuccessful long-term results in eight patients receiving DMET. They experienced corneal clearance in eyes with Fuchs endothelial dystrophy (FED) but not PBK, suggesting that the mechanism of DMET might be to stimulate host endothelial migratory response rather than to supply functional cells .One year after the surgery, our patient developed ICK, probably because of the combination of topical corticosteroids and bandage contact lens, as ICK typically occurs after an epithelial defect from a surgical procedure and is potentiated by localized immunosuppression, commonly steroids. Many pathogens can cause ICK although Although rare, malignant glaucoma can be a complication after DMEK surgery, and we must suspect it in postoperative patients with high IOP and flat AC in the presence of a patent iridotomy. Although steroid dosage is usually lower after DMEK compared to other keratoplasty techniques, it remains a risk factor for the growth of stromal biofilm that could ultimately lead to ICK, especially if bandage contact lens is worn.DisclosuresPatient informed consent was obtained.Conflict of interestConflicts of interest and source of funding: none.AcknowledgementsNone.


International Brazilian Journal of Urology. It will complete 45 years of uninterrupted publication of our magazine. This is a landmark, since Int Braz J Urol is a journal always supported by the Brazilian Society of Urology, without the aid of any other publishing company. Also, this is an open access journal that has never charged the authors for any publishing fee.This is the first issue of 2019, a special year for In the end of the current year, my second and last term as Editor in Chief will end, according to the Internal Regiment of SBU. We will begin the process of selecting a new Editor, who will edit Int Braz J Urol with me during the second semester and will assume the position in January, 2020.It has been an honor and a pleasure to edit this Journal, along with an excellent group of Associated Editors, with renowned and participative Consulting Members, and a high quality technical group for support. In 2018, we received 762 papers from Brazil and several parts of the Word. Our 1,232 reviewers evaluated and accepted 169 articles. Many authors of good papers could not publish their work at Int Braz J Urol due to limited space, and we thank them all for choosing our Journal to publicize their research.The reviewers deserve a special attention. They are the soul of any scientific journal, since it is a voluntary and most of the times anonymous job, almost exclusively performed in the name of the art of Science and scientific publication. To all of them, my sincere acknowledgment.As in every year, Int Braz J Urol announces the most effective reviewers. Evaluation is based on the number of evaluated papers, number of accepted invitations for revision, time to review and quality of revision. In 2018, these are the six more effective reviewers: Victor Espinheira Santos, MD, ; Sarica Kemal, MD, ; Giovani Marchini, MD, Divisão de Urologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, SP, Brazil); Aley Talans, MD, ; Victor Tonso, MD, . Trushar Patel, MD,  was elected the best reviewer of the videos section.The former “Jornal Brasileiro de Urologia” was founded in 1974 as a scientific periodic were the Brazilian urologists could publish their work. Over the years, it became an International Journal, changed its name, was indexed in the main international database and gained the World, maintaining its Brazilian characteristics. I am sure that every Brazilian urologist must be proud of these 45 years!


The 44th annual Great American Smokeout will occur on November 21, 2019.The American Cancer Society’s Great American Smokeout is an annual event that encourages smokers to make a plan to quit smoking  and the National Cancer Institute’s smokefree.gov (https://smokefree.gov/) offer additional resources.Smoking remains the leading preventable cause of disease, disability, and death in the United States (


Starch can represent 70–80% of the cereals grains (on a dry matter basis) used for livestock feeding. Several methods have been developed to estimate the feed starch contents of energy feed sources. However, the efficiency of these methods to evaluate the starch content in other feed sources, as well as other types of samples used to evaluate starch availability in the gastrointestinal tract, such as digesta and faeces, remains unclear. Furthermore, most of the currently used starch analysis methods have not been effectively evaluated, being only applied to samples of sporadic experiments, without a wide-ranging validation of the procedures and results. Here, we propose a modification of a method for analysing the starch content in different organic matrices normally evaluated in ruminant nutrition studies. The evaluated organic matrices were: soybean meal, soybean hull, Tifton 85 Bermuda grass hay, abomasal digesta, and faeces.•The modified method is more feasible than the original procedures.•The modified method estimates the starch contents in different organic matrices with accuracy and precision. Specification TableThe experiment was carried out in the Department of Animal Science at the Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The care and handling of the experimental animals followed protocols that were approved by the Institutional Animal Care and Use Committee of the Universidade Federal de Viçosa (protocol number 32/2018).1Weigh 200 mg (air-dried basis) of sample, previously grounded to pass through a 2-mm screen sieve in a knife mill, into a 20-mL screw-cap test tube;2Add 10 mL of distilled water and 10 mL of the buffer solution ;3Add 67 units of amyloglucosidase (1 mg of enzyme) and 1 drop of toluene;4Tightly cap the tube, gently shake it and incubate at 39 °C for 2 h in shaking water bath;5Transfer 1 mL of the starch hydrolisate solution and 4 mL of the trichloroacetic acid solution  into a 10-mL centrifuge tube, then vortex briefly;6Keep tube at room temperature for 5 min and then centrifuge at 6000 rpm for 10 min;7o-toluidine solution  and 400 μL of TCA to the supernatant solution in a separate test tube. Then, cap and incubate it at 100 °C in a water bath for 10 min;Add 4 mL of 8Remove the tube from the water bath and place it in an ice bath for 5 min;9Read absorbance at 630 nm.This study proposes several modifications to the starch analysis method proposed by Zinn , which hCompromising points regarding these analysis procedures and method accuracy were properly studied and will be further discussed.A starch recovery test was performed using five different matrices: soybean meal, soybean hulls, Tifton 85 hay, and cattle abomasal digesta and faeces. Samples of abomasal digesta and faeces were collected from one Nellore bull (330 kg of body weight) fed a Tifton 85 grass hay-based diet, and oven dried (55 °C). All materials were ground to pass through a 1-mm screen sieve . Five levels of soluble starch  were added over each matrix: 0, 20, 40, 60, and 80% (as-is basis). For the calculations, the amount of starch added was corrected according to its moisture content (10%). Starch analyses were performed in triplicate for each matrix/starch level.ij is the observed starch content of the ith level of starch inclusion in the jth replicate; β0 is the intercept, which represents the basal content of starch in the matrix; β1 is the slope, which corresponds to the recovery of added starch; Xi is the level of starch inclusion; and e(i)j is the random error assumed to have a normal distribution [e(i)j ∼ N ].Results for each matrix were evaluated using a simple linear regression model of the measured (dependent variable) over actual (independent variable) starch added, according to the following model:Model (1) was evaluated using the following hypotheses:P <  0.05.The acceptance of the null hypothesis described in Eq. After the evaluation of modified method on the different organic matrices, a comparative evaluation was performed where six samples were analysed following the original Zinn  method aInitially, in our laboratory, several samples of feeds, faeces, and digesta were analysed using the original method . HoweverFirst, the sample amount was increased to 250 mg (air-dried basis) to improve the absorbance reading. In the evaluations routinely performed in our laboratory, 200-mg samples were not enough to assure an adequate absorbance with a reliable signal-to-noise ratio. Additionally, all samples were ground to pass through a 1-mm screen sieve to increase specific surface area for more effective enzymatic action, which is expected to improve the precision of results.Steps 2–4 were restructured to optimise the amyloglucosidase action. First, 10 mL of distilled water was added into the test tubes, as described in the original method. Nonetheless, 0.5 mL of a thermostable α-amylase solution  was also added into the tubes. Then, the tubes were incubated at 90 °C for 2 h in a water bath and subsequently ice bathed for 10 min. The inclusion of this step aimed to establish a partial process of starch hydrolysis, which prevents its gelatinisation , was choRegarding step 5 of the original method, the 4 mL of TCA solution used for protein precipitation was replaced with 2 mL of 15% zinc sulphate solution , which io-toluidine solution  was used for glucose quantification, since the original method does not specify a reagent for this procedure [o-toluidine solution (4 mL) was added to 400 μL of filtrate, according to the original method [o-toluidine solution was added to this diluted solution.The P ≥  0.67, P >  0.99). Thus, the modified method estimated the starch added to the evaluated organic matrices with accuracy. Similarly, the relative standard deviations for the starch content had a small range (from 2.2–5.3%; All organic matrices had a complete recovery of added starch when the modified method was performed (o-toluidine addition. Such a pattern should indicate a high concentration of the analyte in the solution, which was outlined by the additional dilution with distillate water. Actually, as analyte concentration increases, the intermolecular distances in a given sample solution will decrease, eventually reaching a point at which neighbouring molecules mutually affect the charge distribution of the other. This perturbation may significantly affect the ability of the analyte to capture photons of a given wavelength; that is, it may alter analyte absorptivity [The shapes of the glucose standard curves obtained by both methods were quite different to each other . While trptivity . This wirptivity ,9. This rptivity  standardGeneral, our arguments are supported by the estimates of starch contents in maize and sorghum grain samples . The conFor all evaluated samples the precision of the starch content estimates was higher for the modified method . Such imIn summary, the modified method has estimated the starch content in different organic matrices with accuracy, precision, and feasibility. Therefore, this modified method might be recommended for the evaluation of the starch contents of feeds and different materials obtained in digestion trials with ruminants, such as abomasum digesta and faeces.


The preparation of highly porous magnetic crosslinked aggregates (pm-CLEA) of porcine pancreas lipase (PPL) is reported. Some strategies to improve the volumetric activity of the immobilized biocatalyst were evaluated, such as treatment of PPL with enzyme surface-modifying agents , co-aggregation with protein co-feeders , use of silica magnetic nanoparticles functionalized with amino groups (SMNPs) as separation aid, and starch as pore-making agent. The combination of enzyme surface modification with dodecyl aldehyde, co-aggregation with SMNPs and soy protein, in the presence of 0.8% starch (followed by hydrolysis of the starch with α-amylase), yielded CLEAs expressing high activity (immobilization yield around 100% and recovered activity around 80%), high effectiveness factor  and high stability at 40 °C and pH 8.0, i.e., PPL CLEAs co-aggregated with SMNPs/bovine serum albumin or SMNPs/soy protein retained 80% and 50% activity after 10 h incubation, respectively, while free PPL was fully inactivated after 2 h. Besides, highly porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches. Lipases  are enzymes whose natural function is the hydrolysis of triglycerides at the water-lipid interface releasing free fatty acids, diglycerides, monoglycerides and glycerol ,3,4. HowHowever, the use of enzymes in their soluble form for large-scale industrial processes is not very attractive, because of their high production cost and low operational stability . A stratThere are a great number of techniques and supports suitable for enzyme immobilization. In general, some criteria are followed in their choice regarding to high activity of the immobilized enzyme, high stability against temperature and organic solvents, low cost of immobilization, and low toxicity of the immobilization reagents and supports ,23,24,25In the case of lipases, a popular technique that has been widely reported is their immobilization by hydrophobic adsorption on highly hydrophobic surfaces ,30,31,32−1) as co-feeder, followed by crosslinking with 1% w/w glutaraldehyde, reaching 75% catalytic retention. Ramos et al. [The immobilization of enzymes as CLEAs is a simple technique. In a first step, the enzyme is aggregated and precipitated by a precipitant , and in a second step the aggregates are crosslinked with bifunctional  or multifunctional  agents usually via the amino groups on the enzyme surface ,42,43,44s et al.  reportedw/v) yielded 100% catalytic potential, demonstrating the feasibility of this CLEA preparation method. Recently, Cui et al. [An important drawback to be considered in the use of CLEAs is the mass transfer limitation within their highly compact supramolecular structure . Some woi et al.  reportedOther problem of CLEAs is the difficulty of their handling , problemIn this context, this work evaluated some strategies to prepare porous magnetic CLEAs of PPL, aiming to produce biocatalysts with decreased mass transfer limitation, ease of recovery without separation by centrifugation, and increased thermal and mechanical stabilities. A set of experiments was carried out to choose the protein co-feeders, mass ratio between magnetic nanoparticles and protein co-feeder, concentration of starch as pore making agent, and modification of the enzyme surface using polyethyleneimine or hydrophobic polyaldehydes. For the best PPL CLEAs, their activities and thermal stabilities, performance in the tributyrin hydrolysis, and reusability were characterized.Bovine serum albumin (BSA) and soy protein (SP) were evaluated as co-feeders, and silica magnetic nanoparticles functionalized with amino groups (SMNPs) were employed to produce magnetic CLEAs. CLEAs prepared with SMMPs also showed an increase in the recovered activity (8.4 times), with no significant decrease in the immobilization yield (from 65% to 63%). However, a combination of protein co-feeders and SMNPs (mass ratio of 1:1) showed to be the best strategy, particularly in the case of BSA/SMMPs, which yielded CLEAs with immobilization yield around 70% and recovered activity of 25.43% (around 17 times higher than that in the absence of co-feeders (where recovered activity was only 1.46%)). Besides the improvement in the recovered activity, these CLEAs have magnetic properties, which allow their easy recovery applying an external magnetic field .Several studies have showed that CLEAs co-aggregated with protein co-feeders (BSA or SP) exhibit better performance regarding to enzyme leaching during washing steps and reduced mass transfer limitations during the reaction, mainly using macromolecular substrates ,45,54,63N-terminal domain (residues from 1 to 336) contains the catalytic triad , and the C-terminal domain (residues from 337 to 449) is involved in the colipase binding, a small protein (~10 kDa) which anchors the lipase to the water-lipid interface [N- and C-terminal domains . This property of the N-terminal domain to move independently from the lid (Cys238–Cys262 segment), colipase, and the C-terminal domain may have functional implications as the lipase molecule binds the water-lipid interface [240 and Lys81, respectively). The involvement of these Lys residues in covalent links with glutaraldehyde may negatively affect the enzyme activity.As the combination of BSA and SMNPs  did provide better results in terms of recovered activity and immobilization yield, other BSA/SMNPs mass ratios were also evaluated. nterface . This linterface . BesidesThus, the mass ratios enzyme/(BSA + SMNPs) of 1:3 and BSA/SMNPs of 1:1 were chosen for further set of experiments.N- and C-terminal domains) has 22 Lys residues and the amino terminal, while chain B (colipase) has 4 Lys residues (both number of Lys residues was obtained from the primary sequence of the lipase-colipase complex—PDB code 1ETH). Thus, particularly the low modification of amino groups by dodecyl aldehyde does not compromise the crosslinking of the enzyme with glutaraldehyde in the CLEA preparation because of the free primary amino groups still available in the enzyme surface. Despite that, the modified lipases were more stable than the non-modified LPP (The modification of the PPL surface with polyethyleneimine (PEI) and dodecyl aldehyde did lead to a loss of around 30% of the activity, where the specific activities (U/mg protein) were 27.54 ± 0.67, 19.44 ± 0.53 and 19.63 ± 0.18, for the unmodified enzyme, PEI or aldehyde treated enzyme, respectively. fied LPP . ParticuThe CLEAs prepared with PPL treated with PEI yielded the lowest recovered activity (7.17%), probably due to the formation of more compact structures promoted by the intense crosslinking. Besides, the cationic character of this polymer could form a hydrophilic layer on the enzyme surface, impairing substrate partition from the bulk to the active site. On the other hand, the treatment of PPL surface with dodecyl aldehyde  allowed a recovered activity of around 20%, because this compound probably did created a hydrophobic layer on the enzyme surface, aiding in the aggregation step, even although the amino groups in the enzyme surface will be decreased for the crosslinking step. As the combination of both treatments did allow a small increase in the recovered activity, only the surface treatment with dodecyl aldehyde was chosen for further experiments.w/v), but only the concentrations of 0.8 and 1% (w/v) yielded 100% immobilization. The recovered activity increased for all starch concentrations, reaching maximum catalytic retention of 67% (more than doubling of the activity recovered without starch) when 0.8% starch was used during CLEA preparation. Thus, 0.8% (w/v) of starch was selected for the preparation of CLEAs of PPL.The influence of the starch concentration on the immobilization yield and recovered activity of CLEAs of PPL is shown in Saccharomyces cerevisiae and found that the use of starch led to the formation of a non-uniform porous surface, resulting in better mass transfer and minimization of diffusion problems. Besides that, they concluded that 0.4% (w/v) starch concentration yielded 100% catalytic potential, demonstrating the feasibility of this enzyme aggregate preparation method.Talekar et al.  reportedThe influence of the glutaraldehyde treatment time of the PPL CLEAs on the recovered activity was analyzed, and results pointed that this has not influence on the recovered activities, which were statistically equal by the Tukey test around 66%). The incubation of the PPL CLEAs in phosphate buffer at high ionic strength for 12 h showed high stability against leaching (80 to 90% recovered activity). Despite this, 15 h crosslinking time was chosen for further assays [%. The inw/v) as pore-making agent is shown in The influence of the presence of protein feeders  and silica magnetic nanoparticles (SMNPs) in the preparation of PPL CLEAs using starch  is shown in At 60 °C, pm-BSA-CLEA and pm-SP-CLEA presented specific activities of 28.0 ± 0.6 U/mg protein and 20.0 ± 0.8 U/mg protein, respectively, while free PPL showed an activity of 2.0 ± 0.1 U/mg protein. Again, at these conditions the immobilized enzyme becomes much more active than the free enzyme. Differences increase at higher temperature, e.g., at 70 °C the free enzyme is fully inactive while pm-BSA-CLEA retained more than 20% of the activity. These results show that the pm-BSA-CLEA presented greater stability in its structure under very drastic conditions than that of the free PPL since the immobilization produced a strong rigidification of the enzymatic structure.Thus, the new biocatalyst presented better catalytic performance under drastic conditions than the free enzyme and is a new example on how the recovered activity may be altered depending on the activity determination conditions .CLEAs prepared in presence of starch as pore-making agent were evaluated as their effectiveness factors. On the other hand, images of CLEAs after starch hydrolysis show non-uniform and porous surface due to removal of the starch molecules from the structure of the enzyme aggregate, which could explain the higher effectiveness factors (around η = 0.65), mainly for CLEAs prepared in presence of soy protein and SMNPs. On the other hand, PPL co-aggregated with BSA and SMNPs (pm-BSA-CLEAs) did not show significant increase in the observed activity, even using starch as pore-making agent. These results showed that the combination of SMNPs and soy protein as aids in the aggregation step and starch as pore-making agent was a good strategy to reduce mass transfer problems within the supramolecular structure of the CLEA.It should be considered that free enzyme was quite unstable just at 3 °C more , while ttert-butyl alcohol, tributyrin, polyethyleneimine , dodecyl aldehyde, and Bradford reagent were purchased from Sigma-Aldrich . Glutaraldehyde solution (25% in H2O) was purchased from Vetec Química Fina . Soluble starch was purchased from Panreac Química . Anhydrous ethanol (99.8% P.A.) was purchased from Synth . Soy protein was acquired from local market. Silica magnetic nanoparticles (SMNPs) functionalized with amine groups derived from 3-aminopropyltriethoxysilane (APTS) (136 ± 10 µmol amino/g) were purchased from Kopp Technologies .Lipase from porcine pancreas type II (PPL), bovine serum albumin (BSA), −1) and co-feeders (15 mg mL−1) prepared in 5.0 mM sodium phosphate buffer (pH 7.0). The mixture was maintained at 4 °C in an orbital shaker at 150 rpm stirring for 30 min. After this time, glutaraldehyde  was added and the crosslinking step proceeded for 2.5 h [i is the initial activity; ADerivative is the activity of the CLEAs, Asuper is the activity of the supernatant and Awashes is the activity of the washing supernatants.In the precipitation step, ethanol (3.0 mL) was added to 1.0 mL of a homogeneous mixture containing PPL (5.0 mg of protein mLor 2.5 h . CLEAs p−1), protein feeder/additive (7.5 mg of BSA or soy protein and 7.5 mg of SMNPs), and soluble starch  prepared in 5.0 mM sodium phosphate buffer (pH 7.0). The mixture was maintained at 4 °C in an orbital shaker stirred at 150 rpm for 30 min. After, glutaraldehyde  was added and the crosslinking step proceeded for 2.5 h. The precipitate was recovered by magnetic separation, washed, and resuspended in 3.0 mL of 5 mM phosphate buffer (pH 7.0). A volume of 100 μL α-amylase was added and the suspension was incubated at 25 °C for 2 h in order to hydrolyze starch into dextrins, maltose and glucose, which can be easily washed away [Porous magnetic CLEAs (pm-CLEAs) were prepared by the general procedure described above but adding to the enzyme solution SMNPs and soluble starch as pore forming agent. In the precipitation step, 3.0 mL of ethanol were added to 1 mL of a homogeneous mixture containing PPL  and/or dodecyl aldehyde.−1) in a homogeneous PPL solution (5 mg mL−1) prepared in 5 mM phosphate buffer . The reaction medium was incubated at 25 °C and stirred at 150 rpm for 60 min. At the end, the enzyme solution was dialyzed in a dialysis tubing cellulose membrane  at 4 °C for 16 h against excess of water to remove excess of surface modifying agent.The treatment with PEI was performed according to Wilson et al. , adding −1) were added to 30 mL of a PPL solution (5 mg mL−1) prepared in 100 mM sodium carbonate buffer (pH 10.0) to give a dodecyl aldehyde:PPL mass ratio of 1:1. The solution was incubated at 25 °C under 150 rpm stirring for 180 min. In this case, both non-modified PPL and PPL treated with PEI were modified with dodecyl aldehyde. After, sodium borohydride (1 mg mL-1 solution) was added to the solution and the reaction proceeded for 30 min. At the end, the enzyme solution was dialyzed at 4 °C for 16 h against excess of water to remove excess of surface modifying and reducing agents.For the treatment with dodecyl aldehyde, 181 μL of this aldehyde solution (831 mg mLv/v) TNBS containing modified and non-modified PPL (0.01 mg mL−1) were prepared in 100 mM sodium borate pH 9.0 and incubated at 25 °C for 30 min. After, the absorbance was measured at 420 nm and it was related to amino group concentration using a standard curve constructed with glycine as standard amino acid  was utilized to discount acid or alkaline chemical hydrolyses.To determinate the optimum activity temperature of free or immobilized lipase, the enzymatic activity was measured using 100 mM sodium phosphate at pH 7.5 in a temperature range from 10 to 70 °C.For stability assays, free and immobilized PPL were incubated at 40 °C and 100 mM phosphate buffer pH 8.0 for 10 h under 500 rpm stirring. At regular time intervals, samples were withdrawn for measurement of hydrolytic activity.imm and Vfree are the rates of the reaction catalyzed by the same amount of enzyme .Initial reaction rates were measured at 40 °C and pH 7.5 using different tributyrin concentrations  for free and immobilized PPL. Effectiveness factor (η) for each form of PPL and tributyrin concentration was determined using the following equation:The performance of free PPL and pm-SP-CLEA was evaluated in the hydrolysis of a 74.2 mM tributyrin solution at 37 °C and pH 7.5 for 4 h under stirring at 500 rpm. The reaction medium was composed by 6 mL sodium phosphate buffer , 16.5 mL distilled water, 0.5 mL tributyrin, and an enzyme load of 70 TBU/g tributyrin (for free and immobilized PPL).Reuse assays were carried out at the same conditions above using pm-SP-CLEAs as biocatalyst. Between each cycle, the CLEAs were recovered by magnetic separation and washed with distilled water.The surface morphology of the CLEAs and pm-CLEAs was studied by scanning electron microscopy with field emission gun (SEM-FEG) using a JEOL JSM6701F (Germany) electron microscope operated at 2 kV. The samples were impregnated with silicon and dried in a desiccator for 24 h before being scanned under vacuum.All experiments were performed in triplicate. The results were expressed as an average ± standard deviation (σ). Analyses of variance between averages were performed by Tukey test at 5% significance.The co-aggregation of porcine pancreas lipase (PPL) with protein feeder (mainly soy protein) and silica magnetic nanoparticles activated with amino groups, using starch as pore-making agent, yielded porous magnetic CLEAs (pm-CLEAs) of PPL with high retained activity (around 80% recovered activity) and high effectiveness factor  due to their porous structures. The pm-CLEAs were high stable (around 80% of retained activity after 10 h at 40 °C and pH 8.0) and easily separated by an external magnetic field, thus avoiding the formation of large clusters conventionally observed by centrifugation separation, which can aggravate mass transfer problems in the compacted CLEA structures. Besides, porous magnetic CLEAs co-aggregated with soy protein and magnetic nanoparticles (pm-SP-CLEAs) showed good performance and reusability in the hydrolysis of tributyrin for five 4h-batches. The use of magnetic nanoparticles can permit to immobilize the enzyme on its surface providing some extra-rigidification via multipoint covalent attachment , also ma


The patient underwent coaxial phacoemulsification with posterior chamber intraocular lens implantation through a 2.2 mm clear corneal incision under peribulbar anesthesia. During the hydration of the incisions, the main port incision was noted to be leaky. A possible disruption in the graft–host lamellar interface was suspected intraoperatively, due to the inner lip of the main port incision opening in the lamellar interface. The incision was closed with two interrupted sutures of 10-0 nylon, and the globe was well-pressurized by forming the anterior chamber (AC) with balanced salt solution followed by the injection of an air bubble. On the postoperative day 1, PVA was 20/400 with mild graft edema, AC was well formed, and the air bubble was in the upper third of the AC. On the follow-up at two weeks, the patient reported further diminution of vision for seven days. PVA had decreased to “hand motion” close to the face, and there was significant graft edema along with    presence of fluid between the anterior lamellar graft and posterior lamella leading to the formation of double AC, as observed on anterior segment optical coherence tomography (OCT)  [Figure 1]. Intracameral 14% perfluoropropane (C3F8) gas was injected, and supine posture was advised for one day. On the subsequent day, PVA was “counting fingers” at 1 m. The gas bubble was partially in the AC and partially entrapped between the corneal lamellae [Figures 2(a) and (b)]. The interface gas bubble was drained on the slit-lamp with a 26-gauge needle entering through the mid-peripheral cornea. This immediately led to the reattachment of the lamellae  and improvement in graft clarity with only a small residual interface gas bubble away from the visual axis [Figures 2(c) and (d)]. A final uncorrected VA of 20/60 and best-corrected VA of 20/40 with a refraction of +0.5 DC A 39-year-old female with a history of automated anterior lamellar keratoplasty for a nebulomacular corneal opacity in the right eye presented with a gradually progressive decrease in vision two years ago. Presenting visual acuity (PVA) was 20/200, and a grade 3 nuclear cataract was noted.,3 The probable reason for lamellar detachment occurring twice in a short span, in this case, could be the clear corneal incision traversing the lamellar interface, leading to the opening of this potential space by the continuous infusion of fluid from the AC during phacoemulsification and in the early post-operative period and by the C3F8 bubble later on. Performing phacoemulsification through a scleral tunnel instead of a clear corneal incision may help avoid this complication.Even after complete healing, the tensile strength of corneal wound is less than that of the intact cornea. The lamellar interface heals with weak adhesions as observed clinically by the ease of lifting of LASIK flap even years after surgery.Nil.There is no conflict of intrest.


This report presents a review of the Malaysian Journal of Medical Sciences’ (MJMS) performance status throughout 2017, which covers the submission pattern of original manuscripts by month, the geographical distribution of submitting authors, the types of manuscripts and overall acceptance/rejection rates.As the years progress, MJMS continues to receive an escalating number of manuscript submissions. This contributes to an ever-increasing workload, which makes administrative tasks continually more challenging. Although the manuscript submission platform seeks to minimise the pre-publication workload of the journal administrator, it is still a time-consuming task, particularly when authors seek exclusive attention for their submitted manuscripts. As expected, the number of original manuscripts sent to the Malaysian Journal of Medical Sciences (MJMS) via our electronic submission system, ScholarOne Manuscripts™, has shown an increase of 17.5%, from 301 manuscripts in 2016 . Out of 365 manuscripts submitted in 2017, ‘Original Article’ manuscripts were the most common, with 273 submissions in 2017 compared to 213 submissions in 2016 .® used by publishers all over the world (Submission of research manuscripts is a much easier process today because of online submission systems like ScholarOne, Editorial Manager and Evisehe world . These sOn the other side of things, however, the journal administrator faces increasing challenges along the journey of journal publication, when finding solutions and technical troubleshooting become additional tasks alongside juggling the responsibilities of being a copy editor, production editor and system administrator.One recurring difficulty is time pressure. This is defined as the subjective feeling of having less time than required to complete a task. It arises each time the journal administrator has to perform a long list of tasks which must keep moving in order to meet deadlines. This includes having to respond to queries by authors through emails while the queue of new submitted manuscripts continues to build up, and keeping authors updated when they insist on exclusive attention. The reality is that everyone wants their manuscript to be entertained first. Last but not least, the administrator must deal with imperfect manuscripts which do not meet the journal’s requirements as given in its guidelines.Based on a study by Moore and Tenney , time pr