The gaps in organ supply and demand necessitate the use of expanded criteria donor (ECD) kidneys.To identify which pre-transplant and post-transplant predictors are most informative regarding short- and long-term ECD transplant outcomes.Retrospective cohort study.Single center, Quebec, Canada.The patients were 163 consecutive first-time ECD kidney only transplant recipients who underwent transplantation at McGill University Health Centre (MUHC) between January 1, 2008 and December 31, 2014 and had frozen section wedge procurement biopsies.Short-term graft outcomes, including delayed graft function and 1-year estimated glomerular filtration rate (eGFR), as well as long-term outcomes including all-cause graft loss .Pre-transplant donor, recipient, and transplant characteristics were assessed as predictors of transplant outcomes. The added value of post-transplant predictors, including longitudinal eGFR, was also assessed using time-varying Cox proportional hazards models.In univariate analyses, among the pre-transplant donor characteristics, histopathologic variables did not show evidence of association with delayed graft function, 1-year post-transplant eGFR or all cause graft loss. Recipient age was associated with all-cause graft loss . Inclusion of time-dependent post-transplant eGFR improved the model’s prediction accuracy . Pre-transplant ECD characteristics were not associated with long-term survival, whereas post-transplant characteristics allowed better model discrimination.Single-center study, small sample size, and potential incomplete capture of all covariate data.Incorporation of dynamic prediction models into electronic health records may enable timely mitigation of ECD graft failure risk and/or facilitate planning for renal replacement therapies. Histopathologic findings on preimplantation biopsies have a limited role in predicting long-term ECD outcomes.Not applicable. To bridge the gap between organ supply and demand, the practice of accepting expanded criteria donor (ECD) kidneys for transplantation has become more prevalent. Clinical donor characteristics and histopathology are used to inform on allograft quality.Long-term ECD transplant outcomes are not informed by donor characteristics alone; rather, an interplay with recipient characteristics predicts ECD transplant outcomes. Of the pre-transplant characteristics, recipient age was most predictive of long-term transplant outcomes with longitudinal post-transplant eGFR improving model performance.14 To bridge the gap between organ supply and demand, the practice of accepting expanded criteria donor (ECD) kidneys for transplantation has become more prevalent. In this context, it is crucial to understand the determinants of outcomes in kidney transplant recipients (KTR) who receive ECD transplants.5Over the years, there has been a universal shortage and an ever-increasing need for donor kidneys. Donor and recipient age have also been steadily rising.6 The KDRI considers donor age, height, weight, ethnicity, history of hypertension or diabetes, cause of death, serum creatinine, history of hepatitis C, and donation after cardio-circulatory death (DCD).7Various donor characteristics measured at the time of organ transplantation have been linked to long-term kidney transplant outcomes. The Kidney Donor Risk Index (KDRI), for example, estimates the relative risk of post-transplant kidney graft failure  associated with each donor.8 In a recent systematic review, Wang et al9 reported on findings from 47 manuscripts published between 1994 and 2014. Quality assessment established that many studies were prone to selection bias because decisions on utilization or discard of ECD kidneys often relied on the perceived quality of procurement biopsies. Studies were also prone to bias related to inconsistent conduct of biopsies and incomplete account for confounders. Some studies reported on paraffin-embedded implantation biopsies rather than FSWB and some studies did not exclusively evaluate ECD transplants but also included biopsies from living donors or very young deceased donors.In addition to clinical characteristics, frozen section wedge procurement biopsies (FSWB) have been used to guide decisions on organ utilization.10 our objective was to identify which donor , transplant, and recipient characteristics were independent predictors of the short-term  and long-term outcomes (graft failure and death). Although current standards for long-term monitoring rely on eGFR, the role of dynamic eGFR monitoring post-transplant in determining transplant outcomes has not been defined to date. For this reason, in addition to baseline donor, recipient, and transplant characteristics, we also assessed the incremental value of longitudinal eGFR measurements in predicting long-term transplant outcomes.In the absence of a clear consensus on what should determine the utilization of ECD kidneys,A total of 238 patients underwent negative cross-match kidney transplants at McGill University Health Centre (MUHC) between 2008 and 2014. After application of the exclusion criteria, 163 unique recipients and 135 unique donors (kidneys from 28 donors were transplanted into 2 recipients) were included in the analytic cohort . RecipieAmong the transplants, 46% were conducted on or before 2011. The recipients were primarily white (72%), males (67%) with an average age of 62 years (range: 27-80 years). The cause of end-stage renal disease (ESRD) was diabetic nephropathy (31%), cystic kidney disease (15%), or other (53%), and the average time on dialysis was 43 months.The donors were primarily white (96%), males (55%) with an average age of 65 years (range: 50-85 years). Hypertension and diabetes were present in 56% and 19% of donors, respectively. Only 6% were DCD. Seventy-three percent of donors had a history of smoking. Average terminal donor creatinine was 66 mmol/L (range: 32-190) corresponding to an eGFR of 91 mL/min (range: 32.32-127.13). The mean KDRI for donors was 1.81 (range: 1.22-3.49).Most grafts (90%) underwent pulsatile machine perfusion. Mean cold ischemia time was 18.74 hours. Eighty-nine percent of KTR received lymphocyte-depleting induction agents and 61% were on a triple-agent maintenance immunosuppression regimen.11 we chose to include at most 3 characteristics, and these were chosen by univariate screening with a P value threshold of .2, resulting in a multivariate logistic regression model for DGF including recipient age and sex as well as donor age, sex, and DCD status. The odds ratios (ORs) and corresponding 95% confidence intervals (CIs) are shown in Forty recipients experienced DGF. Recipient age and sex were included in the model a priori. Continuous variables  and categorical variables , arteriolar hyalinosis (AH), interstitial fibrosis (CI), tubular atrophy (CT), arteriolosclerosis (CV), Remuzzi and Karpinski scores]) were considered for inclusion in the model. To avoid model overfitting,P value threshold of .2) in the univariate selection. These characteristics included donor age, height, KDRI, sex, history of diabetes, DCD, history of smoking, histological lesions (GS and CT), and histological summary scores (Remuzzi and Karpinski). Parameter estimates, standard errors (SEs), and 95% CIs from the multivariate model are presented in When modeling 1-year post-transplant eGFR, we included subjects who survived past 1-year post-transplant (N = 145 recipients). The eGFR data are presented in Supplementary Information 1. In addition to recipient age and sex, we considered the same donor characteristics as for the DGF model. The multivariate linear regression model included donor characteristics that showed association with the eGFR  linear regression modelOver the duration of the study, 50 KTR experienced all-cause graft loss events (27 graft failures and 23 deaths with a functioning graft). The median graft survival time was 6.91 years. The Kaplan-Meier survival curve for all-cause graft failure is presented in When assessing the association between pre-transplant recipient, donor, and transplant characteristics with all-cause graft loss using univariate Cox proportional hazards models, recipient age  and diabetic nephropathy as the cause of ESRD  were the pre-transplant variables that demonstrated a strong association with all-cause graft loss. Histologic lesions and KDRI  were not associated with this outcome. In addition, the scaled Schoenfeld residual analysis showed strong nonproportionality of hazard, but no clear biologically plausible association pattern. Therefore, the final multivariate Cox proportional hazards model included recipient age, sex, and diabetic nephropathy as causes of ESRD along with donor pre-procurement eGFR and cold ischemia time .When assessing the association between pre-transplant characteristics with death-censored graft failure (DCGF), none of the characteristics (including histopathology and KDRI) were statistically significantly associated with DCGF. Following our modeling strategies for all-cause graft loss, we built a pre-transplant model with recipient age and sex as covariates. The model demonstrated limited discrimination .To account for post-transplant predictors of long-term kidney transplant outcomes, we also assessed the incremental value of DGF and longitudinal post-transplant eGFR estimates over and above pre-transplant characteristics by fitting a multivariate Cox proportional hazards model. The HRs and 95% CIs are presented in When assessing the association between pre- and post-transplant characteristics with DCGF using univariate models, only DGF was statistically significantly associated with DCGF . Thus, our final model for DCGF included recipient age, sex, and DGF as covariates .In our exploratory analysis, we noted that the longitudinally updated eGFR was highly predictive of outcome. The Weighted Mean Rank (WMR) estimate of the longitudinal eGFR sustained a high predictive value (WMR above 0.65 for most of the study duration), whereas the WMR estimate of baseline eGFR was not useful (WMR close to 0.5 for most of the study duration). The WMR curve based on the longitudinal eGFR and baseline eGFR as well as the difference in their prediction accuracy (and 95% bootstrap CI) are displayed in Because Transplant Quebec policy dictated that all donors who were ≥60 years old underwent procurement biopsies during the study period, we conducted a sensitivity analysis to verify the validity of our observations in this subgroup. An analysis restricted to this subgroup did not differ meaningfully from the observations in the entire ECD cohort .pre-transplant donor, recipient, and transplant characteristics. Among the pre-transplant characteristics, in most multivariate analyses, we found that histopathologic features on ECD procurement biopsies were poor predictors of both short- and long-term graft outcomes. For long-term outcomes, inclusion of donor eGFR, cold ischemia time, recipient age, sex, and diabetic nephropathy as causes of ESRD marginally improved model discrimination. A model including the post-transplant characteristics DGF and longitudinal eGFR, in addition to pre-transplant characteristics, increased the prediction accuracy of overall graft survival from 0.590 (pre-transplant model) to 0.711 (post-transplant model). Moreover, this dynamic model demonstrated a high degree of prediction accuracy over time.This study sought to address one of the greatest challenges in decision-making when offered ECD transplants—how to predict which combination of donor, recipient, and transplant characteristics will ensure the best possible kidney transplant outcome? To this end, we fit Cox regression models with 13Prediction models for all-cause graft loss of ECD transplant recipients were developed in a conventional step-by-step fashion. Of the pre-transplant recipient characteristics, age was the most important predictor of all-cause graft loss. This finding aligns with a recent SRTR (Scientific Registry of Transplant Recipients) analysis that demonstrated how the impact of donor quality on long-term outcomes might vary by candidate condition, measured by the Estimated Post-Transplant Survival (EPTS) score. The EPTS relies on data acquired at wait-listing and considers time on dialysis, diagnosis of diabetes, prior solid organ transplant, and age.14 This observation may be explained by high interobserver variability when pathologists score chronic lesions, dependence on biopsies being examined by an experienced renal pathologist or not, overrepresentation of lesions such as GS and CI in the outer cortex of the kidney,1518 and tendency toward underrepresentation of vascular pathology including arteriosclerosis or AH .9 Interestingly, markedly higher discard rates for biopsied kidneys than nonbiopsied kidneys were observed in the United States in 2016, with nearly one-third of biopsied kidneys discarded, despite declining KDRI of biopsied kidneys over the past decade (from 1.61 in 2005 to 1.45 in 2016).19 Given the limitations and poor predictive value of donor kidney histopathology, it is hard to justify using information from procurement biopsies to decide between organ utilization and discard.Our finding that ECD kidney quality assessment on FSWB has limited utility as a predictor of long-term outcomes is consistent with a growing body of evidence.20 who found the ECD classification useful for risk stratification of donor kidneys outside of the United States. The same study, however, reported that the predictive value of the ECD system did not exceed that provided by donor age. Moreover, terminal MDRD  eGFR did not appear to drive the association between ECD status and graft loss.Our finding that donor terminal eGFR is not predictive of long-term transplant outcomes aligns with Young et al,2123 While generalizable, its predictive role above and beyond a smaller number of variables, and specifically, donor age, is not convincing. Inability to observe an association between KDRI and long-term graft outcomes in our study may be related to insufficient power. In addition, our study focused on ECD KTR, resulting in a higher median KDRI in comparison with the originally used U.S. donor pool.6 Our analysis suggests that beyond the ECD status, which is already deemed higher risk, KDRI may not provide any additional information. It is also important to note the KDRI was reported to have insufficient prediction accuracy of long-term graft outcomes even in the SRTR,7 from which it had been originally derived. Given accuracy of prediction models tends to be overestimated in the population they are derived from, it is not surprising that in our patient population, KDRI was a less important predictor. Moreover, a growing body of literature finds KDRI and its derivative, Kidney Donor Profile Index (assigning a percentile score of 0-100 based on kidney donors recovered in previous years), may have resulted in overlabeling of organs as high risk leading to excessive organ discard,7 suggesting that caution must be exercised when considering organ discard based on clinical ECD characteristics.In our cohort of ECD KTR, we found that donor characteristics associated with the short-term outcomes DGF and 1-year eGFR included donor history of diabetes and DCD status . A sensitivity analysis using a LASSO model found only KDRI to be a statistically significant predictor of 1-year eGFR. The comprehensive KDRI score based on 10 donor characteristics was not predictive of long-term transplant outcomes when considered in Cox regression models. The U.S.-developed KDRI was studied in several Canadian populations, including Ontario, BC, and Alberta.14 The discrepancy between studies may be explained by the small percentage of DCD-ECD donors, the relatively short cold ischemia time, and the protective effect of pulsatile machine perfusion, which was used for most of the transplants in the Quebec cohort only.Of the baseline transplant characteristics considered as predictors of all-cause graft loss, surprisingly, our analysis did not find any evidence of impact of cold ischemia time. This differs from a recent study of ECD transplants in France.2426 and eGFR slopes were previously shown to indicate graft failure secondary to immune-mediated injuries.28 Although post-transplant longitudinal eGFR measurements are not included in most prediction models evaluating graft outcomes, a “dynamic” approach to risk prediction for progression to kidney failure has been proposed in the context of chronic kidney disease.29 It is rational to assume that recipient characteristics and events influencing eGFR over the post-transplant course can similarly result in irreversible damage and affect long-term graft outcomes. Because this approach more closely simulates transplant nephrologists’ clinical monitoring practices, we believe that integration of our prediction model into electronic health records can facilitate reevaluation of ECD KTRs’ risk of graft loss on an ongoing basis. This information can facilitate timely interventions to mitigate risk of premature graft loss, and when this is not possible, to allow patient-provider communication for appropriate ESRD management planning.A key finding of our study is that dynamic post-transplant eGFR is the most important predictor of all-cause graft loss. Analysis of eGFR decline has been shown to predict development of ESRD30 This is because, during the study period, Quebec Transplant policy mandated that procurement biopsies be conducted in all donors ≥60 years and decisions on organ utilization did not rely primarily on biopsy findings. Some limitations must also be noted. First, our effort to capture all pertinent pre-transplant determinants of donor quality in ECD KTR, including histology on FSWB, resulted in a modest sample size that was not amenable to subgroup analyses. Second, Quebec allocation practices of ECD kidneys resulted in the analytical cohort being composed of relatively older KTR with an associated higher comorbidity burden. We expect this practice to be consistent with that of other jurisdictions. Our analyses account for relevant measurable donor, recipient, and transplant characteristics. Yet, the observational nature of the study makes it vulnerable to residual confounding. For example, post-transplant donor-specific antibody (DSA) screening was implemented at our center toward the end of the study period. Consequently, while the practice in Quebec was to pursue transplantation in the absence of preformed DSA (complement-dependent cytotoxicity and/or flow cross-match negative transplants), de novo DSAs were not included among the dynamic predictors of long-term transplant outcomes.We report on predictors of transplant outcomes in one of the largest single-center ECD KTR cohorts studied to date. In contrast to prior studies using preimplantation biopsies to judge donor kidney quality, our study is less vulnerable to selection bias.It is likely that more ECD donors will become available given our aging population. In our study, of the pre-transplant characteristics, recipient age was most predictive of long-term transplant outcomes and longitudinal post-transplant eGFR improved model accuracy. While our models require validation in external cohorts, these findings suggest that caution should be exercised when considering organ utilization or discard based on clinical and histopathologic ECD donor characteristics alone.We conducted a retrospective cohort study in consecutive first-time KTR who underwent ECD transplants at MUHC between January 1, 2008 and December 31, 2014. The KTRs for whom donor procurement biopsies were not conducted or for whom FSWB were not available; who underwent multiorgan transplants, or retransplantation; who had PNF; or were missing key predictor variables, were excluded from the study. Importantly, during the study period, the policy of the Quebec organ procurement organization, Transplant Quebec, was to conduct procurement biopsies in all ECD donors older than 60 years.Data were obtained from the MUHC Transplant Database and Transplant Quebec donor charts. Ethics approval was obtained from the MUHC research ethics board (15-192-MUHC), which granted permission to access retrospective patient and donor information. The manuscript was written in adherence with the STROBE guidelines.The primary long-term endpoint was time to all-cause graft loss . Death-censored graft failure, defined as return to dialysis or retransplantation, was also studied. Patients were administratively censored at the end of study follow-up .Short-term endpoints included DGF, defined as the need for dialysis within the first week post-transplant, and eGFR calculated by the CKD-EPI formula using creatinine measurements at 12-months (±6 weeks) post-transplant.We defined “baseline” variables as any donor, recipient, and/or transplant characteristics available at the time of transplantation. These characteristics included donor demographics , donor comorbidities , DCD, pre-procurement estimated glomerular filtration rate , the donor-only KDRI, and histologic features on FSWB, including GS, AH, CI, CT, and CV. On-call pathologists in the procurement centers graded these features from 0 to 3 based on increasing severity. Summary histologic scores such as Karpinski and Remuzzi scores  were also considered. Baseline recipient characteristics included demographics , time on dialysis (in days), and cause of ESRD. Baseline transplant characteristics included transplant era , cold ischemia time, pulsatile machine perfusion, type of induction therapy , maintenance immunosuppression regimen , dual agent  or other, and human leukocyte antigen (HLA) mismatch . Finally, among the post-transplant characteristics, we considered DGF, defined as the need for dialysis during the first week after transplant, and recipient post-transplant longitudinal eGFR.We performed descriptive analyses on the baseline variables  and Kaplan-Meier survival curve for time to all-cause graft loss. To estimate the role of pre-transplant donor characteristics in determining post-transplant outcomes, we fit several prediction models.one-year post-transplant eGFR.When considering studying short-term outcomes, we found that only 3 participants experienced PNF, which was deemed too small to study this endpoint and were excluded from the analysis. We conducted logistic regression to estimate risk for DGF and linear regression to predict We fit a Cox proportional hazards model to characterize all-cause graft loss and DCGF. Very few donors or recipients had missing values for important covariates see . Therefo3133 To identify additional predictors, we first assessed univariate associations via Cox proportional hazards models between the outcome and several donor , recipient (cause of ESRD), and transplant characteristics . The variables that had a strong association with the outcome  were retained for further exploration in a multivariate Cox proportional hazards model. To maintain an expected number of events per variable between 5 and 10,11 we restricted the number of variables in the multivariate prediction model to be between 5 and 10, including levels of categorical variables. The validity of proportional hazards assumption was assessed using scaled Schoenfeld residuals. Overall prediction accuracy of the models was assessed using Harrell’s C-index and a novel accuracy measure C*-index.34 C*-index is a recently proposed accuracy index, particularly suitable for longitudinal covariates as predictors of a survival outcome.Our objective was to provide a prediction model that would accurately predict time to all-cause graft loss or DCGF, be easy to interpret, and be useful to support decisions on organ utilization pre-transplant. Considering transplant outcomes are also dependent on recipient characteristics, we decided a priori to include recipient age and sex in the multivariable model. Other donor and transplant variables that were included in the prediction model regardless of their strength of association in univariate analysis were donor pre-procurement eGFR and cold ischemia time.29 In this context, we were interested to characterize the incremental value of post-transplant longitudinal eGFR measurements for predicting time to all-cause graft loss. The longitudinal eGFR was based on serum creatinine measured repeatedly starting from the first post-transplant day. To impute missing creatinine measurements and to standardize the measurement to a 3-month measurement window, we carried the last observation forward.Because long-term ECD transplant outcomes may be influenced by post-transplant factors, we also wanted to provide a prediction model that would be able to dynamically capture changes in the risk of graft failure post-transplant. Recently, dynamic changes in eGFR have been incorporated in prediction models informing risk of ESRD.35 to show the prediction accuracy of a model that includes longitudinal eGFR measurements. The nonparametric WMR curve is a generalization of the conventional C-index and can characterize the accuracy in a longitudinal fashion to reveal potentially important time-dependent variation in prediction accuracy.We included the longitudinal eGFR in a Cox proportional hazards model that already included baseline characteristics to formally assess the incremental accuracy of post-transplant eGFR in predicting time to all-cause graft loss. We used Harrell’s C-index and C*-index to assess the overall prediction accuracy of the model. As an added visual tool, we used the recently proposed WMR estimatorP value of <.05 was considered statistically significant.Finally, given Quebec Transplant policy to perform biopsies in all donors who were ≥60 years old, we performed a sensitivity analysis to verify the validity of our findings in this subgroup. For all models, Click here for additional data file.Supplemental material, SUPLLEMENTARY_INFORMATION_1 for Predicting Clinical Outcome in Expanded Criteria Donor Kidney Transplantation: A Retrospective Cohort Study by Paramita Saha-Chaudhuri, Carly Rabin, Jean Tchervenkov, Dana Baran, Justin Morein and Ruth Sapir-Pichhadze in Canadian Journal of Kidney Health and Disease


It was established on 20th Falgun, 2007 BS (4th March 1951). It has been publishing an open access scientific publication, the Journal of Nepal Medical Association (JNMA) since 1963.th to 21st Magh, 2076 and we accepted it. We also helped NMA during the pre-election procedure that needs to be done before going for the election. We think that was the work where we got the trust of NMA officers.Our volunteering began being a part of the editorial support team in the Journal of Nepal Medical Association. This has really built up our confidence to work as a team. During our days in Journal Office, a sudden knock of another opportunity struck our door. The Administrative Officer of Nepal Medical Association asked us about volunteering in the Election of Nepal Medical Association which was held from 17Time management is one of the important factors when we do multiple works and it is a difficult thing to do. Attending the lectures and going to the election site just after you finish the lectures with some biscuits in your hand. If we think of those days, we will surely ask ourselves a question, “how did we actually manage?” This taught us to manage our time which will surely help in our upcoming days. We, volunteers, have worked as a team during this election, we have done volunteering on a shift basis. As mentioned earlier, it’s a regular day volunteering so shifts were on the same day. Working as a team has really helped us in every challenge we faced during our volunteering period. Nothing can help us until we have the courage to perform it and there should always be someone who provides us that courage. During the time period of volunteering, the continuous guidance from our seniors was beneficial.Working in an election was one of the best experiences that we had in medical school life. The election voting was done for 5 days and the voting system was well managed so that the voters can vote easily. Also, the system for voting was managed well to prevent double-voting with a single name. We performed digital entry, manual entry with the voters signature so as to prevent the double voting with a single name. We volunteers also helped the elder doctors to reach the secret room for filling up the ballot paper. Small tokens with serial numbers were provided to each voter so that the numbering of voters can be done later on and to cross-match it with the number of ballot papers inside the ballot box. We also didn’t allow the voters to take photos or videos inside the secret room as per the guideline of Election Commission. We checked the voters name in excel file which was prepared before the election and once the voters have voted, we ticked the name in the list as voted. CCTV surveillance was also present which shows the alertness of the election team. Identifcation, verifcation, providing the ballot paper, taking voters to the secret room and making them drop their paper in a systematic manner into the ballot box is actually the individual steps that we performed in this election. Besides these, there were so many legal procedures that our seniors performed to prevent future complications. For example, once the double paper was mistakenly provided to a single person but we could not identify the person to whom we gave it, in such condition our seniors called the candidate representative and wrote what actually happened and it was signed by the candidate representative and was sealed.Usually, the vote-counting starts after the voting finishes. But, few doctors demanded a live telecast of the vote counting for the purpose of fairness. Many meetings were held to allow the vote counting. The vote counting was withheld for two days to ensure the system of vote counting among the representatives and the election committee. Candidates and candidate representatives were only allowed to seat in the vote-counting place. Counting six thousand votes and naming or registering 13 names for a single ballot paper continuously for 4 days with some breaks in between, this was actually what we did while counting votes. Google forms were created and the digital entry was done in the forms. We also counted the votes manually using the tally method. Basically, it was done for double checking the mistakes. Management of each ballot paper, showing the ballot paper to each of the representatives present inside the room and reciting it aloud, manual entry, digital entry and putting the bundles of ballot paper with signature into another box. During this phase, a lot of invalid ballot papers and invalid votes were seen. For example, two tick marks in two different candidate names. This way, we suggest to pre-inform the voters the way of casting votes in the upcoming election. We had minimal problems using google forms, although, it would be better if we use good software which makes the work more efficient. In an organization with above 10,000 members who are doctors by profession, it would be efficient if the vote casting system was made online. These are some of our suggestions and our learning curve from the election experience.Through our continuous work, we gained trust from our seniors and a lot of opportunities have come to us like attending different medical conferences inside the country. Recently we attended the ORTHOCON 2020 where we learned a diverse range of things from academics to manage the program. Hence, we came to know that possibilities are endless. We are acknowledged to the NMA officers, the Election Committee and the JNMA team.Work experience: We gain real-world skills which are very helpful in developing our career.Teamwork: Working in a team deals with lots of things among which group dynamics is a thing which when maintained makes teamwork much more efficient and easy.Ability to assess situations: We are exposed to a number of social and political issues while volunteering in various events and programs. We gain the ability to assess various situations in a compatible way.2Employability: If we can manage to volunteer and perform well even when no one is paying you, it makes a good impact on our commitment to do quality work.A good networking tool: Through volunteering, we are taking a step forward and opening ourselves up to people who are looking for candidates for jobs or other opportunities. Contacts are useful - you never know when you might need them.A sense of purpose: Most of us feel a bit ‘lost’ at university which isn’t good for our mental health. Therefore, volunteering fills our time and gives us a sense of purpose. We practiced self-reflection and mindfulness which gave us a sense of purpose.3 As a volunteer, we gain the skills that make us to do more. Finally, we get to know possibilities are endless. To find opportunities we never knew existed: By volunteering and grabbing one opportunity, we open ourselves up much more.Planning semester in advance: Going through the syllabus and figuring out what we need to accomplish when. This makes us able to plan for our life bit by bit. It is important for maintaining a positive perspective on all areas of life.Squeezing our time for studies: As the saying goes time and tides wait for no man, we got to know the value of each and every time and made each time valuable for our studies.4 Above all these are the primary traits to gain so that we can be able to live our lives in a new place far from home country. It also helps set you up for a future career.2Learning how to be independent, how to manage time and how to be responsible. These are skills that will shape our lives in the years to come, like an outline on how to live.Volunteering in a field related to our career can help us gain experience in our area of interest and meet people in the field. Even if we are not planning on changing careers, volunteering gives us an opportunity to practice important skills used in the workplace - like teamwork, communication, problem-solving, planning and task management.As we are donating our valuable time, so it’s important that we enjoy and beneft from our volunteering.5Asking questions is a good way to make sure that the experience is right for our skills, our goals, and the time we want to spend.We should know what’s expected. You should be comfortable with the organization and understand the time commitment. Starting small is good so that we don’t over-commit ourselves at first. We should be flexible to change our focus if needed.We should not be afraid to make a change. We should not force ourselves into a bad fit or feel compelled to stick with a volunteer role or program we dislike. Id something bad happens we should talk to the organization about changing our focus or look for a different organization that’s a better fit.The best volunteer experience benefits both the volunteer and the organization- ultimately, it’s a win-win game!To make sure that our volunteer position is a good fit:These are the main things that made us able to work in the journal team and various other opportunities like election, election vote counting, and conferences.None.


This finding was consistent for the overall follow-up. Variables associated with NLR decrease included preoperative NLR > 4, model for end-stage liver disease score, intraoperative inotropic infusion and red blood cell transfusion, and operative duration. The least absolute shrinkage and selection operator model yielded similar results. NLR decrease during LDLT appeared to be independently associated with graft survival. Further studies are needed to confirm our findings.Preoperative neutrophil–lymphocyte ratio (NLR), has shown a predictive value in living donor liver transplantation (LDLT). However, the change in the NLR during LDLT has not been fully investigated. We aimed to compare graft survival between the NLR increase and decrease during LDLT. From June 1997 to April 2019, we identified 1292 adult LDLT recipients with intraoperative NLR change. The recipients were divided according to NLR change: 103 (8.0%) in the decrease group and 1189 (92.0%) in the increase group. The primary outcome was graft failure in the first year. In addition, variables associated with NLR change during LDLT were evaluated. During 1-year follow-up, graft failure was significantly higher in the decrease group (22.3% vs. 9.1%; hazard ratio 1.87; 95% confidence interval 1.10–3.18;  The NLR has been shown to have a predictive value in various clinical situations including cardiovascular disease, cancer, and liver cirrhosis4. These findings were also consistently reported in surgical patients, and a perioperative high NLR was associated with adverse events after cardiac surgery and recurrence after cancer resection10.Neutrophil–lymphocyte ratio (NLR) is a simple, inexpensive, readily available, and reproducible index that reflects the systemic inflammatory respone12. In liver transplant candidates, high NLR is a predictor of mortality13, and preoperative elevation of NLR also showed significant correlations with higher mortality and recurrence rate after liver transplantation for HCC15. Based on these previous findings, preoperative NLR was suggested to be helpful in selecting adequate HCC recipients for LDLT14. However, the current clinical value of NLR in LDLT seems to be limited to using preoperative value to predict outcome.Liver transplantation is an established treatment modality for end-stage liver disease and unresectable hepatocellular carcinoma (HCC) with or without cirrhotic change. Living donor liver transplantation (LDLT) provides a survival benefit as well as reduced waiting time for the candidates17. Furthermore, previous studies showed an efficacy of controlling intraoperative inflammation as indicated by the NLR to improve postoperative outcomes18. Therefore, we hypothesized that the change in NLR during LDLT could reflect operative burden and be associated with graft survival. In this study, we enrolled LDLT recipients with available NLR change data during the surgery and compared the incidence of graft failure. We also calculated the attributable fraction (AF) of each variable on graft failure and evaluated the variables that are associated with NLR change during LDLT using logistic regression and the least absolute shrinkage and selection operator (LASSO) models. Our finding may provide valuable information on the association between intraoperative NLR change and clinical outcome in LDLT and a direction for perioperative management and future studies.Postoperative NLR independently predicts mortality and adverse outcomes such as acute kidney injury and cardiovascular events in various surgeries and is associated with recurrence rate after cancer resectionFrom June 1997 to April 2019, a total of 1349 cases of adult-to-adult LDLT were performed in our institution. From the entire cohort, 1306 cases of LDLT in which pre- and post-surgical NLR data were available were included, and 14 re-transplantation cases were excluded. Finally, 1292 LDLT recipients were enrolled for the study. Smooth plots for the change of odds ratio (OR) was construced and showed a significant association when the reference value was an absolute NLR change of 0 . As shown in the 1-year follow-up, the incidence of graft failure in the decrease group was higher during the overall follow-up period  , MELD score , no use of inotropic continuous infusion , intraoperative packed red blood cell transfusion , and operative duration   the NLR value decreased during LDLT in 8.0% of the recipients, (2) the incidence of graft failure was significantly higher in the recipients with NLR decrease during LDLT, and (3) NLR decrease during LDLT was associated with preoperative variables such as NLR > 4 and MELD score and intraoperative variables such as use of inotropic infusion, packed red blood cell transfusion, and operative duration. These findings indicate that the change in NLR during LDLT may be associated with pre- and intraoperative conditions and may independently predict graft failure regardless of an absolute value.1. NLR has the advantage of being inexpensive and readily available by daily measurements, and substantial evidence supports the association with clinical outcomes of acute and chronic conditions21. In previous studies on surgical patients, postoperative elevation of NLR has consistently shown associations with higher incidence of complication in cardiac and non-cardiac surgeries and recurrence after cancer surgeries16. Because there has been no previous report regarding postoperative NLR in LDLT, we initially evaluated the change of risk for graft failure according to postoperative NLR and different reference values of NLR change during LDLT. Based on our initial analysis, the recipients were divided into the increase and decrease groups by using dynamics of NLR during the surgical procedures instead of using an absolute value of postoperative NLR. Indeed, inflammatory markers have shown limited clinical utilities when using an absolute value in the postoperative period because an inflammatory response during the surgical procedure is inevitable. This response is characterized by a rise in circulating neutrophils accompanied by a fall in lymphocytes22.Several biomarkers have been identified to detect and measure systemic inflammation, most of which consume additional time and cost24, and the decrease of NLR during the surgical procedure, on the other hand, may be a surrogate marker that indicates generally poor condition of recipients who could not adequately respond to metabolic stress from LDLT procedures. Indeed, hematologic abnormalities are known to be associated with poor prognosis in cirrhotic patients25, and the cause of graft failure also supports our hypothesis. Compared with the increase group, the incidence of bleeding associated with graft failure was higher for the decrease group, and more graft failure in the decrease group was caused by pulmonary and cardiac complications, which are more closely related to general condition of patients, rather than graft rejection or surgical complication. However, determination of the degree of NLR increase that could be considered as normal requires further investigation. This is also true for the determination of whether an enormous increase in NLR is associated with adverse outcomes.In this study, we evaluated the change of NLR during surgical procedures instead of an absolute value and demonstrated that decreased NLR during LDLT was associated with increased incidence of graft failure despite the lower absolute value of postoperative NLR. Our explanation for this result is that, the increase of NLR could be regarded as a normal reaction to surgical injuries26. In contrast, the effect of the inflammatory response on carcinogenesis plays a key role in the reoccurrence of HCC15. In previous studies, high NLR was associated with mortality of the overall liver transplant candidates13. However, in the recipients who actually underwent transplantation, preoperative NLR was mainly associated with outcomes of liver transplantation for HCC15. In the subgroup analysis, no significant interaction was observed for the association between NLR decrease and graft failure; however, considering a low incidence of NLR decrease, a larger study is needed to evaluate the generalizability of this finding.In addition to the aforementioned explanation on cirrhotic recipients, a separate explanation may be needed for the recipients with non-cirrhotic HCC. The prognostic impact of inflammatory response has discrete underlying mechanisms for cirrhotic disease and HCC. In cirrhotic disease, the alterations in the intestinal barrier along with increased luminal aerobic gram-negative bacilli, bacterial translocation, bacteremic events, and endotoxemia lead to increased inflammatory cytokine production which has prognostic significance18, but whether prevention of intraoperative NLR decrease could improve outcome in LDLT or whether postoperative immunosuppression should be adjusted in the recipients with postoperative NLR decrease also requires investigation.Preoperative NLR elevation seems to play a complex role in our analysis. Preoperative NLR elevation was a strong predictor of NLR decrease during LDLT, but the calculated AF on graft failure was not significant in the multivariable model after retaining the intraoperative NLR decrease. Variables associated with NLR decrease during LDLT also included MELD score, supporting our explanation that the progression of disease and underlying state of the recipients may be involved in this association. Of note, intraoperative use of inotropic infusion and packed red blood cell transfusion also appeared to affect the incidence of NLR decrease. This finding suggests the possibility that adequate intraoperative management could prevent NLR decrease, but this requires further investigation. The control of perioperative inflammation resulted in improved outcomes of various cancer surgeriesOur results should be appraised considering the following limitations. First, as a single-center retrospective study, our results may have been affected by confounding factors. Although relevant variables were adjusted for, the effect of unmeasured variables may have remained. A well-designed, prospective study with a larger number of patients may be needed to confirm our finding. Also, due to the long study period, advancements in surgical techniques and postoperative management could also have biased the results. Another limitation is that due to the low incidence of NLR decrease during LDLT, our results may have been biased despite the statistical adjustments and the larger number of the recipients than previous studies. Despite these limitations, this is the first study to demonstrate the negative impacts of NLR decrease during LDLT.The incidence of graft failure was significantly increased in the recipients with a NLR decrease during LDLT. Further studies are needed to confirm our findings.This retrospective observational cohort study was approved by the Institutional Review Board at our institution  and was conducted according to the Declaration of Helsinki. The need for written informed consent was waived by the Institutional Review Board at our institution considering the minimal risk for the participants and retrospective nature of the study.We reviewed the entire cohort of liver transplantation at our institution between June 1997 and April 2019 and identified adult-to-adult LDLT recipients. Cases were required to have available NLR data for pre- and postoperative period. This data were required to be obtained between before the anesthetic induction and immediately after arrival into the ICU. In recipients with multiple liver transplantations, only the first transplantation was enrolled for analysis. In the enrolled recipients, smooth plots of the change of OR for 1-year graft failure were constructed according different values of an absolute NLR change and NLR percent change, defined as (postoperative NLR − preoperative NLR)/preoperative NLR. According to the results of smooth plots, recipients were divided according to an absolute NLR change value of 0 into NLR increase or decrease groups. Clinical, laboratory, and outcome data were collected by a trained study coordinator who was not otherwise involved in this study, and NLR and its change were calculated by another investigator who was blinded to clinical outcomes. The data from all recipients were analyzed anonymously.27. Graft rejection was confirmed by biopsy when clinically suspected.The primary endpoint of this study was graft failure during 1-year follow-up. Graft failure was defined as death or re-transplantation, and recipients who underwent re-transplantation before death were regarded as both death and re-transplantation in the table. One secondary endpoint was graft failure during the overall follow-up. Another secondary endpoint was the composite of complications according to Clavien–Dindo classification and graft rejection during overall follow-up. The classification was briefly defined as follows: IIIa, complications requiring interventions without general anesthesia; IIIb, complications requiring interventions under general anesthesia; IV, life-threatening complications; V, death28. In brief, our criteria included adult younger than 65 years old, a body mass index lower than 35, biochemistries within normal range, and adequate size of graft and expected remnant liver of more than 30%. The presence of any conditions related to increased risk to the donor were excluded.Donor selection criteria and surgical procedures of our institution have been previously describedMost of the grafts were from the right side and consisted of 5 to 8 segments according to the Couinaud’s classification system. The surgical margin of the graft was determined after considering anatomical characteristics. For donors, full mobilization of the liver was achieved by dissecting the ligaments around the liver, and cholecystectomy was performed. After identifying bifurcations of the hepatic duct, portal vein, and hepatic artery, the intraparenchymal hepatic vein was identified with the aid of intraoperative ultrasound. The bile duct was transected after completing parenchymal dissection, and the graft liver was removed.Harvested graft liver was implanted into the recipient using a piggyback technique. The right hepatic vein was initially anastomosed; if necessary, anastomosis of the inferior hepatic vein followed. After portal vein anastomosis, the hepatic vein and portal vein were unclamped to reperfuse the graft liver. After reperfusion, segment veins were anastomosed to the inferior vena cava using a cryopreserved allovascular graft, and the hepatic artery and biliary tract were then anastomosed. All recipients were routinely transferred to the ICU.Standardized anesthesia according to the institutional protocol was performed in all recipients. Under the standard monitoring of vital signs , the induction of anesthesia was achieved using thiopental sodium and maintained with isoflurane titrated to a bispectral index of 40 to 60. The radial artery, femoral artery and vein, and internal jugular vein were cannulated for direct hemodynamic monitoring and blood tests. The initial arterial blood gas analysis and complete blood cell test were performed before the surgical incision and repeated throughout the surgical procedure. Mechanical ventilation was set with a tidal volume of 8 to 10 mL/kg based on ideal body weight using a mixture of medical air and oxygen at a fresh gas flow rate of 2 L/min, and respiratory rate was continuously adjusted to maintain normocapnea. Remifentanil was used in response to hemodynamic changes. Intravenous fluids were infused to maintain the central venous pressure at a minimum of 5 mmHg. Continuous infusion of inotropic drugs, such as dopamine, norepinephrine, and vasopressin, was administered to maintain mean arterial pressure at a minimum of 70 mmHg. Bolus injection of epinephrine was given when sudden hemodynamic instability was anticipated or had occurred. Indication for intraoperative transfusion of packed red blood cell was blood hemoglobin < 8.0 g/dL. To maintain normothermia, a warm blanket and a fluid warmer were used; the room temperature was thermostatically set at 24 °C.The full blood test was immediately performed at the arrival into the ICU, and daily routine blood tests were performed during the hospital stay. Oxygenation, nutritional support, and early feeding and ambulation were encouraged while being closely monitored for early detection of postoperative complications such as bleeding, thrombosis, and biliary leakage or stricture.χ2 or Fishers exact test. Kaplan–Meier estimates were used to construct survival curves for each group and compared with the log-rank test. We used a multivariable Cox regression analysis to adjust for variables between the two groups. Variables for the multivariable analysis were selected based on clinical relevance or p < 0.05. Retained variables include age, sex, MELD score, operative duration, alcoholics, hepatorenal syndrome, hypertension, preoperative ICU treatment, viral disease, cirrhotic disease, HCC, preoperative NLR > 4, donor sex, and donor age. The results were reported as HR with 95% CI. For sensitivity analysis, we estimated the potential impact of unmeasured confounders29. Considering the long study period, a sensitivity analysis was also performed for the former and latter cases. We stratified the recipients in a chronological order and divided them at the median number. The significance of the observed association between NLR change and 1-year graft failure was evaluated in recipients before and after November 2010.To divide the recipients according to NLR change during LDLT, we constructed six smooth plots of the change of OR for 1-year graft failure when the reference value was absolute NLR change of 0, median value of absolute NLR change, 80% of absolute NLR change, NLR percentage change of 0, median value of NLR percentage change, and 40% of NLR percentage change. NLR percent change defined as [postoperative NLR − preoperative NLR]/preoperative NLR. Differences between the groups for continuous data were compared using the t-test or the Mann–Whitney test when applicable and presented as mean ± standard deviation. Categorical data were presented numerically and compared by using 30. This measure represents the proportional reduction of the outcome within a population that would occur if the incidence of the variable was reduced to zero. Multivariable logistic regression analysis was used to identify variables associated with NLR change during LDLT and was reported as odds ratio (OR) with 95% CI. The LASSO model, a regression analysis that fits a generalized linear model via penalized maximum likelihood and select variables, was used for more accurate evaluation31. All statistical analyses were performed with R 3.6.1 . All tests were 2-tailed, and p < 0.05 was considered statistically significant.Subgroup analysis was also conducted to reveal hidden interaction with relevant variables. To compare the impact of NLR decrease on graft failure with other variables, we calculated the AF for each variable based on the results of the Cox proportional hazards modelSupplementary Informations.


The dynamic recrystallization (DRX) behavior in the hot working of TB8 titanium alloy was studied by using the experiment and finite element simulation (FEM) method. The results showed that the DRX behavior of TB8 titanium alloys was drastically affected by the hot processing parameters. The rising deformation temperature and reducing strain rate led to an augmentation in the grain size ( It is well known that the aim of metal forming is primarily to obtain components with excellent mechanical properties, in addition to the shape and dimension of the parts. The mechanical properties of the parts are remarkably associated with the microstructure formed during hot working, which is affected by hot processing parameters. The in-depth investigation of the microstructure evolution of metals during hot working is very helpful to obtain the optimal processing parameters of hot working. DRX is generally considered to be a dominant phenomenon during the microstructure evolution of metals, which can significantly refine the grain size of metals and enhance the mechanical property of metals. Thus, it is quite critical to reveal the DRX behavior of metals during hot working for the control of the final microstructures of the components. Recently, a great number of investigations into the kinetic behavior of the DRX of metals has been carried out ,2,3. Nevi et al.  investigi et al.  predicten et al.  implanten et al.  establisTB8 titanium alloy presents ultra-high strength, good ductility and exceptional corrosion resistance, which have been extensively employed to manufacture aircraft fasteners in the aerospace industry ,13,14. TThe objective of the present paper is to construct a kinetics model of the DRX of TB8 titanium alloys via the process of hot working based on the experiment data, and then implant the established kinetics model of DRX into the FE model to characterize dynamically the DRX behavior of the TB8 titanium alloys during hot compression tests. The effects of deformation parameters on the The material employed in this paper was a forged TB8 titanium alloy with a diameter of 60 mm, and the composition of TB8 was shown in −1. The samples were heated to a given temperature at a rate of 20 °C/s for 5 min so that the temperature in the center of the deformed samples was the same as that on the surface of the deformed samples prior to deformation, and then all samples were compressed to a true strain of 0.8. To keep the deformed microstructure at the elevated temperature, the deformed sample was immediately water-quenched to 25 °C. Moreover, some samples were selected to be deformed to 0.2, 0.4, 0.6 and 0.8 under the hot processing parameters of 900 °C/0.001 s−1, respectively, for analyzing the effect of strain on the DRX behavior of TB8 titanium alloy. In order to examine the microstructural evolution of TB8 titanium alloy during hot compression by using a Leica DMI5000M , all deformed samples were cut along the radial direction of the deformed samples. For optical observation, the deformed samples were grinded, mechanically polished and chemically etched using a Kroll reagent of 10 mL HF, 30 mL HNO3 and 60 mL H2O. To analyze the characteristic of grain boundaries during compression deformation by using an Electron Backscatter Diffraction  technique, EBSD samples were ground and electro-polished in a solution of 70 mL CH3OH, 10 mL HClO4 and 20 mL (CH2OH)2 at the voltage of 20 V for 10 s. EBSD detection was performed at the voltage of 20 keV. The step size for EBSD observation was set as 0.8 µm.Cylindrical samples with ø8 × 12 mm were prepared from the heat-treated bars. To thoroughly understand the DRX evolution of TB8 titanium alloy, the hot compression test was operated on a Gleeble 3500 (Dynamic Systems Inc. (DSI), Poestenkill, NY, USA). The testing temperature was varied from 850 to 1000 °C and the strain rate was varied from 0.001 to 1 s−1, the flow stress rapidly rose to a peak stress with the increase of strain, then hardly changed, showing that flow curves present a characterization of dynamic recovery. This is attributed to the deformation time being too short to permit the dynamic recrystallization at a high strain rate. When the strain rate was less than 0.1 s−1, a typical characterization of DRX was visible. At a given strain rate, it was found that the flow stress was lowered with the augmentation of temperature. This is associated with the promotion of grain boundary mobility with the elevation of the temperature during hot working.−1, a full dynamic recrystallization was observed  and strain rate εpIn order to compute the values of Based on the linear relationship between The relationship between ribed by ,22(5)εcDuring the hot working of metal materials, the DRX behavior, including nucleation and growth, is dramatically dependent on the density of dislocation. In general, the nucleation of DRX preferentially occurs near to the grain boundaries of deformed grains, with a high strain gradient , and theequation ,10,25,26T can be described as [According to the flow stress data of TB8 titanium alloy, as well as the value of ribed as :(10)ε0.5To compute the values of The average values of rized by :(14)dDRXThe values of According to the flow curve gained from experimental tests and the above developed models for the DRX of TB8 titanium alloy, the isothermal forging process of the cylindrical samples was simulated under various hot working parameters with the DEFORM 3D software. In the process of establishing the FE model, the elastic deformation of the workpiece can usually be ignored under the high-temperature plastic deformation condition. Therefore, the workpiece was considered as a plastic body, while the tools were regarded as rigid bodies ,29,30. SN is the total amount of data employed in this study. It can be observed from −1, 900 °C/1 s−1, 950 °C/1 s−1 and 850 °C/0.1 s−1 were lower than 5%, which can be ignored in the calculation processing of the ∆ value. Thus, the ∆ value for the So as to confirm the accuracy of the FEM results obtained from the derived microstructure evolution models, the correlation coefficient ( formula :(17)R2=∑(1)The DRX behavior of TB8 titanium alloys was drastically influenced by the processing parameters. The rising deformation temperature and reducing strain rate resulted in an increase in the (2)Based on the true stress–strain curves obtained from experimental tests, the (3)In terms of the true stress–strain curve gained from isothermal tests and the developed models for the DRX of TB8 titanium alloy, the isothermal forging process of the cylindrical samples was simulated by the DEFORM-3D software. The distributions of effective strain and DRX volume fraction were analyzed for TB8 titanium alloys during hot deformation. The maximum values of the effective strain and (4)The value of the correlation coefficient was respectively 0.973 and 0.994 for the In the present paper, the DRX behavior of TB8 titanium alloy during hot working was investigated by the experiment and FEM method. The main conclusions are as follows:


Brazilian Journal of Occupational Medicine . In this issue, we introduce some changes that aim to help the journal to overcome the challenges of a scientific publication, including, but not limited to, improving the quality of the journal and expanding the list of collaborators.We proudly present the second issue of volume 18 of the Thus, we are proud to inform that we have increased the number of reviewers registered in the system, which will allow us to reduce the authors’ waiting time for approval of submitted articles. In parallel with that, we have observed a significant increase in the number of articles submitted for publication, a phenomenon that reflects the journal’s growing prestige.Furthermore, in order to meet the demands for improvement in journal publishing processes imposed by indexing bodies, we have decided to change the publisher responsible for producing the journal. Our new partner is Scientific Linguagem, a renowned Brazilian publisher that has been operating in the scientific publishing market for over 20 years, supporting both authors and editors. We would also like to highlight that the RBMT website, designed by GN1, has been renovated, having now a new layout that is modern, clean, and more comfortable to navigate.All of those actions are, in the view of the editors, required to improve the journal, and they take time to be implemented. However, we believe that we are on the right path and that we will soon be reaping the rewards of those decisions.We are continuously improving to attract more authors, from both national and international audiences, to increase the journal’s visibility, and to achieve the goals proposed by the current administration.


Bombyx mori silkworm, is a biocompatible and biodegradable polymer with excellent mechanical properties and an amphiphilic chemistry that make it a promising candidate as a controlled release drug system. The aim of this work is to synthesize naringenin-loaded silk fibroin nanoparticles (NAR-SFNs) by dissolving the SF in the ionic liquid 1-ethyl-3-methylimidazolium acetate, using high-power ultrasounds and rapid desolvation in methanol followed by the adsorption of NAR. The NAR-SFNs were characterized by dynamic light scattering, Fourier transform infrared spectroscopy and thermogravimetric analysis. The drug loading content and encapsulation efficiency were calculated. The drug release profile best fitted a first order equation. The cytotoxicity effects of free NAR, bare silk fibroin nanoparticles (SFNs) and NAR-SFNs were assessed on HeLa and EA.hy926 cells via 3--2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated the higher in vitro anticancer potential of synthesized NAR-SFNs than that of free NAR in HeLa cancer cells.Naringenin (NAR), a flavonoid present in a variety of fruits, vegetables and herbs, exhibits a wide range of pharmacological effects, including anticancer activity. Nevertheless, its application in cancer therapy is limited due to its low bioavailability at the tumour site because of its poor solubility in water and slow dissolution rate. To improve the therapeutic efficacy of NAR, emergent research is looking into using nanocarriers. Silk fibroin (SF), from the  Naringenin chroman-4-one, NAR) is a natural aglycone flavonoid and metabolite of naringin. It is widely distributed in a variety of fruits, vegetables and herbs. As a flavonoid compound, its beneficial pharmacological effects on biological systems as an anticancer, antioxidant, anti-inflammatory, hepatoprotective and anti-mutagenic compound have been described . Among tIn order to improve the therapeutic efficiency and bioavailability of drugs, scientists have developed a great variety of NDDS. Among these, synthetic or natural nanocarriers, such as solid lipid nanoparticles , polymerBombyx mori cocoons. Its use as biomaterial and nanocarrier is of great interest due to its relatively inexpensive nature, wide availability and inherent properties, including biocompatibility, environmental stability, non-toxicity and a controllable rate of biodegradation [acetate−]) for the silk dissolution. Previously, the SF was dissolved in other ionic liquids (ILs) such as 1-butyl-3-methylimidazolium chloride ([bmim+][Cl−]) to form a stable SF solution [acetate−] by high-power ultrasounds, as previously reported [Silk of Bombyx Mori was obtained from the cocoons of silkworms reared in the sericulture facilities of IMIDA  and raised on a diet of fresh Morus alba L. leaves. This silk was treated to remove the sericin with an aqueous solution of Nareported . The ion+][acetate−] (SIL) solution (10 wt. %) was prepared by adding 0.5 g of SF to 4.5 g of [emim+][acetate−] and dissolved with the aid of the ultrasonicator. A program of alternating pulses of 30 s was used for a time of approximately 20 min, with an amplitude of 30% and maximum temperature of 75–80 °C to prevent degradation of the protein (measured with the temperature probe that incorporates the equipment). The SF was added in small portions, checking each time that it was completely dissolved. Using the vortex mixer, 3 mL of ultrapure water was slowly added to this solution (freshly prepared or kept at 40 °C in the oven beforehand) to reduce viscosity, reaching a final SF concentration of 6.66 wt. % in the SIL solution. The sample was placed in a thermostatic bath at a temperature of 60 °C and then pumped and precipitated through a thermostatically controlled 0.7 mm two-fluid nozzle  into 100 mL of methanol (−20 °C) while stirring (1800–2200 rpm). The feed rate of the SIL solution into the spray was maintained at 13.64 mL/min . The nitrogen pressure was maintained at 1 bar throughout the process. Once the precipitation process was completed, the precipitated solution was stirred for two hours. The process used to synthesize SFNs was based on the technique previously described by Lozano-Pérez et al.  with mod+][acetate−] from the methanolic fractions, a BÜCHI RE-111 rotary evaporator, at 80 °C and 80 mbar, was used. As mentioned above, due to the very low vapour pressure of the IL, it can be separated from the methanol and both later reused. The recycled IL was filtered through 0.22 µm pore diameter, then maintained for 24 h in the oven at 100 °C to remove water, after which it was kept in a desiccator under vacuum and phosphorus pentoxide atmosphere until reuse.To recovery the  of NAR (Y) to NAR concentration (X) being as follows: Y = −61.674X (correlation coefficient 2R = 0.9998).High-performance liquid chromatography (HPLC) was used to measure the NAR concentration in the NAR/ethanol drug-loading solutions. The HPLC equipment  was equipped with an e2695 separation module, an injector valve with a 100 µL loop, and a Waters 2998 photodiode array (PDA) detector. The results were processed by Empower 3, Waters software . Separations were performed on a SunfireY) and NAR concentration (X) in the range 0.005–0.043 mM (Y = 16.232X), with a correlation coefficient of 2R = 0.9983. The spectrophotometric detection was determined at an absorption maximum of 290 nm.The drug content in PBS  solutions was measured by Ultraviolet-Visible (UV-Vis) spectroscopy. Analyses were carried out using a UV-Vis HELIOSα spectrophotometer  and good linear correlations were obtained between absorbance  in natural light, while the other was refrigerated (4 °C) in darkness. The NAR concentration of both was measured daily by HPLC over 12 days.After lyophilisation was completed for each batch of particles (loaded and unloaded), the mean hydrodynamic diameter (Z-average), the polydispersity index (PdI), the zeta potential and the electrophoretic mobility were measured by DLS using a Zetasizer Nano ZS instrument . All measurements were obtained in ultrapure water at 25 °C and at a 173° angle relative to the source. Before the measurements, each sample was sonicated for 3 min at 30% amplitude with pulses of 15 s ON and 15 s OFF; the NAR-SFNs concentration was 0.66 mg/mL. All measurements were performed in triplicate and values expressed as mean ± SD.−1 in the spectral range of 1750–900 cm−1 with a zero-filling factor of 2 and Fourier-transformed using the Blackman-Harris 3-term apodization function. Each measured spectrum was averaged from 64 scans. A background spectrum without sample with the same number of scans was collected before each measurement.Infrared spectral data of the SFNs, NAR and NAR-SFNs were obtained by ATR-FTIR in a Nicolet iS5 spectrometer coupled to a diamond crystal iD7 ATR module . OMNIC Software V9.9.471  was used for the control and processing spectral data. Interferograms were recorded at a resolution of 2 cmSpectra of the samples were acquired by placing 2.5 µL of the nanoparticle dispersion or NAR solution (15 mg/mL) on the top of the ATR-crystal after drying under a gentle stream of nitrogen.TM microscope . The NAR-SFNs sample was deposited as a powder and coated with a thin gold layer.FESEM was used for the morphological characterization of NAR-SFNs, using a FEI SciosThe thermal behaviour of NAR-SFNs was determined by TGA and compared with the thermal profiles of the SFNs and NAR individually. The measurements were carried out by a thermal gravimetric analyser , in a temperature range of 25−800 °C and heating rate of 10 °C/min under an inert nitrogen dynamic atmosphere. Weight loss was recorded and plotted against temperature. The derivative of the weight loss curves (DTGA) was calculated and also plotted versus temperature.DLC and EE quantify the success of the loading with respect to the mass of the nanoparticles and the mass of the drug, respectively. These were calculated according to Equations (2) and (3):2 for both cell lines. Cells were subcultured using a solution of 0.25% trypsine-0.25 mM ethylenediaminetetraacetic acid (EDTA) and the medium was changed twice a week. It was checked that all cell lines were mycoplasma-free, before and after the experiments.Human cervical cancer cells (HeLa) and Human umbilical immortalized cells (EA.hy926) were purchased from the American Type Culture Collection . The culture medium used was Dulbecco’s Modified Eagle Medium (DMEM) with a low content of glucose (1 g/L). The medium was supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM glutamax, 1% antibiotics (penicillin-streptomycin) and 1 mM pyruvate at 37 °C under a humidified atmosphere containing 5% CO3 cells/well were seeded onto a 96-well plate and incubated at 37 °C. After 24 h, the culture medium of each well was replaced with fresh medium and cells were treated with different concentrations of free NAR (0.39–50 µg/mL), SFNs (1.95–250 µg/mL) and NAR-SFNs (1.95–250 µg/mL). In each experiment, growth medium without nanoparticles was used as a control. Cells were incubated at 37 °C for 48 h. Then, the media was removed and 200 µL of MTT -2,5-diphenyltetrazolium bromide) solution at a final concentration of 1nmg/mL were added and left in darkness for 4 h, after which the MTT was removed and 100 µL of dimethyl sulfoxide (DMSO) were added. Absorbance was measured in a microplate reader (Floustar Omega) spectrophotometer at 560 nm. Each sample was tested in three independent sets with triplicate points.A total of 5 × 10® vials containing a mixture of 100 μL of water and 800 μL of methanol and 10.83 mg of NAR-SFNs. The reaction mixture was incubated for 30 min at 25 °C in darkness and the absorbance was recorded at 517 nm using a UV-Vis HELIOSα spectrophotometer . As standard control, ascorbic acid solutions (0–2.5 nmol/mL in methanol) were used. The radical scavenging activity is presented as Ascorbic Acid Equivalents per milligram of NAR in the NAR-SFNs (nmol AAE/mg NAR). The ability to scavenge the DPPH radical was also expressed in terms of the inhibition percentage calculated with Equation (4):0A is the absorbance of the control and 1A is the absorbance of the sample of NAR-SFNs. Apart from for the sample of NAR-SFNs, inhibition percentage of the ascorbic acid solutions was also calculated from their respective absorbances and represented versus their concentrations.As stated above, NAR is widely considered as an antioxidant compound with activity against oxidative damage of human cells. The ability of the NAR-SFNs to annihilate the DPPH radical  was investigated using the method described by Blois . CompounThe experiment was repeated three times and the results are expressed as mean ± standard deviation (SD). In order to study the drug release properties of the NAR-SFNs, drug release experiments were carried out with the three NAR-SFNs (obtained with different NAR/SFN loading solution ratios) in PBS 1x (pH = 7.4) with 0.5% of TWEEN 80. The concentration and pH of this medium is similar to those of blood. Thirty milligrams of NAR-SFNs were dispersed in 1 mL of PBS 1x by ultrasonication and incubated at 37 °C in a tube shaker for 2 days. At predetermined time points , the samples were centrifuged for 15 min at 13400 rpm. The concentration of NAR in the supernatant was measured by UV-Vis spectrophotometry as described in p > 0.05) and homoscedasticity  were met, the statistical significance was determined using the parametric tests of Tukey (p < 0.05) and ANOVA (p < 0.05) for the comparisons of two or more groups, respectively.Data were presented as mean ± SD (standard deviation), calculated from three independent samples per condition by using GraphPad Prism 8.0.1 software . As normality , and darkness in a refrigerator (4 °C). Over the course of 12 days, the concentration of the NAR/ethanol solution exposed to light and room temperature decreased from 0.279 to 0.235 g/L, while the concentration of the solution kept in the fridge decreased from 0.279 to 0.271 g/L. Although the decrease in NAR concentration was small in both cases, all the solutions containing NAR or NAR-SFNs were kept refrigerated throughout the study and were never exposed to light or ambient room temperature for more than one hour.The SFNs and NAR-SFNs  were characterized by DLS to ascertain their hydrodynamic diameter (expressed as Z-average), zeta potential, PdI and electrophoretic mobility. As can be seen in In summary, the NAR-SFNs increased in size with the NAR concentration in the loading solution, but still retained an average particle size of <200 nm, while maintaining a relatively high absolute zeta potential value. In terms of their physical characteristics, therefore, they appear to be entirely suitable as potential nanocarriers.ATR-FTIR was carried out to obtain information about the secondary structure of the proteins based on the position of the signals and relative intensity. −1, denoting amide I, II and III, respectively [acetate−] using high power ultrasounds and via atomization in methanol, followed by the adsorption of NAR during incubation. The DLC increased with increasing mass ratios of NAR/SFN during incubation in the range studied. As the DLC increased, the hydrodynamic diameter increased, and the zeta potential decreased. Both factors contribute negatively to colloidal stability, but hydrodynamic diameter <180 nm and zeta potential >30(in absolute terms) make both SFNs and NAR-SFNs suitable systems for therapeutic applications. By means of FTIR, the characteristic bands of SF were observed from the three large peaks denoting amide I, II and III. The antiparallel β-sheet secondary structure of SF was regenerated upon contact with organic polar solvent. Furthermore, the TGA study revealed that the thermal decomposition of the NAR-SFNs occurs at around 295 °C. We also found that the NAR release profiles of NAR-SFNs made from different loading solutions showed good agreement with a first order equation. Finally, it was demonstrated that NAR-SFNs retain their antioxidant activity and antitumor activity towards HeLa cells, while the viability of healthy cells hardly decreases. This feature is highly promising, since it broadens the possibility of using these nanoparticles for cancer treatment, diminishing the side effects that frequently occur with currently used therapies.NAR-SFNs were successfully synthesized by dissolving SF in the ionic liquid [emim


To evaluate the surgical outcomes of cryopreserved and dehydrated human amniotic membrane (hAM) graft transplantation for macular hole (MH) and macular hole retinal detachment (MHRD) repair.  This retrospective, interventional case series was conducted in two hospitals. Two types of hAM grafts, namely, the dehydrated form  and the cryopreserved form , were consecutively used in MH surgeries. Anatomical and functional outcomes between the 2 types of hAM grafts were compared. p=0.03). In the other 4 cases with persistent MH, 3 had graft dislocation and 1 had a reopened MH with graft contraction. There were no significant differences in closure rate  or VA improvement  between the 2 kinds of hAM graft.  Seventeen patients  males) were enrolled. Of them, 11 patients had persistent MH, 3 had MH without prior surgery, and 3 had MHRD. A cryopreserved hAM graft was used in 10 patients, and a dehydrated hAM graft was used in 8 patients. One patient used a cryopreserved hAM in the first MH surgery and a dehydrated hAM in the second surgery for extramacular hole with retinal detachment. After a 6-month follow-up, 13 (76.5%) patients had sealed MHs. The average visual acuity (VA) of cases with sealed MHs improved from 1.38 ± 0.62 to 1.12 ± 0.47 logMAR ( This preliminary case series showed that both cryopreserved hAM and dehydrated hAM are feasible alternative grafts for either persistent or recurrent MH. Both approaches have similar anatomical and functional outcomes. Persistent macular holes (MHs) and macular hole retinal detachment (MHRD) or primary MHs and MHRD with large holes are very challenging to manage and often cause severe visual impairment, especially in highly myopic eyes –4. The rβ signal transduction [Human amniotic membrane (hAM) grafts have been widely applied in the treatment of ocular surface disorders for a long time, as they serve as a scaffold for cell growth and migration, facilitate re-epithelialization by providing growth factors, and reduce inflammation and scarring by inhibiting TGF-sduction –17. Recesduction –20. Cryoμm.Two types of commercial hAM graft products are now available for medical use: cryopreserved and dehydrated amniotic membranes. They differ in morphology and structure due to differences in processing . Both tyThis retrospective, consecutive case series was conducted in Taipei Veterans General Hospital and National Yang-Ming University Hospital. This study adhered to the Declaration of Helsinki and was approved by the Institutional Review Boards of Taipei Veterans General Hospitals (2019-06-023CC) and National Yang-Ming University Hospital (2019A026). Informed consent for surgery was obtained from all the patients.We retrospectively reviewed the medical records of patients who received vitrectomy and hAM graft transplantation from October 2018 to August 2019. All patients had MH with or without retinal detachment before surgery and were followed up for at least 6 months. All surgeries were performed by the same experienced surgeons (S. J. Chen and D. C. Tsai). Detailed ophthalmic histories were taken preoperatively, including previous ocular diagnoses, duration of MH, previous surgeries, or traumatic injury. All patients underwent a thorough ophthalmic examination before and after the surgery , including best-corrective visual acuity , axial length, slit-lamp biomicroscopy, fundus color photography, optical coherence tomography (OCT), and optical coherence tomography angiography . The size of the macular hole was defined as the minimum diameter of the hole measured on the OCT image using the caliper function.μm larger than the MH size calculated according to OCT before surgery. One of either the dehydrated  graft or the cryopreserved hAM  graft was randomly selected for each patient according to the availability of the graft at the time of surgery. For easier identification of the two sides of the graft, the epithelial side of the graft was stained with Brilliant Blue G  or indocyanine green  before cutting. The trimmed hAM graft was then gently implanted into the MH with intraocular forceps and secured by trapping the graft edge beneath the retina 360 degree, if possible, with the stromal side in contact with the retinal pigmented epithelium (RPE) or at least under the retina as much as possible and left the rest of the graft above the retina in patients with large MHs and concomitant RD. To make sure that the graft was not inserted upside down, we identified the sticky stromal side with the intraocular forceps before implantation into the MH, as described by Caporossi et al. [6) or silicone oil. All patients maintained the prone position immediately after the surgery and kept the face-down position for one to two weeks. They stayed in the hospital for 3–5 days and were then discharged. We instructed the patients to maintain the prone position at home for at least 16 hours a day for 1 week for patients with MH and 2 weeks for patients with MHRD.All cases underwent standard 23-gauge 3-port microincision vitrectomy with a Constellation Vision System . After completing vitrectomy and internal limiting membrane peeling for primary MH and MHRD, the hAM graft was trimmed according to the size of the MH by a 1 mm- or 1.5 mm-skin biopsy punch or simply cut using scissors with the aid of a caliper. The size of the graft was designed to be approximately 300–500 i et al. . When thPathological exam of the 2 types of commercial hAM graft products was performed to find out the differences in their structures at cellular level.U test. p < 0.05 was considered to indicate statistical significance.The statistical analysis was performed using SPSS software . Descriptive statistics were used to obtain mean and standard deviation. Univariate analyses were conducted using Pearson's chi-square test or the Mann–Whitney In the pathological exam, both types of hAM graft have a single layer of devitalized epithelial cells Figures . Cryopreμm (range 230–3285 μm). Cryopreserved hAM grafts were used in 10 patients, and dehydrated grafts were used in 8 patients   including 3 cases of MHRD had sealed MHs. There was no significant difference in the MH closure rate between patients with cryopreserved and dehydrated hAM grafts  . Among tp=0.68) .p=0.03). When comparing the final visual outcome or visual gain of patients receiving cryopreserved or dehydrated hAM grafts, there were no significant differences between the two groups . In the other 4 cases of persistent MH, 3 cases (16.7%) had hAM graft dislocation (p=0.27) in these patients. There was no significant difference in visual gain between highly myopic  and nonhighly myopic  patients . No significant difference in visual gain was found between patients with large MHs (≥1000 μm) or small MHs (<1000 μm) . After excluding those with MHRD and silicone oil tamponade, MH sizes were significantly larger in patients with graft dislocation, reopened MH, and partial closure  than in those with sealed MHs , while age and axial length were not significantly different between the 2 groups.The average BCVA of cases with sealed MHs improved from 1.38 ± 0.62 to 1.12 ± 0.47 logMAR  for both types of grafts. In these cases, sealed MHs could be observed at the first follow-up (2 weeks postoperatively). Although cryopreserved hAMs have better preserved structural and biochemical integrity than dehydrated hAMs, we found no significant difference in healing rates or in the regeneration of the outer retina. These grafts acted as scaffolds for glial cell proliferation above the graft as in case 14  and alsoBritish Journal of Ophthalmology, submitted).In this short-term study, graft contracture with parafoveal atrophy was found around the hole in four highly myopic cases  in the first month after surgery. We hypothesized that the potential contributing factors may include preexisting myopic maculopathy, graft insertion-related surgical trauma, and hAM graft contracture. To minimize the surgical trauma to the RPE, we slid the graft into the subretinal space as gently as possible and avoided excessive manipulation in all cases. Parafoveal atrophy did not develop in the other 3 cases with graft dislocation, 2 of which were highly myopic. Thus, we do not think that surgical trauma itself can fully explain this phenomenon. We hypothesized that the strong adhesion of the stromal side of the hAM graft to RPE may drag the pigment and cause parafoveal atrophy during graft contracture. The mechanism of atrophy in these cases still needs further investigation . The cryopreserved grafts were used for two cases, and the dehydrated graft was used for one case; all graft dislocation was noted on the first postoperative day. All 3 dislocated cases had grafts inserted 360° underneath the retina and used SFOur study was limited by the small sample size. Further studies with longer follow-up periods are needed to confirm the clinical application of hAM grafts and evaluate the differences between the two types of hAM graft.This preliminary case series showed that both dehydrated and cryopreserved hAM grafts were feasible alternatives for MH and MHRD. Both approaches have similar anatomical and functional outcomes. The surgical procedure provides a high sealing rate for persistent MH, MHRD, and giant MHs. However, grafts may dislodge when the size of the MH is large.