Patients waiting for a kidney transplant by far exceed available organs. AB0 incompatible living donor kidney transplantation (AB0i LDKT) represents an additional therapeutic strategy, but with higher risk for complications. We aimed at evaluating outcomes of AB0i LDKTs compared to compatible (AB0c) controls at our Institution.Retrospective matched case – control study (1:2) comparing AB0i vs. AB0c LDKTs from March 2012 to September 2021. Considered outcomes: graft function, acute rejection, sepsis, CMV infection, BK virus reactivation, death-censored graft survival, patient survival.2) of 67 vs. 66 at 1 year (p = 0.41), 63 vs. 64 at 3 years (p = 0.53). AB0i recipients had a statistically significant higher incidence of acute rejection, acute antibody-mediated rejection and sepsis within 30 days , 1 year  and 3 years  after surgery. There was no difference in CMV infection, BK virus reactivation, death-censored graft survival between the two groups. Patient survival was inferior in AB0i group at 1 and 3 years  due to early death for opportunistic infections. AB0i LDKTs spent longer time on dialysis (p = 0.04) and 82.3 vs. 38.3% controls had blood group 0 (p = 0.003).Seventeen AB0i LDKTs matched to 34 AB0c controls. We found excellent graft function, comparable in the two groups, at all considered intervals, with an eGFR (ml/min/1.73 mAB0i LDKT is an effective therapeutic strategy with graft function and survival comparable to AB0c LDKTs, despite higher rates of acute rejection and sepsis. It is an additional opportunity for patients with less chances of being transplanted, as blood group 0 individuals. End stage renal disease (ESRD) represents a constantly growing health-care burden worldwide. Kidney transplantation stands as the best therapeutic option for patients with ESRD, with lower morbidity and mortality rates compared to dialysis . HoweverAll AB0i LDKTs performed at the Kidney Transplant Center of the University and Hospital Trust of Verona from March 2012 to September 2021 were enrolled as cases and matched to two AB0c LDKT controls. Matching was based on donor and recipient age, parental affiliation , number of HLA mismatches and time of transplantation. Exclusion criteria for matching eligibility were: combined-organ transplant, a positive cross-match, a follow up shorter than 6 months, unavailability of complete data.IgM and IgG isoagglutinin titers were measured using a conventional tube test for agglutination . Titers 2) was administered 30 days before the procedure. Sessions of PP were started two to three weeks before transplantation, on alternate days, aiming at achieving a titer ≤8. For PP, a centrifuge-driven cell separator was used, with 1 plasma volume removed per each session and replaced with albumin 5%. To reduce the risk of bleeding, fresh frozen plasma was used in the 2 sessions before transplantation and in additional PP after surgery. From January 2017, routine PP after transplantation were omitted and performed “on demand” in case of titer rebound (≥16) or acute antibody-mediated rejection (aAMR). The day after PP, high dose IVIg  were administered until transplantation. Two weeks before transplantation immunosuppression therapy with tacrolimus , mycophenolate sodium , and methylprednisolone (16 mg per day) was started.All AB0i LKDTs received Rituximab, standard non-selective plasmapheresis (PP), IVIg and standard immunosuppression before transplantation. Rituximab  for 3 months after transplantation. No routine systemic fungal prophylaxis was used.All AB0i and AB0c LDKTs received, as induction therapy, either Basiliximab  or rabbit anti-thymocyte globulin , and methylprednisolone . Tacrolimus, mycophenolate sodium and steroids were used as maintenance therapy. All patients received valganciclovir (Roche) for 3–5 months as prophylaxis for cytomegalovirus (CMV) infection, according to donor–recipient serological status, and trimethoprim/sulfamethoxazole or dapsone as prophylaxis for For each donor – recipient couple we registered: age, sex, blood group, number of HLA mismatches, parental relationship. For all donors: baseline serum creatinine (SCr), estimated glomerular filtration rate (eGFR), hypertension. For all recipients: cause of ESRD, presence of diabetes mellitus, type and duration of renal replacement therapy, panel-reactive antibody (PRA), donor-specific antibodies (DSA), anti-HLA non DSA antibodies, previous transplants.3 copies/mL. As secondary outcomes, we considered death-censored graft survival and patient survival at 1 and 3 years of follow up.We considered as primary outcomes graft function and the following complications: delayed graft function (DGF), acute rejection (AR), sepsis, viral infections . For graft function, SCr and eGFR were considered at 1, 3, 6 months, 1, 3 years of follow-up. eGFR was calculated with CKD-EPI 2021 equation . All “coT-test/one-way ANOVA was used to compare means. Chi-squared test and Fisher exact test were used to compare categorical variables. Survival functions were plotted on Kaplan–Meier curves and compared using log-rank test. A p value < 0.05 was considered for statistical significance. All data were processed using Stata 14.1 for Windows .Means and standard deviations were used for the description of continuous variables. Categorical variables were expressed in numbers and percentages and divided in classes. Student's p = 0.04). Blood type was 0 in 14 AB0i patients (82.3%) vs. 13 (38.2%) AB0c controls (p = 0.003). The following combinations of AB0i transplants were performed: 10 A to 0 (58.8%), 4 B to 0 (23.5%), 2AB to A (11.7%) and 1 AB to B (5.9%). Complete data on donors and recipients are summarized in Between March 2012 and September 2021, 155 LDKTs were performed at our Institution. Seventeen were AB0i LDKTs, enrolled as cases and matched to 34 AB0c controls. There was no difference in donors' basal kidney function between the two groups. Two AB0i recipients had a history of previous transplant, two had a positive PRA, and one had positive DSA at the time of transplantation. One AB0c recipient had a positive PRA, none had DSA nor had a previous transplant. Eleven recipients (64.7%) in AB0i vs. 13 (38.2%) in the AB0c group were on hemodialysis, three (17.6%) vs. seven (20.6%) on peritoneal dialysis, three (17.6%) vs. 14 (41.2%) had a pre-emptive transplant. Mean time on dialysis before transplantation was 24.2 months in AB0i group vs. 9.9 months in AB0c group (≥64 in 14 patients (82.3%), being 256 in three patients (17.6%) and 128 in other three (17.6%). All patients achieved a titer ≤8 within the day before surgery , 118 vs. 119 at 1 year (p = 0.53), 123 vs. 116 at 3 years (p = 0.47) in AB0i and AB0c LDKTs, respectively. Mean eGFR (ml/min/1.73 m2) was 57 vs. 60 at 1 month (p = 0.28), 67 vs. 66 at 1 year (p = 0.41), and 63 vs. 64 at 3 years (p = 0.53) in AB0i vs. AB0c LDKTs. Complete data are shown in There was no statistically significant difference in graft function between the two groups at all considered time points, with excellent functional outcomes. Mean SCr (μmol/L) was 132 vs. 130 at 1 month  and aAMR. Patient's isoagglutinin titer was 128 at baseline and 8 at the time of surgery. Therapy with Eculizumab, PP, and methylprednisolone boluses was effective, with a regain of function and an eGRF of 34 ml/min 1.73 mp = 0.016), 4 (23.5%) vs 1 (2.9%) aAMR (p = 0.02). Within 1 year, 8 (47%) vs 5 (14.7%) AR events were registered in AB0i vs AB0c controls (p = 0.012), 5 (29.4%) vs 2 (5.9%) aAMR (p = 0.02). After 3 years, 9 (52.9%) vs 5 (14.7%) AR events occurred in AB0i vs AB0c controls (p = 0.004), 6 (35.2%) vs 2 (5.9%) aAMR (p = 0.006). All AR events in both groups were responsive to therapy and did not lead to graft loss. None of the AR events in the AB0i group was associated with a rebound in isoagglutinin titer.AB0i recipients had a statistically significant higher rate of AR and aAMR at 30 days, 1 and 3 years after transplantation, compared to AB0c controls. Within 30 days, 7 (41.2%) vs 4 (11.8%) AR events occurred in AB0i vs AB0c LDKTs (p = 0.001) within 30 days after surgery, 8 (47%) vs. 2 (5.9%) at 1 year (p = 0.0004), and 8 (47%) vs. 5 (14.7%) at 3 years (p = 0.012). Urosepsis was the most frequent recorded infection in both groups. Six of the 8 AB0i LDKTs who had a sepsis, had an AR and had received additional immunosuppressive therapy before sepsis onset.Sepsis events, as well, were significantly more frequent in AB0i cases vs. AB0c controls at all considered time-points. Six (35.3%) AB0i vs. 1 (2.9%) AB0c LDKTs developed a sepsis , as two AB0i recipient died in the first 6 months after transplantation due to infection. The first patient died after 47 days due to P. jirovecii pneumonia, while the second patient died after 138 days for disseminated fungal infection.No patient experienced graft loss due to rejection, infection or other causes, with an identical death-censored graft survival of 100% in the two groups at 3 years. Patient survival was inferior in AB0i group at 1 and 3 years  , one of In our investigation, AB0i LDKTs had an inferior patient survival due to early death for opportunistic infections within the first year after transplantation, a finding reported also by other studies in the literature and to be considered as directly related to higher immunosuppression implied in AB0i transplantation. In De Weerd et al. meta-analysis, AB0i recipients had a lower 1-year patient survival (96% vs. 98%), with 49% of deaths caused by infectious events compared to only 13% in AB0c controls . The CTSFinally, in our study, AB0i recipients had mostly blood group 0 (82.3%), spent longer time on dialysis before transplantation, had a lower rate of pre-emptive transplantation, a history of sensitization. All these data underline how AB0i transplantation represents an additional therapeutic option for patients with reduced chances of receiving an organ .Considering risks of AB0i LDKT, an alternative option for AB0i pairs to be mentioned is kidney paired donation (KPD), a well-established strategy to overcome AB0 and HLA incompatibility barriers without desensitization . In KPD,Our study has some limitations, being a single-center, retrospective, observational study with a limited number of cases and a medium-term follow up. However, as point of strength, the matching criteria, the analogous immunosuppressive induction therapy and prophylaxis for infection made the comparison between the two groups more robust.In conclusion, we observed excellent outcomes in terms of graft function and graft survival in AB0i LDKTs on a medium term follow up, comparable to AB0c controls. During the first month and first year after transplantation, AB0i recipients experienced a higher burden of AR and sepsis, which, however, did not lead to graft loss. AB0i transplantation represents a viable therapeutic option for individuals with an available, but incompatible living donor, and for patients with reduced chances of being transplanted, including blood group 0 individuals. Further investigation and development in this field is warranted, as AB0i LDKTs represent one of the possible strategies to answer a constantly growing demand for transplantation.The raw data supporting the conclusions of this article will be made available by the authors at request, without reservation.The study was conducted ethically in line with the principles of the Declaration of Helsinki.MC, LB, and PP: research idea and study design. MC, PD, GU, RN, FN, ZB, and PP: data acquisition. MC, LB, PP, and GGam: data analysis/interpretation. LB: statistical analysis. MC and LB: wrote the manuscript. All authors reviewed the article and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Community Eye Health Journal.First-line management of and preparation for the following eye emergencies are addressed in this issue of the


A data-driven approach to characterizing the risk of cyanobacteria-based harmful algal blooms (cyanoHABs) was undertaken for the Ohio River. Twenty-five years of river discharge data were used to develop Bayesian regression models that are currently applicable to 20 sites spread-out along the entire 1579 km of the river’s length. Two site-level prediction models were developed based on the antecedent flow conditions of the two blooms that occurred on the river in 2015 and 2019: one predicts if the current year will have a bloom (the occurrence model), and another predicts bloom persistence (the persistence model). Predictors for both models were based on time-lagged average flow exceedances and a site’s characteristic residence time under low flow conditions. Model results are presented in terms of probabilities of occurrence or persistence with uncertainty. Although the occurrence of the 2019 bloom was well predicted with the modeling approach, the limited number of events constrained formal model validation. However, as a measure of performance, leave-one-out cross validation returned low misclassification rates, suggesting that future years with flow time series like the previous bloom years will be correctly predicted and characterized for persistence potential. The prediction probabilities are served in real time as a component of a risk characterization tool/web application. In addition to presenting the model’s results, the tool was designed with visualization options for studying water quality trends among eight river sites currently collecting data that could be associated with or indicative of bloom conditions. The tool is made accessible to river water quality professionals to support risk communication to stakeholders, as well as serving as a real-time water data monitoring utility. Most The expanding awareness, if not actual number, of freshwater cyanoHABs reported over the last couple of decades has stimulated an intense amount of research to better understand their ecology for management and prevention ,10. ThisCyanoHABs appear to be impacting lentic systems the most, but lotic systems are also susceptible ,35. LargAn unprecedented bloom event on the Ohio River in 2015 brought the potential risks of cyanoHABs in larger rivers of the United States into the spotlight –43. It wThere are 32 municipal drinking water treatment plants (DWTPs) on the Ohio River, serving 5 million people. Over the course of the 2015 cyanoHAB, 20 of the DWTPs had to take precautions by providing additional treatment, increasing monitoring, or closing their intakes. The extra treatment cost was estimated at $2 million USD based on a survey of utilities conducted by staff of the Ohio River Valley Water Sanitation Commission (ORSANCO), which administers the provisions of an interstate compact that includes a responsibility of monitoring water quality in the Ohio River. The Greater Cincinnati Water Works, alone, reported additional treatment costs of 7000.00 USD per day for two months. The 2015 cyanoHAB caught water resource professionals and drinking water utility managers off-guard, creating much interest in understanding the cause of the event and in implementing risk management procedures and practices to be better prepared for subsequent ones. Risk management and mitigation approaches used for larger lakes may not easily translate to flowing water systems. For instance, the long and narrow configurations of river systems limit the use of satellite-based risk characterization at present –47. ThisThis research was initiated in response to this HABs risk management need. The study goal was to produce a tool that could be used by ORSANCO for assessing the potential for cyanoHABs in real time and at multiple locations along the river’s length. When the project started, the effort was constrained by the fact that there was only one cyanoHAB event ever documented on the Ohio River. However, in 2019, the river experienced another bloom. This event was smaller (ca. 482 km) and did not last quite as long: it was first reported on 11 September and lasted about 30 days. Prior to the 2015 and 2019 events, the largest algal bloom on the Ohio River was believed to have occurred in 2008, covering 48 km and lasting about 10 days. However, this event could not be verified as a cyanoHAB based on the available grab sampling data . We hypo2.2.1.2 watershed. The river is also strongly impacted by industrial discharges [The Ohio River receives direct discharges of nutrients from 182 wastewater treatment plants that are permitted for a combined daily load of nearly 1 billion gallons, although typically individual plants operate lower than their permitted level. There are forty-nine communities along the river with combined sewers, which may release untreated wastewater during large rainfall/runoff events through 965 outfalls. Additionally, there are 69 communities with municipal separate storm sewer systems draining urban runoff to the river. Furthermore, nutrients enter the river from the roughly 40% agricultural land cover of the 528,204 kmscharges .The model development component of the risk characterization tool needed to be tied to current and actively maintained water data acquisition. However, it would have to depend on historic data of the same type as that currently acquired, as both current and older data would be used to test assumptions and hypotheses about the controlling factors and ecological responses related to the river’s major cyanoHAB events. Several entities have water monitoring stations on the mainstem of the Ohio River. Of these, we focused attention on sources that were likely to have consistent, long-term, and current information available. These included the U.S. Geological Survey (USGS), the U.S. Army Corps of Engineers (USACE), The Ohio River Forecast Center (OHRFC), ORSANCO, and several drinking water treatment utilities. 2.2.Starting with 25 years of river stage data, we focused our attention on identifying locations where discharge estimates over the whole range of stages were likely to be the most accurate. There were 18 L&D locations that met the data requirements . Each on2.3With the time series of discharges, we wanted to determine if the 2015 cyanoHAB could be related to unique flow conditions, which, in turn, could be used as a basis for risk modeling. We produced a conceptual cause and effect model to guide the analysis of the empirical data . The con2.4First, we had to decide how a bloom would be defined for modeling. When the effort began, we had either zero or one bloom occurrence at each site, as only the 2015 event had taken place at this time. Defining the precise beginning and end of a bloom anywhere on the river is uncertain in practice: Blooms thus far have been first reported by river observers on seeing atypically green waters or surface scums. Once reported, the suspected bloom is subsequently sampled to verify if toxin-producing cyanobacteria are present and to test for toxins. If toxins are detected, then “bloom condition” status remains in effect from a risk management perspective until two consecutive samples collected a week apart are toxin-free. This means that, in practice, bloom status is operationally disconnected from actual dynamics of cyanobacteria cell densities. Therefore, we had no choice other than to model a binary response , one based on when an observational report of a bloom was first received on the front end and the date when river sections were considered toxin-free on the back end . TherefoOther key modeling considerations including both assumptions and data characteristics were the availability of 25 years of flow data at 25 locations on the river; spatial and temporal correlations; that there was no “true” replication; and an assumption that years are independent. We desired a model that would estimate the probability that the current year at a given site would be a bloom year, and that could be quantified as such on any given day during the period that a cyanoHAB might occur on the river, which we qualified as May through October. We acknowledge that because blooms have occurred so infrequently on the Ohio River , this constrains the reliability of the model for future occurrences. With this acknowledgment, our approach to model development proceeded with the notion that the modeling component of the risk characterization tool would be updated annually as more blooms occur in the future.2.4.1To normalize flow rates across sites, we used “exceedances”, which are rankings based on the percentage of maximum flow for the period of record. The lowest flow at a site has an exceedance of 100%, and the highest flow has an exceedance of 0%, so high flows are associated with low exceedances, and low flows are associated with high exceedances. During an exploratory phase, we developed temporal lag terms using the exceedances to reflect the hypothesized flow conditions required for a cyanoHAB. The first lag term was meant to capture the period of low flows preceding a bloom. We started with averaging the previous 30 days of daily average flow exceedances at a site for all days in the site’s time series. When average flow rates are very low over this lag period, the exceedances are high, and, therefore, the value of this term is high. A second lag term was developed to reflect the period of high flows prior to the 30 day low flow period. To start, we calculated an average exceedance based on a 30 to 75 day lag period. The lower this average exceedance, the higher the average flows would have been for this period 30 days prior to the bloom. We then took a ratio of these two lag terms to characterize the hypothesized prerequisite conditions as one variable. A relatively high value of the ratio of the early lag term over the later one indicates a period preceding any given point in the time series when very low flows were preceded by very high flows.Plotting the ratio of lagged exceedance terms suggested that the ratio entered a rapidly increasing phase that appeared unique for the 2015 bloom year. We hypothesized that producing another predictor variable that quantified the extent the ratio was increasing prior to a bloom event would likely improve model fit.In the preliminary model fitting phase, we used a mixed effects binary logistic regression approach for rapid testing. Because the averaging periods of the exploratory phase’s lag terms were arbitrary based on visualizing the discharge time series , we systmaxratio” as the maximum 1–19:21–55 ratio that occurred prior to the 2015 cyanoHAB start date at a site. For other years, the maxratio is defined as the maximum ratio that occurred at any time during which a cyanoHAB is deemed possible . We defined the predictor variable “inc15” as the number of days in the 15 days prior to the day the maxratio occurred over which the ratio increased. The predictors are summarized yearly, but probabilities are estimated daily by using daily 1–19:21–55 day ratio and number of increasing ratio days.Using the slope rather than a simple count to characterize the ratio’s increasing phase did not improve the model fit, and the 1 to 19 day and 21 to 55 day ratio significantly outperformed all other lag term combinations . meanrt” was calculated as the mean residence time during the first (1–19 day) lag period in 2015. It is an integer between 0 and 15 and it does not change year to year.We also considered an estimate of a site’s residence time as a potentially important predictor. This was based on our conceptual model  and the 2.4.2.We used Bayesian hierarchical regression for modeling. With Bayesian inference, the credible interval defines bloom prediction probability. The hierarchy is defined by year and site-specific predictors. The occurrence model responses are yearly summaries of whether a bloom occurred or not at each location for the years 1995 to 2020. Responses are assumed to follow a Bernoulli distribution, and the model is defined with a logit link function. The logit is the logarithm of the odds of bloom occurrence where odds are defined by the probability of bloom occurrence divided by the probability of bloom absence si is a vector of yearly probabilities of bloom occurrence for location s; 0, …, β4 are the associated regression coefficients  would increase the odds of occurrence by a factor of e15.8 or about 7 million. Similarly, an effect of −15.8 translates to a decrease of about 7 million. A similar argument applies to the choice of the half-Cauchy for variance parameters, which has positive support yet does not restrict the magnitude of the location effect. Markov chain Monte Carlo (MCMC) sampling was used to fit the model using R 3.5.1 [The prior parameter distributions are chosen to be non-informative or weakly informative. The Cauchy distribution is a common choice for a non-informative prior because it has support over the whole real line, with infinite mean and variance . The 5th R 3.5.1 , the STA R 3.5.1 . Three c2.4.3.s is a vector of daily probabilities of bloom occurrence for location s. 0, …, β7 are the associated regression coefficients  after the date on which the maximum ratio occurred, and X3, which is a binary indicator of a sharp increase in flow , providing a mechanism for bloom cessation. The selection of 15 and 19 was based on the data during the end of the 2015 and 2019 blooms during which a decrease of this magnitude was observed at almost all locations. Boundary conditions and prior distributions are the same or similar to the occurrence model. As above, MCMC sampling was used to fit the model with four chains and 6000 iterations each. The first 2000 iterations of each chain were discarded as warmup, and the initial values were again obtained from a preliminary mixed effects model. Modeling assumptions are discussed, and convergence statistics are provided in A second model was developed in response to the experiences gained while trying to monitor and manage risks posed from the second cyanoHAB occurring in 2019. We realized then that the occurrence model lacked utility when a bloom was actively occurring. We desired a predictive model that could produce a probability of bloom conditions persisting: the persistence model. The responses of the persistence model are daily summaries of whether a bloom is occurring or not at each location for the 100 days following the maximum ratio day. Responses are assumed to follow a Bernoulli distribution, and the model is defined with a logit link function. The logit is the logarithm of the odds of bloom persistence where odds are defined by the probability of bloom persistence divided by the probability of bloom absence ficients . Predict2.5.Although WQ data were not used directly in the model development, we incorporated visualization and download options for eight sites currently reporting continuous WQ data into the same application meant to serve the results of the occurrence and persistence models. It varies among sites, but WQ variables generally include water temperature, dissolved oxygen, specific conductance, and pH, among others and depending on the site. More details on the characteristics of the available WQ data are provided in 2.6.https://shiny.rstudio.com/ accessed on 23 June 2020). The risk characterization tool is a Shiny app that was developed under a framework of (1) providing a platform for serving the risk probability modeling results and housing Statistical analyses and risk characterization tool development were completed using the R programming language . R softw3.3.1We include nutrient loads estimated from the ORSANCO data in tributary and main river sites collected during the month of July among 16 years of monitoring as supporting evidence of the supposition that excess nutrients in the river in the period just before the development of the bloom in 2015 are potentially an important controlling factor for cyanoHABs see . Total NFollowing the period of high nutrient loading, the postulated requisite period of low flows begins, which results in higher pool residence time and opportunity for the cyanobacteria biomass to build to a bloom condition. We offer supporting evidence for the supposed importance of residence time by plotting estimates for L&Ds in the upper river see . As the 3.2.Model inputs for the bloom occurrence model are location, the current day’s lagged exceedance ratio, and the number of days the ratio has been increasing. Model output includes the probability that the current year will be a bloom year with 95% credible interval. We plot the model results for bloom probability at the Greenup site in maxratio was observed for a 10 year period at the Markland site, which experienced a cyanoHAB in both 2015 and 2019, suggested the 2019 bloom was well-predicted  from when the maxratio occurred . In contrast, had we estimated the probability 25 days earlier or later, the probability of persistence would have dropped to zero.The leave-one-out cross validation of the persistence model had a misclassification rate of 3.4% . The ris3.4.https://orsanco-hab.shinyapps.io/shiny-ohio-river/# accessed on 29 December 2021). The landing page is an interactive map that serves model results in real time  the assumed limitations posed by the fact that only one or two bloom events had occurred on the river in the past at any of the data reporting locations; (2) the limited availability of data directly linked to the phytoplankton community at any site; and (3) the expressed need of the end users to be able to assess the risk of cyanoHABs over the entire river at once and in real time. These drove the predictive modeling to focus on the interpretation of historical flow dynamics and the configuration of the Shiny app that reports prediction probabilities with additional diagnostics for interpreting the water quality from the limited number of sites with continuously reported data. Here, we offer the scientific rationale for why we consider this a sound approach for handling cyanoHAB risk characterization in a regulated river like the Ohio.First, much of the research on cyanoHAB forecasting to date has benefited from satellite-based data. Good examples of this in the United States come from the Western Basin of Lake Erie ,54 and tSecond, while direct measurements of the phytoplankton community are invaluable to assessing community structure and the presence of toxin producing taxa, these data come from a grab sampling effort that requires a physical presence to take the sample and have processing times that can range from several hours to many days before getting the results. Nevertheless, we did consider opportunities for incorporating grab sampling data into our framework more directly. As described in Planktothrix and Microcystis were the most abundant among the cyanobacteria, but two weeks later, while the bloom toxicity remained high, neither genus was observed. Then a week after this, Microcystis was again a community dominant (see The results suggested that, even under low flow conditions, the river hydraulics imparted variability that would constrain the utility of grab sampling for predictive modeling. While cyanobacteria dominated the community coincident with the timing of the Markland bloom report in 2019 , they hanant see . This teThird, we considered the in situ sensing of chlorophyll and phycocyanin pigments as more direct and time relevant indicators of cyanobacteria biomass . Indeed,As a case in point, we can demonstrate such ineffectual in situ sensor data from a suite deployed in the Greenup pool on the Ohio River. The data from the sensors produced ambiguous results on bloom status during the 2019 cyanoHAB, with the in situ signals of both chlorophyll and phycocyanin suggesting no indications of increasing or excess phytoplankton biomass prior to or during the period of the bloom see . This laFinally, while rare, we note that there are a few examples of cyanoHAB prediction models developed for other regulated river systems outside of the United States. Primary examples come from hypereutrophic rivers in South Korea and China, e.g., ,67–69 anThe results from these studies provide supporting evidence that low flow conditions must be of long duration for cyanobacteria to concentrate to a bloom status on a large, regulated river. However, unique to our treatment was the use of the historical flow time series data and accounting for the magnitude and timing of the preceding high flow conditions that was necessary to fit a significant model. We hypothesized that the controlling mechanism behind the significance of the high flow period had to do with nutrient bioavailability. Even though average nutrient concentrations from grab sampling suggest non-limiting conditions for phytoplankton growth in the Ohio River see , we knowWith the real-time flows among 25 sites used to derive the main predictors in the occurrence and persistence models coupled with the configuration of the visualization options for the continuous water quality data, the Ohio River cyanoHABs risk characterization tool provides a sound framework for assessing river conditions related to cyanoHAB ecology. The content organization of the Shiny app was based on the practices used by water quality managers to make decisions about when and where samples should be taken and to communicate potential risks of river conditions to stakeholders. When a user arrives at the Shiny app’s opening page, they can determine the bloom occurrence probability of all sites simultaneously. Clicking on a site of interest adjusts the embedded data window and provides options for visualizing data trends. If a site on the entry page returns a concerning bloom prediction, the “Water Quality” tab can be selected to visualize recent trends at the site and relative to what is being reported at other sites or has been observed in previous years from the same site. These comparisons offer additional diagnostics for gauging risk and help to inform appropriate management actions. Finally, using the Shiny app offers river managers a means of studying river conditions within the context of factors known to be important to cyanoHAB dynamics. This fosters experiential learning that can be used to guide updates and tool improvements over time.Supplementary File


In bread, the negative association of kernel intactness and resistant starch with GI was seen in vitro but not in vivo. Bread GIiv values were: roller milled flour, 67.4; stoneground flour 61.1; kibbled grain, 53.0; kibbled + intact kernel, 49.5; but correlation with clinical values was low (R2 = 0.47), and variability in the clinical results was high . Low glycaemic potency of wheat by minimal processing was achieved by maintaining particle size, avoiding hydrothermal treatment, avoiding crushing and using a food matrix requiring little chewing for ingestion. Use of in vitro digestive analysis for high precision measurement of starch fractions with potential secondary health benefits was validated by accurate concurrent prediction of the glycaemic index but needed to account for effects of chewing.Eight wheat products differing in texture (porridge vs. bread), grain fineness , and cooking (raw vs. cooked), with pre-measured glycaemic indexes (GI), were analysed by in vitro amylolytic digestion to determine effects of processing to reduce GI on quantities of starch fractions differing in digestibility. The accuracy and precision of the in vitro analysis was assessed from its ability to concurrently predict clinical GI. In porridges, kernel intactness and lack of cooking reduced GI while increasing Type 1 (inaccessible) and Type 2 (ungelatinised) resistant starch. Porridge in vitro GI values (GI Two recent papers showed how different degrees of cooking and milling of wheat grain could be used to modify the glycaemic response in eight products ,2. AssocHaving timed the in vitro and in vivo responses for two texturally contrasting types of wholegrain products, porridges, and bread, also provides an opportunity to examine the effect of product type on glycaemic analysis. Porridges, such as mueslis, are grains ingested as slurries, require little chewing to swallow, and their starch component is derived wholly from partially intact grain fragments. In bread, on the other hand, grain particles are embedded in a relatively dry starch-based matrix which requires chewing and bolus formation to be easily swallowed. The inhibitory effect of coarse wheat grain structure on the glycaemic response to bread is largely eliminated during normal ingestion because of the effects of mastication . TherefoA number of factors determine the effects of minimal processing on starch digestibility and have been exploited to make products of reduced glycaemic potency . Native Establishing a valid association between changes in starch fractions and glycaemic impact would require measurement of starch digestion in the same material as was responsible for the glycaemic response, with a sampling of intestinal contents. However, if an in vitro digestion method used to determine starch fractions releases digestion product at a rate that correlates closely with the glycaemic response, it is likely to provide an accurate estimate of the starch fractions in vivo associated with the response and would validate the method. A method based on clinical data has been developed. It provides simulated blood glucose response curves by progressively subtracting estimated blood glucose disposal from available carbohydrates released in the course of in vitro digestion . The areAn aim of the present research was to determine how the content of nutritionally relevant starch fractions in wheat grain products would change as a result of processing to reduce the glycaemic potency of the products. A second aim was to test the correlation between GIs of the products predicted from in vitro digestion and GIs determined from the incremental area under the postprandial blood glucose response curves, as a test of the accuracy of the in vitro digestive analysis, and as a concurrent validation of the starch, fractions measured. A further aim was to determine whether the type of product in which kibbled wheat structure was ingested affected the survival of the low glycaemic and resistant starch properties of the kibbled grains. Two auxiliary experiments were conducted on the effects of cutting, crushing and cooking, respectively, on the digestibility of starch in cereal grains to demonstrate the effect of the individual physical processes on the glycaemic potency of cereal products. Finally, the research illustrates how processing steps taken to reduce the glycaemic impact of cereal products may lead to secondary changes in starch fractions considered to be of potential benefit to gut health .Four bread and four porridge samples  were pre1.The cooked porridges were made by heating the grain preparations in water at 85 °C for 15 min and served at 65 °C to prevent starch retrogradation. The porridges were served with yoghurt in portions required to deliver a 50 g carbohydrate dose The in vitro digestion has been described in detail elsewhere . BrieflyThe digestions were carried out in duplicate in 70 mL specimen pots  placed in a custom-built heating block on a 15-place magnetic stirrer. A sub-sample (2.5 ± 0.01 g) of each bread was accurately weighed into each pot, 30 mL of water added, followed by 1.0 mL of 1 M HCl and 1 mL of 10% pepsin protease  dissolved in 0.05 M HCl. The samples were incubated at pH 2.5 and 37 °C for 30 min to simulate gastric digestion.3 and 5 mL of 0.2 M Na maleate buffer pH 6.3 and made to the full (53 mL) mark with distilled water. Intestinal digestion, during which carbohydrate digestion was monitored, was initiated by adding 0.1 mL of amyloglucosidase  and 1.0 mL of 1% pancreatin  solution in maleate buffer to the digestion pots, which were stirred at 130 rpm and 37 °C. Samples (0.5 mL) were removed to ethanol (2.0 mL) at 0, 10, 20, 40, 60 and 120 min and immediately mixed to stop digestion and precipitate any undigested starch. At 120 min, the digests were homogenised (Omni-GLH with an S18N-19G dispersing tool) to a slurry in the digestion pots, a further 0.1 mL of amyloglucosidase added, and the samples digested for a further 30 min to capture any starch protected by food structure before a final 0.5 mL sample was removed to ethanol.The intestinal phase was initiated by adjusting the samples to pH 6.3 by adding 2 mL of 1 M NaHCOThe ethanolic samples were centrifuged and a sub-sample of the supernatant given a secondary digestion, with 1% amyloglucosidase and 1% invertase, to simulate brush border processing and convert all 80% ethanol-soluble fragments from digestion of starch  to glucose for analysis. Soluble sugars were then measured as grams of glucose equivalents (GE) using the standard dinitrosalicylic acid colourimetric procedure .Starch fractions determined in the above analysis were:Rapidly digested starch (RDS)—starch digested in vitro up to 20 min. RDS refers solely to the time period of digestion, although it will contain glucose from starch species that differ in their intrinsic rates of digestion.Slowly digested starch (SDS)—starch digested between 20 and 120 min in vitro.Resistant starch Type 1(RS1)—starch that is not digested because amylase access is restricted by food structure. It was measured as the increase in sugar release at 150 min when the sample had been given secondary digestion after homogenising at 120 min.Resistant starch Type 2 (RS2)—starch resistant to digestion as a result of native molecular structure. Hydrothermal processing converts Type 2 RS to digestible gelatinised starch as long as the starch is able to hydrate. RS2 was measured as difference between total starch in the fully cooked dispersed as the difference between total starch (TS) digested in the dispersed fully cooked sample and the sum of the preceding fractions (RS2 = TS − (RDS + SDS + RS1)).iv) was determined from the digestion curves as previously described [iv of the product:In vitro glycaemic index  The role of cutting and crushing in digestibility of cooked wheat grain; (B) The role of cooking in digestibility of crushed grain, using wholegrain rolled oats.In Experiment A, wheat grains were allowed to hydrate overnight and cooked for 10 min in a glass tube in a boiling water bath. The cooked grains were digested individually either intact, sliced equatorially with a sharp razor blade, or crushed to 1 mm between glass sides, with 5 grains for each treatment. A scaled-down version of the in vitro digestion (above) was used with sugar release measured at 20 min (RDS), 120 min (RDS + SDS) and after homogenising with further digestion (RDS + SDS + RS1).In Experiment B commercially available wholegrain rolled oats  were digested using the full-scale in vitro digestion procedure  either as is or after cooking for 10 min in a boiling water bath for determination of RDS, RDS + SDS, and after homogenising, total starch.Mean values were calculated for all time points in the digestion. Precision of the analyses was estimated as the mean absolute deviation between duplicate values for sugar release per 100 g sample.Values for total potentially available carbohydrates in the products were obtained from the 150 min value for the homogenised cooked version of the product (bread or porridge) made from the finest particle size flour. This would correspond to a standard available carbohydrate analysis.GI) were calculated from the given SDs of the IAUC values used to calculate GI using the formula:Standard deviations (SD) of the clinical GI values  but not for the bread   . For allThe in vitro GI values were determined with much greater precision than the clinical values because they were unaffected by individual differences in glycaemic responsiveness.2 = 0.99, y = 1.03x − 2.05), but less closely for the bread . Across all products the percentage of starch that was RDS was a strong predictor of both the clinical and in vitro GI values .RDS as a percentage of TS (x) closely predicted clinical GI (y) for porridges  grain that has been crushed during rolling. In the form of muesli, its starch was only slowly digested, but upon cooking as hot porridge, there was a large increase with in vitro digestible starch fractions B, with RThe results have shown the extent to which increased particle size and reduced gelatinisation, which may be part of minimal processing of whole grains for reduced glycaemic potency, lead to changes in starch fractions that could have secondary health benefits. Amongst these changes, an increase in RS may make an important contribution to gut health through its prebiotic effects in the colon, with numerous secondary health benefits . ProcessA typical serving of porridge or muesli containing 50 g of wholegrain, with a “carbohydrate”  content of about 35 g would, according to An important advantage of consuming resistant starch in the form of intact and partially intact cereal kernels in whole grain products is that it is accompanied by bran. While RS may improve gut health by acting as a prebiotic substrate for the microbiota , it addsThe two supplementary experiments on cooked wheat kernels A and rolThe high correlation between in vitro and in vivo GI values for the porridges suggests that in vitro digestion gives a true indication of the functional states of starch in vivo because the starch fractions and the in vitro GI values were based on the same starch digestion data. Furthermore, the physical form in vivo and in vitro would have been similar because bolus formation was not required for ingestion. The reason for the weaker correlation between the in vitro and in vivo GI values in the bread is probably because the population density of wholegrains was reduced by the flour-based bread matrix, The “hydrothermal” process of baking would have converted most of the RS1 to RDS, and the data were subject to large errors intrinsic to clinical GI determinations . The cliIn an earlier study of bread containing fully hydrated, cooked, coarse grain, swallowing the bread without chewing lowered glycaemic impact, but the effect was largely eliminated when the bread was ingested normally . PresumaThe results of the present study indicate that if coarsely kibbled grains are used to lower the glycaemic potency of foods, the foods containing them should be moist enough or textured to avert the need and urge to form a well-chewed bolus containing crushed particles before swallowing. Secondly, as RS2 formed such a substantial portion of the products with the lowest in vitro and clinical GIs , Table 22 = 0.88, consistent with the value of R2 = 0.90 obtained with 24 carbohydrate foods of diverse types [The present study has also shown how in vitro digestive analysis can be a useful tool in developing whole grain products for low glycaemic potency and/or improved prebiotic potential based on restricting the conversion of RS to digestible starch. The correlation between in vitro GI and clinical GI across all eight products was Rse types , and witse types . The preHowever, as the present study has shown, if in vitro digestion is to be used to gauge the relative glycaemic impact of whole grain foods, such as experimental preclinical cereal products, it is important that the food be disintegrated in a way that accurately replicates the physical effects of ingestion, such as crushing . For fooIf the in vitro procedure is to be applied beyond simple wholegrain cereal products, to produce low glycaemic impact composite products, the influence of food components that affect gastric emptying rate may need to be considered. Components such as fat, highly viscous polysaccharides, and organic acids may need to be made constant across comparisons so that effects of cereal and starch structure are not confounded by separate effects of food components on digestion and glucose absorption. Once the role of cereal structure has been defined under controlled conditions, other factors may be included to determine their additional contribution to an overall improvement in the nutritional attributes of a product.This paper, by showing the substantial impact of factors, such as particle size, pre-hydration, cutting, crushing, and gelatinisation on the glycaemic potency of wholegrains, has highlighted a set of variables that can be systematically varied during minimal processing of grains. The products generated could create a matrix of minimally processed wholegrains for use in numerous composite products of predictable glycaemic impact.The present study suffered from some limitations imposed by the fact that the primary aim of the study was to measure the effects of milling and cooking of wheat on human glycaemic responses to wheat products, with a necessarily limited number of samples. The design of the clinical study meant that changes in starch fractions and glycaemic responses as a detailed function of isolated processing variables could not be determined. However, the availability of the clinical data and the products responsible provided an excellent opportunity to retest in vitro digestive analysis and obtain some explanation for the clinical effects in terms of the effects of processing on starch fractions.Minimal processing to preserve structure in kernel fragments and whole grains, and molecular organisation in starch granules, may moderate the rate of digestion of starch in cereal products, leading to low glycaemic potency and an increase in resistant starch fractions. A dual benefit of improved glycaemic control and improved gut health may therefore be obtained. In vitro digestive analysis can be an accurate and economical tool in the preclinical development of healthier wholegrain products, provided the effects of in vivo ingestion on food structure are allowed.


The skin epidermis and appendages undergo ongoing renewal throughout life. Stem cells residing in the epidermis and hair follicles are pivotal for sustaining skin homeostasis. The self-renewal ability of stem cells significantly decreases during skin aging but actively increases during wound repair. Residential stem cells reside in niches that provide spatially distinct microenvironments for stem cell maintenance and function. Cell-extracellular matrix (ECM) adhesion is essential for the establishment of niche architecture. Collagen XVII (COL17), as a transmembrane protein constituting hemidesmosomes (HDs), mediates the interactions of stem cells with surrounding cells and the matrix to regulate skin homeostasis, aging and wound repair. This review focuses on the pivotal role of the niche component COL17 in stem cell maintenance and its function in regulation of skin aging and wound repair. The transmembrane protein collagen XVII (COL17/BP180/BPAG2) is a structural component of hemidesmosomes (HDs). It was first named BP180 due to its initial discovery in bullous pemphigoid (BP) in the 1980s COL17 is mainly expressed by basal keratinocytes, and its ICD binds to keratin intermediate filaments through plectin and BP230 COL17 has been reported to be involved in multiple skin diseases. In autoimmune skin diseases, such as BP, mucous membrane pemphigoid, gestational pemphigoid, and linear IgA bullous dermatosis, COL17 acts as an autoantigen Since the skin of JEB patients is also characterized by skin atrophy and fragility, alopecia, dyspigmentation, and delayed wound healing The skin shows profound structural and functional changes with age, including epidermal and dermal thinning, loss of dermal elasticity and wrinkling, and greying and loss of hair Accumulating evidence has confirmed that cell-cell and cell-extracellular matrix adhesion is essential for the establishment and maintenance of niche architecture high ESCs are constantly outcompeting COL17A1low cells to eliminate stressed or unfit cells from the skin. Lineage tracing for aged skin presents an expanding single-type cell clone consisting of noncompetitive COL17A1-MCM2- cells in the basal layer instead of the conspicuous heterogeneity of COL17A1 expression in young skin. Cell competition is possibly driven by the two types of cell divisions mediated by differential COL17A1 expression. COL17A1low cells exhibit an increased ratio of perpendicular cell divisions to generate a basal cell and an apically located differentiating suprabasal cell, whereas COL17A1high cells divide in parallel to generate two identical basal daughter cells. Therefore, the COL17-mediated symmetric cell divisions (SCDs) mechanistically push out COL17A1low cells by causing a reduced number of hemidesmosomes allowing for microdetachment from the basement membrane. In addition to the cell competition theory, COL17 can directly increase the self-renewal capacity of epidermal cell colonies by increasing the ratio of parallel divisions The exact function of COL17 in aging was recently reported The mechanism by which COL17 regulates cell division is discussed in subsequent studies Collectively, age-induced COL17 proteolysis leads to the imbalance of SCDs and ACDs in ESCs through PKC signaling, resulting in the loss of cell competition, self-renewal capacity, and stem cell maintenance, ultimately causing age-associated epidermal atrophy and fragility and nonhealing wounds, which will be discussed in the following section.Hair loss is one of the common manifestations of skin aging HF aging features miniaturization of hair follicles and the absence of dermal papillae, sebaceous glands, and the infundibulum. Preceding HF miniaturization is stem cell dysregulation, including the loss of HFSC markers and HFSC maintenance markers. Similar to ESCs, HFSCs are committed to epidermal terminal differentiation and keratinization during aging; these cells are distributed in the junctional zone located above the bulge area; they then move to the suprabasal epidermis and eventually to the skin surface to be eliminated from the stem cell pool and from the skin The expression of COL17 in HFSCs has been observed. COL17 is specifically reduced in aged quiescent HFSCs, and the expression and distribution of non-HD COL17A1 in HFSCs changes greatly during aging, negating the contribution of HD COL17-induced skin detachment in hair loss MSCs are pigment-producing melanocytes residing around ESCs and HFSCs in the interfollicular epidermis or the follicular bulge-subbulge area COL17 deficiency induces relatively mild dyspigmentation in the tail skin, as well as hair greying. However, COL17 is not expressed in MSCs, indicating the indirect effect of COL17 on MSCs. As envisaged, differentiated melanocytes colocated with differentiated ESCs and HFSCs exist early in preaging skin and hair follicles, owing to the niche created by ESCs and HFSCs through TGF-β signaling Wound healing occurs through distinct overlapping phases: hemostasis, inflammation, proliferation, and remodeling Stem cells are mainly involved in three biological processes during re-epithelialization, including stem cell migration and proliferation, stem cell population dynamics, and stem cell plasticity Apart from preaging of skin, decreased wound closure is induced by COL17 KO and increased wound closure by inhibition of COL17 shedding high subcluster with top markers, such as COL17A1 and TP63, a gene enriched in quiescent bulge HF stem cells, is present in both wounded and unwounded skin. The COL17A1high state scores the lowest for inflammation and EMT genes but the highest for genes of a “quiescence and sternness” signature derived from tissue quiescent stem cells, which are more quiescent, persist longer and could give rise to more rapidly cycling committed progenitors with a shorter lifespan. This portion of cells differentiates directly or indirectly into spinous cells or a specific pool of proliferating basal cells, thus maintaining skin renewal during homeostasis. In wound repair, such cells differentiate into suprabasal cells at the wound periphery or convert into cells of higher motility and subsequently migrate into the wound.In single-cell analysis research Two kinds of colonies are formed in human keratinocyte culture, expanding colonies and stacking colonies The migration of epidermal basal cells requires the remodeling of the cell-cell and cell-substratum contacts to allow the cells to detach from the intact basement membrane in the unwounded epidermis. As the cells move over the wound, they degrade the provisional matrix while depositing new matrix proteins, including laminin-332. In keratinocytes, matrix proteins and their receptors are clustered into two distinct protein complexes: focal adhesions and hemidesmosomes The features of cell motility can be roughly grouped into the velocity and the directions of migration For studies in which the velocity shows no correlation with COL17, the directions of migration are considered. To migrate efficiently in a directed manner, cells must establish and maintain an asymmetric morphology with defined leading and trailing edges In addition to the different objects of regulation, whether COL17 itself or COL17 shedding plays a role in wound repair has been discussed. A scenario can be visualized in the wound in which shedding of the COL17 ectodomain loosens or releases the cell from some of its present binding partners and allows it to embark on other functions, as required Thus far, it is not clear whether the lack of soluble ectodomain, the concomitantly generated membrane-tethered endodomain of COL17 or the combination of both caused accelerated re-epithelialization in nonshedding COL17 skin. There is evidence that the released ectodomain has a direct suppressive effect on keratinocyte motility because 1) deposits of the highly stable released COL17 ectodomain are mainly found in the trails of migrating cells incorporated with laminin-332, leading to stabilization of the substrate and cell immobilization but directed cell motility In terms of the upstream regulatory mechanism of COL17, BP-IgG in BP induces the internalization of COL17 to inhibit its function and reduce cell motility As introduced above, the active restoration of stem cell pools by the use of residual intact stem cells before the irreversible advancement of tissue architectural changes may be essential for successful antiaging and tissue regeneration in the skin. COL17, as a good marker for stem cells reflecting individual cellular potential and quality for self-renewal, represents an interesting therapeutic target in antiaging and wound treatment.in vitro study showed that the forced maintenance of COL17 rescues both epidermal thinning and hair loss in aged mice for more than 24 months, suggesting that the stimulation of COL17 expression is a potential therapy for skin aging An For patients with COL17 deficiency, genetic therapy has been introduced to solve the skin aging problem Regarding wound repair, forced expression of COL17 by basal keratinocytes in mice promotes wound healing Collectively, upregulation of COL17 is a common solution for skin rejuvenation and wound healing. Therefore, studies on the mechanism of pre- and posttranscriptional regulation of COL17 set the basis for more therapeutic strategies. Moreover, the development of synthetic peptides that stimulate COL17 synthesis and high-throughput screening for drugs that upregulate COL17 and subsequent clinical trials of such drugs will promote the clinical translation of these findings.Taken together, this body of work provides previously unappreciated insights into the cellular and molecular mechanisms by which COL17 regulates skin aging and wound repair, focusing on the perspective of stem cell niches.high state in lineage tracing of wounded skin, the exploration of the function of COL17 in stem cell plasticity will be an important challenge for the future.However, many open questions remain regarding the detailed upstream mechanisms modulating COL17 level. We know little about the signaling pathway regulating the gradual or rapid decline of COL17 during aging and the high expression and distribution of COL17 to the wound edges. Downstream mechanisms also remain to be discovered. It is also necessary to elucidate how the multiple mechanisms introduced above interact with each other; for example, whether the functions of HD COL17 and non-HD COL17 overlap or are independent and the paradoxical role of COL17 in the regulation of cell migration has not yet been verified. In addition to the deepening of existing studies, the research direction can be further broadened. For instance, considering the regulation of COL17 on directed cell migration and its distribution to the wound edge, it will be interesting to further detect the potential role of COL17 in collective cell migration. Moreover, due to the dynamic change in COL17 expression and the return of cells in differentiated states to the COL17A1When the functions and mechanisms of COL17 are fully studied and corresponding clinical trials are performed, it will be possible to propose future therapeutic strategies for skin aging and wound repair.


The unfolding COVID-19 pandemic, and the unprecedented social and economic costs it has inflicted, provide an important opportunity to scrutinize the interplay between the resilience of small and medium-sized enterprises (SMEs) and the resilience of the communities they are embedded in. In this article, we articulate the specific ways that SMEs play a crucial, and underappreciated role in building resilience to human and natural hazards, and provide new opportunities to accelerate the adoption of sustainability practices through the configuration of ‘enabling ecosystems’ geared towards promoting sustainability in the private sector. We argue that capacity-building and experimentation are not only required within companies, but also throughout this emerging supportive ecosystem of policies, resources , governance actors, and intermediaries to adequately focus investment, technical capabilities and innovation. Ultimately, we call for a new transdisciplinary action research agenda that centers on SMEs as pivotal actors and amplifiers of community resilience; while recognizing that these firms are themselves in need of support to secure their own capacity to respond to, and transform in light of, crises. This research program calls for recognizing and applying the lessons that the pandemic presents to the urgent need for accelerated climate action. This will be enabled by developing more targeted approaches to collaborative capacity-building activities in SMEs that feed into experimentation and allow for the accelerated adoption of deliberate and strategic resilient business practices and models. An emerging agenda for policy and practice should center on SMEs as pivotal actors and amplifiers for community resilienceCapacity must be built within the supportive ‘ecosystem’ of policies, resources (i.e. finance) governance actors, and intermediaries, not simply within SMEsTo enhance the transformative potential of SMEs, support programs and policies should target the underlying business model rather than only on resource efficiencyThe impact of the COVID-19 pandemic on small and medium-sized enterprises (SMEs)Similar challenges occur in the context of disasters and natural hazards, which are becoming more frequent and severe in a changing climate. SMEs are disproportionately affected by environmental risks compared to larger firms due to financial constraints, limited management and human resources, focus on short-term planning and survival with a hierarchical management culture, and lack of time and/or skills to prepare for environmental challenges , governance actors, and intermediaries. SMEs act as pivotal amplifiers that can enhance resilience in the communities they serve and are embedded in. While many definitions of community exist, we think of community as the dynamic interplay among the place-based physical and natural environment, and the constellation of civil society organizations, individuals, government actors, and broader private sector entities within which a business operates  enable SMEs to access partners’ innovation resources and overcome SMEs’ intrinsic resource constraints, while developing resilience-specific innovations within and beyond the SMEs , universities , or consultancies   case studies on resilient SMEs and their pathway to resilience (cf. Burch et al. Transdisciplinary scholars will need to engage in more meaningful and sustained ways with SMEs to better understand the steps, sequences, and limits in their pursuit of sustainability and resilience-building practices.


Annals of Clinical and Translational Neurology, and I had the privilege of being the first editor‐in‐chief. The beginning of the journal is so fresh in my mind that it is amazing that more than 9 years have passed, and that the time has come to pass the baton to a new EIC. In an subsequent editorial the next EIC, Ahmet Hoke, will introduce himself and outline his vision and plans for the future of the journal.In 2013, the ANA launched a new open‐access journal, the ACTN was blessed with the imprimatur and prestige of the ANA, the support of its sister journal, the Annals of Neurology, and an exceptional group of founding associate editors who built the journal. I would like to thank the first group of editors, Flint Beal, Andrew Cole, Anne Cross, Leon Epstein, Ahmet Hoke, and Steven Warach, for their efforts in making ACTN a success. ACTN also was fortunate to be able to attract Emmanuelle Waubant, Puneet Opal, Jennifer Graves, Raquel Sanchez‐Valle, and Sean Savitz as editors who maintained the editorial excellence established at the onset of the journal.Like all new ventures, the beginnings for ACTN were uncertain with no guarantee of success either academically or fiscally. Convincing authors to submit strong manuscripts to an unknown journal with an unclear future was difficult. But ACTN is now a well‐established and recognized journal that helps to position the ANA well for the open‐access future of publishing. Its success reflects the support and contributions of many people to whom I am grateful. First, I would like to thank the ANA Board and Eva Feldman, the President when ACTN was conceptualized, for giving me the opportunity to be the EIC. I have been fortunate that there has been unwavering support from both the ANA Board and our Editorial Board over the past 9 years. I also would like to thank Clif Saper, and more recently Ken Tyler, for their help and insights as EIC of the Annals of Neurology since the relationship between the two journals was an instrumental part of the success of ACTN. In 2016 a unique and now popular interactive clinical case series, InterACTN, was started with Danny Bega as the editor, and I believe that InterACTN's success will lead to additional interactive features in the coming years. We also have been fortunate to have the support and expertise of our publisher, Cathy Krendel, and the staff at John Wiley and Sons as well as our assistant editors, Emily Hammond and Keegan Brewster. ACTN also benefited greatly from the expertise of Kathleen Gaffney, an outside editorial consultant who helped negotiate our new publishing contract, and of Morna Conway who coordinated the selection of a new EIC and who is overseeing the transition. And I am deeply grateful to the cadre of Reviewers who helped to assure the quality of what we publish, and I am particularly indebted to those Reviewers who helped before ACTN was well recognized by the neurological community.My experiences as EIC have been exciting, complicated, arduous, sometimes frustrating, occasionally painful, but always personally rewarding. I am very fortunate to have had the opportunity to help establish the journal. I am deeply invested emotionally in ACTN's success, and it was very important to me for the ANA to appoint a new EIC who can continue to expand the footprint and impact of the journal. I am delighted that someone as outstanding as Ahmet Hoke was chosen to lead ACTN into the future.


Purpose: Neuroinflammation often initiates iron overload in the pathogenesis of neurological disorders. Chemokine-driven neuroinflammation is required for central sensitization and chronic allodynia following fractures, but specific molecular modulations are elusive. This present study explored whether hydrogen-rich saline, as one potent anti-inflammatory pharmaceutical, could alleviate fracture-caused allodynia by suppressing chemokine CXCL1 expression and iron overload. Methods: A mouse model of tibial fracture with intramedullary pinning was employed for establishing chronic allodynia. Three applications of hydrogen-rich saline  were administrated intraperitoneally on a daily basis from days 4 to 6 following fractures. Spinal CXCL1 and its receptor CXCR2 levels, transferrin receptor 1 (TfR1) expression and iron concentration were examined. Recombinant CXCL1, a selective CXCR2 antagonist and an iron chelator were used for verification of mechanisms. Results: Repetitive injections of hydrogen-rich saline (5 and 10 mL/kg but not 1 mL/kg) prevent fracture-caused mechanical allodynia and cold allodynia in a dose-dependent manner. Single exposure to hydrogen-rich saline (10 mL/kg) on day 14 after orthopedic surgeries controls the established persistent fracture allodynia. Furthermore, hydrogen-rich saline therapy reduces spinal CXCL1/CXCR2 over-expression and TfR1-mediated iron accumulation in fracture mice. Spinal CXCR2 antagonism impairs allodynia and iron overload following fracture surgery. Intrathecal delivery of recombinant CXCL1 induces acute allodynia and spinal iron overload, which is reversed by hydrogen-rich saline. Moreover, iron chelation alleviates exogenous CXCL1-induced acute pain behaviors. Conclusions: These findings identify that hydrogen-rich saline confers protection against fracture-caused chronic allodynia via spinal down-modulation of CXCL1-dependent TfR1-mediated iron accumulation in mice. Due in large part to an increasing amount of traffic trauma and osteoporosis, the financial expenditure and epidemiological incidence of bone fractures are growing worldwide . FracturInteraction between chemokines and their receptors is considered one of the most pivotal steps in the neuroinflammatory responses underlying the pro-nociception-related process ,9,10. ChDysregulation of iron homeostasis is central for nociceptive synaptic transmission in remifentanil-induced hyperalgesia and fracture allodynia ,16. IronRecent advances have updated our understanding in the reduction of inflammation, oxidative insult and infection by molecular hydrogen in several pathological conditions . IntriguThis study investigated the possible properties of intraperitoneal (i.p.) hydrogen-rich saline in fracture-caused chronic allodynia using the mouse model of tibia bone fractures with orthopedic surgeries. The CXCL1/CXCR2 expression, iron content and iron metabolism-related protein transferrin receptor 1 (TfR1) in the spinal dorsal horn were evaluated for the verification of anti-nociceptive mechanisms of hydrogen-rich saline. Additionally, recombinant CXCL1, a selective CXCR2 antagonist and an iron chelator were employed to identify the interaction between CXCL1/CXCR2 and iron hyper-concentration in nociceptive transmission.Adult C57BL/6J mice  were raised in an artificially regulated 12 h light/dark environment at 23 ± 2 °C with free access to food and water. All animals were provided from the experimental animal center of the Chinese Academy of Military Medical Science. All experimental studies and protocols were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethical and Welfare Committee of The Affiliated Hospital of Qingdao University .The mouse model of tibial fracture associated postoperative allodynia was established as in previous reports ,21. In sHydrogen-rich saline was produced as in a previous description . HydrogeA selective CXCR2 antagonist SB225002  and recombinant CXCL1  was dissolved in 0.9% normal saline and administration was carried out via intrathecal injection. Deferoxamine  was dissolved in 1% dimethyl sulfoxide  for intrathecal injection. Under brief anesthesia with sevoflurane, drug injections with 30G needles through intrathecal routes were conducted at the L4-5 spinal segment .The baseline peripheral mechanical and cold sensitivity were tested 1 day before any experimental treatments, and all animals were habituated 2 h per day in the testing circumstance for 3 days prior to the basal pain examinations. For evaluating mechanical allodynia, the plantar surface of the left hind paw underwent stimulation with perpendicularly presented von Frey hairs, with exponentially increasing stiffness from 0.02 g to 2.56 g; Stoelting, Wood Dale, IL, USA). Then, 50% PW’s mechanical threshold was determined by the Dixon’s up and down method ,21. For g for 10 min and the supernatant was collected. BCA Protein Assay (Pierce) was employed to determine protein concentrations. For each reaction in a 96-well plate, 100 μg of proteins from the samples was used. All ELISA experiments followed the manufacturer’s protocol. The optical densities of samples were measured using an ELISA plate reader  at a wavelength of 450 nm and the levels of CXCL21 and CXCR2 were calculated using the standard curves and normalized to the total protein levels.An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of CXCL1 , and CXCR2  in the L4-5 levels of left spinal cord . Spinal All the animals were sacrificed under deep anesthesia of sevoflurane (3%). The left L3-5 segments of spinal dorsal horn were removed rapidly and homogenized in ice-cold RIPA buffer containing PMSF . The lysate was centrifuged, and the supernatant was collected as the total protein. The protein content was determined using the bicinchoninic acid assay method. The equivalent amount of proteins was resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane and probed with monoclonal mouse anti-β-actin antibody  and polyclonal rabbit antibody against transferrin receptor 1 , followed by incubation with horseradish peroxidase-conjugated secondary antibodies . The membrane-bound secondary antibodies were visualized with enhanced chemiluminescence  and quantified using Gene Tools Match software .4.The iron content of the spinal dorsal horn was detected by flame atomic absorption spectrophotometer  at 248.3 nm with the digestion of tissues . To obtap < 0.05.All data were analyzed with SPSS 19.0 . Results are shown in box-and-whiskers plots with the “box” depicting the median and the 25th and 75th quartiles and the “whiskers” showing the 5th and 95th percentiles. Individual data points were superimposed on the box-and-whiskers plots. Behavioral data analysis was carried out by two-way ANOVA with post hoc Bonferroni test. Differences of biochemical data were compared using one-way ANOVA with post hoc Bonferroni test. The criterion for statistical significance was p > 0.05, n = 6, p > 0.05, n = 6, First, there were no significant differences in basal mechanical sensitivity and cold response to acetone between sham and fracture animals  daily on days 4, 5 and 6 (in the early phase) following orthopedic operations. Von Frey and the acetone tests revealed that hydrogen-rich saline at the dose of 5 and 10 mL/kg (but not 1 mL/kg) prevented the production of fracture-caused mechanical allodynia and cold allodynia, as demonstrated by the long-lasting increase in paw withdrawal mechanical threshold  = 70.53, p < 0.05, n = 4, one-way ANOVA; p < 0.05, n = 4, one-way ANOVA; p < 0.05, n = 4, one-way ANOVA; In general, expression variations in chemokines and their receptors in the spinal dorsal horn are a key step for the pathophysiology of fracture-caused chronic allodynia developments ,25,26. Ep < 0.0001, n = 6, two-way ANOVA; p < 0.0001, n = 6, two-way ANOVA; p < 0.05, n = 4, one-way ANOVA; p < 0.0001, n = 6, two-way ANOVA; p = 0.0226, n = 6, two-way ANOVA; To investigate whether CXCL1/CXCR2 cascade is important in chronic allodynia phenotypes following fracture operations, the selective CXCR2 antagonist SB225002 was employed. First, mice received three intrathecal injections of SB225002 (10 μg) daily on days 4, 5 and 6 following tibial fracture and intramedullary pin insertion. No changes in basal mechanical and cold peripheral sensitivities were witnessed in sham mice with SB225002 administration A,B. Intrp < 0.05, n = 6, two-way ANOVA; p < 0.0001, n = 6, two-way ANOVA; p < 0.0001, n = 6, two-way ANOVA; p = 0.0113, n = 4, one-way ANOVA; p = 0.0004, n = 4, one-way ANOVA; Further, recombinant CXCL1 was utilized for determining whether CXCL1/CXCR2 cascade was implicated in nociception sensation and hydrogen-rich saline analgesia. Notably, intrathecal delivery of recombinant CXCL1 (100 ng) evoked a robust decrease in paw withdrawal mechanical threshold and increase in cold response to acetone in naive mice from 1 h to 12 h after spinal application  was intrathecally injected 1 h prior to recombinant CXCL1 (100 ng) administration. Interestingly, we found that DFO reduced exogenous CXCL1-evoked mechanical allodynia and cold allodynia, as indicated by a dramatic elevation of paw withdrawal mechanical threshold  = 49.4, The present study, for the first time, reports that hydrogen-rich saline alleviates tibial fracture-caused mechanical allodynia and cold allodynia through spinal reduction of CXCL1/CXCR2 expression and iron overload. Furthermore, it is indicated that pharmacological inhibition of CXCL1/CXCR2 attenuates chronic fracture allodynia through down-regulating TfR1-dependent iron overload in the spinal dorsal horn. Intrathecal  exposure to exogenous CXCL1 elicits acute allodynia behaviors and spinal CXCR2 over-expression and iron overload, which was impaired by systemic hydrogen-rich saline therapy. Moreover, spinal iron chelation prevents CXCL1-induced acute allodynia.The requirement of neuroinflammation in spinal dorsal horn for pro-nociceptive sensations has been well elucidated ,6,7. CheNeuronal iron accumulation is indispensable for the functional plasticity of excitatory glutaminergic synapses ,30,31. TIn spite of medical developments and clinical practice for many years, effective strategies for controlling chronic fracture pain remain insufficient ,26. OpioThis is the first study wherein repetitive applications of hydrogen-rich saline (5 and 10 mL/kg but not 1 mL/kg) prevent fracture-caused mechanical allodynia and cold allodynia in a dose-dependent manner. Single delivery of hydrogen-rich saline (10 mL/kg) is effective against the established fracture allodynia. Moreover, systemic hydrogen-rich saline interventions reduce the CXCL1/CXCR2 expression and TfR1-dependent iron accumulation in the spinal dorsal horn of mice with chronic fracture allodynia. Additionally, hydrogen-rich saline treatment impairs exogenous CXCL1-evoked acute allodynia behaviors and spinal iron overload. These results elucidated for the first time that systemic hydrogen-rich saline therapies protect against fracture-caused chronic allodynia via inhibiting CXCL1/CXCR2-mediated neuroinflammation and TfR1-dependent iron overload, suggesting that molecular hydrogen might be utilized for developing potential therapeutic strategies in pain conditions. However, certain concerns are raised regarding how hydrogen modulates CXCL1 cascades following fractures and orthopedic repairs. One limitation is that we did not evaluate the distribution of CXCL1, CXCR2 and TfR1 proteins in the spinal dorsal horn using immunohistochemistry staining in our fracture pain models, which should be addressed in future. In addition, previous reports revealed that divalent metal transporter 1 (DMT1) is one of the most important regulators in neural iron overload during opioid-induced acute hyperalgesia and fracture-caused chronic allodynia ,16,19. GIn summary, the present findings highlight an innovative pharmacological property of molecular hydrogen in the amelioration of tibial fracture-caused chronic allodynia through the spinal reduction of CXCL1/CXCR2 cascade-mediated TfR1-dependent iron overload. These data also suggest that hydrogen therapy and CXCR2 antagonism may be novel and neurotherapeutic strategies for fracture patients with chronic pain.


We ended 2013 with a historic achievement in Otorhinolaryngology. The flagship journal of the national scientific community has received an impact factor, a unique achievement in Latin America.The Brazilian Journal of Otorhinolaryngology (BJORL) has been in an upbeat path in the last decade. Having overcome the steps of successive indexations our challenge now reaches international standards. Our journal ceases to be a leading national publication to join the great journals of the world. To maintain and increase its impact factor, it will be necessary to attract relevant papers that generate citations in other publications. We will have to attract authors from other countries, as do most internationally known journals.Like the federal government, which implemented a program of sending about 100 thousand students abroad to encourage scientific production in BrazilThis is no simple task, considering the large number of existing journals and the tradition many of them have earned.Over the next 12 months, the BJORL will be regularly sent to 100 scientists and opinion leaders around the world, pursuing a higher international visibility strategy.In order to evaluate our potential, we perform an extensive comparative study between the BJORL traits and those from the leading international journals. Laryngology and voice was our field of choice.Between 2009 and 2013, we assessed the relevant publications in the BJORL and in the five most traditional journals in this field: Laryngoscope, Otolaryngology-Head and Neck Surgery, JAMA Archives of Otolaryngology-Head Neck Surgery, Journal of Voice and the European Archives of Otorhinolaryngology. These journals were selected by impact factor - obtained from the ranking list of impact factor provided by the journals themselvesWe read 1,487 abstracts published between January 2009 and November 2013, from the Laryngology sections of the Journals or those with laryngology-related titles from the journals that did not have such section.The abstracts were classified according to their main content within one of the following themes: Vocal Assessment, stem cells, wound healing, dysphagia, dystonia, benign disease, stenosis, diagnostic tests, growth factors, pathophysiology, phonomicrosurgery, cancer, laryngeal papillomatosis, vocal fold paralysis and pharyngolaryngeal reflux.The scientific production of the last five years was evaluated as to the type of study, level of evidence and country of origin, as well as its distribution by subject. The scientific evidence level of each paper was classified according to the Oxford Centre for Evidence-Based MedicineThe results revealed that Brazil is the second country in the generation of scientific papers in Laryngology and Voice, published in five selected newspapers, second only to the United States, accounting for 41% of the papers published during the assessment period. Brazil is ahead of European and Asian nations such as Germany, Japan, England, France and other traditional strongholds of scientific production .Figure 2From this statement we have excluded the papers published by the BJORL, considering that many countries also publish their own journals, where they publish much of their scientific production. Brazilian publications, besides the BJORL, are concentrated in the Journal of Voice, possibly due to its specificity in the subjects of the matter .Figure 3The number of laryngology publications did not vary much along the years in the study, showing a stable BJORL interest in the area .Figure 4The most publish subjects in the BJORL were neoplasia and vocal assessment. There were no publications on stem cells or growth factor in the study period .Figure 5The BJORL focuses on level-C-recommendation publications. Publications with level B recommendation focus on papers with evidence levels 2C and 3B. The main types of studies were retrospective descriptive; prospective descriptive and case reports. Others, less common types were case-control studies, experimental studies and epidemiological studies on prevalence. In comparison with the other five international journals, there is a higher number of Level-4 publications and no publication with Recommendation Level A in the BJORL .Figure 6Although this study was specific to the field of Laryngology, we believe that this information can be useful to guide future directions to our journal.We conclude that Brazil boasts a significant scientific output in laryngology; we are the second country in the number of publications in the most prestigious international journals. We need to qualitatively improve this large scientific production, producing more studies with evidence Levels A and B.Henrique Furlan Pauna Fernando Laffitte Fernandes Carlos Correa


The courses of starch digestion with individual α-amylase (AA), amyloglucosidase (AMG), and AA/AMG bi-enzyme system were performed and analyzed by first-order-reaction equations in the absence and presence of tannic acid (TA). An antagonistic effect between AA and AMG occurred at the digestion phase of readily-digestible starch due to the higher catalytic efficiency of AMG for starchy-substrates with more complex structures. This effect caused a faster rate of glucose production with AMG than with AA/AMG bi-enzyme system at this phase both in the absence and presence of TA. TA had a higher binding affinity to AA than to AMG as accessed by several methods, such as inhibition kinetics, fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking. Besides, differential scanning calorimetry (DSC) indicated that the change in the thermal and structural stabilities of enzymes in the presence of TA was related to the enzyme residues involved in binding with TA, rather than the inhibitory effects of TA. The binding characters of TA to both enzymes resulted in more “free” AMG without TA binding in AA/AMG bi-enzyme system than that in individual AMG. This binding property caused more and faster rate of glucose production at the digestion phase of slowly digestible starch (SDS) in the bi-enzyme system. However, the standard substrate of yeast α-glucosidase is manufactured as p-nitrophenyl-α-D-glucopyranoside (pNPG), instead of starch naturally-occurring in plants  is a polyphenol composed of 10 galloyl moieties linked with 1 glucosyl . It is aor of AA , 21.In this study, TA was selected to investigate its influence on starch digestion with the individual enzymes  and AA/AMG bi-enzyme system using a first-order reaction analysis. Then, the binding of TA to AA and AMG was characterized using inhibition kinetics, fluorescence quenching, ITC, DSC, and molecular docking methodologies to determine how the interactions inhibited starch digestion differently.Aspergillus niger, phosphate-buffered saline (PBS) tablets, and p-hydroxybenzoic acid hydrazide (PAHBAH) were purchased from Sigma-Aldrich Co. . Normal maize starch was obtained from Yuanye Biotech. Co. . A D-glucose assay kit (GOPOD-FORMAT) was obtained from Megazyme® (Megazyme E-AMGDF). Other chemicals were of analytical grade.Tannic acid, AA (EC 3.2.1.1) from porcine pancreas, AMG  from The courses of starch digestion with AA, AMG, or AA/AMG in the absence and presence of TA were carried out according to previous studies –24. Spec2CO3 to stop the reaction . The tubes containing the stopped reaction solution were centrifuged at 7,300 × g force for 10 min, and the supernatants were withdrawn for product analyses as follows.To initiate the digestion process, 2.5 ml of gelatinized starch was added to the mixture of TA and enzymes, and the reaction tubes were incubated at 37°C with a magnetic stirring speed of 600 rpm. As for starch digestion with the individual enzymes, AA or AMG was replaced by a buffer solution with the equivalent volume, respectively. Then, at each interval during the digestion process, some amount of reaction solution was withdrawn into a tube containing a solution of NaFor the starch digestion with AA, the reducing sugar contents in the supernatants were determined using the PAHBAH method . The fraCt is the fraction of digested starch at digestion time t; C∞ is the fraction of digested starch at digestion termination; k is the digestion rate constant; t is the digestion time.where k value, the equation  for a series of TA concentrations. In detail, starch digestion procedures were conducted with 4 ml of 10 mg/ml starch, 50 μl of TA and 50 μl of 4 U/ml AA for AA inhibition determination; with 2.5 ml of 10 mg/ml starch, 80 μl of TA and 80 μl of 14 U/ml AMG for AMG inhibition determination. At the digestion interval of 3 min (up to 12 min), 300 μl of AA catalyzing hydrolysate and 150 μl of AMG catalyzing hydrolysate were withdrawn for the determination of reducing sugar and glucose contents according to the PAHBAH and GOPOD methods, respectively. The initial digestion velocity (v) was obtained from the slope of the linear relationship between reducing sugar or glucose contents and the digestion time (t). Then, the inhibition (I) was calculated according to equation  were determined as described above with 4 ml of gelatinized starch , 50 μl of TA , and 50 μl of 2.5 U/ml AA for AA inhibition kinetics; with 2.5 ml of gelatinized starch , 80 μl of TA , and 80 μl of 11 U/ml AMG for AMG inhibition kinetics. Notably, the concentrations of TA and starch were selected according to the excepted linear characters of the equations applied in the inhibition kinetics analysis.Lineweaver–Burk, Dixon, and Cornish–Bowden plots were applied to describe the kinetics of inhibition. The values of initial digestion velocity  and the uncompetitive inhibition constant (Kiu) were obtained from the respective intersection points of Dixon  and the Michalis constant (Km) were obtained according to the intercept (1/vmax) and slope (Km/vmax) of Lineweaver–Burk equation  was obtained from the Stern-Volmer equation , the modified exponential Stern–Volmer equation  was appl follows , 32:A TA® NanoITC isothermal titration calorimeter instrument was used to detect the enthalpy changes caused by the binding interactions between TA and the enzymes , 34. SpeQi is the total heat collected after the injection i; V0 is the volume of the ITC sample cell (170 μl); [M] is the total concentration of enzymes; [P] is the total concentration of TA; n is the molar ratios of interacting molecules (stoichiometry); ΔH is the enthalpy changes; Kd is the equilibrium dissociation constant of TA-enzyme complex.where Td, °C) and denaturation enthalpy  required to fully denature the enzymes were obtained.The thermal stability of enzymes in the absence and presence of TA was determined by use of a TA® Q2000 differential scanning calorimeter , 34. SpeEb was calculated based on the equation  followed by Tukey's test (Graphpad Prism 6) was applied to analyze the significant difference between the selected constants. The data were considered statistically significant at k) at each phase were also calculated  and thus a higher catalytic activity of the respective enzymes. Interestingly, although both AMG and AA were able to digest starch substrate, the catalytic velocity of AA was higher than that of AMG (etc.)  , both ofk (digestion rate constant) in the presence of TA to that in the absence of TA suggests a higher inhibition effect of TA on starch digestion catalyzed by the enzymes. Therefore, the strongest inhibitory effect of TA was observed on starch digestion by AA among the three enzymes   increased with the increasing TA concentration  . The 1/vhibition . Besidesntration , suggestvmax and Km parameters, the Lineweaver–Burk kinetics analysis showed that TA competitively inhibited both enzymes , non-covalent π-π conjugations (stackings) between the aromatic rings of polyphenols and that of the enzymes would form. The non-covalent interactions are able to “cover” the fluorescence property of the enzyme fluorophores  against TA concentrations were shown with a character that concaved toward y-axis (kq) of TA were calculated. Notably, the kq value for a specific dynamic quenching is approximately 1 × 1010 M−1s−1, and the kq values of TA for AA and AMG quenching were 115 and 22 times of this value, respectively  of TA indicated that the binding interactions of TA with the fluorophores of AA were stronger than with that of AMG  caused by TA-enzyme binding interactions were obtained  for both the titration processes  of TA with the respective enzymes were calculated as the reciprocal of Kd (1/Kd)  to AA than to AMG was observed (n) that indicates the molar ratio of the ligand to the enzyme required to saturate the enzyme binding sites, as well as the entropy changes per molar titration (ΔSitc) that indicates the degree of the order of the ligand-enzyme complex system, are two constants highly related with the number of binding sites and enzyme molecular conformation    of AA and AMG were 92.54 and 76.49°C  of AA and two fluorescent residues (Tyr580 and Trp597) of AMG were involved in π-conjugations with the aromatic rings of TA, respectively, such as parallel conjugations (red dashed lines) and vertical conjugations (red full curves) (It was found that the 12 amino acid residues of AA were involved in interactions through hydrogen bondings (yellow dash lines) and π-conjugations (red lines and curves) with TA , compare curves) . These c curves) . In gene curves) .Eb) indicates the docking efficiency of a ligand to a protein  value for the titration of TA to AA as described in the ITC result .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.